CA2580313A1 - Insulin-oligomer conjugates, formulations and uses thereof - Google Patents

Insulin-oligomer conjugates, formulations and uses thereof Download PDF

Info

Publication number
CA2580313A1
CA2580313A1 CA002580313A CA2580313A CA2580313A1 CA 2580313 A1 CA2580313 A1 CA 2580313A1 CA 002580313 A CA002580313 A CA 002580313A CA 2580313 A CA2580313 A CA 2580313A CA 2580313 A1 CA2580313 A1 CA 2580313A1
Authority
CA
Canada
Prior art keywords
insulin compound
complex
insulin
pharmaceutical composition
modifying moiety
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002580313A
Other languages
French (fr)
Other versions
CA2580313C (en
Inventor
Balasingam Radhakrishnan
Diti Aggarwal
Michelle Ferro
Kenneth D. James
Navdeep B. Malkar
Mark A. Miller
Leo Pavliv
Karen Polowy
Monica Puskas
Nnochiri N. Ekwuribe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biocon Ltd
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to CA2910494A priority Critical patent/CA2910494C/en
Publication of CA2580313A1 publication Critical patent/CA2580313A1/en
Application granted granted Critical
Publication of CA2580313C publication Critical patent/CA2580313C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/28—Mercury; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The invention provides a complex including a cation and an insulin compound conjugate. The insulin compound conjugate includes insulin compound, such as human insulin or an analog thereof, conjugated to a modifying moiety, such as a polyethylene glycol moiety. The invention also includes solids and pharmaceutical compositions including such complexes, methods of making such complexes, and methods of using such complexes in the treatment of insulin compound deficiencies and other ailments. Further, the invention includes novel insulin compound conjugates and modifying moieties for use in making novel insulin compound conjugates. The invention also includes fatty acid compositions for administration of of pharmaceutical agents, such as the novel insulin compound conjugates, and/or the cation-insulin compound conjugate complexes of the invention.

Description

Insulin-oligomer conjugates, formulations and uses thereof 1 Related Applications This application claims priority to and incorporates by reference the entire disclosures of U.S.
Patent Application Nos. 60/589,058 filed July 19, 2004, 60/619,153 filed October 15, 2004, 60/632,578 filed December 2, 2004, and 60/655,838 filed February 24, 2005, and 60/655,803 filed February 24, 2005. This application also incorporates by reference the following applications filed herewith on 19-Jul-05 by Radhakrishnan et al.: U.S. Patent Application No.
11/184,668, entitled "Cation complexes of insulin compound conjugates, formulations and uses thereof"; U.S. Patent Application No. 11/184,594, entitled "Insulin-oligomer compound conjugates, formulations and uses thereof'; U.S. Patent Application No.
11/184,528, entitled "Fatty acid formulations for oral delivery of proteins and peptides, and uses thereof."
2 Field The invention relates to novel insulin compound conjugates in which an insulin or insulin analog is coupled to a modifying moiety. The invention also relates to cation complexes of such insulin compound conjugates and to pharmaceutical formulations including such insulin compound conjugates and/or modifying moieties.
3 Background Zinc complexed insulin compound is commercially available, for example, under the trade names HUMUL.IN and HUMALOG . Zinc complexed insulin typically exists in a hexameric form.

Various methods have been described for the use of zinc in the crystallization of acylated insulin.
For example, U.S. Patent Publication 20010041786, published on 15-Nov-01, by Mark L. Brader et al., entitled "Stabilized acylated insulin formulations" describes a formulation with an aqueous solution for parenteral delivery, particularly as an injectable formulation, with a pH of 7.1 to 7.6, containing a fatty acid-acylated insulin or a fatty acid-acylated insulin analog and stabilized using zinc and preferably a phenolic compound. U.S. Patent 6,451,970, issued on 17-Sept-02 to S~~~~~~~ ~ ~ ~~EEF (RULE 21JI

Schaffer et al., assigned to Novo Nordisk A/S, entitled "Peptide derivatives"
describes derivatives of insulin compound and insulin analogs where the N-tenninal amino group of the B-chain and/or the e-amino group of Lys in position B28, B29 or B30 is acylated using long chain hydrocarbon group having from 12 to 22 carbon atoms and zinc coinplexes thereof.

Protamines and phenolic compounds have been described for use in the crystallization of acylated insulin. U.S. Patents 6,268,335 (31-Jul-01) and 6,465,426 (10-Oct-02) to Brader, both entitled "Insoluble insulin compositions," describe insoluble compositions comprised of acylated insulin a protamine coinplexing compound, a hexamer-stabilizing phenolic compound, and a divalent metal cation.

Existing approaches are especially tailored for crystallization of native insulin coinpound or insulin compound analogs or for acylated insulin compounds having increased lipophilicity relative to non-acylated insulin compounds. There is a need in the art for pharmaceutically acceptable complexes including derivatized insulin compounds, other than acylated insulin compound, such as hydrophilic and/or amphiphilic insulin compound derivatives, and for stabilizing non-acylated lipophilic insulin compound analogs. There is also a need in the art for new protein conjugates having increased bioavailability or other improved pharmaceutical attributes relative to existing conjugates. There is a need in the art for new formulations that facilitate oral delivery of proteins and protein conjugates. Finally, there is a need for a combined approach to improving the oral bioavailability of a protein, such as insulin compound, which incorporates an improved oral protein conjugate provided as a solid in an improved formulation to maximize the benefits for the oral delivery of proteins.
4 Summary of the Invention In general, the invention provides a complex including an insulin compound conjugate with an insulin compound conjugated to a modifying moiety, and a cation, where the insulin compound conjugate is complexed with the cation. The insulin compound may, for example, be a native insulin or an insulin analogs. Examples of insulin compounds include human insulin, lyspro insulin, des30 insulin, native proinsulin, artificial proinsulins, etc. The cation component may, for exainple, be a divalent metal cation selected from the group consisting of Zn++, Mn++, Ca++, Fe++, Ni++, Cu++, Co++ and Mg++.

The modifying moiety may be selected to render the insulin coinpound conjugate more, less or equally soluble as compared to the corresponding unconjugated insulin compound. The modifying moiety is preferably selected to render the insulin compound conjugate at least 1.05, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 times more soluble than a corresponding unconjugated insulin coinpound in an aqueous solution at a pH of about 7.4. Preferably the modifying moiety is selected to render an insulin coinpound conjugate having an aqueous solubility that exceeds about 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 10 g/L, 15 g/L, 20 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, or 150 g/L at a pH of about 7.4. Further, the the modifying moiety is selected to render the insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin compound, and the water solubility of the insulin compound conjugate is decreased by the addition of zinc. In another embodiment, the modifying moiety is selected to render the insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin compound;
the water solubility of the insulin compound conjugate is decreased by the addition of zinc; and water solubility of the complex is greater than the water solubility of insulin compound. In still another embodiment, the relative lipophilicity of the insulin compound conjugate as compared to corrsesponding parent insulin compound (krel) is 1 or less than 1.

The invention also provides novel insulin compound conjugates having an insulin coinpound conjugated to a modifying moiety. 'For example, the invention provides insulin compounds coupled to a modifying moiety having a forinula:

-X-R1-Y-PAG-Z-R2 (Formula VI) where, X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond, at least one of R' and R2 is present, and is lower alkyl and may optionally include a carbonyl group, RZ is a capping group, such as -CH3, -H, tosylate, or an activating group, and PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties (i.e., oxyalkalene moieties), and optionally incorporating one or more additional moieties selected from the group consisting of -S-, -0-, -N-, and -C(O)-, and where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavy atoms.

In embodiments of the invention, any one or more of X, Y and Z may be absent.
Further, when present, X, Y and/or Z may be independently selected from -C(O)-, -0-, -S-, -C-and -N-. In one embodiment, Z is -C(O)-. In another embodiment, Z is not present.

In some einbodiinents, R' is lower alkyl, and R 2 is not present. In other embodiments, R2 is lower alkyl, and R' is not present.

In another embodiment, the modifying moiety may include a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -0-, =0, -S-, -N-, -Si-. The heavy atoms will typically include one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -0-, -S-, -N-, and =0. The carbon atoms and non-carbon heavy atoms are typically present in a ratio of at least 1 carbon atom for every non-carbon heavy atom, preferably at least 2 carbon atoms for every non-carbon heavy atom, more preferably at least 3 carbon atoms for every non-carbon heavy atom. The carbon atoms and oxygen atoms are typically present in a ratio of at least 1 carbon atom for every oxygen atom, preferably at least 2 carbon atoms for every oxygen atom, more preferably at least 3 carbon atoms for every oxygen atom. The modifying moiety may include one or more capping groups, such as branched or linear C1_6, branched or linear, or a carbonyl.
The modifying moiety will typically include hydrogens, and one or more of the hydrogens may be substituted with a fluorine (which is a heavy atom but should not be counted as a heavy atom in the foregoing formula). The modifying moiety may in some cases specifially exclude unsubstituted alkyl moieties. The modifying moiety may, for exainple, be coupled to an available group on an amino acid, such as an amino group, a hydroxyl group or a free carboxyllic acid group the polypeptide, e.g., by a linking group, such as a carbamate, carbonate, ether, ester, amide, or secondary amine group, or by a disulfide bond. The molecules in the linking group are counted as part of the modifying inoiety. In a preferred embodiment, the molecular weight of the modifying moiety is less than the molecular weight of the HIM2 modifying moiety.

The invention includes includes insulin compound conjugates having modifying moieties with a formula:

O
HO O m n (Formula VII), where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5; and/or O
HO O n O\\W
(Formula VIII), where n is 1, 2, 3, 4 or 5, and m is 1, 2, 3 or 4.

It will be appreciated that the novel modifying moieties, as well as the use of such moities to modfy insulin and other polypeptides are themselves aspects of the invention.

The invention also provides novel formulations including the insulin compound conjugates and/or cation-insulin compound conjugates of the invention. The inventors have surprisingly discovered that certain fatty acid compositions are particularly useful, especially for oral delivery of the polypeptides and polypeptide conjugates, such as insulin and insulin compound conjugates and/or oral delivery of the cation-insulin compound conjugate complexes of the invention. In one aspect, the invention provides fatty acid compositions with one or more saturated or unsaturated C4, C5, C6, C72 Cs, Cg or Clo fatty acids and/or salts of such fatty acids. Preferred fatty acids are caprylic, capric, myristic and lauric. Preferred fatty acid salts are sodium salts of caprylic, capric, myristic and lauric acid. The fatty acid content of the composition is typically within a range having as a lower limit of about 0.1, 0.2, 03, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0 % w/w. In yet another embodiment, the fatty acid content of the composition is within a range having as a lower limit about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0 % w/w, and the fatty acid content of the composition is typically greater than about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fatty acid, preferably caprylic, capric, myristic or lauric, or a salt thereof.

The invention also provides method of treating insulin deficiencies or otherwise supplementing insulin in a subject using the insulin compound conjugates, cation-insulin compound conjugate complexes, and/or formulations of the invention. The methods generally include administering a therapeutically effective amount of one or inore of the the insulin coinpound conjugates, cation-insulin compound conjugate coinplexes, and/or formulations of the invention to a subject in need thereof.

5 Brief Description of the Figures Figures 1-15B show photomicrographs of various crystalline solids of the invention. Figures 1 and 2 are photomicrographs taken using a Zeiss Axiovert microscope showing T-type Zn complex of of HIM2 30 g/L concentration, crystals grown for 24 hours. Figure 3 is a photomicrograph taken using a Zeiss Axiovert microscope showing T-type Zn complex of of HIM2 30 g/L concentration, crystals grown for 5 days. Figure 4 is a photomicrograph taken using a Zeiss Axiovert microscope showing R-type Zn complex of HIM2 at 30 g/L
crystals grown for 4 days. Figure 5 shows photomicrograph of R-type crystalline Zn complex of 1N105 containing 30% organic. Figures 6A-lOB show photomicrographs of various R-type Zn complexes of HIM2 made using organic solvent. Figures 11A-14B show photomicrographs of crystals of various R-type co-crystallized Zn complexes of HIM2 and IN105.
Figures 15A-15B
show photomicrographs of crystals of various R-type co-crystallized Zn complexes of HIM2 and human insulin. The invention includes crystals having the morphologies shown in any of Figures 1-15B.

Figures 16-20 show Mouse Blood Glucose Assay results for HIM2 and various Zn-coznplexes. Figure 16 shows MBGA biopotency profiles for HIM2. Figure 17 shows MBGA

biopotency profiles for Zn HIM2 insulin compound product R type. Figure 18 shows MBGA
biopotency profiles for Zn HIM2 insulin compound product T-type. Figure 19 shows MBGA
biopotency profiles for Zn HIM2 insulin compound product with protamine.
Figure 20 shows glucose lowering effect of R type protamine complex at 30 and 90 minutes post dose.

Figures 21-24 show MBGA biopotency profiles for IN-186, IN-192, IN-190, IN-191, IN-189, IN-178, IN-193, [N-194, IN-185, IN-196 and IN-197.

Figures 25 and 26 show dog clamp study results for Zn-HIM2 complexes of the invention.
Figures 27 and 28 show dog clamp study results for Zn-IN 105 complexes of the invention.
Figures 29 and 30 show dog clamp study results for dogs dosed with IN105 in 3%
w/v capric acid sodium salt in a phosphate buffer without additional excipients.

Figures 31-33 show show dog clamp study results for dogs dosed with tablets containing 6mg of IN105 and 150mg Mannitol, 30mg Exlotab with 143mg Caparte with or with out 143 mg laurate.
Figures 34-37 show show dog clamp study results for dogs dosed with prototype tablet 150mg and 280mg caprate tablets and with 140mg/140mg caprate/laurate tablets.

6 Definitions The following are definitions of the terms as used throughout this specification and claims. The definitions provided apply throughout the present specification unless otherwise indicated. Terms not defined herein have the meaning commonly understood in the art to which the term pertains.
"Additioia," when used in reference to an amino acid sequence, includes extensions of one or more amino acids at either or both ends of the sequence as well as insertions within the sequence.
"Cofizplex" refers to a molecular association in which one or more insulin compounds or insulin compound conjugates form coordinate bonds with one or more metal atoms or ions. Complexes may exist in solution or as a solid, such as a crystal, microcrystal, or an amorphous solid.
"Coinplex mixtui=e" means a mixture having two or more different complexes, whether in solution or in solid form. Complexes mixtures may, for example, include complexes with different insulin compounds, different insulin compound conjugates, different hybrid complexes, different cations, combinations of the foregoing, and the like. "Hybrid covaplex" means a cation-insulin compound conjugate complex having two or more different insulin compounds and/or insulin compound conjugates.

"Cozzzplexiizg agent" means a molecule that has a multiplicity of charges and that binds to or complexes with insulin compound conjugates. Examples of complexing agents suitable for use in the present invention include protamines, surfen, globin proteins, spermine, spermidine albumin, amino acids, carboxylic acids, polycationic polymer compounds, cationic polypeptides, anionic polypeptides, nucleotides, and antisense. See Brange, J., Galenics of Insulin compound, Springer-Verlag, Berlin Heidelberg (1987), the entire disclosure of which is incorporated herein by reference.

"Coizservative" used in reference to an addition, deletion or substitution of an amino acid means an addition, deletion or substitution in an amino acid chain that does not completely diminish the therapeutic efficacy of the insulin compound, i.e., the efficacy may be reduced, the same, or enhanced, relative to the therapeutic efficacy of scientifically acceptable control, such as a corresponding native insulin coinpound.

"Hydrophilic" means exhibiting characteristics of water solubility, and the tenn "Izydrophilic moiety" refers to a moiety wliich is hydrophilic and/or which when attached to another chemical entity, increases the hydrophilicity of sucli chemical entity. Examples include, but are not limited to, sugars and polyalkylene moieties such as polyethylene glycol. "Lipophilic"
means exhibiting characteristics of fat solubility, such as accumulation in fat and fatty tissues, the ability to dissolve in lipids and/or the ability to penetrate, interact with and/or traverse biological membranes, and the term, "lipophilic ><zzoiety" means a moiety which is lipophilic and/or which, when attached to another chemical entity, increases the lipophilicity of such chemical entity.
"Anzphiphilic" means exhibiting characteristics of hydropilicity and lipophilicity, and the term "aniphiphilic vzoiety" means a moiety which is amphiphilic and/or which, when attached to a polypeptide or non-polypeptide drug, increases the amphiphilicity (i.e., increases both the hydrophilicity and the amphiphilicity) of the resulting conjugate, e.g., certain PEG-fatty acid modifying moieties, and sugar-fatty acid modifying moieties.

"Lower alkyl" means substituted or unsubstituted, linear or branched alkyl moieties having from one to six carbon atoms, i.e., C1, C2, C3, C4, C5 or C6. "Higher alkyl" means substituted or unsubstituted, linear or branched alkyl moieties having six or more carbon atoms, e.g., C7, C8, C9, C10~ Cl1e C12~ C13~ C142 C15v C16~ C17P C18v C1% C20v etC=

"Moizodispersed" describes a mixture of compounds where about 100 percent of the compounds in the mixture have the same molecular weight. ""Substantially zzzoszodispersed" describes a mixture of compounds where at least about 95 percent of the compounds in the mixture have the same molecular weight. "Purely mofzodispersed" describes a mixture of compounds where about 100 percent of the compounds in the mixture have the same molecular weight and have the same molecular structure. Thus, a purely monodispersed mixture is a monodispersed mixture, but a monodispersed mixture is not necessarily a purely monodispersed mixture.
"Substantially purely fnonodispersed" describes a mixture of compounds where at least about 95 percent of the compounds in the mixture have the same molecular weight and same molecular structure. Thus, a substantially purely monodispersed mixture is a substantially monodispersed mixture, but a substantially monodispersed mixture is not necessarily a substantially purely monodispersed mixture. The insulin compound conjugate components of the cation-insulin compound conjugate compositions are preferably monodispersed, substantially monodispersed, purely monodispersed or substantially purely monodispersed, but may also be polydispersed.
"Polydispea=sed" means having a dispersity that is not monodispersed, substantially monodispersed, purely monodispersed or substantially purely monodispersed.

"Native itzsuliia co zpound" as specifically used herein means mainmalian insulin coinpound (e.g., human insulin, bovine insulin compound, porcine insulin compound or whale insulin compound), provided by natural, synthetic, or genetically engineered sources.
Human insulin is comprised of a twenty-one ainino acid A-chain and a thirty-amino acid B-chain which are cross-linked by disulfide bonds. A properly cross-linked human insulin includes three disulfide bridges: one between A7 and B7, a second between A20 and B 19, and a third between A6 and All. Human insulin possesses three free amino groups: B1-Phenylalanine, Al-Glycine, and B29-Lysine. The free amino groups at positions Al and Bl are a-amino groups.
The free amino group at position B29 is an e-amino group. "lusulin analog" means a polypeptide exhibiting soine, all or enhanced activity relative to a corresponding native insulin or which is converted in in vivo or in vitro into a polypeptide exhibiting ome, all or enhanced activity relative to a corresponding native insulin, e.g., a polypeptide having the structure of a human insulin with one or more conservative amino acid additions, deletions and/or substitutions.
Insulin analogs can be identified using known techniques, such as those described in U.S. Patent Publication No.
20030049654, "Protein design automation for protein libraries," filed 18-Mar-02 in the name of Dahiyat et al. Proinsulins, pre-proinsulins, insulin precursors, single chain insulin precursors of humans and non-human animals and analogs of any of the foregoing are also referred to herein as insulin analogs, as are non-mammalian insulins. Many insulin analogs are known in the art (see discussion below). Unless context specifically indicates otherwise (e.g., were a specific insulin is referenced, such as "human insulin" or the like), the term "zU2sulzn cotnpound" is used broadly to include native insulins and insulin analogs.

"Polyallrylene glycol" or PAG refers to substituted or unsubstituted, linear or branched polyalkylene glycol polymers such as polyethylene glycol (PEG), polypropylene glycol (PPG), and polybutylene glycol (PBG), and combinations thereof (e.g., linear or branched polymers including combinations of two or more different PAG subunits, such as two or more different PAG units selected from PEG, PPG, PPG, and PBG subunits), and includes the monoalkylether of the polyalkylene glycol. The term PAG subunit means a single PAG unit, e.g., "PEG subuuit"
refers to a single polyethylene glycol unit, e.g., -(CH2CHZO)-, "PPG subuuit"
refers to a single polypropylene glycol unit, e.g., -(CHZCH2CH,O)-, and "PBG subunit" refers to a single polypropylene glycol unit, e.g., -(CHzCHZCHZCHZO)-. PAGs and/or PAG subunits also include substituted PAGs or PAG subunits, e.g., PAGs including alkyl side chains, such as methyl, ethyl or propyl side chains, or carbonyl side chains, as well as PAGs including one or more branched PAG subunits, such as iso-PPG or iso-PBG.

"Proiusuliu co zpouud" means an insulin compound in which the C-terminus of the B-chain is coupled to the N-terminus of the A-chain via a natural or artificial C-peptide having 5 or more amino acids. "Preproinsulirz conzpoutzd" means a proinsulin compound further including a leader sequence coupled to the N-terminus of the B-chain, such as a sequence selected to promote excretion as a soluble protein, or a sequence selected to prevent conjugation of the N-terminus, or a sequence selected to enhance purification (e.g., a sequence with binding affinity to a purification column). "Sifzgle chaiu uzsuhn cosnpound precursor" or "tniuiproinsulin conzpouud" means an insulin compound in which the C-terminus of the B-chain (or a truncated B-chain having 1, 2, 3 or 4 amino acids removed from the C-terminus) is coupled to the N-terminus of the A-chain or a truncated A-chain shortened at the N-terininus by 1, 2, 3 or 4 amino acids, without an intervening C-peptide, or via a shortened C-peptide having 1, 2, 3 or 4 amino acids.

"Protanziize" refers to a mixture of strongly basic proteins obtained from natural (e.g., fish sperm) or recombinant sources. See Hoffmann, J. A., et al., Protein Expression and Purification, 1:127-133 (1990). The Protamine composition can be provided in a relatively salt-free preparation of the proteins, often called "protamine base" or in a preparation including salts of the proteins.

"Proteiu" "peptide" and "polypeptide" are used interchangeably herein to refer to compounds having amino acid sequences of at least two and up to any length.

"R-type" means a complex conformation formed in the presence of insulin compound conjugate, a cation and a stabilizing compound, such as phenol. "T-type" means a complex conformation formed in the presence of insulin compound conjugate and a cation without a stabilizing compound, such as phenol. A T-type or R-type complex may include or exclude protamine.

"Scieiztifically acceptable control" means an experimental control that is acceptable to a person of ordinary skill in the art of the subject matter of the experiment.

"Solid" means a state of matter in which there is three-dimensional regularity of structure; the term is used broadly herein to refer to both ciystalline solids, amorphous solids, and combinations of crystalline solids and amorphous solids. "Cation-insuliu cosnpouud coujugate solid," refers to a solid that includes a cation-insulin compound conjugate, preferably coordinated with a monovalent or multivalent cation. "Ciystal" means a solid with a regular polyhedral shape.
"Clystalline" refers to solids having the characteristics of crystals.
"Microciystal" means a solid that is coinprised primarily of matter in a crystalline state that is microscopic in size, typically of longest dimension within the range 1 micron to 100 microns. In some cases, the individual crystals of a microcrystalline composition are predominantly of a single crystallographic composition. In some embodiments, the crystals of the invention are not microcrystals. The terin "snicrocrystallifae" refers to the state of being a microcrystal.
"Atizofphous" refers to a solid material that is not crystalline in form. The person of ordinary skill in the art can distinguish ciystals from amorphous materials using standard techniques, e.g., using x-ray crystallographic techniques, scanning electron microscopy or optical microscopy. "Solid mixture" means a mixture of two different solids. "Cfystal mixture" means a mixture of two different crystals.
"Co-crystaP' means a crystal having two or more different insulin compounds and/or insulin compound conjugates. The cation-insulin compound conjugate complexes of the invention may be provided in any of the foregoing forms or in mixtures of two or more of such forms.
"Substitutiou" means replacement of one or more amino acid residues within the insulin compound sequence with another amino acid. In some cases, the substituted amino acid acts as a functional equivalent, resulting in a silent alteration. Substitutions may be conservative; for example, conservative substitutions may be selected from other meinbers of the class to which the substituted amino acid belongs. Examples of nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Examples of polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of positively charged (basic) amino acids include arginine, lysine and histidine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

"Water solubility" or "aqueous solubility" unless otherwise indicated, is detennined in an aqueous buffer solution at a pH of 7.4.

7 Detailed Description of the Invention The invention provides cation-insulin compound conjugate complexes and various compositions including such complexes, as well as methods of making and using such complexes and compositions. The complexes are useful for administering insulin compound for the treatment of various medical conditions, such as conditions characterized by insulin compound deficiency.
The complexes generally include a cation component and an insulin compound conjugate component. The insulin coinpound conjugate component generally includes an insulin compound coupled to a modifying moiety. Examples of other suitable components of the complexes and/or compositions include stabilizing agents, complexing agents, and other components known in the art for use in preparing cation-protein complexes. The invention also provides novel insulin compound conjugates and fatty acid formulations including such insulin compound conjugates and/or cation-insulin compound conjugate complexes.

7.1 Insulin Compound The cation-insulin compound conjugate includes an insulin compound component.
The insulin compound may, for example, be a mammalian insulin compound, such as human insulin, or an insulin compound analog.

A wide variety of insulin compound analogs are known in the art. Preferred insulin compound analogs are those which include a lysine, preferably a lysine within 5 amino acids of the C-terminus of the B chain, e.g., at position B26, B27, B28, B29 and/or B30. A
set of suitable analogs is described in EP-A 1227000107 (the entire disclosure of which is incorporated herein by reference), having the sequence of insulin compound, except that the amino acid residue at position B28 is Asp, Lys, Leu, Val, or Ala; the amino acid residue at position B29 is Lys or Pro;
the amino acid residue at position B 10 is His or Asp; the amino acid residue at position B 1 is Phe, Asp, or deleted alone or in combination with a deletion of the residue at position B2; the amino acid residue at position B30 is Thr, Ala, or deleted; and the amino acid residue at position B9 is Ser or Asp; provided that either position B28 or B29 is Lys.

Other exainples of suitable insulin compound analogs include AspBZ$ human insulin, Lysa23 human insulin, LeuBZS human insulin, Va1B28 human insulin, AlaB28 human insulin, AspB28Pro$Z9 human insulin, LysB28ProB29 human insulin, LeuB28ProB29 human insulin, Va1B28ProB29 human insulin, AlaB28ProB29 human insulin, as well as analogs provided using the substitution guidelines described above. Insulin compound fragments include, but are not limited to, B22-B30 human insulin, B23-B30 human insulin, B25-B30 human insulin, B26-B30 human insulin, human insulin, B29-B30 human insulin, B1-B2 human insulin, B1-B3 human insulin, B1-B4 human insulin, B1-B5 human insulin, the A chain of human insulin, and the B
chain of human insulin.

Still other examples of suitable insulin compound analogs can be found in U.S.
Patent Publication No. 20030144181A1, entitled "Insoluble compositions for controlling blood glucose," 31-Jul-03; U.S. Patent Publication No. 20030104983A1; entitled "Stable insulin formulations," 5-Jun-03; U.S. Patent Publication No. 20030040601A1, entitled "Method for making insulin precursors and insulin analog precursors," 27-Feb-03; U.S.
Patent Publication No. 20030004096A1, entitled "Zinc-free and low-zinc insulin preparations having iinproved stability," 2-Jan-03; U.S. Patent 6,551,992B1, entitled "Stable insulin fonnulations," 22-Apr-03;
U.S. Patent 6,534,288B1, entitled "C peptide for improved preparation of insulin and insulin analogs," 18-Mar-03; U.S. Patent 6,531,448B1, entitled "Insoluble compositions for controlling blood glucose," 11-Mar-03; U.S. Patent RE37,971E, entitled "Selective acylation of epsilon-amino groups," 28-Jan-03; U.S. Patent Publication No. 20020198140A1, entitled "Pulinonary insulin crystals," 26-Dec-02; U.S. Patent 6,465,426B2, entitled "Insoluble insulin compositions," 15-Oct-02; U.S. Patent 6,444,641B1, entitled "Fatty acid-acylated insulin analogs," 3-Sep-02; U.S. Patent Publication No. 20020137144A1, entitled "Method for making insulin precursors and insulin precursor analogues having improved fennentation yield in yeast,"
26-Sep-02; U.S. Patent Publication No. 20020132760A1, entitled "Stabilized insulin formulations," 19-Sep-02; U.S_ Patent Publication No. 20020082199A1, entitled "Insoluble insulin compositions," 27-Jun-02; U.S. Patent 6,335,316B1, entitled "Method for administering acylated insulin," 1-Jan-02; U.S. Patent 6,268,335B1, entitled "Insoluble insulin compositions,"
31-Jul-01; U.S. Patent Publication No. 20010041787A1, entitled "Method for making insulin precursors and insulin precursor analogues having improved fermentation yield in yeast,"
15-Nov-01; U.S. Patent Publication No. 20010041786A1, entitled "Stabilized acylated insulin formulations," 15-Nov-01; U.S. Patent Publication No. 20010039260A1, entitled "Pulmonary insulin crystals," 8-Nov-01; U.S. Patent Publication No. 20010036916A1, entitled "Insoluble insulin compositions," 1-Nov-01; U.S. Patent Publication No. 20010007853A1, entitled "Method for administering monomeric insulin analogs," 12-Jul-01; U.S. Patent 6,051,551A, entitled "Method for administering acylated insulin," 18-Apr-00; U.S. Patent 6,034,054A, entitled "Stable insulin formulations," 7-Mar-00; U.S. Patent 5,970,973A, entitled "Method of delivering insulin lispro," 26-Oct-99; U.S. Patent 5,952,297A, entitled "Monoineric insulin analog formulations," 14-Sep-99; U.S. Patent 5,922,675A, entitled "Acylated Insulin Analogs,"
13-Jul-99; U.S. Patent 5,888,477A, entitled "Use of monomeric insulin as a means for improving the bioavailability of inhaled insulin," 30-Mar-99; U.S. Patent 5,873,358A, entitled "Method of maintaining a diabetic patient's blood glucose level in a desired range," 23-Feb-99; U.S. Patent 5,747,642A, entitled "Monomeric insulin analog formulations," 5-May-98; U.S.
Patent 5,693,609A, entitled "Acylated insulin compound analogs," 2-Dec-97; U.S.
Patent 5,650,486A, entitled "Monomeric insulin analog formulations," 22-Jul-97; U.S. Patent 5,646,242A, entitled "Selective acylation of epsilon-amino groups," 8-Jul-97; U.S. Patent 5,597,893A, entitled "Preparation of stable insulin analog crystals," 28-Jan-97; U.S. Patent 5,547,929A, entitled "Insulin analog formulations," 20-Aug-96; U.S. 5,504,188A, entitled "Preparation of stable zinc insulin compound analog crystals," 2-Apr-96; U.S. 5,474,978A, entitled "Insulin analog formulations," 12-Dec-95; U.S. Patent 5,461,031A, entitled "Monomeric insulin analog formulations," 24-Oct-95; U.S. Patent 4,421,685A, entitled "Process for producing an insulin,"
20-Dec-83; U.S. Patent 6,221,837, entitled "Insulin derivatives with increased zinc binding"
24-Apr-01; U.S. Patent 5,177,058, entitled "Pharmaceutical formulation for the treatment of diabetes mellitus" 5-Jan-93 (describes pharmaceutical formulations including an insulin compound derivative modified with a base at B31 and having an isoelectric point between 5.8 and 8.5 and/or at least one of its physiologically tolerated salts in a pharmaceutically acceptable excipient, and a relatively high zinc ion content in the range from above 1 Etg to about 200 g of zinc/IU, including insulin compound-B31-Arg-OH and human insulin-B31-Arg-B32-Arg-OH).
The entire disclosure of each of the foregoing patent documents is incorporated herein by' reference, particularly for teaching about the making, using and compositions of various insulin compound analogs.

Insulin compound used to prepare the cation-insulin compound conjugates can be prepared by any of a variety of recognized peptide synthesis techniques, e.g., classical (solution) methods, solid phase methods, seini-synthetic methods, and recombinant DNA methods. For exainple, Chance et al., U.S. Patent Application No. 07/388,201, EP0383472, Brange et al., EP0214826, and Belagaje et al., U.S. Patent 5,304,473 disclose the preparation of various proinsulin compound and insulin compound analogs and are herein incorporated by reference. The A and B
chains of the insulin compound analogs may also be prepared via a proinsulin compound-like precursor molecule or single chain insulin compound precursor molecule using recombinant DNA techniques. See Frank at al., "Peptides: Synthesis-Structure-Function,"
Proc. Seventh Ana.
Pept. Syrnp., Eds. D. Rich and E. Gr6ss (1981); Bernd Gutte, Peptides :
Synthesis, Stizictures, and Applications, Academic Press (October 19, 1995); Chan, Weng and White, Peter (Eds.), F77zoc Solid Phase Peptide Syiathesis: A Practical Approach, Oxford University Press (March 2000); the entire disclosures of which are incorporated herein by reference for their teachings concerning peptide synthesis, recombinant production and manufacture.

7.2 Modifying Moiety The cation-insulin compound conjugate complexes include a modifying moiety coupled (e.g., covalently or ionically) to the insulin compound to provide the insulin compound conjugate.
Modifying moieties are moieties coupled to the insulin compound that provide the insulin compound with desired properties as described herein. For example, the modifying moiety can reduce the rate of degradation of the insulin compound in various environments (such as the GI
tract, and/or the bloodstream), such that less of the insulin coinpound is degraded in the modified fonn than would be degraded in the absence of the modifying moiety in such environments.
Preferred modifying moieties are those which permit the insulin compound conjugate to retain a therapeutically significant percentage of the biological activity of the parent insulin compound.
Further, preferred modifying moieties are those which are amphiphilic or hydrophilic, and/or which render the insulin compound conjugate amphiphilic or hydrophilic or less lipophilic than a scientifically acceptable control, such as a corresponding insulin compound, or a corresponding unconjugated insulin compound.

Examples of suitable modifying moieties and insulin coinpound conjugates useful in the cation-insulin compound conjugate compositions can be found in the following patents, the entire disclosures of which are incorporated herein by reference: U.S. Patent 6,303,569, entitled "Trialkyl-lock-facilitated polymeric prodrugs of amino-containing bioactive agents," 16-Oct-01;
U.S. Patent 6,214,330, "Coumarin and related aromatic-based polymeric prodrugs," 10-Apr-01;
U.S. Patent 6,113,906, entitled "Water-soluble non-antigenic polymer linkable to biologically active material," 05-Sep-00; U.S. Patent 5,985,263, entitled "Substantially pure histidine-linked protein polymer conjugates," 16-Nov-99; U.S. Patent 5,900,402, entitled "Method of reducing side effects associated with administration of oxygen-carrying proteins," 04-05-99; U.S. Patent 5,681,811, "Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same" 28-Oct-97; U.S.
Patent 5,637,749, entitled "Aryl imidate activated polyalkylene oxides," 10-Jun-97; U.S. Patent 5,612,460, entitled "Active carbonates of polyalkylene oxides for modification of polypeptides,"
18-Mar-97; U.S. Patent 5,567,422, entitled "Azlactone activated polyalkylene oxides conjugated to biologically active nucleophiles," 22-Oct-96; U.S. Patent 5,405,877, entitled "Cyclic imide thione activated polyalkylene oxides," 11-Apr-95; and U.S. Patent 5,359,030, entitled "Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same," 25-Oct-94.
Additional examples of conjugated polypeptides useful in the formulations of the instant invention can be found in the following U.S. patent applications, the entire specifications of which are incorporated herein by reference: U.S. Patent Application Nos.
09/134,803, filed 14-Aug-98; 10/018,879, filed 19-Dec-01; 10/235,381, filed 05-Sep-02;
10/235,284, filed 05-Sep-02; and 09/873,797, filed 04-Jun-01. The entire disclosure of each of the foregoing patents and patent applications is incorporated herein by reference for their teachings concerning moieties used to modify polypeptides.

The modifying moieties may include weak or degradable linkages in their backbones. For example, the PAGs can include hydrolytically unstable linkages, such as lactide, glycolide, carbonate, ester, carbamate and the like, wllich are susceptible to hydrolysis. This approach allows the polymers to be cleaved into lower molecular weight fragments.
Examples of such polymers are described, for example, in U.S. Patent 6,153,211, entitled, to Hubbell et al., the entire disclosure of which is incorporated herein by reference. See also U.S.
Patent 6,309,633, to Ekwuribe et al., the entire disclosure of which is incorporated herein by reference.

The modifying moiety can include any hydrophilic moieties, lipophilic moieties, amphiphilic moieties, salt-forming moieties, and combinations thereof. Representative hydrophilic, amphiphilic, and lipophilic polymers and modifying moieties are described in more detail below.
7.2.1 Hydrophilic Moieties Examples of suitable hydrophilic moieties include PAG moieties, other hydrophilic polymers, sugar moieties, polysorbate moieties, and combinations thereof.

7.2.2 Polyalkylene Glycol Moieties PAGs are compounds with repeat alkylene glycol units. In some embodiments, the units are all identical (e.g., PEG or PPG). In other embodiments, the alkylene units are different (e.g., polyethylene-co-propylene glycol, or PLURONICSO). The polymers can be random copolymers (for example, where ethylene oxide and propylene oxide are co-polymerized) or branched or graft copolymers.

PEG is a preferred PAG, and is useful in biological applications because it has highly desirable properties and is generally regarded as safe (GRAS) by the Food and Drug Administration. PEG
generally has the formula H-(CH2CH2O),,-H, where n can range from about 2 to about 4000 or more, though the capping moieties may vary, e.g., mono-methoxy or di-hydroxy.
PEG typically is colorless, odorless, water-soluble or water-miscible (depending on molecular weight), heat stable, cheinically inert, hydrolytically stable, and generally nontoxic. PEG
is also biocompatible, and typically does not produce an immune response in the body.
Preferred PEG
moieties include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more PEG subunits.

The PEG may be monodispersed, substantially monodispersed, purely monodispersed or substantially purely monodispersed (e.g., as previously described by the applicants in U.S. Patent 09/873,731 and U.S. Patent 09/873,797, both filed 04-Jun-01, the entire disclosures of which are incorporated herein by reference) or polydispersed. One advantage of using the relatively low molecular weight, monodispersed polymers is that they form easily defined conjugate molecules, which can facilitate both reproducible synthesis and FDA approval.

The PEG can be linear with a hydroxyl group at each terminus (before being conjugated to the remainder of the insulin compound). The PEG can also be an alkoxy PEG, such as methoxy-PEG
(or mPEG), where one terminus is a relatively inert alkoxy group (e.g., linear or branched OC1_6), while the other terminus is a hydroxyl group (that is coupled to the insulin compound).

The PEG can also be branched, which can in one embodiment be represented as R(-PEG-õOH)m in which R represents a central (typically polyhydric) core agent such as pentaerytlu-itol, sugar, lysine or glycerol, n represents the number of PEG subunits and can vary for each arm and is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 and m represents the number of arms, and ranges from 2 to the maximum nuinber of attachment sitesz on the core agent. Each branch can be the same or different and can be terminated, for example, with ethers and/or esters. The number of arms m can range from three to a hundred or more, and one or more of the terminal hydroxyl groups can be coupled to the remainder of the insulin compound, or otherwise subject to chemical modification.

Other branched PEGs include those represented by the formula (CH3O-PEG-)PR-Z, where p equals 2 or 3, R represents a central core such, as lysine or glycerol, and Z
represents a group such as carboxyl that is subject to ready chemical activation. Still another branched form, the pendant PEG, has reactive groups, such as carboxyls, along the PEG backbone rather than, or in addition to, the end of the PEG chains. Forked PEG can be represented by the formula PEG(-LCHXz),,, where L is a linking group and X is an activated terminal group.

7.2.3 Sugar Moieties The modifying moieties described herein can include sugar moieties. In general, the sugar moiety is a carbohydrate product of at least one saccharose group. Representative sugar moieties include, but are not limited to, glycerol moieties, mono-, di-, tri-, and oligosaccharides, and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums.
Specific monosaccharides include C6 and above (preferably C6 to C8) sugars such as glucose, fructose, mannose, galactose, ribose, and sedoheptulose; di- and trisaccharides include moieties having two or three monosaccharide units (preferably C5 to C8) such as sucrose, cellobiose, maltose, lactose, and raffinose. Conjugation using sugar moieties is described in US Patents 5,681,811, 5,438,040, and 5,359,030, the entire disclosures of which are incorporated herein by reference.

7.2.4 Polysorbate Moieties The modifying moieties may include one or more polysorbate moieties. Examples include sorbitan esters, and polysorbate derivatized with polyoxyethylene. Conjugation using polysorbate moieties is described in US Patents 5,681,811, 5,438,040, and 5,359,030, the entire disclosures of which are incorporated herein by reference.

7.2.5 Biocompatible Water-soluble Polycationic Moieties In some embodiments, biocompatible water-soluble polycationic polymers can be used.
Biocompatible water-soluble polycationic polymers include, for example, any modifying moiety having protonated heterocycles attached as pendant groups. "Water soluble" in this context means that the entire modifying moiety is soluble in aqueous solutions, such as buffered saline or buffered saline with small amounts of added organic solvents as cosolvents, at a temperature between 20 and 37 C. In some embodiments, the modifying moiety itself is not sufficiently soluble in aqueous solutions per se but is brought into solution by grafting with water-soluble polymers such as PEG chains. Examples include polyamines having amine groups on either the modifying moiety backbone or the modifying moiety side chains, such as poly-L-Lys and other positively charged polyamino acids of natural or synthetic amino acids or mixtures of amino acids, including poly(D-Lys), poly(ornithine), poly(Arg), and poly(histidine), and nonpeptide polyamines such as poly(aminostyrene), poly(aminoacrylate), poly (N-methyl aminoacrylate), poly (N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate), poly(N,N-diethylaminoacrylate), poly(aminoinethacrylate), poly(N-methyl amino-methacrylate), poly(N-ethyl aminomethacrylate), poly(N,N-dimethyl aminomethacrylate), poly(N,N-diethyl aminomethacrylate), poly(ethyleneimine), polymers of quaternary amines, such as poly(N,N,N-trimethylaminoacrylate chloride), poly(inethyacrylamidopropyltrimethyl ammonium chloride), and natural or synthetic polysaccharides such as chitosan.

7.2.6 Other Hydrophilic Moieties The modifying moieties may also include other hydrophilic polyiners. Examples include poly(oxyethylated polyols) such as poly(oxyethylated glycerol), poly(oxyethylated sorbitol), and poly(oxyethylated glucose); poly(vinyl alcohol) ("PVA"); dextran; carbohydrate-based polymers and the like. The polymers can be homopolymers or random or block copolymers and terpolymers based on the monomers of the above polymers, linear chain or branched.

Specific examples of suitable additional polymers include, but are not limited to, poly(oxazoline), difunctional poly(acryloylmorpholine) ("PAcM"), and poly(vinylpyrrolidone)("PVP"). PVP and poly(oxazoline) are well known polymers in the art and their preparation will be readily apparent to the skilled artisan. PAcM and its synthesis and use are described in U.S.
Patent 5,629,384 and U.S. Patent 5,631,322, the disclosures of which are incorporated herein by reference in their entirety.

7.2.7 Bioadhesive Polyanionic Moieties Certain hydrophilic polymers appear to have potentially useful bioadhesive properties. Examples of such polymers are found, for example, in U.S. Patent 6,197,346, to Mathiowitz, et al. Those polymers containing carboxylic groups (e.g., poly(acrylic acid)) exhibit bioadhesive properties, and are also readily conjugated with the insulin compounds described herein.
Rapidly bioerodible polymers that expose carboxylic acid groups on degradation, such as poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, are also bioadhesive polymers.
These polymers can be used to deliver the insulin compounds to the gastrointestinal tract. As the polymers degrade, they can expose carboxylic acid groups to enable them to adhere strongly to the gastrointestinal tract, and can aid in the delivery of the insulin compound conjugates.

7.2.8 Lipophilic Moieties In some embodiments, the modifying moieties include one or more lipophilic moieties. The lipophilic moiety may be various lipophilic moieties as will be understood by those skilled in the art including, but not limited to, alkyl moieties, alkenyl moieties, alkynyl moieties, aryl moieties, arylalkyl moieties, alkylaryl moieties, fatty acid moieties, adamantantyl, and cholesteryl, as well as lipophilic polymers and/or oligomers.

The alkyl moiety can be a saturated or unsaturated, linear, branched, or cyclic hydrocarbon chain.
In some embodiments, the alkyl moiety has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more carbon atoms. Exainples include saturated, linear alkyl moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; saturated, branched alkyl moieties such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; and unsaturated alkyl moieties derived from the above saturated alkyl moieties including, but not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl, and 2-propynyl. In other einbodiments, the alkyl moiety is a lower alkyl moiety. In still other embodiments, the alkyl moiety is a Ci to C3 lower alkyl moiety. In some embodiments, the modifying moiety specifically does not consist of an alkyl moiety, or specifically does not consist of a lower alkyl moiety, or specifically does not consist of an alkane moiety, or specifically does not consist of a lower alkane moiety.

The alkyl groups can either be unsubstituted or substituted with one or more substituents, and such substituents preferably either do not interfere with the methods of synthesis of the conjugates or eliminate the biological activity of the conjugates. Potentially interfering functionality can be suitably blocked with a protecting group so as to render the functionality non-interfering. Each substituent may be optionally substituted with additional non-interfering substituents. The tenn "non-interfering" characterizes the substituents as not eliminating the feasibility of any reactions to be performed in accordance with the process of this invention.

The lipophilic moiety may be a fatty acid moiety, such as a natural or synthetic, saturated or unsaturated, linear or branched fatty acid moiety. In some embodiments, the fatty acid moiety has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more carbon atoms. In some embodiments, the modifying moiety specifically does not consist of a fatty acid moiety; or specifically does not consist of a fatty acid moiety having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more carbon atoms.

When the modifying moiety includes an aryl ring, the ring can be functionalized with a nucleophilic functional group (such as OH, or SH) that is positioned so that it can react in an intrainolecular fashion with the carbamate moiety and assist in its hydrolysis. In some einbodiinents, the nucleophilic group is protected with a protecting group capable of being hydrolyzed or otherwise degraded in vivo, with the result being that when the protecting group is deprotected, hydrolysis of the conjugate, and resultant release of the parent insulin compound, is facilitated.

Other examples of suitable modifying moieties include -C(CH2OH)3i -CH(CHZOH)2; -C(CH3)3; -CH(CH3)2.

7.2.9 Amphiphilic Moieties In some embodiments, the modifying moiety includes an amphiphilic moiety. Many polyiners and oligomers are amphiphilic. These are often block co-polymers, branched copolymers or graft co-polymers that include hydrophilic and lipophilic moieties, which can be in the form of oligomers and/or polymers, such as linear chain, branched, or graft polymers or co-polymers.

The amphiphilic modifying moieties may include combinations of any of the lipophilic and hydrophilic moieties described herein. Such modifying moieties typically include at least one reactive functional group, for example, halo, hydroxyl, amine, thiol, sulfonic acid, carboxylic acid, isocyanate, epoxy, ester, and the like, which is often at a terminal end of the modifying moiety. These reactive functional groups can be used to attach a lipophilic linear or branched chain alkyl, alkenyl, alkynyl, arylalkyl, or alkylaryl group, or a lipophilic polyiner or oligomer, thereby increasing the lipophilicity of the modifying moiety (and thereby rendering them generally amphiphilic).

The lipophilic groups can, for example, be derived from mono- or di-carboxylic acids, or where appropriate, reactive equivalents of carboxylic acids such as anhydrides or acid chlorides.
Examples of suitable precursors for the lipophilic groups are acetic acid, propionic acid, butyric acid, valeric acid, isobutyric acid, trimethylacetic acid, caproic acid, caprylic acid, heptanoic acid, e"
capric acid, pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, lignoceric acid, ceratic acid, montanoic acid, isostearic acid, isononanoic acid, 2-ethylhexanoic acid, oleic acid, ricinoleic acid, linoleic acid, linolenic acid, ei-ucic acid, soybean fatty acid, linseed fatty acid, dehydrated castor fatty acid, tall oil fatty acid, tung oil fatty acid, sunflower fatty acid, safflower fatty acid, acrylic acid, methacrylic acid, maleic anhydride, orthophthalic anhydride, terephthalic acid, isophthalic acid, adipic acid, azelaic acid, sebacic acid, tetrahydrophthalic anhydride, hexahydrophthalic anhydride, succinic acid and polyolefin carboxylic acids.

The terminal lipophilic groups need not be equivalent, i.e., the resulting copolymers can include terminal lipophilic groups that are the same or different. The lipophilic groups can be derived from more than one mono or di-functional alkyl, alkenyl, alkynyl, cycloalkyl, arylalkyl or alkylaryl group as defined above.

7.2.10 PAG-alkyl Modifying Moieties The modifying moiety may be a linear or branched polyineric moiety having one or more linear or branched PAG moieties and/or one or more linear or branched, substituted or unsubstituted alkyl moieties. In certain cases, such moieties are considered amphiphilic;
however, the PAG and alkyl moieties may be varied to render such moieties more lipophilic or more hydrophilic. In certain embodiments, the modifying moiety specifically does not consist of an alkyl moiety and in other embodiments, the modifying moiety specifically does not consist of an alkane moiety.
The PAG moieties in some einbodiments include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 PAG subunits arranged in linear or branched form. The PAG
moieties in some embodiments include PEG, PPG and/or PBG subunits. The alkyl moieties in some embodiments preferably have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkyl moieties are preferably alkane moieties. The modifying moiety may include a capping moiety, such as -OCH3. Further, the modifying moiety may include a hydrophobic group, such as a pivaloyl group.

In one embodiment, the modifying moiety has a formula:

-~-(CH2)0- X-(CH2CH20)P Y-(CH2)q-Z-R
(Formula I) where o, p and q are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, and at least one of o, p and q is at least 2. X, Y and Z are independently selected from -C-, -0-, -C(O)-, -C(0)0-, -OC(O)-, -NH-, -NHC(O)-, and -C(O)NH-, and R is H
or an alkyl, preferably a lower alkyl, more preferably methyl. The variables o, p and q are preferably selected to yield a hydrophilic or amphiphilic modifying moiety, and are preferably selected in relation to the insulin compound to yield a hydrophilic or amphiphilic insulin compound conjugate, preferably a monoconjugate, diconjugate or triconjugate.
In one preferred embodiment for an insulin compound conjugate which is to be used for basal insulin compound maintenance, o, p and q are selected to yield a PAG which is proximal to the insulin compound and an alkyl moiety which is distal to the insulin compound. Alternatively, 0, P and Q may be selected to yield a PAG which is distal to the insulin compound and an alkyl which is proximal to the insulin. In an alternative embodiment, R is a pivaloyl group or an alkyl-pivaloyl group.

In a related embodiment, the modifying moiety has a formula:

-~- C- (CH2)m- X(C2Hq0)n-Y
(Formula II), where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 and n is from 2 to 100, preferably 2 to 50, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, X is -C-, -0-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-, and Y is lower alkyl or -H. X is preferably 0 and Y is preferably -CH3. In some cases the carbonyl group (-C(O)-) may be absent, and the -(CH2)- moiety may be coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.

In a preferred embodiment, the modifying moiety has a structure selected from the following:

I I
'Z~ ?_ ~C~~\ O
~
Me and (when the immediately preceding modifying moiety is coupled to human insulin at B29, the resulting monoconjugate is referred to as IN105).

II
C Me (when the iminediately preceding modifying moiety is coupled to human insulin at B29, the resulting monoconjugate is referred to as HIM2). Any of the foregoing moieties may, for example, be coupled to human insulin at a nucleophilic residue, e.g., Al, B1 or B29. In some cases the carbonyl group (-C(O)-) may be absent or replaced with an alkyl moiety, preferably a lower alkyl moiety, and the -(CH2)- moiety may be coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.

In another embodiment, the modifying moiety has a formula:
O
O /-, Crõ-X-PAGõ PAGr,-X-Cm (Formula III), wliere each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each PAG
is independently selected and is a PAG moiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; each X is independently selected and is a linking moiety coupling PAG to C, and is preferably -C-, -0-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-. In some embodiments the C,,; X moiety is absent, and the PAGõ moiety is terminated with an -OH
moiety or an -OCH3 moiety. For example, the PAG may be methoxy-terminated or hydroxy-terminated PAG, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG subunits, including PEG, PPG, and/or PBG subunits. In some cases the carbonyl group (-C(O)-) may be replaced with an alkyl moiety, preferably a lower alkyl moiety, which may be coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.

The modifying moiety may, for example, have a formula:

O O

o PAGn X

o (Formula IV), where each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and each PAG
is independently selected and is a PAG moiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; X is -0-, or -NH-; each o is independently selected and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. For example, the PAG may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG subunits, including PEG, PPG, and/or PBG subunits. In some cases the carbonyl group (-C(O)-) proximal to the point of attachment may be absent or replaced with an alkyl moiety, preferably a lower alkyl moiety, and the -(CH2)- moiety may be coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.

The modifying moiety may, for example, have a formula:
O

o O
Cm-X-PAGn PAGn-X"Cm (Formula V), where each C is independently selected and is an alkyl moiety having m carbons and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and each PAG
is independently selected and is a PAG moiety having n subunits and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25; each X is independently selected and is a linking moiety coupling PAG to C, and is preferably -C-, -0-, -C(O)-, -NH-, -NHC(O)-, or -C(O)NH-;
each o is independently selected and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In soine embodiments the Cm-X inoiety is absent, and the PAGõ moiety is terminated with an -OH moiety or an -OCH3 moiety. For example, the PAG may be methoxy-terininated or hydroxy-terminated PAG, having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 PAG subunits, including PEG, PPG, and/or PBG subunits. In some cases the carbonyl group (-C(O)-) proximal to the point of attachment may be absent, and the -(CH2)- moiety may be coupled to an available group on an amino acid, such as a hydroxyl group or a free carboxyllic acid group.

In another embodiment, the modifying moiety may have a formula:
-X-R1-Y-PAG-Z-R2 (Formula VI) where, X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond, at least one of R' and R2 is present, and is lower alkyl and may optionally include a carbonyl group, RZ is a capping group, such as -CH3, -H, tosylate, or an activating group, and PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties (i.e., oxyalkalene moieties), and optionally incorporating one or more additional moieties selected from the group consisting of -S-, -0-, -N-, and -C(O)-, and -where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavy atoms.

In embodiments of the invention, any one or more of X, Y and Z may be absent.
Further, when present, X, Y and/or Z may be independently selected from -C(O)-, -0-, -S-, -C-and -N-. In one embodiment, Z is -C(O)-. In another embodiment, Z is not present.

In some embodiments, R' is lower alkyl, and R2 is not present. In other embodiments, R2 is lower alkyl, and Rl is not present.

In another embodiment, the modifying moiety may include a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -0-, =O, -S-, -N-, -Si-. The heavy atoms will typically include one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -0-, -S-, -N-, and =0. The carbon atoms and non-carbon heavy atoms are typically present in a ratio of at least I carbon atom for every non-carbon heavy atom, preferably at least 2 carbon atoms for every non-carbon heavy atom, more preferably at least 3 carbon atoms for every non-carbon heavy atom. The carbon atoms and oxygen atoms are typically present in a ratio of at least 1 carbon atom for every oxygen atom, preferably at least 2 carbon atoms for every oxygen atom, more preferably at least 3 carbon atoms for every oxygen atom. The modifying moiety may include one or more capping groups, such as branched or linear Cj_6, branched or linear, or a carbonyl.
The modifying moiety will typically include hydrogens, and one or more of the hydrogens may be substituted with a fluorine (which is a heavy atom but should not be counted as a heavy atom in the foregoing formula). The modifying moiety may in some cases specifially exclude unsubstituted alkyl moieties. The modifying moiety may, for example, be coupled to an available group on an amino acid, such as an amino group, a hydroxyl group or a fi-ee carboxyllic acid group the polypeptide, e.g., by a linking group, such as a carbamate, carbonate, ether, ester, amide, or secondary amine group, or by a disulfide bond. The molecules in the linking group are counted as part of the modifying moiety. In a preferred embodiment, the molecular weight of the modifying moiety is less than the molecular weiglit of the HIM2 modifying moiety.

The invention includes modifying moieties having a formula:
O

HO O
n (Formula VII), where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5.

The invention includes modifying moieties having a formula:
O
O
HO O
m (Formula VIII), where n is 1, 2, 3, 4 or 5, andmis 1, 2, 3 or 4.

The invention includes modifying moieties having a formula:

O O O
<
NH
HO ~C~n O

(Forinula IX), where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The invention also includes modifying moieties having a formula:
O O
HO O(PAG)m W
(Formula X), where PAG is a PAG moiety having m subunits and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

Other preferred modifying moieties include:
O

O\

O

O\ HO O 4 O

O

O

O

O

and ~

.=~''~'~' y"'' ~ ..~'''a.~",,~ ~ 1~, ~ ~ '~..

The following modifying moieties can be particularly preferred for use in a basal insulin compound replacement regimen.

O

O(mPEG)7 HO , O

O(mPEG)7 HO

O O
HO O(PEG)3 O O

HO O(PPG)3 O O

HO O(PEG)3 HO "lk O(PEG)4 HO O(PEG)6 O
HO
N
H

, and O O "~~/
.
o Q ' 0~~ o N H~
Still other modifying moieties include the following:

R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-CHz-CH2-CHa-CH2-CH2-CH2-CH2-CHa-CH2-CH2-CH2-CH2-O-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H a-C H2-C H Z-C H2-C H2-C H Z-C H Z-C H 2-C H a-C H 2-C H 3, R-C H 2-C H2-C H2-C H2-C H 2-C H a-C H a-C H 2-C H 2-C H 2-C H 2-C H 2-O-C H2-C H 3, R-C H 2-C H2-C H2-C H2-C H a-C H 2-C H 2-C H 2-C H 2-C H 2-C H a-C H 2-O -C H
3i R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CHa-CH2-CHZ-CHZ-CH2-CHZ-CH2-CH2-CH2-CH2-CH2-O-CHa-CH2-CH3, R-C H 2-C H 2-C H 2-C H 2-C H2-C H 2-C H a-C H2-C H 2-C H 2-C H Z-O-C H 2-C H
3, R-CH2-CHZ-CHZ-CHZ-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3, R-CH2-CH2-CH2-C H2-CH2-C H2-CH2-C HZ-CHZ-CHZ-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-C H 2-C H 2- C H 2-C H 2-C H2-C H2-C H z-O-C H Z-C H Z-C H
2-C H 3, R-C H 2-C H 2-C H 2-C H 2-C H2-C H 2-C H Z-C H2-C H2-C H2-O-C H a-C H 2-C H 3, R-CHZ-CH2-CH2-CH2-CH2-CH2-CHa-CHa-CH2-CH2-O-CH2-CH2-O-CH3, R-C H 2-C H 2-C H 2-C H 2-C H 2-C H Z-C H2-C H2-C H 2-C H 2-O-C H 2-C H 3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3, R-C HZ-CHZ-CHZ-CH2-C H2-C H2-CH2-CHa-C H2-CH 3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CHZ-CHZ-CH2-CH2-CH2-CHZ-CH2-CH2-CH2-O-CHa-CH2-CH2-CHZ-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3, R-CHZ-CH2-CH2-CH2-CHZ-CHZ-CHZ-CHa-CHZ-O-CHa-CH2-CH2-CH3, 3, R-CHZ-CH2-CH2-CH2-CHZ-CH2-CHZ-CH2-CH2-O-CHa-CHz-CH3, C H 3, R-CH2-CH2-CH2-CH2-CH2-C H2-CH2-C Ha-CH2-O-C H2-CHa-O-CH3, R-CH2-CH2-CH2-CHa-CH2-CHa-CH2-CH2-CH2-O-CH2-CH3, R-C H 2-C H 2-C H 2-C H 2-C H 2-C H 2-C H 2-C H 2-C H a-O-C H 3, R-CH2-CH2-CH2-C H2-C HZ-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H2-C H Z-C H a-C H a-C H 2-C H 2-C H 3, R-C H 2-C H 2-C H 2-C H 2-C H2-C H2-C H 2-C H2-O-C H 2-C H 2-C H 2-C H 2-C H z-C H 3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CHZ-O-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H z-C H 2-C H a-C H 2-C H 2-C H2-C H a-O -C H a-C H a-C H2-C H 3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH3, R-C H 2-C H 2-C H 2-C H 2-C H Z-C H 2-C H a-C H 2-O -C H 2-C H 2-C H 3, R-CHZ-CH2-CHa-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH3, R-CH2-CH2-CH2-CH2-CH2-CHa-CH2-CH2-O-CHa-CH2-O-CHz-CHZ-CH3, R-CHZ-CHZ-CHZ-C H2-C H2-CH2-CH2-CH2-O-CH2-CH2-O-C H2-C H3, R-CH2-CH2-CH2-CHZ-CH2-CH2-CH2-CH2-O-CH2-CHZ-O-CH3, R-C H 2-C H 2-C H 2-C H Z-C H 2-C H Z-C H 2-C H2-O -C H 2-C H 3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-CH2-CH2-C Hz-C HZ-CH2-CH2-CH3, R-C HZ-CH2-C H 2-C H 2-C HZ-C H 2-CH2-O-CHZ-C H2-C H2-C H2-CH2-CH 2-CH3, R-CHa-CH2-CH2-CH2-CHa-CHZ-CH2-O-CH2-CH2-CH2-CH2-CHZ-CH3, R-CH2-CH2-CH2-CH2-CH2-CHZ-CH2-O-CHZ-CH2-CH2-CH2-CH3, R-C H Z-C H2-C H2-C H2-C H 2-C H Z-C H 2-O-C H2-C H 2-C H Z-C H 3, R-C H 2-C H a-C H 2-C H Z-C H 2- C H Z-C H 2-O-C H a-C H Z-C H 2-O -C H2-C H a-C H 3, R-C H 2-C H Z-C H 2-C H 2-C H2-C H 2-C H 2-O-C H 2-C H 2-C H Z-O-C H 2-C H 3, R-C H a-C H a-C H a-C H 2-C H 2- C H2-C H 2-O-C H Z-C H 2-C H 2- O-C H 3, R-CH2-CH2-CH2-CH2-CHZ-CH2-CHa-O-CH2-CH2-CH3, R-C H Z-C H 2-C H a-C H a-C H 2-C H 2-C H 2-O-C H 2-C H 2-O-C H2-C H 2-C H 2-C
H 3, R-C H 2-C H 2-C H 2-C H a-C H 2- C H2-C H 2-O-C H 2-C H Z-O -C H Z-C H 2-C H
3, R-C HZ-CH2-CHa-CH2-CH2-C H2-CH2-O-CHa-CHZ-O-CH 2-CH2-O-C H3, R-C H2-CHZ-CHa-CH2-C H2-CH2-CHa-O-CHa-CH2-O-CH 2-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3, R-C H Z-C H 2-C H a-C H2-C H 2-C H 2-C H 2-O-C H 2-C H 3, R-C H2-C H2-C H2-C H 2-C H2-C H2-C H 2-O-C H 2-C H 3, R-C H2-CH 2-C H2-CHa-C H2-CHZ-CH2-O-C H3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-C H 2-C H a-C H Z-O -C H2-C H z-C H Z-C H 2-C H Z-C H 2-C
H2-C H 3, R-CH2-CH2-CH2-CH2-CHZ-CHa-O-CH2-CH2-CH2-CH2-CH2-CHZ-CH3i R-C H 2-C H 2-C H Z-C H 2-C H 2-C H 2-O-C H2-C H 2-C H a-C H 2-C H 2-C H 3, R-C H 2-C H 2-C H2-C H2-C H Z- C H 2-O-C H Z- C H 2-C H 2-C H 2-C H3, R-CHZ-CH2-CHZ-CHZ-CHa-CH2-O-CH2-CHZ-CH2-CH3, R-CH2-CH2-CHa-CH2-CHZ-CH2-O-CHa-CH2-CH2-O-CH2-CHZ-CH2-CH3, R-C H Z-C H Z-C H 2-C H 2-C H a- C H 2-O -C H2-C H 2-C H2-O-C H2-C H 2-C H 3, R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3, R-C H 2-C H 2-C H 2-C H2-C H 2-C H a-O-C H 2-C H2-C H 3, R-CH2-CH2-CHa-CHZ-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CHZ-CHZ-CH3, R-CH2-CHZ-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHZ-CHa-CH3, R-C H Z-C H Z-C H 2-C H2-C H 2-C H 2-O -C H 2-C H2-O -C H Z-C H 2-C H 2-C H 3, R-C H Z-C H Z-C H 2-C H 2-C H2- C H 2-O-C H 2-C H Z-O -C H 2-C H2-C H a-O -, R-C H z-C H 2-C H 2-C H2-C H 2- C H 2-O-C H2-C H Z-O- C H 2-C H a-C H 3, R-CH2-CH2-CH2-CH2-CHZ-CH2-O-CHZ-CH2-O-CH2-CH2-O-CH2-CH3, R-C H2-C H2-C H 2-C H 2-C Ha- C H2-O-C H 2-C H2-O-C H a-C H 2-O-C H 3, R-C H Z-C H a-C H Z-C H2-C H Z- C H2-O-C H 2-C H Z-O-C H Z-C H 3, R-C H 2-C H 2-C H 2-C H 2-C H 2-C H 2-O-C H2-C H 2-O-C H 3, R-C H2-CH2-CH2-CHz-CH2-CH2-O-C H2-CH 3, R-CH2-CH2-CH2-CH2-CH2-CH2-O-CH3, R-C H 2-C H 2-C H Z-C H 2-C H 2-C H 3, R-C H Z-C H 2-C H a-C H2-C H 2-O-C H 2-C H 2-C H 2-C H 2-C H Z-C H 2-C H2-C H
Z-C H 3, R-C H2-C H a-C H 2-C H 2-C H z-O-C H2-C H 2-C H 2-C H 2-C H2-C H 2-C H2-C H 3, R-CH2-CH2-CH2-CH2-CH2-O-C H2-CH2-CH2-CH 2-C H2-CH2-CH3, R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-C H2-C H2-O-C H2-C H Z-C H 2-C H 2-C H 3, R-C H Z-C H2-C H z-C H 2-C H 2-O-C H 2-C H2-C H2-C H 3, R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CHa-CH3, R-C H Z-C H2-C H 2-C H Z-C H 2-O-C H z-C H2-C H 2-O-C H 2-C H Z-C H a-C H2-C H
3, R-CHa-CH2-CH2-CH2-CHZ-O-CHZ-CH2-CH2-O-CH2-CH2-CH2-CH3, R-C H Z-C H 2-C H 2-C H2-C H 2-O-C H 2-C H Z-C H 2-O -C H 2-C H Z-C H 3, R-C H a-C H 2-C H 2-C H 2-C H 2-O-C H 2-C H 2-C H Z-O-C H Z-C H2-O-C H a-C H
3, R-C H 2-C H 2-C H 2-C H 2-C H z- O-C H2-C H Z-C H 2-O-C H Z-C H Z-O-C H 3, R-C H2-C H2-C H Z-C H 2-C H 2-O-C H z-C H Z-C H 2-O-C H Z-C H 3, R-CH2-CH2-CHZ-CH2-CH2-O-CH2-CH2-CH2-O-CH3, R-C H 2-C H 2-C H2-C H2-C H Z-O-C H 2-C H2-C H 3, R-C H 2-CH2-CH2-CH2-C HZ-O-CH2-C HZ-O-C H2-C HZ-C H 2-CHZ-C H2-C H3, R-C H2-C HZ-CH2-C H2-C HZ-O-C H2-C HZ-O-C H2-C H2-C H 2-CH2-C H3, R-C H2-C H 2-CH2-C HZ-C H2-O-C H2-C H2-O-C H2-C H2-C H2-CH3, R-CH2-CHZ-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3, R-CH2-CH2-CHa-CH2-CH2-O-CH2-CH2-O-CHZ-CH2-CH2-O-CH3, R-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3, R-C H 2-C H 2-C H Z-C H 2-C H 2- O-C H2-C H2-O-C H 2-C H2-C H 3, R-CH2-CH2-C H2-C H2-C H2-O-C H2-CH2-O-C H2-CH2-O-C H2-CH2-CH 3, R-CH2-CH2-CH2-C H2-C H2-O-C HZ-CH2-O-CH2-C H2-O-C HZ-CH3, R-CHa-CHZ-CHa-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3, R-C H2-C H2-C H2-C H Z-C H 2- O-C H 2-C H 2-O-C H 2-C H 3, R-C H 2-C H2-C H 2-C H2-C H a-O-C H a-C H2-O-C H 3, R-C H2-C H 2-C H2-C H 2-C H 2-O-C H 2-C H 3, R-CH2-CH2-CHZ-CH2-C HZ-O-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-, R-CH2-CH2-CH2-CH2-O-CH2-C Ha-C Hz-CH2-C H2-C H2-C H2-CH2-CH2-CH3, R-C H2-CH2-C H2-C H2-O-CHZ-C H2-CHa-CHZ-CH2-CH2-C H2-CH2-CH 3i R-C H a- C H 2-C H Z-C H a-O-C H 2-C H2-C H 2-C H Z-C H 2-C H 2-C H Z-C H 3, R-C HZ-C H2-C H2-C H2-O-CH 2-C HZ-C H2-C H 2-CH2-C H2-C H3, R-CH2-CH2-CHZ-CH2-O-CHZ-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H Z-C H2-O -C H2-C H 2-C H 2-C H Z-C H 3, R-C H 2-C H 2-C H Z-C H 2-O -C H 2-C H 2-C H Z-C H 3, R-C H2-CH 2-CH2-CH2-O-C H 2-CH2-C H2-O-C HZ-CH2-CH2-C H2-CHZ-C H3, R-C H2-C H2-C H 2-C H 2-O -C H 2-C H2-C H2-O-C H 2-C H2-C H z-C H2-C H 3, R-CH2-CH2-CHa-CH2--O-CH2-CH2-CH2-O-CHZ-CH2-CHz-CH3, R-CH2-CH2-CHz-CH2-O-CHa-CHa-CH2-O-CHZ-CH2-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3, R-C H2-CH2-CH2-CH2-O-C H2-C H2-C H2-O-CH2-CH2-O-C H2-C H3, R-CH2-CH2-CH2-C H2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH3, R-C HZ-CH2-C H2-C HZ-O-CH2-C HZ-C H2-O-CH3, R-C H 2-C H Z-C H a-C H 2-O-C H2-C H2-C H 3, R-C H2-C H 2-C H 2-C H 2-O -CH 2-C H 2-O-C H a-C H2-C H 2-C H a-C H Z-C H2-C H
3, 3, R-CH2-CHZ-CH2-CHa-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-CHZ-CH2-CH2-CH2-O-CHZ-CH2-O-CH2-CH2-CH2-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3, R-C H 2-C H Z-C H 2-C H 2-O -C H Z-C H 2-O-C H z-C H 2-C H 2-O-C H Z-C H 2-C H
3, R-C H 2-C H 2-C H 2-C H z-O-C H 2-C H 2-O -C H z-C H Z-C H2-O-C H 2-C H 3, R-CH2-CH2-CHZ-CH2-O-CHZ-CH2-O-CH2-CH2-CH2-O-CH3, R-C H2-CH2-CH2-CH2-O-CH2-C H2-O-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-C H2-O-C H 2-C H 2-O -C H 2-C H a-C H 3, R-C H 2-C H 2-C H 2-C H2-O-C H a-C H 2-O -C H Z-C H Z-O-C H2-C H 2-C H 2-C H
3, R-C H 2-C H 2-C H2-C H2-O-C H2-C H 2-O-C H2-C H a-O-C H 2-C H 2- C H 3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHa-O-CH2-CH2-O-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3, R-CH2-CH2-CH 2-CH2-O-CH2-C H2-O-CH2-CHZ-O-CH3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH3, R-C H 2-C H Z-C H Z-C H 2-O -C H2-C H 2-O-C H 2-C H 3, R-CH2-CH2-CH2-CH2-O-CH2-CH2-O-CH3i R-C H 2-C H Z-C H2-C H2-O -C H 3, R-C H 2-C H 2-C H 2-O-C H 2-C H 2-C H a-C H 2-C H 2-C H 2-C H a-C H 2-C H2-C
H2-C H 3, R-C H 2-C H Z-C H 2-O-C H Z-C H 2-C H 2-C H a-C H a-C H2-C H z-C H2- C H 2-C H
3, R-C H 2-C H 2-C H Z-O-C H 2-C H Z-C H Z-C H Z- C H2-C H2-C H z-C H z-C H 3, R-CHa-CH2-CH2-O-CHz-CH2-CHZ-CHa-CHa-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-O-C H Z-C H Z-C H Z-C H2-C H 2-C H2-C H 3, R-C H2-CHZ-C HZ-O-CH2-C H 2-C H2-C H2-CHZ-C H 3, R-CH2-CH2-CH2-O-CH2-CH2-CHZ-CH2-CH3, R-C H 2- C H 2-C H 2-O -C H 2-C H2-C H Z-C H Z-O-C H Z- C H 2-C H z-, R-CH2-CH2-CH2-O-CHZ-CHa-CH2-C H2-O-CHZ-CH2-CHZ-CH2-, R-CH2-CH2-CH2-O-CH2-CH2-CH2-CHZ-O-CH2-CH2-O-CH2-, R-C H2-CH2-CH a-O-CH2-CH 2-C H2-C H2-O-C Ha-CH3, R-C H2-C H a-C H 2-O-C H 2-C H 2-C H Z-C H Z-O-C H 3:
R-C H a- C H2-C H 2-O-C H 2-C H 2-C H2-C H 3, R-CHZ-CH 2-CH2-O-C H 2-C H2-C HZ-O-CH 2-CHa-CH2-CH 2-C H2-CHZ-C H3, R-CH2-CH2-CHa-O-CH2-CHZ-CH2-O-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-O-C H 2-C H 2-C H 2-O -C H 2-C H a-C H 2-C H2-C H 3, R-CH2-CH2-CH 2-O-CH2-CH2-C H2-O-C H2-CHa-CHZ-CH3, R-CH2-CHa-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH3, R-CH2-CHZ-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3, R-CH2-CHZ-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3, R-C H 2-C H 2-C H 2-O-C H 2-C H 2-C H 2-O-C H 2-C H 2-O-C H 2-C H2- O-C H 3, R-C H 2-C H 2-C H Z-O-C H a-C H 2-C H 2-O -C H2-C H 2-O-C H 2-C H 3, R-C H2-C H a-C H 2-O-C H 2-C H 2-C H Z-O -C H 2-C H 2-O-C H 3, R-CH2-CH2-CH2-O-CHZ-CH2-CH2-O-CH2-CH3, R-CHZ-CH2-CH2-O-CH2-CH2-CHa-O-CH3, R-C H Z-C H 2-C H a-O-C H a-C H Z-C H 3, H 3, R-CH2-C Ha-C H 2-O-CH2-CH2-O-CH2-CHa-C H2-CHz-CH2-CH2-C H3, R-C H 2- C H 2-C H 2-O-C H 2-C H 2-O-C H 2-C H 2-C H 2-C H 2-C H 2-C H 3, R-CH2-CH2-CH2-O-CHZ-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C H2-C H 2-C H 2-O-C H 2-C H Z-O-C H a-C H 2-C H Z-C H 3, R-CHa-CH2-CH2-O-CH2-CHz-O-CH2-CHa-CHZ-CH3, 3, R-C H 2- C H 2-C H 2-O-C H2-C H 2-O-C H 2-C H 2-C H 2-O-C H a-C H 2-C H 3, R-CHZ-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CHZ-O-CH3, R-C H 2-C H2-C H 2-O -C H Z-C H 2-O-C H2-C H 2-C H Z-O-C H2-C H 3, R-C H2-C H Z-C H 2-O-C H 2-C H2-O-C H Z-C H2-C H2-O-C H 3, R-CHZ-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C H3, R-C H2-C H 2-C H2-O -C H 2-C H 2-O-C H2-C H 2-C H 3, R-CH2-CHZ-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CHZ-CH3, R-C HZ-CH2-CH2-O-CH2-CH 2-O-CH2-CH2-O-C H 2-CH2-CH2-C H3, R-C H2-CH2-C H2-O-C H2-CH2-O-CHa-CH2-O-CHZ-CH z-C H3, R-C H2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CHa-O-CH2-CH3, R-C HZ-CH2-CH2-O-CHZ-CH 2-O-CHa-CH2-O-CHa-CH2-O-CH3, R-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH3, R-CH2-CHZ-CH2-O-CHZ-CH2-O-CH2-CH2-O-CH3, R-C H 2-C H 2-C H 2-0- C H z-C H2-O -C H2-C H 3, R-C H Z-C H 2-C H 2-O-C H2-C H2-O-C H 3, R-C H 2-C H2-C H 2-O-C H Z-C H 3, R-CHa-CH2-CH2-O-CH3, R-C H2-CH2-O-C H2-CH2-C H2-CHZ-CHz-CH2-CH Z-C H2-C H2-CH2-CH2-CH3, R-CH2-CH2-O-CHZ-CH2-CH2-CH2-CH2-CH2-CHZ-CHZ-CH2-CH2-CH3, R-C H a-C H 2-O -C H2-C H 2-C H 2-C H 2-C H 2-C H Z-C H Z-C H 2-C H2-C H 3, R-C H 2-C H 2-O-C H 2-C H2-C H 2-C H a-C H 2-C H 2-C H 2-C H2-C H 3, R-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-O-C H 2-C H2-C H 2-C H a-C H2-C H 3, R-C H Z-C H 2-O-C H 2-C H 2-C H 2-C H a-C H 3, R-C H 2-C H 2-O-C H 2-C H 2-C H 2-C H 2-O -C H 2-C H a-C Ha-C H2-C H 3, R-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CHZ-CHa-CH2-CH3, R-C H 2-C H a-O-C H 2-C H 2-C H 2-C H 2-O -C H2-C H 2-C H 2-O-C H 3, R-CH2-C HZ-O-C H2-CH 2-C H2-C H2-O-CH2-C HZ-CH3, R-CH2-CH2-O-CH2-CH2-CHZ-CH2-O-CHZ-CH2-O-CHZ-CH3, R-C H 2-C H 2-O-C H Z-C H 2-C H 2-C H 2-O -C H 2- C H a- O-C H 3, R-C H 2-C H 2-O -C H 2-C H 2-C H 2-C H 2-O-C H2-C H 3, R-CH2-CH2-O-CH2-CH2-CH2-CH2-O-CH3, R-C H2-C H 2-O-C H 2-C H 2-C H Z-C H 3, H 3, R-C H2-C H Z-O-C H 2-C H 2-C H 2-O-C H 2-C H 2-C H a-C H 2-C H2-C H 2-C H 3, R-C H2-C H 2-O-C H Z-C H 2-C H2-O-C H a-C H 2-C H Z-C H Z-C H 2-C H 3, R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C Ha-CH 2-O-CH2-CH2-CH2-O-CHZ-CH 2-CH2-CH3, R-CH2-CHa-O-CHZ-CH2-CH2-O-CH2-CHa-CH3, R-CH2-CHa-O-CH2-CHZ-CH2-O-CH2-CH2-O-CH2-CHZ-CH2-CH2-CH3, R-C H a-C H 2-O-C H 2-C H 2-C H 2-O -C H 2-C H 2-O -C H2-C H2-C H2-C H 3, R-CH2-CH2-O-CHZ-CH2-CH2-O-CHZ-CH2-O-CH2-CH2-CH3, R-C H2-C H 2-O-C H2- C H a-C H 2-O-C H2-C H 2-O-C H 2-C H 2-O -C H 2-C H 3, R-C H Z-C H 2-O-C H2-C H2-C H 2-O-C H2-C H 2-O -C H2-C H 2-O -C H 3, R-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CHZ-CH3, R-C H 2-C H 2-O-C H 2- C H 2-C H 2-O-C H2-C H 2-O-C H 3, R-CHZ-CH2-O-CH2-CH2-CH2-O-CH2-CH3, R-C H2-C H 2-O-C H Z-C H 2-C H 3, R-C H 2-C H z-O-C H2-C H a-O -C H 2-C H 2-C H2-C H 2- C H 2-C H 2-C H 2-C H Z-C H 3, R-C H Z-C H 2-O -C H2-C H2-O-C H2-C H 2-C H 2-C H Z-C H Z-C H 2-C H2-C H 3, R-C H2-C H Z-O-C H Z-C H 2-O-C H 2-C H2-C H 2-C H a-C H2-CH 2-C H 3, R-CHa-CH2-O-CHZ-CH2-O-CH2-CH2-CH2-CH2-CHa-CH3, R-CH2-CHa-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C H 2-C H 2-O-C H a- C H2-O-C H z-C H2-C H 2-C H 3, R-C Ha-C H 2-O-C H 2-C H 2-O-C H 2-C H2-C H Z-C H 3, R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-CHZ-CH2-O-CHz-CH2-O-CHZ-CH2-CH2-O-CH2-CH2-CHa-CH3, R-C H a-C H 2-O-C H 2-C H 2-O-C H Z-C H 2-C H 2-O -C H 2-C H 2-C H 3, R-CH2-CH2-O-C HZ-CHZ-O-CH2-CH2-CH 2-O-CH 2-CH2-O-CH2-CH 3, R-C H2-C H Z-O-C H2-C H Z-O-C H 2-C H 2-C H2-O -C H a-C H2-O-C H 3, R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CHZ-O-CH2-CH3, R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH3i R-CHa-CH2-O-CH2-CH2-O-CHZ-CH2-CH3, R-CH2-CH2-O-CH2-CH2-O-CHZ-CH2-CH3, R-CH2-CH2-O-C H2-CH2-O-CH2-CHZ-O-CH2-CH2-CH2-CH2-CHZ-CH3, R-C H2-CHZ-O-CHZ-CH2-O-C H2-CHZ-O-CH2-CHZ-CH2-CHa-CH3, R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH3, R-CH2-CH2-O-C H2-C H2-O-CH2-CH2-O-CH2-CH2-CH3, R-C H2-CH2-O-CH2-CH2-O-CH2-C H2-O-CH2-CH2-O-CHz-CH2-CH3, R-CH2-CH2-O-CHa-CH2-O-CH2-CH2-O-CH2-CH2-O-CH Z-CH3, R-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3, R-CH2-CH2-O-CHZ-CH2-O-CH2-CH2-O-CHZ-CH3, R-C H 2-C H 2-O -C H 2-C H a-O-C H Z-C H 2-O-C H 3, R-CH2-CH2-O-CH2-CHa-O-CH2-CH3, R-C H 2-C H 2-O-C H 2-C H z-O-C H 3, R-C H2-C H 2-O -C H 2-C H 3, R-CH2-CH2-O-CH3i C H 3, R-C H2-O -C H2-C H 2-C H 2-C H 2-C H2-C H a-C H 2-C H 2-C H 2-C H 2-C H Z-C H
3, R-C H 2-O-C H 2-C H 2-C H 2-C H2-C H 2-C H 2-C H Z-C H 2-C H 2-C H 2-C H 3, R-C H 2- O-C H Z-C H Z-C H 2-C H Z-C H Z-C H 2-C H2-C H 2-C H 2-C H 3, R-CHz-O-CH2-CH2-CHa-CHZ-CH2-CHa-CH2-CH2-CH2-O-CH3, R-C H Z-O-C H Z-C H a-C H 2-C H 2-C H Z-C H 2-C H 2-C H 2-C H 3, R-C H 2-O-C H Z-C H 2-C H z-C H Z-C H 2-C H Z-C H 2-C H2-O-C H2-C H 3, R-C H 2-O -C H 2-C H 2-C H2-C H a-C H 2-C H 2-C H2-C H Z-O -C H 3, R-C H 2-O-C H Z-C H a-C H 2-C H 2-C H 2-C H 2- C H 2-C H 3, R-C H2-O -C H2-C H2-C H 2-C H 2-C H 2-C H 2-C H 2-O-C H 2-C H2-C H 3, R-CHZ-O-CH2-CHZ-CH2-CH2-CHZ-CH2-CH2-O-CH2-CH3, R-C H2-O-C H2-C H2-C H2-C H a-C H Z-C H 2-C H 2-O-C H 3, R-C H 2-O -C H z-C H2-C H 2-C H2-C H 2-C H 2-C H 3, R-CH2-O-CH2-CH2-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH3, R-C H2-O-C H2-C H2-CH2-CH2-CH2-C H2-O-C H2-CHZ-CH3, R-CH2-O-CH2-CHa-CH2-CH2-CHa-CHZ-O-CH2-CH2-O-CH3, R-C H 2-O-C H 2-C H 2-C H Z-C H Z-C H2-C H Z-O-C H Z- C H 3, R-C H2- O-C H2-C H2-C H2-C H2-C H a-C H Z-O-C H3, R-C H 2-O-C H 2-C H 2-C H 2-C H2-C H2-C H 3, R-CH2-O-CHZ-CHZ-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-CH2-O-CH2-CH2-CHa=CH2-CHZ-O-CH2-CH2-CH2-CH2-CH3, R-CH2-O-CH2-C H2-CH2-CH2-CH2-O-CH2-CHa-CH2-CH3, R-C H 2-O -C H2-C H Z-C H 2-C H 2-C H2-O -C H 2-C H 2-C H2-O-C H 3, R-CHa-O-CH2-CH2-CH2-CH2-CHZ-O-CH2-CHa-CH3, R-C H2-O-CH2-C H2-CHZ-CH2-CH2-O-CH2-C H2-O-CH2-C H3, R-CH2-O-CH2-CH2-CH2-CH2-CH2-O-CH2-CHa-O-CH3, R-C H 2-O-C H2-C H Z-C H 2-C H Z-C H Z-O -C H 2-C H 3, R-C H 2-O-C H 2-C H Z-C H Z-C H 2-C H 3, R-C H 2-O-C H2-C H 2-C H 2-C H Z-O-C H 2-C H Z-C H 2-C H2-C H Z-C H 3, R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CH2-CHZ-CH2-O-CH3, R-CHa-O-CH2-CH2-CHZ-CH2-O-CH2-CH2-CH2-CH3, R-CH2-O-CH2-CH2-CH2-CH2-O-CH2-CHa-CH2-O-CH2-CH3, R-C H2-O-C H2-CH2-CH Z-CH2-O-CH2-CH2-CHz-O-CH3, R-C H2-O-C H 2-C H 2-C H2-C H 2-O-C H 2-C H Z-C H 3, R-CHa-O-CH2-CHZ-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH3, R-CH2-O-CH2-CH2-CH2-CH2-O-CHZ-CH2-O-CH2-CH3, R-C H 2-O-C H2-C H 2-C H 2-C H Z-O-C HZ-C H2-O -C H 3, R-C H 2-O-C H 2-C H Z-C H Z-C H 2-O-C H Z-C H 3, R-C H 2-O-C H2-C H Z-C H 2-C H 2-O-C H 3, R-CH2-O-CHa-CHZ-CH2-CH3, R-CH2-O-CH2-CH2-CH2-O-CH2-C H2-CH a-CH2-CH2-CH2-C H2-C HZ-CH3, R-C H Z-O-C H 2-C H 2-C H 2-O -C H a-C H 2-C H 2-C H 2-C H 2-C H 2-C H2-C H 3, R-C H Z-O -C H z-C H2-C H2-O-C H z-C H 2-C H a-C H 2-C H 2-C H 2-C H 3, R-CH2-O-C HZ-C H2-C H2-O-CH2-CHZ-CH2-C H2-CH2-CH3, R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C H 2-O-C H Z-C H 2-C H 2-O-C H a-C H 2-C H 2-C H 3, R-C H 2-O-C H Z-C H 2-C H2-O-C H 2-C H Z-C H 3, R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CHZ-CH2-CH2-CH2-CH3, R-CH2-O-CH2-CH2-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-CH2-O-CH2-CHZ-CH2-O-CH2-CHZ-O-CH2-CH2-CH2-CH3, R-C H 2-O-C H z-C H Z-C H 2-O -C H 2-C H Z-O-C H 2-C H 2-C H 3, R-C H2-O-CH2-CH2-CH2-O-CH2-CHz-O-CHz-CH2-O-CH2-CH2-CH3, R-C HZ-O-CHZ-CH2-CH2-O-CHZ-C H2-O-CH2-CH2-O-CH2-CH3i R-C H Z-O -C H 2-C H 2-C H2-O-C H2-C H 2-O-C H2-C H 2-O-C H 3, R-CH2-O-CH2-CH2-CH2-O-CHa-CH2-O-CH2-CH3, R-C H2-O-C H2-C H2-CH 2-O-CH2-CH2-O-CH3, R-C H 2-O -C H Z-C H z-C H 3, R-C H2-O -C H 2-C H 2- O-C H 2-C H Z-, H 3, R-CH2-O-CH2-CHZ-O-CHZ-CHZ-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3, R-CH2-O-C H2-CH2-O-C H2-CH2-CH2-CH2-CH2-CH 2-CH 2-CH2-CH3, R-C H Z-O-C H2-C H2-O-C H2-C H 2-C H2-C H 2-C H 2-C H Z-C H 2-C H 2-C H 3, R-C H 2-O-C H 2-C H2-O-C H a-C H 2-C H z-C H2-C H 2-C H 2-C H 2-C H 3, R-C H 2- O-C H 2-C H 2-O-C H2-C H 2-C H 2-C H2-C H 2-C H 2-C H 3, R-C H 2-O-C H 2-C H 2-O-C H 2-C H 2-C H 2-C H 2-C H 2-C H 3, R-CHa-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C H 2-O-C H 2-C H2-O-C H2-C H 2-C H Z-C H 3, R-C H2-O-C H2-C H2-O-C HZ-C H2-CH2-CH 3, R-CH2-O-CH2-C H2-O-C H2-CHZ-CHa-O-CHa-C H2-CH2-C H2-CH2-CH3, R-C H2-O-C HZ-C H2-O-C H2-CH2-CH Z-O-CH2-C H2-C H2-C H2-CH 3, R-CH2-O-CH2-CHZ-O-CHZ-CH2-CH2-O-CH2-CHa-CH2-CH3, R-C H 2-O-C H 2-C H 2- O-C H 2-C H 2-C H 2-O-C H 2-C H a-C H 3, R-C H2-O-C H 2-C H 2-O-C H Z-C H2-C H 2-O-C H 2-C H z-O -C H2-C H 2-C H 3, R-C H 2-O-C H a-C H2- O-C H Z-C H 2-C H2-O-C H a-C H 2-O-C H 2-C H 3, R-C H 2-O-C H 2-C H 2-O-C H2-C H Z-C H 2-O-C H a-C H 2-O -C H 3, R-CH2-O-CH2-CHZ-O-CH2-CHZ-CH2-O-CH2-CH3, R-CH2-O-CH2-CH2-O-CH2-CH2-CH2-O-CH3, R-C H 2- O-C H2-C H2-O-C H 2-C H 2-C H 3, R-C H2- O-C H 2-C H Z-O -C H 2-C H2-O-C H 2-C H 2-C H 2-C H2-C H 2-C H2-C H 3, R-CH2-O-C HZ-C H2-O-C Hz-CHZ-O-CH2-CH2-CH 2-CH2-CH2-C H3, R-CH2-O-CHZ-CH2-O-CH2-CH2-O-CH2-CH2-CH2-CH2-CH3, R-C H2-O-C H 2-C H 2-O-C H 2-C H2-O-C H 2-C H 2-C H Z-C H 3, R-CH2-O-C H2-CH2-O-CH2-CH2-O-C H2-CH2-CH3, R-C H2-O-C H 2-C H 2-O-C H Z-C H2-O-C H 2-C H Z-O-C H 2-C H2-C H2-C H 3, R-CH2-O-CH2-C H2-O-C Ha-CH2-O-CH2-CH2-O-CH2-CHZ-CH3, R-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH3, R-CH2-O-CH2-CH2-O-CH2-CHZ-O-CHZ-CH2-O-CH2-CH3, R-CHZ-O-CH2-CH2-O-CHZ-CHa-O-CHZ-CH2-O-CH3, R-C H2-O-C H2-C H 2-O-C H 2-C H Z-O-C H Z-C H 3, R-C H 2-O-C H2-C H2-O-C H2-C H Z-O-C H 3, R-CH2-O-CHZ-CH2-O-CH3, R-CH2-O-CH2-CH3i R-CH2-O-CH3, where R is -H, -OH, -CHZOH, -CH(OH)2, -C(O)OH, -CHZC(O)OH, or an activating moiety, such as a carbodiimide, a mixed anhydride, or an N-hydroxysuccinimide, or a capping group. The invention also includes such moieties attached to a protein or peptide, preferably to an insulin compound. Specific conjugation strategies are discussed in more detail below.
Of these modifying moieties, prefered moieties are those which render the insulin compound less lipophilic and/or more hydrophilic than the corresponding unconjugated insulin compound. The invention includes such modifying moieties further including one or more carbonyl groups, preferably 1, 2, 3, 4, or 5 carbonyl groups; the carbonyl groups may be inserted into the modifying moiety, or an -0- or -CH2- may be replaced with a carbonyl. Further, any of the -CH2-or -CH3 moieties may be substituted, e.g., with a lower alkyl or an -OH or a PAG chain having 1, 2, 3, 4, or 5 PAG subunits, which may be the same or different. Preferably R
is selected so that each -0- is separated from the nearest -0- by at least 2 carbons. The invention also includes branched modifying moieties in which two or more of the moieties are attached to a branching moiety, such as a lysine.

The pharmaceutical characteristics, such as hydrophilicity/lipophilicity of the conjugates according to embodiments of the invention, can be varied by, for example, adjusting the lipophilic and hydrophilic portions of the modifying moieties, e.g., by increasing or decreasing the number of PAG monomers, the type and length of alkyl chain, the nature of the PAG-peptide linkage, and the number of conjugation sites. The exact nature of the modifying moiety-peptide linkage can be varied such that it is stable and/or sensitive to hydrolysis at physiological pH or in plasma. The invention also includes any of the foregoing modifying moieties coupled to a polypeptide, preferably to insulin compound. Preferably, the modifying moiety renders the polypeptide more soluble than a corresponding unconjugated polypeptide, e.g., by a multiplier of at least 1.05, 1.25. 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15. A modifying moiety of the invention may be coupled, for example, to an insulin compound, such as a human insulin, at any available point of attachment. A preferred point of attachment is a nucleophilic residue, e.g., Al, B1 and/or B29.
Moreover, it will be appreciated that one aspect of the invention includes novel modifying moieties, such as but not limited to the moieties of Formulas VII and VIII, in a carboxylic acid form. Further, where the modifying moiety includes a carboxyl group, it can be converted to a mixed anhydride and reacted with an amino group of a peptide to create a conjugate containing an amide bond. In another procedure, the carboxyl group can be treated with water-soluble carbodiimide and reacted with the peptide to produce conjugates containing amide bonds.
Consequently, the invention includes activated forms of the novel moieties presented herein, such as activated forms of the modifying moieties of Formulas VII and VIII and other novel oligomers of the invention, such as carbodiimides, mixed anhydrides, or N-hydroxysuccinimides.

In some cases, the modifying moiety may be coupled to the polypeptide via an amino acid or series of 2 or more amino acids coupled to the C-terminus, or a side chain of the polypeptide. For example, in one embodiment, the modifying moiety is coupled at the -OH or -C(O)OH of Thr, and the mm-modified Thr is coupled to a polypeptide at the carboxy terminus.
For example, in one embodiment, the modifying moiety is coupled at the -OH or -C(O)OH of Thr, and the modified Thr is coupled to the B29 amino acid (e.g., a B29 Lys for liuman insulin) of des-Thr insulin compound. In another example, the mm is coupled at the -OH or -C(O)OH
of Thr of a terminal octapeptide from the insulin compound B-chain, and the mm-modified octapeptide is coupled to the B22 amino acid of des-octa insulin compound. Other variations will be apparent to one skilled in the art in light of this specification.

7.2.11 Salt-forming Moieties In some embodiments, the modifying moiety comprises a salt-forming moiety. The salt-foiming moiety may be various suitable salt-forming moieties as will be understood by those skilled in the art including, but not limited to, carboxylate and ammonium. In some embodiments where the modifying moiety includes a salt forming moiety, the insulin compound conjugate is provided n salt form. In these embodiments, the insulin compound conjugate is associated with a suitable pharmaceutically acceptable counterion as will be understood by those skilled in the art including, but not limited to, negative ions such as chloro, bromo, iodo, phosphate, acetate, carbonate, sulfate, tosylate, and mesylate, or positive ions such as sodium, potassium, calcium, lithium, and ammoniuin.

The foregoing examples of modifying moieties are intended as illustrative and should not be taken as limiting in any way. One skilled in the art will recognize that suitable moieties for conjugation to achieve particular functionality will be possible within the bounds of the chemical conjugation mechanisms disclosed and claimed herein. Accordingly, additional moieties can be selected and used according to the principles as disclosed herein.

7.3 Conjugation Strategies Factors such as the degree of conjugation with modifying moieties, selection of conjugation sites on the molecule and selection of modifying moieties may be varied to produce a conjugate which, for example, is less susceptible to in vivo degradation, and thus, has an increased plasma half life.
For example, the insulin compounds may be modified to include a modifying moiety at one, two, three, four, five, or more sites on the insulin compound structure at appropriate attachment (i.e., modifying moiety conjugation) sites suitable for facilitating the association of a modifying moiety thereon. By way of example, such suitable conjugation sites may comprise an amino acid residue, such as a lysine amino acid residue.

In some einbodiments, the insulin compound conjugates are monoconjugates. In other embodiments, the insulin compound conjugates are multi-conjugates, such as di-conjugates, tri-conjugates, tetra-conjugates, penta-conjugates and the like. The number of modifying moieties on the insulin compound is limited only by the number of conjugation sites on the insulin compound. In still other embodiments, the insulin compound conjugates are a mixture of mono-conjugates, di-conjugates, tri-conjugates, tetra-conjugates, and/or penta-conjugates.

Preferred conjugation strategies are those which yield a conjugate relating some or all of the bioactivity of the parent insulin compound.

Preferred attachment sites include Al N-terminus, Bl N-terminus, and B29 lysine side chain.
The B29 monoconjugate and B1, B29 diconjugates are highly preferred. Another preferred point of attachment is an amino functionality on a C-peptide component or a leader peptide component of the insulin compound.

One or more modifying moieties (i.e., a single or a plurality of modifying moiety structures) may be coupled to the insulin compound. The modifying moieties in the plurality are preferably the same. However, it is to be understood that the modifying moieties in the plurality may be different from one another, or, alternatively, some of the modifying moieties in the plurality may be the same and some may be different. When a plurality of modifying moieties are coupled to the insulin compound, it may be preferable to couple one or more of the modifying moieties to the insulin compound with hydrolyzable bonds and couple one or more of the modifying moieties to the insulin compound with non-hydrolyzable bonds. Alternatively, all of the bonds coupling the plurality of modifying moieties to the insulin compound may be hydrolyzable but have varying degrees of hydrolyzability such that, for example, one or more of the modifying moieties may be relatively rapidly removed from the insulin compound by hydrolysis in the body and one or more of the modifying moieties is more slowly removed from the insulin compound by hydrolysis in the body.

7.3.1 Coupling of Modifying Moiety to Insulin Compound The modifying moiety is preferably covalently coupled to the insulin compound.
More than one moiety on the modifying moiety may be covalently coupled to the insulin compound. Coupling may employ hydrolyzable or non-hydrolyzable bonds or mixtures of the two (i.e., different bonds at different conjugation sites).

In some embodiments, the insulin compound is coupled to the modifying moiety using a hydrolyzable bond (e.g., an ester, carbonate or hydrolyzable carbamate bond).
Use of a hydrolyzable coupling will provide an insulin compound conjugate that acts as a prodrug. A
prodrug approach may be desirable where the insulin compound-modifying moiety conjugate is inactive (i.e., the conjugate lacks the ability to affect the body through the insulin compound's primary mechanism of action), such as when the modifying moiety conjugation site is in a binding region of insulin compound. Use of a hydrolyzable coupling can also provide for a time-release or controlled-release effect, administering the insulin compound over a given time period as one or more modifying moieties are cleaved from their respective insulin coinpound-modifying moiety conjugates to provide the active drug.

In other embodiments, the insulin coinpound is coupled to the modifying moiety utilizing a non-hydrolyzable bond (e.g., a non-hydrolyzable carbamate, amide, or ether bond). Use of a non-hydrolyzable bond may be preferable when it is desirable to allow therapeutically significant amounts of the insulin compound conjugate to circulate in the bloodstream for an extended period of time, e.g., at least 2 hours post administration. Bonds used to covalently couple the insulin compound to the modifying moiety in a non-hydrolyzable fashion are typically selected from the group consisting of covalent bond(s), ester moieties, carbonate moieties, carbamate moieties, amide moieties and secondary amine moieties.

The modifying moiety may be coupled to the insulin compound at various nucleophilic residues, including, but not limited to, nucleophilic hydroxyl functions and/or amino functions.
Nucleophilic hydroxyl functions may be found, for exainple, at serine and/or tyrosine residues, and nucleophilic amino functions may be found, for example, at histidine and/or Lys residues, and/or at the one or more N-terminus of the A or B chains of the insulin compound. When a modifying moiety is coupled to the N-terminus of the natriuretic peptide, coupling preferably forms a secondary amine.

The modifying moiety may be coupled to the insulin compound at a fiee -SH
group, e.g., by forming a thioester, thioether or sulfonate bond.

The modifying moiety may be coupled to the insulin compound via one or more amino groups.
Examples in human insulin include the amino groups at Al, Bl and B29. In one embodiment, a single modifying moiety is coupled to a single amino group on the insulin compound. In another embodiment, two modifying moieties are each connected to a different amino group on the insulin compound. Where there are two modifying moieties coupled to two amino groups, a preferred arrangement is coupling of at BI and B29. Where there are inultiple polymers, the polymers may all be the same or or one or more of the polymers may be different from the others.
Various methods and types of coupling of polymers to insulin coinpounds are described in U.S.
Patent Application No. 09/873,899, entitled "Mixtures of insulin compound conjugates comprising polyalkylene glycol, uses thereof, and methods of making same,"
filed on 04-Jun-01, the entire disclosure of which is incorporated herein by reference.

In still other embodiments, a partial prodrug approach may be used, in which a portion of the modifying moiety is hydrolyzed. For example, see U.S. Patent 6,309,633 to Ek-wuribe et al. (the entire disclosure of which is incorporated herein by reference), which describes modifying moieties having hydrophilic and lipophilic components in which the lipophilic components hydrolyze in vivo to yield a micropegylated conjugate.

7.3.2 Selection of Modifying Moiety and Properties of the Insulin- Compound Conjugate and Complexes Thereof The modifying moiety may be selected to provide desired attributes to the insulin compound conjugate and complexes thereof. Preferred modifying moieties are selected to render the insulin coinpound more soluble in an aqueous solution than the aqueous solubility of the insulin compound in the absence of the modifying moiety, preferably at least 1.05, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 tiines more soluble than the parent insulin compound (i.e., the corresponding unconjugated insulin compound) in an aqueous solution. For example, uncomplexed native human insulin has a solubility of -18mg/ml at a pH of about 7.4. The inventors have surprisingly discovered a method of complexing human insulin conjugates that are more soluble than human insulin by a multiplier of at least 1.05, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15.

In certain embodiments, the modifying moiety is selected to render an insulin compound conjugate having an aqueous solubility that exceeds 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 20 g/L, 50 g/L, 100 g/L, or even 150 g/L at a pH ranging from about 4 to about 8, preferably preferably a pH
ranging from about 5 to about 7.5, ideally pH of about 7.4.

The insulin compound conjug'e can be more orally bioavalable in a mammal than a scientifically acceptable control, such as a corresponding unconjugated insulin compound. In other embodiments, the insulin coinpound conjugate is more orally bioavalable in a human than a scientifically acceptable control, such as a corresponding unconjugated insulin compound. In certain embodiments, absorption of the insulin compound conjugate, e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorption of an unconjugated insulin compound control.

It will be appreciated that while in some aspects of the invention the modifying moiety is selected to render the insulin compound conjugate more soluble than a corresponding unconjugated insulin compound, in other aspects the modifying moiety may also or alternatively be selected to render the insulin compound conjugate equally or more hydrophilic than a corresponding unconjugated insulin compound. Further, the modifying moiety may be selected to render the insulin compound conjugate more amphiphilic than a corresponding unconjugated insulin compound.

In some embodiments, the cation-insulin compound conjugate coinplex is equally as water soluble or more water soluble than .(a) a corresponding uncomplexed insulin compound conjugate, (b) a corresponding uncomplexed and unconjugated insulin compound, and/or (c) a corresponding complexed but unconjugated insulin compound.

In a preferred embodiment, the water solubility of the insulin compound conjugate is decreased by the addition of Zn++. In some embodiments, the modifying moiety is selected to render the insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin compound, and the water solubility of the insulin compound conjugate is decreased by the addition of zinc. In other embodiments, the modifying moiety is selected to render the insulin coinpound conjugate equally or more soluble than a corresponding unconjugated insulin coinpound, the water solubility of the insulin compound conjugate is decreased by the addition of zinc, and the water solubility of the cation complex is greater than the water solubility of insulin coinpound. In another aspect, the insulin compound conjugate is a fatty acid acylated insulin compound, the cation is zinc, and the water solubility of the insulin compound conjugate is decreased by the addition of the zinc. In still another embodiment, the insulin compound conjugate is a fatty acid acylated insulin compound that is equally or more water soluble than a corresponding unconjugated insulin compound, the cation is zinc, and the water solubility of the insulin compound conjugate is decreased by the addition of the zinc.

In certain preferred embodiments, the lipophilicity of the insulin compound conjugate relative to the corrsesponding parent insulin compound is 1 or less than 1. The relative lipophilicity of the insulin compound conjugate as compared to corrsesponding parent insulin compound (krd) can, for exainple, be determined as follows: krel =(teoõjõgate - to)/(th,,,,,a. -to), where relative lipophilicity is measured on an LiChroSorb RP18 (5 m, 250 X 4 mm) high performance liquid chromatography column by isocratic elution at 40 C. The following mixtures can be used as eluents: 0.1M sodium phosphate buffer at pH 7.3 containing 10% acetonitrile, and 50%
acetonitrile in water. Void time (to) is identified by injecting 0.1 mM sodium nitrate. Retention time for human insulin is adjusted to at least 2to by varying the ration between the mixtures of (c)(i) and (c)(ii). Preferably, in these embodiments, the relative lipophilicity is about equal to 1 or is less than 1 or substantially less than 1. In a preferred embodiment, the insulin compound is human insulin, and the relative lipophilicity is less than 1. I'referably the relative lipophilicity is less than about 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, or 0.90.
Discussion of techniques for determining solubility and/or lipophilicity of insulin and insulin conjugates are set forth in the U.S. Patent 5,750,499 entitled "Acylated insulin" issued to Harelund et al., on 12-May-98, the entire disclosure of which is incorporated herein by reference.

In one embodiinent, the relative lipophilicity is as described above and the modifying moiety is a carbon chain having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbons, wherein the carbon chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 oxy groups inserted therein. In another embodiment, the relative lipophilicity is as described above and the modifying moiety is a carbon chain having 5, 6, 7, 8, 9 or 10 carbons, wherein the carbon chain comprises 2, 3 or 4 oxy groups inserted therein. In a related embodiment, the relative lipophilicity is as described above and the modifying moiety comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 polyalkalene glycol units. In another related embodiment, relative lipophilicity is as described above and the modifying moiety comprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3 polypropylene glycol units.

7.4 Metal Cation Component and Characteristics of Complexes The cation-insulin compound conjugate complexes include a metal cation.
Suitable metal cations for use as the cation component include any metal cation capable of complexing, aggregating, or crystallizing with the insulin compound conjugate. It is preferred that the metal cation be complexed to the insulin compound conjugate. Single or multiple cations can be used. The cation is preferably not significantly oxidizing to the insulin compound conjugate, i.e., not oxidizing to the extent that the coinplexes are rendered useless for their intended purpose.

In some embodiments, the metal cation is biocompatible. A metal cation is biocompatible if the cation presents no unduly significant deleterious effects on the recipient's body, such as a significant immunological reaction at the injection site. However, it will be appreciated that in some circumstances, the risks of toxity and other deleterious effects may be outweighed by the benefits of the cation-insulin compound conjugate composition, and therefore may be acceptable under such circumstances.

The suitability of metal cations for stabilizing biologically active agents and the ratio of metal cation to biologically active agent needed can be determined by one of ordinary skill in the art by performing a variety of tests for stability such as polyacrylamide gel electrophoresis, isoelectric focusing, reverse phase chromatography, and HPLC analysis on particles of metal cation-stabilized biologically active agents prior to and following particle size reduction and/or encapsulation.

The metal cation component suitably includes one or more monovalent, divalent, or trivalent metal cations, or combinations thereof. In a preferred embodiment, the metal cation is a Group II
or transition metal cation. Examples of suitable divalent cations include Zn++, Mn++, Ca++, Fe++, Ni++, Cu++, Co++ and/or Mg++. Where a monovalent cation is included, it is preferably Na+, Li+, or K+. The cation is preferably added as a salt, such as a chloride or acetate salt, most preferred are ZnCIZ and ZnAc.

The molar ratio of insulin compound conjugate to cation is typically between about 1:1 and about 1:100, preferably between about 1:2 and about 1:12, and more preferably between about 1:2 and about 1: 7 or about 1:2, 1:3, 1:4, 1:5, 1:6, or 1:7. In a particular embodiment, Zn++ is used as the cation component, it is provided at a zinc cation component to insulin compound conjugate molar ratio of about 1:1 and about 1:100, preferably between about 1:2' and about 1:12, and more preferably between about 1:2 and about 1:7 or about 1:2, 1:3, 1:4, 1:5, 1:6, or 1:7.

The cation component is preferably greater than about 90% a single cation, such as Zn++.
Preferably, the cation is greater than about 95%, 99%, or 99.9% Zn++.

Preferably resistance of the complexed insulin compound conjugate to chymotrypsin degradation is greater than the chymotrypsin degradation of the corresponding uncomplexed insulin compound conjugate. Preferably resistance of the complexed insulin compound conjugate to chymotrypsin degradation is greater than the chymotrypsin degradation of the corresponding complexed but uncof jugated insulin compound.

The complexed insulin compound conjugate can be more orally bioavalable in a mammal than a scientifically acceptable control, such as a corresponding unconiplexed insulin compound conjugate. In other embodiments, the complexed insulin compound conjugate is more orally bioavalable in a human than a scientifically acceptable control, such as a corresponding uncomplexed insulin compound conjugate. In certain embodiments, absorption of the complexed insulin compound conjugate, e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorption of an uncoinplexed insulin compound conjugate.

The coinplexed insulin compound conjugate can be more orally bioavalable in a mammal than a scientifically acceptable control, such as a corresponding complexed bait uraconjugated insulin compound. In other embodiments, the complexed insulin compound conjugate is more orally bioavalable in a human than a scientifically acceptable control, such as a corresponding complexed but unconjugated insulin compound.. In certain embodiments, absorption of the complexed insulin compound conjugate, e.g., as measured by plasma levels of the conjugate, is at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater that the absorption of an complexed but unconjugated insulin coinpound..

7.5 Complexing agents In some embodiments, the cation-insulin compound conjugatecompositions include one or more complexing agents. Examples of coinplexing agents suitable for use in the present invention include protamines, surfen, globin proteins, spermine, spermidine albumin, amino acids, carboxyllic acids, polycationic polymer coinpounds, cationic polypeptides, anionic polypeptides, nucleotides, and antisense. See Brange, J., Galenics of Insulin compound, Springer-Verlag, Berlin Heidelberg (1987), the entire disclosure of which is incorporated herein by reference. The suitability of complexing agent's for stabilizing the compositions can be determined by one of ordinary skill in the art in the light of the present disclosure. In some embodiments, the cation-insulin compound conjugatecompositions specifically exclude or are substantially devoid of a complexing agent.

A preferred complexing agent is protainine. In a solid form, the protamine will preferably be present in about 3:1 to about 1:3 molar ratio of insulin compound to protamine, more preferably about 2:1 to about 1:2 molar ratio, ideally about 1:1 molar ratio. In some embodiments, the cation-insulin compound conjugatecompositions specifically exclude or are substantially devoid of protamine.

Amino acids may also be used as complexing agents, e.g., glycine, alanine, valine, leucine, isoleucine, serine threonine, phenyl alanine, proline, tryptophan, asparagine, glutamic acid, and histidine, and oligopeptides, such as diglycine.

Carboxylic acids are also suitable for use as complexing agents; examples include acetic acid, and hydroxycarboxylic acids, such as citric acid, 3-hydroxybutyric acid, and lactic acid.

7.6 Stabilizing agents In some embodiments, the cation-insulin compound conjugate compositions include one or more stabilizing agents. Preferred stabilizing agents include phenolic compounds and aromatic compounds. Preferred phenolic compounds are phenol, m-cresol and m-paraben or mixtures thereof. The stabilizing agent may be provided in any amount that improves stability of the cation-insulin compound conjugate compositions relative to a scientifically acceptable control, such as a corresponding cation-insulin compound conjugate composition in the absence of the stabilizing agent.

7.7 Presentation of Complexes The complexes may be provided as a dry solid, such as a substantially pure powder of cation-insulin compound conjugate, or a powder including a cation-insulin compound conjugate solid along with other pharmaceutically acceptable coinponents. The complexes may also be provided in a dissolved state in aqueous or organic medium, and/or as undissolved solids in such mediums.

7.7.1 Solid Compositions The cation-insulin compound conjugate complexe may be provided as as a solid.
The solid may, for example be in a dried state or in an undissolved state in an aqueous solution, organic solvent, emulsion, microemulsion, or oher non-dried form.

In one embodiment, the cation-insulin compound conjugate complexe is provided as a pure processed solid composition. In a pure processed solid compostion, the molar ratio of insulin compound conjugate to cation is typically about 3:4 to about 3:0.5 (insulin compound conjugate:cation), about 3:3.5 to about 3:1, or ideally about 3:1.

In a processed pure,solid T-type compostion (with cation, insulin compound conjugate and without protamine), the molar ratio of insulin compound conjugate to cation is typically about is typically about 3:4 to about 3:0.5 (insulin compound conjugate:cation), about 3:3.5 to about 3:1, or ideally about 3:1. In a processed pure solid T-type protamine compostion (with cation, insulin compound conjugate and protamine), the molar ratio of insulin compound conjugate to cation is typically about 3:6 to about 3:0.5 (insulin compound conjugate:cation), about 3:5 to about 3:1, or ideally about 3:2.

In, a processed pure solid R-type (lente) compostion (with cation, insulin compound and stabilizing compound (e.g., a phenolic compound), and without protamine), the molar ratio of insulin compound conjugate to cation can typically range from about 3:4.5 to about 3:0.9, preferably about 3:3.9 to about 3:2.4. In a processed pure solid R-type (ultralente) compostion (with cation, insulin compound and stabilizing compound (e.g., a phenolic compound), and without protamine), the molar ratio of insulin compound conjugate to cation can typically range from about 3:12 to greater than about 3:4.5, preferably about 3:9 to about 3:4.8, more preferably about 3:6 to about 3:5.4. In a processed pure solid R-type protamine compostion (with cation, insulin compound and stabilizing compound (e.g., a phenolic compound), and protamine), the molar ratio of insulin compound conjugate to cation can typically range from about 3:12 to about 3:3, preferably about 3:9 to about 3:4.5, more preferably about 3:6.9 to about 3:5.4.

For a monovalent cation, such as Na+, the solid would be expected to have an insulin compound conjugate to cation ratio of about 3:6 to about 3:3.

Solid compositions of the invention may, for example, include coinpositions, such as powders, including insulin coinpound conjugates and/or cation-insulin compound conjugate complexes of the invention. Preferably the solid compositions are provided at a pharmaceutically acceptable level of purity, i.e., free of contaminants which would unacceptably diminish the suitability of the compositions for use in liumans.

In some embodiments, compositions are provided in which the cation-insulin compound conjugate component is greater than about 90% crystalline, preferably greater than about 95%
crystalline, more preferably greater than about 99% crystalline. In other embodiments, compositions are provided in which the cation-insulin compound conjugate component is greater than about 90% amorphous solids, preferably greater than about 95% amorphous solids, more preferably greater than about 99% amorphous solids.

In still other embodiments, compositions are provided in which the cation-insulin compound conjugate coinponent is present in a mixture of amorphous solids and crystalline solids. For exainple, the ratio of amorphous solid to crystalline solid may be from about 1:10 to about 10:1, or about 1:9 to about 9:1, or about 1:8 to about 8:1, or about 1:7 to about 7:1, or about 1:6 to about 6:1, or about 1:5 to about 5:1, or about 1:4 to about 4:1, or about 1:3 to about 3:1, or about 1:2 to about 2:1, or about 1:1.

Furthennore, compositions can be provided using mixtures of cation-insulin compound solids having different insulin compounds, such as a solid including native insulin compound with a solid including insulin compound conjugates, or solids including one insulin compound conjugate with a solid including a different insulin compound conjugate.

Moreover, the solid type and insulin compound/insulin compound conjugate component may all vary. For example, compositions can be provided wliich include Zn-insulin compound crystals using native insulin compound and amorphous insulin compound conjugates, or compositions can be provided which include amorphous Zn-insulin compound solids using native insulin compound and crystalline Zn-insulin compound conjugates. Such mixtures may be used to achieve variations in physical characteristics, such as dissolution profile and/or variations in pharmacokinetic profile.

The average particle size of the solids are preferably in the range of about 0.1 to about 100 microns, more preferably 1-50 microns, still more preferably 1-25 microns, ideally 1-15 microns.
Small particle sizes may be obtained by microcrystallization conditions, spray drying, milling, vacuum drying, freeze drying and the like.

In one embodiment the composition, when dried, contains greater than about 96%
w/w insulin compound conjugate and from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc. In another embodiment the coinposition, when dried, contains greater than about 91% w/w insulin compound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, and from about 0.2 to about 5% w/w phenol. In yet another embodiment the composition, when dried, contains greater than about 82% w/w insulin compound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, from about 0.2 to about 14 % w/w protamine. In yet another embodiment the composition, when dried, contains greater than about 71 % w/w insulin compound conjugate, from about 0.05, 0.1, 0.15, or 0.2 to about 4% w/w zinc, from about 0.2 to about 14 % w/w protamine, and from about 0.2 to about 15% w/w phenol.

In another embodiment the composition, when dried, includes from about 0.1 to about 2% w/w Zn and from about 0.08 to about 1% w/w phenol, preferably from about 0.5 to about 1.3%
w/w Zn++, andfiom about 0.1 to about 0.7% w/w phenol, more preferably from greater than or equal to 1 to about 3.5% w/w Zn', and from about 0.1 to about 3% w/w phenol, and still more preferably from greater than or equal to 1.3 to about 2.2% w/w Zn++, and from about 0.4 to about 2% w/w phenol.

The complexes can be provided in a lente-type preparation. For example, in a preferred dried lente-type preparation, Zn is provided in an amount ranging from about 0.1 to about 2% w/w and phenol is present in an amount ranging from about 0.08 to about 1% w/w, with the remaining %
w/w being insulin compound conjugate. Ideally, for a dried lente-type preparation, Zn is provided in an amount ranging from about 0.5 to about 1.3% w/w and phenol is present in an amount ranging from about 0.1 to about 0.7% w/w, with the remaining % w/w being insulin compound conjugate.

The coinplexes can be provided in an ultralente-type preparation. For example, in a preferred dried ultralente-type preparation, Zn is provided in an amount ranging from greater than or equal to 1 to about 3.5% w/w, and phenol is present in an amount ranging from about 0.1 to about 3%
w/w, with the remaining % w/w being insulin compound conjugate. Ideally, for a dried ultralente-type preparation, Zn is provided in an amount ranging from greater than or equal to 1.3 to about 2.2% w/w, and phenol is present in an amount ranging from about 0.4 to about 2% w/w, with the remaining % w/w being insulin compound conjugate.

7.7.2 Liquid Compositions The cation-insulin compound conjugate complexes may be provided as components undissolved components of a liquid. For example, the liquid may be an aqueous solution including a cation-insulin coinpound conjugate as a precipitate, or the cation-insulin compound conjugate may be provided as a component of a suspension, emulsion or microemulsion. The liquid may also include dissolved components or complexes, along with the undissolved components.

7.7.3 Mixtures and Co-crystals The compositions of the invention may, for example, include complex mixtures, solid mixtures, hybrid complexes and co-crystals.

Thus, for example, the invention provides compositions which include two or more insulin compound conjugates and/or unconjugated insulin compounds. Further, where the compositions include solids, the solids may have different forms. Thus, for example, on solid may be crystalline and another solid may be an amorphous solid. As noted elsewhere, the solids may be provided in a dried form or may be provided as solid components of a liquid mixture. In a preferred embodiment, the mixture of the invention includes two or more different insulin compound conjugates, and the different insulin compound conjugates have different solubilities.
In one embodiment, one of the complexes comprises a lipophilic insulin compound conjugate and the other coinprises a hydrophilic insulin compound conjugate. In still another embodiment, the complexes may include different insulin compound conjugates, where one ore more of the coinplexes has a circulation half-life of from about 1 to about 4 hours, and one or more the complexes has a circulation half-life that is significantly greater than the circulation half-life of the first complex. In a related embodiment, one of the complexes has a rapid-acting profile and another of the complexes has a medium-to-long acting profile. Furthemore, one of the complexes may have profile suitable for basal insulin compound control while anotlier has a profile suitable for post-prandial glucose control. Preferred mixtures are mixtures of HIM2 and insulin, mixtures of HIM2 and IN105, mixtures of IN105 and insulin compound, mixtures of IN105 and fatty acid acylated insulin, mixtures of HIM2 and fatty acid acylated insulin. Suitable fatty acid acylated insulins are described in the following U.S. patents, the entire disclosures of which are incoiporated herein by reference: U.S. Patent 6,531,448, entitled "Insoluble compositions for controlling blood glucose," issued 11-Mar-03; U.S. Patent RE37,971, entitled "Selective acylation of epsilon-ainino groups," issued 28-Jan-03; U.S. Patent 6,465,426, entitled "Insoluble insulin compositions," issued 15-Oct-02; U.S. Patent 6,444,641, entitled "Fatty acid-acylated insulin analogs." issued 03-Sep-02; U.S. Patent 6,335,316, entitled "Method for administering acylated insulin," issued 01-Jan-02; U.S. Patent 6,268,335, entitled "Insoluble insulin compositions," issued 31-Jul-01; U.S. Patent 6,051,551, entitled "Method for administering acylated insulin," issued 18-Apr-00; U.S. Patent 5,922,675, entitled "Acylated Insulin Analogs,"
issued 13-Jul-99; U.S. Patent 5,700,904, entitled "Preparation of an acylated protein powder,"
issued 23-Dec-97; U.S. Patent 5,693,609, entitled "Acylated insulin analogs Granted," issued 02-Dec97; U.S. Patent 5,646,242, entitled "Selective acylation of epsilon-amino groups," issue 08-Jul-97; U.S. Patent 5,631,347, entitled "Reducing gelation of a fatty acid-acylated protein," issued 20-May-97; U.S. Patent 6,451,974, entitled "Method of acylating peptides and novel acylating agents," issued 17-Sep-02; U.S. Patent 6,011,007, entitled "Acylated insulin,"
issued 04-Jan-00;
U.S. Patent 5,750,497, entitled "Acylated insulin Granted: 12-May-98; U.S.
Patent 5,905,140, entitled "Selective acylation method," issued May 18, 1999; U.S. Patent 6,620,780, entitled "Insulin derivatives," issued Sep. 16, 2003; U.S. Patent 6,251,856, entitled "Insulin derivatives,"
issued Jun. 26, 2001; U.S. Patent 6,211,144, entitled "Stable concentrated insulin preparations for pulmonary delivery," issued Apr. 3, 2001; U.S. Patent 6,310,038, entitled "Pulmonary insulin crystals," issued Oct. 30, 2001; and U.S. Patent 6,174,856, entitled "Stabilized insulin compositions," issued Jan. 16, 2001. Especially preferred mono-fatty acid acylated insulins having 12, 13, 14, 15, or 16-carbon fatty acids covalently bound to Lys(B29) of human insulin.

In one embodiment, the invention provides a co-crystal having two different insulin compounds and/or insulin compound conjugates. Preferably the co-crystal exhibits one or more of the following characteristics: substantially homogenous dissolution, a single in vivo dissolution curve, and/or a single peak pharmacodynamic profile. Preferred co-crystals are co-crystals of HIM2 and insulin, co-crystals of HIM2 and 1N105, co-crystals of IN 105 and insulin compound.

In one embodiment, the co-crystal includes human insulin, and co-crystallization with human insulin reduces the solubility of the crystal relative to the solubility of a corresponding crystal of the insulin compound conjugate. In another embodiment, the co-crystal includes human insulin, and co-crystallization with human insulin decreases the solubility of the crystal relative to the solubility of a corresponding crystal of the insulin compound conjugate.

In another embodiment, the co-crystal includes a rapid acting, rapid clearing, and/or highly potent insulin compound conjugate, and a long-acting, slow clearing, and/or poorly potent insulin compound conjugate. Preferably the co-crystal has a PK/PD profile suitable for post-prandial glucose control or for overnight basal insulin compound control.

In another embodiment, the invention provides a mixture or co-crystal in which an insulin compound conjugate is included with human insulin or lyspro insulin. The mixtures of the invention may include two different insulin compound conjugates. The mixtures may include an insulin compound conjugate and an unconjugated insulin compound. The mixtures may include different insulin compound conjugates with different insulin compounds.

Further, the invention provides coinplexes having two different insulin compound conjugates and/or an insulin compound conjugate and an unconjugated insulin compound. The invention provides hybrid co-crystals of two, three or more different insulin compound conjugates. The invention provides a complex having an insulin compound conjugate with an unconjugated insulin compound. The invention provides a co-crustal with two or more different hydrophilic insulin compound conjugates; two or more different hydrophobic insulin compound conjugates;
two or more different amphiphilic insulin compound conjugates; a hydrophilic insulin compound conjugate and a lipophilic insulin compound conjugate; a hydrophilic insulin compound conjugate and an unconjugated insulin compound; HIM2 together with an unconjugated insulin compound; IN105 together with an unconjugated insulin compound; HIM2 together with IN105;
HIM2 together with insulin compound and 1N105; and otlier coinbinations of the foregiong elements. As mentioned elsewhere, the complexes may be provided as dried solids, as dissolved complexes in solution and/or as undissolved complexes in solution. Various combinations may, for example, be employed to provide a complex or co-crystal having an extended profile.

7.8 Solubility of Complexes of the Invention Preferably the aqueous solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from about 1/15, 1/14, 1/13, 1/12, 1/11, 1/10, 1/9, 1/8, 1/7, 1/6, 1/5 up to about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or <10 times the aqueous solubility of the uncomplexed insulin compound conjugate. Any combination of the foregoing upper and lower limits is within the scope of the invention. However, a preferred range is from about 1/15 to <5, more preferred is about 1/10 to about 2, ideal is about 1/10 to <0. In a particularly surprising aspect of the invention, the aqueous solubility of the cation-insulin compound conjugate in solution at a pH of about 7.4 is often substantially less than the aqueous solubility of the insulin compound conjugate in solution at a pH of about 7.4. However, it will be appreciated that in certain embodiments, the aqueous solubility of the cation-insulin compound conjugate in solution at a pH of about 7.4 may be the same as, greater than, or substantially greater than, the aqueous solubility of the insulin compound conjugate in solution at a pH of about 7.4.

In one surprising embodiment, the aqueous solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the corresponding uncomplexed insulin compound conjugate in solution at a pH of about 7.4, and the cation-insulin compound conjugate complex remains soluble at greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L in aqueous solution across a pH range beginning at about 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.1, or 6.9 and ending at about 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9. In yet another embodiment, the aqueous solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the corresponding insulin compound conjugate in solution at a pH of about 7.4, and the cation-insulin compound conjugate complex remains soluble at greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L in aqueous solution across a pH range from about 5.8 to about 8.5, preferably across a pH range from about 6.5 to about 8, more preferably across a pH range from about 6.9 to about 7.8. [QUESTION: Is the g/E
solubility nzeasui=ecl using the weiglzt of tlze cofizplex of= the weight of the insuliu cofzzpouud coizjugate?]

Preferably the insulin compound conjugates of the invention are selected to produce crystals in aqueous solution at a pH which is equal to pI +/-anout 2.5, where the concentration of insulin compound conjugate is from about 0.5 mghnl to about 50 mg/ml, preferably about 5 mghnl to about 30 mg/ml, more preferably about 15 mghnl to about 30 mg/ml, and the crystal formulation begins to occur at about 3, 4 or 5% w/w/ cation to insulin coinpound conjugate, where the cation is preferable Z-'+. Preferably crystals are present for a monoconjugate without protamine in an aqueous solution at a pH ranging from about 4, 4.1, 4.2, 4.3 or 4.4 to about 5.2., 5.3, 5.4, 5.5, 5.6, 5.7 or 5.8, preferably at pH of about 4 to <6.5, preferably about 4 to <5.8, preferably about 4.2 to about 5.5, more preferably about 4.4 to about 5.2. Preferably crystals are present for a diconjugate without protamine at pH of about 3.5 to <5.8, preferably about 3.8 to about 5.5, more preferably about 4.0 to about 5.2. Preferably crystals are present for a triconjugate without protamine at pH of about 3 to <5.5, preferably about 3.3 to about 5, more preferably about 3.8 to about 4.8.

7.8.1 R-type Complexes Preferably the aqueous solubility of the R type Zn complex of the insulin compound conjugate at a pH of about 7.4 has a range of about 10 to about 150 g/L, more preferably about 20 to about 130 g/L, more preferably about 30 to about 110 g/L, more preferably about 35 to about 60 g/L.
Preferably the aqueous solubility of the R type Zn complex of the insulin compound conjugate with protamine at a pH of about 7.4 has a range of about 10 to about 110 g/L, more preferably about 20 to about 85 g/L, more preferably about 30 to about 70 g/L.
7.8.2 T-type Complexes Preferably the aqueous solubility of the T-type Zn complex of the insulin compound conjugate at a pH of about 7.4 has a range of about 30 to about 175 g/L, more preferably about 50 to about 160 g/L, more preferably about 70 to about 150 g/L.

Preferably the aqueous solubility of the T-type Zn complex of the insulin compound conjugate with protainine at a pH of about 7.4 has a range of about 10 to about 150 g/L, more preferably about 20 to about 130 g/L, more preferably about 30 to about I 10 g/L, more preferably about 35 to about 60 g/L.

7.8.3 NPH-Type Complexes Preferably the aqueous solubility of the NPH-type complex, of the insulin compound conjugate at a pH of about 7.4 has a range of about about I to about 150 g/L, more preferably about 5 to about 120 g/L, still more preferably about 10 to about 90 g/L.

7.9 Pharmaceutical Properties Complexation of the insulin compound conjugate with cation generally results in improved pharmaceutical properties of the insulin compound conjugate, relative to a scientifically acceptable control, such as a corresponding uncomplexed insulin compound conjugate.

In some cases, the complexed insulin compound conjugate will exhibit an extended or otherwise altered pK profile relative to a scientifically acceptable control, such as a corresponding uncoinplexed insulin compound conjugate. In certain cases, the pK profile will exhibit a lispro-like profile. pK profile can be assessed using standard in vivo experiments, e.g., in mice, rats, dogs, or humans. Assays described herein for assessing the attributes of cation-insulin compound conjugate complexes are an aspect of the invention.

The complexes may exhibit improved chemical stability. Various attributes of stability can be assessed by exposing the complex to various assay conditions such as the presence of plasma, the presence of proteases, the presence of liver homogenate, the presence of acidic conditions, and the presence of basic conditions. Stability is improved relative to uncomplexed insulin compound conjugate when stability of the complexed insulin compound conjugate in any one or more of these assay conditions is greater than stability of the uncomplexed insulin compound conjugate in the same conditions. A preferred assay for determining stability in an acidic environment involves exposing the complexed insulin coinpound conjugate to a solution having a pH of 2 for at least 2 hours, where decreased degradation of the complexed insulin compound conjugate relative to a scientifically acceptable control, such as a corresponding uncomplexed insulin compound conjugate, is indicative of improved stability. In vivo assays can also be used to test stability. For example, stability of the complexed insulin compound conjugate can be tested by exposure to the gastrointestinal tract of a subject and comparison with an appropriate control.
7.10 Method of Making The invention also provides a method of making cation-insulin compound conjugate compositions described herein. The method generally involves contacting one or more insulin compound conjugates, as described herein, with one or more cations, as described herein, to form a solid.

For a divalent cation, such as Zn#, the molar ratio of insulin compound conjugate to cation used to make the coinposition in an aqueous solution with an insulin compound concentration ranging from about 2 mg/ml to about 50 mg/ml can typically range from about 1:15 (insulin compound conjugate:cation) to about 1:0.4, preferably about 1:9 to about 1:2.

To make T-type solid (with cation and insulin compound conjugate and without protamine) in the aqueous solution conditions described above, the molar ratio of insulin compound conjugate to cation is preferably about 1:1.5 to 1:3, ideally about 1:2. To make R-type solid (with cation, insulin compound and stabilizing compound (e.g., a plienolic compound), and without protamine) in the aqueous solution conditions described above, the molar ratio of insulin compound conjugate to cation is preferably about 1:4 to 1:9, preferably about 1:7 to about 1:9 ideally about 1:8.

To make T-type protamine solid (with cation and insulin compound conjugate and protamine) in the aqueous solution conditions described above, the molar ratio of insulin compound conjugate to cation is preferably about 1:1.5 to 1:9, ideally about 1:2. To make R-type protamine solid (with cation, insulin compound and stabilizing compound (e.g., a phenolic coinpound), and protamine) in the aqueous solution conditions described above, the molar ratio of insulin compound conjugate to cation is preferably about 1:2 to 1:15, preferably about 1:7 to about 1:9 ideally about 1:8.

The insulin compound conjugate is preferably added to the buffer in an amount which is calculated to achieve a concentration in the range of from greater than 2 to about 100 g/L, preferably from about 3, 4, 5, 6, 7, 8, 9 or 10 to about 40 g/L, more preferably from about 10 to about 30 g/L.

Where the cation is divalent (e.g., Zn++,Can, it is preferably added in an amount which calculated to achieve a concentration in the range of from about 0.04 to about 10 g/L, preferably from about 0.1 to about 5 g/L, more preferably from about 0.2 to about 4 gIL.
For T-type crystals or T-type protamine crystals, the cation concentration is preferably in the range of from about 0.04 to about 1 g/L, more preferably about 0.1 to about 0.3 g/L. For R-type crystals or R-type protamine crystals, the cation concentration is preferably in the range of from about 1 to about 5 g/L, more preferably about 1.5 to about 4 g/L.

Where the cation is monovalent, it is preferably added in an amount which calculated to achieve a concentration in the range of from about 0.08 to about 40 g/L, preferably from about 0.4 to about 20 g/L, more preferably from about 0.8 to about 16 g/L.

The method may further include combining a stablizing agent with the cation and insulin compound conjugate. Preferred stablizing agents are described above. When used, the stabilizing agent is added in an amount sufficient to provide a greater degree of solid formation than is achieved using the same reagents and reaction conditions in the absence of the stabilizing agent. Where the stabilizing agent is a phenolic compound (e.g., phenol, m-cresol, m-paraben), can be added in an amount ranging from about 10 to about 50% w/w, more preferably from about 20 to about 40% w/w, still more preferably from about 25 to about 35% w/w. In a more preferred embodiment, the stabilizing agent is a phenolic compound (e.g., phenol, m-cresol, m-paraben), can be added in an amount ranging from about 0.01 to about 10% w/w, more preferably 0.01 to about 5% w/w, still more preferably 0.01 to about 1% w/w. Thus, in one embodiment, the method involves combining insulin compound conjugate, a cation and a stabilizing agent in an aqueous solution to yield the cation-insulin compound conjugate coinposition, wliere the combination may yield solublized complexes and/or crystalline or non-crystalline solids.

The method may further include the use of a complexing agent, such as protamine, which is combined with the cation and insulin compound conjugate, and optionally also includes a stabilizing agent.

To prepare a solid in an aqueous solution having a pH in the range of about 5 to about 8, protamine is preferably provided in an amount relative to insulin compound conjugate of about 4 to about 45% w/w (protamine/insulin compound), preferably about 8 to about 25%
w/w, more preferably about 9 to about 20% w/w, ideally about 10 to about 12% w/w. For T-type solids, a preferred pH range is from about 5 to about 6, more preferably about 5 to about 5.5, still more preferably about 5.1 to about 5.3, ideally about 5.2. For R-type solids, a preferred pH range is from about 6 to about 7, more preferably about 6.2 to about 6.8, still more preferably about 6.4 to about 6.6, ideally about 6.5.

The inventors have surprisingly discovered that T-type complexes can be converted to protamine T-type complexes in the absence of a stabilizing agent, such as phenol. The T-type complex is made by complexing Zn with the insulin compound molecule in aqueous solution in the absence of phenol. Protamine is then added to convert the T-type complex into a protamine T-type complex. Amounts and pH ranges are as described above.

Thus, in one embodiment, the method involves combining insulin compound conjugate, a cation and a coinplexing agent in an aqueous solution to yield the cation-insulin compound conjugate composition, where the combination may yield solublized complexes and/or crystalline or amorphous solids. In another embodiment, the method involves coinbining insulin compound conjugate, a cation, a complexing agent, and a stabilizing agent in an aqueous solution to yield the cation-insulin compound conjugate composition, where the combination may yield solublized complexes and/or crystalline or amorphous solids.

In some embodiments, the compositions can include preservatives. Examples of suitable preservatives include benzyl alcohol, p-hydroxybenzoic acid esters, glycerol.
Stabilizing agents, such as phenol, m-cresol, and m-paraben, can also be used as preservatives.
Glycerol and phenol are suitably added together to enhance antimicrobial effectiveness.

Other components useful in preparing the solids include isotonic agents, such as NaCl, glycerol, and monosaccharides.

The cation insulin compound conjugate solids can typically be formed relatively quickly. For example, solid fonnation is typically complete within three days, often within 24 hours. It may be desirable in some instances to slow the reaction down in order to improve crystal formation.

In one embodiment of the invention, the solids are formed at room temperature (25 C) without requiring temperature reduction for inducing precipitation of solids. For example, room temperature is effective for R-type and T-type crystals. The temperature for solid formation is preferably about 0 to about 40 C, preferably about 17 to about 30 C, and more preferably about 22 to about 27 C, ideally about 25 C.

In one embodiment, the method includes combining in an aqueous solution an insulin compound conjugate and a metal cation to provide a crystalline or amorphous solid. The aqueous solution containing the insulin compound conjugate to which the cation will be added is preferably a buffered solution having a pH in the range of pI of the insulin compound conjugate +/- about 1.5, preferably pI +/- about 1, more preferably pI +/- about 0.75. These ranges also apply to T-type, R-type and protamine coinplexes. However, for neutral protamine complexes (NPH-type), the preferred pH is about 7 to about 8.5, more preferably about 7.5 to about 8.
Once the metal cation is added, the pH may change slightly, and the pH may be adjusted to target the pH ranges described above. With phenolic compounds, there may be a minor pH change, and an acid or base can be used to adjust to the preferred ranges.

pI values for insulin compound conjugates typically require a pH of less than about 7, preferably less than about 6, more preferably less than about 5.5. Human insulin monoconjugates with neutral modifying moieties typically have a pI range of about 4.75 +/-.25. For human insulin diconjugates, the pI range is typically 4.25 +/-.25. For human insulin triconjugates, the pI range is typically 3.5 +/-.25.

Examples of suitable buffer systems include ammonium acetate buffer, sodium phosphate buffer, tris buffer, mixture of sodium phosphate and ammonium acetate, sodium acetate buffer, mixture of sodium acetate and ammonium acetate, and citric acid buffer, and any of the foregoing buffer systems [A] also containing ethanol and/or acetonitrile [B] (e.g., at percent ratio of A:B of about 1:1 to about 10:1). It is a surprising aspect of the invention that the cation-insulin compound conjugate solid can be fonned in an aqueous mixture containing an organic solvent, such as ethanol or acetonitrile.

One unique feature of the invention is that in addition to providing useful cation-insulin compound conjugate complexes, the invention also provides a method of separating cation-insulin coinpound conjugates from unconjugated insulin compound in the manufacturing process. In this process, the cation-insulin compound conjugates can be precipitated out of solution and the solubilized unconjugated insulin compound can be removed by filtration, for example. This feature eliminates 2 steps in the manufacture of insulin compound conjugates: the concentration step and the lyophilization step.

Processed pure solid composition may be formed using standard techniques, such as centrifugation and/or filtration, followed by washing (e.g., with ethanol/water), and lyophilization or vacuum drying. Multiple washings may be used to adjust phenol and/or cation content.

7.11 Formulation The complexes may be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Alfonso R. Gennaro, Remington: The Sciefice and Practice of Pltarnzacy, Lippincott Williams & Wilkins Publishers (June 2003), and Howard C. Ansel, Plaafmaceutical Dosage Forins and Drug Deliveiy Systeins, Lippincott Williams & Wilkins Publishers, 7th ed. (October 1999), the entire disclosures of which are incorporated herein by reference for their teachings concerning the selection, making and using of pharmaceutical dosage forms.

The complexes, typically in the form of an amorphous or crystalline solid, can be combined with a pharmaceutically acceptable carrier. The carrier must be acceptable in the sense of being compatible with any other ingredients in the pharmaceutical composition and should not be unduly deleterious to the subject, relative to the benefit provided by the active ingredient(s). The carrier may be a solid or a liquid, or both. It is preferably formulated as a unit-dose formulation, for example, a tablet. The unit dosage form may, for example, contain from about 0.01 or 0.5%
to about 95% or 99% by weight of the cation-insulin compound coinplex. The pharmaceutical compositions may be prepared by any of the well known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
Examples of suitable pharmaceutical compositions include those made for oral, rectal, inhalation (e.g., via an aerosol) buccal (e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous, intramuscular, intradermal, intraarticular, intrapleural, intraperitoneal, inracerebral, intraarterial, or intravenous), topical, mucosal surfaces (including airway surfaces), nasal surfaces, and transdermal administration. The most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular cation-insulin compound complexes being used. Preferred oral compositions are compositions prepared for ingestion by the subject. Ideally, the oral compositions are prepared to survive or substantially survive passage through the stoinach and to completely or substantially completely dissolve in the intestine for delivery of the active ingredient. Examples of suitable transdermal systems include ultrasonic, iontophoretic, and patch delivery systems.

In one aspect, the invention provides fatty acid compositions comprising one or more saturated or unsaturated C4, C5, C6, C7, C8, Cg or Clo fatty acids and/or salts of such fatty acids. Preferred fatty acids are caprylic, capric, myristic and lauric. Preferred fatty acid salts are sodium salts of caprylic, capric, myristic and lauric acid.

Preferred fatty acid compositions include a single fatty acid or a single fatty acid salt and do not include substantial amounts of other fatty acids or fatty acid salts. In one aspect of the invention, the fatty acid content of the composition is greater than about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fatty acid. In another embodiment, the fatty acid content of the composition is within a range having as a lower limit of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit of about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0 % w/w. In yet another embodiment, the fatty acid content of the composition is within a range having as a lower limit about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 % w/w, and having as an upper limit about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, or 12.0 %
w/w, and the fatty acid content of the composition is greater than about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, or 99.9% w/w a single fatty acid.

Active components of these formulations may include conjugated or unconjugated, complexed or uncomplexed proteins and/or peptides. Preferred proteins and/or peptides are those described herein. Preferred conjugates are those described herein. Preferred complexes are those described herein. Preferred oral compositions are compositions prepared for ingestion by the subject.
Ideally, the oral coinpositions are prepared to survive or substantially survive passage through the stomach and to completely or substantially completely dissolve in the intestine for delivery of the active ingredient. The formulation may in some cases include an enteric coating, and in some cases, the formulation will specifically exclude an enteric coating. The composition is preferably provided as a tablet, powder, hard gelatin capsule, or soft gelatin capsule, though other forms described herein are suitable as well.

The fatty acid compositions of the invention may include fatty acid acylated insulins. Examples of suitable fatty acid acylated insulins are described in the following U.S.
patents, the entire disclosures of which are incorporated herein by reference: U.S. Patent 6,531,448, entitled "Insoluble compositions for controlling blood glucose," issued 11-Mar-03; U.S.
Patent RE37,971, entitled "Selective acylation of epsilon-amino groups," issued 28-Jan-03; U.S.
Patent 6,465,426, entitled "Insoluble insulin compositions," issued 15-Oct-02; U.S. Patent 6,444,641, entitled "Fatty acid-acylated insulin analogs." issued 03-Sep-02; U.S. Patent 6,335,316, entitled "Method for administering acylated insulin," issued 01-Jan-02; U.S. Patent 6,268,335, entitled "Insoluble insulin compositions," issued 31-Jul-01; U.S. Patent 6,051,551, entitled "Method for administering acylated insulin," issued 18-Apr-00; U.S. Patent 5,922,675, entitled "Acylated Insulin Analogs," issued 13-Jul-99; U.S. Patent 5,700,904, entitled "Preparation of an acylated protein powder," issued 23-Dec-97; U.S. Patent 5,693,609, entitled "Acylated insulin analogs Granted," issued 02-Dec97; U.S. Patent 5,646,242, entitled "Selective acylation of epsilon-amino groups," issue 08-Jul-97; U.S. Patent 5,631,347, entitled "Reducing gelation of a fatty acid-acylated protein," issued 20-May-97; U.S. Patent 6,451,974, entitled "Method of acylating peptides and novel acylating agents," issued 17-Sep-02; U.S. Patent 6,011,007, entitled "Acylated insulin," issued 04-Jan-00; U.S. Patent 5,750,497, entitled "Acylated insulin Granted: 12-May-98;
U.S. Patent 5,905,140, entitled "Selective acylation method," issued May 18, 1999; U.S. Patent 6,620,780, entitled "Insulin derivatives," issued Sep. 16, 2003; U.S. Patent 6,251,856, entitled "Insulin derivatives," issued Jun. 26, 2001; U.S. Patent 6,211,144, entitled "Stable concentrated insulin preparations for pulmonary delivery," issued Apr. 3, 2001; U.S. Patent 6,310,038, entitled "Pulmonary insulin crystals," issued Oct. 30, 2001; and U.S. Patent 6,174,856, entitled "Stabilized insulin compositions," issued Jan. 16, 2001. Especially preferred are mono-fatty acid acylated insulins having 12, 13, 14, 15, or 16-carbon fatty acids covalently bound to Lys(B29) of human insulin.

Pharmaceutical compositions suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tables, each containing a predetermined amount of the mixture of insulin compound conjugates; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the mixture of insulin compound conjugates and a suitable carrier (which may contain one or more accessory ingredients as noted above).
Fonnulations may include suspensions of solids, complexed cation-insulin coinpound conjugates, uncoinplexed active ingredient (e.g., native insulin compound, insulin compound conjugates), and mixtures of the foregoing.

In general, the pharmaceutical compositions of the invention are prepared by unifonnly and intimately admixing the complexes with a liquid or solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the mixture of insulin compound conjugates, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the mixture in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered composition moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual) administration include lozenges comprising the mixture of insulin compound conjugates in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the mixture of insulin compound conjugates in an inert base such as gelatin and glycerin or sucrose and acacia. Exainples of suitable formulations can be found in U.S. Patent Publication Nos. 20030229022 ("Pharmaceutical formulation");
20030236192 ("Method of modifying the release profile of sustained release compositions");
20030096011 ("Method of producing submicron particles of a labile agent and use thereof');
20020037309 ("Process for the preparation of polymer-based sustained release compositions");
20030118660 ("Residual solvent extraction method and microparticles produced thereby"); as well as U.S. Patents 6,180,141 ("Composite gel microparticles as active principle carriers");
6,737,045 ("Methods and compositions for the pulmonary delivery insulin compound");
6,730,334 ("Multi-arm block copolymers as drug delivery vehicles"); 6,685,967 ("Methods and coinpositions for pulmonary delivery of insulin compound"); 6,630,169 ("Particulate delivery systems and methods of use"); 6,589,560 ("Stable glassy state powder formulations; 6,592,904 ("Dispersible macromolecule compositions and methods for their preparation and use");
6,582,728 ("Spray drying of macromolecules to produce inhaleable dry powders"); 6,565,885 ("Methods of spray drying pharmaceutical compositions"); 6,546,929 ("Dry powder dispersing apparatus and methods for their use"); 6,543,448 ("Apparatus and methods for dispersing dry powder medicaments"); 6,518,239 ("Dry powder compositions having improved dispersivity");
6,514,496 ("Dispersible antibody compositions and methods for their preparation and use");
6,509,006 ("Devices compositions and methods for the pulmonary delivery of aerosolized medicaments"); 6,433,040 ("Stabilized bioactive preparations and methods of use"); 6,423,344 ("Dispersible macromolecule compositions and methods for their preparation and use");
6,372,258 ("Methods of spray-drying a drug and a hydrophobic amino acid");
6,309,671 ("Stable glassy state powder formulations"); 6,309,623 ("Stabilized preparations for use in metered dose inhalers"); 6,294,204 ("Method of producing morphologically uniform microcapsules and microcapsules produced by this method"); 6,267,155 ("Powder filling systems, apparatus and methods"); 6,258,341 ("Stable glassy state powder formulations"); 6,182,712 ("Power filling apparatus and methods for their use"); 6,165,463 ("Dispersible antibody compositions and methods for their preparation and use"); 6,138,668 ("Method and device for delivering aerosolized medicaments"); 6,103,270 ("Methods and system for processing dispersible fine powders"); 6,089,228 ("Apparatus and methods for dispersing dry powder medicaments");
6,080,721 ("Pulmonary delivery of active fragments of parathyroid hormone");
6,051,256 ("Dispersible macromolecule compositions and methods for their preparation and use");
6,019,968 ("Dispersible antibody compositions and methods for their preparation and use");
5,997,848 ("Methods and compositions for pulmonary delivery of insulin compound"); 5,993,783 ("Method and apparatus for pulmonary administration of dry powder.alpha.1-antitrypsin");
5,922,354 ("Methods and system for processing dispersible fine powders");
5,826,633 ("Powder filling systems, apparatus and methods"); 5,814,607 ("Pulmonary delivery of active fraginents of parathyroid hormone"); 5,785,049 ("Method and apparatus for dispersion of dry powder medicaments"); 5,780,014 ("Method and apparatus for pulmonary administration of dry powder alpha 1-antitrypsin"); 5,775,320 ("Method and device for delivering aerosolized medicaments");
5,740,794 ("Apparatus and methods for dispersing dry powder medicaments");
5,654,007 ("Methods and system for processing dispersible fine powders"); 5,607,915 ("Pulmonary delivery of active fragments of parathyroid hormone"); 5,458,135 ("Method and device for delivering aerosolized medicaments"); 6,602,952 ("Hydrogels derived from chitosan and poly(ethylene glycol) or related polymers"); and 5,932,462 ("Multiarmed, monofunctional, polymer for coupling to molecules and surfaces"). Further, Suitable sustained release formulations are described in Cardinal Health's US Patent 5,968,554, entitled "A sustained release pharmaceutical preparation," issued 19-Oct-99, the entire disclosure of which is incorporated herein by reference.
Suitable microparticle formulations are described in Spherics, Inc.'s International Patent Publication WO/2003-049,701, entitled "Methods and products useful in the formation and isolation of microparticles," published 30-Oct-03. Suitable bioadhesive formulations are described in Spherics, Inc.'s International Patent Publication WO/2003-051,304, entitled "Bioadhesive drug delivery systein with enhanced gastric retention", published 06-May-04.
Pharmaceutical compositions according to embodiments of the invention suitable for parenteral administration comprise sterile aqueous and non-aqueous injection solutions of the complexes, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions may include suspending agents and thickening agents. The compositions may be presented in unit\dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water-for-injection immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile composition with a mixture of complexes in a unit dosage form in a sealed container may be provided. The mixture of complexes can be provided in the form of a lyophilizate which is capable of being reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The parenteral unit dosage form typically comprises from about 1 microgram to about 10 mg of the mixture of complexes. When the complexes are substantially water-insoluble, a sufficient amount of emulsifying agent which is physiologically acceptable may be einployed in sufficient quantity to emulsify the coinplexes in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

A solid dosage form for oral administration typically includes from about 2 mg to about 500 mg, preferably about 10 mg to about 250 ing, ideally about 20 mg to about 110 mg of the coinplexes.
Pharmaceutical compositions suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the complexes with one or more conventional solid carriers, for exainple, cocoa butter, and then shaping the resulting mixture.
Pharmaceutical compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include petroleum jelly, lanoline, PEGs, alcohols, transdermal enhancers, and combinations of two or more thereof.

Phannaceutical compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Compositions suitable for transdei-mal administration may also be delivered by iontophoresis (see, for example, Plzarinaceutical Research 3 (6):318 (1986)) and typically take the form of an optionally buffered aqueous solution of the mixture of insulin compound conjugates. Suitable forinulations comprise citrate or bis/tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2M active ingredient.

In a preferred embodiment, the complexes are administered as components of solid fatty acid formulations as described in U.S. Patent Application No. 60/494,821, filed on 13-Aug-03, by Opawale et al., the entire disclosure of which is incorporated herein by reference.

In certain embodiments, the insulin compound conjugate may be provided separately from the cation and/or other components needed to form the solids. For exainple, the insulin compound conjugate may be provided as a dried solid, and the buffer solution including the cation, stabilizing agent, preservative and/or other component may be provided separately, so that the user may combine the separate components to produce the cation-insulin compound conjugate complexes.

7.12 Methods of treatment The cation-insulin compound conjugate compositions and formulations thereof are useful in the treatment of conditions in which increasing the amount of circulating insulin compound (relative to the amount provided by the subject in the absence of administration of insulin compound from an exogenous source) yields a desirable therapeutic or physiological effect.
For example, the condition treated may be Type I oR-type II diabetes, prediabetes and/or metabolic syndrome. In one einbodiment, the compositions are administered to alleviate symptoms of diabetes. In another embodiment, the compositions are administered to a prediabetic subject in order to prevent or delay the onset of diabetes.

The effective amount of the cation-insulin compound conjugate composition for administration according to the methods of the invention will vary somewhat from mixture to mixture, and subject to subject, and will depend upon factors such as the age and condition of the subject, the route of delivery and the condition being treated. Such dosages can be determined in accordance with routine pharmacological procedures known to those skilled in the art.

As a general proposition, an oral dosage from about 0.025 to about 10 mg/kg of active ingredient (i.e., the conjugate) will have therapeutic efficacy, with all weights being calculated based upon the weight of the mixture of insulin compound conjugates. A more preferred range is about 0.06 to about 1 mg/kg, and an even more preferred range is about 0.125 to about 0.5 mg/kg A parenteral dosage typically ranges from about 0.5 g/kg to about 0.5 mg/kg, with all weights being calculated based upon the weight of the mixture of insulin compound conjugates. A more preferred range is about 1 g/kg to about 100 g/kg.

The frequency of administration is usually one, two, or three times per day or as necessary to control the condition. Alternatively, the cation-insulin compound conjugate compositions may be administered by continuous infusion. The duration of treatment depends on the type of insulin compound deficiency being treated and may be for as long as the life of the subject. The complexes may, for exainple, be administered within 0 to 30 minutes prior to a meal. The complexes may, for example, be administered within 0 to 2 hours prior to bedtime.

8 Synthesis Examples The following examples are presented to illustrate and explain the invention.

8.1 Synthesis of protected MPEG6C3 oligomer (3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionic acid tert-butyl ester) O Na OH + O
THF
4h, 58%
O

Methyl hexaethylene glycol (1.0 g, 3.37 inmol) and tert-butyl acrylate (0.216 g, 1.69 mmol) were dissolved in dry THF (10 mL). Sodium metal 0.4 mg, 0.016 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1 M HCI (15 mL). The quenched reaction mixture was then extracted with CHZCl2 (1 x 50 mL, 1 x mL). The organic layer was dried (MgSO4) and concentrated. After purification by silica gel chromatography (ethyl acetate as eluent), the product was obtained as an oil (0.832 g, 58%).

8.2 Synthesis of the MPEG6C3 oligomer acid (3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy)-ethoxy)-ethoxy]-ethoxy}-propionic acid) TFA
O
45 min, 87%

The tert-butyl ester (0.165 g, 0.0389 mmol) was deprotected by stirring at room temperature in trifluoroacetic acid (2.0 mL). The contents were then concentrated to a constant weight (0.125 g, 87%).

8.3 Synthesis of the activated MPEG6C3 oligomer (3-{2-[2-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester) O EDC
HO~
+ N

O
O
O
The acid (0.660 g, 1.79 mmol) and N-hydroxysuccinimide (0.2278 g, 1.97 mmol) were dissolved in dry CH2C12 (15 mL). Ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC, 0.343 g, 1.79 mmol) was added. After stirring at room temperature overnight, the reaction mixture was diluted with CH2C12 and was washed with water (2 x 45 mL). The organic layer was dried (MgSO4) and concentrated to a constant weight. The product was an oil (0.441 g, 53%).

8.4 Synthesis of the protected MPEG4C3 oligomer (3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxy)-ethoxy}-ethoxy)-propionic acid tert-butyl ester) O Na O-,,,O,,,--,O,-~OH + O
THF
4 h, 79 /a O

Methyl tetraethylene glycol (1.0 g, 4.80 mmol) and tert-butyl acrylate (0.308 g, 2.40 mmol) were dissolved in dry THF (10 mL). Sodium metal 0.6 mg, 0.024 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1 M HCI (15 mL). The quenched reaction mixture was then extracted with CH2ClZ (1 x 50 mL, 1 x 25 ml.,). The organic layer was dried (MgS04) and concentrated. After purification by silica gel chromatography (ethyl acetate as eluent), the product was obtained as an oil (1.28 g, 79%).

8.5 Synthesis of the MPEG6C3 oligomer acid (3-(2-{2-[2-(2-Methoxy-ethoxy)-ethoxyl-ethoxy}-ethoxy)-propionic acid) TFA
O+
O
45 min, 91 %

The tert-butyl ester (1 g, 3.42 mmol) was deprotected by stirring at room teinperature in trifluoroacetic acid (6.0 mI.,). The contents were then concentrated to a constant weight (0.87 g, 91%).

8.6 Synthesis of the activated MPEG4C3 oligomer (3-(2-{2-(2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester) O EDC
HO~
N

O

O
O
O
The acid (0.6 g, 2.14 mmol) and N-hydroxysuccinimide (0.271 g, 2.35 mmol) were dissolved in dry CH2ClZ (20 mL). Ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC, 0.409 g, 2.14 mmol) was added. After stirring at room temperature overnight, the reaction mixture was diluted with CH2C12 and was washed with water (2 x 45 mL). The organic layer was dried (MgSO4) and concentrated to a constant weight. The product was an oil (0.563 g, 69%).

8.7 Synthesis of the protected MPEG4C3 oligomer (3-(2-Methoxy-ethoxy)-propionic acid tert-butyl ester) 0 Na O"'-~ OOH + O
THF
4h, 89%
0~~ O+
O
Methyl tetraethylene glycol (5.0 g, 41.6 mmol) and tert-butyl acrylate (2.66 g, 20.8 mmol) were dissolved in dry THF (20 mL). Sodium metal 0.47mg, 20.8 mmol) was added to the solution.
After stirring for 4 h at room temperature, the reaction mixture was quenched by the addition of 1 M HCl (30 mL). The quenched reaction mixture was then extracted with CH2C12 (1 x 100 mL, 1 x 50 mL). The organic layer was dried (MgSO4) and concentrated. After purification by silica gel chromatography (ethyl acetate as eluent), the product was obtained as an oil (7.5 g, 89%).

8.8 Synthesis of the MPEG6C3 oligomer acid (3-[2-(2-Methoxy-ethoxy)-ethoxy]-propionic acid) TFA
IOI

45 min, 91%

The tert-butyl ester (1 g, 4.90 mmol) was deprotected by stirring at room temperature in trifluoroacetic acid (6.0 mL). The contents were then concentrated to a constant weight (0.652 g, 89%).

8.9 Synthesis of 2-[2-(2-Propoxy-ethoxy)-ethaxy]-ethanol (1) Triethylene glycol (19.5 g, 0.13 mol) was dissolved in tetrahydrofuran (150 mL) and sodium hydride (2.60 g, 0.065 mol) was added portion wise over 0.5 h and the reaction was stirred for an additional 1 h. Then 1-bromopropanol (8.0 g, 0.065 mol) dissolved in tetrahydrofuran (30 mL) was added dropwise via addition funnel and the reaction was stirred overnight at room temperature. Crude reaction mixture was filtered tlirough Celite, washed CH2C12, and evaporated to dryness. The resultant oil was dissolved in CH2CI2 (250 mL), washed sat.
NaCI (250 mL), H20 (250 mL), dried MgSO4, and evaporated to dryness. Column chromatography (Silica, ethyl acetate) afforded 1 a yellowish oil (2.24 g, 18% yield).

8.10 Syntheis of carbonic acid 4-nitro-phenyl ester 2-[2-(2-propoxy-ethoxy)-ethoxy]-ethyl ester NO, JI~I I

4-Nitrochloroformate (3.45 g, 17.1 mmol) and 1 (2.2 g, 11.4 mmol) were dissolved in CH2C12 (20.
mL). After stirring for 10 min, TEA (2.1 mL, 15 mmol) was added and reaction stirred overnight at room temperature. Crude reaction was diluted with CH2CI2 (50 mL), washed 1M
HCl (50 mL), H20 (50 mL), dried MgSO4, and evaporated to dryness. Column chromatography (silica, etllyl acetate / hexanes, 3:2) afforded 2 a yellowish oil (2.57 g, 63% yield).

8.11 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester 2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl ester Triethylene glycol monomethyl ether (1.0 g, 6.1 mmol) and N,N'-disuccinimidyl carbonate (1.87 g, 7.3 mmol) were dissolved in acetonitrile (10 mL). Then triethylamine (1.3 mL, 9.15 mmol) was added and the reaction stirred overnight at room temperature. Crude reaction was evaporated to dryness, dissolved in sat. NaHCO3 (50 mL), washed ethyl acetate (2 x 50 mI.,), dried MgSO4, and evaporated to dryness. Column chromatography (Silica, ethyl acetate) afforded 1 a clear oil (0.367 g, 20% yield).

8.12 Synthesis of carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester 2-{2-[2-(2-methoxy-ethoxy)-ethoxy]-ethoxy}-ethyl ester (1).

Tetraethylene glycol monomethyl ether (1.0 g, 4.8 mmol) and N,N'-disuccinimidyl carbonate (1.48 g, 5.8 mmol) were dissolved in acetonitrile (10 mL). Then triethylamine (1.0 mL, 7.2 mmol) was added and the reaction stirred overnight at room temperature. Crude reaction was evaporated to dryness, dissolved in sat. NaHCO3 (30 mL), washed ethyl acetate (2 x 30 mL), dried MgSO4i and evaporated to dryness. Column chromatography (Silica, ethyl acetate / MeOH, 20:1) afforded 1 a clear oil (0.462 g, 28% yield).

8.13 Synthesis of but-3-enoic acid ethyl ester Vinylacetic acid (10.0 g, 0.12 mol) was dissolved in ethanol (200 mL) and conc. sulfuric acid (0.75 mL, 0.014 mol) was added. The reaction was heated to reflux for 4 h.
Crude reaction was diluted with ethyl acetate (200 mL), washed H20 (200mL), sat. NaHCO3 (200 mL), dried MgSO4, and evaporated to dryness to afford 1 a clear oil (3.17 g, 23%).

8.14 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid ethyl ester Triethylene glycol monomethyl ether (4.27 g, 0.026 mol) and But-3-enoic acid ethyl ester (1.5 g, 0.013 mol) were dissolved in tetrahydrofuran (10 mL). Then lump Na (0.030 g, 0.013 mol) was added and the reaction was stirred for 4 h. Crude reaction was quenched with 1M HCl (20 mL), washed ethyl acetate (3 x 20 mL). Organic layers were combined and washed with H20 (2 x 10 mL), dried MgSO4, and evaporated to dryness to afford 2 a yellowish oil (1.07 g, 30% yield).
8.15 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid \O/ v O/ ~ OH

4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid ethyl ester (1.07 g, 4.0 mmol) was dissolved in 1M NaOH (10 mL) and the reaction was stirred for 2 h. Crude reaction was diluted with sat. NaCl (40 mL), acidified to pH -2 with conc. HCI, washed CHZClZ (2 x 50 mL), dried MgSO4, and evaporated to dryness to afford 3 a clear oil (0.945 g, 94% yield).

8.16 Synthesis of 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid 2,5-dioxo-pyrrolidin-1-yl ester N-hydroxysuccinimide (0.55 g, 4.8 mmol) and EDCI (1.15 g, 6.0 mmol) were dissolved in CH2C12 (7 mL). Then 4-{2-[2-(2-Methoxy-ethoxy)-ethoxy]-ethoxy}-butyric acid (0.940 g, 3.8 mmol), dissolved in CH2C12 (2 mL), was added. Reaction stirred overnight at room temperature.
Crude reaction was diluted with CH2C12 (21 mL), washed 1M HCl (30 mL), HzO (30 inL), dried MgSO4, and evaporated to dryness. Coluinn chromatography (Silica, ethyl acetate) afforded 4, a clear oil (0.556 g, 43% yield).

9 Preparation of Complexes Methods were investigated for the preparation of zinc complexes of insulin compound conjugates. New methods, exceptional to published methods used for complexation/crystallization of insulin coinpound and insulin compound analogs, were developed to make zinc complex of HIM2. HIM2 is a human insulin monoconjugate with a modifying moiety coupled at B29, where the modifying moiety has the following structure:

, c\-o\\

Further coinplexes were prepared using IN105, a human insulin monoconjugate with a modifying moiety coupled at B29, where the modifying moiety has the following structure:

!I
~ Z/Me The methods provided three main types, "T-type" and "R-type" and "protainine"
cation complexes of insulin compound conjugate solids.

9.1 Preparation and Analysis of T-type Solids 9.1.1 Attempted Preparation of T-type Zn Complex of HIM2 (2 g/L) A HIM2 solution at approximately 2 g/L was prepared having a final pH -3 with 10% HCI.
Glacial acetic acid was added to a lOmL aliquot (20mg protein) of the above solution to a final concentration of 0.25M. Twenty (or forty) L of a 2% w/w ZnC12 solution was added to the sample. The pH was adjusted to 5.1 (or 5.5) with concentrated ammonium hydroxide. The solution stirred for 15 minutes at room temperature (or +5 C) and then stood for one day at room temperature (or +5 C) to allow solid formation. No crystals or precipitation formed after allowing the reaction to stand one day at room temperature (or at +5 C). See Example 2 of U.S.
Patent 5,504,188, entitled "Preparation of stable zinc insulin compound analog crystals."

9.1.2 T-type Zn Complex of HIM2 (10 g/L Concentration) A HIM2 solution at approxiinately 10 g/L was prepared having a final pH -3 with 10% I-1C1.
Glacial acetic acid was added to a 10 mL (100ing protein) aliquot of the above solution to a final concentration of 0.25M. Forty L of a 10% w/w ZnC12 solution was added to the sample. The pH was adjusted to 5.20 with concentrated ammonium hydroxide. The solution was stirred for 15 minutes at +5 C and then allowed to stand for five days at +5 C to allow solid formation.

The reaction inixture was transferred to a centrifuge tube and centrifuged at 2000 RPM for 10minutes. The solution was decanted and the solid was washed with 5 mL cold DI water. This solution was centrifuged at 2000 RPM for 10 minutes before the water was decanted and the solids were washed with another 5 mL cold DI water. Again, the sample was centrifuged at about 2000 RPM for about 10 minutes before the H20 was decanted. The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at 2000 RPM for 10 minutes before the EtOH was decanted. The sample was dried in a lyophilizer to provide white solid.

9.1.3 T-type Zn Complex of HIM2 (20 g/L Concentration) A HIM2 solution at approximately 20 g/L was prepared having a final pH -3 with 10% HCI.
Glacial acetic acid was added to a 10 mL (200mg protein) aliquot of the above solution to a final concentration of 0.25 M. Eighty L of a 10% w/w ZnCl2 , solution was added to the sample. The pH was adjusted to 5.37 with concentrated ammonium hydroxide. The solution stirred for 15 minutes at +5 C and then stood for four days at +5 C to allow solid formation.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20minutes. The solution was decanted and the solid was washed with 5 mL cold DI water. This solution was centrifuged at 2600 RPM for 20 minutes before the water was decanted and the solids were washed with another 5mL cold DI water. Again, the sample was centrifuged at about 2600 RPM for about 20 minutes before the H20 was decanted. The sample was washed with 5 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. The sample was dried in a lyophilizer to provide white solid.

9.1.4 T-type Zn Complex of of HIM2 (30 g/L Concentration) A HIM2 solution at approximately 30 g/L is prepared having a final pH -3 with 10% HCl.
Glacial acetic acid was added to a 50 mL (1.5g protein) aliquot of the above solution to a final concentration of 0.25 M. Six hundred L of a 10% w/w ZnC12 solution was added to the sample.

The pH was adjusted to 5.34 with concentrated ammonium hydroxide. The solution stood at +5 C for five days to allow solid formation.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed three times with 10 mL cold DI
water, centrifuging and decanting the H20 each wash. The sample was then washed three times with 10 mL 200 proof cold EtOH. It was centrifuged at 2800 RPM for 15 minutes and decanted after each wash. The sample was dried in a lyophilizer to provide white solid.

Figures 1 and 2 are photomicrographs taken using a Zeiss Axiovert microscope showing crystals grown for 24 hours. In Figure 1, the crystal size is approximately 11.3 M in length and approximately 5.3 M in diameter. In figure 2, the size of the crystal on the left is approximately 15.1 M in length and approximately 5.9 M in diameter, and the size of the crystal on the right is approximately 9.1 M in length and approximately 5.3 M in diameter. Figure 3 is a photoinicrograph taken using a Zeiss Axiovert microscope showing crystals grown for 5 days. In one aspect, the invention includes crystals having a morphology as shown in Figure 1, 2 or 3.

9.1.5 T-type Zn Complex of of HIM2 (50 g/L Concentration) A HIM2 solution at approximately 50 g/L was prepared to a final pH -3 with 10%
HCl. Glacial acetic acid was added to a 10 mL aliquot of the above solution to a final concentration of 0.25 M.
Two hundred L of a 10% ZnC12 solution was added to the sample. The pH was adjusted to 5.23 with concentrated ammonium hydroxide. The solution was stirred at +5 C for 15 minutes and then stood at +5 C for four days to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 5mL cold DI
HZO. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted and the solid was washed with another 5 mL cold DI H20. Again, the sample was centrifuged at for 20 minutes before the H20 was decanted. The sample was washed with 5 mL
200 proof cold EtOH and centrifuged at 2600 RPM for 20 ininutes before the EtOH was decanted.
The sample was dried in a lyophilizer for three days.

9.1.6 T-type Zn Complex of of HIM2 (1 g Scale) A HIM2 solution at approximately 10 g/L was prepared to a final pH N3 with 10%
HCI. Glacial acetic acid was added to a 50 mL (500mg protein) aliquot of the above solution to a final concentration of 0.25 M. Two hundred L of a 10% ZnC12 solution was added to the sample.
The pH was adjusted to 5.49 with concentrated ammonium hydroxide. The solution was stirred at +5 C for 15 minutes and then stood at +5 C for seven days to allow solid formation to occur.
The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 10 mL cold DI
H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted. The water washes were repeated two additional times. The sample was then washed with 10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two more EtOH washes were carried out the same way before the sample was placed on the lyophilizer to dry for four days.

9.1.7 T-type Zn Complex of of HIM2 at Neutral pH (5 g scale) A HIM2 solution at approximately 10 g/L was prepared to a final pH -3 with 10%
HC1. Two mililiters of a 10% ZnC12 solution was added to the sample. The pH was adjusted to 7.05 with concentrated aminonium hydroxide. The solution was stirred at room temperature overnight to allow solid formation to occur.

The milky Zn-HIM2 reaction mixture (500mL) was added, in parts, to a 350mL
fine-fritted (4.5-5um) disc =funnel (ChemGlass CG1402-28, 90mm diameter). The filtrate was collected in a side-arm flask while applying vacuum for about 4-6 hours. As an option, the cake may be washed with 100 mL cold 1% ZnC12 and the filtrate collected separately. The cake was washed with 100 mL ice-cold water, and the filtrate was again collected. The cake was also washed with an additional 100 mL ice-cold 100% ethanol and the filtrate was collected once again. The final wash of the cake was 100mL of fresh ice-cold water and the final filtrate collected. The cake was dried under vacuum and/or air-dried over 12-18 hours. After drying, the cake was scraped off the funnel, weighed, and moisture/protein contents were measured via HPLC. The collected filtrates from the various wash steps were also analyzed using HPLC to determine the concentration of the lost Zn-HIM2 during the process. The filtration yielded a 2.5% w/w Zn content with an overall yield of 98%.

9.1.8 T-type Zn Complex of HIM2 at neutral pH (500mg scale) A HIM2 solution at approximately 10 g/L was prepared to a final pH -3 with 10%
HC1. Two hundred gL of a 10% ZnC12 solution was added to the sample. The pH was adjusted to 7.06 with concentrated ammonium hydroxide. The solution was stirred at +5 for 15 minutes and then stood at +5 for two days to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed with 10 mL cold DI
H20. This solution was centrifuged at 2800 RPM for 15 minutes before the H20 was decanted. The water washes were repeated two additional times. The sample was then washed with 10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted. Two more EtOH washes were carried out the same way before the sample was placed on the lyophilizer to dry for two days.

9.1.9 Results for T-type Solid Compositions Reaction Observations Solubility (mg/mL) % w/w Zn 9.1.1 No solid formed N/A N/A
9.1.2 White solid NEM NEM
9.1.3 White solid NEM NEM
9.1.4 White solid NEM 0.53 9.1.5 White solid 146 0.66 9.1.6 White solid 109 0.55 9.1.7 White solid ND 2.50 9.1.8 White solid ND 1.63 NEM = Not enough material ND = No data 9.2 Preparation and Analysis of R-type Solids 9.2.1 R-type Zn complex of HIM2 with Phenol at 2 g/L
A HIM2 solution at approximately 2 g/L was prepared to a final pH -3 with glacial acetic acid.
Thirty three mircoliters of liquefied phenol was added to a 10 mL aliquot of the above solution.
The pH was adjusted to 5.89 with concentrated ammonium hydroxide. One hundred sixty L of a 10% w/w ZnCl2 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature for three days to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 3400 RPM for l5minutes. The supeinatant was decanter and the solid was washed with 5 mL
cold DI water.
This solution was centrifuged at 3200 RPM for 15 minutes before the HZO was decanted. The sample was then washed with 5 mL 200 proof cold EtOH and centrifuged at 3200 RPM for 15 minutes before the EtOH was decanted. Again the sample was washed with 5 inL
of cold EtOH, however, it was not centrifuged. The solid was allowed to settle to the bottom of the tube and then placed in the speed vacuum to dry.

9.2.2 Preparation of R-type Zn Complex of HIM2 at 20 g/L
A HIM2 solution at approximately 20 g/L was prepared to a final pH -3 with 10%
HCI. Sixty six gL of liquefied phenol was added to a 10 mL aliquot of the above solution. The pH was adjusted to 6.43 with concentrated ammonium hydroxide. Three hundred twenty .L of a 10% ZnCl2 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature for four days to allow more precipitate to forin.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 5 mL cold DI
H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted and the solid was washed with another 5mL cold DI H20. Again, the sample was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted. The sample was washed witll 5 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
The sample was lyophilized for three days.

9.2.3 Preparation of R-type Zn Complex of HIM2 at 30 g/L
A HIM2 solution at approximately 30 g/L was prepared to a final pH -3 with 10%
HCI. Ninety nine mircoliters of liquefied phenol was added to a 10 mL aliquot of the above solution. The pH
was adjusted to 6.47 with concentrated ammonium hydroxide. Then, 480 L of a 10% ZnC12 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature for four days to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20minutes. The solution was decanted and the solid was washed with 5 mL cold DI H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted and the solid was washed with another 5 mL cold DI H20. Again, the sample was centrifuged at for 20 minutes before the H20 was decanted. The sample was washed with 5 mL
200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
The sainple was lyophilized for three days.

Figure 4 shows solid grown for 4 days. The picture was taken using a Zeiss Axiovert microscope. The average length of the crystals is approximately 9.7 M.

9.2.4 Preparation of R-type Zn Complex of HIM2 at 50 g/L
A HIM2 solution at approximately 50 g/L was prepared to a final pH -3 with 10%
HCI. One hundred sixty five mircoliters of liquefied phenol was added to a 10 mL
aliquot of the above solution. The pH was adjusted to 6.82 with concentrated ammoniuin hydroxide.
Eight hundred L of a 10% ZnC12 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature for four days to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20minutes. The solution was decanted, and the solid was washed with 5 mL cold DI H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted and the solid was washed with another 5 mL cold DI H20. Again, the sample was centrifuged at for 20 minutes before the H20 was decanted. The sample was washed with 5 mL
200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
The sample was lyophilized for three days.

9.2.5 Preparation of R-type Zn Complex of HIM2 at 1 g Scale A HIM2 solution at approximately 10 g/L was prepared to a final pH -3 with 10%
HC1. One hundred sixty five mircoliters of liquefied phenol was added to a 50 mL
aliquot of the above solution. The pH was adjusted to 6.42 with concentrated ammonium hydroxide.
Eight hundred L of a 10% ZnC12 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature for seven days to allow more precipitate to form.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 10 mL cold DI
H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted. Two more water washes occurred the same way. The sample was then washed with 10 inL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
Two more EtOH washes were carried out the same way before the sainple was placed on the lyophilizer for four days.

9.2.6 R-type Zn complex of HIM2 at 5 g Scale A HIM2 solution at approximately 10 g/L was prepared to a final pH -3 with 10%
HCI. Fifteen hundred mircoliters of liquefied phenol was added to 450 mL of the above solution. The pH was adjusted to 7.1 with concentrated ammonium hydroxide. Eighteen hundred L of a 10% ZnC12 solution was added to the sample. The solution was stirred at room temperature for 15 minutes and then stood at room temperature overnight to allow more precipitate to fonn.

The reaction performed above was split into three filtration trials. In trial one, the reaction mixture was filtered through a fine fritted funnel and then washed with a 1%
ZnC12 solution. The material was dried overnight via vacuum filtration. The second trial was filtered over a medium fritted filter which also contained filter paper. The substance was then washed with ethanol and water and dried overnight via vacuum filtration. Finally, the third trial was filtered through a fine fritted funnel, washed with a 1% ZnC12 solution and also washed with ethanol and water. This material was also dried overnight under vacuum filtration.

Trial 1(fine-frit, Trial 2 (filter paper, Trial 3 (fine-frit, ZnC12 wash, not medium frit, EtOH/H20 washes) H20/EtOH wash EtOH/H20 wash) Yield 74% 93% 58%
w/w% Zn 1.99 2.83 2.06%
w/w% Phenol 0.033 0.45 1.28 9.2.7 R-type Zn Complex of HIM2 at Neutral pH
A HIM2 solution at approximately 10 g/L was prepared to a final pH -3 with 10%
HCI. One hundred sixty five microliters of liquefied phenol was added to 50 mL of the above solution.
Then, two hundred microliters of a 10% ZnC12 solution was added to the sample.
The pH was adjusted to 7.18 with concentrated ammonium hydroxide. The solution sat at room temperature for two days to allow precipitate to form.

The reaction mixture was transferred to a centrifuge tube and centrifuged 'at 2800 RPM for 20 minutes. However, the material did not settle to the bottom of the tube initially and was therefore, centrifuged for about 2 hours. The solution was decanted and the solid was washed with 5 mL cold DI H20. This solution was centrifuged at 2800 RPM for 60 minutes before the H20 was decanted. The water wash was repeated two more times. The sample was then washed with 5 mL 200 proof cold EtOH and centrifuged at 2800 RPM for 60 minutes before the EtOH
was decanted. Two more EtOH washes were carried out the same way. Material was cloudy after the third EtOH wash and was placed in the refrigerator overnight to allow the reactions to settle more. The solvent was decanted and the material was placed on the lyophilizer for 2 days.
9.2.8 Results for R-type Solid Compositions Reaction Observations Solubility % w/w Zn Phenol (mg/mL) 9.2.1 White solid NEM NEM NEM
9.2.2 White solid 44.75 1.21 0.097 9.2.3 White solid 50.49 1.74 0.41 9.2.4 White solid 36.24 2.32 0.52 9.2.5 White solid 47.7 1.06 0.16 9.2.6 White solid ND See above See above 9.2.7 White solid ND 1.74 1.62 NEM = Not enough material ND = No data 9.3 Preparation and Analysis of Protamine Solids 9.3.1 Preparation of T-type Zn Complex of HIM2 with Protamine at Acidic pH
Protamine was added to a 10 g/L stock solution of HIM2 that had a final pH -3 with 10% HCl.
Glacial acetic acid was added to a 10 mL aliquot (100 mg protein) of the above solution to a final concentration of 0.25 M. Two hundred microliters of a 10% ZnC12 solution was added to the sample. The pH was adjusted with concentrated ammonium hydroxide to a pH -5.
The solution was stirred at + 5 C for 15 minutes and then stood at +5 C for two days to allow solid forination to occur.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 10 mL cold DI
H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted. Two more H20 washed occurred the same way. The sample was then washed with 10 inL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
Two more EtOH washes were carried out the same way before the sample was placed on the lyophilizer for two days.

9.3.2 Preparation of T-type Zn Complex of HIM2with Protamine at Neutral pH
A HIM2 solution at approximately 30 g/L was prepared to a final pH - 3 with 10% HCI. One milliliter of glacial acetic acid was added to a 50 mL aliquot (1.5 g protein) of the above solution.
Six hundred microliters of a 10% ZnC12 solution was added to the reaction followed by the addition of 225 milligrams of protamine. The pH was adjusted to 6.95 with concentrated ammonium hydroxide and the reaction stood for two days at +5 C to allow solid fonnation to occur.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 10 mL cold DI
H20. This solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted. Two more H2? washed occurred the same way. The sample was then washed with 10 mL 200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
Two more EtOH washes were carried out the same way before the sample was placed on the lyophilizer for three days 9.3.3 Preparation of R-type Zn Complex of HIM2with Protamine at Acidic pH
A HIM2 solution at approximately 10 g/L was prepared to a final pH - 3 with 10% HCI.
Liquified phenol (2.48mL) was added to a 150 mL aliquot (1.5 g protein) of the above solution.
The pH of the reaction was adjusted with concentrated aminoniuin hydroxide to a pH - 6.57.
Twelve microliters of a 10% ZnC12 solution was added to the reaction followed by the addition of 225 milligrams of protamine. The reaction mixture stirred at room temperature for 15 minutes before it stood for two days at room temperature to allow solid formation to occur.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed with 50 mL cold DI
H2?. This solution was centrifuged at 2800 RPM for 15 minutes before the H20 was decanted. Two more H20 washed occurred the same way. The sample was then washed with 10 mL 200 proof cold EtOH and centrifuged at 2800 RPM for 15 minutes before the EtOH was decanted.
Two more EtOH washes were carried out the same way before the sainple was placed on the lyophilizer for two days.

9.3.4 Preparation of R-type Zn Complex of HIM2with Protamine at Neutral pH
A HIM2 solution at approximately 10 g/L was prepared to a final pH - 3 with 10% HCI.
Liquefied phenol (495 mL) was added to 150 mL reaction. Then, 600 mililiters of a 10% ZnC12 solution was added to reaction followed by the addition of 75 ing protamine.
The pH was adjusted with concentrated ammonium hydroxide to a pH of 7.01. The reaction stood for three days at room temperature to allow solid formation.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed with 50 mL cold DI
H20. This solution was centrifuged at 2800 RPM for 15 minutes before the H20 was decanted. Two more H20 washes occurred the same way. The sample was then washed with 50 mL 200 Proof cold EtOH and centrifuged at 2800RPM for 15 minutes before the EtOH was decanted.
Two more EtOH washes were carried out the same way before the sample was placed on the lyophlizer for two days.

9.3.5 Results for Protamine Solid Compositions Reaction Observations Solubility % whv Zn Phenol (mg/mL) 9.3.1 White solid ND 0.66 N/A
9.3.2 White solid ND 2.47 N/A
9.3.3 White solid 36.78 1.22 9.87 9.3.4 White solid NEM NEM NEM
NEM = Not enough material ND = No data 9.4 Preparation and Analysis of Complexes of Insulin Compound Diconjugates 9.4.1 T-type Zn Complex at Al and B29 Insulin Compound Diconjugate An insulin compound diconjugate having a modifying moiety -C(O)(CH2)5(OCH2CH2)70CH3 coupled at B29 and Al of human insulin (DICON-1) was added to solution at approximately 10 g/L and prepared to a final pH 3.15 with 10% HC1. Glacial acetic acid was added to a 3.75 mL
aliquot of the above solution to a final concentration of 0.25 M. Then 15 L
of a 10% ZnC12 solution was added to the sample. The pH was adjusted to 4.90 with concentrated ainmonium hydroxide. The solution stirred for 15 minutes at +5 C and then stood for six days at +5 C to allow solid formation (yielded a white solid).

9.4.2 R-type Zn Complex at Al and B29 Insulin Compound Diconjugate DICON-1 was added to solution at approximately 10 g/L and prepared to a final pH 3.15 with 10% HC1. About 12 L of liquefied phenol was added to a 3.75 mL aliquot of the above solution. The pH was adjusted to 5.75 with concentrated ammonium hydroxide.
Sixty L of a 10% ZnC12 solution was added to the sample. The solution was stirred at room temperature for minutes and then stood at room temperature for six days to allow more precipitate to form (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2600 RPM for 20 minutes. The solution was decanted and the solid was washed with 5 mL cold DI
H20. This 10 solution was centrifuged at 2600 RPM for 20 minutes before the H20 was decanted and the solid was washed with another 5 mL cold DI H20. Again, the sample was centrifuged at for 20 minutes before the H20 was decanted. The sample was washed with 5 mL
200 proof cold EtOH and centrifuged at 2600 RPM for 20 minutes before the EtOH was decanted.
The sample was lyophilized for six days.

15 9.4.3 Diconjugate B1, B29 (lOmg/mL) DICON-1 was added to solution at approximately 10 g/L and 33 L of liquified phenol was added. The pH was adjusted to 5.34 with concentrated ammonium hydroxide. Then 160 L of a 10% ZnC12 solution was added to the sample. The solution stood at room teinperature for two weeks to allow solid formation to occur (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed three times with 5 mL cold DI
H20. The solution was centrifuged for 15 minutes at 2800 RPM and decanted after each wash.
The sample was then washed three times with 5 mL 200 proof cold EtOH. Again the sample was centrifuged at 2600 RPM for 15 minutes and decanted after each wash. The sample was lyophilized for two days.

9.4.4 Diconjugate B1, B29 (20mg/mL) DICON-1 was added to solution at approximately 20 g/L and 66 microliters of liquified phenol was added. The pH was adjusted to 7.65 with concentrated ammonium hydroxide.
Then 320 L
of a 10% ZnClZ solution was added to the sample. The solution stood at room temperature for two weeks to allow solid formation to occur (yielded a white solid).

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution was decanted and the solid was washed three times with 5 mL cold DI
H20. The solution was centrifuged for 15 minutes at 2800 RPM and decanted after each wash.
The sainple was then washed three times with 5 mL 200 proof cold EtOH. Again the sample was centrifuged at 2600 RPM for 15 minutes and decanted after each wash. The sample was lyophilized for two days.

9.5 Preparation and Analysis of T-type IN105 solids 9.5.1 T-type Zn Complex of IN105 Monoconjugate (10 g/L concentration) A IN105 solution at approximately 10 g/L (100mg) was prepared having a final pH -3 with 10%
HC1. 50 L of a 10% w/w ZnC12 solution was added to the sample. The pH was adjusted to 7.52 with concentrated ammonium hydroxide. The cloudy solution was stirred and then allowed to stand for five days at room temperature to allow solid fonnation.

The reaction mixture was transferred to a centrifuge tube and centrifuged at 2900 RPM for 15 minutes. The solution was decanted and the solid was washed wit113x10 mL cold DI water. This solution was centrifuged at 2900 RPM for 10 minutes before the water was decanted and the solids were washed with another portion of cold DI water. The sample then was washed with 3x10 mL 200 proof cold EtOH and centrifuged at 2900 RPM for 10 minutes before the EtOH was decanted. The sample was vacuum dried to provide white solid (90mg).

9.5.2 T-type Zn Complex of IN105 Monoconjugate (1 g Scale) A IN105 solution at approximately 10 g/L (lg) was prepared to a final pH -3 with 10% HCl.
Five hundred L of a 10% ZnClZ solution was added to the sample. The pH was adjusted to -7.4 with concentrated ammonium hydroxide. The cloudy solution was stirred for 15 minutes and then allowed to stand at room temperature for -2 days before filtration.

The reaction mixture was filtered through sintered glass funnel (fine) under house vacuum. The sintered glass funnel with filtered material was placed under vacuum in a glass dessicator over night to result a white fine powder (900mg).

9.5.3 T-type Zn Complex of IN105 Monoconjugate at Neutral pIi (5 g scale) A IN105 solution at approximately 10 g/L (5g, lot#Nobex040706L) was prepared to a final pH -3 with 10% HCI. Two mL of a 10% ZnC12 solution was added to the sample. The pH
was adjusted to -7.4 with concentrated ammonium hydroxide. The cloudy solution was then allowed to stand at room temperature overnight to allow solid formation before filtration.

The reaction performed above was split into 4x50 mL centrifuge tubes and initially centrifuged at 3200 RPM for a total 2 hours. The material was then centrifuged at 9000 RPM
for 20 minutes and stored at 5 C over night. The supernatant was decanted and the solid was washed with 10 mL
cold DI H20 from each tube. The tubes were inverted and centrifuged at 3200 RPM for - 1 hour before the H20 was decanted and the solids were washed with another 10 mL cold DI I-IZO.
Again, the sample was centrifuged at 3200 RPM for - 1 hour before the H20 was decanted. The sample was washed with 2x10 mL 200 proof cold EtOH and centrifuged at 3200 RPM
for 1 hour before the EtOH was decanted. The sample was vacuumn dried for two days to give 1.64g (lot#Nobex040730L-A) of white powder.

9.5.4 Results for T-type IN105 Solid Compositions Solubility (mg/mL) in Reaction Observations a pH of about 7.4 , % w/w Zn 0.1M phosphate buffer 9.5.1 White solid 80-85 0.0 9.5.2 White solid 10-20 1.67 9.5.3 White solid ND 1.88 9.6 Preparation and Analysis of R-type IN105 solids 9.6.1 R-type Zn Complex at IN105 Conjugate with Phenol at Neutral pH
A IN105 solution at approximately 10 g/L (500mg) was prepared to a final pH -3 with 10% HCl.
Two hundred L of 10% ZnC12 and 165 L of liquefied phenol was added to the above solution.
The pH was adjusted to 7.37 with concentrated aminonium hydroxide. The cloudy solution sat at room temperature for 2 days to allow solid formation before filtration.

The reaction mixture was filtered through sintered glass funnel (fine) under house vacuum. The sintered glass funnel with filtered material was placed in under vacuum in a glass dessicator over night to result in a white fine powder (440mg).

9.6.2 R-type Zn Complex of IN105 Conjugate at 5 g Scale A IN105 solution at approximately 10 g/L (4.2g, lot#Nobex040706L) was prepared to a final pH
-3 with 10% HCI. 1.5 liquefied phenol and 1.8 mL of 10% ZnC12 solution was added to the above solution. The pH was adjusted to -7.4 with concentrated ammonium hydroxide. The very cloudy solution stood at room temperature overnight to allow more precipitate to form.

The reaction performed above was split into 4x50 mL centrifuge tubes and initially centrifuged at 3200 RPM for 2 hours. The material was then centrifuged at 9000 RPM for 20 minutes and stored at 5 C over night. The supernatant was decanted and the solid was washed with 10 mL
cold DI H20 from each tube. The tubes were inverted and centrifuged at 3200 RPM for - 1 hour before the H20 was decanted and the solids were washed with another 10 mL cold DI H20.
Again, the sample was centrifuged at 3200 RPM for - 1 hour before the H20 was decanted. The sample was washed with 2x10 mL 200 proof cold EtOH and centrifuged at 3200 RPM
for 1 hour before the EtOH was decanted. The sample was vacuum dried for 2 days to give 2.34g of white powder.

9.6.3 Results for R-Type IN105 Solid Compositions Reaction Observations (mg/mL)Smlubility* % w/w Zn % w/w Phenol 9.6.1 White solid ND 1.85 2.37 9.6.2 White solid 10-25 1.71 2.66 ND = No data *In a pH of about 7.4 phosphate buffer 9.7 Preparation and Analysis of Protamine IN105 solids 9.7.1 Preparation of R-type Zn Complex of IN105 Monoconjugate with Protamine at Acidic pH
A IN105 solution at approximately 10 g/L is prepared to a final pH - 3 with 10% HCI. Liquified phenol (248 uL) is added to a 15 mL aliquot (150 mg protein) of the above solution. The pH of the reaction is adjusted with concentrated ammonium hydroxide to a pH - 6.50.
One microliter of a 10% ZnC12 solution is added to the reaction followed by the addition of 22.5 milligrams of protamine. The reaction mixture is stirred at room temperature for 15 minutes before it stood for two days at room temperature to allow solid formation to occur.

The reaction mixture is transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution is decanted and the solid is washed with 5 mL cold DI
H20. This solution is centrifuged at 2800 RPM for 15 minutes before the H20 is decanted. Two more H20 wash is occurred the same way. The sample is then washed with 10 mL 200 proof cold EtOH and centrifuged at 2800 RPM for 15 minutes before the EtOH is decanted. Two more EtOH washes are carried out the same way before the sample is vacuum dried over two days.

9.7.2 Preparation of R-type Zn Complex of IN105 Conjugate With Protamine at Neutral pH
A IN105 solution at approximately 10 g/L is prepared to a final pH - 3 with 10% HCI. Liquefied phenol (49.5 uL) is added to 15 mL reaction. Then, 60 microliters of a 10%
ZnC12 solution is added to reaction followed by the addition of 7.5 mg protamine. The pH is adjusted with concentrated ammonium hydroxide to a pH of 7.00. The reaction is allowed to stand for three days at room temperature to allow solid formation.

The reaction mixture is transferred to a centrifuge tube and centrifuged at 2800 RPM for 15 minutes. The solution is decanted and the solid was washed with 5.0 mL cold DI
H"O. This solution is centrifuged at 2800 RPM for 15 minutes before the H20 is decanted.
Two more H20 washes are occurred the same way. The sainple is then washed with 50 mL 200 Proof cold EtOH
and centrifuged at 2800 RPM for 15 minutes before the EtOH is decanted. Two more EtOH
washes are carried out the same way before the sample is vacuum dried over two days.

9.7.3 Preparation of R-type Crystalline Zn Complex of IN105 A crude 15mg/mL IN105 solution containing 25% organic was pH adjusted to 3.47 using 1M
HCI. Solid phenol was melted in a 40-60 C water bath and 0.218mL was added to reaction flask.
Then 0.4mL of 4% acidified aqueous ZnC12 solution was added to reaction. The pH of the solution was adjusted with 1M NaOH to a final pH of 6.6. While adjusting the pH, lOmL
aliquots were pulled at the following pH values: 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4 and 6.6.
The samples were allowed to sit without stirring for 24 hours. Needle-like crystals were observed under a microsope.

9.7.4 Preparation of R-type Crystalline Zn Complex of IN105 Containing 30%
Organic A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 250mM
ammonium acetate buffer and the pH was adjusted to 2.81 with 1M HCI. Liquefied phenol, 0.040mL, and 95% EtOH, 4.25mL, were added to the solution. Then, 0.400mL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.7 to 5.4 using 50% NH4OH and pulling 1mL aliquots at each of the following desired pH: 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals (see Figure 5) from the pH
range 4.0 to 5.2.

9.7.5 Preparation of R-type Crystalline Zn Complex of IN105 in 100mM
ammonium Acetate Buffer (30, 20 and 10% EtOH) A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 100mM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HCI. Liquefied phenol, 0.040mL, and 95% EtOH, 4.25mL, were added to the solution. Then, 0.400mL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 2.9 to 5.6 using 5M NH4OH and pulling 0.5mL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit witl7out stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals from the pH range 4.4 to 4.8.

A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 100mM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HCI. Liquefied phenol, 0.040mL, and 95% EtOH, 2.25mL, were added to the solution. Then, 0.400mL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted froin 2.9 to 5.6 using 5M NH4OH and pulling 0.5mL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The sainples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.8 to 5.4.

A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 100mM
ammonium acetate buffer and the pH was adjusted to 2.8 with 5M HCI. Liquefied phenol, 0.040mL, and 95% EtOH, 1.15inL, were added to the solution. Then, 0.400mL of a 4% acidified ZnClz solution was added to the reaction mixture. The pH of the solution was adjusted from 2.8 to 5.6 using 5M NH4OH and pulling 0.5mL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals from the pH range 5.0 to 5.6.

9.7.6 Preparation of R-type Crystalline Zn Complex of IN105 in 20% Organic with 0.1 and 0.2% Phenol A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 100mM
ammonium acetate buffer and the pH was adjusted to 3.0 with 5M HCl. Liquefied phenol, 0.010mL, and 95% EtOH, 2.5mL, were added to the solution. Then, 0.400mL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.2 to 5.6 using 5M NH4OH and pulling 0.5mL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.4 to 5.4.
A fresh 15mg/mL solution of MPEG3 propionyl insulin compound was prepared in 100mM
ammonium acetate buffer and the pH was adjusted to 3.0 with 5M HC1. Liquefied phenol, 0.020mL, and 95% EtOH, 2.5mL, were added to the solution. Then, 0.400mL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.3 to 5.6 using 5M NH4OH and pulling 0.5mL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed circle-like crystals from the pH range 4.4 to 5.2.
9.7.7 Preparation of R-type Crystalline Zn Complex of IN105 at 8.0 gram scale, pH 4.8 and Room Temperature A fresh 15mg/hnL solution of MPEG3 propionyl insulin compound was prepared in 250mM
ammonium acetate buffer and the pH was adjusted to 2.0 with 5M HCl. Liquefied phenol, 2.13mL, and 95% EtOH, 225mL, were added to the solution. Then, 21.3mL of a 4%
acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted to 4.8 using 5M NH4OH. The solution was allowed to sit without stirring for 24 hours before the crystals were harvested. Needle-like crystals were observed at the T=0 microscope picture.

The crystals were harvested by splitting the reaction mixture into 6X250mL
centrifuge tubes.
The tubes were spun at 10,000RPM for 8 minutes at 10 C before the supematant was decanted.
Then, to each tube, was added l OmL cold H20 before consolidating the 6 tubes into 2 tubes. The centrifuge process was repeated once more with cold water and twice more with cold EtOH. The crystals were then dried with a desktop lyophilizer for 2 days. The procedure produced 93%
yield (w/w) relative to the starting material.

9.7.8 Preparation of R-type Crystalline Zn Complex of IN105 at 1.5 gram scale, pH 4.8 and Room Temperature A fresh solution of MPEG3 Propionyl Insulin compound (IN105) was prepared by dissolving 1.52g of solid IN105 in 100 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.8 using 5M HC1 / 5M NH~OH. Solid phenol was melted in a 40-60 C warm water bath. 400 L of melted phenol and 42.5mL of 95% EtOH were added to the reaction flask.
Then 4mL of 4% acidified aqueous ZnC12 was added to the reaction flask. The resulting solution was then adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit without stirring for 48 hours before crystals were harvested. Needle-like crystal formation was observed after 21 hours via microscope.

The crystals were harvested by splitting of the reaction slurry among 4 x 50mL
centrifuge tubes.
The tubes were spun initially at 1000 RPM for 8 min. The supernatant was then decanted. The crystals in each tube were washed with 1 x 5mL aliquot of ice-cold H20 then spun at 3000 RPM
for 8 min. The supematant was then decanted. Repeated the washing/ spinning procedure with 1 x 5mL aliquot of ice-cold H20 then with 1 x 5 mL aliquot of ice-cold EtOH. The crystals were then dried in a vacuum dessicator overnight. The procedure produced 73% yield (w/w) relative to the starting material.

9.7.9 Preparation of R-type Crystalline Zn Complex of IN105 at 1.5 gram scale, pH 4.4 and Room Temperature A fresh solution of MPEG3 Propionyl Insulin compound (IN105) was prepared by dissolving 1.50 g of solid IN105 in 100 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.6 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath.
400 L of melted phenol and 42.5mL of 95% EtOH were added to the reaction flask. Then 4mL
of 4% acidified aqueous ZnC12 was added to the reaction flask. The resulting solution was then adjusted to pH 4.4 using 5M NH4OH. The reaction was then allowed to sit without stirring for 22 hours before crystals were harvested. A mixture of needle-like crystal formation and precipitate was observed after 2 hours via microscope. The reaction mixture appeared to be completely crystalline after 21 hours via microscope.

The crystals were harvested by transferring the reaction slurry to a I x 250mL
centrifuge tube.
The tube was spun initially at 10,000 RPM for 8 min. The supernatant was then decanted. The crystals were washed with 1 x 20 mL aliquot of ice-cold H20 then spun at 10,000 RPM for 8 min.
The supernatant was then decanted. Repeated the washing/ spinning procedure with 1 x 20 mL
aliquot of ice-cold H20 then with 2 x 20 mL aliquots of ice-cold EtOH and a final 1 x 20 mL
aliquot of ice-cold H20. The crystals were then dried in a vacuum dessicator overnight. The procedure produced 67% yield (w/w) relative to the starting material.

9.7.10 Preparation of R-type Crystalline Zn Complex of IN105 at 8.0 Gram Scale, pH 4.8 and Room Temperature A fresh solution of MPEG3 Propionyl Insulin compound (IN105) was prepared by dissolving 7.98 g of solid IN105 in 533 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.4 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 2.13 mL of melted phenol and 225 mL of 95% EtOH were added to the reaction flask.
Then 21.3 mL
of 4% acidified aqueous ZnC12 was added to the reaction flask. The resulting solution was then adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit without stirring for 21 hours before crystals were harvested. The reaction mixture appeared to be completely crystalline after 2 hours via microscope.

The crystals were harvested by splitting the reaction slurry among 6 x 250mL
centrifuge tubes.
The tubes were spun initially at 10,000 RPM for 8 min. The supernatant was then decanted. The crystals in each tube were washed with 1 x 10 mL aliquots of ice-cold H20 then spun at 10,000 RPM for 8 min. The supematant was then decanted Repeated the washing/ spinning procedure with 1 x 10 mL aliquot of ice-cold H20 then with 2 x 10 mL aliquots of ice-cold EtOH and a final 1 x 10 mL aliquot of ice-cold H20. The crystals were then dried in a vacuuin dessicator for 2 days. The procedure produced 87% yield (w/w) relative to the starting material.

9.7.11 Preparation of R-type Crystalline Zn Complex of IN105 at 10.0 Gram Scale, pH 4.8 and Room Temperature A fresh solution of MPEG3 Propionyl Insulin compound (IN105) was prepared by dissolving 10.06 g of solid IN105 in 670 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.6 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 2.7 mL of melted phenol and 285 mL of 95% EtOH were added to the reaction flask.
Then 27 mL of 4% acidified aqueous ZnC12 was added to the reaction flask. The resulting solution was then adjusted to pH 4.8 using 5M NH4OH. The reaction was then allowed to sit witliout stirring for 21 hours before crystals were harvested. The reaction mixture appeared to be coinpletely crystalline after 2.5 hours via microscope.

The crystals were harvested by splitting the reaction slurry among 6 x 250mL
centrifuge tubes.
The tubes were spun initially at 10 C , 10,000 RPM for 8 min. The supematant was then decanted. The crystals in each tube were washed with 1 x 10 mL aliquots of ice-cold H20, and consolidated into 2 x 250mL centrifuge tubes then spun at 10 C , 10,000 RPM
for 8 min. The supernatant was then decanted Repeated the washing/ spinning procedure with 1 x 30 mL aliquot of ice-cold H-,O then with 2 x 30 mL aliquots of ice-cold EtOH and a final 1 x 30 mL aliquot of ice-cold HZO. The crystals were then dried using a benchtop lyopholizer for 3 days. The procedure produced 89% yield (w/w) relative to the starting material.

9.8 Preparation and analysis of cryatalline Zn compex of HIM2 using organic solvent 9.8.1 Preparation of R-type Zn Complexes of HIM2 A fresh l5mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were added to the solution. Then, 600uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted froin 3.14 to 6.0 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4 (See Figure 6A), 4.6, 4.8, 5.0, 5.2, 5.4 (See Figure 6B), 5.6, 5.8, 6Ø The samples were allowed to sit without stirring for 24 hours.
The microscope pictures taken after 24 hours showed needle-like crystals at pH
4.4. The pH
range from 4.6- 6.0 show large, crystalline like solids of various shapes and sizes.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were added to the solution. Then, 400uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.22 to 6.0 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2 (See Figure 7A), 5.4, 5.6, 5.8, 6Ø The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours show crystalline-like solids from pH 4.2-6.0 of various shapes and sizes.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 3.5mL, were added to the solution. Then, 200uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted froin 3.19 to 6.0 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0 (See Figure 7B), 5.2, 5.4, 5.6, 5.8, 6Ø The samples were allowed to sit without stin-ing for 24 hours. The microscope pictures taken after 24 hours showed crystalline-like solids from pH 4.4-4.6 of various shapes and sizes. The pH range of 4.8-5.2 show more uniform, needle-like crystals.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 2.6mL, were added to the solution. Then, 600uL of a 4% acidified ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.04 to 6.0 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See Figure 8A), 5.6, 5.8, 6Ø The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed flat, snowflake-like crystals from pH 4.6-5.4.

A fresh l5mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 2.6mL, were added to the solution. Then, 400uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.05 to 6.0 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8, 5.0 (See Figure 8B), 5.2, 5.4, 5.6, 5.8, 6Ø The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals at pH5.0, crystal-like solids at pH5.2 and flat, snowflake-like crystals at pH5.4.

Rxfz 6 A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.95 with 5M HCI. Liquefied phenol, 40uL, and 95%
EtOH, 2.6mL, were added to the solution. Then, 200uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.09 to 6.0 using 5M NH4OH and pulling 500uL aliquots at each of the following desired pH: 4.2, 4.4, 4.6, 4.8 (See Figure 9A), 5.0, 5.2, 5.4, 5.6, 5.8, 6Ø The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals and crystal like solid at pH4.8-5.6.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 250uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted fiom 2.97 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed crystal-like precipitation from pH 4.6-5.8 A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCI. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 200uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See Fi~ure 9B), 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed crystal-like precipitation from pH 4.6-5.6.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 150uL of a 4% acidified ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed crystals of various sizes and shapes from pH 5.0-5.2.

A fresh 15ing/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HC1. Liquefied phenol, 40uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 100uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.09 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0 (See Fi,;ure l0A), 5.2, 5.4 (See Figure 10B), 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals at pH 5.0 and various shapes and sizes of crystalline material from pH 5.2-5.6.

A fresh l5mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCI. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 250uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.08 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed crystal-like precipitation from pH 4.8-5.8.

A fresh 15ing/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCI. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 200uL of a 4% acidified ZnCI2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed very little crystal-like solids.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCI. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 200uL of a 4% acidified ZnC12 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.05 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed very little crystal-like solids.

A fresh 15mg/mL solution of HIM2 was prepared in 250mM ammonium acetate buffer and the pH was adjusted to 2.76 with 5M HCl. Liquefied phenol, 20uL, and 95% EtOH, 4.25mL, were added to the solution. Then, 100uL of a 4% acidified ZnCl2 solution was added to the reaction mixture. The pH of the solution was adjusted from 3.06 to 5.8 using 5M NH4OH
and pulling 500uL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed very little crystal-like solids.

9.9 Co-crystallization of HIM2 and IN105 with zinc 9.9.1 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and IN105 9.9.2 50:50 (HIM2:IN105) A fresh solution of HIM2 and IN105 was prepared by dissolving 37.3mg HIM2 and 36.4mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.84 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 80p,L of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted froin 3.19 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 4 hours. The microscope pictures taken after 4 hours show various sizes and shapes of crystals from the pH
range 4.4 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 37.Img HIM2 and 35.9mg IN105 in 4 inL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.03 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnCIZ was added to the reaction flask. The pH of the solution was then adjusted from 3.38 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0 (See Figure 11A), 5.2 (See Fi~ure 11B), 5.4 and 5.6 (See Figure 12A). The samples were allowed to sit without stirring for 4 hours. The microscope pictures taken after 4 hours show mostly short, needle-like crystals from the pH range 4.6 to 5.6.

9.9.3 70:30 (HIM2:IN105) A fresh solution of HIM2 and IN105 was prepared by dissolving 53.4mg HIM2 and 23.2 mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.62 using 5M HC1. Solid phenol was melted in a 40-60 C warm water batli. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 80uL of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted from 3.02 to 5.60 using 5M NHdOH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2 (See Figure 12B), 5.4 and 5.6. The samples were allowed to sit without stirring for 1 hour. The microscope pictures taken after 1 hour show various sizes and shapes of crystal-like precipitation from the pH range 4.4 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 53.6mg HIM2 and 24.5mg IN105 in 4 mL of 250 mM ainmonium acetate pH 7.5. The solution was adjusted to pH 2.89 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted from 3.28 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2 (See Figure 13A), 5.4 and 5.6. The samples were allowed to sit without stirring for 1 hour. The microscope pictures taken after 1 hour show mostly various sizes and shapes of crystal-like precipitation from the pH range 4.6 to 4.8 and many, short, needle-like crystals from the pH range 5.0 to 5.4.

9.9.4 30:70 (HIM2:IN105) A fresh solution of HIM2 and IN105 was prepared by dissolving 23.3mg HIM2 and 54.7mg IN105 in 4 mL of 250 mM ammoniuin acetate pH 7.5. The solution was adjusted to pH 2.84 using 5M HC1. Solid phenol was melted in. a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 8011L of 4%
acidified aqueous ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted fiom 3.27 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 1 hour. The microscope pictures taken after 1 hour show mostly various sizes and shapes of crystal-like precipitation from the pH range 4.4 to 5.0 and few, needle-like crystals from the pH range 5.2 to 5.6.

A fresh solution of HIM2 and IN105 was prepared by dissolving 24.8mg HIM2 and 54.9mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.09 using 5M HCl. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40 L of 4%
acidified aqueous ZnClz was added to the reaction flask. The pH of the solution was then adjusted from 3.47 to 5.60 using 5M NH4OH and pulling 0.500inL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 and 5.6 (See Figure 13B). The samples were allowed to sit without stirring for 1 hour. The microscope pictures taken after 1 hour show mostly various circular sizes of crystal-like precipitation from the pH range 4.4 to 5.0 and crystals various shapes and sizes from the pH range 5.2 to 5.6.

9.9.5 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and IN105 9.9.6 50:50 (HIM2:IN105) A fresh solution of HIM2 and IN105 was prepared by dissolving 37.4mg HIM2 and 35.9mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.60 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted from 2.15 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed crystal solids of various shapes and sizes from pH=4.6-5.6.

9.9.7 70:30 (HIM2:IN105) A fresh solution of HIM2 and IN 105 was prepared by dissolving 57.0mg HIM2 and 24.5mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.43 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnCl2 was added to the reaction flask. The pH of the solution was then adjusted from 2.92 to 5.60 using 5M NHdOH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6 (See Fi~ure 14A), 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals from pH 5.0-5.2.

9.9.8 30:70 (HIM2:IN105) A fresh solution of HIM2 and IN105 was prepared by dissolving 24.1mg HIM2 and 53.8mg IN105 in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.35 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40 L of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted from 2.60 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0 (See Figure 14B), 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed needle-like crystals from pH 5.0-5.2.

9.9.9 Preparation of R-type Co-crystallized Zn Complexes of HIM2 and Human Insulin 9.9.10 50:50 (HIM2:Insulin) A fresh solution of HIM2 and Insulin was prepared by dissolving 39.2mg HIM2 and 36.7mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 2.53 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnClz was added to the reaction flask. The pH of the solution was then adjusted from 2.82 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2 (See Figure 15A), 5.4 and 5.6. The samples were allowed to sit without stirring loo for 24 hours. The microscope pictures taken after 24 hours showed various shapes and sizes of crystal-like solid from pH 5.2 and 5.4. Many tiny, needle-like crystals were observed at pH 5.6.
9.9.11 70:30 (HIM2:Insulin) A fresh solution of HIM2 and Insulin was prepared by dissolving 56.5mg HIM2 and 20.2mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.23 using 5M HCI. Solid phenol was melted in a 40-60 C warm water bath. 16 L of melted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnClz was added to the reaction flask. The pH of the solution was then adjusted from 2.82 to 5.60 using 5M NH4OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 and 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed various shapes and sizes of crystal-like solid from pH 5.2 and 5.6.

9.9.12 30:70 (1HM2:Insulin) A fresh solution of HIM2 and Insulin was prepared by dissolving 21.8mg HIM2 and 49.2mg Insulin in 4 mL of 250 mM ammonium acetate pH 7.5. The solution was adjusted to pH 3.23 using 5M HC1. Solid phenol was melted in a 40-60 C warm water bath. 16 L of rimelted phenol and 1.75mL of 95% EtOH were added to the reaction flask. Then, 40uL of 4%
acidified aqueous ZnC12 was added to the reaction flask. The pH of the solution was then adjusted from 2.93 to 5.60 using 5M NH~OH and pulling 0.500mL aliquots at each of the following desired pH: 4.4, 4.6, 4.8, 5.0, 5.2, 5.4 (See Figure 15B) and 5.6. The samples were allowed to sit without stirring for 24 hours. The microscope pictures taken after 24 hours showed flat, snowflake-like crystals at pH 4.8. At pH 5.0, there was a mix of needle-like and snowflake-like crystals. From pH 5.2-5.6 there were many tiny, needle-like crystals observed.

10 Aqueous Solubility of Zn Complexes Two hundred microliters of 0.1M Phosphate Buffer Saline (PBS, filtered, pH =
7.4) was added to a 1 mL conical reaction vial. To this vial, a small amount of sample was added slowly until saturation is observed. Periodically, the solution was vortexed. Upon saturation, the vial was placed in a small centrifuge tube and the sample was centrifuged at 2000RPM
for 3min at RT.
After centrifugation, 10 L of the sample was removed from the supernatant and diluted in 490 L
buffer (0.1M PBS). This diluted sample was analyzed via HPLC to determine its' concentration.

Conjugate Solubility Zn Phenol IN105 -26mg/mL N/A N/A
ZnIN105 15mg/mL - 20mg/mL 0.44% 0.76%
ZnIN 105 -24mg/mL 0.61% 1.11%
ZnIN105 15mg/mL - 20mg/mL 0.63% 1.04%
11 In vitro Enzyme Resistance Examples for Zn IN105 Complexes insulin compound conjugates (IN105) were provided in 10 mM sodium phosphate buffer (a pH of about 7.4) and their concentrations were detennined by HPLC (the solutions are diluted with buffer so that equimolar comparisons can be made between parent and conjugates -0.6 mg/mL).
Lyophilized chymotrypsin enzyme was resuspended in 1 mM HC1 to a concentration of -7.53 U/mL. A 1.53 mL aliquot of each sample was added to sample tubes and 0.850 mL
into control tubes. Samples were tested in duplicate along with four control tubes per sample. Aliquots were incubated at 37 C in a thermomixer for 15 minutes. Then 17 L of chymotrypsin enzyme was added to each sample tube. Five L of 1 mM HCl was added to each control tube.
Immediately following the additions, 200 L was removed from the sample and the control tubes and placed into 50 p.L of 1% TFA previously aliquoted out into centrifuge tubes. This sample serves as T=O.
The sampling procedure for Insulin compound (Zn fi-ee), IN105 (Zn free) and Insulin compound (regular insulin compound) was repeated at the following intervals: 0, 2, 5, 8, 12, 15, and 30 minutes. The control procedure was repeated at the following intervals: 0, 8, 15, 30 minutes. For T-type and R-type sainples, the procedure was repeated at the following intervals: 0, 5, 8, 12, 30, 40 and 60 minutes. The control procedure for the Zn complexes was repeated at the following intervals: 0, 12, 40 and 60 minutes. Samples were stored at -20 C until analysis can occur via HPLC. HPLC was performed to determine percent degradation relative to the respective T= 0 minute for each digest. The natural log of the percent remaining was plotted versus time and a linear regression run for each digest. The half life was calculated using the equation: t~,:
-0.693/slope.

Results at 0.6 m2/ml protein:

Sample T half Zinc content Phenol content Insulin compound 4.9 mins 0.0 --(zinc free) IN105 12.5 mins 0.0 --(zinc free) IN105 11.1 mins 0.0 (zinc free) Insulin compound, USP 11.6 mins 0.3 to 1% w/w ---(Regular insulin compound) IN105 55.3 mins 1.85 % w/w 2.37 % w/w (R-type zinc complex) IN105 54.8 mins 1.88 % w/w ---(T-type zinc complex) 12 Formulation Examples 12.1 Liquid Formulation Examples 12.1.1 Buffer Solution for R6 type Zn-HIM2 Buffer Study Components Amount in 1 mL solution Dibasic Sodium Phosphate 1.88 mg Insulin component 3.7 mg (100 units) Glycerol 16.0 mg Phenol (or m-Cresol) 3.0 mg Zinc 0.037mg (1 % w/w of insulin)*
pH 7.4 to 7.8**
* Adjustment of the amount of Zinc to 0.037mg/ml per 3.7mg/ml insulin by adding Zinc chloride and to be based the zinc content in the Zinc HIM2 solid.
** pH may be adjusted with HCI 10% and/or sodium hydroxide 10%

12.1.2 Preparation of Capric Acid/Lauric Acid Formulation Oral Liquid Diluent We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine, citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using IN sodium hydroxide or 1N

hydrochloric acid. We then adjusted the temperature to 45 - 50 C by warming on a hotplate and maintained this temperature. We then added the capric acid to the warm solution and mixed until the capric acid was dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 - 7.9 using 1N sodium hydroxide or 1N hydrochloric acid. We then mixed the solution for 5 minutes.
We added appropriate amount of sterile water to equal 100% of the required volume and mixed well.
Component Percentage (%w/v) Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0.50 Lauric Acid 0.50 Sodium Hydroxide, 1N As Needed to Adjust pH 7.7 - 7.9 Hydrochloric Acid, 1N As Needed to Adjust pH 7.7 - 7.9 Sterile Water Dilute to Required Volume IN105, HIM2 or ZnHIM2 was weighed out in amounts necessary to achieve appropriate concentration for dosing studies, e.g., 1 mg 1N105 (of protein) was weighed out and coinbined with 1 mL of formulation to yield a 1 mg/mL 1N105 in formulation.

12.1.3 Preparation of Oleic acid/capric Acid/Lauric Acid/Cholate Formulation Oral Liquid Diluent An oral liquid formulation of R-type Zn HIM2 was prepared having the components shown in the following table:

Component Percentage (%w/v) Tromethamine 4.24 Trol amine 5.22 Citric Acid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Sodium Cholate 3.00 Oleic Acid 1.00 Capric Acid 0.50 Lauric Acid 0.50 Sucralose Solution, 25% 0.80 Strawberry Flavor 0.40 Sodium Hydroxide, IN As Needed to Adjtist pH 7.7- 7.9 Hydrochloric Acid, IN As Needed to Adjust pH 7.7 - 7.9 Sterile Water Dilute to Required Volume Oral liquid samples were prepared to contain 1 mg/mL protein equivalent of R-type Zn HIM2 (ZnHIM2-R). The ZnHIM2-R was removed from the Freezer (-20 C), placed in a dessicator and allowed to come to room temperature. A 1 mg/mL protein equivalent of ZnHIM2-R
was prepared in oral liquid diluent solution as follows. 6.4 mg of ZnHIM2-R was weighed. Then, 5.0 mL of oral liquid diluent was transferred into the container and gently swirled to mix. The solution took approximately 45 minutes to dissolve. The resulting solution was a suspension (cloudy appearance). Prior to dosing, the solution was gently swirled for 60 seconds to ensure the solution was a homogeneous solution. For ZnHIM2-R, the protein content was 78.6%, 1 mg/mL
protein equivalent, quantity = 5 mL. Amount of ZnHIM2-R =(1 mg/mL) /(0.786)}
X(5.0 mL) =
6.4 mg. ZnHIM2-R Concentration = (6.4 mg)/(5.0 mL) = 1.28 mg/mL (equivalent to 1 mg/mL
adjusted for protein content).

12.1.4 Preparation of Capric Acid Liquid Formulations We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine, citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using 1N sodium hydroxide or 1N
hydrochloric acid. We then adjusted the temperature to 45 - 50 C by warming on a hotplate and maintained this temperature. We then added the capric acid to the warm solution and mixed until the capric acid was dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 - 7.9 using 1N sodium hydroxide or iN hydrochloric acid. We then mixed the solution for 5 minutes.
We added appropriate amount of sterile water to equal 100% of the required volume and mixed well.

Component % w/v Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0.9, 1.5, 3.0 or 6.0 Sodium Hydroxide, 1N As Needed to Adjust pH
Hydrochloric Acid, IN As Needed to Adjust pH
Sterile Water To Required Volume IN105 was weighed out in amounts necessary to achieve appropriate concentration for dosing studies, e.g., 1 mg IN105 (of protein) was weighed out and combined with 1 mL
of formulation to yield a 1 mg/mL IN105 in formulation.

12.1.5 Caprate and/or Laurate in Phosphate Buffer Liquid Formulations Preparation of 100 inM Sodium Phosphate Buffer, pH 7.8 or 8.2. We transferred 1.17 grams of Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was added and mixed well until dissolved. We then added 24.58 grams of Sodium Phosphate Dibasic Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed well. Filtered through a 0.22 m filter. We adjusted the pH to 7.8 or 8.2 with 1N HCI or 1N
NaOH.

For IN105, we transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 or 8.2 into a suitable container. We then added an amount of caprate calculated to produce 3% w/v of the final solution and mixed well until dissolved. We then adjusted the pH to 7.8 or 8.2 with 1N
HCI or 1N NaOH. We diluted to the appropriate volume (e.g., 100 inI.,) with phosphate buffer pH 7.8 or 8.2.

Component % w/v Sodium Caprate 3.0 100 mM Sodium Phosphate Buffer, pH 7.8 or 8.2 QS to 100%

For BN-054, we weighed 400 grams of 100 mM Sodium Phosphate Buffer, pH 7.8 in a suitable container. We added 9.7 grams of Sodium Caprate and 11.1 grams Sodium Laurate and mixed well until dissolved. We added appropriate amount of 100 mM Sodium Phosphate Buffer, pH
7.8, to equal a net weight of 500 grams.

Component % w/w Sodium Caprate 1.94 Sodium Laurate 2.22 100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%
12.1.6 Liquid Formulation with Arginine or Trolamine Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred 1.17 grams of Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was added and mixed well until dissolved. We then added 24.58 grains of Sodium Phosphate Dibasic Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed well. Filtered through a 0.22 in filter. We adjusted the pH to 7.8 with 1N
HC1 or 1N NaOH.

We transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 into a suitable container. We added the appropriate amount (as indicated in the table below) of arginine or trolamine to the container and mixed well until dissolved. We then added an amount of caprate calculated to produce 3% w/v of the final solution and mixed well until dissolved. We then adjusted the pH to 7.8 with 1N HCI or 1N NaOH. We diluted to the appropriate volume (e.g., 100 mL) with phosphate buffer pH 7.8.

Component % w/v Sodium Caprate 3.0 Arginine or Trolamine 0.4 or 1.2 100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%

IN105 was weighed out in amounts necessary to achieve appropriate concentration for dosing studies, e.g., 1 mg IN 105 (of protein) was weighed out and combined with 1 mL
of formulation to yield a 1 mg/mL IN105 in formulation.

12.1.7 Liquid Formulation with Caprylic Acid We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolainine, citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using 1N sodium hydroxide or 1N
hydrochloric acid. We then adjusted the temperature to 45 - 50 C by warming on a hotplate and maintained this temperature. We then added the caprylic acid to the warm solution and mixed until the caprylic acid was dissolved. We adjusted the teinperature to 21 - 25 C (or room teinperature) and measured the pH of the liquid. As needed, we adjusted the pH
to 7.7 - 7.9 using 1N sodium hydroxide or 1N hydrochloric acid. We then mixed the solution for 5 minutes.
We added appropriate amount of sterile water to equal 100% of the required volume and nuxed well.

Component % w/v Tromethamine 4.24 Trol amine 5.22 Citric Acid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Caprylic Acid 3.0 Sodium Hydroxide, 1N As Needed to Adjust pH
Hydrochloric Acid, 1N As Needed to Adjust pH
Sterile Water To Required Volume IN105 was weighed out in amounts necessary to achieve appropriate concentration for dosing studies, e.g., 1 mg IN105 (of protein) was weighed out and combined with 1 inL
of formulation to yield a 1 mg/mL IN105 in formulation.

12.1.8 Liquid Formulation with Linoleic Acid Preparation of 100 mM Sodium Phosphate Buffer, pH 7.8. We transferred 1.17 grams of Monosodium Phosphate Monohydrate to a 1-L flask. Approximately 500 mL of sterile water was added and mixed well until dissolved. We then added 24.58 grams of Sodium Phosphate Dibasic Heptahydrate and mixed well until dissolved. Diluted to volume with sterile water and mixed well. Filtered through a 0.22 in filter. We adjusted the pH to 7.8 with 1N
HC1 or 1N NaOH.

We transferred 60% of the appropriate volume of the phosphate buffer pH 7.8 into a suitable container. We then added an amount of linoleic acid sodium salt calculated to produce 3% w/v of the final solution and mixed well until dissolved. We then adjusted the pH to 7.8 with 1N HCI or 1N NaOH. We diluted to the appropriate volume (e.g., 100 mL) with phosphate buffer pH 7.8.

Component % w/v Linoleic Acid Sodium Salt 3.0 100 mM Sodium Phosphate Buffer, pH 7.8 QS to 100%

12.1.9 Preparation of Capric Acid and Lauric Acid Liquid Formulations We transfered approximately 60% of the required sterile water volume into a suitable container.
We added the appropriate amount (as indicated in the table below) of tromethamine, trolamine, citric acid anhydrous, and sodium hydroxide pellets to the container and mixed well until dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. We adjusted the pH to 7.7 - 7.9 as necessary using 1N sodium hydroxide or 1N
hydrochloric acid. We then adjusted the temperature to 45 - 50 C by warming on a hotplate and maintained this temperature. We then added the capric acid and/or lauric acid to the warm solution.and mixed until the capric acid and/or lauric acid were dissolved. We adjusted the temperature to 21 - 25 C (or room temperature) and measured the pH of the liquid. As needed, we adjusted the pH to 7.7 - 7.9 using 1N sodium hydroxide or 1N hydrochloric acid. We then mixed the solution for 5 minutes. We added appropriate amount of sterile water to equal 100% of the required volume and mixed well.

Component % w/v Tromethamine 4.24 Trolamine 5.22 Citric Acid Anhydrous 6.72 Sodium Hydroxide Pellets 1.88 Capric Acid 0, 0.1 or 0.9 Lauric Acid 0, 0.9 or 0.1 Sodium Hydroxide, 1N As Needed to Adjust pH
Hydrochloric Acid, 1N As Needed to Adjust pH
Sterile Water To Required Volume IN105 was weighed out in amounts necessary to achieve appropriate concentration for dosing studies, e.g., 1 mg IN105 (of protein) was weighed out and combined with 1 mL
of formulation to yield a 1 mg/mL IN105 in formulation.

12.2 Solid Dosage Formulation Examples 12.2.1 Preparation and dissolution profile of caprate/laurate solid dosage formulation using Nobex-IN105-[8541-Transfer approximately 58 mg of sodium caprate, 57 mg of sodium laurate, 286 mg mannitol, 30 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly. Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-[8541 58 mg Caprate and 57 m~
Laurate per Tablet Component mg per Tablet Sodium Caprate 58 Sodium Laurate 57 Mannitol 286 Explotab (sodium starch glycolate) 30 Nobex-IN 105 (protein) 6 The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1 % TFA
(mobile phase A) and acetonitrile with 0.1 % TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and column was a C18 (150 x 2 mm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets Formulation [854] containing 6 mg Zn-IN105 (protein), 286 mg Mannitol, 58 ing Sodium Caprate, 57 mg Sodium Laurate, and 30 mg sodium starch glycolate (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8541, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets (8541, %
Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8541, %
Laurate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 12.2.2 Solid Dosage Form (Tablet) Formulation Preparation 143 mg Caprate and 140 mg Laurate per Tablet Preparation of Formulation Nobex-IN105- f 8561 10 Transfer approximately 143 mg of sodium caprate, 140 mg of sodium laurate, 150 mg mannitol, 30 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly. Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosa2e Form (Tablets) Formulation Nobex-IN105-f8561 143 mR Caprate and 140 m~
Laurate per Tablet Component mg per Tablet Sodium Caprate 143 Sodium Laurate 140 Mannitol 150 Explotab (sodium starch glycolate) 30 Nobex-IN105 (protein) 6 The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and column was a C18 (150 x 2 mm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mg Mannitol, 143 mg Sodium Caprate, 140 mg Sodium Laurate, and 30 mg sodium starch glycolate (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8561, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets 18561, %
Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8561, %
Laurate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 12.2.3 Solid Dosage Form (Tablet) Formulation Preparation 143 mg Caprate per Tablet Preparation of Formulation Nobex-IN105-18591 Transfer approximately 143 mg of sodium caprate, 150 mg mannitol, 30 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
10 Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105- f 8591 143 mg Caprate per Tablet Component mg per Tablet Sodium Caprate 143 Mannitol 150 Explotab (sodium starch glycolate) 30 Nobex-IN105 (protein) 6 The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples 15 were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1 % TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and column was a C18 (150 x 2 mm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mg Mannitol, 143 mg Sodium Caprate, and 30 mg sodium starch glycolate (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets 18591, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 5 Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8591, %
Caprate Dissolved Sample Time Vesset 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 12.2.4 Solid Dosage Form (Tablet) Formulation Preparation 286 mg Caprate per Tablet Preparation of Formulation Nobex-IN105-18601 Transfer approximately 286 mg of sodium caprate, 150 mg mannitol, 30 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosa2e Form (Tablets) Formulation Nobex-IN10548601 286m2 Caprate per Tablet Component mg per Tablet Sodium Caprate 286 Mannitol 150 Explotab (sodium starch glycolate) 30 Nobex-IN105 (protein) 6 The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and coluinn was a C18 (150 x 2 mm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mg Mannitol, 286 mg Sodium Caprate, and 30 mg sodium starch glycolate (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets 18601, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets f8601, %
Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 12.2.5 Solid Dosage Form (Tablet) Formulation Preparation 100 mg Caprate per Tablet Preparation of Formulation Nobex-IN105- f 8611 Transfer approximately 100 mg of sodium caprate, 150 mg mannitol, 25 mg of sodium starch glycolate, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105-(8611 100 m2 Caprate per Tablet Component mg per Tablet Sodium Caprate 100 Mannitol 150 Explotab (sodium starch glycolate) 25 Nobex-IN105 (protein) 6 The dissolution testing was carried out using a USP apparatus 2 dissolution unit. The medium was water; paddle speed 50 ipm, and the medium volume was 500 mL. The dissolution samples were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1% TFA
(mobile phase A) and acetonitrile with 0.1% TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% inobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and column was a C18 (150 x 2 inm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mg Mannitol, 100 mg Sodium Caprate, and 25 mg sodium starch glycolate (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8611, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets f8611, %
Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 12.2.6 Solid Dosage Form (Tablet) Formulation Preparation 150 mg Caprate per Tablet Preparation of Formulation Nobex-IN105-18621 Transfer approximately 150 ing of sodium caprate, 150 mg mannitol, 25 mg of croscarmellose sodium, and 6 mg (protein) of Nobex-IN105 onto a piece of weigh paper and blend thoroughly.
Transfer the blend to the press and compress at approximately 350 psi to form a tablet.

Solid Dosage Form (Tablets) Formulation Nobex-IN105- f 8621 150 mg Caprate per Tablet Component mg per Tablet Sodium Caprate 150 Mannitol 150 Explotab (Croscarmellose Sodium) 25 Nobex-IN105 (protein) 6 The dissolution testing was can-ied out using a USP apparatus 2 dissolution unit. The medium was water, paddle speed 50 rpm, and the medium volume was 500 mL. The dissolution samples were analyzed by HPLC using a gradient system. The mobile phases were water with 0.1 % TFA
(mobile phase A) and acetonitrile with 0.1 % TFA (mobile phase B). The gradient utilized was: 0 minutes 100% mobile phase A, 11 minutes 65% mobile phase A, 15 minutes 20%
mobile phase A, 16 minutes 20% mobile phase A, 17 minutes 100% mobile phase A. The wavelength was 214 nm and coluinn was a C18 (150 x 2 mm). The following tables and graphs summarize the dissolution data obtained for the dissolution testing of Nobex-Zn-IN105 Tablets containing 6 mg Zn-IN105 (protein), 150 mg Mannitol, 150 mg Sodium Caprate, and 25 mg Croscarmellose Sodium (Explotab):

Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets [8621, %

Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) Data Summary for the Dissolution Profile of Nobex-Zn-IN105 Tablets f8621, %
Caprate Dissolved Sample Time Vessel 1 Vessel 2 Average (Minutes) (% Dissolved) (% Dissolved) (% Dissolved) 13 bi vitro Enzyme Resistance Examples 5 insulin coinpound conjugates (HIM2) were provided in 10 mM sodium phosphate buffer (a pH of about 7.4) and their concentrations are determined by HPLC (the solutions are diluted with buffer so that equimolar comparisons can be made between parent and conjugates -0.6 mg/mL).
Lyophilized chymotrypsin enzyme was resuspended in 1 mM HCl to a concentration of 7.53 U/mL. A 1.53 mL aliquot of each sample was added to sample tubes and 0.850 mL
into control 10 tubes. Samples were tested in duplicate along with four control tubes per sample. Aliquots were incubated at 37 C in a thermomixer for 15 ininutes. Then 17 L of chyinotrypsin enzyme was added to each sample tube. Five L of 1 mM HCl was added to each control tube.
Immediately following the additions, 200 L was removed from the sample and the control tubes and placed into 50 .L of 1% TFA previously aliquoted out into centrifuge tubes. This sample serves as T=O.

15 The sampling procedure for Insulin (Zn free), HIM2 (Zn free) and Insulin (regular insulin compound) was repeated at the following intervals: 0, 2, 5, 8, 12, 15, and 30 minutes. The control procedure was repeated at the following intervals: 0, 8, 15, 30 minutes. For T-type and R-type samples, the procedure was repeated at the following intervals: 0, 5, 8, 12, 30, 40 and 60 minutes. The control procedure for the Zn complexes was repeated at the following intervals: 0, 12, 40 and 60 minutes. Samples were stored at -20 C until analysis can occur via HPLC. HPLC
was performed to determine percent degradation relative to the respective T= 0 minute for each digest. The natural log of the pecent remaining was plotted versus time and a linear regression run for each digest. The half life was calculated using the equation: t yz: =-0.693/slope.

Results at 0.6 mg/ml protein Sample T half Zinc content Phenol content Insulin 7-9 mins -- --(zinc free) HIM2 12-15 mins -- --(zinc free) Insulin, USP 26-29 mins 0.3 to 1% w/w ---(Regular insulin compound) HIM2 51-78 mins 1.1 % w/w 0.1 to 0.25% w/w (R-type zinc complex) HIM2 51 mins 0.55 % w/w ---(T-type zinc complex) HIM2 95-120 mins 2.0 to 2.1 % w/w 5.3 to 6.2% w/w (R-type zinc/protamine complex) DICON-1 24-26 mins ND ND
(R-type zinc complex) Results at 0.3 me/mL protein Sample T half Zinc content Phenol content Insulin 4-5 mins -- --(zinc free) HIM2 7-9 mins -- --(zinc free) Insulin, USP 7-8 mins 0.4 to 1% w/w ---(regular insulin compound) HIM2 19-21 mins 1.1 % w/w 0.1 to 0.25% w/w (R-type zinc complex) HIM2 12-15 mins 0.55 % w/w ---(T-type zinc complex) 14 In vivo Examples 14.1 Extended Mouse Blood Glucose Assay (MBGA) Six paired-dose groups of 5 male CF-1 mice (Charles River Laboratories; 25-30 g) received subcutaneous injections of either the insulin compound conjugate (test article) or recombinant human insulin. The test article was reconstituted with phosphate buffer (0.O1M, a pH of about 7.4) containing 0.1% w/w bovine serum albumin and dosed at 100, 66.6, 43.3, 30, 20, and 13.3 ltg/kg. Insulin was reconstituted with phosphate buffer (0.01 M, a pH of about 7.4) containing 0.1% w/w bovine serum albumin and dosed at 50, 33.3, 21.7, 15, 10, and 6.7 g/kg. After receiving a subcutaneous dose in the pocket fonned by the thigh and groin, animals were returned to their cages for 30 minutes at room temperature and then were quickly anesthetized and terminally bled. Blood samples were collected in heparin tubes for glucose assay. If glucose assay was delayed, the tubes were stored in ice water and re-warmed to room temperature before assay.

Plasma glucose was measured with a glucometer (e.g., One Touch Basic;
Lifescan), which was calibrated at the beginning of each day of use according to the manufacturer's instructions. The potency of the insulin compound conjugate was then calculated relative to the standard curve that was generated for the recoinbinant human insulin response. Calculations were based upon the assumption that recombinant huinan insulin has a potency of 27.4 IU/mg.

Results are shown in Fipures 16-20. Figure 16 shows MBGA biopotency profiles for HIM2.
Figure 17 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product R-type.
Figure 18 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product T-type.
Figure 19 shows MBGA biopotency profiles for Zn-HIM2 insulin compound product with protainine. Figure 20 shows glucose lowering effect of R-type protamine complex at 30 and 90 minutes post dose. These results show that the biopotency of HIM2 is not significantly reduced by complexation with Zn++. The R-type protamine complex (see Figure 17 [71) shows greater glucose reduction at 30 minutes than 90 minutes.

Further, Figures 21-24 show MBGA biopotency profiles for IN-186, IN-192, IN-190, IN-191, IN-189, IN-178,1N-193, IN-194, IN-185, IN-196 and IN-197 having structures as follows:

B1 monoconiugate, IN-186:
O

H0 J~'~~o 0 B29 monoconhugate, IN-194:

HO~~ 0 B29 monoconiui4ate, IN- 197:

B29 monoconjugate, IN-178:

õ~~
H~ y''~~

a 6~ ~
B29 monoconjulZate, IN-190:

HO

B29 monoconjugate, IN-196:

B29 monoconjugate, IN-191:
C

B29 monoconjumate, IN- 189:

Ha 0 4 0 14.2 Dog Clamp Studies 14.2.1 Initial HIM2 Studies Dogs (n=3 or 6) were prepared surgically (isoflurane anesthesia) by placing a catheter in a femoral artery. The animals were allowed to recover for 16-17 days after which they were fasted overnight and studied in the conscious state. After a 60 min equilibration period, there was a 20 min control period after which the drug was given by mouth. Zn++ -HIM2 R-type Insulin coinpound was tested in a buffer solution, prepared as shown in Example Error!
Reference source not found.. In addition, R-type and NPH-type complexes were tested in oral liquid formulation that contains caprate acid and laurate acid, prepared as shown in Example Error!
Reference source not found..

All 3 test samples were tested at only one dose level (the dose level were identified based on the previous experimental results). The plasma glucose level was then be clamped at a euglycemic value by infusion of D-20 through a leg vein for 4 h. Blood samples (4 ml) were taken at -20, 0, 5, 10, 20, 30, 45, 90, 120, 180 and 240 min for measurement of glucose, insulin compound and C-peptide. Arterial blood samples were obtained as required to clamp the plasma glucose level.
A total of 72 ml of blood was taken in each experiment.

The following measurements were performed: glucose infusion rate, insulin compound concentration, C-peptide concentration, and plasma C-peptide levels (to allow an estimation of endogenous insulin compound release). The glucose infusion rate required to maintain euglycemia provides an index of insulin compound action.

Following the experiment the free end of the catheter was buried subcutaneously and the dogs were allowed to recover for two weeks prior to another study in which a different test article was used. Animals were randomized to dose and used a total of 3 times. Total number of dogs was 6.
Figures 25 and 26 show the results.

14.2.2 Initial IN105 Studies The study was conducted on six (6) overnight fasted conscious mongrel dogs which had been fed a diet of 34% protein, 46% carbohydrate, 14.5% fat and 5.5% fiber based on dry weight. Each animal had a silastic catheter inserted into the femoral artery as described elsewhere (1) approximately three weeks prior to the experiment. On the day of experiment the catheter was removed from its subcutaneous pocket under local anesthesia. Test article:
Nobex-IN105 (Lot.#
KJ-173-095 & KJ-173-116) was provided in the oral fatty acid fonnulation (Nobex-IN-[753]-040422) at a concentration of 1.0 mg/ml. Each dog received 0.25 mg/kg oral dose of Nobex-IN105 (1.0 mg/ml@0.25 ml/kg dosing volume). Nobex-IN105 was given at t=0 and glucose (D-20) was infused through a cephalic vein in order to maintain euglycemia.
Arterial blood samples were drawn for the measurement of insulin and glucose as previously described (1). After the experiment was coinpleted, the arterial catheter was buried subcutaneously as it was during the initial surgery.

During the experiment, one dog vomited immediately after the dosing and only a portion of the dose administered. Therefore, the results from this experiment were reported with and without the data obtained from this dog.

Arterial plasma insulin levels rose in all six dogs, including outlier (Oral-2i), following the oral administration of Nobex-IN105. Mean arterial insulin rose from 6.0 1.4 U/ml (6.3 - 1.7 U/ml, n=5) to a peak of 109.4 =L 31.4 gU/ml (127.8 30.1 gU/ml, n=5) at 10 min post-administration and then fell so that by 150 min all dogs returned to baseline insulin levels (Figures 27 and 28).

Euglycemia was maintained by glucose infusion. The glucose infusion rate required to maintain euglycemia as greatest in the animals with larger rise in arterial insulin (Figures 27 and 28).

Mean Area-Under-The-Glucose Infusion Rate Curve (AUCO-240) was 578.5 144.5 mg/kg/min (669.4 137.7 mg/kg/min, n=5).

14.2.3 Solid Formulations Formulation screening studies using the glucose-clamp model were conducted on overnight fasted conscious mongrel dogs which had been fed a diet of 34% protein, 46%
carbohydrate, 14.5% fat and 5.5% fiber, based on dry weight. Each animal had a silastic catheter inserted into the femoral artery as described elsewhere (reference 1) approximately three weeks prior to the experiment. On the day of experiment the catheter was removed from its subcutaneous pocket under local anesthesia. The test article contained l.Omg/mL Zn IN105 in different liquid formulations or 5-6mg of IN105 per capsule or tablet. Each dog in every experiment received an oral liquid dose of approximately 0.25 mg/kg or a capsule or tablet containing 5 or 6mg of IN105 twice in succession, the first at t = 0 and the second at t= 120 minutes.
Glucose (D-20) was infused through a cephalic vein in order to maintain euglycemia. In some cases study time was extended after dosing where the effect lasted beyond 120 minutes. Arterial blood samples were drawn for the measurement of insulin, glucose and C-peptide as previously described (reference 1). After the experiment was completed, the arterial catheter was replaced into the subcutaneous tissue.

Formulations, both solution and solid dosage forms, ware prepared with different levels of fatty acids, buffers, diluents and disintegrants. To minimize variables, the liquid and the solid formulations contained consistent levels of IN105 and each excipient (Capric, Lauric, Caprylic, Myristic, Linoleic), thus varying only the relative amounts of fatty acid content, buffer, diluents (mannitol or micro crystalline cellulose), and/or disintegrant (Explotab). The glucose infusion rate and IN105 absorption (plasma insulin immuno-reactivity) data were evaluated and coinpared with each dosed formulation.

Initially, experiments were carried out to simplify and refine the optimized liquid formulation (reference 2) to a liquid formulation that would be more readily converted to a solid dosage form.
This was carried out by substituting the free fatty acid with the corresponding sodium salt (e.g.
capric acid replaced by sodium caprate) as well as removing the buffer components (citric acid, trolamine, tromethamine, sodium hydroxide) that were deemed to be no longer required.
Additionally, the effect of other fatty acids such as linoleic, caprylic and myristric acids and the amino acid, arginine, were examined for their effects on the absorption of IN105.

After an initial set of prototype screens using one to two dogs per experiments few prototype formulations were selected and tested in additional dogs to better determine the variability and consistency between formulations and individual animals.

A dissolution method was developed and dissolution studies were carried out on a variety of candidate formulations to evaluate dissolution profiles of IN105 and the fatty acids contents.
RESULTS
In the initial experiments, dogs dosed with IN105 in 3% w/v capric acid sodium salt in a phosphate buffer without additional excipients, showed (Figures 29-30) a similar response compared to the optimized liquid formulation containing 3% w/v capric acid in Trolamine/Citric acid/Tromethamine/Sodiuin Hydroxide buffer. This demonstrated that the sodium salt of the fatty acid form behaves comparably to the acid form and that the additional buffer components did not contribute to the formulation.

In separate studies evaluating alternative fatty acids substituting 3% caprate with either 3%
caprylic acid or 3% linoleic acid, neither of the alternatives exhibited significant effects. The use of caprylic acid resulted in the need for low to moderate levels of glucose while the use of linoleic acid did not result in requiring any glucose infusion suggesting lack of effect. Both fonnulations showed relatively low levels of arterial insulin. A liquid formulation containing arginine showed relatively no benefit on GIR or IN105 levels.

In the primary studies, solid forinulations were evaluated as both powder blend filled into hard gelatin capsules and tablets compressed by hand using a Carver Press. The capsules, powder blend of 6mg of IN105 (insulin equivalent, 0.25mg/Kg) with 57mg of caprate /
57 mg laurate showed no significant effect in the first dosing up to 120mins, and in the second dosing a significant effect was observed with the GIR concurrent with IN105 absorption where the levels were well above the base line form 0 to 120 mins. This data suggests a variable but potential delayed response in IN105 in the capsule dosage form. Dissolution was only slightly delayed with the capsule relative to the tablets although there may be little to no in-vitro/in-vivo correlation.

In the initial prototype tablet screening studies, tablets containing 6mg of 1N105 and 150mg Mannitol , 30mg sodium starch glycolate with 143mg Caparte with or with out 143 mg laurate showed significantly higher GIR and IN105 absorption than the same tablets with 54mg Caprate or/ and 54mg Laurate (Figures 31, 32, and 33). This suggests a reasonable dose response of higher GIR and fN105 levels relative to increasing levels of caprate and laurate. The tables showed an early and consistent GIR response time to the IN 105 filled capsules. Additionally, the 1N105 tablets showed an arterial plasma rise in insulin in all dogs dosed.

In a series of final studies, three prototype tablet formulations (Formuluation [856] contained 143 ing caprate and 140 mg laurate, [860] contained 286 mg caprate, and [862]]
contained 150 mg caprate] were selected for evaluation in 5 dogs (3 different dogs for each formulation) to assess the consistency of performance (Figures 34-37). Arterial plasma insulin levels rose in all 3 dogs and at all 18 doses (3 dogs X 3 tablets X 2 doses each ) with corresponding GIR response following the oral administration of 6mg (Insulin equivalent) of IN105 formulated in the tablets containing either 150mg or 280 mg caprate or 143mg/140mg caparte/laurate (Figares 31-32 and 38-42 . The c-peptide levels (ng/ml), with 150mg and 280mg caprate tablets, the average (n=2), showed a decrease from an initial level of 0.30 0.05 to 0.22 0.02 during the first dosing and 0.1 1 0.05 to 0.02 1 0.0 in the second dosing, and with 140mg/140mg caprate/laurate tablets, showed 0.21 0.05 to 0.05 0.02 during the fist dosing and 0.18 0.05 to 0.18 0.01. This is indicative of suppression of C-peptide secretion from the pancreas as result of the exogenous IN105 insulin.

All three prototype tablet formulations of IN105 showed consistent levels of 1N105 absorption and resultant glucose infusion rate among doses and within and between dogs, including on different days During the final studies, in which sets of 6 dogs were utilized, one dog (dog #3) experienced less response with all liquid and solid dosages. To more accurately represent the results, the data is presented with and without results from dog #3. Data from dogs that did not receive a complete dose (bad gavage, vomiting, etc) or had endogenous insulin are omitted.

Dissolution study: Representative samples of tablets and capsules were subject to dissolution testing (described above).

Discussion These studies demonstrate that the prototype IN105 tablet containing caprate or caprate and laurate sodium salts with mannitol and the disintegrant sodium starch glycolate and containing 6mg IN105 (approximately 0.25 mg/kg) delivered orally resulted in significant and consistent elevation of arterial plasma insulin that required glucose infusion to preserve euglycemia.

These prototype tablets resulted in IN105 levels and GIR rates at least as good and likely-to be better than the liquid formulations containing comparable levels of caprate or laurate. The prototype tablets forms maintain the absorption profile of the oral liquid formulation. The relative oral bio-efficacy of the selected prototype tablet formulations (e.g., 280mg and 150mg caprate containing tablets, n=6, AUC for GIR= 496 117 and 500 275) appears to be better than liquid formulations (e.g., 3% w/v capric acid liquid formulation, n=5, AUC for GIR
=182 92 and 198 119).

The data suggests that the tablets containing sodium caprate as the only fatty acid along with mannitol and the disintegrant, sodium starch glycolate would be useful in the further development of solid dosage forms for use in clinical studies. Data also suggest that Insulin levels following the oral administration of IN 105 in the selected prototype caprate tablets forms (sodium caprate at either 150mg or 286mg) peaked steadily with a typical Tmax at around 20 min post-dose and a Cmax of about 59.0 20.1 and 62.9. 25.4 Units/ml, in both doses. The plasma insulin levels remained elevated close to the Cmax level for 10-15 minutes and above basal levels throughout 120 min following each dose. The GIR required maintaining euglycemia using these tablets reached Tmax at or around 30 to 40 min in both doses and GIR Cmax reached an average of 8.4 1.99 and 7.41 2.18 mg/kg/min. The tablet dosage fonns required higher GIR
Cmax (7.4-8.4 vs.
4.5-5.4) and required glucose infusion for a longer duration (100-120 mins vs.
60-90min) to maintain euglycemia then the optimized liquid formulation.

In comparison with arterial plasma insulin levels of historical SQ and inhaled insulin, it appears that these prototype tablets provide maximum insulin levels similar to SQ and inhaled delivery and resembles an insulin profile comparable to that of inhaled insulin (Figures 34-37).

The prototype tablet experiments suggests the selected prototype tablets are suitable as solid dosage formulations for further evaluation of IN105 with future development to focus on producing a clinical formulation that can be produced using a tablet press.

This specifzcatiou is divided ilzto sectious with subject for ease of refereuce olzly. Sections aud subject headings are lzot ilztelzded to lilzzit the scope of the ilzvelztiolz.
The enzbodimelzts described lzereiu are for the pulpose of illustratilzg the uzalzy aspects alzd attributes of tlze invelztiolz alzd are lzot ilztelzded to lilzzit tlze scope of the ilzvelztion.

Claims (353)

1. A complex comprising:

(a) an insulin compound conjugate comprising an insulin compound conjugated to a modifying moiety, and (b) a cation, where the insulin compound conjugate is complexed with the cation.
2. The complex of claim 1 where the insulin compound conjugate comprises human insulin.
3. The complex of claim 1 where the insulin compound conjugate comprises an insulin compound analog.
4. The complex of claim 3 where the insulin compound analog is human insulin with Lys28 and Pro29
5. The complex of claim 3 where the insulin compound analog is a native proinsulin compound.
6. The complex of claim 3 where the insulin compound analog is an artificial proinsulin compound.
7. The complex of claim 3 where the insulin compound analog is a miniproinsulin compound comprising a C-peptide having 5 or less amino acids.
8. The complex of claim 3 where the insulin compound analog is a native proinsulin compound having a leader peptide which is cleavable or non-cleavable.
9. The complex of claim 3 where the insulin compound analog is an artificial proinsulin compound having a leader peptide which is cleavable or non-cleavable.
10. The complex of claim 3 where the insulin compound analog is a miniproinsulin compound comprising:

(a) a C-peptide having 5 or less amino acids;

(b) a leader peptide which is cleavable or non-cleavable.
11. The complex of claim 3 where the insulin compound analog is a pre-proinsulin compound comprising proinsulin compound and a leader peptide or carrier protein joined at the C-terminus or N-terminus of the proinsulin compound.
12. The complex of claim 3 where the insulin compound analog is a single chain insulin compound precursor comprising insulin compound A-chain-B-chain, where the A
chain and/or B chain may be truncated at the point of attachment to the other by 1, 2 or 3 amino acids.
13. The complex of claim 3 where the insulin compound analog is an insulin compound comprising a leader peptide or a carrier protein.
14. The complex of claim 1 where the modifying moiety is selected to render the insulin compound conjugate more water soluble than a corresponding unconjugated insulin compound.
15. The complex of claim 1 where the modifying moiety is selected to render the insulin compound conjugate equally or more hydrophilic than a corresponding unconjugated insulin compound.
16. The complex of claim 1 where the modifying moiety is selected to render the insulin compound conjugate more amphiphilic than a corresponding unconjugated insulin compound.
17. The complex of claim 14 where the modifying moiety is selected to render the insulin compound conjugate at least 1.05, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 times more soluble than a corresponding unconjugated insulin compound in an aqueous solution at a pH of about 7.4.
18. The complex of claim 14 where the modifying moiety is selected to render an insulin compound conjugate having a solubility that exceeds about 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, g/L, 15 g/L, 20 g/L, 25 g/L, 50 g/L, 75 g/L, 100 g/L, 125 g/L, or 150 g/L at a pH ranging from about 4 to about 8.
19. The complex of claim 1 where the water solubility of the insulin compound conjugate is decreased by the addition of zinc.
20. The complex of claim 1 where:

(a) the modifying moiety is selected to render the insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin compound;
and (b) the water solubility of the insulin compound conjugate is decreased by the addition of zinc.
21. The complex of claim 1 where:

(a) the modifying moiety is selected to render the insulin compound conjugate equally or more soluble than a corresponding unconjugated insulin compound;

(b) the water solubility of the insulin compound conjugate is decreased by the addition of zinc; and (c) water solubility of the complex is greater than the water solubility of insulin compound.
22. The complex of claim 1 where:

(a) the insulin compound conjugate is an acylated insulin compound;
(b) the cation is zinc; and (c) the water solubility of the insulin compound conjugate is decreased by the addition of the zinc.
23. The complex of claim 1 where:

(a) the insulin compound conjugate is an acylated insulin compound that is equally or more water soluble than a corresponding unconjugated insulin compound;
and (b) the cation is zinc; and (c) the water solubility of the insulin compound conjugate is decreased by the addition of the zinc.
24. The complex of claim 1 where the relative lipophilicity of the insulin compound conjugate relative to the corrsesponding parent insulin compound is 1 or less than 1.
25. The complex of claim 1 where the relative lipophilicity of the insulin compound conjugate as compared to corrsesponding parent insulin compound (k rel) is 1 or less than 1 wherein:

(a) k rel=(t conjugate - t0)/(t human - t0);

(b) relative lipophilicity is measured on an LiChroSorb RP18 (5µm, 250 X 4 mm) high performance liquid chromatography column by isocratic elution at 40° C
(c) the following mixtures are used as eluents:

(i) 0.1M sodium phosphate buffer at pH 7.3 containing 10% acetonitrile, and (ii) 50% acetonitrile in water;

(d) void time (to) is identified by injecting 0.1 mM sodium nitrate;

(e) retention time for human insulin is adjusted to at least 2t0 by varying the ratio between the mixtures of (c)(i) and (c)(ii).
26. The complex of claim 25 wherein the relative lipophilicity is about 1 or less than 1.
27. The complex of claim 25 wherein:

(a) the insulin compound is human insulin, and (b) the relative lipophilicity is less than 1.
28. The complex of claim 25 wherein the relative lipophilicity is less than 0.99, 0.98, 0.97, 0.96, 0.95, 0.94, 0.93, 0.92, 0.91, or 0.90.
29. The complex of claim 25 wherein the modifying moiety is not an alkyl moiety.
30. The complex of claim 25 wherein the modifying moiety is a carbon chain comprising oxy groups inserted therein.
31. The complex of claim 25 wherein the modifying moiety is a carbon chain having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbons, wherein the carbon chain comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 oxy groups inserted therein.
32. The complex of claim 25 wherein the modifying moiety is a carbon chain having 5, 6, 7, 8, 9 or 10 carbons, wherein the carbon chain comprises 2, 3 or 4 oxy groups inserted therein.
33. The complex of claim 25 wherein the modifying moiety comprises 2, 3, 4, 5, 6, 7, 8, 9 or polyalkalene glycol units.
34. The complex of claim 25 wherein the modifying moiety comprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3 polypropylene glycol units.
35. The complex of claim 25 wherein:
(a) the cation is zinc, and (b) aqueous solubility of the insulin compound conjugate is decreased by addition of the zinc.
36. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is less than or equal to the solubility of the insulin compound conjugate.
37. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is greater than the solubility of the insulin compound conjugate.
38. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is greater than 5 times the solubility of the insulin compound conjugate.
39. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from about 1/15 to 5 times the solubility of the insulin compound conjugate.
40. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from about 1/10 to about 2 times the solubility of the insulin compound conjugate.
41. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from about 1/10 to < 0 times the solubility of the insulin compound conjugate.
42. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from greater than about 1/10 the solubility of the insulin compound conjugate at a pH of about 7.4 to substantially less than the solubility of the insulin compound conjugate at a pH of about 7.4.
43. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is from greater than about 1/10 the solubility of the insulin compound conjugate at a pH of about 7.4 to equal to or less than the solubility of the insulin compound conjugate at a pH of about 7.4.
44. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially greater than the solubility of the insulin compound conjugate in solution at a pH of about 7.4.
45. The complex of claim 1 where the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the insulin compound conjugate in solution at a pH of about 7.4.
46. The complex of claim 1 where:

(a) the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the insulin compound conjugate in solution at a pH of about 7.4, and (b) the cation-insulin compound conjugate remains soluble at greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or 130 g/L in aqueous solution across a pH range from about 5 to about 8.5.
47. The complex of claim 1 where:

(a) the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the insulin compound conjugate in solution at a pH of about 7.4, and (b) the cation-insulin compound conjugate remains soluble at greater than about 1 g/L in aqueous solution across a pH range from 5.8 to about 8.5.
48. The complex of claim 1 where:

(a) the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the insulin compound conjugate in solution at a pH of about 7.4, and (b) the cation-insulin compound conjugate remains soluble at greater than about 1, 5, 10 or 20 g/L in aqueous solution across a pH range from 6.5 to about 8.
49. The complex of claim 1 where:

(a) the solubility of the cation-insulin compound conjugate complex at a pH of about 7.4 is substantially less than the solubility of the insulin compound conjugate in solution at a pH of about 7.4, and (b) the cation-insulin compound conjugate remains soluble at greater than about 1, 5, 10 or 20 g/L in aqueous solution across a pH range from 6.9 to about 7.8.
50. The complex of claim 1 where:

(a) the complex is an R-type complex; and (b) at a pH of about 7.4, the aqueous solubility of the complex is from about to about 150 g/L. from about 20 to about 130 g/L, from about 30 to about 110 g/L, or from about 35 to about 60 g/L.
51. The complex of claim 1 where:

(a) the complex is an NPH-type complex; and (b) at a pH of about 7.4, the aqueous solubility of the complex is from about 1 to about 150 g/L, from about 5 to about 120 g/L, or from about 10 to about 90 g/L.
52. The complex of claim 1 where:

(a) the complex is a T-type complex; and (b) at a pH of about 7.4, the aqueous solubility of the complex is from about to about 175 g/L, from about 50 to about 160 g/L, or from about 70 to about 150 g/L.
53. The complex of claim 1 where:

(a) the complex is an R-type Zn complex comprising protamine; and (b) at a pH of about 7.4, the aqueous solubility of the complex is from about to about 110 g/L, from about 20 to about 85 g/L, or from about 30 to about 70 g/L.
54. The complex of claim 1 where:

(a) the complex is a T-type Zn complex comprising protamine; and (b) at a pH of about 7.4, the aqueous solubility of the complex is from about to about 150 g/L, from about 20 to about 130 g/L, from about 30 to about 110 g/L, or from about 35 to about 60 g/L.
55. The complex of claim 1 where:

(a) the insulin compound conjugate is a monoconjugate; and (b) the complex produces crystals in an aqueous solution at pH of about 4 to <

6.5, from about pH of about 4 to < 5.8, from about 4.2 to about 5.5.
56. The complex of claim 1 where:

(a) the insulin compound conjugate is a monoconjugate;
(b) the cation is zinc; and (c) complex produces crystals in an aqueous solution at about 4.4 to about 5.2.
57. The complex of claim 1 where the insulin compound conjugate produces crystals in aqueous solution at a pH which is equal to the pI plus or minus 2.5.
58. The complex of claim 1 where:

(a) the modifying moiety comprises PAG and/or a PAG component coupled to a lipophilic component, where the lipophilic component is selected from the group consisting of alkyls, fatty acids and linear or branched lipids, and (b) either the PAG component or the lipophilic component is coupled to the insulin compound.
59. The complex of claim 58 where the modifying moiety is coupled to one or more amino groups on the insulin compound.
60. The complex of claim 58 where a modifying moiety is coupled at the caboxy terminus of the A-chain and/or a modifying moiety is coupled at the caboxy terminus of the B-chain of the insulin compound.
61. The complex of claim 58 where a modifying moiety is coupled at the N-terminus of a leader peptide which is coupled to the B-chain of the insulin compound.
62. The complex of claim 58 where a modifying moiety is coupled at an amino acid side chain functional group.
63. The complex of claim 58 where:

(a) the insulin compound comprises an amino group at A1, B1, and/or B29; and (b) a single modifying moiety is coupled to a single amino group on the insulin compound at a position selected from the group consisting of A1, B1, and B29.
64. The complex of claim 58 where:

(a) the insulin compound comprises an amino group at B1, and/or B29, (b) a first modifying moiety is coupled to an amino group at B1, and (c) a second modifying moiety is coupled to an amino group at B29.
65. The complex of claim 58 where:

(a) the insulin compound comprises an amino group at B29, and (b) a single modifying moiety is coupled to an amino group at B29.
66. The complex of claim 58 where a modifying moiety is coupled at a free amino group in the insulin compound to form a carbamate bond.
67. The complex of claim 58 where:

(a) the insulin compounds comprises a free hydroxyl group, and (b) a modifying moiety is coupled to the free hydroxyl group to form a carboxyllic, ether, or carbonate bond.
68. The complex of claim 58 where:

(a) the insulin compounds comprises a free -SH group, and (b) a modifying moiety is coupled to the -SH group to form a thioester, thioether, or sulfonate bond.
69. The complex of claim 1 where:

(a) the conjugated insulin compound molecule is acylated to the modifying moiety; and (b) the modifying moiety does not consist of a fatty acid, an alkyl having more than 2 carbons, or an amino acid.
70. The complex of claim 1 where:

(a) the conjugated insulin compound molecule is acylated to the modifying moiety; and (b) the modifying moiety does not does not comprise a fatty acid, an alkyl having more than 2 or 3 carbons, or an amino acid.
71. The complex of claim 1 where the cation component comprises a monovalent or multivalent metal cation.
72. The complex of claim 1 where the cation component comprises a monovalent metal cation selected from Na+, Li+, and K+.
73. The complex of claim 1 where the cation component comprises a divalent metal cation selected from the group consisting of Zn++, Mn++, Ca++, Fe++, Ni++, Cu++, Co++
and Mg++.
74. The complex of claim 1 where the cation component comprises Zn++.
75. The complex of claim 1 where the cation component is greater than about 90, 95, 99, or 99.9% Zn++.
76. The complex of claim 1 where molar ratio of insulin compound conjugate to cation component is between about 1:1 and about 1:100, about 1:2 and about 1:12, or about 1:2 and about 1:7.
77. The complex of claim 1 where resistance of the complexed insulin compound conjugate to chymotrypsin degradation is greater than the chymotrypsin degradation of the corresponding uncomplexed insulin compound conjugate.
78. The complex of claim 1 where resistance of the complexed insulin compound conjugate to chymotrypsin degradation is greater than the chymotrypsin degradation of the corresponding complexed but unconjugated insulin compound.
79. The complex of claim 1 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

80. The complex of claim 1 where the insulin compound conjugate consists of human insulin coupled at B29 to a modifying moiety having a structure:

81. The complex of claim 1 where the insulin compound conjugate consists of human insulin coupled at A1 and B29 to a modifying moiety having a structure:

82. The complex of claim 1 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

83. The complex of claim 1 where the modifying moiety has a formula:

where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
84. The complex of claim 1 where the modifying moiety has a formula:

where PAG is a PAG moiety having m subunits and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
85. The complex of claim 1 where the modifying moiety has a structure:

86. The complex of claim 1 where the modifying moiety has a structure:

87. The complex of claim 1 where the modifying moiety has a structure:

88. The complex of claim 1 where the modifying moiety has a structure:

89. The complex of claim 1 where the modifying moiety has a structure:

90. The complex of claim 1 where the modifying moiety has a structure:

91. The complex of claim 1 where the modifying moiety has a structure:

92. The complex of claim 1 where the modifying moiety has a structure:

93. The complex of claim 1 where the modifying moiety has a structure:

94. The complex of claim 1 where the modifying moiety has a structure:

95. The complex of claim 1 where the modifying moiety has a structure:

96. The complex of claim 1 where the modifying moiety has a structure:

97. The complex of claim 1 where the modifying moiety has a structure:

98. The complex of claim 1 where the modifying moiety has a structure:

99. The complex of claim 1 where the modifying moiety has a structure:

100. The complex of claim 1 where the modifying moiety has a structure:

101. The complex of claim 1 where the insulin compound conjugate is coupled at A1, B1 and/or B29 to a linear or branched PEG moiety having from 2 to 20 PEG
subunits, from 2 to 15 PEG subunits, or from 2 to 10 PEG subunits.
102. A complex mixture comprising the complex of claim 1.
103. A solid mixture comprising the complex of claim 1.
104. A hybrid complex comprising the complex of claim 1.
105. A co-crystal comprising the complex of claim 1.
106. A composition comprising the complex of claim 1 wherein the composition is provided as a lyophilized powder.
107. The complex of claim 1 in a mixture having a pharmaceutically acceptable level of purity.
108. A composition comprising:

(a) the complex of claim 1; and (b) a second insulin compound conjugate or unconjugated insulin compound.
109. The composition of claim 108 where the complex of 108(a) and/or 108(b) is provided as a solid.
110. The composition of claim 108 where:

(a) one of the complexes of 108(a) and 108(b) is provided as a crystalline solid;
and (b) the other is provided as an amorphous solid.
111. The composition of claim 108 where the complex of 108(a) and/or 108(b) is provided as a crystalline solid.
112. The composition of claim 108 further comprising a third complex comprising:

(a) an insulin compound conjugate comprising an insulin compound coupled to a third modifying moiety, and (b) a cation component, where the insulin compound conjugate is complexed with the cation.
113. The composition of claim 108 where the insulin compound conjugates of the complexes of 108(a) and 108(b) comprise different insulin compounds.
114. The composition of claim 108 where the complexes of 108(a) and 108(b) comprise different insulin compound conjugates.
115. The composition of claim 108 where:

(a) the complexes of 108(a) and 108(b) comprise different insulin compound conjugates, and (b) the different insulin compound conjugates have different solubilities.
116. The composition of claim 108 where:

(a) the complexes of 108(a) and 108(b) comprise different insulin compound conjugates, and (b) one of the complexes comprises a lipophilic insulin compound conjugate and the other of the complexes comprises a hydrophilic insulin compound conjugate.
117. The composition of claim 108 where:

(a) the complexes of 108(a) and 108(b) comprise different insulin compound conjugates, and (b) one of the complexes has a circulation half-life of from about 1 to about hours; and (c) the other of the complexes has a circulation half-life that is greater than the circulation half-life of the first complex.
118. The composition of claim 108 where:

(a) the complexes of 108(a) and 108(b) comprise different insulin compound conjugates, and (b) one of the complexes has a rapid-acting profile; and (c) the other of the complexes has a medium-to-long acting profile.
119. The composition of claim 108 where:

(a) the complexes of 108(a) and 108(b) comprise different insulin compound conjugates, and (b) one of the complexes has profile suitable for basal insulin compound control;
and (c) the other of the complexes has a profile suitable for post-prandial glucose control.
120. The composition of claim 108 where the composition is a crystal exhibiting one or more of the following characteristics: substantially homogenous dissolution, a single in vivo dissolution curve, and/or a single peak pharmacodynamic profile.
121. The composition of claim 108 where the composition is a co-crystal of HIM2 and insulin compound.
122. The composition of claim 108 where the composition is a co-crystal of HIM2 and IN105.
123. The composition of claim 108 where the composition is a co-crystal of IN105 and insulin compound.
124. The composition of claim 108 where the second active ingredient is insulin compound, and wherein co-crystallization with insulin compound reduces the solubility of the crystal relative to the solubility of a crystal of the insulin compound conjugate.
125. The composition of claim 108 where the second active ingredient is insulin compound, and wherein co-crystallization with insulin compound increases the solubility of the crystal relative to the solubility of a crystal of the insulin compound conjugate.
126. The composition of claim 108 where co-crystallization the insulin compound conjugate and the second active ingredient are selected to achieve a co-crystal having a desired therapeutic activity.
127. The composition of claim 108 where the composition is a co-crystal comprising:
(a) a rapid acting, rapid clearing, or highly potent insulin compound conjugate, (b) a long-acting, slow clearing, or poorly potent insulin compound conjugate.
128. The composition of claim 108 where the composition is a co-crystal has a PK/PD
profile suitable for post-prandial glucose control.
129. The composition of claim 108 where the composition is a co-crystal has a PK/PD
profile suitable for overnight basal insulin compound control.
130. A composition comprising:

(a) the complex of claim 1; and (b) an unconjugated insulin compound.
131. The composition of claim 130 where the insulin compound is human insulin or lys-pro insulin compound.
132. The complex of claim 1 comprising two different insulin compound conjugates.
133. The complex of claim 1 comprising an insulin compound conjugate and an unconjugated insulin compound.
134. The complex of claim 1 comprising two different insulin compound conjugates comprising two different insulin compounds.
135. The complex of claim 1 comprising two different insulin compound conjugates.
136. The complex of claim 1 comprising an insulin compound conjugate and an unconjugated insulin compound.
137. The complex of claim 1 provided as a component of a hybrid co-crystal of two different insulin compound conjugates.
138. The complex of claim 1 provided as a component of a hybrid co-crystal of three different insulin compound conjugates.
139. The complex of claim 1 provided as a component of a hybrid co-crystal of a different insulin compound conjugates.
140. The complex of claim 1 comprising at least two different insulin compound conjugates.
141. The complex of claim 1 comprising a co-crystal of at least two different insulin compound conjugates.
142. The complex of claim 1 comprising one insulin compound conjugate and further comprising an unconjugated insulin compound.
143. The complex of claim 1 comprising comprising the insulin compound conjugate in a complex together with an unconjugated insulin compound.
144. The complex of claim 1 provided as a component of a co-crystal comprising two different hydrophilic insulin compound conjugates.
145. The complex of claim 1 provided as a component of a co-crystal comprising a hydrophilic insulin compound conjugate and a lipophilic insulin compound conjugate.
146. The complex of claim 1 provided as a component of a co-crystal comprising a hydrophilic insulin compound conjugate and an unconjugated insulin compound.
147. The complex of claim 1 provided as a component of a co-crystal comprising crystallized together with an unconjugated insulin compound.
148. The complex of claim 1 provided as a component of a co-crystal comprising crystallized together with an unconjugated insulin compound.
149. The complex of claim 1 provided as a component of a co-crystal comprising crystallized together with IN105.
150. The complex of claim 1 provided as a component of a co-crystal comprising crystallized together with insulin compound and IN105.
151. A co-crystal comprising the complex of claim 1 and having a extended profile.
152. The complex of claim 1 further comprising a complexing agent.
153. The complex of claim 152 where the complexing agent is selected from the group consisting of protamines, surfen, globin proteins, spermine, spermidine albumin, amino acids, carboxyllic acids, polycationic polymer compounds, cationic polypeptides, polylysine, anionic polypeptides, nucleotides, and antisense.
154. The complex of claim 1 substantially devoid of protamine.
155. The complex of claim 1 in the form of a solid.
156. A composition comprising the complex of claim 155 in the form of a crystalline solid.
157. A composition comprising the complex of claim 155 in the form of rod shaped crystal.
158. A composition comprising the complex of claim 155 in the form of a crystal having irregular morphology.
159. A composition comprising the complex of claim 155 in the form of a crystals having an averege diameter ranging from about 0.5 to about 40 µm.
160. A composition comprising the complex of claim 155 in the form of crystals having a morphology as shown in Figure 1, 2, 3, 4 or 5.
161. A composition comprising the complex of claim 155 in the form of a dry solid.
162. A composition comprising the complex of claim 155 in the form of a mixture of amorphous and crystalline solids.
163. A composition comprising the complex of claim 155 in the form of a substantially pure powder.
164. A composition comprising the complex of claim 155 in the form of a powder comprising a cation-insulin compound conjugate solid and one or more additional pharmaceutically acceptable components.
165. A composition comprising the complex of claim 155 as an undissolved component of a liquid.
166. A composition comprising the complex of claim 155 as a precipitate in a liquid.
167. A composition comprising the complex of claim 135 as a component of a suspension, emulsion or microemulsion.
168. The complex of claim 155 where in a dried state, the solid comprises:
(a) greater than about 96% w/w insulin compound conjugate, and (b) from about 0.05 to about 4% w/w Zn++.
169. The complex of claim 155 where in a dried state, the solid comprises:
(a) greater than about 91% w/w insulin compound conjugate, (b) from about 0.3 to about 4 % w/w Zn++, and (c) from about 0.2 to about 5% w/w phenol.
170. The complex of claim 155 where in a dried state, the solid comprises:
(a) greater than about 82% w/w insulin compound conjugate, (b) from about 0.5 to about 4% w/w Zn+, and (c) from about 0.2 to about 14 % w/w protamine.
171. The complex of claim 155 where in a dried state, the solid comprises:
(a) greater than about 71% w/w insulin compound conjugate, (b) from about 0.5 to about 4% w/w Zn++, (c) from about 0.2 to about 14% w/w protamine, and (d) from about 0.2 to about 15% w/w phenol.
172. The complex of claim 155 where in a dried state, the solid comprises:
(a) from about 0.1 to about 2% w/w Zn++, and (b) from about 0.08 to about 1% w/w phenol.
173. The complex of claim 155 where in a dried state, the solid comprises:
(a) from about 0.5 to about 1.3% w/w Zn++, (b) from about 0.1 to about 0.7% w/w phenol.
174. The complex of claim 155 where in a dried state, the solid comprises:
(a) from greater than or equal to 1 to about 3.5% w/w Zn++, and (b) from about 0.1 to about 3% w/w phenol.
175. The complex of claim 155 where in a dried state, the solid comprises:
(a) from greater than or equal to 1.3 to about 2.2% w/w Zn++, and (b) from about 0.4 to about 2% w/w phenol.
176. The complex of claim 155 where the solid is greater than about 90, 95, or 99%
crystalline.
177. The complex of claim 155 where the solid is greater than about 90, 95, or 99%
amorphous.
178. The complex of claim 155 where the solid comprises a mixture of amorphous solids and crystalline solids.
179. The complex of claim 155 where the solid comprises conjugated and unconjugated insulin compounds.
180. A pharmaceutical composition comprising the complex of claim 155.
181. The pharmaceutical composition of claim 180 where the complex comprises a solid present in a liquid.
182. The pharmaceutical composition of claim 181 where the solid is present in a buffer.
183. The pharmaceutical composition of claim 180 further comprising a stabilizing agent.
184. The pharmaceutical composition of claim 183 where the stabilizing agent is a phenolic compound.
185. The pharmaceutical composition of claim 183 where the stabilizing agent is selected from the group consisting of phenol, m-cresol and paraben.
186. A method of making a cation-insulin compound conjugate complex, the method comprising combining in an aqueous solution an insulin compound conjugate and a cation.
187. The method of claim 186 where the aqueous solution comprises an organic solvent dissolved therein.
188. A method of treatment comprising administering to a subject in need thereof a pharmaceutically acceptable amount of a complex of claim 1.
189. The method of claim 188 where the subject is a diabetic.
190. The method of claim 188 where the composition is administered by a route selected from the group consisting of oral, oral ingestion, parenteral, buccal, sublingual, nasal, inhalation and transdermal.
191. The method of claim 188 where the composition is administered as a component of a pharmaceutical composition is formulated for administration by a route selected from the group consisting of oral, oral ingestion, parenteral, buccal, sublingual, nasal, inhalation and transdermal.
192. A pharmaceutical composition comprising the composition of claim 1 and a carrier.
193. The pharmaceutical composition of claim 192 formulated for administration by a route selected from the group consisting of oral, oral ingestion, parenteral, buccal, sublingual, nasal, inhalation and transdermal.
194. The pharmaceutical composition of claim 192 formulated in a form selected from the group consisting of solid dosage form, suspension, and film.
195. An insulin compound coupled to a modifying moiety having a formula:
-X-R1-Y-PAG-Z-R2 (Formula VI) where, X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond, either R1 or R2 is is a lower alkyl, optionally including a carbonyl group, and when R1 is a lower alkyl, R2 is a capping group, and PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties, and optionally incorporating one or more additional moieties selected from the group consisting of -S-, -O-, -N-, and -C(O)-, and where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavy atoms.
196. A polypeptide conjugate comprising a polypeptide compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
197. An insulin compound conjugate comprising an insulin compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
198. The insulin compound conjugate of claim 197 where the backbone comprises one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, -S-, -N-, and =O.
199. The insulin compound conjugate of claim 197 where the carbon chain moiety does not include any ring structures.
200. The insulin compound conjugate of claim 197 where the carbon chain moiety does not includes a single branch.
201. The insulin compound conjugate of claim 197 where the carbon chain moiety is not an alkyl.
202. The insulin compound conjugate of claim 197 where the backbone includes one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, N-, and =O.
203. The insulin compound conjugate of claim 197 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and 2, 3, 4 or 5-O- moities coupled between carbon atoms.
204. The insulin compound conjugate of claim 197 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O- and =O.
205. The insulin compound conjugate of claim 198 where the carbon atoms and non-carbon heavy atoms are present in a ratio of at least 1 carbon atom for every non-carbon heavy atom.
206. The insulin compound conjugate of claim 198 where the carbon atoms and non-carbon heavy atoms are present in a ratio of at least 2 carbon atoms for every non-carbon heavy atom.
207. The insulin compound conjugate of claim 198 where the carbon atoms and non-carbon heavy atoms are present in a ratio of at least 3 carbon atoms for every non-carbon heavy atom.
208. The insulin compound conjugate of claim 198 comprising carbon atoms and oxygen atoms in a ratio of at least 1 carbon atom for every oxygen atom.
209. The insulin compound conjugate of claim 198 comprising carbon atoms and oxygen atoms in a ratio of at least 2 carbon atoms for every oxygen atom.
210. The insulin compound conjugate of claim 198 comprising carbon atoms and oxygen atoms in a ratio of at least 3 carbon atoms for every oxygen atom.
211. The insulin compound conjugate of claim 198 including an -C(O)O- group in which the O- is coupled to the insulin compound and the -C is coupled to a linear carbon chain having 3, 4, 5, 6, 7, 8, or 9 carbon atoms and 2, 3, or 4 oxygen atoms in the form of oxy groups.
212. The insulin compound conjugate of claim 197 where modifying moiety comprises one or more capping groups.
213. The insulin compound conjugate of claim 197 where modifying moiety comprises one or more capping groups selected from the group consisting of branched or linear C1-6, and a carbonyl.
214. The insulin compound conjugate of claim 197 wherein the modifying moiety comprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3 polypropylene glycol units.
215. The insulin compound conjugate of claim 197 modifying moiety is selected to render the insulin compound conjugate equally as soluble or more soluble than a corresponding unconjugated insulin compound.
216. The insulin compound conjugate of claim 197 wherein the water solubility of the insulin compound conjugate is decreased by addition of the zinc.
217. An insulin compound coupled to a modifying moiety having a formula:

where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5.
218. The insulin compound of claim 217 where the modifying moiety is:
219. The insulin compound of claim 217 where the modifying moiety is:

220. The insulin compound of claim 217 where the modifying moiety is:
221. The insulin compound of claim 217 where the modifying moiety is:
222. The insulin compound of claim 217 where the modifying moiety is coupled at:
(a) B29 of human insulin;

(b) B1 of human insulin; or (c) B1 and B29 of human insulin.
223. An insulin compound coupled to a modifying moiety having a formula:

where n is 1, 2, 3, 4 or 5, and m is 1, 2, 3 or 4.
224. The insulin compound of claim 223 where the modifying moiety is:
225. The insulin compound of claim 223 where the modifying moiety is:

226. The insulin compound of claim 223 where the modifying moiety is coupled at B29 of human insulin.
227. The insulin compound of claim 223 where the modifying moiety is coupled at:
(a) B1 of human insulin; or (b) B1 and B29 of human insulin.
228. A method of treating an insulin deficiency in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an insulin compound of claim 195.
229. The method of claim 228 wherein the insulin deficiency has a cause comprising:
(a) diabetes;

(b) pre-diabetes; or (c) metabolic syndrome.
230. The method of claim 228 wherein the insulin compound conjugate is administered to effect post-prandial glucose control.
231. The method of claim 228 wherein the insulin insulin compound is admistered within one hour prior to a meal.
232. The method of claim 228 wherein the insulin compound conjugate is administered to effect overnight glucose control.
233. A modifying moiety having a formula:
where n is 1, 2, 3 or 4, and m is 1, 2, 3, 4 or 5.
234. The modifying claim 233 having a formula:
235. The modifying claim 233 having a formula:

236. The modifying claim 233 having a formula:
237. The modifying claim 233 having a formula:
238. A modifying moiety having a formula:

where n is 1, 2, 3, 4 or 5, and m is 1, 2, 3 or 4.
239. The modifying moiety of claim 238 having a formula:
240. The modifying moiety of claim 238 having a formula:

241. An activated form of the modifying moiety of claim 233.
242. A method of conjugating an insulin compound, the method comprising activating a modifying moiety of claim 233 and coupling the activated modifying moiety to the insulin compound.
243. A solid pharmaceutical composition formulated for oral administration by ingestion, comprising:

(a) from about 0.1 to about 75% w/w fatty acid component, where the fatty acid component comprises saturated or unsaturated C4, C5, C6, C7, C8, C9, C10, C11, or C12 fatty acids and/or salts of such fatty acids;

(b) a therapeutic agent.
244. The pharmaceutical composition of claim 243 comprising from about 5 to about 65%
w/w fatty acid component.
245. The pharmaceutical composition of claim 243 comprising from about 40 to about 60% w/w fatty acid component.
246. The pharmaceutical composition of claim 243 where the therapeutic agent is a protein or peptide.
247. The pharmaceutical composition of claim 243 where the therapeutic agent is a protein or peptide having from 2 to 200 amino acids, from 10 to 65 amino acids, or from 25 to 55 amino acids.
248. The pharmaceutical composition of claim 243 where the therapeutic agent is a selected from the group consisting of insulin compounds, calcitonins, interferons, growth hormones, growth factors, GLP-1, PTH, somatostatins, LCRF, natriuretic peptides, BNP, ANP, exenatide, erythropoetins having less than 200 amino acids, antibody fragments, vaccines, and analogs of any of the foregoing.
249. The pharmaceutical composition of claim 243 where the therapeutic agent is a conjugated protein or peptide, an acylated protein or peptide, or a PEGylated protein or peptide.
250. The pharmaceutical composition of claim 243 where the therapeutic agent is a conjugated insulin compounds, calcitonins, interferons, growth hormones, growth factors, GLP-1, PTH, somatostatins, LCRF, natriuretic peptides, BNP, ANP, exenatide, erythropoetins having less than 200 amino acids, antibody fragments, vaccines, and analogs of any of the foregoing.
251. The pharmaceutical composition of claim 243 where the therapeutic agent is an insulin compound.
252. The pharmaceutical composition of claim 243 where the therapeutic agent is a complexed protein or peptide or a complexed conjugated protein or peptide.
253. The pharmaceutical composition of claim 243 where the therapeutic agent is a complexed insulin compound or a complexed conjugated insulin compound.
254. The pharmaceutical composition of claim 243 where the ionized or non-ionized fatty acid content of the composition is greater than about 90% w/w a single fatty acid, about 95%
w/w a single fatty acid, or about 99% w/w a single fatty acid.
255. The pharmaceutical composition of claim 243 prepared to survive passage through the stomach without substantial loss of the therapeutic agent and to substantially completely dissolve in the intestine for delivery of the therapeutic agent.
256. The pharmaceutical composition of claim 243 prepared to survive passage through the stomach without substantial loss of the therapeutic agent and to substantially completely dissolve in the intestine for delivery of the active ingredient, where the composition is substantially devoid of enteric coating.
257. The pharmaceutical composition of claim 243 further comprising an enteric coating.
258. The pharmaceutical composition of claim 243 lacking or substantially lacking an enteric coating.
259. The pharmaceutical composition of claim 243 comprising a diluent selected from the group consisting of binders, disintegrants, fillers, diluents, lubricants, glidants, flow enhancers, compression aids, colors, sweeteners, preservatives, suspensing agents, dispersing agents, film formers, coatings, flavors, printing inks, celluloses, sugars, mannitols, lactoses, dissolution enhancers, and crosscarmaloses.
260. The pharmaceutical composition of claim 243 prepared in a form selected from the group consisting of tablets, minitabs, powders, hard gelatin capsules, or soft gelatin capsules.
261. The pharmaceutical composition of claim 243, wherein the fatty acid component is capric acid and/or lauric acid and/or salts of capric acid and/or lauric acid.
262. The pharmaceutical composition of claim 243 comprising a buffer salt selected from the group consisting of phosphate buffer, sudium phosphate, tris buffer, citric acid buffer, and ethanolamine buffer.
263. The pharmaceutical composition of claim 243 comprising a buffer salt selected to achieve a buffering capacity at the site of absorbtion to maintain a local pH
of from about 4.8 to about 9.5, or from about 5 to about 8.
264. The pharmaceutical composition of claim 243 provided as a capsule comprising a semi-solid fill.
265. The pharmaceutical composition of claim 243 comprising an insulin compound conjugate comprising an insulin compound coupled to a modifying moiety.
266. The pharmaceutical composition of claim 243 comprising an insulin compound coupled to a modifying moiety having a formula:

-X-R1-Y-PAG-Z-R2 (Formula VI) where, X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond, either R1 or R2 is is a lower alkyl, optionally including a carbonyl group, and when R1 is a lower alkyl, R2 is a capping group, and PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties, and optionally incorporating one or more additional moieties selected from the group consisting of -S-, -O-, -N-, and -C(O)-, and where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavy atoms.
267. The pharmaceutical composition of claim 243 comprising a polypeptide conjugate comprising a polypeptide compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
268. The pharmaceutical composition of claim 243 comprising an insulin compound conjugate comprising an insulin compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
269. The pharmaceutical composition of claim 268 comprising where the backbone comprises one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, -S-, -N-, and =O.
270. The pharmaceutical composition of claim 234 where the carbon chain moiety does not include any ring structures.
271. The pharmaceutical composition of claim 234 where the carbon chain moiety is not branched.
272. The pharmaceutical composition of claim 234 where the carbon chain moiety is not an alkyl.
273. The pharmaceutical composition of claim 234 where the backbone includes one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, N-, and =O.
274. The pharmaceutical composition of claim 234 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and 2, 3, 4 or 5-O- moities coupled between carbon atoms.
275. The pharmaceutical composition of claim 234 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O- and O.
276. The pharmaceutical composition of claim 234 where the carbon atoms and non-carbon heavy atoms are present in a ratio of at least 1, 2, 3 or 4 carbon atom for every non-carbon heavy atom.
277. The pharmaceutical composition of claim 234 comprising carbon atoms and oxygen atoms in a ratio of at least 1, 2, 3 or 4 carbon atom for every oxygen atom.
278. The pharmaceutical composition of claim 234 including an -C(O)O- group in which the O- is coupled to the insulin compound and the -C is coupled to a linear carbon chain having 3, 4, 5, 6, 7, 8, or 9 carbon atoms and 2, 3, or 4 oxygen atoms in the form of oxy groups.
279. The pharmaceutical composition of claim 265 where modifying moiety comprises one or more capping groups.
280. The pharmaceutical composition of claim 265 where modifying moiety comprises one or more capping groups selected from the group consisting of branched or linear C1-6, and carbonyl.
281. The pharmaceutical composition of claim 234 wherein the modifying moiety comprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3 polypropylene glycol units.
282. The pharmaceutical composition of claim 234 modifying moiety is selected to render the insulin compound conjugate equally as soluble or more soluble than a corresponding unconjugated insulin compound.
283. The pharmaceutical composition of claim 234 wherein the water solubility of the insulin compound conjugate is decreased by addition of the zinc.
284. The pharmaceutical composition of claim 234 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

285. The pharmaceutical composition of claim 234 where the insulin compound conjugate consists of human insulin coupled at B29 to a modifying moiety having a structure:

286. The pharmaceutical composition of claim 234 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

287. The pharmaceutical composition of claim 234 where the modifying moiety has a formula:

where PAG is a PAG moiety having m subunits and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
288. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

289. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

290. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

291. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

292. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

293. The pharmaceutical composition of claim 234 where the modifying moiety has a structure:

294. The pharmaceutical composition of claim 243 where the insulin compound conjugate is coupled at A1, B1 and/or B29 to a linear or branched PEG moiety having from 2 to 15 PEG
or from 2 to 10 PEG subunits.
295. The pharmaceutical composition of claim 243 having a pharmaceutically acceptable level of purity.
296. A liquid pharmaceutical composition formulated for oral administration by ingestion, comprising:

(a) from about 0.1 to about 10% w/v fatty acid component, where the fatty acid component comprises saturated or unsaturated C4, C5, C6, C7, C8, C93 C10, C11, or C12 fatty acids and/or salts of such fatty acids;

(b) a therapeutic agent.
297. The pharmaceutical composition of claim 296 comprising from about .25 to about 6%
w/v fatty acid component or from about 1.5 to about 3.5% w/v fatty acid component.
298. The pharmaceutical composition of claim 296 where the therapeutic agent is a protein or peptide.
299. The pharmaceutical composition of claim 296 where the therapeutic agent is a protein or peptide having from 2 to 200 amino acids, from 10 to 65 amino acids, or 25 to 55 amino acids.
300. The pharmaceutical composition of claim 296 where the therapeutic agent is a selected from the group consisting of insulin compounds, calcitonins, interferons, growth hormones, growth factors, GLP-1, PTH, somatostatins, LCRF, natriuretic peptides, BNP, ANP, exenatide, erythropoetins having less than 200 amino acids, antibody fragments, vaccines, and analogs of any of the foregoing.
301. The pharmaceutical composition of claim 296 where the therapeutic agent is a conjugated protein or peptide, an acylated protein or peptide, or a PEGylated protein or peptide.
302. The pharmaceutical composition of claim 296 where the therapeutic agent is a conjugated insulin compounds, calcitonins, interferons, growth hormones, growth factors, GLP-1, PTH, somatostatins, LCRF, natriuretic peptides, BNP, ANP, exenatide, erythropoetins having less than 200 amino acids, antibody fragments, vaccines, and analogs of any of the foregoing.
303. The pharmaceutical composition of claim 296 where the therapeutic agent is an insulin compound.
304. The pharmaceutical composition of claim 296 where the therapeutic agent is a complexed protein or peptide or is a complexed conjugated protein or peptide.
305. The pharmaceutical composition of claim 296 where the therapeutic agent is a complexed insulin compound or a complexed conjugated insulin compound.
306. The pharmaceutical composition of claim 296 where the ionized or non-ionized fatty acid content of the composition is greater than about 90% w/w a single fatty acid, greater than about 95% w/w a single fatty acid, or greater than about 99% w/w a single fatty acid.
307. The pharmaceutical composition of claim 296 prepared in a form selected from the group consisting of semisolids, suspensions, microemulsions, and emulsions.
308. The pharmaceutical composition of claim 296, wherein the fatty acid component is capric acid and/or lauric acid and/or salts of capric acid and/or lauric acid.
309. The pharmaceutical composition of claim 296 comprising a buffer salt selected from the group consisting of phosphate buffer, sudium phosphate, tris buffer, citric acid buffer, ethanolamine buffer, and triethylamine buffer.
310. The pharmaceutical composition of claim 296 comprising a buffer salt in an amount calculated to achieve a buffering capacity at the site of absorbtion to maintain a local pH of from about 4.8 to about 9.5 or about 5 to about 8.
311. The pharmaceutical composition of claim 296 comprising an insulin compound coupled to a modifying moiety.
312. The pharmaceutical composition of claim 311 wherein the modifying moiety has a formula:

-X-R1-Y-PAG-Z-R2 (Formula VI) where, X, Y and Z are independently selected linking groups and each is optionally present, and X, when present, is coupled to the insulin compound by a covalent bond, either R1 or R2 is is a lower alkyl, optionally including a carbonyl group, and when R1 is a lower alkyl, R2 is a capping group, and PAG is a linear or branched carbon chain incorporating one or more alkalene glycol moieties, and optionally incorporating one or more additional moieties selected from the group consisting of -S-, -O-, -N-, and -C(O)-, and where the modifying moiety has a maximum number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 heavy atoms.
313. The pharmaceutical composition of claim 296 wherein the therapeutic agent comprises a polypeptide conjugate comprising a polypeptide compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
314. The pharmaceutical composition of claim 296 wherein the therapeutic agent comprises an insulin compound conjugate comprising an insulin compound coupled to a linear or branched, substituted carbon chain moiety having a backbone of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24 or 25 atoms selected from the group consisting of -C, -C-, -O-, =O, -S-, -N-, -Si-.
315. The pharmaceutical composition of claim 314 where the backbone comprises one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, -S-, -N-, and =O.
316. The pharmaceutical composition of claim 296 where the carbon chain moiety does not include any ring structures.
317. The pharmaceutical composition of claim 296 where the carbon chain moiety is not branched.
318. The pharmaceutical composition of claim 296 where the carbon chain moiety is not an alkyl.
319. The pharmaceutical composition of claim 234 where the backbone includes one or more carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O-, N-, and =O.
320. The pharmaceutical composition of claim 234 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and 2, 3, 4 or 5 -O- moities coupled between carbon atoms.
321. The pharmaceutical composition of claim 234 where the backbone includes 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms and one or more non-carbon heavy atoms selected from the group consisting of -O- and =O.
322. The pharmaceutical composition of claim 234 where the carbon atoms and non-carbon heavy atoms are present in a ratio of at least 1, 2, 3 or 4 carbon atom for every non-carbon heavy atom.
323. The pharmaceutical composition of claim 234 comprising carbon atoms and oxygen atoms in a ratio of at least 1, 2, 3 or 4 carbon atom for every oxygen atom.
324. The pharmaceutical composition of claim 234 including an -C(O)O- group in which the O- is coupled to the insulin compound and the -C is coupled to a linear carbon chain having 3, 4, 5, 6, 7, 8, or 9 carbon atoms and 2, 3, or 4 oxygen atoms in the form of oxy groups.
325. The pharmaceutical composition of claim 234 where modifying moiety comprises one or more capping groups.
326. The pharmaceutical composition of claim 234 where modifying moiety comprises one or more capping groups selected from the group consisting of branched or linear C1-6, and a carbonyl.
327. The pharmaceutical composition of claim 234 wherein the modifying moiety comprises 1, 2 or 3 polyethylene glycol units and 1, 2 or 3 polypropylene glycol units.
328. The pharmaceutical composition of claim 234 modifying moiety is selected to render the insulin compound conjugate equally as soluble or more soluble than a corresponding unconjugated insulin compound.
329. The pharmaceutical composition of claim 234 wherein the water solubility of the insulin compound conjugate is decreased by addition of the zinc.
330. The pharmaceutical composition of claim 311 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

331. The pharmaceutical composition of claim 311 where the insulin compound conjugate consists of human insulin coupled at B29 to a modifying moiety having a structure:

332. The pharmaceutical composition of claim 234 where the insulin compound conjugate consists of human insulin coupled at A1, B1 and/or B29 to a modifying moiety having a structure:

333. The pharmaceutical composition of claim 311 where the modifying moiety has a formula:

where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
334. The pharmaceutical composition of claim 311 where the modifying moiety has a formula:

where PAG is a PAG moiety having m subunits and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
335. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

336. The complex of claim 311 where the modifying moiety has a structure:

337. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

338. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

339. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

340. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

341. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

342. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

343. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

344. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

345. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

346. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

347. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

348. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

349. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

350. The pharmaceutical composition of claim 311 where the modifying moiety has a structure:

351. The pharmaceutical composition of claim 311 where the insulin compound conjugate is coupled at A1, B1 and/or B29 to a linear or branched PEG moiety having from 2 to 20 PEG
subunits.
352. The pharmaceutical composition of claim 311 where the insulin compound conjugate is coupled at A1, B1 and/or B29 to a linear or branched PEG moiety having from 2 to 15 PEG
subunits.
353. The pharmaceutical composition of claim 311 where the insulin compound conjugate is coupled at A1, B1 and/or B29 to a linear or branched PEG moiety having from 2 to 10 PEG
subunits.
CA2580313A 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof Expired - Fee Related CA2580313C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CA2910494A CA2910494C (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof

Applications Claiming Priority (11)

Application Number Priority Date Filing Date Title
US58905804P 2004-07-19 2004-07-19
US60/589,058 2004-07-19
US61915304P 2004-10-15 2004-10-15
US60/619,153 2004-10-15
US63257804P 2004-12-02 2004-12-02
US60/632,578 2004-12-02
US65583805P 2005-02-24 2005-02-24
US65580305P 2005-02-24 2005-02-24
US60/655,838 2005-02-24
US60/655,803 2005-02-24
PCT/US2005/025644 WO2006014673A2 (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CA2910494A Division CA2910494C (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof

Publications (2)

Publication Number Publication Date
CA2580313A1 true CA2580313A1 (en) 2006-02-09
CA2580313C CA2580313C (en) 2016-03-15

Family

ID=35787693

Family Applications (2)

Application Number Title Priority Date Filing Date
CA2910494A Expired - Fee Related CA2910494C (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof
CA2580313A Expired - Fee Related CA2580313C (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CA2910494A Expired - Fee Related CA2910494C (en) 2004-07-19 2005-07-19 Insulin-oligomer conjugates, formulations and uses thereof

Country Status (21)

Country Link
US (6) US7605123B2 (en)
EP (2) EP2626368B1 (en)
JP (3) JP5220410B2 (en)
KR (2) KR101276754B1 (en)
CN (3) CN105801686B (en)
AU (1) AU2005269753B2 (en)
BR (1) BRPI0513508B1 (en)
CA (2) CA2910494C (en)
DK (2) DK1773878T3 (en)
ES (2) ES2618028T3 (en)
HK (1) HK1187826A1 (en)
IL (3) IL180762A (en)
MX (1) MX2007000883A (en)
NO (2) NO345613B1 (en)
NZ (1) NZ553263A (en)
PL (2) PL1773878T3 (en)
PT (2) PT1773878E (en)
RU (2) RU2527893C2 (en)
UA (2) UA103758C2 (en)
WO (1) WO2006014673A2 (en)
ZA (1) ZA200701446B (en)

Families Citing this family (106)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7658938B2 (en) 1999-02-22 2010-02-09 Merrion Reasearch III Limited Solid oral dosage form containing an enhancer
US7060675B2 (en) * 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
JP2005524657A (en) * 2002-02-27 2005-08-18 ファーメイン， エルティーディー． Compositions for delivering therapeutic agents and other substances, and methods of making and using said compositions
US7635463B2 (en) * 2002-02-27 2009-12-22 Pharmain Corporation Compositions for delivery of therapeutics and other materials
US20050260259A1 (en) * 2004-04-23 2005-11-24 Bolotin Elijah M Compositions for treatment with glucagon-like peptide, and methods of making and using the same
EP2085406A1 (en) * 2003-07-25 2009-08-05 ConjuChem Biotechnologies Inc. Long lasting insulin derivatives and methods thereof
EP1933862B1 (en) * 2005-09-06 2014-03-26 Oramed Pharmaceuticals Inc. Methods and compositions for oral administration of proteins
WO2007075534A2 (en) 2005-12-16 2007-07-05 Nektar Therapeutics Al, Corporation Polymer conjugates of glp-1
AU2006330610B2 (en) 2005-12-19 2012-08-16 Pharmain Corporation Hydrophobic core carrier compositions for delivery of therapeutic agents, methods of making and using the same
MX2008012678A (en) * 2006-04-07 2008-12-17 Merrion Res Iii Ltd Solid oral dosage form containing an enhancer.
US20090306337A1 (en) * 2006-07-31 2009-12-10 Novo Nordisk A/S Pegylated, Extended Insulins
KR101729986B1 (en) * 2006-09-22 2017-04-25 노보 노르디스크 에이/에스 Protease resistant insulin analogues
WO2008049711A1 (en) 2006-10-27 2008-05-02 Novo Nordisk A/S Peptide extended insulins
WO2008132224A2 (en) * 2007-04-30 2008-11-06 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
JP5675347B2 (en) * 2007-06-01 2015-02-25 ノボ・ノルデイスク・エー／エス Stable non-aqueous pharmaceutical composition
EP2017288A1 (en) * 2007-07-16 2009-01-21 Novo Nordisk A/S Protease stabilized, pegylated insulin analogues
CN101743252A (en) 2007-07-16 2010-06-16 诺沃-诺迪斯克有限公司 protease stabilized, pegylated insulin analogues
DK2180787T3 (en) 2007-08-01 2014-02-03 Univ Pittsburgh NITROOLIC ACID MODULATION OF TYPE II DIABETES
US7960336B2 (en) 2007-08-03 2011-06-14 Pharmain Corporation Composition for long-acting peptide analogs
US8563527B2 (en) * 2007-08-20 2013-10-22 Pharmain Corporation Oligonucleotide core carrier compositions for delivery of nucleic acid-containing therapeutic agents, methods of making and using the same
ES2664822T3 (en) * 2007-10-16 2018-04-23 Biocon Limited A solid pharmaceutical composition orally administrable and a process thereof
CN107412742B (en) 2007-11-16 2022-03-15 诺沃—诺迪斯克有限公司 Pharmaceutical composition comprising a GLP-1 peptide or exendin-4 peptide and a basal insulin peptide
JP5547083B2 (en) * 2007-11-20 2014-07-09 アンブルックス，インコーポレイテッド Modified insulin polypeptides and their use
US8598311B2 (en) * 2007-11-21 2013-12-03 The University Of The Sciences In Philadelphia Complexation of fatty acid-conjugated molecules with albumin
FR2925333B1 (en) * 2007-12-19 2012-04-13 Farid Bennis PHARMACEUTICAL COMPOSITIONS COMPRISING AT LEAST ONE PROTEIN ACTIVE INGREDIENT PROTECTS DIGESTIVE ENZYMES
US20090176892A1 (en) * 2008-01-09 2009-07-09 Pharmain Corporation Soluble Hydrophobic Core Carrier Compositions for Delivery of Therapeutic Agents, Methods of Making and Using the Same
ES2618073T3 (en) * 2008-03-14 2017-06-20 Novo Nordisk A/S Insulin analogs stabilized against proteases
US8691759B2 (en) 2008-03-18 2014-04-08 Novo Nordisk A/S Protease stabilized, acylated insulin analogues
EP2279244B1 (en) * 2008-03-26 2020-07-01 Oramed Ltd. Methods and compositions for oral administration of proteins
DK2280928T3 (en) 2008-05-01 2018-11-05 Complexa Inc Vinyl-substituted fatty acids
WO2009136392A2 (en) 2008-05-05 2009-11-12 Oramed Pharmaceuticals, Ltd. Methods and compositions for oral administration of exenatide
RU2517135C2 (en) * 2008-05-07 2014-05-27 Меррион Рисерч Iii Лимитед Peptide compositions and methods for production thereof
KR101430627B1 (en) 2008-05-13 2014-08-14 유니버시티 오브 캔사스 Metal Abstraction Peptide(MAP) Tag and Associated Methods
CN102099024B (en) 2008-06-19 2015-11-25 犹他大学研究基金会 The purposes of nitrated lipid in the treatment of the side effect of toxicity medical therapies
US20140024713A1 (en) 2008-06-19 2014-01-23 University Of Utah Research Foundation Use of nitrated lipids for treatment of side effects of toxic medical therapies
BRPI0823084B1 (en) * 2008-07-14 2019-07-16 Biocon Limited SUMMARY METHOD OF A SUBSTANCIALLY MONODISPHERE OLIGOMER MIX
EP2334335A1 (en) * 2008-09-19 2011-06-22 Nektar Therapeutics Polymer conjugates of cd-np peptides
EP2391217A4 (en) 2009-01-28 2015-05-20 Smartcells Inc Synthetic conjugates and uses thereof
CA2750269A1 (en) 2009-01-28 2010-08-05 Smartcells, Inc. Crystalline insulin-conjugates
WO2010088268A1 (en) 2009-01-28 2010-08-05 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
JP5508438B2 (en) 2009-01-28 2014-05-28 スマートセルズ・インコーポレイテツド Complex-based system for controlled drug delivery
US20100215743A1 (en) * 2009-02-25 2010-08-26 Leonard Thomas W Composition and drug delivery of bisphosphonates
US9095623B2 (en) * 2009-03-20 2015-08-04 Smartcells, Inc. Terminally-functionalized conjugates and uses thereof
AU2010226243A1 (en) * 2009-03-20 2011-09-22 Smartcells, Inc. Terminally-functionalized conjugates and uses thereof
EP2408470A4 (en) 2009-03-20 2012-08-29 Smartcells Inc Soluble non-depot insulin conjugates and uses thereof
WO2010120365A2 (en) * 2009-04-16 2010-10-21 Wu Nian Protein-carrier conjugates
EP2451472A1 (en) * 2009-07-06 2012-05-16 Sanofi-Aventis Deutschland GmbH Heat- and vibration-stable insulin preparations
EP3045167A1 (en) 2009-07-31 2016-07-20 University of Pittsburgh - Of the Commonwealth System of Higher Education Keto fatty acids as anti-inflammatory agents
WO2011041639A2 (en) 2009-10-02 2011-04-07 Miller Raymond A Heteroatom containing substituted fatty acids
CA2776302A1 (en) 2009-10-13 2011-04-21 F. Hoffmann-La Roche Ag Neuropeptide-2 receptor (y-2r) agonists
US8865647B2 (en) * 2009-11-02 2014-10-21 Novo Nordisk A/S Pharmaceutical solution of non covalently bound albumin and acylated insulin
WO2011084618A2 (en) 2009-12-16 2011-07-14 Nod Pharmaceuticals, Inc. Compositions and methods for oral drug delivery
US20110142889A1 (en) * 2009-12-16 2011-06-16 Nod Pharmaceuticals, Inc. Compositions and methods for oral drug delivery
US20110182985A1 (en) * 2010-01-28 2011-07-28 Coughlan David C Solid Pharmaceutical Composition with Enhancers and Methods of Preparing thereof
US9089484B2 (en) * 2010-03-26 2015-07-28 Merrion Research Iii Limited Pharmaceutical compositions of selective factor Xa inhibitors for oral administration
EP2590643A4 (en) * 2010-06-28 2014-01-01 Complexa Inc Multi-component pharmaceuticals for treating diabetes
US9068013B2 (en) 2010-07-28 2015-06-30 Smart Cells, Inc. Recombinant lectins, binding-site modified lectins and uses thereof
US8933207B2 (en) 2010-07-28 2015-01-13 Smartcells, Inc. Drug-ligand conjugates, synthesis thereof, and intermediates thereto
JP2013535467A (en) 2010-07-28 2013-09-12 スマートセルズ・インコーポレイテツド Recombinantly expressed insulin polypeptide and uses thereof
KR20140026354A (en) 2011-01-07 2014-03-05 메리온 리서치 Ⅲ 리미티드 Pharmaceutical compositions of iron for oral administration
CN102614498B (en) * 2011-01-28 2014-12-17 四川科伦药物研究有限公司 Insulin nanoparticle and preparation method thereof
CN102675452B (en) * 2011-03-17 2015-09-16 重庆富进生物医药有限公司 Tool continues the conjugate of insulin human that is hypoglycemic and that combined by height and analogue
CN107397953A (en) * 2011-06-02 2017-11-28 韩美科学株式会社 Non-peptide based polyalcohol insulin multimer and its method of production
US9260503B2 (en) 2011-06-15 2016-02-16 Novo Nordisk A/S Multi-substituted insulins
EP2744491B1 (en) 2011-08-19 2020-07-29 The University of Utah Research Foundation Combination therapy with nitrated lipids and inhibitors of the renin-angiotensin-aldosterone system
ES2644962T3 (en) 2012-01-03 2017-12-01 Oramed Ltd. Capsules containing oil-based liquid compositions of combined therapeutic agents to treat diabetes
WO2013114369A1 (en) 2012-02-01 2013-08-08 Oramed Ltd Protease inhibitor-containing compositions, compositions comprising same, and methods for producing and using same
CN104364260B (en) 2012-04-11 2017-02-22 诺和诺德股份有限公司 insulin formulations
US9187735B2 (en) 2012-06-01 2015-11-17 University Of Kansas Metal abstraction peptide with superoxide dismutase activity
UA116217C2 (en) 2012-10-09 2018-02-26 Санофі Exendin-4 derivatives as dual glp1/glucagon agonists
US9096679B2 (en) 2012-12-03 2015-08-04 The Board Of Regents Of The University Of Oklahoma Peptide compounds and methods of production and use thereof
JP6377070B2 (en) 2012-12-03 2018-08-22 ザ ボード オブ リージェンツ オブ ザ ユニヴァーシティ オブ オクラホマ Peptide compound, method for producing the same and use thereof
WO2014096149A1 (en) 2012-12-21 2014-06-26 Sanofi Exendin-4 Derivatives
CN104902921A (en) 2013-01-03 2015-09-09 奥拉姆德有限公司 Methods and compositions for treating NAFLD, hepatic steatosis, and sequelae thereof
AU2014254016B2 (en) 2013-04-16 2018-02-22 The Board Of Regents Of The University Of Oklahoma Peptide compounds and methods of production and use thereof
US20200157159A1 (en) 2013-04-16 2020-05-21 The Board Of Regents Of The University Of Oklahoma Peptide compounds and compositions thereof
EP3052134B1 (en) 2013-10-04 2021-05-05 Merck Sharp & Dohme Corp. Glucose-responsive insulin conjugates
AU2014333979B2 (en) 2013-10-07 2018-02-15 Novo Nordisk A/S Novel derivative of an insulin analogue
US9822158B2 (en) 2013-12-04 2017-11-21 Merck Sharp & Dohme Corp. Method for preparing crystalline insulin
WO2015086730A1 (en) 2013-12-13 2015-06-18 Sanofi Non-acylated exendin-4 peptide analogues
EP3080154B1 (en) 2013-12-13 2018-02-07 Sanofi Dual glp-1/gip receptor agonists
TW201609795A (en) 2013-12-13 2016-03-16 賽諾菲公司 EXENDIN-4 peptide analogues as dual GLP-1/GIP receptor agonists
WO2015086733A1 (en) 2013-12-13 2015-06-18 Sanofi Dual glp-1/glucagon receptor agonists
AR099569A1 (en) 2014-02-28 2016-08-03 Novo Nordisk As INSULIN DERIVATIVES AND THE MEDICAL USES OF THESE
TW201625670A (en) 2014-04-07 2016-07-16 賽諾菲公司 Dual GLP-1/glucagon receptor agonists derived from EXENDIN-4
TW201625669A (en) 2014-04-07 2016-07-16 賽諾菲公司 Peptidic dual GLP-1/glucagon receptor agonists derived from Exendin-4
TW201625668A (en) 2014-04-07 2016-07-16 賽諾菲公司 Exendin-4 derivatives as peptidic dual GLP-1/glucagon receptor agonists
US9932381B2 (en) 2014-06-18 2018-04-03 Sanofi Exendin-4 derivatives as selective glucagon receptor agonists
JP6574556B2 (en) * 2014-08-27 2019-09-11 日東電工株式会社 Intraoral film-form base and preparation
DK3006045T3 (en) 2014-10-07 2017-07-17 Cyprumed Gmbh Pharmaceutical formulations for oral administration of peptide or protein drugs
CN104447981B (en) * 2014-12-25 2015-07-08 重庆浦诺维生物科技有限公司 Conjugate of hydroxyl-terminated pegylated human insulin and analogues thereof
WO2016120378A1 (en) 2015-01-29 2016-08-04 Novo Nordisk A/S Tablets comprising glp-1 agonist and enteric coating
JP6737793B2 (en) * 2015-01-29 2020-08-12 ノヴォ ノルディスク アー／エス Pharmaceutical composition for oral insulin administration comprising a tablet core and a polyvinyl alcohol coating
AR105319A1 (en) 2015-06-05 2017-09-27 Sanofi Sa PROPHARMS THAT INCLUDE A DUAL AGONIST GLU-1 / GLUCAGON CONJUGATE HIALURONIC ACID CONNECTOR
DK3865484T3 (en) 2015-07-07 2024-01-29 H Lundbeck As PDE9 INHIBITOR WITH IMIDAZOPYRAZINONE BACKBONE FOR THE TREATMENT OF PERIPHERAL DISEASES
TW201706291A (en) 2015-07-10 2017-02-16 賽諾菲公司 New EXENDIN-4 derivatives as selective peptidic dual GLP-1/glucagon receptor agonists
CN113440506A (en) 2015-10-02 2021-09-28 康普莱克夏公司 Prevention, treatment and reversal of disease using therapeutically effective amounts of activated fatty acids
AU2016335287A1 (en) 2015-10-07 2018-04-12 Cyprumed Gmbh Pharmaceutical formulations for the oral delivery of peptide drugs
EP3373921B1 (en) 2015-11-13 2019-08-28 The University of Massachusetts Bifunctional molecules containing peg for use in inhibiting cataracts and presbyopia
EP3384935A4 (en) * 2015-12-02 2019-08-21 Hanmi Pharm. Co., Ltd. Protein complex using fatty acid derivative, and preparation method therefor
US11021430B2 (en) 2016-04-05 2021-06-01 Moresco Corporation Oxa acid compound
US10458409B2 (en) * 2016-06-06 2019-10-29 Emerson Climate Technologies, Inc. Compressor having a sleeve guide assembly
ES2930149T3 (en) 2016-12-16 2022-12-07 Novo Nordisk As Pharmaceutical compositions containing insulin
TWI688406B (en) * 2017-09-19 2020-03-21 免疫功坊股份有限公司 Pharmaceutical constructs with enhanced binding affinity with albumin
WO2019243980A1 (en) * 2018-06-18 2019-12-26 Biocon Limited Method and use of controlling postprandial glucose levels in a subject
WO2020144606A1 (en) * 2019-01-10 2020-07-16 Biocon Limited Preparative crystallization of recombinant human insulin

Family Cites Families (292)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2626228A (en) * 1945-05-17 1953-01-20 Novo Terapeutisk Labor As Method of producing crystalline insulin
US2882203A (en) * 1951-06-26 1959-04-14 Novo Terapeutisk Labor As Injectable insulin preparation with protracted effect
DK78069C (en) * 1952-06-23 1954-09-06 Novo Terapeutisk Labor As Process for the production of crystalline insulin.
US2789080A (en) * 1952-08-14 1957-04-16 Christensen Henry Marinus Insulin-albumin compositions
US2849370A (en) * 1953-06-04 1958-08-26 Novo Terapeutisk Labortorium A Injectable insulin preparations with protracted effect and process of producing same
US3102077A (en) * 1953-08-19 1963-08-27 Christensen Henry Marinus Preparation of insulin containing 2.75 to 8 percent zinc content
US2819999A (en) * 1953-11-13 1958-01-14 Novo Terapeutisk Labor As Process for crystallization of insulin using freeze dried insulin as seeding material
US2799622A (en) * 1953-11-14 1957-07-16 Novo Terapeutisk Labor As Process of producing insulin crystals of substantially uniform size and compositions thereof
US3058885A (en) * 1957-06-08 1962-10-16 Novo Terapeutisk Labor As Process for treatment of insulin crystals and for production of insulin preparationstherefrom
US3060093A (en) * 1957-07-18 1962-10-23 Nordisk Insulinlab Slowly acting insulin preparation in crystalline form and method of preparation
US3014842A (en) * 1957-08-10 1961-12-26 Novo Terapeutish Lab A S Injectable bovine insulin crystal suspensions and process of producing same
US3091573A (en) * 1958-05-12 1963-05-28 Novo Terapeutisk Labor As Quick acting insulin preparation
US3256153A (en) * 1963-02-08 1966-06-14 Smith Kline French Lab Method of stabilizing wax-fat coating materials and product thereof
US4003792A (en) * 1967-07-01 1977-01-18 Miles Laboratories, Inc. Conjugates of acid polysaccharides and complex organic substances
US3950517A (en) * 1970-05-08 1976-04-13 National Research Development Corporation Insulin derivatives
GB1381274A (en) * 1971-01-28 1975-01-22 Nat Res Dev Insulin derivatives
US3919411A (en) * 1972-01-31 1975-11-11 Bayvet Corp Injectable adjuvant and compositions including such adjuvant
US4044196A (en) * 1972-03-30 1977-08-23 Bayer Aktiengesellschaft Crosslinked copolymers of α,β-olefinically unsaturated dicarboxylic anhydrides
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
FR2408387A2 (en) * 1975-06-30 1979-06-08 Oreal COMPOSITIONS BASED ON AQUEOUS DISPERSIONS OF LIPID SPHERULES
US4087390A (en) * 1977-02-02 1978-05-02 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4093574A (en) * 1977-02-02 1978-06-06 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4087930A (en) * 1976-10-20 1978-05-09 O. F. Mossberg & Sons, Inc. Magazine cap retaining means for tubular magazine firearms
US4223163A (en) * 1976-12-10 1980-09-16 The Procter & Gamble Company Process for making ethoxylated fatty alcohols with narrow polyethoxy chain distribution
JPS53116315A (en) * 1977-03-17 1978-10-11 Ueno Seiyaku Oyo Kenkyujo Kk Powder or granular containing improved sorbinic acid
US4100117A (en) * 1977-04-21 1978-07-11 Eli Lilly And Company Somatostatin analogs and intermediates thereto
US4253998A (en) * 1979-03-09 1981-03-03 American Home Products Corporation Peptides related to somatostatin
JPS54148722A (en) * 1978-05-12 1979-11-21 Takeda Chem Ind Ltd Nonapeptide and its preparation
US4277394A (en) * 1979-04-23 1981-07-07 Takeda Chemical Industries, Ltd Tetrapeptidehydrazide derivatives
GB2051574B (en) * 1979-05-10 1984-01-18 Kyoto Pharma Ind Adjuvant for promoting absorption of pharmacologically active substances through the rectum
US4348387A (en) * 1979-07-31 1982-09-07 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
US4469681A (en) * 1979-07-31 1984-09-04 The Rockefeller University Method and system for the controlled release of biologically active substances to a body fluid
FR2465486A1 (en) * 1979-09-21 1981-03-27 Roussel Uclaf NEW APPLICATION USING LH-RH OR AGONISTS
JPS5692846A (en) 1979-12-27 1981-07-27 Takeda Chem Ind Ltd Tetrapeptide derivative and its preparation
US4554101A (en) * 1981-01-09 1985-11-19 New York Blood Center, Inc. Identification and preparation of epitopes on antigens and allergens on the basis of hydrophilicity
IT1144743B (en) * 1981-07-09 1986-10-29 Mario Cane PERFECTED INSULIN INFUSER APPARATUS
US4654324A (en) * 1981-08-27 1987-03-31 Eli Lilly And Company Human proinsulin pharmaceutical formulations
US4476113A (en) * 1981-10-27 1984-10-09 Union Oil Company Of California Stabilized fumigant composition comprising an aqueous solution of ammonia, hydrogen sulfide, carbon disulfide and sulfur
FI78616C (en) 1982-02-05 1989-09-11 Novo Industri As Process for preparing an infused stabilized insulin solution having an elevated zinc content
US4698264A (en) * 1982-08-02 1987-10-06 Durkee Industrial Foods, Corp. Particulate composition and process for making same
IL68769A (en) * 1983-05-23 1986-02-28 Hadassah Med Org Pharmaceutical compositions containing insulin for oral administration
US4602043A (en) * 1983-07-18 1986-07-22 Technology Unlimited Inc. Treatment for hypoglycemia
JPS6032714A (en) * 1983-08-01 1985-02-19 Teijin Ltd Stabilized powdery pharmaceutical composition for application to nasal mucous membrane
JPS60130003A (en) * 1983-12-16 1985-07-11 日本特殊陶業株式会社 High frequency dielectric porcelain composition
US4717566A (en) * 1984-03-19 1988-01-05 Alza Corporation Dosage system and method of using same
US4684524A (en) * 1984-03-19 1987-08-04 Alza Corporation Rate controlled dispenser for administering beneficial agent
US4704394A (en) * 1984-04-25 1987-11-03 Technology Unlimited, Inc. Treatment for hyperactivity
US4849405A (en) * 1984-05-09 1989-07-18 Synthetic Blood Corporation Oral insulin and a method of making the same
US4963367A (en) * 1984-04-27 1990-10-16 Medaphore, Inc. Drug delivery compositions and methods
US4863896A (en) * 1984-05-03 1989-09-05 Technology Unlimited, Inc. Diabetic control by combined insulin forms
US4761287A (en) * 1984-05-03 1988-08-02 Technology Unlimited, Inc. Diabetes control by serotonin
US4963526A (en) * 1984-05-09 1990-10-16 Synthetic Blood Corporation Oral insulin and a method of making the same
US4839341A (en) * 1984-05-29 1989-06-13 Eli Lilly And Company Stabilized insulin formulations
US4622392A (en) * 1984-06-21 1986-11-11 Health Research Inc. (Roswell Park Division) Thiophospholipid conjugates of antitumor agents
US4629621A (en) * 1984-07-23 1986-12-16 Zetachron, Inc. Erodible matrix for sustained release bioactive composition
US4797288A (en) * 1984-10-05 1989-01-10 Warner-Lambert Company Novel drug delivery system
US4946828A (en) * 1985-03-12 1990-08-07 Novo Nordisk A/S Novel insulin peptides
US5157021A (en) * 1985-03-15 1992-10-20 Novo Nordisk A/S Insulin derivatives and pharmaceutical preparations containing these derivatives
US4917888A (en) * 1985-06-26 1990-04-17 Cetus Corporation Solubilization of immunotoxins for pharmaceutical compositions using polymer conjugation
PH25772A (en) 1985-08-30 1991-10-18 Novo Industri As Insulin analogues, process for their preparation
SE457326B (en) * 1986-02-14 1988-12-19 Lejus Medical Ab PROCEDURES FOR PREPARING A QUICK SUBSTANTIAL CANDLES CONTAINING BLA MICROCRISTALLIN CELLULOSA
CA1257199A (en) * 1986-05-20 1989-07-11 Paul Y. Wang Preparation containing bioactive macromolecular substance for multi-months release in vivo
US4801575A (en) * 1986-07-30 1989-01-31 The Regents Of The University Of California Chimeric peptides for neuropeptide delivery through the blood-brain barrier
CA1339955C (en) 1986-10-14 1998-07-14 Richard Eugene Heiney Process for transforming a human insulin precursor to human insulin
GB8706313D0 (en) * 1987-03-17 1987-04-23 Health Lab Service Board Treatment & prevention of viral infections
US4764592A (en) * 1987-04-23 1988-08-16 Eli Lilly And Company Crystalline human proinsulin and process for its production
US5093198A (en) * 1987-06-19 1992-03-03 Temple University Adjuvant-enhanced sustained release composition and method for making
US4822337A (en) * 1987-06-22 1989-04-18 Stanley Newhouse Insulin delivery method and apparatus
DE3721721C1 (en) * 1987-07-01 1988-06-09 Hoechst Ag Process for coating granules
US5080891A (en) 1987-08-03 1992-01-14 Ddi Pharmaceuticals, Inc. Conjugates of superoxide dismutase coupled to high molecular weight polyalkylene glycols
US5034415A (en) * 1987-08-07 1991-07-23 Century Laboratories, Inc. Treatment of diabetes mellitus
JPH01308231A (en) * 1988-06-03 1989-12-12 Takeda Chem Ind Ltd Stabilized pharmaceutical composition and production thereof
US5055300A (en) * 1988-06-17 1991-10-08 Basic Bio Systems, Inc. Time release protein
DK336188D0 (en) * 1988-06-20 1988-06-20 Nordisk Gentofte propeptides
DE3827533A1 (en) * 1988-08-13 1990-02-15 Hoechst Ag PHARMACEUTICAL PREPARATION FOR TREATING THE DIABETES MELLITUS
US5349052A (en) * 1988-10-20 1994-09-20 Royal Free Hospital School Of Medicine Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor
US5162430A (en) * 1988-11-21 1992-11-10 Collagen Corporation Collagen-polymer conjugates
US5306500A (en) 1988-11-21 1994-04-26 Collagen Corporation Method of augmenting tissue with collagen-polymer conjugates
WO1990007522A1 (en) 1988-12-23 1990-07-12 Novo Nordisk A/S Human insulin analogues
US4994439A (en) * 1989-01-19 1991-02-19 California Biotechnology Inc. Transmembrane formulations for drug administration
US5089261A (en) * 1989-01-23 1992-02-18 Cetus Corporation Preparation of a polymer/interleukin-2 conjugate
PT93057B (en) 1989-02-09 1995-12-29 Lilly Co Eli PROCESS FOR THE PREPARATION OF INSULIN ANALOGS
US5182258A (en) 1989-03-20 1993-01-26 Orbon Corporation Systemic delivery of polypeptides through the eye
US5324844A (en) 1989-04-19 1994-06-28 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
US5286637A (en) 1989-08-07 1994-02-15 Debiopharm, S.A. Biologically active drug polymer derivatives and method for preparing same
US5013556A (en) * 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5650388A (en) 1989-11-22 1997-07-22 Enzon, Inc. Fractionated polyalkylene oxide-conjugated hemoglobin solutions
US5312808A (en) * 1989-11-22 1994-05-17 Enzon, Inc. Fractionation of polyalkylene oxide-conjugated hemoglobin solutions
CA2030174C (en) 1990-01-10 1996-12-24 Anthony H. Cincotta Process for the long term reduction of body fat stores, insulin resistance, hyperinsulinemia and hypoglycemia in vertebrates
US5545618A (en) 1990-01-24 1996-08-13 Buckley; Douglas I. GLP-1 analogs useful for diabetes treatment
US5126324A (en) 1990-06-07 1992-06-30 Genentech, Inc. Method of enhancing growth in patients using combination therapy
IE912365A1 (en) * 1990-07-23 1992-01-29 Zeneca Ltd Continuous release pharmaceutical compositions
IL99699A (en) 1990-10-10 2002-04-21 Autoimmune Inc Pharmaceutical oral, enteral or by-inhalation dosage form for suppressing an autoimmune response associated with type i diabetes
US5468727A (en) 1990-12-13 1995-11-21 Board Of Regents, The University Of Texas System Methods of normalizing metabolic parameters in diabetics
US5595732A (en) 1991-03-25 1997-01-21 Hoffmann-La Roche Inc. Polyethylene-protein conjugates
US5321009A (en) * 1991-04-03 1994-06-14 American Home Products Corporation Method of treating diabetes
WO1992018147A1 (en) 1991-04-19 1992-10-29 Affinity Biotech, Inc. Convertible microemulsion formulations
FR2675807B1 (en) 1991-04-23 1994-07-01 Medgenix Group Sa CONJUGATE OF CALCITONIN AND POLYETHYLENE GLYCOL.
US5304473A (en) 1991-06-11 1994-04-19 Eli Lilly And Company A-C-B proinsulin, method of manufacturing and using same, and intermediates in insulin production
JP3230056B2 (en) 1991-07-02 2001-11-19 インヘイル・インコーポレーテッド Device for forming an aerosolized dose of a drug
CH683149A5 (en) 1991-07-22 1994-01-31 Debio Rech Pharma Sa Process for the preparation of microspheres of a biodegradable polymeric material.
JPH06509796A (en) 1991-07-26 1994-11-02 スミスクライン・ビーチャム・コーポレイション W/O microemulsion
US5206219A (en) * 1991-11-25 1993-04-27 Applied Analytical Industries, Inc. Oral compositions of proteinaceous medicaments
US5693769A (en) 1991-12-13 1997-12-02 Transcell Technologies, Inc. Glycosylated steroid derivatives for transport across biological membranes and process for making and using same
US5320094A (en) 1992-01-10 1994-06-14 The Johns Hopkins University Method of administering insulin
US5876754A (en) * 1992-01-17 1999-03-02 Alfatec-Pharma Gmbh Solid bodies containing active substances and a structure consisting of hydrophilic macromolecules, plus a method of producing such bodies
ATE142484T1 (en) 1992-01-17 1996-09-15 Alfatec Pharma Gmbh SOLID BODY CONTAINING ACTIVE INGREDIENTS WITH A FRAMEWORK OF HYDROPHILIC MACROMOLECLES AND METHOD FOR THE PRODUCTION THEREOF
GB9212511D0 (en) 1992-06-12 1992-07-22 Cortecs Ltd Pharmaceutical compositions
US5262172A (en) 1992-06-19 1993-11-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing buffered bile acids
US5415872A (en) 1992-06-22 1995-05-16 Digestive Care Inc. Compositions of gastric acid-resistant microspheres containing salts of bile acids
US6509006B1 (en) 1992-07-08 2003-01-21 Inhale Therapeutic Systems, Inc. Devices compositions and methods for the pulmonary delivery of aerosolized medicaments
US6582728B1 (en) 1992-07-08 2003-06-24 Inhale Therapeutic Systems, Inc. Spray drying of macromolecules to produce inhaleable dry powders
US5785049A (en) 1994-09-21 1998-07-28 Inhale Therapeutic Systems Method and apparatus for dispersion of dry powder medicaments
US5420108A (en) * 1992-09-14 1995-05-30 Shohet; Isaac H. Method of controlling diabetes mellitus
BR9307141A (en) 1992-09-29 1999-03-30 Inhale Therapeutic Syst Process for the pulsatile sistance release of an active fragment of parathyroid hormone (PTH) to a mammalian host and to a patient and pharmaceutical composition
US6093391A (en) 1992-10-08 2000-07-25 Supratek Pharma, Inc. Peptide copolymer compositions
GB9316895D0 (en) 1993-08-13 1993-09-29 Guy S And St Thomas Hospitals Hepatoselective insulin analogues
US5298643A (en) 1992-12-22 1994-03-29 Enzon, Inc. Aryl imidate activated polyalkylene oxides
US5349001A (en) 1993-01-19 1994-09-20 Enzon, Inc. Cyclic imide thione activated polyalkylene oxides
US5364838A (en) 1993-01-29 1994-11-15 Miris Medical Corporation Method of administration of insulin
US5321095A (en) 1993-02-02 1994-06-14 Enzon, Inc. Azlactone activated polyalkylene oxides
US5298410A (en) 1993-02-25 1994-03-29 Sterling Winthrop Inc. Lyophilized formulation of polyethylene oxide modified proteins with increased shelf-life
US5681811A (en) 1993-05-10 1997-10-28 Protein Delivery, Inc. Conjugation-stabilized therapeutic agent compositions, delivery and diagnostic formulations comprising same, and method of making and using the same
US6191105B1 (en) 1993-05-10 2001-02-20 Protein Delivery, Inc. Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin
US5359030A (en) * 1993-05-10 1994-10-25 Protein Delivery, Inc. Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
US5621039A (en) 1993-06-08 1997-04-15 Hallahan; Terrence W. Factor IX- polymeric conjugates
TW402506B (en) 1993-06-24 2000-08-21 Astra Ab Therapeutic preparation for inhalation
US5830853A (en) 1994-06-23 1998-11-03 Astra Aktiebolag Systemic administration of a therapeutic preparation
IS1796B (en) 1993-06-24 2001-12-31 Ab Astra Inhaled polypeptide formulation composition which also contains an enhancer compound
US5747445A (en) 1993-06-24 1998-05-05 Astra Aktiebolag Therapeutic preparation for inhalation
US20010003739A1 (en) * 1993-06-24 2001-06-14 Astrazeneca Ab Systemic administration of a therapeutic preparation
US5506203C1 (en) * 1993-06-24 2001-02-06 Astra Ab Systemic administration of a therapeutic preparation
US6342225B1 (en) 1993-08-13 2002-01-29 Deutshces Wollforschungsinstitut Pharmaceutical active conjugates
US5534488A (en) 1993-08-13 1996-07-09 Eli Lilly And Company Insulin formulation
KR100310122B1 (en) 1993-09-17 2002-04-24 한센 핀 베네드, 안네 제헤르, 웨이콥 마리안느 Acylated Insulin
US5919455A (en) 1993-10-27 1999-07-06 Enzon, Inc. Non-antigenic branched polymer conjugates
US5605976A (en) 1995-05-15 1997-02-25 Enzon, Inc. Method of preparing polyalkylene oxide carboxylic acids
US5643575A (en) 1993-10-27 1997-07-01 Enzon, Inc. Non-antigenic branched polymer conjugates
US5951974A (en) 1993-11-10 1999-09-14 Enzon, Inc. Interferon polymer conjugates
JPH09510182A (en) 1993-11-17 1997-10-14 エルディーエス・テクノロジーズ・インコーポレーテッド Encapsulated transparent liquid for drug delivery
DE69535897D1 (en) 1994-03-07 2009-01-22 Nektar Therapeutics Method and composition for the pulmonary delivery of insulin
US6051256A (en) 1994-03-07 2000-04-18 Inhale Therapeutic Systems Dispersible macromolecule compositions and methods for their preparation and use
GB9406094D0 (en) 1994-03-28 1994-05-18 Univ Nottingham And University Polymer microspheres and a method of production thereof
HU227280B1 (en) 1994-05-10 2011-01-28 Nicox Sa Nitro compounds and their compositions having anti-inflammatory, analgesic and antithrombotic acitivities
WO1995032219A1 (en) 1994-05-20 1995-11-30 Hisamitsu Pharmaceutical Co., Inc. Protein or polypeptide, process for producing the same, and intermediate compound tehrefor
US5474978A (en) 1994-06-16 1995-12-12 Eli Lilly And Company Insulin analog formulations
US5504188A (en) * 1994-06-16 1996-04-02 Eli Lilly And Company Preparation of stable zinc insulin analog crystals
US5461031A (en) 1994-06-16 1995-10-24 Eli Lilly And Company Monomeric insulin analog formulations
US6165976A (en) 1994-06-23 2000-12-26 Astra Aktiebolag Therapeutic preparation for inhalation
US5730990A (en) 1994-06-24 1998-03-24 Enzon, Inc. Non-antigenic amine derived polymers and polymer conjugates
US5543414A (en) * 1994-07-28 1996-08-06 Syntex (Usa) Inc. Achiral amino acid acyl esters of ganciclovir and its derivatives
PE32296A1 (en) * 1994-07-28 1996-08-07 Hoffmann La Roche L-MONOVALINE ESTER DERIVED FROM 2- (2-AMINO-1,6-DIHYDRO-6-OXO-PURIN-9-IL) METOXI-1,3-PROPANDIOL AND ITS PHARMACEUTICALLY ACCEPTABLE SALTS
US5840891A (en) * 1994-07-28 1998-11-24 Syntex (U.S.A.) Inc. 2-(2-amino-1,6-dihydro-6-oxo-purin-9-yl) methoxy-1,3-propanediol derivative
GB9417524D0 (en) 1994-08-31 1994-10-19 Cortecs Ltd Pharmaceutical compositions
CA2555600C (en) 1994-09-21 2008-01-29 Nektar Therapeutics Apparatus and methods for dispersing dry powder medicaments
CN1080575C (en) * 1994-10-14 2002-03-13 蛋白质传送股份有限公司 Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same
US5738846A (en) 1994-11-10 1998-04-14 Enzon, Inc. Interferon polymer conjugates and process for preparing the same
US5693609A (en) 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
US5646242A (en) * 1994-11-17 1997-07-08 Eli Lilly And Company Selective acylation of epsilon-amino groups
EP0796324A1 (en) 1994-12-07 1997-09-24 Novo Nordisk A/S Polypeptide with reduced allergenicity
GB9424902D0 (en) 1994-12-09 1995-02-08 Cortecs Ltd Solubilisation Aids
SE9404468D0 (en) 1994-12-22 1994-12-22 Astra Ab Powder formulations
US5843866A (en) 1994-12-30 1998-12-01 Hampshire Chemical Corp. Pesticidal compositions comprising solutions of polyurea and/or polyurethane
US5932462A (en) 1995-01-10 1999-08-03 Shearwater Polymers, Inc. Multiarmed, monofunctional, polymer for coupling to molecules and surfaces
US5907030A (en) 1995-01-25 1999-05-25 University Of Southern California Method and compositions for lipidization of hydrophilic molecules
KR0150565B1 (en) 1995-02-15 1998-08-17 김정재 A process for preparing human proinsulin by recombinant dna technology
US6251856B1 (en) 1995-03-17 2001-06-26 Novo Nordisk A/S Insulin derivatives
YU18596A (en) 1995-03-31 1998-07-10 Eli Lilly And Company Analogous formulations of monomer insulin
US5606038A (en) 1995-04-10 1997-02-25 Competitive Technologies, Inc. Amphiphilic polyene macrolide antibiotic compounds
US6019968A (en) 1995-04-14 2000-02-01 Inhale Therapeutic Systems, Inc. Dispersible antibody compositions and methods for their preparation and use
US5780014A (en) 1995-04-14 1998-07-14 Inhale Therapeutic Systems Method and apparatus for pulmonary administration of dry powder alpha 1-antitrypsin
US6258341B1 (en) 1995-04-14 2001-07-10 Inhale Therapeutic Systems, Inc. Stable glassy state powder formulations
US6309671B1 (en) 1995-04-14 2001-10-30 Inhale Therapeutic Systems Stable glassy state powder formulations
US6165463A (en) 1997-10-16 2000-12-26 Inhale Therapeutic Systems, Inc. Dispersible antibody compositions and methods for their preparation and use
ES2093593T1 (en) 1995-05-05 1997-01-01 Hoffmann La Roche RECOMBINANT OBESE PROTEINS (OB).
US5824638A (en) 1995-05-22 1998-10-20 Shire Laboratories, Inc. Oral insulin delivery
US5704910A (en) 1995-06-05 1998-01-06 Nephros Therapeutics, Inc. Implantable device and use therefor
US5654007A (en) 1995-06-07 1997-08-05 Inhale Therapeutic Systems Methods and system for processing dispersible fine powders
US20010041786A1 (en) * 1995-06-07 2001-11-15 Mark L. Brader Stabilized acylated insulin formulations
US5631347A (en) 1995-06-07 1997-05-20 Eli Lilly And Company Reducing gelation of a fatty acid-acylated protein
US5700904A (en) 1995-06-07 1997-12-23 Eli Lilly And Company Preparation of an acylated protein powder
US5714519A (en) 1995-06-07 1998-02-03 Ergo Science Incorporated Method for regulating glucose metabolism
ATE252913T1 (en) 1995-06-30 2003-11-15 Novo Nordisk As PREVENTION OF DIABETES-TYPE DISEASE
GB9516268D0 (en) 1995-08-08 1995-10-11 Danbiosyst Uk Compositiion for enhanced uptake of polar drugs from the colon
EP1568771A3 (en) 1995-09-21 2006-05-17 Genentech, Inc. Human growth hormone variants
AU730969B2 (en) 1995-10-19 2001-03-22 University Of Washington Discrete-length polyethylene glycols
US5766620A (en) 1995-10-23 1998-06-16 Theratech, Inc. Buccal delivery of glucagon-like insulinotropic peptides
DE19545257A1 (en) 1995-11-24 1997-06-19 Schering Ag Process for the production of morphologically uniform microcapsules and microcapsules produced by this process
US6451970B1 (en) 1996-02-21 2002-09-17 Novo Nordisk A/S Peptide derivatives
FR2746035B1 (en) 1996-03-15 1998-06-12 COMPOSITE GEL MICROPARTICLES LIKELY TO BE USED AS VECTOR (S) OF ACTIVE INGREDIENT (S), ONE OF THEIR PREPARATION METHODS AND THEIR APPLICATIONS
US5826633A (en) 1996-04-26 1998-10-27 Inhale Therapeutic Systems Powder filling systems, apparatus and methods
US6103270A (en) 1996-06-07 2000-08-15 Inhale Therapeutic Systems Methods and system for processing dispersible fine powders
US5866538A (en) 1996-06-20 1999-02-02 Novo Nordisk A/S Insulin preparations containing NaCl
US5948751A (en) 1996-06-20 1999-09-07 Novo Nordisk A/S X14-mannitol
GB9613858D0 (en) 1996-07-02 1996-09-04 Cortecs Ltd Hydrophobic preparations
US5905140A (en) 1996-07-11 1999-05-18 Novo Nordisk A/S, Novo Alle Selective acylation method
DE59711533D1 (en) 1996-07-26 2004-05-27 Aventis Pharma Gmbh Insulin derivatives with increased zinc binding
US5856369A (en) 1996-07-30 1999-01-05 Osi Specialties, Inc. Polyethers and polysiloxane copolymers manufactured with double metal cyanide catalysts
DE19632440A1 (en) 1996-08-12 1998-02-19 Basf Ag Easily prepared and separated catalyst giving pure alkoxylation product with narrow molecular weight distribution
US6180604B1 (en) 1996-08-21 2001-01-30 Micrologix Biotech Inc. Compositions and methods for treating infections using analogues of indolicidin
US5874111A (en) 1997-01-07 1999-02-23 Maitra; Amarnath Process for the preparation of highly monodispersed polymeric hydrophilic nanoparticles
US6011008A (en) 1997-01-08 2000-01-04 Yissum Research Developement Company Of The Hebrew University Of Jerusalem Conjugates of biologically active substances
US5830918A (en) 1997-01-15 1998-11-03 Terrapin Technologies, Inc. Nonpeptide insulin receptor agonists
ZA98753B (en) 1997-02-05 1998-08-05 Hoffmann La Roche Use of gastrointestinal lipase inhibitors
US5898028A (en) 1997-03-20 1999-04-27 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
US6043214A (en) * 1997-03-20 2000-03-28 Novo Nordisk A/S Method for producing powder formulation comprising an insulin
US6310038B1 (en) 1997-03-20 2001-10-30 Novo Nordisk A/S Pulmonary insulin crystals
KR19980075132A (en) * 1997-03-28 1998-11-05 민병무 Pharmaceutical composition for the treatment of dental caries and periodontal disease, using as an active ingredient medium-chain fatty acids
ZA984697B (en) 1997-06-13 1999-12-01 Lilly Co Eli Stable insulin formulations.
JPH1112177A (en) 1997-06-25 1999-01-19 Teikoku Seiyaku Co Ltd Stable aspirin-containing preparation for external use
US6068993A (en) 1997-07-02 2000-05-30 Biobras Sa Vector for expression of heterologous protein and methods for extracting recombinant protein and for purifying isolated recombinant insulin
US20020115609A1 (en) 1997-07-14 2002-08-22 Hayat Onyuksel Materials and methods for making improved micelle compositions
IL134084A0 (en) 1997-07-18 2001-04-30 Infimed Inc Biodegradable macromers for the controlled release of biologically active substances
US6182712B1 (en) 1997-07-21 2001-02-06 Inhale Therapeutic Systems Power filling apparatus and methods for their use
GB9715444D0 (en) * 1997-07-22 1997-09-24 Scotia Holdings Plc Therapeutic and dietary compositions
RU2117488C1 (en) * 1997-07-30 1998-08-20 Институт нефтехимического синтеза им.А.В.Топчиева РАН Solid insulin-containing drug
US6309623B1 (en) 1997-09-29 2001-10-30 Inhale Therapeutic Systems, Inc. Stabilized preparations for use in metered dose inhalers
US6433040B1 (en) 1997-09-29 2002-08-13 Inhale Therapeutic Systems, Inc. Stabilized bioactive preparations and methods of use
US6565885B1 (en) 1997-09-29 2003-05-20 Inhale Therapeutic Systems, Inc. Methods of spray drying pharmaceutical compositions
US6444641B1 (en) * 1997-10-24 2002-09-03 Eli Lilly Company Fatty acid-acylated insulin analogs
AU1111799A (en) 1997-10-24 1999-05-17 Eli Lilly And Company Fatty acid-acylated insulin analogs
DK1025125T3 (en) * 1997-10-24 2003-10-06 Novo Nordisk As Aggregates for human insulin derivatives
ZA989744B (en) 1997-10-31 2000-04-26 Lilly Co Eli Method for administering acylated insulin.
US5955459A (en) 1997-11-26 1999-09-21 Neuromedica, Inc. Fatty acid-antipsychotic compositions and uses thereof
US6197764B1 (en) 1997-11-26 2001-03-06 Protarga, Inc. Clozapine compositions and uses thereof
US5985263A (en) 1997-12-19 1999-11-16 Enzon, Inc. Substantially pure histidine-linked protein polymer conjugates
US5981709A (en) 1997-12-19 1999-11-09 Enzon, Inc. α-interferon-polymer-conjugates having enhanced biological activity and methods of preparing the same
CO4970787A1 (en) 1997-12-23 2000-11-07 Lilly Co Eli INSOLUBLE COMPOSITIONS OF INSULIN AND INSULIN DERIVATIVES THAT CONTROL BLOOD GLUCOSE
KR100253916B1 (en) 1997-12-29 2000-05-01 김충환 A process for preparing human proinsulin
AU1870099A (en) * 1998-01-09 1999-07-26 Novo Nordisk A/S Stabilised insulin compositions
US6495514B1 (en) * 1998-01-21 2002-12-17 Mercer University Method for reducing inflammation and inducing an analgesic effect and compounds thereof
US6153655A (en) * 1998-04-17 2000-11-28 Enzon, Inc. Terminally-branched polymeric linkers and polymeric conjugates containing the same
US6192887B1 (en) * 1998-05-19 2001-02-27 The Pennsylvania State University Broad spectrum microbicidal and spermicidal compositions and methods having activity against sexually transmitted agents including papillomaviruses
US6257233B1 (en) 1998-06-04 2001-07-10 Inhale Therapeutic Systems Dry powder dispersing apparatus and methods for their use
DE19825447A1 (en) 1998-06-06 1999-12-09 Hoechst Marion Roussel De Gmbh New insulin analogues with increased zinc formation
RU2205188C2 (en) 1998-06-30 2003-05-27 Ново Нордиск А/С Seed crystals for preparing peptides or proteins
US5968554A (en) 1998-07-07 1999-10-19 Cascade Development, Inc. A Subsidiary Of Cardinal Health, Inc. Sustained release pharmaceutical preparation
US6211144B1 (en) 1998-10-16 2001-04-03 Novo Nordisk A/S Stable concentrated insulin preparations for pulmonary delivery
US20030049654A1 (en) 1998-10-16 2003-03-13 Xencor Protein design automation for protein libraries
EP1131089B1 (en) 1998-11-18 2004-02-18 Novo Nordisk A/S Stable aqueous insulin preparations without phenol and cresol
ES2180511T3 (en) * 1999-01-26 2003-02-16 Lilly Co Eli MONODISPERSE FORMULATIONS OF HEXAMERIC INSULIN ANALOGS.
GB9901809D0 (en) 1999-01-27 1999-03-17 Scarista Limited Highly purified ethgyl epa and other epa derivatives for psychiatric and neurological disorderes
US7658938B2 (en) * 1999-02-22 2010-02-09 Merrion Reasearch III Limited Solid oral dosage form containing an enhancer
WO2000050012A1 (en) * 1999-02-22 2000-08-31 Elan Corporation, Plc Solid oral dosage form containing an enhancer
DE19908041A1 (en) 1999-02-24 2000-08-31 Hoecker Hartwig Covalently bridged insulin dimers
US6248363B1 (en) 1999-11-23 2001-06-19 Lipocine, Inc. Solid carriers for improved delivery of active ingredients in pharmaceutical compositions
US6451974B1 (en) * 1999-03-17 2002-09-17 Novo Nordisk A/S Method of acylating peptides and novel acylating agents
US6630169B1 (en) 1999-03-31 2003-10-07 Nektar Therapeutics Particulate delivery systems and methods of use
US6444223B1 (en) 1999-05-28 2002-09-03 Alkermes Controlled Therapeutics, Inc. Method of producing submicron particles of a labile agent and use thereof
WO2001000246A2 (en) 1999-06-11 2001-01-04 Shearwater Corporation Hydrogels derived from chitosan and poly(ethylene glycol)
US6309633B1 (en) 1999-06-19 2001-10-30 Nobex Corporation Amphiphilic drug-oligomer conjugates with hydroyzable lipophile components and methods for making and using the same
EP2280020B1 (en) 1999-06-29 2016-02-17 MannKind Corporation Pharmaceutical formulations comprising a peptide complexed with a diketopiperazine
KR100345214B1 (en) 1999-08-17 2002-07-25 이강춘 The nasal transmucosal delivery of peptides conjugated with biocompatible polymers
US6323311B1 (en) 1999-09-22 2001-11-27 University Of Utah Research Foundation Synthesis of insulin derivatives
TWI310688B (en) 1999-10-29 2009-06-11 Nektar Therapeutics Dry powder compositions having improved dispersivity
WO2001052937A1 (en) 2000-01-24 2001-07-26 Medtronic Minimed, Inc. Mixed buffer system for stabilizing polypeptide formulations
CN1312080A (en) * 2000-02-18 2001-09-12 日本脏器制药株式会社 Composition containing fatty acid
CN100345535C (en) * 2000-06-27 2007-10-31 株式会社Mitech The Controlled release preparation of insulin and its method
US6479065B2 (en) 2000-08-10 2002-11-12 Alkermes Controlled Therapeutics, Inc. Process for the preparation of polymer-based sustained release compositions
US6824822B2 (en) 2001-08-31 2004-11-30 Alkermes Controlled Therapeutics Inc. Ii Residual solvent extraction method and microparticles produced thereby
EP2316490A3 (en) 2000-10-31 2012-02-01 PR Pharmaceuticals, Inc. Methods and compositions for enhanced delivery of bioactive molecules
TWI246524B (en) 2001-01-19 2006-01-01 Shearwater Corp Multi-arm block copolymers as drug delivery vehicles
ATE349469T1 (en) 2001-02-09 2007-01-15 Genentech Inc CRYSTALIZATION OF IGF-1
US7060675B2 (en) 2001-02-15 2006-06-13 Nobex Corporation Methods of treating diabetes mellitus
US6867183B2 (en) 2001-02-15 2005-03-15 Nobex Corporation Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
DE10114178A1 (en) 2001-03-23 2002-10-10 Aventis Pharma Gmbh Zinc-free and low-zinc insulin preparations with improved stability
US6558702B2 (en) 2001-04-13 2003-05-06 Alkermes Controlled Therapeutics, Inc. Method of modifying the release profile of sustained release compositions
AP1763A (en) * 2001-05-21 2007-08-02 Nektar Therapeutics Pulmonary administration of chemically modified insulin
US6835802B2 (en) 2001-06-04 2004-12-28 Nobex Corporation Methods of synthesizing substantially monodispersed mixtures of polymers having polyethylene glycol moieties
US6858580B2 (en) * 2001-06-04 2005-02-22 Nobex Corporation Mixtures of drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828305B2 (en) 2001-06-04 2004-12-07 Nobex Corporation Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6828297B2 (en) * 2001-06-04 2004-12-07 Nobex Corporation Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US6713452B2 (en) * 2001-06-04 2004-03-30 Nobex Corporation Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same
US20030040601A1 (en) 2001-06-08 2003-02-27 Ivan Diers Method for making insulin precursors and insulin analog precursors
US6913903B2 (en) 2001-09-07 2005-07-05 Nobex Corporation Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same
PE20030536A1 (en) * 2001-09-07 2003-07-03 Biocon Ltd METHODS FOR SYNTHESIZING INSULIN-OLIGOMER-POLYPEPTIDE CONJUGATES AND PROINSULIN-OLIGOMER-POLYPEPTIDE CONJUGATES AND METHODS OF SYNTHESIZING SUCH CONJUGATES
US6770625B2 (en) * 2001-09-07 2004-08-03 Nobex Corporation Pharmaceutical compositions of calcitonin drug-oligomer conjugates and methods of treating diseases therewith
US7196059B2 (en) * 2001-09-07 2007-03-27 Biocon Limited Pharmaceutical compositions of insulin drug-oligomer conjugates and methods of treating diseases therewith
CN1170851C (en) * 2001-09-27 2004-10-13 华中科技大学 Fatty diacylamino acid insulin as oral medicine for treating diabetes and its synthesizing process
US20030147965A1 (en) 2001-12-10 2003-08-07 Spherics, Inc. Methods and products useful in the formation and isolation of microparticles
CA2469205A1 (en) 2001-12-15 2003-06-26 Spherics, Inc. Bioadhesive drug delivery system with enhanced gastric retention
WO2003053401A2 (en) * 2001-12-19 2003-07-03 Alza Corporation Formulation and dosage form for increasing oral bioavailability of hydrophilic macromolecules
WO2004060347A2 (en) 2002-09-03 2004-07-22 Transform Pharmaceuticals, Inc. Pharmaceutical propylene glycol solvate compositions
WO2003086361A1 (en) 2002-04-18 2003-10-23 Dr. Reddy's Laboratories Ltd. Rapidly dispersing solid oral compositions
MXPA05000414A (en) 2002-07-09 2005-07-22 Sandoz Ag Liquid formulations with high concentration of human growth hormone (hgh) comprising phenol.
DE10250297A1 (en) 2002-10-29 2004-05-19 Aventis Pharma Deutschland Gmbh Insulin analog crystals and process for their preparation
US20040235948A1 (en) * 2003-03-05 2004-11-25 Solvay Pharmaceuticals Gmbh Treatment of diabetic patients with omega-3-fatty acids
WO2004082402A1 (en) * 2003-03-18 2004-09-30 Novartis Ag Compositions comprising fatty acids and amino acids
WO2005016312A1 (en) * 2003-08-13 2005-02-24 Nobex Corporation Micro-particle fatty acid salt solid dosage formulations for therapeutic agents
WO2005034874A2 (en) * 2003-10-08 2005-04-21 The Mclean Hospital Corporation Enhanced efficacy of omega-3 fatty acid therapy in the treatment of psychiatric disorders and other indications
EP1701714A2 (en) * 2004-01-07 2006-09-20 Nektar Therapeutics Improved sustained release compositions for pulmonary administration of insulin
ES2308056T3 (en) * 2004-03-06 2008-12-01 Cognis Ip Management Gmbh USE OF INSATURED FATTY ACIDS FOR THE REDUCTION OF THE APPETITE OR FOOD INGESTION.

Also Published As

Publication number Publication date
NO20170341A1 (en) 2007-01-22
JP5220410B2 (en) 2013-06-26
US20110124553A1 (en) 2011-05-26
JP5721266B2 (en) 2015-05-20
US8563685B2 (en) 2013-10-22
US9101596B2 (en) 2015-08-11
EP2626368A2 (en) 2013-08-14
UA103758C2 (en) 2013-11-25
IL180762A (en) 2015-06-30
ES2618028T3 (en) 2017-06-20
IL227117A (en) 2017-02-28
PT1773878E (en) 2015-06-05
US20110118179A1 (en) 2011-05-19
US20100105605A1 (en) 2010-04-29
CN105801686B (en) 2020-04-07
CN103223160B (en) 2015-02-18
BRPI0513508A (en) 2008-05-06
US7872095B2 (en) 2011-01-18
CN101027318B (en) 2016-05-25
CN101027318A (en) 2007-08-29
AU2005269753A1 (en) 2006-02-09
BRPI0513508B1 (en) 2021-06-01
RU2393168C2 (en) 2010-06-27
HK1187826A1 (en) 2014-04-17
NO20070859L (en) 2007-04-12
CA2910494A1 (en) 2006-02-09
IL209494A0 (en) 2011-01-31
US20060019873A1 (en) 2006-01-26
NO345613B1 (en) 2021-05-10
RU2527893C2 (en) 2014-09-10
JP2011236233A (en) 2011-11-24
US7605123B2 (en) 2009-10-20
CN103223160A (en) 2013-07-31
RU2007105824A (en) 2008-08-27
DK2626368T3 (en) 2017-02-27
EP2626368A3 (en) 2013-10-16
NZ553263A (en) 2010-06-25
PL1773878T3 (en) 2015-07-31
ZA200701446B (en) 2008-09-25
ES2535823T3 (en) 2015-05-18
RU2010105921A (en) 2011-08-27
PT2626368T (en) 2017-03-10
CA2910494C (en) 2018-10-23
EP1773878A2 (en) 2007-04-18
US9102758B2 (en) 2015-08-11
CN105801686A (en) 2016-07-27
US7875700B2 (en) 2011-01-25
PL2626368T3 (en) 2017-06-30
NO340825B1 (en) 2017-06-26
KR101309770B1 (en) 2013-09-30
KR20070059060A (en) 2007-06-11
US20060019874A1 (en) 2006-01-26
UA91512C2 (en) 2010-08-10
EP2626368B1 (en) 2016-12-21
JP2008506782A (en) 2008-03-06
IL209494A (en) 2015-07-30
KR20120120985A (en) 2012-11-02
AU2005269753A2 (en) 2006-02-09
EP1773878A4 (en) 2012-06-20
US20060018874A1 (en) 2006-01-26
AU2005269753B2 (en) 2011-08-18
JP5750494B2 (en) 2015-07-22
IL180762A0 (en) 2007-06-03
CA2580313C (en) 2016-03-15
JP2014043470A (en) 2014-03-13
MX2007000883A (en) 2007-04-02
KR101276754B1 (en) 2013-06-19
WO2006014673A2 (en) 2006-02-09
EP1773878B1 (en) 2015-03-04
DK1773878T3 (en) 2015-05-11
WO2006014673A3 (en) 2006-08-17

Similar Documents

Publication Publication Date Title
AU2005269753B2 (en) Insulin-oligomer conjugates, formulations and uses thereof
AU2014200247B2 (en) Insulin-oligomer conjugates, formulations and uses thereof

Legal Events

Date Code Title Description
EEER Examination request
MKLA Lapsed

Effective date: 20220719