WO2012054822A1 - Pharmacologically active polymer-glp-1 conjugates - Google Patents

Abstract

The invention provides peptides that are chemically modified by covalent attachment of a water-soluble oligomer. A conjugate of the invention, when administered by any of a number of administration routes, exhibits characteristics that are different from the characteristics of the peptide not attached to the water-soluble oligomer.

Description

PHARMACOLOGICALLY ACTIVE POLYMER -GLP-1 CONJUGATES

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. § 1 19(e) to U.S.

Provisional Patent Application No. 61/405,951, filed October 22, 2010, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] Among other things, the present invention relates generally to

pharmacologically active GLP-1 conjugates and compositions comprising the same.

BACKGROUND OF THE INVENTION

[0003] Glucagon-like peptide- 1 (GLP-1) is a peptide proteolytically processed from pro-glucagon and is secreted from intestinal L cells. GLP-1 indirectly controls blood glucose levels by regulating insulin sensitivity and production, decreasing food intake, and restoring pancreatic beta cell functions. The biological functions of GLP-1 have therapeutic benefit for the treatment of diabetes and numerous variants or formulations of the peptide have been approved for clinical use or are in late-stage clinical trials. The in vivo circulation half-life of the peptide is very short, typically only a few minutes, and most efforts to improve the therapeutic properties of GLP-1 have focused on increasing its in vivo half-life.

[0004] Covalently attaching one or more water-soluble polymers to GLP-1 to thereby form a GLP-1 conjugate represents one approach proposed to increase the in vivo half-life of this peptide. Various GLP-1 conjugates are described in the literature and in, for example, International Publication Nos. WO 99/43707, WO 2004/093823, WO 2007/075534 and WO 2010/033207 and U.S. Patent Application Publication Nos. 2005/0009988 and

2006/293499. An inherent risk with preparing a conjugate of any peptide, including GLP-1, is eliminating or substantially decreasing the activity of the peptide caused by the presence of the covalently attached water-soluble polymers (which may block access to ligand binding sites). Thus, even if the conjugate form of the peptide has an improved in vivo half-life, the activity of the conjugate can be so substantially reduced so as to render the conjugate unsuitable for clinical use. [0005]

has been proposed as one approach to address the problem of a GLP-1 conjugate having substantially reduced binding activity. See, for example, International Publication No.

WO 2007/075534. By having the water-soluble polymer release or detach from the otherwise inactive GLP-1 conjugate, the GLP-1 peptide— along with its activity ~ is restored. This approach, however, suffers from drawbacks associated with ensuring the release of the water-soluble polymer from the GLP-1 conjugate is not only reproducible across the patient populations, but follows a rate to provide sustained activity over time.

[0006] Thus, for GLP-1 conjugates where it is desired to avoid the complications associated with releasable linkages, the artisan remains faced with the risk that a conjugate form exhibits limited binding affinity. As is appreciated in the pharmaceutical industry, however, the conventional approach for identifying and developing promising pharmaceutical compounds is to only pursue those compounds that exhibit relatively high in vitro binding affinities in a model of interest; those compounds exhibiting no or limited activity are effectively abandoned.

[0007] Thus, while GLP-1 conjugates have been described (some of which exhibit no or limited in vitro binding activity), there remains a need in the art to provide GLP-1 conjugates that show good activity in vivo.

[0008] The present invention addresses this and other needs in the art.

SUMMARY OF THE INVENTION

[0009] Accordingly, in one or more embodiments of the invention, a pharmaceutical is provided, the pharmaceutical composition comprising a dose of polymer-GLP-1 conjugates and a pharmaceutically acceptable excipient, wherein (i) each conjugate in the dose of polymer-GLP-1 conjugates comprises a water-soluble, non-peptidic polymer having a molecular weight of greater than 5,000 Daltons covalently attached through an

amide-containing linkage to an amino group of a GLP-1 moiety, (ii) the pharmaceutical composition, upon administration to a mammal, has a glucose-lowering effect, and (iii) the dose of polymer-GLP-1 conjugates achieving the glucose-lowering effect is less than an amount of the GLP-1 moiety in an unconjugated form required to achieve the glucose lowering effect. [0010] In one or more embodiments of the invention, a polymer-GLP-1 conjugate is provided, wherein— in an in vivo model of unfed db/db mice— the polymer-GLP-1 conjugate provides a glucose level at that is at least 50% lower at four hours following intraperitoneal administration than an equimolar amount of the GLP-1 in unconjugated form.

[0011] In one or more embodiments of the invention, a method is provided, the method comprising administering to a patient a dose of polymer-GLP-1 conjugates, wherein following administration, the dose of polymer-GLP-1 conjugates provides at least a 25% lower glucose level after eight hours compared to the expected glucose level at that time absent administration of the therapeutically effective dose of pharmacologically active GLP-1 conjugates.

[0012] In one or more embodiments of the invention, a method is provided, the method comprising administering to a patient a dose of polymer-GLP-1 conjugates, wherein following administration, the dose of polymer-GLP-1 conjugates provides preprandial capillary plasma glucose within the range of 90- 180 mg/dl.

[0013] Additional embodiments of the present conjugates, compositions, methods, and the like will be apparent from the following description, examples, and claims. As can be appreciated from the foregoing and following description, each and every feature described herein, and each and every combination of two or more of such features, is included within the scope of the present disclosure provided that the features included in such a combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention. Additional aspects and advantages of the present invention are set forth in the following description and claims, particularly when considered in conjunction with the accompanying examples and drawings.

BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 1 is a typical SP-HP cation exchange purification profile of

PEG₂-ru-40K-Nter-GLP-l, as further described in Example 1.

[0015] FIG. 2 is a reverse phase HPLC chromatogram of a purified composition of

PEG₂-ru-40K-Nter-GLP-l, as further described in Example 1.

[0016] FIG. 3 is a typical SP-HP cation exchange purification profile of

PEG₂-ru-40K-Lys26/34-GLP-l , as further described in Example 2. [0017] FIG. 4 is a reverse phase HPLC chromatogram of a purified composition of

PEG₂-ru-40K-Lys26/34-GLP-l, as further described in Example 2.

[0018] FIG. 5 is a typical SP-HP cation exchange purification profile of PEG-30K-

Nter-ButyrALD-GLP-1, as further described in Example 3.

[0019] FIG. 6 is a reverse phase HPLC chromatogram of a purified composition of

PEG-30K-Nter-ButryALD-GLP- 1 , as further described in Example 3.

[0020] FIG. 7 is a plot of the in vivo glucose lowering activity of saline, 75 μg of GLP

(amino acid residues 9-36), 75 μg of GLP-1 (amino acid residues 7-36), 75 μg of Conjugate A (Example 1), 75 μg of Conjugate B (Example 2), and 75 μg of Comparative Conjugate 1 (Example 3) in unfed db/db mice as further described in Example 2. It is noted that all doses of conjugates are referenced in terms of GLP-1, wherein, for example, 75 μg of Conjugate A is not 75 μg of Conjugate A, but rather an amount of Conjugate A that provides 75 μg of GLP-1 (and ignoring the weight of polymeric component of the conjugatge). See Example 6.

DETAILED DESCRIPTION

[0021] As used in this specification and the intended claims, the singular forms "a,"

"an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymer" includes a single polymer as well as two or more of the same or different polymers; reference to "an optional excipient" or to "a pharmaceutically acceptable excipient" refers to a single optional excipient as well as two or more of the same or different optional excipients, and the like.

[0022] In describing and claiming one or more embodiments of the present invention, the following terminology will be used in accordance with the definitions described below.

[0023] As used herein, the terms "peptide," "polypeptide," and "protein," refer to polymers comprised of amino acid monomers linked by amide bonds. For use herein, each of "peptide," "polypeptide" and "protein" will be referred to as "peptide." Peptides may include the standard 20 a-amino acids that are used in protein synthesis by cells (i.e. natural amino acids), as well as non-natural amino acids (non-natural amino acids may be found in nature, but not used in protein synthesis by cells, e.g., ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetics. The amino acids may be D- or L-optical isomers. Peptides may be formed by a condensation or coupling reaction between the a-carbon carboxyl group of one amino acid and the amino group of another amino acid. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. Alternatively, the peptides may be non-linear, branched peptides or cyclic peptides. Moreover, the peptides may optionally be modified or protected with a variety of functional groups or protecting groups, including on the amino and/or carboxy terminus.

[0024] Amino acid residues in peptides are abbreviated as follows: Phenylalanine is

Phe or F; Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C;

Tryptophan is Tip or W; Arginine is Arg or R; and Glycine is Gly or G.

[0025] The terms "therapeutic peptide fragment" or "fragments of therapeutic peptides" refer to a peptide that comprises a truncation at the amino-terminus and/or a truncation at the carboxyl-terminus of a therapeutic peptide as defined herein. The terms "therapeutic peptide fragment" or "fragments of therapeutic peptides" also encompasses amino-terminal and/or carboxyl-terminal truncations of therapeutic peptide variants and therapeutic peptide derivatives. Therapeutic peptide fragments may be produced by synthetic techniques known in the art or may arise from in vivo protease activity on longer peptide sequences. It will be understood that therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides.

[0026] As used herein, the terms "therapeutic peptide variants" or "variants of therapeutic peptides" refer to therapeutic peptides having one or more amino acid

substitutions, including conservative substitutions and non-conservative substitutions, amino acid deletions (either internal deletions and/or C- and/or N- terminal truncations), amino acid additions (either internal additions and/or C- and/or N- terminal additions, e.g., fusion peptides), or any combination thereof. Variants may be naturally occurring (e.g. homologs or orthologs), or non-natural in origin. The term "therapeutic peptide variants" may also be used to refer to therapeutic peptides incorporating one or more non-natural amino acids, amino acid analogs, and peptidomimetics. It will be understood that, in accordance with the invention, therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides. [0027] "PEG," "polyethylene glycol" and "poly(ethylene glycol)" as used herein, are interchangeable and encompass any non-peptidic water-soluble poly(ethylene oxide).

Typically, PEGs for use in accordance with the invention comprise the following structure ^M-(OCH₂CH₂)n-" where (n) is 2 to 4000. As used herein, PEG also includes

"-CH₂CH₂-0(CH₂CH₂0)_n-CH₂CH₂-" and ^M-(OCH₂CH₂)_nO-," depending upon whether or not the terminal oxygens have been displaced. Throughout the specification and claims, it should be remembered that the term "PEG" includes structures having various terminal or "end capping" groups and so forth. The term "PEG" also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH₂CH₂- repeating subunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as "branched," "linear," "forked," "multifunctional," and the like, to be described in greater detail below.

[0028] The terms "end-capped" and "terminally capped" are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.

Typically, although not necessarily, the end-capping moiety comprises a hydroxy or Ci_₂₀ alkoxy group, more preferably a CMO alkoxy group, and still more preferably a C_1-5 alkoxy group. Thus, examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. It must be

remembered that the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH₃0(CH₂CH₂0)_n- and CH₃(OCH₂CH₂)_n-]. In addition, saturated, unsaturated, substituted and unsubstituted forms of each of the foregoing are envisioned. Moreover, the end-capping group can also be a silane. The end-capping group can also advantageously comprise a detectable label. When the polymer has an end-capping group comprising a detectable label, the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector. Such labels include, without limitation, fluorescers, chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive moieties, gold particles, quantum dots, and the like. Suitable detectors include photometers, films, spectrometers, and the like. The end-capping group can also

advantageously comprise a phospholipid. When the polymer has an end-capping group comprising a phospholipid, unique properties are imparted to the polymer and the resulting conjugate. Exemplary phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines. Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin.

[0029] "Non-naturally occurring" with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature. A non-naturally occurring polymer, however, may contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.

[0030] The term "water soluble" as in a "water-soluble polymer" is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85% (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water.

[0031] Molecular weight in the context of a water-soluble polymer, such as PEG, can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, and osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight average molecular weight. The polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.

[0032] The term "active" or "activated" when used in conjunction with a particular functional group refers to a reactive functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive" or "inert" group).

[0033] As used herein, the term "functional group" or any synonym thereof is meant to encompass protected forms thereof as well as unprotected forms.

[0034] The terms "spacer moiety," "linkage" and "linker" are used herein to refer to an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymer segment and a therapeutic peptide or an electrophile or nucleophile of a therapeutic peptide. The spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of a therapeutic peptide and a water-soluble polymer that can be attached directly or indirectly through a spacer moiety).

[0035] "Alkyl" refers to a hydrocarbon, typically ranging from about 1 to 15 atoms in length. Such hydrocarbons are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl" includes cycloalkyl as well as cycloalkylene-containing alkyl.

[0036] "Lower alkyl" refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, /-butyl, and t- butyl.

[0037] "Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms. "Cycloalkylene" refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.

[0038] "Alkoxy" refers to an -O-R group, wherein R is alkyl or substituted alkyl, preferably C_1-6 alkyl {e.g., methoxy, ethoxy, propyloxy, and so forth).

[0039] The term "substituted" as in, for example, "substituted alkyl," refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl; C_3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like. "Substituted aryl" is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i. e. , ortho, meta, or para).

[0040] "Noninterfering substituents" are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.

[0041] "Aryl" means one or more aromatic rings, each of 5 or 6 core carbon atoms.

Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl" includes heteroaryl.

[0042] "Heteroaryl" is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.

[0043] "Heterocycle" or "heterocyclic" means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon. Preferred heteroatoms include sulfur, oxygen, and nitrogen.

[0044] "Substituted heteroaryl" is heteroaryl having one or more noninterfering groups as substituents.

[0045] "Substituted heterocycle" is a heterocycle having one or more side chains formed from noninterfering substituents.

[0046] An "organic radical" as used herein shall include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.

[0047] "Electrophile" and "electrophilic group" refer to an ion or atom or collection of atoms, which may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.

[0048] "Nucleophile" and "nucleophilic group" refers to an ion or atom or collection of atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic center or with an electrophile.

[0049] A "physiologically cleavable" or "hydrolyzable" or "degradable" bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions. The tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms. Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

[0050] "Releasably attached," e.g., in reference to a therapeutic peptide releasably attached to a water-soluble polymer, refers to a therapeutic peptide that is covalently attached via a linker that includes a degradable linkage as disclosed herein, wherein upon degradation (e.g., hydrolysis), the therapeutic peptide is released. The therapeutic peptide thus released will typically correspond to the unmodified parent or native therapeutic peptide, or may be slightly altered, e.g., possessing a short organic tag. Preferably, the unmodified parent therapeutic peptide is released.

[0051] An "enzymatically degradable linkage" means a linkage that is subject to degradation by one or more enzymes.

[0052] A "hydrolytically stable" linkage or bond refers to a chemical bond, typically a covalent bond, which is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time. Examples of hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like. Generally, a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1 -2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks. It must be pointed out that some linkages can be hydrolytically stable or hydrolyzable, depending upon (for example) adjacent and neighboring atoms and ambient conditions. One of ordinary skill in the art can determine whether a given linkage or bond is hydrolytically stable or hydrolyzable in a given context by, for example, placing a linkage-containing molecule of interest under conditions of interest and testing for evidence of hydrolysis (e.g., the presence and amount of two molecules resulting from the cleavage of a single molecule). Other approaches known to those of ordinary skill in the art for determining whether a given linkage or bond is hydrolytically stable or hydrolyzable can also be used.

[0053] The terms "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. [0054] "Pharmacologically effective amount," "physiologically effective amount," and

"therapeutically effective amount" are used interchangeably herein to mean the amount of a polymer-(therapeutic peptide) conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated therapeutic peptide) in the bloodstream or in the target tissue. The precise amount will depend upon numerous factors, e.g. , the particular therapeutic peptide, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.

[0055] "Multi-functional" means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different. Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, or from 3-50 functional groups, or from 3-25 functional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone. A "difunctional" polymer means a polymer having two functional groups contained therein, either the same (i.e., homodifunctional) or different (i.e., heterodifunctional) .

[0056] The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals, and pets.

[0057] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not.

[0058] "Substantially" (unless specifically defined for a particular context elsewhere or the context clearly dictates otherwise) means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80%) or greater, 90% or greater, and 95%> or greater of the condition.

[0059] Unless the context clearly dictates otherwise, when the term "about" precedes a numerical value, the numerical value is understood to mean the stated numerical value and also ± 10% of the stated numerical value.

[0060] Turning now to one or more embodiments of the invention, a pharmaceutical is provided, the pharmaceutical composition comprising a dose of polymer-GLP-1 conjugates and a pharmaceutically acceptable excipient, wherein (i) each conjugate in the dose of polymer-GLP-1 conjugates comprises a water-soluble, non-peptidic polymer having a molecular weight of greater than 5,000 Dal tons covalently attached through an

amide-containing linkage to an amino group of a GLP- 1 moiety, (ii) the pharmaceutical composition, upon administration to a mammal, has a glucose-lowering effect, and (iii) the dose of polymer-GLP-1 conjugates achieving the glucose-lowering effect is less than an amount of the GLP-1 moiety in an unconjugated form required to achieve the glucose lowering effect.

[0061] With respect to the dose of polymer-GLP-1 conjugates contained within the pharmaceutical compositions of the invention, each conjugate making up the dose having at least one (and in the majority of cases only one) water-soluble, non-peptidic polymer covalently attached to a GLP-1 moiety. Aspects of the various components that comprise the polymer-GLP-1 conjugates are discussed herein.

[0062] With respect to the GLP-1 moiety, the term "GLP-1 moiety," as used herein, refers to those peptides, polypeptides and proteins having GLP-1 activity (and includes GLP-1 activity- containing peptides derived through site-directed mutagenesis or other mutations), including (for example) GLP-1. Prior to conjugation, the GLP-1 moiety has at least one electrophilic group or nucleophilic group suitable for reaction with a water soluble polymer. In addition, the term "GLP-1 moiety" encompasses both the GLP-1 moiety prior to conjugation as well as the GLP-1 moiety residue following conjugation. It will be

understood, however, that when the GLP-1 moiety is covalently attached to a water-soluble polymer, the GLP-1 moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer (or linker that is attached to the polymer), due to reaction of one of more reactive groups of the GLP-1 moiety (e.g., an amino, carboxyl, etc.), with the water soluble polymer. In some instances, this slightly altered form of the GLP-1 moiety attached to another molecule, such as a water-soluble polymer, is referred to as a "residue" of the GLP-1 moiety. As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has GLP-1 activity.

[0063] With respect to "GLP- 1 " itself (and not in a conjugate form), "GLP- 1 " shall be understood to designate the peptide having the truncated "7-36" amino acid sequence (SEQ ID NO: l): NH₂-His⁷-Ala⁸-Glu⁹-G^

Leu^-Glu^-Gly^-Gln^-Ala²⁴-^

Gly³⁵-Arg³⁶-NH₂). GLP-1 (7-37)OH, an exemplary GLP-1 moiety, shall be understood to designate the peptide having the following amino acid sequence (SEQ ID NO:l): NH₂-His⁷- Ala⁸-Glu⁹-Gly¹⁰-Thrⁿ-Phe^,2-Tl^

Gln²³-Ala²⁴-Ala²⁵-Lys²^^

Other exemplary GLP-1 moieties for use in connection with the present invention include GLP-1 (1-36), GLP-1 analogs (such as those described in WO 91/1 1457), GLP-1 derivatives, GLP-1 moieties described in U.S. Published Patent Application No. 2004/0235710, GLP-1 biologically active fragments, extended GLP-1 (see, for example, WO 03/058203, in particular with respect to the extended glucagon-like peptide- 1 analogs described therein), N-terminal truncated fragments of GLP-1 (such as those described in EP 0 699 686, and exendins (including, for example, exendin-4 and analogs thereof).

[0064] With respect to exendins, exendins are peptides that were first isolated from the salivary secretions of the Gila-monster and the Mexican Beaded Lizard. The exendins have a degree of similarity to several members of the GLP family, with the highest homology, 53%, to GLP-1 (7-36)NH₂ (Goke, et al, J. Biol. Chem., 268: 19650-55, 1993). Particular exendins for use in the present invention include exendin-3 and exendin-4 (synthetic extendin-4 is also known as Exenatide). Exendin-3 (1-39) has the following amino acid sequence (SEQ ID NO:3): His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂. The amino acid sequence of exendin-4 (1-39) corresponds to (SEQ ID NO:4): His-Gly-Glu- Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile- Glu-T -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser wherein the C-terminus serine is amidated.

[0065] Regardless of the GLP-1 moiety used in connection with the present invention, the GLP-1 moiety may be obtained from either non-recombinant methods or from

recombinant methods, and the invention is not limited in this regard. Moreover, several GLP- 1 moieties are commercially available, e.g., hGLP-1 , rExtendin-4, and rHuGLP-1 are available from ProSpecTany Techno Gene LTD (Rehovot, Israel); and (Ser8)GLP-l(7- 36)amide, hGLP-1 amide, and hGLP-1 (7-36)Lys(biotin)amide are available from American Peptide Co., Sunnyvale, CA. Methods for preparing GLP-1 moieties are well-known, and are described, e.g., in U.S. Patent Nos. 5,118,666; 5,120,712; and 5,523,549.

[0066] Briefly, however, GLP-1 moieties can be prepared using standard methods of solution or solid phase peptide synthesis such as those described in Dugas H., Penny, C, Bioorganic Chemistry, Springer Verlag, New York, p. 54-92 (1981); Merrifield (1962) Chem Soc. 85:2149, and Stewart and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco, p. 24-66 (1969). Peptide synthesizers are available from, e.g., Applied Biosystems, Foster City, CA. Solid phase synthesizers are typically used according to manufacturers' instructions for blocking interfering groups, protecting certain amino acids, coupling, decoupling, and capping unreacted amino acids. BOC-amino acids and other reagents are commercially available from Applied Biosystems, Foster City CA. Sequential BOC

(tert-butoxycarbonyl or "BOC") chemistry using double coupling protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM

(oxymethylphenylacetamidomethyl or "PAM") resin is used. Asn, Gin, an Arg are coupled using preformed hydroxybenzotriazole esters. Suitable side chain protecting groups include: Arg (tosyl), Asp (cyclohexyl), Glu (cyclohexyl), Ser (benzyl), Thr (benzyl), and Tyr (4- bromocarbobenzoxy). BOC deprotection may be carried out with trifluoroacetic acid in methylene chloride. Following synthesis, the resulting peptide may be deprotected and cleaved from the resin using, e.g., anhydrous HF containing 10% meta-cresol.

[0067] Exemplary recombinant methods used to prepare a GLP-1 moiety (whether a human GLP-1 or a different peptide having GLP-1 activity) include the following, among others as will be apparent to one skilled in the art. Typically, a GLP-1 moiety as described herein is prepared by constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria such as Escherichia coli, yeast such as Saccharomyces cerevisiae, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment. The expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression. Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art. See, for example, U.S. Patent No. 4,868,122. Often, GLP-1 is typically expressed in E. coli (since it doesn't require glycosylation for activity).

[0068] Yet further methods for making recombinant GLP-1 moieties are described in

WO 03/099847 and U.S. Patent No. 7,498,148.

[0069] To facilitate identification and purification of the recombinant polypeptide, nucleic acid sequences that encode an epitope tag or other affinity binding sequence can be inserted or added in- frame with the coding sequence, thereby producing a fusion protein comprised of the desired peptide and a polypeptide suited for binding. Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein. These and other methods for identifying and purifying recombinant peptides are known to those of ordinary skill in the art. In one or more embodiments of the present invention, the GLP-1 moiety is not in the form of a fusion protein. See, for example, Dillon et al. (1993) Endocrinology 133: 1907- 1910.

[0070] For any given moiety, it is possible to determine whether that moiety possesses

GLP-1 activity. Various assays may be used to assess bioactivity, including in-vitro and in- vivo assays that measure GLP-1 receptor binding activity or receptor activation. See, for example, EP 0 619 322 and U.S. Patent No. 5,120,712 for descriptions of assessing GLP-1 activity. A receptor-signaling assay may also be used to assess GLP-1 activity, such as described in Zlokarnik et al. (1998) Science 279:84-88.

[0071] Other methods known to those of ordinary skill in the art can also be used to determine whether a given moiety has GLP-1 activity. Such methods are useful for determining the GLP-1 activity of both the moiety itself (and therefore can be used as a "GLP- 1 moiety"), as well as that of the corresponding polymer-moiety conjugate. For example, one can determine whether a given moiety is an agonist of the human GLP-1 receptor by assessing whether that moiety stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor. The potency of such moiety is determined by calculating the EC50 value from a dose response curve. As an example, BHK cells (baby hamster kidney cells) expressing the cloned human GLP-1 receptor can be grown in DMEM media containing penicillin, streptomycin, calf serum, and Geneticin. The cells are then washed in phosphate buffered saline and harvested. Plasma membranes are then prepared from the cells by homogenization, and the homogenate is then centrifuged to produce a pellet. The resulting pellet is suspended by homogenization in a suitable buffer, centrifuged, and then washed. The cAMP receptor assay is then carried out by measuring cyclic AMP (cAMP) in response to the test insulinotropic moiety. cAMP can be quantified using the AlphaScreen™ cAMP Kit (Perkin Elmer). Incubations are typically carried out in microtiter plates in buffer, with addition of, e.g., ATP, GTP, IBMX (3-isobutyl-l-methylxanthine, Tween-20, BSA, acceptor beads, and donor beads incubated with biotinylated cAMP. Counting may be carried out, e.g., using the Fusion™ instrument (Perkin Elmer). Concentration-response curves are then plotted for the individual insulinotropic moieties under evaluation, and their EC₅₀ values determined.

[0072] Nonlimiting examples of GLP-1 moieties for use in connection with the present invention include any of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; and SEQ ID NO:4; truncated versions thereof; hybrid variants, and peptide mimetics having GLP-1 activity. Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of GLP-1 activity can also serve as a GLP-1 moiety in the conjugates of the invention.

[0073] Still further GLP-1 moieties for use in connection with the present invention include any of the GLP-1 moieties described herein modified via methylation, N-terminal modification and/or glycosylation.

[0074] With respect to methylation, a GLP-1 moiety may possess one or more methyl or other lower alkyl groups at one or more positions of the GLP-1 sequence. Examples of such groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, and so forth. Sites of modification include residues corresponding to positions 7, 8, 9, and/or 10 [based on a GLP-1 (7-36) numbering convention], with the 7 and/or 9 positions being preferred.

Introduction of one or more methyl groups is expected to modify the dipeptidyl peptidase IV (DPP IV) recognition site, such that the modified GLP-1 is protected against degradation by DPP IV.

[0075] Introduction of N-terminal modifications to GLP-1 may be carried out as described by Gallwitz et al. (2000) Regulatory Peptides 86(1-3): 103-111. Illustrative GLP-1 moieties with alterations at the N-terminus include N-methylated GLP-1 (N-me-GLP-1), alpha-methylated GLP-1 (alpha-me-GLP-1), desamidated GLP-1 (desamino-GLP-1), and imidazole-lactic acid substituted GLP-1 (imi-GLP-1), among others, and are suitable for use in the present invention.

[0076] Methods for synthesizing various other analogues of GLP-1 moieties, including methyl-derivatives as described above, as well examples of such additional GLP-1 analogues are described in WO 00/34332, WO 2004/074315 and WO 2005/058955.

[0077] With respect to glycosylation, the GLP-1 moieties described herein may also contain one or more glycosides. Although any glycoside can be used, the GLP-1 moiety is preferably modified by introduction of a monosaccharide, a disaccharide or a trisaccharide. Although any site on the GLP-1 moiety may be modified by introduction of a saccharide, preferably, the saccharide is introduced at a residue or residues corresponding to any one or more of positions 7, 8, or 9 [based on a GLP-l(7-36) numbering convention] to protect the peptide against DPP IV proteolysis. Further, additional glycosides may be introduced, e.g., at any one or more of positions 22, 23 and 24 [again, based on a GLP- 1(7-36) numbering convention] to increase the helicity through the central portion of the peptide, as well as provide additional resistance to proteolysis.

[0078] Glycosylated GLP-1 moieties are prepared using conventional Fmoc chemistry and solid phase peptide synthesis techniques, where the desired protected glycoamino acids are prepared prior to peptide synthesis and then introduced into the peptide chain at the desired position during peptide synthesis. Preparation of amino acid glycosides is described in U.S. Patent No. 5,767,254. Briefly, alpha and beta selective glycosylations of serine and threonine residues are carried out using the Koenigs-Knorr reaction and Lemieux's in situ anomerization methodology with Schiff base intermediates. Deprotection of the Schiff base glycoside is then carried out using mildly acidic conditions or hydrogenolysis.

[0079] Monosaccharides that may be used for introduction at one or more amino acid residues of GLP-1 include glucose (dextrose), fructose, galactose, and ribose. Additional monosaccharides suitable for use include glyceraldehydes, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose, as well as others. Glycosides, such as mono-, di-, and trisaccharides for use in modifying a GLP-1 moiety, may be naturally occurring or may be synthetic.

[0080] Disaccharides that may be used for introduction at one or more amino acid residues of GLP-1 include sucrose, lactose, maltose, trehalose, melibiose, and cellobiose, among others. Trisaccharides include acarbose, raffinose, and melezitose.

[0081] With respect to the water-soluble, non-peptidic polymer used in the polymer-GLP-1 conjugates in connection with the present invention, the water-soluble, non-peptidic polymer is hydrophilic, non-peptidic, and biocompatible. A substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such a therapeutic peptide) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician. A substance is considered nonimmunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g. , the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician. Typically, the water-soluble polymer is hydrophilic, biocompatible and nonimmunogenic.

[0082] Further the water-soluble polymer is typically characterized as having from 2 to about 300 termini, preferably from 2 to 100 termini, and more preferably from about 2 to 50 termini. Examples of such polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly( vinyl alcohol), polyphosphazene,

polyoxazoline, poly(N-acryloylmorpholine), and combinations of any of the foregoing, including copolymers and terpolymers thereof.

[0083] The water-soluble polymer is not limited to a particular structure and may possess a linear architecture (e.g., alkoxy PEG or bifunctional PEG), or a non-linear architecture, such as branched, forked, multi-armed (e.g., PEGs attached to a polyol core), or dendritic (i.e. having a densely branched structure with numerous end groups). Moreover, the polymer subunits can be organized in any number of different patterns and can be selected, e.g., from homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer. Preferred in connection with the present invention is a water-soluble, non-peptidic polymer that is branched and/or a homopolymer.

[0084] One particularly preferred type of water-soluble, non-peptidic polymer is a polyalkylene oxide, and in particular, polyethylene glycol (or PEG). Generally, a PEG used to prepare a therapeutic peptide polymer conjugate of the invention is "activated" or reactive. That is to say, the activated PEG (and other activated water-soluble polymers collectively referred to herein as "polymeric reagents") used to form a conjugate comprises an activated functional group suitable for coupling to a desired site or sites on the therapeutic peptide. Thus, a polymeric reagent for use in preparing a conjugate includes a functional group for reaction with the therapeutic peptide.

[0085] Representative polymeric reagents and methods for conjugating such polymers to an active moiety are known in the art, and are, e.g., described in Harris, J.M. and Zalipsky, S., eds, Poly (ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997; Veronese, F., and J.M Harris, eds., Peptide and Protein PEGylation, Advanced Drug Delivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al, "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky (1995) Advanced Drug Reviews .157-182, and in Roberts, et al, Adv. Drug Delivery Reviews, 54, 459 >-4Ί '6 (2002).

[0086] PEG reagents suitable for use in the present invention are available from commercial sources and can be prepared synthetically. Descriptions of polymeric reagents, as well as methods for making polymeric reagents, can be found in, for example, U.S. Patent Nos. 5,252,714, 5,650,234, 5,739,208, 5,932,462, 5,629,384, 5,672,662, 5,990,237,

6,448,369, 6,362,254, 6,495,659, 6,413,507, 6,376,604, 6,348,558, 6,602,498, 7,026,440, 7,157,546 and in U.S. Patent Application Publication No. 2005/0009988.

[0087] Typically, the weight- average molecular weight of the water-soluble polymer in the conjugate is from about 5,000 Daltons to about 150,000 Daltons. Exemplary ranges include weight-average molecular weights in the range of from about 5,000 Daltons to about 80,000 Daltons, from 5,000 Daltons to about 80,000 Daltons, from about 5,000 Daltons to about 65,000 Daltons, from about 5,000 Daltons to about 40,000 Daltons, from 5,000 Daltons to about 40,000 Daltons, from greater than 5,000 Daltons to about 80,000 Daltons, from about 10,000 Daltons to about 80,000 Daltons, from about 15,000 Daltons to about 45,000 Daltons, from about 20,000 Daltons to about 45,000 Daltons, from about 30,000 Daltons to about 50,000 Daltons, and from about 35,000 Daltons to about 45,000 Daltons.

[0088] Exemplary weight-average molecular weights for the water-soluble polymer include about about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons.

[0089] Branched versions of the water-soluble polymer (e.g. , a branched 40,000

Dalton water-soluble polymer comprised of two 20,000 Dalton polymers or the like) having a total molecular weight of any of the foregoing can also be used. In one or more particular embodiments, depending upon the other features of the subject therapeutic peptide polymer conjugate, the conjugate is one that does not have one or more attached PEG moieties having a weight-average molecular weight of less than about 6,000 Daltons. [0090]

typically comprise a number of (OCH₂CH₂) monomers. As used herein, the number of repeat units is typically identified by the subscript "n" in, for example, "(OCH₂CH₂)_n." Thus, the value of (n) typically falls within one or more of the following ranges: from 113 to about 2050; from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900. For any given polymer in which the molecular weight is known, it is possible to determine the number of repeating units (i.e., V) by dividing the total weight-average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0091] A polymer for use in the invention may be end-capped, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower alkoxy group (i.e., a Ci_₆ alkoxy group) or a hydroxyl group. One frequently employed end-capped polymer is methoxy-PEG (commonly referred to as mPEG), wherein one terminus of the polymer is a methoxy (-OCH₃) group. The -PEG- symbol used in the foregoing generally represents the following structural unit: -CH₂CH₂0-(CH₂CH₂0)_n-CH₂CH₂-, where (n) generally ranges from about zero to about 4,000.

[0092] Multi-armed or branched PEG molecules, such as those described in U.S.

Patent No. 5,932,462, are also suitable for use in the present invention. For example, the PEG may be described generally according to the structure:

Pob/a— P

R' — C—

polyb— ^Q

where poly_a and poly_b are PEG backbones (either the same or different), such as methoxy poly(ethylene glycol); R" is a non-reactive moiety, such as H, methyl or a PEG backbone; and P and Q are non-reactive linkages. In one embodiment, the branched PEG molecule is one that includes a lysine residue, such as the following reactive PEG suitable for use in forming a therapeutic peptide conjugate. Although the branched PEG below is shown with a reactive succinimidyl group, this represents only one of a myriad of reactive functional groups suitable for reacting with a therapeutic peptide. lysine residue

Lysine Branched mPEG Succinimidyl Derivative

[0093] In some instances, the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a lysine residue in which the polymeric portions are connected to amine groups of the lysine via a "-OCH₂CONHCH₂CO-" group. In still other instances, the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a branched water-soluble polymer that includes a lysine residue (wherein the lysine residue is used to effect branching).

[0094] Additional branched PEGs for use as polymeric reagents to prepare the polymer-GLP-1 conjugates include those polymer reagents described in U.S. Patent

Application Publication No. 2005/0009988. Representative branched polymers described therein include those having the following generalized structure:

R¹ O

POLY¹ (X¹ )a-N-C-0-(X²)_b

R¹ O _/ ^r5— (^χ7)₉— (ΟΗ₂ΟΗ₂0)|-(Χ¾— Z

POLY² (X⁵)e-N-C-0-(X^b)_f

wherein: POLY¹ is a water-soluble polymer; POLY² is a water-soluble polymer; (a) is 0, 1, 2 or 3; (b) is 0, 1, 2 or 3; (e) is 0, 1, 2 or 3; (f) is 0, 1 , 2 or 3; (g') is 0, 1, 2 or 3; (h) is 0, 1, 2 or 3; (j) is 0 to 20; each R¹ is independently H or an organic radical selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl; X , when present, is a spacer moiety; X , when present, is a spacer moiety;

6 7

X , when present, is a spacer moiety; X , when present, is a spacer moiety; X , when present, is a spacer moiety; X^s, when present, is a spacer moiety; R⁵ is a branching moiety; and Z is a reactive group for coupling to a therapeutic peptide, optionally via an intervening spacer. POLY¹ and POLY² in the preceding branched polymer structure may be different or identical, i.e., are of the same polymer type (structure) and molecular weight. [0095] A exemplary branched construct used in a branched polymer corresponds to the following structure:

9

H₃CO-(CH₂CH₂0)_n— CH₂CH₂-NH-C-0-

9

H₃CO-(CH₂CH₂0)_n-CH2CH2-NH-C -0- wherein each n is from 113 to about 2050. Within this exemplary branched construct, exemplary branched polymeric reagents can have the following structure:

wherein each n is from 113 to about 2050 and Z is an electrophile-containing

[0096] Branched polymers suitable for preparing conjugates useful in connection with the present invention also include those represented more generally by the formula R(POLY)_y, where R is a central or core molecule from which extends 2 or more POLY arms such as PEG. The variable y represents the number of POLY arms, where each of the polymer arms can independently be end-capped or alternatively, possess a reactive functional group at its terminus. A more explicit structure in accordance with this embodiment of the invention possesses the structure, R(POLY-Z)_y, where each Z is independently an end-capping group or a reactive group, e.g., suitable for reaction with a therapeutic peptide. In yet a further embodiment when Z is a reactive group, upon reaction with a therapeutic peptide, the resulting linkage can be hydrolytically stable, or alternatively, may be degradable, i.e., hydrolyzable. Typically, at least one polymer arm possesses a terminal functional group suitable for reaction with, e.g. , a therapeutic peptide. Branched PEGs such as those represented generally by the formula, R(PEG)_y above possess 2 polymer arms to about 300 polymer arms (i.e., n ranges from 2 to about 300). Preferably, such branched PEGs typically possess from 2 to about 25 polymer arms, such as from 2 to about 20 polymer arms, from 2 to about 15 polymer arms, or from 3 to about 15 polymer arms. Multi-armed polymers include those having 3, 4, 5, 6, 7 or 8 arms.

[0097] Core molecules in branched PEGs as described above include polyols, which are then further functionalized. Such polyols include aliphatic polyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxyl groups, including ethylene glycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,

4,8-bis(hydroxymethyl)tricyclodecane, cycloalkylidene diols, dihydroxyalkanes,

trihydroxyalkanes, and the like. Cycloaliphatic polyols may also be employed, including straight chained or closed-ring sugars and sugar alcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol, ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like. Additional aliphatic polyols include derivatives of glyceraldehyde, glucose, ribose, mannose, galactose, and related stereoisomers. Other core polyols that may be used include crown ether,

cyclodextrins, dextrins and other carbohydrates such as starches and amylose. Typical polyols include glycerol, pentaerythritol, sorbitol, and trimethylolpropane.

[0098] Alternatively, the polymer may possess an overall forked structure as described in U.S. Patent No. 6,362,254. This type of polymer is useful for reaction with two therapeutic peptide moieties, where the two therapeutic peptide moieties are positioned a precise or predetermined distance apart.

[0099] In any of the representative structures provided herein, one or more degradable linkages may additionally be contained in the polymer, POLY, to allow generation in vivo of a conjugate having a smaller PEG chain than in the initially administered conjugate.

Appropriate physiologically cleavable (i.e., releasable) linkages include but are not limited to ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such linkages when contained in a given polymer segment will often be stable upon storage and upon initial administration.

[0100] The PEG polymer used to prepare a conjugate may comprise a pendant PEG molecule having reactive groups, such as carboxyl or amino, covalently attached along the length of the PEG rather than at the end of the PEG chain(s). The pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.

[0101] One of ordinary skill in the art can determine the proper molecular size of the water-soluble, non-peptidic polymer. For example, one of ordinary skill in the art, using routine experimentation, can determine a proper molecular size by first preparing a variety of conjugates with different weight-average molecular weights of the polymer and then obtaining the clearance profile for each conjugate by administering the conjugate to a patient and taking periodic blood and/or urine samples. Once a series of clearance profiles has been obtained for each tested conjugate, a conjugate or mixture of conjugates having the desired clearance profile(s) can be determined.

[0102] Those of ordinary skill in the art will recognize that the foregoing discussion describing water-soluble polymers for use in forming a conjugate is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated. As used herein, the term "polymeric reagent" generally refers to an entire molecule, which can comprise a water-soluble polymer segment, as well as additional spacers and functional groups.

[0103] Each conjugate in the dose of polymer-GLP-1 conjugates contained with the pharmaceutical composition has the water-soluble, non-peptidic polymer covalently attached via an amide-containing linkage to an amino group of the GLP-1 moiety. Thus, although the overall linkage between the residue of the GLP-1 moiety and the water-soluble, non-peptidic polymer will depend on a number of factors, the linkage will nevertheless contain a amide (i.e., a -NHC(O)- or -C(O)NH-) group. Such factors include, for example, the particular linkage chemistry employed, the particular atoms (if any) surrounding the functional groups effecting the linkage, and so forth. The amide- containing linkage is preferably relatively stable.

[0104] Typically, the nitrogen atom within the amide-containing linkage is contributed by an amine group associated with the GLP-1 moiety. When an amine group associated with the GLP-1 moiety contributes the nitrogen for the amide-containing, usually it is the amine acting as a nucleophile for an electrophilically activated polymeric reagent (e.g., a

water-soluble, non-peptidic polymer bearing an electrophile). In this way, this nitrogen atom effectively becomes the point of attachment for the water-soluble, non-peptidic polymer.

[0105] Exemplary electrophilically activated polymeric reagents include

water-soluble, non-peptidic polymers bearing an electrophile selected from the group consisting of acetals, esters (such as succinimidyl esters of carboxylic acids) and carbonates. For example, an electrophilically activated polymeric reagent useful in connection with the present invention is encompassed by the following structure:

H wherein each n is from 113 to about 2050. Use of this polymeric reagent, produces the exemplary polymer-GLP-1 conjugate of the formula (shown with the location of the amide-containing linkage):

wherein each n is from 113 to about 2050 and (GLP-1) is a residue of a GLP-1 moiety.

[0106] Other exemplary polymeric reagents and the corresponding conjugate resulting therefrom for use in connection with the present invention are provided in the table below. In the table, the variable (n) represents the number of repeating monomeric units and "GLP-1 " represents a GLP-1 moeity following conjugation to the polymeric reagent. While each polymeric portion [e.g., (OCH₂CH₂)_n or (CH₂CH₂0)_n] presented in the terminates in a "CH₃" roup, other groups (such as H and benzyl) can be substituted therefore.

mPEG-Thioester Reagent

[0107] Conjugation of a polymeric reagent to a nitrogen atom within a GLP-1 moiety can be accomplished by a variety of techniques. In one approach, the GLP-1 moiety is conjugated to a polymeric reagent ranctionalized with an active ester such as a succinimidyl derivative (e.g., an N-hydroxysuccinimide ester). In this approach, the polymeric reagent bearing the reactive ester is reacted with the GLP-1 moiety in aqueous media under appropriate pH conditions, e.g. , from pHs ranging from about 3 to about 8, about 3 to about 7, or about 4 to about 6.5. Most polymer active esters can couple to a target peptide such as GLP-1 moiety at physiological pH, e.g., at 7.0. However, less reactive derivatives may require a different pH. Typically, activated PEGs can be attached to a peptide such as therapeutic peptide at pHs from about 7.0 to about 10.0 for covalent attachment to an internal lysine. Typically, lower pHs are used, e.g., 4 to about 5.75, for preferential covalent attachment to the N-terminus. Conjugation reactions can often be carried out at room temperature, although lower temperatures may also be used. Reaction times are typically on the order of minutes, e.g., 30 minutes, to hours, e.g., from about 1 to about 36 hours), depending upon the pH and temperature of the reaction. Varying ratios of polymeric reagent to the GLP-1 moiety may be employed, e.g., from an equimolar ratio up to a 10-fold molar excess of polymeric reagent. Typically, up to a 5-fold molar excess of polymeric reagent will suffice.

[0108] Progress of the reaction can be monitored by withdrawing aliquots from the reaction mixture at various time points and analyzing the reaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analytical method. Once a plateau is reached with respect to the amount of conjugate formed or the amount of unconjugated polymer remaining, the reaction is assumed to be complete. The resulting product mixture is preferably, but not necessarily purified, to separate out excess reagents, unconjugated reactants (e.g., GLP-1 in unconjugated form) undesired multi-conjugated species, and free or unreacted polymeric reagent. The resulting conjugates can then be further characterized using analytical methods such as MALDI, capillary electrophoresis, gel electrophoresis, and/or chromatography.

[0109] With respect to the specific nitrogen atom contributed by the GLP-1 moiety for forming an amide- containing linkage (and which represents the point of attachment for the water-soluble, non-peptidic polymer), the nitrogen atom can be associated with the N-terminal amine of the GLP-1 moiety or be associated with the epsilon amine of a lysine residue within the GLP-1 moiety. Based on a GLP-1 (7-36) numbering convention, lysine residues corresponding to positions 26 (i.e., Lys²⁶) and 34 (i.e., Lys³⁴) are preferred, although other nitrogen atom-containing locations (e.g., as may be introduced by aminating the carboxyl terminus or inserting a lysine residue) that may exist in a given GLP-1 moiety are

contemplated as well.

[0110] It is possible to influence at which nitrogen atom within the GLP-1 moiety the polymeric reagent will attach. Techniques to do so include changing pH (to render amines more or less available for attachment based on pKa), using blocking chemistries (wherein one or more amines can be selectively blocked prior to conjugation with a polymeric reagent, followed by a de-blocking step) and incorporating the water-soluble, non-peptidic polymer during peptide synthesis (see, for example, WO 95/00162). In this regard, reference is made to the Experimental section for a description of influencing the location of polymeric reagent attachment.

[0111] Even with the ability to influence the location of attachment, it is seldom the case that the attachment occurs only at an intended location. As a consequence, compositions are formed that result in positional isomers (wherein, among conjugates in the composition, location(s) of attachment change although the number of polymers does not) and numeric isomers (wherein, among conjugates within the composition, the number of polymers may change).

[0112] For polymer-GLP-1 conjugates in which it is intended that the N-terminal amine is the most represented location of polymer attachment within a composition, it is preferred that at least 60%, more preferably at least 70%, still more preferably at least 80%, and yet still more preferably at least 90% of all polymer-GLP-1 conjugates in the composition have only a single attachment of a water-soluble, non-peptidic polymer attached at the N-terminal amine.

[0113] For polymer-GLP-1 conjugates in which it is intended that locations corresponding to lysine residues Lys²⁶ and Lys³⁴ [based on a GLP-l (7-36) numbering convention] are the predominant attachment sites, it is preferred that at least 75%, more preferably at least 85%, still more preferably at least 95%, and yet still more preferably at least 99% of all polymer-GLP-1 conjugates in the composition have one attachments at one or both of locations corresponding to lysine residues Lys and Lys . As between, Lys and Lys³⁴, it is preferred that the compositions have a majority of conjugates wherein attachment occurs at Lys²⁶ (e.g., a Lys²⁶/Lys³⁴ ratio of 60/40.

[0114] The amide- containing linkage that serves to link the GLP-1 moiety to the water-soluble, non-peptidic polymer can include one or more additional atoms in addition to the amide. The one or more additional atoms making up the amide-containing linkage can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof. Nonlimiting examples of amide-containing linkages include those selected from the group consisting of -C(0)-NH-, -NH-C(O)-, -NH-C(0)-NH-, -0-C(0)-NH-, -NH-C(0)-0-, -C(0)-NH-CH₂-, -C(0)-NH-CH₂-CH₂-, -CH₂-C(0)-NH-CH₂-,

-CH₂-CH₂-C(0)-NH-, -C(0)-NH-CH₂-CH₂-CH₂-, -CH₂-C(0)-NH-CH₂-CH₂-,

-CH₂-CH₂-C(0)-NH-CH₂-, -CH₂-CH₂-CH₂-C(0)-NH-, -C(0)-NH-CH₂-CH₂-CH₂-CH₂-, -CH₂-C(0)-NH-CH₂-CH₂-CH₂-, -CH₂-CH₂-C(0)-NH-CH₂-CH₂-,

-CH₂-CH₂-CH₂-C(0)-NH-CH₂-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-,

-CH₂-CH₂-CH₂-CH₂-C(0)-NH-, -NH-C(0)-CH₂-, -CH₂-NH-C(0)-CH₂-,

-CH₂-CH₂-NH-C(0)-CH₂-, -NH-C(0)-CH₂-CH₂-, -CH₂-NH-C(0)-CH₂-CH₂-,

-CH₂-CH₂-NH-C(0)-CH₂-CH₂-, -C(0)-NH-CH₂-, -C(0)-NH-CH₂-CH₂-, -0-C(0)-NH-CH₂-, -0-C(0)-NH-CH₂-CH₂-, -CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-,

-CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-C(0)-,

-CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-C(0)-CH₂-,

-CH₂-CH₂-CH₂-C(0)-NH-CH₂-CH₂-NH-C(0)-CH₂-CH₂-, -0-C(0)-NH-[CH₂]_h-(OCH₂CH₂)_j-, (h) is zero to six, and (j) is zero to 20. Other amide-containing linkages have the following structures: -C(0)-NH-(CH₂)_1-6-NH-C(0)-, -NH-C(0)-NH-(CH₂)_1-6-NH-C(0)-, and

-0-C(0)-NH-(CH₂)i-₆-NH-C(0)-, wherein the subscript values following each methylene indicate the number of methylenes contained in the structure, e.g., (CH₂)i- means that the linkage can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, any of the above

amide-containing linkages may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., -(CH₂CH₂0)_1-2o]. That is, the ethylene oxide oligomer chain can occur before or after the amide-containing linkage. Also, the oligomer chain would not be considered part of the amide-containing linkage if the oligomer is adjacent to a water-soluble, non-peptidic polymer and merely represents an extension of the polymer.

[0115] The polymer-GLP-1 conjugates associated with the present invention can be purified to obtain/isolate different conjugate species. Specifically, a product mixture can be purified to obtain the desired numeric isomer. In one embodiment of the invention, the GLP-1 conjugates that make up the dose of polymer-GLP-1 conjugates are mono-conjugates. The strategy for purification of a conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular GLP-1 moiety, and the desired characteristics of the product - e.g., monomer, dimer, particular positional isomers, and so forth.

[0116] If desired, conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography. Gel filtration

chromatography may be used to fractionate different conjugates (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein "1-mer" indicates one polymer molecule per GLP-1 moeity, "2-mer" indicates two polymers attached to the GLP-1 moiety, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble, non-peptidic polymer). While this approach can be used to separate PEG and other water-soluble, non-peptidic polymer conjugates having different molecular weights, this approach is generally ineffective for separating positional isomers having different polymer attachment sites within the GLP-1 moiety. For example, gel filtration chromatography can be used to separate from each other mixtures of PEG 1-mers, 2- mers, 3-mers, and so forth, although each of the recovered PEG-mer compositions may contain PEGs attached to different reactive amino groups (e.g., lysine residues) or other functional groups of the therapeutic peptide.

[0117] Gel filtration columns suitable for carrying out this type of separation include

Superdex™ and Sephadex™ columns available from Amersham Biosciences (Piscataway, NJ). Selection of a particular column will depend upon the desired fractionation range desired. Elution is generally carried out using a suitable buffer, such as phosphate, acetate, or the like. The collected fractions may be analyzed by a number of different methods, for example, (i) optical density (OD) at 280 nm for protein content, (ii) bovine serum albumin (BSA) protein analysis, (iii) iodine testing for PEG content (Sims et al. (1980) Anal. Biochem, 107:60-63), and (iv) sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS PAGE), followed by staining with barium iodide.

[0118] Separation of positional isomers is typically carried out by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) CI 8 column (Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a DEAE- or CM-Sepharose™ ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-therapeutic peptide isomers having the same molecular weight (positional isomers).

[0119] The resulting purified compositions are preferably substantially free of the non-conjugated GLP-1 moiety. In addition, the compositions preferably are substantially free of all other non-covalently attached water-soluble, non-peptidic polymers. Further, the compositions are preferably substantially free of albumin.

[0120] With respect to pharmaceutical compositions, the pharmaceutical composition will typically satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have one polymer attached to the GLP-1 moiety; at least about 95% of the conjugates in the composition will one polymer attached to the GLP-1 moiety; and at least about 99% of the conjugates in the composition will have one polymer attached to the GLP-1 moiety.

[0121] Moreover, with respect to pharmaceutical excipients, the pharmaceutical composition of the invention may contain only one pharmaceutical excipient or the pharmaceutical composition may contain more than one pharmaceutical excipient. The specific pharmaceutical excipient(s) included in the composition can vary and is influenced by the particular needs of the formulation and route of administration.

[0122] The pharmaceutical compositions of the invention encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids, as well as for inhalation. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic endotoxin-free water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. [0123]

carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

[0124] Representative carbohydrates for use in the compositions of the present invention include sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers. Exemplary carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like. Preferred, in particular for formulations intended for inhalation, are non-reducing sugars, sugars that can form a substantially dry amorphous or glassy phase when combined with the composition of the present invention, and sugars possessing relatively high glass transition temperatures, or Tgs (e.g., Tgs greater than 40°C, or greater than 50°C, or greater than 60°C, or greater than 70°C, or having Tgs of 80°C and above). Such excipients may be considered glass-forming excipients.

[0125] Exemplary protein excipients include albumins such as human serum albumin

(HSA), recombinant human albumin (rHA), gelatin, casein, hemoglobin, and the like. The compositions may also include a buffer or a pH-adjusting agent, typically but not necessarily a salt prepared from an organic acid or base. Representative buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid. Other suitable buffers include Tris, tromethamine hydrochloride, borate, glycerol phosphate, and phosphate. Amino acids such as glycine are also suitable.

[0126] The pharmaceutical compositions of the present invention may also include one or more additional polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and

hydroxypropylmethylcellulose, FICOLLs (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin and sulfobutylether-β- cyclodextrin), polyethylene glycols, and pectin.

[0127] The pharmaceutical compositions may further include flavoring agents, taste- masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g.,

benzalkonium chloride), sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as 'TWEEN 20" and 'TWEEN 80," and pluronics such as F68 and F88, available from BASF), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., zinc and other such suitable cations). The use of certain di-substituted phosphatidylcholines for producing perforated microstructures (i.e., hollow, porous microspheres) may also be employed.

[0128] Other pharmaceutical excipients and/or additives suitable for use in the compositions according to the present invention are listed in "Remington: The Science & Practice of Pharmacy," 21^st ed., Williams & Williams, (2005), and in the "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, N.J. (2006).

[0129] The dose of the polymer-GLP-1 conjugate in a pharmaceutical compositions

(typically present as a pharmaceutical composition having a single dose) is an amount that achieves a glucose-lowering effect, which dose is less (in terms of amount) than the dose of the GLP-1 moiety in unconjugated form that would be required to achieve the same glucose-lowering effect. In addition, a pharmaceutical preparation, if in solution form, can be housed in a syringe.

[0130] The amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.

[0131] Generally, however, the excipient or excipients will be present in the composition in an amount of about 1% to about 99% by weight, from about 5% to about 98% by weight, from about 15 to about 95% by weight of the excipient, or with concentrations less than 30% by weight. In general, a high concentration of the therapeutic peptide is desired in the final pharmaceutical formulation.

[0132] The pharmaceutical compositions described herein can be administered by any of a number of routes including without limitation, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intrathecal, and pulmonary. Preferred forms of administration include parenteral and pulmonary. Suitable formulation types for parenteral administration include ready- for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.

[0133] In one or more embodiments of the invention, a method is provided, the method comprising delivering a conjugate to a patient, the method comprising the step of administering to the patient a pharmaceutical composition as provided herein. Administration can be effected by any of the routes herein described. The method may be used to treat a mammal suffering from diabetes (e.g., Type II diabetes).

[0134] The actual dose of the conjugate to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered. On a weight basis, a therapeutically effective dosage amount of a therapeutic peptide conjugate as described herein will range from about 0.001 mg per day to about 1000 mg per day for an adult. For example, dosages may range from about 0.1 mg per day to about 100 mg per day, or from about 1.0 mg per day to about 10 mg/day. On an activity basis, corresponding doses based on international units of activity can be calculated by one of ordinary skill in the art.

[0135] The unit dosage of any given conjugate (again, such as provided as part of a pharmaceutical composition) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined

experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.

[0136] It is to be understood that while the invention has been described in

conjunction with the preferred specific embodiments thereof, the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. [0137] All articles, books, patents and other publications referenced herein are hereby incorporated by reference in their entireties.

EXPERIMENTAL

[0138] The practice of the invention will employ, unless otherwise indicated, conventional techniques of organic synthesis, biochemistry, protein purification and the like, which are within the skill of the art. Such techniques are fully explained in the literature. See, for example, J. March, Advanced Organic Chemistry: Reactions Mechanisms and Structure, 4th Ed. (New York: Wiley- Interscience, 1992), supra.

[0139] In the following examples, efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.) but some experimental error and deviation should be taken into account. Unless indicated otherwise, temperature is in degrees C and pressure is at or near atmospheric pressure at sea level. Each of the following examples is considered to be instructive to one of ordinary skill in the art for carrying out one or more of the embodiments described herein.

[0140] Recombinant-GLP-1 used in the Experimental was obtained from LGM

Pharma Inc. (Boca Raton, FL 33487, USA), CAS# 107444-51-9 Batch No. 20090326.

[0141] SDS-PAGE was used to analyze the purity and assess the molecular weights of

GLP-1 conjugates. Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Bio-Rad system (Mini-PROTEAN III Precast Gel Electrophoresis System), and Invitrogen system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared samples were loaded onto a gel and run for approximately thirty minutes.

[0142] To analyze the purity of GLP-1 conjugates and monitor the PEGylation reactions, reversed phase high-performance liquid chromatography (RP-HPLC) was performed on an Agilent 1200 HPLC system (Agilent). Samples were analyzed using an Intrada WP-RP column (3 μπι particle size, 150 x 4.6 mm, Silverton, Japan), and mobile phases consisting of 0.09% trifluoroacetic acid in water (buffer A) and 0.04% trifluoroacetic acid in acetonitrile (buffer B). The flow rate for the column was 0.5 ml/min. The protein and PEG-protein conjugates were eluted with a linear gradient 25%-65% over 30 minutes, and were visualized a Diode Array detector at 220, 280 and 320 nm. [0143] Molecular weights GLP-1 compounds were analyzed with MALDI-TOF spectrophotometer, and sites of PEGylation were confirmed with peptide mapping using Trypsin/Glu-C. The digestion product was analyzed by LC/MALDI-TOF.

[0144] When present, dimers identified through RP-HPLC indicate protein dimer aggregates (and lack any polymeric component).

[0145] Cation exchange chromatography was used to purity GLP-1 conjugates. A

HiTrap SP Sepharose HP cation exchange column (Amersham Biosciences) was used with the AKTA Explorer 100 system (GE Bioscience) to purify the PEG-GLP-1 conjugates. For each conjugate solution prepared, the conjugate solution was loaded on a column that was pre-equilibrated in 20 mM NaOAc buffer, pH 4.0 (buffer A) and then washed with ten column volumes of buffer A to remove any unreacted PEG reagent. Subsequently, a linear gradient of buffer A and buffer B (lOmM NaOAc with 1.0 M NaCl buffer, pH 4.0) was applied over 20 column volumes. The eluent was monitored by UV detector at 220 nm, 280 nm and 320 nm. The purity of individuals fractions of GLP-1 conjugates was determined by RP-HPLC and SDS-PAGE.

[0146] The fractions of the purified GLP-1 conjugates from cation exchange chromatography were pooled and further purified with CG-71 S reverse phase

chromatography. When cation exchange chromatography was conducted, a HiTrap SP Sepharose HP cation exchange column (Roam Haas) with the AKTA Explorer 100 system (GE Bioscience). For each conjugate solution prepared, the conjugate solution was loaded on the CG71 S column that was pre-equilibrated in 5 % NaoAc/H₂0 v/v (buffer A) and then washed with ten column volumes of buffer A to remove undesired salt. Subsequently, a linear gradient of buffer A and buffer B (5 % NaoAc/acetonitrile v/v) was applied over 10 column volumes. The eluent was monitored by UV detector at 220, 280, and 320 nm. The purity of individual fractions of GLP-1 conjugates was analyzed by RP-HPLC and SDS-PAGE. The fractions were pooled and lyophilized, and the lyohpilyzed powder of GLP-1 conjugates was re-suspended in 2 mM HC1 for endotoxin assay (LAL assay, Charles River, Inc), and aliquoted for further characterization. Example 1

Preparation of PEG₂-ru-40K-Nter-GLP-l

mPEG₂-ru-40 -N-Hydroxysuccinimide Derivative, 40kDa, ("mPEG₂-ru-40 -NHS")

[0147] mPEG₂-ru-40K-NHS, stored at -80 °C under argon, was warmed to ambient temperature under nitrogen purging. 200 mG/mL stock solutioners were prepared in 2 n M

HCl, and a molar ratio of 2.5:1 (PEG reagent/GLP-1) was added to a GLP-1 solution to reach a final GLP-1 concentration of 1.5 mG/ML (0.45 raM). MES buffer (0.5 M, pH 6.0) was added to the PEG-GLP-1 mixture to a final concentration of 20 mM, and the PEGylation was allowed to react for three hours. A solution containing 1 M Hydroxyamine and 1 M glycine (pH 6.0) was added to the reaction mixture to final concentrations of 100 mM Hydroxyamine and 100 mM glycine to stop the PEGylation reaction and remove undesired imidazole PEG side product. The reaction was allowed to continue for one hour, and was diluted with H₂0 to a conductivity below 0.5 mS/cm (25 °C). pH was then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification.

[0148] A typical SP-HP cation exchange purification profile of PEG₂-ru-40K-

Nter-GLP-1 is provided in FIG. 1. The PEG₂-ru-40K-Nter-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm). Purity analysis of PEG₂-ru-40 -Nter-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 100% at 280 nm. See FIG. 2.

Purity as determined by 4-12% NuPage Bis-Tris SDS-PAGE with Coomassie Blue staining (gel not shown) resulted in an apparent large molecule weight (66-54 kDa) of PEG₂-ru-40K- Nter-GLP-1, likely attributable to the slow mobility of the conjugate through the gel due to a high degree of PEG hydration. Finally, MALDI-TOF analysis (spectrum not shown) evidenced a peak at 43138 Da, which agrees with the calculated molecular weight of the PEG₂-ru-40K-Nter-GLP-l . Another peak at 87563 Da may represent a singly charged conjugate dimer or a di-PEG GLP-1 conjugate. Example 2

Preparation of PEG₂-ru-40K-Lys26/34-GLP-l

mPEG₂-ru-40K-N-Hydroxysuccinimide Derivative, 40kDa, ("mPEG₂-ru-40K-NHS")

[0149] mPEG₂-ru-40K-N-Hydroxysuccinimide Derivative, 40kDa, ("mPEG₂-ru-

40K-NHS"), stored at -80 °C under argon, is warmed to ambient temperature under nitrogen purging. 200 mG/niL stock solutions of were prepared in 2 mM HC1, and a molar ratio of 2.5: 1 (PEG/GLP-1) was added to GLP-1 solution to reach a final GLP-1 concentration of 1.5 mG/mL (0.45 mM). MES buffer (0.5 M, pH 6.0) was added to the PEG-GLP-1 mixture to a final concentration of 20 mM, and the PEGylation was allowed to react for three hours. A solution containing 1 M Hydroxyamine and 1 M glycine (pH 6.0) was added to the reaction mixture to final concentrations of 100 mM Hydroxyamine and 100 mM glycine to stop the PEGylation reaction and remove undesired imidazole PEG side product. The reaction was allowed to continue for one hour, and was diluted with H₂0 to the conductivity below 0.5 mS/cm (25 °C). pH was then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification.

[0150] A typical SP-HP cation exchange purification profile of PEG₂-ru-40K-

Lys26/34-GLP-l is provided in FIG. 3. The PEG₂-ru-40K-Lys26/34-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm). Purity analysis of PEG₂-ru-40K-Lys26/34-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 99.6% at 280 nm. The peaks at 18.7 minutes may represent the di-PEGylated GLP-1 conjugate. See FIG. 4. Purity as determined by 4-12% NuPage Bis-Tris SDS-PAGE with Coomassie Blue staining (gel not shown) resulted in an apparent large molecule weight (66-54 kDa) of PEG₂-ru-40K- Lys26/34-GLP-l , likely attributable to the slow mobility of the conjugate through the gel due to a high degree of PEG hydration. Finally, MALDI-TOF analysis (spectrum not shown) evidenced a peak at 43090 Da, which agrees with the calculated molecular weight of the PEG₂-ru-40K-Lys26/34-GLP-l . Another peak at 87672 Da may represent a singly charged conjugate dimer or a di-PEG GLP-1 conjugate. Example 3 - Comparative Example

Preparation of PEG-30K-N^ter-ButyrALD-GLP-l

CH₃0(CH₂CH₂0)_n-C(0)NH-(CH₂CH₂0)₄CH₂CH₂CH₂CHO Linear mPEG-Butyraldehyde

Derivative, 30kDa ("mPEG-ButyrALD")

[0151] mPEG-ButyrALD, 30kDa, stored at -80 °C under argon, was warmed to ambient temperature under nitrogen purging. 60 mG/mL stock solutions of were prepared in 2 mM HC1, and a molar ratio of 3: 1 (PEG/GLP-1) was added to GLP-1 solution to reach a final GLP-1 concentration of 1.5 mG/mL (0.45 mM). NaoAc buffer (1 M, pH 4.0) was added to the PEG-GLP-1 mixture to a final concentration of 20 mM, and the PEG/GLP-1 mixture was allowed to mix at 4 °C for one hour. Sodium cyanoborohydrate was then added to the mixture to a final concentration of Add 5 mM, and the PEGylation reaction was allowed to occur at 4°C overnight on a stir plate. The reaction was stopped with a final concentration of 100 mM glycine, and was diluted with H₂0 to the conductivity below 0.5 mS/cm (25 °C). pH was then adjusted to 4.0 using glacial acetic acid prior to column chromatography

purification.

[0152] A typical SP-HP cation exchange purification profile of PEG-30K-Nter-

Butyr ALD-GLP- 1 is provided in FIG. 5. The PEG-30K-Nter-ButyrALD-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm). Purity analysis of PEG-30K-Nter-ButyrALD-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 97% at 280 nm with a retention time at 17 minutes. The peak at 15.2 minutes represent GLP-1 in unconjugated form. See FIG. 6. Purity as determined by 4-12% NuPage Bis-Tris SDS- PAGE with Coomassie Blue staining (gel not shown) indicated a PEG-30K-Nter- Butyr ALD-GLP- 1 purity of >95%. Finally, MALDI-TOF analysis (spectrum not shown) evidenced a peak at 33,538.416 Da, which is within the expected range for the molecular weight of PEG-30K-Nter-Butyr ALD-GLP- 1. Example 4

Stability of PEG₂-ru-40K-N^ter-GLP-l (Example 1) and

PEG₂-ru-40K-Lys^{26 34}-GLP-l (Example 2)

[0153] PEG₂-ru-40K-Nter-GLP- 1 and PEG₂-ru-40K-Lys26/34-GLP-l were shown to be stable in not only fresh murine and rodent plasma, but also in fresh murine and rodent blood. When spiked in various biological matrices listed above at 37° C for up to 28 hours, the two conjugates remained stable, as evidenced by no detectable appearance of PEG or GLP-1 from either conjugate at 28 hours when tested via LC/MS system containing Agilentl200 and Qtrap400 TripleQual Spectrophotometer. These results were expected as the amide bond within the amide-containing linker is stable under physiological conditions. The

plasma/blood stability of PEG-30 -N^ter-ButyrALD-GLP-l (Example 3) was not determined but the conjugate is expected to be stable.

[0154] The gradient settings for GLP-1 conjugates and PEG quantification were as follows: mobile phase A - 0.1 % fomic acid/H₂0; and mobile phase B - 0.1 % fomic acid/acetonitrile, wherein the parameters for each step were as follows: step 0, total time - 0.00, flow rate 500 μΐ/min, 80%A 20%B; step 1 , total time - 1.00, flow rate 500 μΐ/min, 80%A/20%B; step 2, total time - 6.00, flow rate 500 μΐ/min, 55%A/45%B; step 3, total time - 1 1.00, flow rate 500 μΐ/min, 35%A/65%B; step 4, total time - 1 1.30, flow rate 500 μΐ/min, 10%A/90%B; step 5, total time - 13.00, flow rate 500 μΐ/min, 10%A/90%B; and step 6, total time - 13.01, flow rate 750 μΐ/min, 80%A/20%B. An Intrada reverse phase column (WP-RP 3 μιη, 5mm x 150 mm) was used.

[0155] The gradient settings for GLP-1 (7-36) and GLP-1 (9-36) quantification were as follows: mobile phase A - 10 mM ammonium acetate/H₂0; and mobile phase B - 10 mM ammonium acetate/acetonitrile, wherein the parameters for each step were as follows: step 0, total time - 0.00, flow rate 500 μΐ/min, 75%A/25%B; step 1, total time - 1.00, flow rate 500 μΐ/min, 75%A/25%B; step 2, total time - 8.00, flow rate 500 μΐ/min, 55%A/45%B; step 3, total time - 8.30, flow rate 750 μΐ/min, 5%A/95%B; step 4, total time - 10.30, flow rate 750 μΐ/min, 5%A/95%B; step 5, total time - 10.31, flow rate 750 μΐ/min, 75%A/25%B; and step 6, total time - 12.30, flow rate 750 μΐ/min, 75%A/25%B. An Intrada reverse phase column (WP-RP 3 μηι, 5mm x 150 mm) was used. [0156]

parameters associated with the auto-injector were as follows: seat capillary volumne - 2.3 μί; eject speed - 20(^L/minute; injection volume - 20.00μΙ_^; syringe volume - ΙΟΟμΙ_^; injection mode - standard; prefetch vial mode - off; draw speed - 20(^L/minute; and the draw position was 0.

[0157] Briefly, the plasma incubation and extraction procedure involved a 5 mL aliquot of freshly collected, heparin treated, EDTA-free pooled s/d rat plasma, or

IXfreeze/thawed rat plasma into a 15 mL corning centrifuge tube. To this was added 1.2 mL of 5X PBS solution to 5 mL plasma, which was then mixed by inverting the tube. The final concentration of PBS in rat plasma was 1 X PBS. The tube was centrifuged and kept on ice until used. Immediately before plasma incubation, the plasma was pre- warmed to 37 °C for five minutes. Plasma incubation time points were set as follows: T=0, 0.25 hour, 0.5 hour, 1 hour, 2 hour, 3 hour, 4 hour, 6 hour, 8 hour, 12 hour, and 24 hour. For each time point, duplicate samples were prepared by labeling 2 1.5 mL Eppendorf tubes. Protease inhibitor mix (25 uL of 10X HALT) prepared according to conventional methods, was added to the tubes and tubes were kept on ice until use.

[0158] Stock solution (50 uL of 100 X) was pippetted into plasma, mixed briefly by inverting the 15 mL centrifuge tube, and incubation started. At each incubation time point, 200 uL was pipetted to the tubes containing 25 uL inhibitor solution, and mixed by pipeting 5 times. Immediately thereafter, 225 uL of 100 % acetonitrile was added to each tube, and mixed by shaking several times. Tubes were then centrifuged at 13,000 rpm for ten minutes, 4 °C. The supernatant (200 uL from each tube) was pippetted to a new tube. A conventional reconstitution solution (200 uL) was added to the tube, and the total volume was 400 uL. The total volume was mixed by pippetting. An aliquot of this solution (200 uL) was added to a polypropylene insert, placed into amber HPLC vials, and labeled.

[0159] With respect to blood sample collection, blood samples were collected into tubes coated with sodium heparin. The sodium heparin coated tubes were previously prepared, allowed to air dry overnight and then stored at 2-8 °C pending use.

[0160] With repect to plasma sample preparation, protease inhibitor mix (100 uL of

10X Halt) solution, or 1/10 volume of the blood to be collected, was placed into pre-chilled, heparin coated tubes. Approximately 1 mL of blood was added to the tube and mixed gently by inverting the tube five times. Following collection, tubes containing samples were centrifuged at approximately 2500rpm for five minutes (at 2-6°C). Thereafter, two aliquots of resulting plasma (approximately 200 uL each) were transferred to pre-labeled tubes. The tubes were frozen on dry ice and stored at -80°C pending analysis.

[0161] Quantification of GLP-1 analytes with LC/MS. Source ions and daughter ions were listed in individual windows, and the quantification of individual analytes were performed with Multiple Reaction Monitoring (MRM). The Limit of Quantification (LOQ) and Quantification range were 10 nG/mL and 10-1000 nG/mL, respectively, for active GLP-1 (7-36), inactive GLP-1 (9-36), PEG-GLP-1, and free PEG.

[0162] Ex vivo plasma stability of GLP-1 peptides. GLP-1 (7-36) and GLP-1 (9-36) were each spiked in fresh rat plasma at the final concentrations of 1000 nG/mL. The plasma was maintained under 37 °C incubation, and fractions of the plasma were taken at pre-set time points for quantification. The stabilities of the two peptides, as revealed by Tl/2 values, obtained from First order, single exponential decay fitting, were 23 minutes and 2.1 hours, respectively.

[0163] Ex vivo plasma stability of PEG₂-ru-40K-Nter-GLP-l (7-36). 1000 nG/mL

(final concentration) PEG₂-ru-40K-Nter-GLP-l (7-36) was spiked in fresh rat plasma. The plasma was maintained was under 37 °C incubation, and fractions of the plasma were taken at pre-set time points for quantification. The conjugate was associated with no detection of free PEG or GLP-1 peptides and underwent no obvious changes (less than 10 % variation, which was within experimental deviation).

Example 5

In Vitro Activity GLP-1, PEG₂-ru-40K-N^ter-GLP-l (Example 1) and

PEG₂-ru-40K-Lys^26/34-GLP-l (Example 2)

[0164] Receptor binding assay: RINm5F cells (rat insulinoma stably expressing GLP-

1 receptor) were maintained in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in a humidified 5% C02 incubator. Cells in passages 6-8 were seeded at 250K cells/well in 96-well plates and cultivated for 72 hours. During the receptor binding assay, cells were equilibrated with assay buffer for one hour at room temperature. The cells were then incubated with competitive ligand mix containing ΙΟΟρΜ [1251] GLP-1 and Test peptides/conjugates at various doses for three hours at room temperature. Cells were washed

1

to remove unbound ligand and solubilized with NaOH. [ I] signal was detected with Perkin Elmer Cobra II gamma counter. IC50 was determined using non-linear regression curve fit analysis on Graph Pad Prism.

[0165] cAMP stimulation assay: Rat insulinoma (RINm5F) cells in passages 12-15 were seeded at 30,000 cells/well in 96-well plates and grown overnight. The cells were washed twice with Dulbecco's phosphate-buffered saline (D-PBS), then pre-incubated for twenty minutes in D-PBS containing 0.1% BSA, 500 μΜ 3-isobutyl-methylxanthine (IBMX), and 100 uM RO 20-1724 at room temperature. Cells were incubated for ten minutes with various peptide concentrations in D-PBS containing 0.1% BSA, 500 μΜ 3-isobutyl- methylxanthine (IBMX), and 100 uM RO 20-1724. The cells were lysed, and intracellular cAMP was determined using the cAMP-Glo assay system (Promega)

[0166] Following this protocol, it was demonstrated PEG₂-ru-40K-N^ter-GLP-l and

PEG₂-ru-40K-Lys^26/34-GLP-l exhibited minimal or no in vitro biological activity. Binding and activation (cAMP stimulation) studies with RINm5F cells, a rat insulinoma cell line expressing the GLP-1 receptor, indicated that PEG₂-ru-40 -N^ter-GLP-l exhibited no binding activity and 0.2% of the activation activity relative to GLP-1. PEG₂-ru-40K-Lys^26/34-GLP-l exhibited 0.9% and 0.1% of the binding and activation activity relative to GLP-1,

respectively. The in vitro biological activities of PEG₂-ru-40K-N^,er-GLP-l and PEG₂-ru-40K- Lys^26/34-GLP-l are shown in Table 1.

Table 1

In vitro activity of GLP-1, PEG₂-ru-40K-N^,er-GLP-l and PEG₂-ru-40K-Lys ^,,m-GLP-l

[0167] The relatively low in vitro activities of PEG₂-ru-40K-N -GLP-1 and PEG₂-ru-

40K-Lys^26/34-GLP-l are expected as conjugation of the N-terminal amine group and lysines within GLP-1 with large polymers (> 10 kDa) are often known to reduce activity. The binding and cAMP stimulation activities of PEG-30 -N^ter-ButyrALD-GLP- 1 were not determined but the activities are expected to be comparable to the activities of the two conjugates containing the amide linkage. Example 6

In Vivo Activity GLP-1, PEG₂-ru-40K-N^ter-GLP-l (Example 1) and

PEG₂-ru-40K-Lys^26/3 -GLP-l (Example 2)

[0168] Diabetic mice (BKS.Cg- Lepr^db/Lepr^db /OlaHsd) were obtained from Harlan

Laboratories and acclimated for two weeks in testing facility before experiments. Male animals 10-12 weeks old and weighing 35-45 g were randomly assigned into different testing groups. ~10μ1 of blood was collected from mouse tail and the baseline blood glucose level in the animal was measured by a glucometer (Onetouch Ultra from LifeScan). Animals were injected intraperitoneally with either saline, GLP-1 native peptide or equimolar of PEGylated GLP-1 conjugates (each equivalent to 75 μg of GLP-1). At different time points after compound administration, animal blood glucose levels were monitored using the glucometer. Food was removed during the whole study.

[0169] In contrast to the minimal in vitro activities exhibited by PEG₂-ru-40K-N^ter-

GLP-1 and PEG₂-ru-40K-Lys^26/34-GLP-l, the two conjugates exhibited very significant and prolonged in vivo glucose lowering activity in db/db mice. See FIG. 7.

[0170] The significant and surprising in vivo activities of PEG₂-ru-40K-N^ter-GLP-l and PEG₂-ru-40K-Lys^26/34-GLP-l are not consistent with their negligible in vitro activities. The presence of a PEG in a GLP-1 -polymer conjugate in itself does not confer greater in vivo activity relative to GLP-1 as PEG-30K-N^ter-ButyrALD-GLP-l exhibits marginal in vivo activity. In addition, PEG-GLP-1 conjugates have been reported in the literature to have reduced activity. See, for example, Lee et al. (2005) Bioconjugate Chemistry 16:377-382, EP 1 062 240, Lee et al. (2006) Diabetologia 49: 1608-1611. Without wishing to be bound by theory, the two conjugates containing the amide linkage may bind to the GLP-1 receptor with a slow on-rate but also a very slow off-rate. The prolonged presence of the conjugates on the GLP-1 receptor may result in a protracted activation of the receptor and an increased biological response. The extended half-life of activity of PEG2-ru-40K-N^ter-GLP-l and PEG₂-ru-40K-Lys^26/34-GLP-l are expected as PEGylation of GLP-1 with a 40 kD PEG is expected to increase the peptide's circulation half-life.

Claims

WHAT IS CLAIMED IS:

1. A. pharmaceutical composition comprising a dose of polymer-GLP-1 conjugates and a pharmaceutically acceptable excipient, wherein (i) each conjugate in the dose of polymer-GLP-1 conjugates comprises a water-soluble, non-peptidic polymer having a molecular weight of greater than 5,000 Daltons covalently attached through an

amide-containing linkage to an amino group of a GLP-1 moiety, (ii) the pharmaceutical composition, upon administration to a mammal, has a glucose-lowering effect, and (iii) the dose of polymer-GLP-1 conjugates achieving the glucose-lowering effect is less than an amount of the GLP-1 moiety in an unconjugated form required to achieve the glucose lowering effect.

2. The composition of claim 1 , wherein the GLP-1 moiety is GLP-l(7-36).

3. The composition of claim 1, wherein the GLP-1 moiety is Lys^{26 34}-GLP-l (7-36).

4. The composition of claim 1, wherein the composition lacks GLP-1 conjugates having covalent attachment of a fatty acid.

5. The composition of claim 1 , wherein the composition lacks GLP-1 conjugates having covalent attachment of a carbohydrate.

6. The composition of claim 1, wherein the composition lacks GLP-1 conjugates having covalent attachment of albumin.

7. The composition of claim 1 , wherein the non-peptidic, water-soluble polymer is linear.

8. The composition of claim 1, wherein the non-peptidic, water-soluble polymer is branched.

9. The composition of claim 1, wherein the non-peptidic, water-soluble polymer is selected from the group consisting of a poly(alkylene oxide), poly(vinyl pyrrolidone), poly( vinyl alcohol), polyoxazoline, and poly(acryloylmorpholine).

10. The composition of claim 1 , wherein the non-peptidic, water-soluble polymer is poly(ethylene oxide).

1 1. The composition of claim 1 , wherein the non-peptidic, water-soluble polymer is terminally capped with methoxy or hydroxy.

12. The composition of claim 1 , wherein the non-peptidic, water-soluble polymer has a weight-average molecular weight in a range of from about 500 Daltons to about 100,000 Daltons.

13. The composition of claim 1, wherein the non-peptidic, water-soluble polymer has a weight-average molecular weight in a range of from about 2,000 Daltons to about 50,000 Daltons.

14. The composition of claim 1, wherein the non-peptidic, water-soluble polymer has a weight-average molecular weight in a range of from about 6,000 Daltons to about 40,000 Daltons.

15. The composition of claim 1, wherein the pharmacologically active GLP-1 conjugate has the following structure:

O

CH₃0(CH₂CH₂0)_nCH₂CH₂-N-C-0— CH₂ O

O HC—0-CH₂—CH₂—CH₂—C— (GLP-1) CH₃0(CH₂CH₂0)_nCH₂CH₂-N-C-0— CH₂ wherein each (n) is independently an integer having a value of from 2 to about 4000 and (GLP-1) is the GLP-1 moiety.

16. The composition of claim 1 , wherein the amino group corresponds to the

N-terminal amine in the GLP-1 moiety.

17. The composition of claim 1, wherein the amino group is an ε-amine of a lysine residue contained with the GLP-1 moiety.

18. The composition of claim 17, wherein the GLP-1 moiety includes two lysine residues, each lysine residue having an ε-amine, and further wherein a non-peptidic, water-soluble polymer is attached at each ε-amine.

19. The composition of claim 1, further comprising a pharmaceutically acceptable excipient.