CA2260967A1 - Temperature sensitive gel for sustained delivery of protein drugs - Google Patents
Temperature sensitive gel for sustained delivery of protein drugs Download PDFInfo
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- CA2260967A1 CA2260967A1 CA002260967A CA2260967A CA2260967A1 CA 2260967 A1 CA2260967 A1 CA 2260967A1 CA 002260967 A CA002260967 A CA 002260967A CA 2260967 A CA2260967 A CA 2260967A CA 2260967 A1 CA2260967 A1 CA 2260967A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/28—Insulins
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
Pharmaceutical compositions for the delivery of pharmacologically active proteins are provided by the present invention. The compositions of the present invention comprise a polymeric matrix having thermal gelation properties in which is incorporated a discrete suspension of at least one biologically active macromolecular polypeptide which retains greater than 90 percent of its biological activity. Furthermore, the concentration of the macromolecular polypeptide is greater than 0.5 percent by weight of the composition.
Description
2 rCT/US97/1347~ -TEMPERATURE SENSITIVE GEL FOR SUSTAINED DELIVERY OF PROTEIN DRUGS
Des~l ;U~ion Terhr ;~1 Field The present invention relates to temperature-sensitive polymers for the sustained delivery of 5 ph~ ological agents and, more particularly, to poloxamers COlll~ illg suspensions of native macromolecular polypeptide agents.
R~ . vu~d Art Recent years have seen major advances in recombinant DNA techniques and consequently the proliferation of many protein ph~rm~ceu~iczll.c available for a variety of therapeutic needs. Indeed~
10 proteins or polypeptides constitute the largest class of drugs wl~ ly being considered for FDA
approval. However, the therapeutic and commercial potential of polypeptide drugs will only be realized if these advances are accomp2nied by formulation designs leading to effective a~lmini.~tration and stability.
Proteins are large organic molecules or macromolecules made up of amino acid residues 15 covalently linked together by peptide bonds into a linear, unbranched polypeptide chain with relative molecular mass ranging from a few thousand to millions. The useful properties of proteins as drugs depend upon the polypeptide chain adopting a unique three-dimensional folded conforrnation, that is~
the tertiary structure of the protein. It is this unique folding that is responsible for the protein being highly selective in the molecules it will recognize. However, the ability to m~int~in a unique three-20 dimensional structure is precisely one of the obstacles that makes the use of polypeptides in human andanimal diseases fraught with problems.
Traditionally, the most widely used method of ~rlmini.ctration of therapeutic agents is by the oral route. However, such delivery is not feasible, in the case of macromolecular drugs, as they are rapidly degraded and deactivated by hydrolytic enzymes in the ~lim~n~ry tract. Even if stable to enzymatic 25 digestion, their molecular weights are too high for absorption through the intestin~l wall.
Cl)n~equ~ontly, they are usually q ~ le~ed parenterally; but, since such drugs often have short half-lives in ViYo, frequent injections are required to produce an effective therapy. Ul~r~ u,lalely, while the palellt~l~l route is the most efficient means of drug introduction, this route has severe drawbacks in that injections are painful; they can lead to infection; and they can lead to severe vascular problems as a 30 result of repeated intravenous injections.
For these reasons, biodegradable polymer matrices have been considered as sustained release delivery systems for a variety of active agents or drugs. Once implanted, the matrix slowly dissolves or erodes, releasing the drug. An alternative approach is to use small implantable pumps, which slowly extrude the drug and matrix components, which dissolve after cont~cting body fluids. With both 35 systems it is crucial that the drug remain evenly distributed throughout the matrix since heterogeneous di~lribulion of the drug (e.g., formation of large clumps and voids) could lead to erratic dosing.
Ful~h.,~ ore, both systems require polymers that remain somewhat fluid so that they can be easily Sl,..~ 111 UTE SHEET (RULE 26) CA 02260967 lsss-ol-ll manipulated prior to implantation or loading into a device.
The use of polymers as solid implants and for use in small implantable pumps for the delivery of several therapeutic agents has been disclosed in scientific publications and in the patent literature.
See, for example, Kent, et al., "In vivo controlled release of an LHl~H analog from injected polymeric microcapsules", Contracept, Deliv. ~yst., 3:58 (1982); Sanders, et al., "Controlled release of a luteinizing hormone releasing hormone analogue from poly (d, l-lactide-co-glycolide) - microspheres", J. Pharm~ceut. Sci., 73: 1294-1297 (1984); Johnston, T.P., et al., "Sl-ct~inPd delivery of Interleukin-2 from a poloxarner 407 gel matrix following intraperitoneal injection in mice", Pharm~ceut. ~es., 9(3):425-434 (1992); Yolks, et al., "Timed release depot for anti-cancer agents II", Acta Pharm. Svec., 15:382-38$ (1978); Krezanoski, "Clear, water-miscible, liquid pharm~elltir~l vehicles and compositions which gel at body temperature for drug delivery to mucous membranes", U.S. Patent No.
4,474,752. However, the polymers having the greatest potential for use in the delivery of protein drugs would exhibit reverse thermal gelation and have good drug release characteristics.
There exists a class of block copolymers that may be generically classified as polyoxyethylene-polyoxypropylene con-len~tPs, namely Pluronic polyols, or poloxamers. They are formed by the condensation of propylene oxide into a propylene glycol nucleus followed by the conrl~ncation of ethylene oxide into both ends of the polyoxypropylene base. The polyoxyethylene hydrophilic groups on the ends of the molecule are controlled in length to constitute anywhere from 10 percent to 80 percent by weight of the final molecule.
Poloxamers, which have the ability to gel as a function of temperature and polymer concentration, may be represented emperically by the formula:
HO(C2H4O)b (C3H6O)a (C2~4O)bH (I) wherein a and b are integers such that the hydrophobe base lep~es~llled by (C3H6O)a has a molecular weight ranging from about 900 to 4,000, as determined by hydroxyl number; and the polyoxyethylene chain consisting of at least 60 percent to 70 percent by weight of the copolymer, and the copolymer having a total average molecular weight ranging from about 4,000 to 15,000. Table 1, below, references the minimnm concentrations of various pol~Y~mPr.s nP.cess~ry to form a gel in water at room tel"l)el a~ul e .
Table 1 Poloxamer* Molecular Weight Minimllm Concentration Pluronic F-68 8,400 50% - 60%
Pluronic F-88 11,400 40%
Pluronic F-108 14,600 30%
Pluronic F-127 12,600 20 * Poloxamers are commercially available under the referenced tra~lem~rkc through the BASF
CA 02260967 lgg9-ol-ll Corporation, Parsippany, N.J.
Poloxamers of low molecular weight, i.e., below 10,000 MW, do not form gels at any concentration in water.
While poloxamers, and more specifically Pluronic F-127 or Poloxamer 407, have been used to deliver nonpeptidic drugs as well as biologically active proteins, see U.S. Patent Nos. 4,100,271 and 5,457,093, respectively, su.~t~inPd delivery of biologically active macromolecules for weeks or months has not been possible for reasons that are two-fold. First, previous references which disclose the incorporation of proteins in a Pluronic matrix only disclose solutions of a protein, with concentrations less than approximately 2 mgiml and second, formulation approaches used to incorporate proteins into polymeric systems often result in irreversible inactivation of the proteins because of the presence of organic solvents, pH changes, and thermal effects. Congeq -.ontly, prior references which teach the use of poloxamers as pharmaceutical vehicles for the delivery of proteins have suffered two serious limit~tion~; (i) low initial concentrations of protein are used, and (ii) an unacceptable percentage of the protein loses its biological activity during use or storage. These two limitations have a direct impact on the ability to produce a polypeptide drug delivery system which can be shelved for long periods of time prior to usage and ~Amini.~tPr controlled dosages of protein for a period of weeks or more preferably months: Furthermore, degraded proteins can have reduced efficacy as a drug, and can also elicit adverse reactions, such as sen.siti7~tion and adverse immune response.
There is still a need, therefore, for a polypeptide drug delivery device or composition having high concentrations of fully native macromolecular polypeptides which may be regularly released over a long period of time.
Disclosure of Invention Accordingly, it is an object of this invention to provide a polypeptide drug delivery system.
It is a further object of this invention to provide a polypeptide drug delivery device or composition having protein or peptide concentrations greater than 5 mg/ml.
It is an ~ liti-~n~l object of the present invention to incorporate protein stabilizers into the drug delivery device.
Ad~lition~l objects, advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrllmP.nt~litiPs, combinations, compositions, and methods particularly pointed out in the appended claims.
To achieve the ~legoing and other objects and in accordance with the purposes of the present invention, as embodied and broadly described therein, the composition of the present invention comprises a polymeric matrix having thermal gelation properties in which is inco,~ul~ted a suspension of at least one CA 02260967 l999-01-ll biologically active macromolecular polypeptide having a concentration of 0.5 percent or greater by weight of the composition.
Brief Des.l;l,liull of the Dl~wi~
The accompanying drawing, which is incorporated in and forms a part of the specification, S illustrates the prepared embo~im~n~c of the present invention, and together with the description serves tO
explain the principles of the invention.
In the Dl awi~
Figure 1 is a graphic lepl~s~ tion of the effects various additives have on the solution-gel (sol-gel) transition temperature of the present invention, wherein each additive is represented by the following 10 symbols:
--- 0.1% PEG ~00, -x- 0.5 M Sucrose, ---- 1.0% PEG 800, -*- 1.0 M Sucrose, -~- Pluronic F-127, ---- 1.5 M Sucrose.
Best Mode for Carryin~ Out the Invention The ph~rm~reutic~l device or composition of the present invention provides a delivery system for 15 the controlled and sn~t~inPd ~ dlion of fully native macromolecular polypeptides or therapeutic agents to a human or animal. The biodegradable, biocompatible matrix for drug delivery is formed by suspending soluble and insoluble particles of fully native macromolecular polypeptides having a concentration of ~ mg/ml or greater, and other protein stabilizing components uniformly and discretely throughout a pharm~e~l~ir~l vehicle or polymer matrix that exhibits reverse therrnal gelation 20 characteristics. As with previously known systems, the suspended particles and other components are released through a combination of diffusion and dissolution m~ch~nigm~ as the device hydrates and subsequently erodes or dissolves. However, unlike known polymeric matrix systems which deliver macromolecules, the composition of this illv~ldiol1 comprises a ~I,ellsioll as opposed to a solution of native polypeptide(s). Con.~equently, high polypeptide concellll~lions are attained as a result of the suspension, 25 thus achieving the ability for sl~ in~d ~-l."~ tion of the therapeutic agent for a time period of days, weeks or months as opposed to hours. Furthermore, stabilizing agents may be incorporated into the composition of the present invention thereby further minimi7ing the degradation of these drugs, which directly impacts the efficacy of the drug and the ability to store or ship the device to worldwide markets.
The ph~rm~reu~ir~l composition of the present invention is a serendipitous discovery that was made 30 during a research project, the goal of which was simply to identify a drug delivery system that was amenable to study the effect of polymeric matrices on protein structure. In that study, Fourier transforrn infrared spectroscopy was employed, because it can be used to analyze protein secondary structure in solutions, suspensions, and solids. Of the various protein drug delivery lllaLlices ~ rl~ sed in the lil~ldlul~, those employing the polymeric detergent, Pluronic F-127, which forms a tem~,~l~lule sensitive gel, were 35 the most attractive for infrared spectroscopic studies, as it did not appear (based on the structures of poloxamer) that poloxamers would have an infrared absorbance that would interfere with the protein CA 02260967 lsss-ol-ll absorbance signal, which is needed for structural evaluation. Furthermore, it had previously been-established that poloxamers, at sufficient concentrations, have the characteristics of being liquid at temperatures below room temperature but will forrn into gel as they are warrned. Thus, a further objective of the study was to determine what effect this liquid to gel transition of the poloxamer would have on the protein's structure. In order to study protein structure with infrared spectroscopy it is nPces~ry to have protein concentrations of at least 15-20 mglml, thus a protein concentration of 20 mg/ml was prepared in the presence of sufficient poloxamer to allow gelling during warming. The resulting protein solution formed a fine, milky ~u~el~ion, which was initially very disappointing, because the formation of such suspensions often inriie~t?s that a solution component (e.g., the polymeric detergent) caused the protein to denature and to form non-native or inactive protein aggregates, thus im1ir~ring failure of the test. However, infrared spectroscopy can, fortunately, be used to analyze protein structures in suspension, so the suspension was analyzed anyway. Surprisingly, when the protein suspension was analyzed with the Fourier Llal~ro~ infrared specL~usco~y, instead of finding the expected non-native or inactive protein aggregates, it was unexpectedly found to be fully native.
In accordance with the present invention, the phqrm~ce~ltir~l composition of the present invention comprises a polymer such as a polyoxyalkylene block copolymer of forrnula:
HO (C2H4O)b (C3H6O)a (C2~4O)bH (I), which has the unique feature, in the preferred embodiment, of being liquid at ambient or lower ,c,atules and existing as a semi-solid gel at m~mm~ n body t~ elalules wherein a and b are integers in the range of 20 to 80 and 15 to 60, respectively. A preferred polyoxyalkylene block copolymer for use as the pharrn~ceu~ical vehicle of this invention is a polyoxyethylene-polyoxyprûpylene block copolymer having the following formula:
~o-(cH2cH~o)b-(c~I2(cH3)cHo)a-(cH2cH2o)b-H (II) wherein a and b are integers such that the hydrophobe base represented by (CH~(CH3)CHO)~ has a mûlecular weight of at least about 4,000, as determined by hydroxyl number; the polyoxyethylene chain co~ lg about 70 percent of the total number of monomeric units in the molecule and where the copolymer has an average molecular weight of about 12,600. Pluronic F-127, also known as Poloxamer 407, is such a material.
The procedures used to prepare aqueous solutions which form gels of polyoxyalkylene block copolymer are well known. For example, either a hot or cold process for forming the solutions can be used. The cold technique involves the steps of dissolving the polyoxalkylene block copolymer at a Le~ ela~ule of about ~~C to 10~C in water or in a buffer, such as a phosphate buffer. The water, if used in forming the aqueous solution, is preferably purified, as by ~ ti~ln7 filtration, ion-exchange or the like. When the solution is Col"pl it is brought to room L~llpe-àLule whereupon it forms a gel. If the hot process of forming the gel is used, the polymer is added to water or a buffer and heated to a temperature of about 75~C to 85~C with slow stirring until a clear homogenous solution is obtained, upon cooling a clear CA 02260967 lgg9-ol-ll WO 98/02142 PCT/US97tl3479 gel forms.
Any macromolecular polypeptide may be mixed with the pharm:lceutic~l vehicle to form the ph~ reutir.~l composition of this invention wherein the concentration of macromolecular polypeptide is in the range of 0.5 to 50 percent by weight of the composition. The choice of polypeptides which can be 5 delivered in accordance with the practice of this invention is limited only by the requirement that they be at least very slightly soluble in an aqueous physiological media such as plasma, interstitial fluid, and the intra and extracellular fluids of the subcutaneous space and mucosal tissues.
Exemplary classes of polypeptides include, among others, proteins, enzymes, nucleoproteins, glycoproteins, liyu~loteills, hormonally active polypeptides, and synthetic analogues including agonists and 10 antagonists of these molecules.
Specific examples of polypeptides suitable for incorporation in the delivery system of the present invention include the following biologically active macromolecules: intelr~lolls, interleukins, insulin, enzyme inhibitors, colony-stim~ ting factors, plasminogen activators, growth factors and polypeptide hormones.
The list of macromolecular polypeptides recited above are provided only to illustrate the types of active agents which are suitable for use in practicing the present invention, and are not intended to limit the scope of the present invention.
The pha m:~ceutir~l composition of the present invention can be readily prepared using any solution forming technique which achieves the concentration of polyoxyalkylene block copolymer nrcec~ry for gelling. Preferably the pharn~reutir.~l vehicle and polypeptide mixture are prepared separately and the polypeptide mixture having a co,lcel-Ll~tion of 5 mg/ml or greater is added thereto at a temperature of about 0~C to lODC. When combined the protein forms a homogenous suspension of fine particles in the polymer solution, which then has a "milky" appearance. By light miclvscol)y the particles are approximately ~-10 microns in diameter. Raising the sample t~ u~laLule above the gel point of the poloxamer results in an even distribution of protein particles throughout the polymer gel. Due to the high viscosity of the gel matrix, the particles remain homogeneously diaLlib.lLed and do not "settle out." The liquid to gel transition is fully reversible upon cooling. Furthermore, when the gel is exposed to an aqueous solution, the gel matrix and protein particles dissolve, releasing the fully native protein which retains greater than 90 percent of its biological activity.
The ph~ relltit~l composition of the present invention can be implanted directly into the body by injecting it as a liquid, whereupon the ph~ relltir.~l composition will gel once inside the body. In the alternative, the ph~rm~reutir~l composition may be introduced into a small i~inplantable pump which is then introduced into the body.
In another embodirnent, protein-stabilizing solutes, can be incorporated into the ph~tm~ceutical device of the present invention described above. Initially, stabilizers were added to the ph~ eutir~l device of the present invention to increase the stability of the macromolecular polypeptides as such CA 02260967 lsss-ol-ll WO 98/02142 PCT/USs7/13479 stabilization would be crucial for use of the present invention for suct~in~d delivery of protein in the body.
However, in doing so it was discovered that protein-stabilizing solutes, such as sucrose, no~ only aid in protecting and stabilizing the protein, but also allow the poloxamer to form suitable gels at lower concentrations than needed in water or buffer alone. Thus, the working range of polymer concentration 5 can be widened. As discussed previously, the concentration of the polyoxyalylene block copolymer is an important parameter. It is known that a gel will not form when the concentration of polyoxyethelene-polyoxypropylene block copolymer in water or dilute buffer is outside of the range of about 20 to 30 percent by weight, as shown in Figure 1 and exemplified by the line having open triangles. However, by introducing protein-stabilizing solutes to the pharm~c.eutic~l device of the present invention the gel-sol 10 transition temperature may be manipulated, while also lowering the concentration of polyoxyethelene-polyoxypropylene block copolymer which is nPcecc~ry to form a gel.
In a third embodiment, polypeptide concentrations at the high end of the 0.5 to 50 percent by weight range can be achieved by centrifuging the pharmaceutical composition of the present invention at low temperatures in the range of -10~C to 10~C, and preferably 04~C for a period of time sufficient to 1~ sediment the protein particles. For example, a sample of the pharmaceutical composition described previously comprising 20 mg/ml protein can be centrifuged at 4~C so that the insoluble protein particles se-iimP.nt Then supernatant equivalent to half the volume could be removed and the se~limP.nf resuspended in the rem~ining liquid. This will result in a suspension containing almost 40 mg/ml.
In the Examples which follow the ph~ ce ltical composition of the present invention was prepared 20 according to the following preparation procedure. Since the polyoxyalkylenes dissolve more completely at reduced temperatures, the preferred methods of solubilization are to add the required amount of copolymer to the amount of water or buffer to be used. Generally after wetting the copolymer by shaking, the mixture is capped and placed in a cold chamber or in a thennost~ti~ container at about 0~C to 10~C in order to dissolve the copolymer. The mixture can be stirred or shaken to bring about a more rapid solution 25 of the polymer. The polypeptides and various additives such as stabilizers can subsequently be added and dissolved to form a suspension.
The following non-limited examples provide methods for l~lepa~ g temperature sensitive polymers for the sustained delivery of ph~rrn~cel-tic~l agents comprising high concentrations of fully native macromolecular polypeptide agents. All scientific and te~hnif~l terms have the mPaning.c as understood 30 by one with ordinary skill in the art. The specific examples which follow illustrate the representative polypeptides and concentrations capable of being achieved by the present invention and are not to be construed as limiting the invention in sphere or scope. The methods may be adapted to variation in order to produce compositions or devices embraced by this invention but not specifically disclosed. Further variations of the mPtho~l.c to produce the same compositions in somewhat different fashion will be evident 35 to one skilled in the art.
All temperatures are understood to be in Centigrade (~C) when not specified. The infrared (IR) CA 02260967 lsss-ol-ll spectral description was measured on a Nicolet Magna-IR 550 Spectrometer. Comrnercially available ch~min~l~ were used without purification.
In the Examples which follow the 27.5 percent (w/w) solution of Pluronic F-127 was prepared in the following manner. 7.59 grams of dry Pluronic F-127 was added to a sterile tube containing 20 grams 5 of an ice cold phosphate buffer (pH 7.4, 30 mM). The tube was capped and the mixture was well shaken prior to being stored overnight at 4~C.
~XAMPLE 1 p~ inn a~d Chara-l~.;Ldtion o~ a Sl-cpf~n~inn of Cl~ s~ ill PlulV-liC F-127 10 a) Preparation of the ~ .y~ inn-When 50~L of a 100 mglmL suspension of chymotrypsin in 30 mM phosphate buffer at pH 7.4 were mixed with 200 ~L of a 27.5 percent (wlw) solution of Pluronic F-127 at 4~C a white, uniform, milky suspension formed imm~ tely. The su~l~el~iull contained 20 mglmL protein and 22 percent (w/w) Pluronic F-127 and was a viscous liquid below about 18~C and a soft, solid gel above about 20~C. The crystalline portion of the liquid suspension settled out of solution when allowed to stand for a day or two at 4~C or when centrifuged in the cold but could be readily resuspended by mixing while cold. The fact that the suspended material can be se-liment~d out and resuspended in a smaller volume than the original suspension permits formation of suspensions of gienifir~ntly higher concentrations, as discussed previously.
In order to ~l~.t~rmin~ the solubility of chymotrypsin in the liquid phase of the suspension, aliquots of the total suspension and of the supernatant following centrifugation at 1000-5000 rpm for 5 minutes at 4~C were assayed spectrophotometrically for enzyme activity. The concentration of chymotrypsin that r~m~in~d soluble in the Pluronic F-127 is shown below in Table 2.
Table 2 Enzyme Solubility (mglmL)' Std. Dev.
Chymotrypsin 0 132 0.06~
D~L~. .";..~d by dividing the activity measured in the ~ by the calculated activity per mg of protein in the whole 2 n=3.
b) S~epen-~e-l enzyme exhibits native-like seC~n~lq~ structure ~s measured by FTIR:
Infrared spectra obtained using a Nicolet Magna-IR 550 Spectrophotometer in a temperature regulated, 6 ~M path length cell were used to examine and compare the structure of chymotrypsin in phosphate buffer (30 mM, pH 7.4) and in 22 percent (w/w) Pluronic F-127 in the same buffer. Protein 35 concentration in these studies was 20 mg/mL. Careful examination of the spectra showed that the secondary structure of the chymotrypsin was not altered due to suspending the protein in the Pluronic F-127. A-l~itinn~l ~;TIR s~e~loscopic ex~min~tions of the structure of chymotrypsin in Pluronic F-127 at CA 02260967 lsss-ol-ll temperatures below and above the gel transition (about 15~C to 18~C) showed that the formation of the ge]
did not alter the structure.
c) Suspended enzyme m~int~inc biDIo ~ ;cal activity:
For this experiment, a chymotrypsin suspension in 22 percent Pluronic F-127 was prepared as 5 described above but at two dirrelellL concentrations, 20 mg/mL and 2 mg/mL. Solutions with the same protein concentrations were prepared using phosphate buffer (30 mM, pH 7.4) with no Pluronic F-127.
Aliquots of each of these were diluted as necessary and assayed for enzymatic activity as described above.
As can be seen from Table 3, below, enzyme incorporated into the gels could be recovered quantitatively and with no loss of activity after the gels were dissolved in buffer.
Table 3 Environment Concentration Dilution Observed Std.Percent of (mg/ml) before Activity Dev.Activity in Assay Buffer Buffer' 20 5000 21.234 1.134 Pluronic2 20 5000 21.6 3.4 102 Buffer 2 500 21.0 0.8 15Pluronic 2 500 25.8 1.4 123 Phosphate, 30mM. pH 7.4 2 2~% (wlw) in 30 mM Phosphate buffer, pH 7.4 Expressed as mAU/min at 410 nm 204 n=9 The biological activity of chymotrypsin was not damaged by inclusion in the Pluronic F-127. A
slight enh~n~çmPnt of activity was observed when low levels of the detergent were present in the enzyme assay mLYtures. It is possible that the difference in percent recovery observed between the 2 mg/mL and 2~ 20 mg/mL samples shown in Table 3 is due to the greater dilution of the gel in the 20 mglmL sample and thus higher levels of Pluronic F-127 in the 2 mg/mL samples.
d) S~l~pPn(le~l enzyme di~lJldy~ in.leased storage stability:
In order to demonstrate that suspending proteins in 22 percent Pluronic F-127 has a stabilizing action, 20 mg/mL chymotrypsin was prepared in either 30 rnM phosphate buffer, pH 7.4 or 22 percent 30 Pluronic F-127 in this buffer as described above. These suspensions were diluted as necessary and - enzyme activity was assayed. The results shown in Table 4 below, demonstrate that the enzyme incubated in the presence of Pluronic F-127 retained more activity than enzyme incubated in the presence of phosphate buffer alone.
.. . . . ..
CA 02260967 l999-01-ll Table 4 Time(hr.) Buffer' Pluronic2 Buffer Pluronic Buffer Pluronic 8 degrees 8 degrees 25 degrees25 degrees 37 37 degrees degrees 1003 ~ 00 100 lO0 100 24 104 79 104 84 na4 59 I Phosphate 2 22% (w/w) in phosphate buffer, 30 mM, pH 7.4 0 3 Expressed as a p~ of the I hour value 4 Value not available l~9 Thus, Pluronic F-127 provides a stabilizing e~ llent, which is more evident at extended times and elevated temperatures, in which peptide and protein containing drugs may be suspended prior to and during delivery. Additionally, other protein stabilizing agents may be added to the form~ tion as described further in Example 6 below.
F~ dtion and Characterization~of a of Sul)lil~l in Pluronic F-127 a) P~ dlion of the ~IJ ..~ n A suspension of subtilisin was made by the same procedure described in detail above for chymotrypsin. The physical properties were the same in terms of formation and behavior of the milky white sll~pen.cion and gel-sol transition telnperatu~e. The solubility of subtilisin in the liquid phase of the 25 sllcpen~inn was elr~min~od using the method described above and found to be dir~le~lL as might be expected for a di~lelll protein. The results are represented in Table 5 below.
Table 5 Enzyme Solubility (mglmL)' Std. Dev.
Subtilisin 6.482 1. l 12 Determined by dividing the activity measured in the ~u~ dl~ull by the calculated activity per mg of protein in the whole .n 2 n=~
35 b) Suspended enzyme exhibits native-like sec~n-l~ry structure as l-le~.,l ed by FT~R:
Subtilisin suspensions, like those of chymotrypsin described above, were e~min~.d by FTIR
spectroscopy to determinP whether inclusion in the gel had any effect on the structure and whether the gel-sol transition influenced structure. As in the case of chymotrypsin, there was no effect on structure.
CA 02260967 lg99-ol-ll Il c) S~cpen-le~l enzyme maintains biologir~l aeLivily;
In experiments similar to those described above for chymotrypsin, subtilisin suspensions were prepared in both phosphate buffer and 22 percent Pluronic F-127 in phosphate buffer and then dissolved by dilution with buffer. In these cases like in the case of chymotrypsin, 100 percent of the enzyme activity 5 was recovered showing that biological activity was stable while incorporated into the gel suspension.
d) S--cp~n~le~ enzyme ~la~ ine~ea3ed storage stability:
In order to demonstrate that suspending subtilisin in 22 percent Pluronic F-127 has a stabilizing action similar to that demonstrated for chymotrypsin, 20 mg/mL subtilisin was prepared in either 30 mM
phosphate buffer, pH 7.4 or 22 percent Pluronic F-127 in phosphate buffer as described above. These suspensions were incubated at temperatures of 8, 25 and 37~C for times up to 118 hours, the gels or suspensions were diluted as necessary and enzyme activity was assayed. The results, shown below in Table 6, demonstrate that even though subtilisin is al~tocat~lytic and looses activity nnore rapidly than chymotrypsin, it still retained more activity when incubated in the presence of Pluronic F-127 than when incubated in the presence of phosphate buffer alone.
Table 6 Time(hr.) Buffer' Pluronic2 Buffer Pluronic Buffer Pluronic 8 de~rees 8 degrees25 degrees25 degrees37 degrees 37 degrees 0.5 1003 100 100 100 100 100 ' Phosphate, 30 mM, pH 7.4 2 22% ~w/w) in rh- $ph~t~ buffer, 30 m M,pH 7.4 3 Expressed as a percentage of the 0.5 hour value Preparation and Char~el~ iLdtiOIl of a SucpPn~;~ n of Lactate Dehydrogenase in Pluronic F-127 30 a) P~epa~aliu~, of the ~ s;om A ~u~tllsion of lactate dehydrogenase was made by the same procedure described in detail above for chymotrypsin. Visual observations inllic~tPd that the physical properties were the same in terms of formation and behavior of the milky white suspension and gel-sol transition temperature.
b) S~ le-l enzyme maintains biological ..-,livily:
In experiments similar to those described above for chymotrypsin, lactate dehydrogenase suspensions were prepared in both phosphate buffer and 22 percent Pluronic F-127 in phosphate buffer and then disso~ved by dilution with buffer. In these cases like in the case of chymotrypsin, 100 percent of the enzyme activity was recovered showing that biological activity was stable while incorporated into the gel suspension.
WO 98/02142 PCTtUS~7113479 c) Suspended enzyme di~lay~ increased storage stability: -Experiments lil~e those carried out with chymotrypsin were conducted to illustrate the effect of Pluronic F-127 in stabilizing the enzyme activity of lactate dehydrogenase during storage at elevated elaLul~s. In this case 0.5 M sucrose was inrhlded in some samples and thus it was necessary to reduce 5 the concentration of Pluronic F-127 to 18 percent to m~inr~in the same gel-sol transition temperature.
Table 7 below, shows the results obtained when lactate dehydrogenase was stored for 48 hours at 37~C.
Table 7 Sample Composition'Enzyme Activity~ Std. Dev. (n=3) 1018% Pluronic 59.3 1.7 18% Pluronic + 0.5 M sucrose 46.1 2.7 0.5 M sucrose 39.1 3.3 buffer alone 40.0 1.7 ' Bllffer used in all was 50 mM Tris:HCI, pH 7.35 with 0.1% NaN3 2 Expressed in arbitrary units ~XAMPLE 4 Preparation and Chara~ alion of a S~cpPn~;c of Bovine Serum Albulmin in Pluronic F-127 a) Preparation of the s..~l,f ..-: n-Several mixtures of Pluronic F-127 and bovine serum albumin were made in attempts to find a 25 combination that would be a clear gel with a protein concentration of 15 mglmL or higher at room t~ul~elalul~. Pluronic F-127 concentrations below 20 percent (w/w) would not gel at 25~C or below and con-.~ntr~ti~-ns of 20 percent and above resulted in the milly, white suspension when bovine serum albumin was added at concentrations of 14 mg/mL and above. Thus, no cuull,ill~lion was found that would provide the desired properties. Subsequent studies of structure and stability on several proteins demonstrated that 30 it was not nl~ce.cc~ry to have a clear gel to have a .c~ticf~rtory protein drug delivery forrnulation with x Pluronic F-127.
b) Su~ ~P-l protein exhibits native-like secnnllqry ~I- ueLul e as ~l~ea~ur~ d by FTIR:
Preliminary FTIR ~e~ osc~p;c e~r~min~tion of the structure of a suspension of 20 mg/mL bovine serum albumin in 22 percent Pluronic F-127 showed no differences from a similar suspension of the 35 protein in buffer alone. This result was similar to resu1ts from more extensive ~ min~ions of the structures of chymotrypsin and subtilisin suspended in the gel.
Preparation and Characterizatio~n of ~ S~.~ S;~., of In..~llin in Pluroriuc F-127 a) Preparation ofthe ~ n A suspension of insulin was made by the same procedure described in detail above for chymotrypsin. Visual observation in~lic~ted that the physical properties were the same in terms of formation and behavior of the milky white suspension and gel-sol transition temperature.
10 b) Suspended protein exhibits native-like secon~l~ry structure as ~.l.ea~ul~d by FTIR:
Insulin suspensions, like those of chymotrypsin described above, were ex~min~d by FTIR
spectroscopy to determine whether inclusion in the gel had any effect on the structure and whether the gel-sol transition influenred structure. As in the case of chymotrypsin, there was no effect on structure.
Protein Stabilizing Agents Used to Manipulate Gel Properties Various concentrations of sucrose were incorporated into Pluronic F-127 solutions as an example 20 of the use of known protein stabilizing agents to manipulate the properties of the gels. Typical results for gel-sol transition temperature are shown in Table 8 below.
Table 8 % Pluronic' 0 M Sucrose0.5 M Sucrose 1.0 M Sucrose1.5 M Sucrose 16 25 20 0.2 ' w/w, in 30 ~nM phosphate, pH 7.4 It is clear from Table 8 that the gel-sol transition ten1p~ldture can be nanipulated in the range from below 0~C to about 25"C as required by the present invention.
The foregoing description is considered as illustrative omy of the principles of the invention.
Furthermore, since numerous mo-lifi~ ns and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and processes shown as described above.
Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the invention as defined by the claims which follow.
.. _ .. . ...
Des~l ;U~ion Terhr ;~1 Field The present invention relates to temperature-sensitive polymers for the sustained delivery of 5 ph~ ological agents and, more particularly, to poloxamers COlll~ illg suspensions of native macromolecular polypeptide agents.
R~ . vu~d Art Recent years have seen major advances in recombinant DNA techniques and consequently the proliferation of many protein ph~rm~ceu~iczll.c available for a variety of therapeutic needs. Indeed~
10 proteins or polypeptides constitute the largest class of drugs wl~ ly being considered for FDA
approval. However, the therapeutic and commercial potential of polypeptide drugs will only be realized if these advances are accomp2nied by formulation designs leading to effective a~lmini.~tration and stability.
Proteins are large organic molecules or macromolecules made up of amino acid residues 15 covalently linked together by peptide bonds into a linear, unbranched polypeptide chain with relative molecular mass ranging from a few thousand to millions. The useful properties of proteins as drugs depend upon the polypeptide chain adopting a unique three-dimensional folded conforrnation, that is~
the tertiary structure of the protein. It is this unique folding that is responsible for the protein being highly selective in the molecules it will recognize. However, the ability to m~int~in a unique three-20 dimensional structure is precisely one of the obstacles that makes the use of polypeptides in human andanimal diseases fraught with problems.
Traditionally, the most widely used method of ~rlmini.ctration of therapeutic agents is by the oral route. However, such delivery is not feasible, in the case of macromolecular drugs, as they are rapidly degraded and deactivated by hydrolytic enzymes in the ~lim~n~ry tract. Even if stable to enzymatic 25 digestion, their molecular weights are too high for absorption through the intestin~l wall.
Cl)n~equ~ontly, they are usually q ~ le~ed parenterally; but, since such drugs often have short half-lives in ViYo, frequent injections are required to produce an effective therapy. Ul~r~ u,lalely, while the palellt~l~l route is the most efficient means of drug introduction, this route has severe drawbacks in that injections are painful; they can lead to infection; and they can lead to severe vascular problems as a 30 result of repeated intravenous injections.
For these reasons, biodegradable polymer matrices have been considered as sustained release delivery systems for a variety of active agents or drugs. Once implanted, the matrix slowly dissolves or erodes, releasing the drug. An alternative approach is to use small implantable pumps, which slowly extrude the drug and matrix components, which dissolve after cont~cting body fluids. With both 35 systems it is crucial that the drug remain evenly distributed throughout the matrix since heterogeneous di~lribulion of the drug (e.g., formation of large clumps and voids) could lead to erratic dosing.
Ful~h.,~ ore, both systems require polymers that remain somewhat fluid so that they can be easily Sl,..~ 111 UTE SHEET (RULE 26) CA 02260967 lsss-ol-ll manipulated prior to implantation or loading into a device.
The use of polymers as solid implants and for use in small implantable pumps for the delivery of several therapeutic agents has been disclosed in scientific publications and in the patent literature.
See, for example, Kent, et al., "In vivo controlled release of an LHl~H analog from injected polymeric microcapsules", Contracept, Deliv. ~yst., 3:58 (1982); Sanders, et al., "Controlled release of a luteinizing hormone releasing hormone analogue from poly (d, l-lactide-co-glycolide) - microspheres", J. Pharm~ceut. Sci., 73: 1294-1297 (1984); Johnston, T.P., et al., "Sl-ct~inPd delivery of Interleukin-2 from a poloxarner 407 gel matrix following intraperitoneal injection in mice", Pharm~ceut. ~es., 9(3):425-434 (1992); Yolks, et al., "Timed release depot for anti-cancer agents II", Acta Pharm. Svec., 15:382-38$ (1978); Krezanoski, "Clear, water-miscible, liquid pharm~elltir~l vehicles and compositions which gel at body temperature for drug delivery to mucous membranes", U.S. Patent No.
4,474,752. However, the polymers having the greatest potential for use in the delivery of protein drugs would exhibit reverse thermal gelation and have good drug release characteristics.
There exists a class of block copolymers that may be generically classified as polyoxyethylene-polyoxypropylene con-len~tPs, namely Pluronic polyols, or poloxamers. They are formed by the condensation of propylene oxide into a propylene glycol nucleus followed by the conrl~ncation of ethylene oxide into both ends of the polyoxypropylene base. The polyoxyethylene hydrophilic groups on the ends of the molecule are controlled in length to constitute anywhere from 10 percent to 80 percent by weight of the final molecule.
Poloxamers, which have the ability to gel as a function of temperature and polymer concentration, may be represented emperically by the formula:
HO(C2H4O)b (C3H6O)a (C2~4O)bH (I) wherein a and b are integers such that the hydrophobe base lep~es~llled by (C3H6O)a has a molecular weight ranging from about 900 to 4,000, as determined by hydroxyl number; and the polyoxyethylene chain consisting of at least 60 percent to 70 percent by weight of the copolymer, and the copolymer having a total average molecular weight ranging from about 4,000 to 15,000. Table 1, below, references the minimnm concentrations of various pol~Y~mPr.s nP.cess~ry to form a gel in water at room tel"l)el a~ul e .
Table 1 Poloxamer* Molecular Weight Minimllm Concentration Pluronic F-68 8,400 50% - 60%
Pluronic F-88 11,400 40%
Pluronic F-108 14,600 30%
Pluronic F-127 12,600 20 * Poloxamers are commercially available under the referenced tra~lem~rkc through the BASF
CA 02260967 lgg9-ol-ll Corporation, Parsippany, N.J.
Poloxamers of low molecular weight, i.e., below 10,000 MW, do not form gels at any concentration in water.
While poloxamers, and more specifically Pluronic F-127 or Poloxamer 407, have been used to deliver nonpeptidic drugs as well as biologically active proteins, see U.S. Patent Nos. 4,100,271 and 5,457,093, respectively, su.~t~inPd delivery of biologically active macromolecules for weeks or months has not been possible for reasons that are two-fold. First, previous references which disclose the incorporation of proteins in a Pluronic matrix only disclose solutions of a protein, with concentrations less than approximately 2 mgiml and second, formulation approaches used to incorporate proteins into polymeric systems often result in irreversible inactivation of the proteins because of the presence of organic solvents, pH changes, and thermal effects. Congeq -.ontly, prior references which teach the use of poloxamers as pharmaceutical vehicles for the delivery of proteins have suffered two serious limit~tion~; (i) low initial concentrations of protein are used, and (ii) an unacceptable percentage of the protein loses its biological activity during use or storage. These two limitations have a direct impact on the ability to produce a polypeptide drug delivery system which can be shelved for long periods of time prior to usage and ~Amini.~tPr controlled dosages of protein for a period of weeks or more preferably months: Furthermore, degraded proteins can have reduced efficacy as a drug, and can also elicit adverse reactions, such as sen.siti7~tion and adverse immune response.
There is still a need, therefore, for a polypeptide drug delivery device or composition having high concentrations of fully native macromolecular polypeptides which may be regularly released over a long period of time.
Disclosure of Invention Accordingly, it is an object of this invention to provide a polypeptide drug delivery system.
It is a further object of this invention to provide a polypeptide drug delivery device or composition having protein or peptide concentrations greater than 5 mg/ml.
It is an ~ liti-~n~l object of the present invention to incorporate protein stabilizers into the drug delivery device.
Ad~lition~l objects, advantages and novel features of this invention shall be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following specification or may be learned by the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrllmP.nt~litiPs, combinations, compositions, and methods particularly pointed out in the appended claims.
To achieve the ~legoing and other objects and in accordance with the purposes of the present invention, as embodied and broadly described therein, the composition of the present invention comprises a polymeric matrix having thermal gelation properties in which is inco,~ul~ted a suspension of at least one CA 02260967 l999-01-ll biologically active macromolecular polypeptide having a concentration of 0.5 percent or greater by weight of the composition.
Brief Des.l;l,liull of the Dl~wi~
The accompanying drawing, which is incorporated in and forms a part of the specification, S illustrates the prepared embo~im~n~c of the present invention, and together with the description serves tO
explain the principles of the invention.
In the Dl awi~
Figure 1 is a graphic lepl~s~ tion of the effects various additives have on the solution-gel (sol-gel) transition temperature of the present invention, wherein each additive is represented by the following 10 symbols:
--- 0.1% PEG ~00, -x- 0.5 M Sucrose, ---- 1.0% PEG 800, -*- 1.0 M Sucrose, -~- Pluronic F-127, ---- 1.5 M Sucrose.
Best Mode for Carryin~ Out the Invention The ph~rm~reutic~l device or composition of the present invention provides a delivery system for 15 the controlled and sn~t~inPd ~ dlion of fully native macromolecular polypeptides or therapeutic agents to a human or animal. The biodegradable, biocompatible matrix for drug delivery is formed by suspending soluble and insoluble particles of fully native macromolecular polypeptides having a concentration of ~ mg/ml or greater, and other protein stabilizing components uniformly and discretely throughout a pharm~e~l~ir~l vehicle or polymer matrix that exhibits reverse therrnal gelation 20 characteristics. As with previously known systems, the suspended particles and other components are released through a combination of diffusion and dissolution m~ch~nigm~ as the device hydrates and subsequently erodes or dissolves. However, unlike known polymeric matrix systems which deliver macromolecules, the composition of this illv~ldiol1 comprises a ~I,ellsioll as opposed to a solution of native polypeptide(s). Con.~equently, high polypeptide concellll~lions are attained as a result of the suspension, 25 thus achieving the ability for sl~ in~d ~-l."~ tion of the therapeutic agent for a time period of days, weeks or months as opposed to hours. Furthermore, stabilizing agents may be incorporated into the composition of the present invention thereby further minimi7ing the degradation of these drugs, which directly impacts the efficacy of the drug and the ability to store or ship the device to worldwide markets.
The ph~rm~reu~ir~l composition of the present invention is a serendipitous discovery that was made 30 during a research project, the goal of which was simply to identify a drug delivery system that was amenable to study the effect of polymeric matrices on protein structure. In that study, Fourier transforrn infrared spectroscopy was employed, because it can be used to analyze protein secondary structure in solutions, suspensions, and solids. Of the various protein drug delivery lllaLlices ~ rl~ sed in the lil~ldlul~, those employing the polymeric detergent, Pluronic F-127, which forms a tem~,~l~lule sensitive gel, were 35 the most attractive for infrared spectroscopic studies, as it did not appear (based on the structures of poloxamer) that poloxamers would have an infrared absorbance that would interfere with the protein CA 02260967 lsss-ol-ll absorbance signal, which is needed for structural evaluation. Furthermore, it had previously been-established that poloxamers, at sufficient concentrations, have the characteristics of being liquid at temperatures below room temperature but will forrn into gel as they are warrned. Thus, a further objective of the study was to determine what effect this liquid to gel transition of the poloxamer would have on the protein's structure. In order to study protein structure with infrared spectroscopy it is nPces~ry to have protein concentrations of at least 15-20 mglml, thus a protein concentration of 20 mg/ml was prepared in the presence of sufficient poloxamer to allow gelling during warming. The resulting protein solution formed a fine, milky ~u~el~ion, which was initially very disappointing, because the formation of such suspensions often inriie~t?s that a solution component (e.g., the polymeric detergent) caused the protein to denature and to form non-native or inactive protein aggregates, thus im1ir~ring failure of the test. However, infrared spectroscopy can, fortunately, be used to analyze protein structures in suspension, so the suspension was analyzed anyway. Surprisingly, when the protein suspension was analyzed with the Fourier Llal~ro~ infrared specL~usco~y, instead of finding the expected non-native or inactive protein aggregates, it was unexpectedly found to be fully native.
In accordance with the present invention, the phqrm~ce~ltir~l composition of the present invention comprises a polymer such as a polyoxyalkylene block copolymer of forrnula:
HO (C2H4O)b (C3H6O)a (C2~4O)bH (I), which has the unique feature, in the preferred embodiment, of being liquid at ambient or lower ,c,atules and existing as a semi-solid gel at m~mm~ n body t~ elalules wherein a and b are integers in the range of 20 to 80 and 15 to 60, respectively. A preferred polyoxyalkylene block copolymer for use as the pharrn~ceu~ical vehicle of this invention is a polyoxyethylene-polyoxyprûpylene block copolymer having the following formula:
~o-(cH2cH~o)b-(c~I2(cH3)cHo)a-(cH2cH2o)b-H (II) wherein a and b are integers such that the hydrophobe base represented by (CH~(CH3)CHO)~ has a mûlecular weight of at least about 4,000, as determined by hydroxyl number; the polyoxyethylene chain co~ lg about 70 percent of the total number of monomeric units in the molecule and where the copolymer has an average molecular weight of about 12,600. Pluronic F-127, also known as Poloxamer 407, is such a material.
The procedures used to prepare aqueous solutions which form gels of polyoxyalkylene block copolymer are well known. For example, either a hot or cold process for forming the solutions can be used. The cold technique involves the steps of dissolving the polyoxalkylene block copolymer at a Le~ ela~ule of about ~~C to 10~C in water or in a buffer, such as a phosphate buffer. The water, if used in forming the aqueous solution, is preferably purified, as by ~ ti~ln7 filtration, ion-exchange or the like. When the solution is Col"pl it is brought to room L~llpe-àLule whereupon it forms a gel. If the hot process of forming the gel is used, the polymer is added to water or a buffer and heated to a temperature of about 75~C to 85~C with slow stirring until a clear homogenous solution is obtained, upon cooling a clear CA 02260967 lgg9-ol-ll WO 98/02142 PCT/US97tl3479 gel forms.
Any macromolecular polypeptide may be mixed with the pharm:lceutic~l vehicle to form the ph~ reutir.~l composition of this invention wherein the concentration of macromolecular polypeptide is in the range of 0.5 to 50 percent by weight of the composition. The choice of polypeptides which can be 5 delivered in accordance with the practice of this invention is limited only by the requirement that they be at least very slightly soluble in an aqueous physiological media such as plasma, interstitial fluid, and the intra and extracellular fluids of the subcutaneous space and mucosal tissues.
Exemplary classes of polypeptides include, among others, proteins, enzymes, nucleoproteins, glycoproteins, liyu~loteills, hormonally active polypeptides, and synthetic analogues including agonists and 10 antagonists of these molecules.
Specific examples of polypeptides suitable for incorporation in the delivery system of the present invention include the following biologically active macromolecules: intelr~lolls, interleukins, insulin, enzyme inhibitors, colony-stim~ ting factors, plasminogen activators, growth factors and polypeptide hormones.
The list of macromolecular polypeptides recited above are provided only to illustrate the types of active agents which are suitable for use in practicing the present invention, and are not intended to limit the scope of the present invention.
The pha m:~ceutir~l composition of the present invention can be readily prepared using any solution forming technique which achieves the concentration of polyoxyalkylene block copolymer nrcec~ry for gelling. Preferably the pharn~reutir.~l vehicle and polypeptide mixture are prepared separately and the polypeptide mixture having a co,lcel-Ll~tion of 5 mg/ml or greater is added thereto at a temperature of about 0~C to lODC. When combined the protein forms a homogenous suspension of fine particles in the polymer solution, which then has a "milky" appearance. By light miclvscol)y the particles are approximately ~-10 microns in diameter. Raising the sample t~ u~laLule above the gel point of the poloxamer results in an even distribution of protein particles throughout the polymer gel. Due to the high viscosity of the gel matrix, the particles remain homogeneously diaLlib.lLed and do not "settle out." The liquid to gel transition is fully reversible upon cooling. Furthermore, when the gel is exposed to an aqueous solution, the gel matrix and protein particles dissolve, releasing the fully native protein which retains greater than 90 percent of its biological activity.
The ph~ relltit~l composition of the present invention can be implanted directly into the body by injecting it as a liquid, whereupon the ph~ relltir.~l composition will gel once inside the body. In the alternative, the ph~rm~reutir~l composition may be introduced into a small i~inplantable pump which is then introduced into the body.
In another embodirnent, protein-stabilizing solutes, can be incorporated into the ph~tm~ceutical device of the present invention described above. Initially, stabilizers were added to the ph~ eutir~l device of the present invention to increase the stability of the macromolecular polypeptides as such CA 02260967 lsss-ol-ll WO 98/02142 PCT/USs7/13479 stabilization would be crucial for use of the present invention for suct~in~d delivery of protein in the body.
However, in doing so it was discovered that protein-stabilizing solutes, such as sucrose, no~ only aid in protecting and stabilizing the protein, but also allow the poloxamer to form suitable gels at lower concentrations than needed in water or buffer alone. Thus, the working range of polymer concentration 5 can be widened. As discussed previously, the concentration of the polyoxyalylene block copolymer is an important parameter. It is known that a gel will not form when the concentration of polyoxyethelene-polyoxypropylene block copolymer in water or dilute buffer is outside of the range of about 20 to 30 percent by weight, as shown in Figure 1 and exemplified by the line having open triangles. However, by introducing protein-stabilizing solutes to the pharm~c.eutic~l device of the present invention the gel-sol 10 transition temperature may be manipulated, while also lowering the concentration of polyoxyethelene-polyoxypropylene block copolymer which is nPcecc~ry to form a gel.
In a third embodiment, polypeptide concentrations at the high end of the 0.5 to 50 percent by weight range can be achieved by centrifuging the pharmaceutical composition of the present invention at low temperatures in the range of -10~C to 10~C, and preferably 04~C for a period of time sufficient to 1~ sediment the protein particles. For example, a sample of the pharmaceutical composition described previously comprising 20 mg/ml protein can be centrifuged at 4~C so that the insoluble protein particles se-iimP.nt Then supernatant equivalent to half the volume could be removed and the se~limP.nf resuspended in the rem~ining liquid. This will result in a suspension containing almost 40 mg/ml.
In the Examples which follow the ph~ ce ltical composition of the present invention was prepared 20 according to the following preparation procedure. Since the polyoxyalkylenes dissolve more completely at reduced temperatures, the preferred methods of solubilization are to add the required amount of copolymer to the amount of water or buffer to be used. Generally after wetting the copolymer by shaking, the mixture is capped and placed in a cold chamber or in a thennost~ti~ container at about 0~C to 10~C in order to dissolve the copolymer. The mixture can be stirred or shaken to bring about a more rapid solution 25 of the polymer. The polypeptides and various additives such as stabilizers can subsequently be added and dissolved to form a suspension.
The following non-limited examples provide methods for l~lepa~ g temperature sensitive polymers for the sustained delivery of ph~rrn~cel-tic~l agents comprising high concentrations of fully native macromolecular polypeptide agents. All scientific and te~hnif~l terms have the mPaning.c as understood 30 by one with ordinary skill in the art. The specific examples which follow illustrate the representative polypeptides and concentrations capable of being achieved by the present invention and are not to be construed as limiting the invention in sphere or scope. The methods may be adapted to variation in order to produce compositions or devices embraced by this invention but not specifically disclosed. Further variations of the mPtho~l.c to produce the same compositions in somewhat different fashion will be evident 35 to one skilled in the art.
All temperatures are understood to be in Centigrade (~C) when not specified. The infrared (IR) CA 02260967 lsss-ol-ll spectral description was measured on a Nicolet Magna-IR 550 Spectrometer. Comrnercially available ch~min~l~ were used without purification.
In the Examples which follow the 27.5 percent (w/w) solution of Pluronic F-127 was prepared in the following manner. 7.59 grams of dry Pluronic F-127 was added to a sterile tube containing 20 grams 5 of an ice cold phosphate buffer (pH 7.4, 30 mM). The tube was capped and the mixture was well shaken prior to being stored overnight at 4~C.
~XAMPLE 1 p~ inn a~d Chara-l~.;Ldtion o~ a Sl-cpf~n~inn of Cl~ s~ ill PlulV-liC F-127 10 a) Preparation of the ~ .y~ inn-When 50~L of a 100 mglmL suspension of chymotrypsin in 30 mM phosphate buffer at pH 7.4 were mixed with 200 ~L of a 27.5 percent (wlw) solution of Pluronic F-127 at 4~C a white, uniform, milky suspension formed imm~ tely. The su~l~el~iull contained 20 mglmL protein and 22 percent (w/w) Pluronic F-127 and was a viscous liquid below about 18~C and a soft, solid gel above about 20~C. The crystalline portion of the liquid suspension settled out of solution when allowed to stand for a day or two at 4~C or when centrifuged in the cold but could be readily resuspended by mixing while cold. The fact that the suspended material can be se-liment~d out and resuspended in a smaller volume than the original suspension permits formation of suspensions of gienifir~ntly higher concentrations, as discussed previously.
In order to ~l~.t~rmin~ the solubility of chymotrypsin in the liquid phase of the suspension, aliquots of the total suspension and of the supernatant following centrifugation at 1000-5000 rpm for 5 minutes at 4~C were assayed spectrophotometrically for enzyme activity. The concentration of chymotrypsin that r~m~in~d soluble in the Pluronic F-127 is shown below in Table 2.
Table 2 Enzyme Solubility (mglmL)' Std. Dev.
Chymotrypsin 0 132 0.06~
D~L~. .";..~d by dividing the activity measured in the ~ by the calculated activity per mg of protein in the whole 2 n=3.
b) S~epen-~e-l enzyme exhibits native-like seC~n~lq~ structure ~s measured by FTIR:
Infrared spectra obtained using a Nicolet Magna-IR 550 Spectrophotometer in a temperature regulated, 6 ~M path length cell were used to examine and compare the structure of chymotrypsin in phosphate buffer (30 mM, pH 7.4) and in 22 percent (w/w) Pluronic F-127 in the same buffer. Protein 35 concentration in these studies was 20 mg/mL. Careful examination of the spectra showed that the secondary structure of the chymotrypsin was not altered due to suspending the protein in the Pluronic F-127. A-l~itinn~l ~;TIR s~e~loscopic ex~min~tions of the structure of chymotrypsin in Pluronic F-127 at CA 02260967 lsss-ol-ll temperatures below and above the gel transition (about 15~C to 18~C) showed that the formation of the ge]
did not alter the structure.
c) Suspended enzyme m~int~inc biDIo ~ ;cal activity:
For this experiment, a chymotrypsin suspension in 22 percent Pluronic F-127 was prepared as 5 described above but at two dirrelellL concentrations, 20 mg/mL and 2 mg/mL. Solutions with the same protein concentrations were prepared using phosphate buffer (30 mM, pH 7.4) with no Pluronic F-127.
Aliquots of each of these were diluted as necessary and assayed for enzymatic activity as described above.
As can be seen from Table 3, below, enzyme incorporated into the gels could be recovered quantitatively and with no loss of activity after the gels were dissolved in buffer.
Table 3 Environment Concentration Dilution Observed Std.Percent of (mg/ml) before Activity Dev.Activity in Assay Buffer Buffer' 20 5000 21.234 1.134 Pluronic2 20 5000 21.6 3.4 102 Buffer 2 500 21.0 0.8 15Pluronic 2 500 25.8 1.4 123 Phosphate, 30mM. pH 7.4 2 2~% (wlw) in 30 mM Phosphate buffer, pH 7.4 Expressed as mAU/min at 410 nm 204 n=9 The biological activity of chymotrypsin was not damaged by inclusion in the Pluronic F-127. A
slight enh~n~çmPnt of activity was observed when low levels of the detergent were present in the enzyme assay mLYtures. It is possible that the difference in percent recovery observed between the 2 mg/mL and 2~ 20 mg/mL samples shown in Table 3 is due to the greater dilution of the gel in the 20 mglmL sample and thus higher levels of Pluronic F-127 in the 2 mg/mL samples.
d) S~l~pPn(le~l enzyme di~lJldy~ in.leased storage stability:
In order to demonstrate that suspending proteins in 22 percent Pluronic F-127 has a stabilizing action, 20 mg/mL chymotrypsin was prepared in either 30 rnM phosphate buffer, pH 7.4 or 22 percent 30 Pluronic F-127 in this buffer as described above. These suspensions were diluted as necessary and - enzyme activity was assayed. The results shown in Table 4 below, demonstrate that the enzyme incubated in the presence of Pluronic F-127 retained more activity than enzyme incubated in the presence of phosphate buffer alone.
.. . . . ..
CA 02260967 l999-01-ll Table 4 Time(hr.) Buffer' Pluronic2 Buffer Pluronic Buffer Pluronic 8 degrees 8 degrees 25 degrees25 degrees 37 37 degrees degrees 1003 ~ 00 100 lO0 100 24 104 79 104 84 na4 59 I Phosphate 2 22% (w/w) in phosphate buffer, 30 mM, pH 7.4 0 3 Expressed as a p~ of the I hour value 4 Value not available l~9 Thus, Pluronic F-127 provides a stabilizing e~ llent, which is more evident at extended times and elevated temperatures, in which peptide and protein containing drugs may be suspended prior to and during delivery. Additionally, other protein stabilizing agents may be added to the form~ tion as described further in Example 6 below.
F~ dtion and Characterization~of a of Sul)lil~l in Pluronic F-127 a) P~ dlion of the ~IJ ..~ n A suspension of subtilisin was made by the same procedure described in detail above for chymotrypsin. The physical properties were the same in terms of formation and behavior of the milky white sll~pen.cion and gel-sol transition telnperatu~e. The solubility of subtilisin in the liquid phase of the 25 sllcpen~inn was elr~min~od using the method described above and found to be dir~le~lL as might be expected for a di~lelll protein. The results are represented in Table 5 below.
Table 5 Enzyme Solubility (mglmL)' Std. Dev.
Subtilisin 6.482 1. l 12 Determined by dividing the activity measured in the ~u~ dl~ull by the calculated activity per mg of protein in the whole .n 2 n=~
35 b) Suspended enzyme exhibits native-like sec~n-l~ry structure as l-le~.,l ed by FT~R:
Subtilisin suspensions, like those of chymotrypsin described above, were e~min~.d by FTIR
spectroscopy to determinP whether inclusion in the gel had any effect on the structure and whether the gel-sol transition influenced structure. As in the case of chymotrypsin, there was no effect on structure.
CA 02260967 lg99-ol-ll Il c) S~cpen-le~l enzyme maintains biologir~l aeLivily;
In experiments similar to those described above for chymotrypsin, subtilisin suspensions were prepared in both phosphate buffer and 22 percent Pluronic F-127 in phosphate buffer and then dissolved by dilution with buffer. In these cases like in the case of chymotrypsin, 100 percent of the enzyme activity 5 was recovered showing that biological activity was stable while incorporated into the gel suspension.
d) S--cp~n~le~ enzyme ~la~ ine~ea3ed storage stability:
In order to demonstrate that suspending subtilisin in 22 percent Pluronic F-127 has a stabilizing action similar to that demonstrated for chymotrypsin, 20 mg/mL subtilisin was prepared in either 30 mM
phosphate buffer, pH 7.4 or 22 percent Pluronic F-127 in phosphate buffer as described above. These suspensions were incubated at temperatures of 8, 25 and 37~C for times up to 118 hours, the gels or suspensions were diluted as necessary and enzyme activity was assayed. The results, shown below in Table 6, demonstrate that even though subtilisin is al~tocat~lytic and looses activity nnore rapidly than chymotrypsin, it still retained more activity when incubated in the presence of Pluronic F-127 than when incubated in the presence of phosphate buffer alone.
Table 6 Time(hr.) Buffer' Pluronic2 Buffer Pluronic Buffer Pluronic 8 de~rees 8 degrees25 degrees25 degrees37 degrees 37 degrees 0.5 1003 100 100 100 100 100 ' Phosphate, 30 mM, pH 7.4 2 22% ~w/w) in rh- $ph~t~ buffer, 30 m M,pH 7.4 3 Expressed as a percentage of the 0.5 hour value Preparation and Char~el~ iLdtiOIl of a SucpPn~;~ n of Lactate Dehydrogenase in Pluronic F-127 30 a) P~epa~aliu~, of the ~ s;om A ~u~tllsion of lactate dehydrogenase was made by the same procedure described in detail above for chymotrypsin. Visual observations inllic~tPd that the physical properties were the same in terms of formation and behavior of the milky white suspension and gel-sol transition temperature.
b) S~ le-l enzyme maintains biological ..-,livily:
In experiments similar to those described above for chymotrypsin, lactate dehydrogenase suspensions were prepared in both phosphate buffer and 22 percent Pluronic F-127 in phosphate buffer and then disso~ved by dilution with buffer. In these cases like in the case of chymotrypsin, 100 percent of the enzyme activity was recovered showing that biological activity was stable while incorporated into the gel suspension.
WO 98/02142 PCTtUS~7113479 c) Suspended enzyme di~lay~ increased storage stability: -Experiments lil~e those carried out with chymotrypsin were conducted to illustrate the effect of Pluronic F-127 in stabilizing the enzyme activity of lactate dehydrogenase during storage at elevated elaLul~s. In this case 0.5 M sucrose was inrhlded in some samples and thus it was necessary to reduce 5 the concentration of Pluronic F-127 to 18 percent to m~inr~in the same gel-sol transition temperature.
Table 7 below, shows the results obtained when lactate dehydrogenase was stored for 48 hours at 37~C.
Table 7 Sample Composition'Enzyme Activity~ Std. Dev. (n=3) 1018% Pluronic 59.3 1.7 18% Pluronic + 0.5 M sucrose 46.1 2.7 0.5 M sucrose 39.1 3.3 buffer alone 40.0 1.7 ' Bllffer used in all was 50 mM Tris:HCI, pH 7.35 with 0.1% NaN3 2 Expressed in arbitrary units ~XAMPLE 4 Preparation and Chara~ alion of a S~cpPn~;c of Bovine Serum Albulmin in Pluronic F-127 a) Preparation of the s..~l,f ..-: n-Several mixtures of Pluronic F-127 and bovine serum albumin were made in attempts to find a 25 combination that would be a clear gel with a protein concentration of 15 mglmL or higher at room t~ul~elalul~. Pluronic F-127 concentrations below 20 percent (w/w) would not gel at 25~C or below and con-.~ntr~ti~-ns of 20 percent and above resulted in the milly, white suspension when bovine serum albumin was added at concentrations of 14 mg/mL and above. Thus, no cuull,ill~lion was found that would provide the desired properties. Subsequent studies of structure and stability on several proteins demonstrated that 30 it was not nl~ce.cc~ry to have a clear gel to have a .c~ticf~rtory protein drug delivery forrnulation with x Pluronic F-127.
b) Su~ ~P-l protein exhibits native-like secnnllqry ~I- ueLul e as ~l~ea~ur~ d by FTIR:
Preliminary FTIR ~e~ osc~p;c e~r~min~tion of the structure of a suspension of 20 mg/mL bovine serum albumin in 22 percent Pluronic F-127 showed no differences from a similar suspension of the 35 protein in buffer alone. This result was similar to resu1ts from more extensive ~ min~ions of the structures of chymotrypsin and subtilisin suspended in the gel.
Preparation and Characterizatio~n of ~ S~.~ S;~., of In..~llin in Pluroriuc F-127 a) Preparation ofthe ~ n A suspension of insulin was made by the same procedure described in detail above for chymotrypsin. Visual observation in~lic~ted that the physical properties were the same in terms of formation and behavior of the milky white suspension and gel-sol transition temperature.
10 b) Suspended protein exhibits native-like secon~l~ry structure as ~.l.ea~ul~d by FTIR:
Insulin suspensions, like those of chymotrypsin described above, were ex~min~d by FTIR
spectroscopy to determine whether inclusion in the gel had any effect on the structure and whether the gel-sol transition influenred structure. As in the case of chymotrypsin, there was no effect on structure.
Protein Stabilizing Agents Used to Manipulate Gel Properties Various concentrations of sucrose were incorporated into Pluronic F-127 solutions as an example 20 of the use of known protein stabilizing agents to manipulate the properties of the gels. Typical results for gel-sol transition temperature are shown in Table 8 below.
Table 8 % Pluronic' 0 M Sucrose0.5 M Sucrose 1.0 M Sucrose1.5 M Sucrose 16 25 20 0.2 ' w/w, in 30 ~nM phosphate, pH 7.4 It is clear from Table 8 that the gel-sol transition ten1p~ldture can be nanipulated in the range from below 0~C to about 25"C as required by the present invention.
The foregoing description is considered as illustrative omy of the principles of the invention.
Furthermore, since numerous mo-lifi~ ns and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and processes shown as described above.
Accordingly, all suitable modifications and equivalents may be resorted to falling within the scope of the invention as defined by the claims which follow.
.. _ .. . ...
Claims (32)
1. A pharmaceutical composition for the controlled and sustained administration of biologically active macromolecular polypeptides comprising a polymeric matrix having thermal gelation properties in which aqueous soluble particles of at least one biologically active macromolecular polypeptide at a concentration sufficient to form a suspension when incorporated into said polymeric matrix are incorporated into said polymeric matrix.
2. The composition of claim 1, wherein said polymeric matrix is a polyoxyalkylene block copolymer.
3. The composition of claim 2, wherein said polyoxyalkylene block copolymer has the formula:
HO(CH2O)~(C3H~O)~(C2H4O)~H
wherein polyoxyethylene moiety constitutes at least about 70 percent by weight of said polyoxyalkylene block copolymer, wherein a and b are integers such that the hydrophobe base represented by (C~H~O)~
has a molecular weight of at least about 4,000 and wherein said copolymer has an average molecular weight of about 12,600.
HO(CH2O)~(C3H~O)~(C2H4O)~H
wherein polyoxyethylene moiety constitutes at least about 70 percent by weight of said polyoxyalkylene block copolymer, wherein a and b are integers such that the hydrophobe base represented by (C~H~O)~
has a molecular weight of at least about 4,000 and wherein said copolymer has an average molecular weight of about 12,600.
4. The composition of claim 2, wherein said polyoxyalkylene block copolymer has the formula;
wherein a is 20 to 80 ant b is 15 to 60.
wherein a is 20 to 80 ant b is 15 to 60.
5. The composition of claim 4, wherein a is 67 and b is 49.
6, The composition of claim 1, wherein said particles of macromolecular polypeptides constitute greater than 0.5 percent by weight of the composition.
7. The composition of claim 1, wherein said incorporated macromolecular polypeptide retains greater than 90 percent of the biological activity which said macromolecular polypeptide possessed prior to being incorporated.
8. The composition of claim 1, wherein a transition temperature exists 50 that if said composition is held at a first temperature below said transition temperature said composition will exist in a liquid phase ant if said composition is held at a second temperature above said transition temperature said composition will exist in a gelatinous phase.
9. The composition of claim 8, further comprising a solute that varies said transition temperature at which said composition will exist in either a liquid or gelatinous phase.
10. The composition of claim 9, wherein said transition temperature is decreased when said solute is sucrose.
11. The composition of claim 10, wherein said sucrose has a molar concentration of 0.05 to 1.5.
12. The composition of claim 9, wherein said transition temperature is increased when said solute is polyethylene glycol,
13. The composition of claim 9, wherein the minimum concentration of said polymeric matrix necessary to gel decreases upon the addition of said solute.
14. A pharmaceutical composition for the controlled and sustained administration of a biologically active macromolecular polypeptide comprising a polymeric matrix in which a suspension of aqueous soluble, macromolecular polypeptide particles is incorporated.
15. The composition of claim 14, wherein said polypeptide retains greater than 90 percent of the biological activity which said polypeptide possessed prior the being incorporated into said polymeric matrix.
16. The composition of claim 14, wherein said polymeric matrix possesses thermal gelation properties.
17. The composition of claim 16, wherein said polymeric matrix is a polyoxyethylene-polyoxypropylene block copolymer.
18. The composition of claim 18, wherein said concentration of said polymeric matrix necessary to form a gel may be decreased with the addition of a solute.
19. The composition of claim 18, wherein said solute is sucrose.
20. The composition of claim 9, wherein said solute is a protein stabilizer.
21. The composition of claim 16, wherein a transition temperature exists wherein said polymeric matrix form a gel if in a liquid phase or alternatively said polymeric matrix will form a liquid if in a gelatinous phase.
22. The composition of claim 21, wherein said transition temperature may be varied with the addition of a solute.
23. The composition of claim 22, wherein said transition temperature is decreased when said solute is sucrose.
24. The composition of claim 22, wherein said transition temperature is increased when said solute is polyethylene glycol.
25. The composition of claim 22, wherein said solute is a protein stabilizer.
26. The composition of claim 22, wherein said solute is sucrose.
27. The composition of claim 22, wherein said solute is polyethylene glycol.
28. A method for preparing the pharmaceutical composition of claim 1, comprising:
preparing a mixture of a polymeric matrix having thermal gelation properties; and adding to said mixture at least one soluble biologically active macromolecular polypeptide.
preparing a mixture of a polymeric matrix having thermal gelation properties; and adding to said mixture at least one soluble biologically active macromolecular polypeptide.
29. The method of claim 28, wherein said polymeric matrix is a polyoxyalkylene block copolymer.
30. The method of claim 29, wherein said polyoxyalkylene block copolymer has the formula:
HO(C2H4O)~(C3H~O)~(C~H4O)~ H wherein a is 20 to 80 and b is 15 to 60.
HO(C2H4O)~(C3H~O)~(C~H4O)~ H wherein a is 20 to 80 and b is 15 to 60.
31. The method of claim 28, further comprising adding sucrose.
32. The method of claim 28, further comprising adding polyethylene glycol.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/679,199 US5861174A (en) | 1996-07-12 | 1996-07-12 | Temperature sensitive gel for sustained delivery of protein drugs |
| US08/679,199 | 1996-07-12 | ||
| PCT/US1997/013479 WO1998002142A1 (en) | 1996-07-12 | 1997-07-11 | Temperature sensitive gel for sustained delivery of protein drugs |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2260967A1 true CA2260967A1 (en) | 1998-01-22 |
Family
ID=24725962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002260967A Abandoned CA2260967A1 (en) | 1996-07-12 | 1997-07-11 | Temperature sensitive gel for sustained delivery of protein drugs |
Country Status (13)
| Country | Link |
|---|---|
| US (2) | US5861174A (en) |
| EP (1) | EP0910342B1 (en) |
| JP (1) | JP2000514811A (en) |
| KR (1) | KR20000023751A (en) |
| CN (1) | CN1230108A (en) |
| AT (1) | ATE417601T1 (en) |
| AU (1) | AU3822497A (en) |
| BR (1) | BR9712969A (en) |
| CA (1) | CA2260967A1 (en) |
| DE (1) | DE69739169D1 (en) |
| ES (1) | ES2318860T3 (en) |
| IL (1) | IL127976A0 (en) |
| WO (1) | WO1998002142A1 (en) |
Families Citing this family (66)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0955063A1 (en) * | 1998-04-01 | 1999-11-10 | Basf Aktiengesellschaft | Use of aqueous compositions in medicaments for subcutaneous or intramuscular administration |
| JP2002512200A (en) * | 1998-04-17 | 2002-04-23 | アンジオジェニックス， インコーポレイテッド | Therapeutic angiogenic factors and methods of use |
| ATE341345T1 (en) | 1999-06-11 | 2006-10-15 | Cytyc Corp | LIQUID GEL FORMULATION FOR DETECTING MILK DUCTS IN THE BREAST PRIOR TO SURGICAL ABLATION OF THE BREAST TISSUE |
| US6488659B1 (en) * | 1999-08-05 | 2002-12-03 | Biocardia, Inc. | System and method for delivering thermally sensitive and reverse-thermal gelation materials |
| AU2001276831A1 (en) | 2000-06-22 | 2002-01-02 | Rxkinetix, Inc. | Delivery vehicle composition and methods for delivering antigens and other drugs |
| BR0111948A (en) * | 2000-06-26 | 2004-08-24 | Rxkinetix Inc | Composition for delivery of hematopoietic growth factor |
| MXPA03004546A (en) * | 2000-11-22 | 2003-09-10 | Rxkinetix Inc | Treatment of mucositis. |
| US6565530B2 (en) | 2001-02-28 | 2003-05-20 | Scimed Life Systems, Inc. | Immobilizing objects in the body |
| US20040185101A1 (en) * | 2001-03-27 | 2004-09-23 | Macromed, Incorporated. | Biodegradable triblock copolymers as solubilizing agents for drugs and method of use thereof |
| CN1253220C (en) * | 2001-06-29 | 2006-04-26 | 贝克顿迪肯森公司 | Intradermal delivery of vaccines and gene therapeutic agents via microcannula |
| US20060073174A1 (en) * | 2001-08-16 | 2006-04-06 | Access Pharmaceuticals, Inc. | Adherent and erodible film to treat a moist surface of a body tissue |
| US6585997B2 (en) | 2001-08-16 | 2003-07-01 | Access Pharmaceuticals, Inc. | Mucoadhesive erodible drug delivery device for controlled administration of pharmaceuticals and other active compounds |
| MXPA04003640A (en) * | 2001-10-16 | 2005-04-11 | Rxkinetix Inc | High-concentration protein formulations and method of manufacture. |
| US7205337B2 (en) * | 2001-12-21 | 2007-04-17 | Isotis Orthobiologics, Inc. | End-capped polymers and compositions containing such compounds |
| KR20040075901A (en) * | 2001-12-21 | 2004-08-30 | 아이소티스 오르토바이올로직스, 인크. | End-capped polyalkylene glycols and compositions containing such compounds |
| WO2003070805A1 (en) * | 2002-02-15 | 2003-08-28 | Nektar Therapeutics Al, Corporation | Hydrolytically degradable alkylene oxide based polymers |
| US7649023B2 (en) | 2002-06-11 | 2010-01-19 | Novartis Ag | Biodegradable block copolymeric compositions for drug delivery |
| US7588774B2 (en) * | 2003-05-12 | 2009-09-15 | Becton, Dickinson And Company | Molecules enhancing dermal delivery of influenza vaccines |
| US20050123550A1 (en) * | 2003-05-12 | 2005-06-09 | Laurent Philippe E. | Molecules enhancing dermal delivery of influenza vaccines |
| DE10326152A1 (en) * | 2003-06-06 | 2005-01-05 | Aventis Pharma Deutschland Gmbh | Method and device for the quantitative analysis of solutions and dispersions by means of near-infrared spectroscopy |
| US20050118261A1 (en) * | 2003-06-12 | 2005-06-02 | Oien Hal J. | Compositions and methods of administering doxepin to mucosal tissue |
| AU2004315509A1 (en) * | 2003-12-05 | 2005-08-18 | Becton, Dickinson And Company | Methods of enhancing immune response in the intradermal compartment and compounds useful in the methods |
| WO2006012536A2 (en) | 2004-07-22 | 2006-02-02 | Ritter Andrew J | Methods and compositions for treating lactose intolerance |
| US20060121055A1 (en) * | 2004-12-06 | 2006-06-08 | Becton, Dickinson And Company, Inc. | Compositions with enhanced immunogenicity |
| CA2635603C (en) * | 2005-11-30 | 2016-01-19 | Endo Pharmaceuticals Inc. | Treatment of xerostomia |
| US20070191816A1 (en) * | 2006-02-13 | 2007-08-16 | Boston Scientific Scimed, Inc. | Radio frequency induced drug elution |
| EA026670B1 (en) * | 2006-03-10 | 2017-05-31 | Лабосвисс Аг | Method for solubilizing, dispersing, and stabilizing materials, products manufactured according to said method, and use thereof |
| CA2656179A1 (en) * | 2006-06-21 | 2007-12-27 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for joining non-conjoined lumens |
| US8608760B2 (en) * | 2006-06-21 | 2013-12-17 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for joining non-conjoined lumens |
| US8197499B2 (en) * | 2006-06-21 | 2012-06-12 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions and methods for joining non-conjoined lumens |
| PL2124556T3 (en) | 2006-10-09 | 2015-02-27 | Charleston Laboratories Inc | Pharmaceutical compositions |
| US8043604B2 (en) * | 2007-04-27 | 2011-10-25 | Pluromed, Inc. | Ultrasonography using time- and temperature-sensitive variable adhesion coupling gels |
| CN101450035B (en) * | 2007-12-07 | 2010-09-29 | 福建医科大学附属协和医院 | Double sustained release biological agent for tumor local ablation therapy |
| EP2224968A2 (en) * | 2007-12-20 | 2010-09-08 | Tautona Group, L.P. | Compositions and methods for joining non-conjoined lumens |
| EP2240022B1 (en) | 2008-01-09 | 2016-12-28 | Charleston Laboratories, Inc. | Bilayered tablets comprising oxycodone and promethazine |
| JP5836593B2 (en) * | 2008-02-29 | 2015-12-24 | プルーロームド インコーポレイテッドＰｌｕｒｏｍｅｄ， Ｉｎｃ． | Local embolization by heating of thermosensitive polymers |
| WO2009132050A2 (en) | 2008-04-21 | 2009-10-29 | Otonomy, Inc. | Auris formulations for treating otic diseases and conditions |
| US8030297B2 (en) | 2008-05-14 | 2011-10-04 | Otonomy, Inc. | Controlled release corticosteroid compositions and methods for the treatment of OTIC disorders |
| US8846770B2 (en) | 2008-06-18 | 2014-09-30 | Otonomy, Inc. | Controlled release aural pressure modulator compositions and methods for the treatment of OTIC disorders |
| WO2010011466A2 (en) | 2008-06-27 | 2010-01-28 | Otonomy, Inc. | Controlled-release cns modulating compositions and methods for the treatment of otic disorders |
| CA2731769C (en) | 2008-07-21 | 2013-09-10 | Otonomy, Inc. | Controlled-release otic structure modulating and innate immune system modulating compositions and methods for the treatment of otic disorders |
| CA2731766C (en) * | 2008-07-21 | 2014-01-28 | Otonomy, Inc. | Controlled release antimicrobial compositions and methods for the treatment of otic disorders |
| US8496957B2 (en) | 2008-07-21 | 2013-07-30 | Otonomy, Inc | Controlled release auris sensory cell modulator compositions and methods for the treatment of otic disorders |
| US8784870B2 (en) | 2008-07-21 | 2014-07-22 | Otonomy, Inc. | Controlled release compositions for modulating free-radical induced damage and methods of use thereof |
| US8318817B2 (en) | 2008-07-21 | 2012-11-27 | Otonomy, Inc. | Controlled release antimicrobial compositions and methods for the treatment of otic disorders |
| US8399018B2 (en) * | 2008-07-21 | 2013-03-19 | Otonomy, Inc. | Controlled release ion channel modulator compositions and methods for the treatment of otic disorders |
| US8563037B2 (en) * | 2009-02-06 | 2013-10-22 | Tautona Group, L.P. | Compositions and methods for joining non-conjoined lumens |
| KR20170053733A (en) | 2009-02-24 | 2017-05-16 | 리터 파마슈티컬즈 인코오포레이티드 | Prebiotic formulations and methods of use |
| WO2011006012A1 (en) | 2009-07-08 | 2011-01-13 | Charleston Laboratories Inc. | Pharmaceutical compositions |
| JP5892940B2 (en) | 2009-11-25 | 2016-03-23 | アリスジェン ソシエテ アノニム | Mucosal delivery composition comprising a peptide complexed with a crown compound and / or counterion |
| KR101812820B1 (en) * | 2010-01-08 | 2017-12-27 | 웨이크 포리스트 유니버시티 헬스 사이언시즈 | Delivery system |
| WO2011137249A1 (en) | 2010-04-28 | 2011-11-03 | Ritter Pharmaceuticals, Inc. | Prebiotic formulations and methods of use |
| US8642502B2 (en) | 2010-10-11 | 2014-02-04 | TRGel, LLC | Reversible gel-forming compositions for controlled delivery of bioactive substances |
| WO2012122105A1 (en) | 2011-03-07 | 2012-09-13 | Wake Forest University Health Sciences | Delivery system |
| CA2829791A1 (en) * | 2011-03-28 | 2012-10-04 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Sustained-release injectable formulation |
| WO2015031393A1 (en) | 2013-08-27 | 2015-03-05 | Otonomy, Inc. | Treatment of pediatric otic disorders |
| WO2015153841A1 (en) | 2014-04-04 | 2015-10-08 | Ritter Pharmaceuticals, Inc. | Methods and compositions for microbiome alteration |
| CA2954170A1 (en) | 2014-07-03 | 2016-01-07 | Otonomy, Inc. | Sterilization of ciprofloxacin composition |
| EP3272360B1 (en) * | 2015-03-17 | 2022-11-23 | Santen Pharmaceutical Co., Ltd. | Pharmaceutical composition comprising polypeptide |
| EP3423041A4 (en) | 2016-03-04 | 2019-09-11 | Charleston Laboratories, Inc. | Pharmaceutical compositions |
| US11040004B2 (en) | 2016-09-16 | 2021-06-22 | Otonomy, Inc. | Otic gel formulations for treating otitis externa |
| CA3088630A1 (en) | 2017-12-15 | 2019-06-20 | Solarea Bio, Inc. | Microbial compositions and methods for treating type 2 diabetes, obesity, and metabolic syndrome |
| CA3111795A1 (en) | 2018-09-05 | 2020-03-12 | Solarea Bio, Inc. | Methods and compositions for treating musculoskeletal diseases |
| EP3986163A2 (en) | 2019-06-19 | 2022-04-27 | Solarea Bio, Inc. | Microbial compositions and methods for producing upgraded probiotic assemblages |
| WO2023092150A1 (en) | 2021-11-22 | 2023-05-25 | Solarea Bio, Inc. | Methods and compositions for treating musculoskeletal diseases, treating inflammation, and managing symptoms of menopause |
| US20230190834A1 (en) | 2021-12-21 | 2023-06-22 | Solarea Bio, Inc. | Immunomodulatory compositions comprising microbial entities |
Family Cites Families (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4188373A (en) * | 1976-02-26 | 1980-02-12 | Cooper Laboratories, Inc. | Clear, water-miscible, liquid pharmaceutical vehicles and compositions which gel at body temperature for drug delivery to mucous membranes |
| US4100271A (en) * | 1976-02-26 | 1978-07-11 | Cooper Laboratories, Inc. | Clear, water-miscible, liquid pharmaceutical vehicles and compositions which gel at body temperature for drug delivery to mucous membranes |
| JPS5821691A (en) * | 1981-07-29 | 1983-02-08 | Mochida Pharmaceut Co Ltd | Purifying method of interferon |
| US5328695A (en) * | 1983-03-22 | 1994-07-12 | Massachusetts Institute Of Technology | Muscle morphogenic protein and use thereof |
| US4474752A (en) * | 1983-05-16 | 1984-10-02 | Merck & Co., Inc. | Drug delivery system utilizing thermosetting gels |
| US4474753A (en) * | 1983-05-16 | 1984-10-02 | Merck & Co., Inc. | Topical drug delivery system utilizing thermosetting gels |
| US4474751A (en) * | 1983-05-16 | 1984-10-02 | Merck & Co., Inc. | Ophthalmic drug delivery system utilizing thermosetting gels |
| US4478822A (en) * | 1983-05-16 | 1984-10-23 | Merck & Co., Inc. | Drug delivery system utilizing thermosetting gels |
| US4702917A (en) * | 1985-11-18 | 1987-10-27 | Research Triangle Institute | Porous bioabsorbable polyesters |
| US4954154A (en) * | 1986-03-26 | 1990-09-04 | The O. M. Scott & Sons Company | Controlled release fertilizer gel composition and process for its production |
| US4962091A (en) * | 1986-05-23 | 1990-10-09 | Syntex (U.S.A.) Inc. | Controlled release of macromolecular polypeptides |
| US4895716A (en) * | 1987-06-09 | 1990-01-23 | Biogen, Inc. | Stabilized formulations of gamma interferons |
| NZ226171A (en) * | 1987-09-18 | 1990-06-26 | Ethicon Inc | Gel formulation containing polypeptide growth factor |
| US5457093A (en) * | 1987-09-18 | 1995-10-10 | Ethicon, Inc. | Gel formulations containing growth factors |
| US5041292A (en) * | 1988-08-31 | 1991-08-20 | Theratech, Inc. | Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents |
| US4925677A (en) * | 1988-08-31 | 1990-05-15 | Theratech, Inc. | Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents |
| US5126141A (en) * | 1988-11-16 | 1992-06-30 | Mediventures Incorporated | Composition and method for post-surgical adhesion reduction with thermo-irreversible gels of polyoxyalkylene polymers and ionic polysaccharides |
| US5324519A (en) * | 1989-07-24 | 1994-06-28 | Atrix Laboratories, Inc. | Biodegradable polymer composition |
| DE4002736A1 (en) * | 1990-01-31 | 1991-08-01 | Hoechst Ag | Poly-amino-di:carboxylic acid co-anhydro-amino-di:carboxylic derivs. - bio:degradable polymeric peptide(s) for controlled release of pharmaceuticals |
| US5300295A (en) * | 1990-05-01 | 1994-04-05 | Mediventures, Inc. | Ophthalmic drug delivery with thermoreversible polyoxyalkylene gels adjustable for pH |
| US5292516A (en) * | 1990-05-01 | 1994-03-08 | Mediventures, Inc. | Body cavity drug delivery with thermoreversible gels containing polyoxyalkylene copolymers |
| US5306501A (en) * | 1990-05-01 | 1994-04-26 | Mediventures, Inc. | Drug delivery by injection with thermoreversible gels containing polyoxyalkylene copolymers |
| US5077033A (en) * | 1990-08-07 | 1991-12-31 | Mediventures Inc. | Ophthalmic drug delivery with thermo-irreversible gels of polxoxyalkylene polymer and ionic polysaccharide |
| CA2044878C (en) * | 1990-08-07 | 2000-12-26 | Tacey X. Viegas | Thermally reversible and irreversible gels |
| US5143731A (en) * | 1990-08-07 | 1992-09-01 | Mediventures Incorporated | Body cavity drug delivery with thermo-irreversible polyoxyalkylene and ionic polysaccharide gels |
| US5346703A (en) * | 1990-08-07 | 1994-09-13 | Mediventures, Inc. | Body cavity drug delivery with thermo-irreversible polyoxyalkylene and ionic polysaccharide gels |
| US5071644A (en) * | 1990-08-07 | 1991-12-10 | Mediventures, Inc. | Topical drug delivery with thermo-irreversible gels |
| US5124151A (en) * | 1990-08-07 | 1992-06-23 | Mediventures Inc. | Drug delivery by injection with thermo-irreversible gels |
| US5330768A (en) * | 1991-07-05 | 1994-07-19 | Massachusetts Institute Of Technology | Controlled drug delivery using polymer/pluronic blends |
| US5411737A (en) * | 1991-10-15 | 1995-05-02 | Merck & Co., Inc. | Slow release syneresing polymeric drug delivery device |
| EP0551626A1 (en) * | 1991-12-19 | 1993-07-21 | LEK, tovarna farmacevtskih in kemicnih izdelkov, d.d. | Thermoreversible gel as a liquid pharmaceutical carrier for a galenic formulation |
| US5468505A (en) * | 1992-02-28 | 1995-11-21 | Board Of Regents, The University Of Texas System | Local delivery of fibrinolysis enhancing agents |
| IT1255460B (en) * | 1992-07-28 | 1995-11-02 | Poli Ind Chimica Spa | PHARMACEUTICAL COMPOSITIONS IN THE FORM OF MICROEMULSIONS OR BIOADHESIVE LIPOSOMIAL DISPERSIONS FOR THE TRANSMUCOSAL ADMINISTRATION OF PEPTIDAL SUBSTANCES AND PHARMACOLOGICALLY ACTIVE PROTEINS |
| FR2694559B1 (en) * | 1992-08-06 | 1994-10-28 | Atta | New amphiphilic derivatives of amino acids or peptides, their preparation process and their use in preparations for biomedical use. |
| JP3698721B2 (en) * | 1993-02-23 | 2005-09-21 | ジェネンテク・インコーポレイテッド | Excipient stabilization of polypeptides treated with organic solvents |
| US5420197A (en) * | 1994-01-13 | 1995-05-30 | Hydromer, Inc. | Gels formed by the interaction of polyvinylpyrrolidone with chitosan derivatives |
| US5554147A (en) * | 1994-02-01 | 1996-09-10 | Caphco, Inc. | Compositions and devices for controlled release of active ingredients |
-
1996
- 1996-07-12 US US08/679,199 patent/US5861174A/en not_active Expired - Lifetime
-
1997
- 1997-07-11 ES ES97935238T patent/ES2318860T3/en not_active Expired - Lifetime
- 1997-07-11 AT AT97935238T patent/ATE417601T1/en not_active IP Right Cessation
- 1997-07-11 AU AU38224/97A patent/AU3822497A/en not_active Abandoned
- 1997-07-11 WO PCT/US1997/013479 patent/WO1998002142A1/en not_active Application Discontinuation
- 1997-07-11 CA CA002260967A patent/CA2260967A1/en not_active Abandoned
- 1997-07-11 IL IL12797697A patent/IL127976A0/en unknown
- 1997-07-11 BR BR9712969-0A patent/BR9712969A/en not_active Application Discontinuation
- 1997-07-11 EP EP97935238A patent/EP0910342B1/en not_active Expired - Lifetime
- 1997-07-11 DE DE69739169T patent/DE69739169D1/en not_active Expired - Lifetime
- 1997-07-11 CN CN97197798A patent/CN1230108A/en active Pending
- 1997-07-11 JP JP10506356A patent/JP2000514811A/en active Pending
-
1999
- 1999-01-12 KR KR1019997000215A patent/KR20000023751A/en not_active Application Discontinuation
- 1999-01-15 US US09/232,288 patent/US6482435B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP0910342B1 (en) | 2008-12-17 |
| IL127976A0 (en) | 1999-11-30 |
| EP0910342A4 (en) | 2006-03-22 |
| KR20000023751A (en) | 2000-04-25 |
| AU3822497A (en) | 1998-02-09 |
| EP0910342A1 (en) | 1999-04-28 |
| US5861174A (en) | 1999-01-19 |
| BR9712969A (en) | 2002-02-05 |
| DE69739169D1 (en) | 2009-01-29 |
| WO1998002142A1 (en) | 1998-01-22 |
| ATE417601T1 (en) | 2009-01-15 |
| US6482435B1 (en) | 2002-11-19 |
| ES2318860T3 (en) | 2009-05-01 |
| CN1230108A (en) | 1999-09-29 |
| JP2000514811A (en) | 2000-11-07 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |