WO1999024062A1 - Nouvelle composition d'igf-i et son utilisation - Google Patents

Nouvelle composition d'igf-i et son utilisation Download PDF

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Publication number
WO1999024062A1
WO1999024062A1 PCT/US1998/023672 US9823672W WO9924062A1 WO 1999024062 A1 WO1999024062 A1 WO 1999024062A1 US 9823672 W US9823672 W US 9823672W WO 9924062 A1 WO9924062 A1 WO 9924062A1
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Prior art keywords
igf
syrup
variant
composition
biologically active
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PCT/US1998/023672
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English (en)
Inventor
Bret A. Shirley
Maninder S. Hora
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Chiron Corporation
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Priority to EP98957637A priority Critical patent/EP1028747A1/fr
Priority to JP2000520150A priority patent/JP2001522813A/ja
Priority to AU13847/99A priority patent/AU1384799A/en
Publication of WO1999024062A1 publication Critical patent/WO1999024062A1/fr

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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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • 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
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Definitions

  • the present invention relates to a novel composition of IGF-I and variants thereof.
  • IGF-I Insulin-like growth factor I
  • IGF-I is a 70-amino-acid polypeptide hormone having insulin-like and mitogenic growth biological activities (Rinderknecht (1978) J. Biol. Chem. 253:2769; Rinderknecht (1978) FEBSLett. 89:283). This hormone enhances growth and/or survival of cells in a variety of tissues including musculoskeletal systems, liver, kidney, intestines, nervous system tissues, heart, and lung.
  • Administration of IGF-I has been indicated for the treatment of a variety of conditions in humans and animals.
  • Various formulations of IGF-I have been made. See, for example, U.S.
  • IGF-I Inclusion bodies containing IGF-I have been formed when IGF-I is expressed as a heterologous protein. See, for example, European Patent Nos. EP 123,228, EP 128,733, EP 135,094, EP 230,869, and EP 288,451. When incorporated into such inclusion bodies, IGF-I is in a generally misfolded and biologically inactive form, and must be reduced, refolded, and resolubilized into an active, solubilized form. See, for example, U.S. Patent Nos. 5,288,931, 5,410,026, 5,663,304, and 5,756,672; and International Publication No. WO 91/02807.
  • IGF-I or variant thereof and methods for its preparation are provided.
  • This novel form of IGF-I which is obtained according to the methods of the invention, has the consistency of a viscous "syrup".
  • This syrup has an IGF-I concentration of at least about 250 mg/ml, a density of about 1.0 g/ml to about 1.2 g/ml, and a viscosity of about 13,000 centipoise (cps) to about 19,000 cps, as measured at ambient temperature (23°C).
  • IGF-I is biologically active without the need for refolding.
  • the IGF-I syrup is prepared by precipitating IGF-I from solution, by, for example, appropriately adjusting the solution pH or by removal of a solubility enhancer.
  • the highly concentrated IGF-I syrup is useful as a means of storing IGF-I in a stable form and as a means for preparing compositions comprising biologically active IGF-I.
  • the IGF-I syrup, or IGF-I reconstituted from this syrup may be incorporated into other substances to form such compositions, as, for example, pharmaceutical preparations such as sustained-release formulations and delivery devices.
  • Pharmaceutical compositions comprising this concentrated IGF-I syrup are provided. Kits comprising IGF-I in this highly concentrated syrup form and a separate pharmaceutically acceptable biological buffer are also provided.
  • the IGF- I syrup, IGF-I reconstituted from the syrup, pharmaceutical compositions, and kits are useful in IGF-I therapy directed to IGF-I-responsive conditions.
  • FIG. 1 shows rhIGF-I solubility as a function of pH.
  • Figure 2 shows rhIGF-I solubility as a function of the concentration of arginine or one of several other compounds, some of which have a guanidinium group and some of which do not.
  • DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a novel form of IGF-I or variant thereof and methods for its preparation.
  • This highly concentrated, low salt-containing, biologically active form of IGF-I or variant thereof has the consistency of a viscous "syrup", hereinafter referred to as a syrup. Methods for preparing this novel form of IGF-I or a variant thereof are disclosed.
  • the IGF-I syrup is prepared by precipitating IGF-I from solution, preferably by appropriately adjusting the solution pH or by addition of an appropriate solubilizing agent to concentrate IGF- I in solution followed by removal of the solubilizing agent.
  • the resulting syrup form of IGF-I or variant thereof provides a means for packaging greater amounts of IGF-I within a given volume. The ramifications of these are the basis for the compositions and other methods disclosed in the present invention that are useful for IGF-I therapy directed to IGF-I-responsive conditions.
  • IGF-I insulin-like growth factor I
  • IGF- I insulin-like growth factor I
  • compositions and methods of the present invention encompass both IGF-I and IGF-I variants as defined below.
  • highly concentrated is intended an IGF-I concentration of at least about 250 mg/ml, for example, at least about 300 mg/ml, or at least about 350 mg/ml, or at least about 425 mg/ml, or about 450 mg/ml to 500 mg/ml, as measured at ambient temperature (23°C).
  • this syrup has a density of about 1.0 g/ml to about 1.2 g/ml, more preferably about 1.1 g/ml, and a viscosity of about 13,000 cps to about 19,000 cps, preferably about 14,000 cps to about 18,000 cps, more preferably about 15,000 cps to about 17,000 cps, still more preferably about 15,500 cps to about 16,500 cps, even more preferably about 16,000 cps. This is a substantially lower viscosity than the viscosity of IGF-I formed in inclusion bodies.
  • the syrup has an IGF-I concentration of about 350 mg/ml, a density of about 1.07 g/ml, and a viscosity of about 15,700 cps, as measured at ambient temperature.
  • low salt- containing is intended an amount of salt that is insufficient to cause precipitation of the protein.
  • Biologically active is intended to mean that the IGF-I or variant, when reconstituted from its syrup form into a solution form, is biologically active without the need for refolding.
  • This highly concentrated IGF-I syrup is obtained by precipitating IGF-I or variant thereof in accordance with the methods of the present invention.
  • This syrup form of IGF-I is flowable and clear to opalescent in appearance, features that distinguish it from salt-precipitated forms of IGF-I, such as IGF-I prepared by precipitation or "salting out” using, for example, ammonium sulfate.
  • salt-precipitated forms of IGF-I such as IGF-I prepared by precipitation or "salting out” using, for example, ammonium sulfate.
  • ammonium sulfate 3.9 M in water at 0°C
  • high ionic strength solutions favoring IGF-I precipitation can readily be achieved. See, for example, Voet and Voet (1995) Biochemistry (John Wiley and Sons, New York), pp. 79-81.
  • This method results in precipitation of a salt-protein complex that is white in appearance, has the consistency of a thick paste, and has a substantially higher viscosity than the IGF-I syrup of the present invention.
  • a precipitated salt-protein complex is not amenable to quick and easy recovery of low salt- containing IGF-I.
  • the precipitate would have to be resolubilized (and thereby made less concentrated), followed by removal of salt from the protein solution.
  • Preparation of the highly concentrated IGF-I syrup of the present invention is preferably carried out in accordance with the methods of the present invention. These methods involve manipulation of solution pH or addition of a solubilizing agent to enhance solubility of IGF-I followed by removal of the solubilizing agent to create the IGF-I syrup. Both of these methods allow for precipitation of IGF-I into a highly concentrated, low salt-containing syrup that can readily be reconstituted to recover a solution of biologically active protein.
  • the first of these methods is based upon observations of the unusual solubility properties of IGF-I.
  • IGF-I is very soluble below pH 5.0, where concentrations of 50-200 mg/ml can be obtained. However, a sharp decrease in solubility is observed between pH 5.0 and pH 5.5. Above pH 5.5, the solubility of IGF-I is less than 10 mg/ml (see Figure 1).
  • This method of preparing the highly concentrated IGF-I syrup comprises reducing the solubility of IGF-I such that IGF-I precipitates from a buffer solution containing IGF-I. Precipitation is achieved by adjusting the pH of the IGF-I- containing buffer solution to a pH above about pH 5.0 as disclosed below.
  • IGF-I is prepared within a suitable buffer solution whose critical characteristic is an initial pH that favors solubility of IGF-I.
  • the buffer solution may be any buffer that provides the desired initial pH.
  • suitable buffers are available in the art, including, but not limited to, succinate buffer, phosphate buffer, citrate buffer, acetic acid buffer, an acetic acid salt buffer such as sodium acetate or potassium acetate, and the like.
  • the buffer is acetic acid. Any buffer can be used as long as the initial pH promotes IGF-I solubility.
  • the buffer solution containing IGF-I will have an initial pH of less than about pH 5.0, preferably about pH 2.0 to about pH 5.0, more preferably about pH 3.0 to about pH 4.5, even more preferably about pH 3.5 to about pH 4.0.
  • the initial concentration of IGF-I in this low-pH buffer solution will determine the amount of the highly concentrated IGF-I syrup obtained following upward adjustment of pH. Thus, a higher initial concentration of IGF-I will yield a greater amount of precipitated IGF-I syrup. Because solubility of IGF-I decreases sharply at solution pH greater than about pH 5.0, an initial solution pH in the range less than about 5.0 is preferable to maximize the initial concentration of IGF-I in the buffer solution and therefore maximize yield of precipitated syrup. Regardless of the initial concentration of IGF-I, the concentration of the precipitated IGF-I syrup is at least about 250 mg/ml as noted above.
  • the initial pH of the buffer solution containing IGF-I is adjusted upward to a final pH greater than about pH 5.0, preferably to a pH of greater than about pH 5.0 to about pH 9.0, more preferably to a pH of greater than about pH 5.0 to about pH 8.0, still more preferably to a pH of about pH 5.5 to about pH 7.0, even more preferably to a pH of about pH 5.5 to about pH 6.5, and most preferably to a pH of about pH 5.5 to about pH 6.0.
  • pH 5.0 preferably to a pH of greater than about pH 5.0 to about pH 9.0
  • a pH of greater than about pH 5.0 to about pH 8.0 still more preferably to a pH of about pH 5.5 to about pH 7.0, even more preferably to a pH of about pH 5.5 to about pH 6.5, and most preferably to a pH of about pH 5.5 to about pH 6.0.
  • pH is increased, IGF-I above the solubility limit at the higher pH conditions precipitates, forming a viscous syrup.
  • pH of the buffer solution may be adjusted by standard titrating procedures well known in the art, such as with addition of sodium hydroxide.
  • solution pH may be adjusted by dialyzing the initial buffer solution containing IGF-I against any suitable buffer solution having the desired final pH above pH 5.0 as disclosed above.
  • buffers include, for example, inorganic (e.g., phosphate) and organic (e.g., acetate) buffers.
  • the IGF-I buffer solution having an initial pH less than or equal to pH 5.0 is dialyzed against a sodium citrate buffer at pH 6.0.
  • IGF-I syrup can be separated from the buffered solution by decanting or suctioning off the solution.
  • This syrup has a concentration of IGF-I of at least about 250 mg/ml, as disclosed above.
  • This highly concentrated IGF-I syrup represents a precipitated form of IGF-
  • the precipitation reaction results from a denaturation and/or aggregation reaction that is irreversible, leading to protein inactivation.
  • the precipitation reaction is reversible.
  • the IGF-I syrup can be reconstituted, and the recovered soluble IGF-I retains full biological activity when compared to the biological activity of IGF-I that has not undergone precipitation by the method of the present invention.
  • Layering a buffer solution over known aliquots of syrup allows for the IGF-I to reconstitute. Any suitable buffer solution may be used for reconstitution, as long as the buffering capacity maintains solution pH in a range that allows for IGF-I solubility.
  • IGF-I solubility is a function of pH
  • greater amounts of soluble IGF-I can be recovered from the concentrated IGF-I syrup using a given volume of buffer solution when solution pH is below about pH 5.0 than can be recovered when solution pH is above about pH 5.0.
  • the highly concentrated IGF-I syrup of the present invention can also be prepared using an appropriate solubilizing agent or so-called solubility enhancer.
  • solubility enhancer refers to a compound that includes a guanidinium group and that is capable of enhancing the solubility of IGF-I or a variant of IGF-I.
  • solubilizing agents include the amino acid arginine, as well as amino acid analogues of arginine that retain the ability to enhance solubility of IGF-I at pH 5.5 or greater.
  • Such analogues include, without limitation, dipeptides and tripeptides that contain arginine.
  • guanidine-containing compounds such as guanidine carbaniedine, guanidine acetate, guanidine amine, guanidine carbonate, guanidine 1 -cyano, guanidine 1,3-diphenyl, guanidine l,3-di(2-toyl), guanidine hydrochloride, guanidine nitrate, 1-nitroguanidine, guanidine picrate, guanidine thiocyanate, guanidine tetraphenyl, guanidine 1,1,3-triphenyl, guanidine 1,2,3-triphenyl, guanidine 1-ureido, agmatine, 4-guanidinobenzoic acid, guanidoacetic acid, guanidinosuccinic acid, guanethidine, 4'acetamidophenyl 4- guanidinobenzoate, 2-iminobiotin, N-(2-aminobiotin, N-(2-
  • enhancing the solubility of IGF-I is intended increasing the amount of IGF-I that can be dissolved in solution at pH 5.5 or greater, pH 6.0 or greater, pH 7.0 or greater, pH 8.0 or greater, or pH 9.0 or greater in the presence of a guanidinium-containing compound compared to the amount of IGF-I that can be dissolved at pH 5.5 or greater, pH 6.0 or greater, pH 7.0 or greater, pH 8.0 or greater, or pH 9.0 or greater in a solution with the same components but lacking the guanidinium-containing compound.
  • the ability of a guanidinium-containing compound to enhance the solubility of IGF-I can be determined using methods well known in the art.
  • the concentration of the solubilizing agent added to solution will be from about 10 mM to about 1 M, preferably about 15mM to about 500 mM, and more preferably, for example, in the case of the compound arginine, in a concentration range of about 20 mM to about 200 mM, as disclosed in the copending application filed concurrently herewith entitled "Compositions Providing for Increased IGF-I Solubility," U.S. Patent Application Serial No.
  • the solubility enhancer is then removed from this IGF-I solution by dialysis or diafiltration. Removal of the solubility enhancer results in precipitation of IGF-I in the highly concentrated syrup form. The soluble portion of IGF-I can then be decanted off and the IGF-I syrup recovered. Again, when reconstituted in solution, the IGF-I is biologically active without the need for refolding.
  • the IGF-I to be prepared in a highly concentrated form according to the methods of the present invention can be from any animal species including, but not limited to, avian, canine, bovine, porcine, equine, and human.
  • the IGF-I is from a mammalian species when the concentrated form is to be used in treatment of a mammalian IGF-I-responsive disorder, and more preferably is from a mammal of the same species as the mammal undergoing treatment for such a disorder.
  • the IGF-I can be made by recombinant methods using the corresponding coding sequence for IGF-I from the animal species of interest. Such recombinant methods are discussed in more detail below.
  • Biologically active variants of IGF-I are also encompassed by the method of the present invention. Such variants should retain IGF-I activities, particularly the ability to bind to IGF-I receptor sites. IGF-I activity may be measured using standard IGF-I bioassays.
  • Representative assays include known radioreceptor assays using placental membranes (see, e.g., U.S. Patent No. 5,324,639; Hall et al. (1974) J. Clin. Endocrinol. and Metab. 39:973-976; and Marshall et al. (1974) J. Clin. Endocrinol. and Metab. 39:283-292), a bioassay that measures the ability of the molecule to enhance incorporation of tritiated thymidine, in a dose-dependent manner, into the DNA of BALB/c 3T3 fibroblasts (see, e.g., Tamura et al. (1989) J. Biol. Chem. 262:5616-5621), and the like; herein incorporated by reference.
  • the variant has at least the same activity as the native molecule.
  • Suitable biologically active variants can be IGF-I fragments, analogues, and derivatives.
  • IGF-I fragment is intended a protein consisting of only a part of the intact IGF-I sequence and structure, and can be a C-terminal deletion or N- terminal deletion of IGF-I.
  • analogues is intended analogues of either IGF-I or an IGF-I fragment that comprise a native IGF-I sequence and structure having one or more amino acid substitutions, insertions, or deletions.
  • Peptides having one or more peptoids are also encompassed by the term analogue (see International Publication No. WO 91/04282).
  • derivatives any suitable modification of IGF-I, IGF-I fragments, or their respective analogues, such as glycosylation, phosphorylation, or other addition of foreign moieties, so long as the IGF-I activity is retained.
  • Methods for making IGF-I fragments, analogues, and derivatives are available in the art. See generally U.S. Patent Nos. 4,738,921, 5,158,875, and 5,077,276; International Publication Nos. WO 85/00831, WO 92/04363, WO 87/01038, and WO 89/05822; and European Patent Nos. EP 135094, EP 123228, and EP 128733; herein incorporated by reference.
  • IGF-I variants will generally have at least 70%, preferably at least 80%, more preferably about 90% to 95% or more, and most preferably about 98% or more amino acid sequence identity to the amino acid sequence of the reference IGF-I molecule.
  • a variant may differ by as few as 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • sequence identity is intended the same amino acid residues are found within the IGF-I variant and the reference IGF-I molecule when a specified, contiguous segment of the amino acid sequence of the variant is aligned and compared to the amino acid sequence of the reference molecule. Methods for determining identity between sequences are well known in the art.
  • the contiguous segment of the amino acid sequence of the variant may have additional amino acid residues or deleted amino acid residues with respect to the amino acid sequence of the reference molecule.
  • the contiguous segment used for comparison to the reference amino acid sequence will comprise at least twenty (20) contiguous nucleotides, and may be 30, 40, 50, 100, or more nucleotides.
  • corrections for increased sequence identity associated with inclusion of gaps in the variant's amino acid sequence can be made by assigning gap penalties.
  • Methods of sequence alignment are well known in the art. When considering percentage of amino acid sequence identity, some amino acid residue positions may differ as a result of conservative amino acid substitutions, which do not affect properties of protein function. In these instances, percent sequence identity may be adjusted upwards to account for the similarity in conservatively substituted amino acids. Such adjustments are well known in the art. See, for example, Meyers and Miller (1988) Computer Applic. Biol. Sci. 4:11- 17.
  • a fragment of IGF-I will generally include at least about 10 contiguous amino acid residues of the full-length molecule, preferably about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably about 20-50 or more contiguous amino acid residues of full-length IGF-I.
  • one of skill in the art can readily determine which modifications to the native protein nucleotide or amino acid sequence will result in a variant that enables preparation of the highly concentrated form of the IGF-I variant in accordance with the methods disclosed in the present invention.
  • IGF-I variants are known in the art and include those described in, for example, Proc. Natl. Acad. Sci. USA 83 (1986):4904-4907; Biochem. Biophys. Res. Commun. 149 (1987):398-404; J Biol. Chem. 263 (1988):6233-6239; Biochem. Biophys. Res. Commun. 165 (1989):766-771; Forsbert et al. (1990) Biochem. J. 271:357-363; U.S. Patent Nos.
  • variants include one with a deletion of Glu-3 of the mature molecule, a variant with up to 5 amino acids truncated from the N-terminus, a variant with a truncation of the first 3 N-terminal amino acids (referred to as des(l-3)-IGF-I, des-IGF-I, tIGF-I, or brain IGF), and a variant including the first 17 amino acids of the B chain of human insulin in place of the first 16 amino acids of human IGF-I.
  • the IGF-I used in making the highly concentrated syrup form of IGF-I according to the present invention can be in its substantially purified, native, recombinantly produced, or chemically synthesized forms.
  • the IGF-I can be isolated directly from blood, such as from serum or plasma, by known methods. See, for example, Phillips (1980) New Eng. JMed. 302:371-380;
  • IGF-I can be synthesized chemically, by any of several techniques that are known to those skilled in the peptide art. See, for example, Li et al. (1983) Proc. Natl. Acad. Sci.
  • IGF-I insulin growth factor-I
  • the human DNA sequence encoding IGF-I is known and can be introduced into host cells for expression.
  • IGF-I can be produced by recombinant DNA techniques in E. coli, yeast, insect, and mammalian cells. Secreted IGF-I can be made by adding a signal sequence to the DNA sequence encoding IGF-I.
  • the DNA sequence encoding IGF-I can be manipulated to make IGF-I fragments, analogues, or derivatives.
  • Such recombinant DNA techniques are generally available in the art. See, for example, International Publication No. WO 96/07424, where recombinant human IGF-I protein is produced in yeast.
  • the syrup itself has several uses as disclosed in the present invention.
  • the syrup provides a means for packaging high concentrations of IGF-I within small volume spaces.
  • the syrup provides an easier means of storage of IGF- I.
  • aliquots of the syrup can be reconstituted using a suitable buffer solution to recover soluble IGF-I that retains its biological activity.
  • Storage of the IGF-I syrup is preferably at a temperature of about 2°C to about 10°C, more preferably about 2°C to about 8°C, most preferably at about 4°C. Storage in this manner provides a shelf life of 18 to 24 months or more.
  • the IGF-I syrup can be formulated with protein stabilizers in order to preserve the activity thereof.
  • protein stabilizers include, e.g., simple salts, buffer salts, polyhydroxylated compounds such as glycerol, mannitol, sucrose and polyethylene glycols, and surfactants. See, e.g., International Publication No. WO 92/11844.
  • Containers comprising the highly concentrated IGF-I syrup can be packaged in kit form for subsequent preparation of pharmaceutical compositions useful in IGF-I therapy.
  • a kit additionally comprises a suitable buffer for reconstituting the highly concentrated syrup form of IGF-I.
  • the resulting reconstituted IGF-I can be used in formulating a pharmaceutical composition as outlined below.
  • the pharmaceutical composition is formulated with a known concentration of IGF-I such that administration of a therapeutically effective dose promotes a desired therapeutic response with respect to a particular IGF-I responsive condition undergoing therapy.
  • desired therapeutic response is intended an improvement in the condition or in the symptoms associated with the condition.
  • the syrup form of IGF-I is useful as a means of preparing compositions that comprise IGF-I.
  • the IGF-I syrup may be directly incorporated into one or more substances to form a composition that comprises biologically active IGF-I in a highly concentrated form.
  • aliquots of the IGF-I syrup may serve as a source for reconstituted IGF-I, which may then be incorporated into one or more substances to form a composition comprising biologically active IGF-I in its reconstituted state.
  • pharmaceutical compositions and compositions comprising IGF-I in an encapsulated state that is useful, for example, in formulating sustained-release pharmaceutical compositions.
  • the highly concentrated IGF-I syrup of the present invention is useful in
  • IGF-I therapy directed to any IGF-I-responsive condition when administered to a therapeutic site undergoing therapy for such a condition.
  • IGF-I in its syrup form, or IGF-I reconstituted from the IGF-I syrup may be incorporated into one or more substances to form a pharmaceutical composition that is then placed in contact with the therapeutic site. Administering a therapeutically effective amount of such a composition promotes a desired therapeutic response with respect to an IGF-I-responsive condition undergoing IGF-I therapy.
  • the preferred form of IGF - I i.e., syrup or reconstituted, depends upon the preferred method of delivery of the pharmaceutical composition, as outlined below.
  • Suitable methods of delivery of the pharmaceutical composition comprising the concentrated IGF-I syrup include, but are not limited to, gel formulations, viscous solutions, sustained-release formulations, implant delivery systems, such as pumps, and the like. Such delivery systems allow for the controlled and concentrated delivery of IGF-I to a therapeutic site.
  • the exact formulation employed will depend on the type of application that is desired. For example, gel formulations may be utilized for topical or incisional wound healing, whereas low viscosity, aqueous formulations may be used for those applications requiring a more fluid formulation having a higher water content.
  • the highly concentrated IGF-I syrup of the invention can be used in gel formulations to provide a controlled-delivery system.
  • the concentrated IGF-I syrup of the present invention can be utilized in formulations to increase the residence time of the growth factor and provide a sustained-release dosage form. This is an important advantage as it permits less frequent application of the formulation to the therapeutic site and thereby permits less disturbance of the wound or site and its cellular components. See, for example, U.S. Patent Nos. 5,705,485, 5,457,093, 3,934,013, and 5,071,644.
  • the gel formulations of the invention include those containing a water soluble, pharmaceutically acceptable polymeric material. See, for example, U.S. Patent No. 5,705,485.
  • the concentrated IGF-I of the invention can also be used in sustained- release pharmaceutical compositions, which prolong the presence of IGF-I in the treated mammal, generally for longer than one day.
  • a sustained-release pharmaceutical composition generally provides the pharmaceutical composition within a polymer, preferably a hydrophilic polymer for sustained-release of the drug.
  • Many methods of preparation of a sustained-release formulation are known in the art and are disclosed in Remington's Pharmaceutical Sciences (18 th ed.; Mack Pub. Co.: Eaton, Pennsylvania, 1990), herein incorporated by reference.
  • the IGF-I can be entrapped in semipermeable matrices of solid hydrophobic polymers.
  • the matrices can be shaped into films or microcapsules.
  • Such matrices include, but are not limited to, polyesters, copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al. (1983) Biopolymers 22:547-556), poly-actides (U.S. Patent No. 3,773,919 and EP 58,481), polylactate polyglycolate (PLGA) hydrogels (see, for example, Langer et al. (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech.
  • microcapsules can also include hydroxymethylcellulose or gelatin-microcapsules and poly-methylmethacylate microcapsules prepared by coacervation techniques or by interfacial polymerization.
  • microemulsions or colloidal drug delivery systems such as liposomes and albumin microspheres may also be used. See Remington 's Pharmaceutical Sciences (18 th ed.; Mack Pub. Co.: Eaton, Pennsylvania, 1990).
  • sustained-release pharmaceutical compositions are described, for example, in U.S. Patent Nos. 4,178,361; 4,404,183; 4,343,789; 5,614,487; 5,422,116; 4,309,405; 4,248,858; 4,524,060; 4,973,470; 4,539,199; 4,309,406; 4,309,404; 4,248,857; and 4,248,856; all of which are herein incorporated by reference.
  • One such sustained-release composition where a polypeptide such as IGF-I is entrapped in biodegradable microp articles is described in the application filed concurrently herewith entitled "Method for Producing Sustained-Release Formulations," U.S. Patent Application Serial No. , the entirety of which is herein incorporated by reference.
  • the pharmaceutical composition comprising the highly concentrated IGF-I syrup is administered via local drug delivery.
  • Local application of the drug affords concentrated delivery of the drug, achieving tissue levels not otherwise obtainable through other administration routes.
  • drug release occurs via diffusion, a time-dependent process, delivery to the target cite will be sustained for days to weeks or beyond, depending upon the delivery system utilized.
  • local delivery reduces systemic drug exposure, thereby limiting systemic side effects. It also allows for delivery of agents that might otherwise be difficult or impossible to deliver via oral or intravenous routes due to problems of solubility or formulation.
  • Local administration also provides the possibility of utilizing agents that might not otherwise be administratable because of dosage range of toxicity limitations encountered with conventional routes of administration.
  • Means for local drug delivery include balloon catheter delivery systems, endovascular polymer-coated stents, facilitated diffusion, polymeric endoluminal paving, and controlled-release matrices. See, for example, Eccleston et al. (1995) Interventional Cardiology Monitor 1:33-40-41, and Slepian (1996) Intervente Cardiol. 1:103-116, herein incorporated by reference.
  • the highly concentrated IGF-I syrup is used in an implantable pump, such as the osmotically driven DUROSTM implantable pump from ALZA (Palo Alto, California).
  • Such pumps are surgical implants that provide for drug delivery over months (e.g., 6-12 months) in a continuous, steady-state fashion at a variety of doses.
  • the IGF-I syrup of the present invention provides a means of packaging high concentrations of IGF-I in the small volumes typical of such implant devices.
  • aliquots of the highly concentrated IGF-I syrup may be incorporated into liquid injectables for parenteral delivery.
  • an aliquot of the syrup is reconstituted as previously described using a pharmaceutically acceptable buffer having a buffering capacity that maintains solution pH in a range that allows for IGF-I solubility, preferably a pH below 5.0.
  • the reconstituted IGF-I is then incorporated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier as described below.
  • the carrier is a carrier favorable for parenteral delivery, and preferably is isotonic with the blood of the recipient.
  • Such carriers include, but are not limited to, water, saline, Ringer's solution, and dextrose solution. Other carriers are described below.
  • the pharmaceutical composition comprising reconstituted IGF-I should be formulated in a unit dosage and in an injectable or infusible form such as solution, suspension, or emulsion. It can also be in the form of lyophilized powder, which can be converted into solution, suspension, or emulsion before administration.
  • the pharmaceutical composition comprising reconstituted IGF-I is preferably sterilized by membrane filtration and is stored in unit-dose or multi-dose containers such as sealed vials or ampules.
  • any of the pharmaceutical compositions comprising the concentrated IGF-I syrup, or IGF-I reconstituted from the IGF-I syrup, as described above may contain other components that modulate the therapeutic treatment with IGF-I.
  • Such components include any of the IGF-I binding proteins, IGF-I receptors, and the acid-labile subunit of the IGF-I binding complex.
  • IGFBP-3 may enhance the stimulatory effect of IGF-I on proteoglycan synthesis (see Chevalier et al. (1996) British J. Rheumat. 35:515-522).
  • an acid labile glycoprotein also has been shown to be associated with the protein complex formed by IGF-I and its binding proteins.
  • the therapeutically effective pharmaceutical composition may contain such acid-labile glycoprotein and IGF-I binding proteins, when proven to facilitate the desired positive response on the IGF-I-responsive disorder undergoing treatment.
  • the amount of IGFBPs to be administered with IGF-I can be determined according to the molar ratio between IGF-I and IGFBPs. This molar ratio can range from about 0.5:1 to about 3:1, preferably about 1:1 (see U.S. Patent No. 5,187,151).
  • the pharmaceutical composition may include agents that disrupt IGF-I binding to IGFBPs and which may be effective in increasing the amount of IGF-I present in the affected physiological site to a therapeutically effective level.
  • the pharmaceutical composition comprising IGF-I may include one or more protease inhibitors.
  • An exemplary protease inhibitor is sodium pentosan polysulfate (PPS), a polysulfated polysaccharide. This protease inhibitor has efficacy in treating osteoarthritis in combination with low dosages of IGF-I (1 ⁇ g IGF-I intra-articularly 3 times per week) (Rogachefsky et al. (1993) Osteoarthritis and Cartilage 1:105-114).
  • PPS pentosan polysulfate
  • This protease inhibitor has efficacy in treating osteoarthritis in combination with low dosages of IGF-I (1 ⁇ g IGF-I intra-articularly 3 times per week) (Rogachefsky et al. (1993) Osteoarthritis and Cartilage 1:105-114).
  • Such a protease inhibitor can be administered by other routes, such as intramuscularly, during administration of the effective dose of I
  • the pharmaceutical composition in accordance with the present invention may further comprise one or more other therapeutic agents that are effective in treating other disorders in the individual, as long as the biochemical actions of the additional therapeutic agents do not interfere with the efficacy of intended action of the IGF-I treatment.
  • additional therapeutic agents include, but are not limited to, antibiotics, anti-inflammatory agents, and the like.
  • a pharmaceutically acceptable carrier should be mixed with the IGF-I and other components used in preparing the pharmaceutical composition.
  • pharmaceutically acceptable carrier is intended a carrier that is conventionally used in the art to facilitate the storage, administration, and/or the healing effect of the therapeutic ingredients.
  • a carrier may also reduce any undesirable side effects of the IGF-I.
  • a suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effect in recipients at the dosages and concentrations employed for treatment. Such carriers are generally known in the art.
  • Suitable carriers for this invention are those conventionally used large stable macromolecules such as albumin, gelatin, collagen, polysaccharide, monosaccharides, polyvinyl- pyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG), and the like.
  • Slow-release carriers such as hyaluronic acid, may also be suitable. See particularly Prisell et al. (1992) Int. J. Pharmaceu. 85:51-56, and U.S. Patent No.
  • hyaluronic acid and other polymers may have an additional beneficial effect on the IGF-I-responsive disorder osteoarthritis. See particularly Bragantini (1987) Clin. Trials J. 24(4):333-340; Dougados et al. (1993) Osteoarthritis and Cartilage 1:97- 103; and Lussier et al. (1996) J. Rheum. 23:1579-1585; herein incorporated by reference.
  • Other acceptable components in the composition include, but are not limited to, buffers that enhance isotonicity such as water, saline, phosphate, citrate, succinate, acetic acid, and other organic acids or their salts.
  • Preferred pharmaceutical compositions may incorporate buffers having reduced local pain and irritation resulting from injection of IGF-I compositions.
  • buffers include, but are not limited to, low phosphate buffers and succinate buffers.
  • International Publication No. WO 94/15584 describes isotonic IGF-I solution at pH 5.5 to 6.5 with phosphate buffer present in an amount less than 50 mmol L, which are reported to result in reduced pain upon injection.
  • the pharmaceutical composition may comprise a succinate buffer with pH in the range of about 4.0 to about 7.5, and succinate in the range of 0.5 mM up to about 100 mM, preferably a range less than about 50 mM, as in the formulation disclosed in the copending application entitled "Injectable Formulation Containing Succinate," U.S. Patent Application Serial No. 60/080,008, filed April 3, 1998.
  • the pharmaceutical composition may additionally comprise a solubilizing agent or so-called solubility enhancer.
  • a solubilizing agent or so-called solubility enhancer.
  • Compounds containing a guanidinium group, most preferably arginine, are suitable solubility enhancers for IGF-I, as described above.
  • IGF-I-responsive condition any condition that responds in the short-term or in the long-term either positively or negatively to IGF-I.
  • IGF-I-responsive conditions may be a normal condition.
  • a mammal may undergo IGF-I therapy to increase normal muscle mass where greater muscle mass is desirable, as in an athlete.
  • the IGF-I responsive condition may be an abnormal condition that is chronic, and thus occurs more or less continuously, or that is acute, as occurs following injury to a site, such as a joint or bone injury.
  • Conditions responsive to IGF-I include acute or chronic conditions including, but not limited to, hyperglycemic disorders, including all forms of diabetes; chronic lung disease; acute and chronic renal disorders; acute and chronic liver failure; hepatic cirrhosis; inflammatory responses, such as rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, and inflammatory bowel disease; short gut; ischemic injuries involving the heart, liver, or brain, or such as results from renal tubular necrosis; immunological disorders, such as immunodeficiencies including decreased immune tolerance or chemotherapy-induced tissue damage; organ rejection after transplantation; diseases or insufficiencies of cardiac structure or function, such as chronic heart conditions, cardiomyopathy, stroke, and congestive heart failure; growth retardation; osteoporosis; wound healing; bone damage; ophthalmic conditions; infertility; neurodegenerative disorders, such as motoneuron disease, multiple sclerosis, muscular dystrophy, diabetic neuropathy, demyelinating peripheral neuropathies, Parkinson's disease,
  • any IGF-I-responsive disorder may benefit from administration of the pharmaceutical compositions comprising the IGF-I syrup or reconstituted IGF-I obtained therefrom of the present invention.
  • “therapy” is intended treatment of an existing normal condition that is enhanced by IGF-I therapy, therapeutic treatment of an existing IGF-I-responsive abnormal condition, and preventive or prophylactic procedures performed before the occurrence of an abnormal disorder.
  • the pharmaceutical compositions comprising the IGF-I syrup or reconstituted IGF-I obtained therefrom may be used in therapy for IGF-I- responsive conditions of any mammal. Exemplary mammals include, but are not limited to, cats, dogs, horses, cows, sheep, pigs, and more preferably humans.
  • the highly concentrated syrup form of IGF-I disclosed in the present invention finds further use as an essentially water-free preparation of IGF-I.
  • the methods of the present invention provide a means of preparing an essentially water-free IGF-I composition.
  • the IGF-I syrup is useful for preparing a dry powder form of IGF-I. Because little water must be removed from the IGF-I syrup during lyophilization as compared to solution formulations, the syrup form of IGF-I may be more efficiently dried in shorter periods of time. The resulting dry powder form of IGF-I is more densely packed (e.g., more IGF-I per unit volume) than other lyophilized IGF-I, and the preparation is low in salt content.
  • the syrup form of IGF-I finds use in other processes that require the removal of water from protein compositions.
  • the IGF-I syrup could be used to encapsulate IGF-I in PLGA (poly(D,L- lactide-co-glycolide)) microspheres using the cryogenic process described by Johnson et al. (1996) Nature Medicine 2:795-799. See also the cryogenic process described in U.S. Patent No. 5,019,400.
  • EXPERIMENTAL IGF-I for use in these experiments was recombinantly produced in the yeast strain Pichia pastoris and purified essentially as described in U.S. Patent Nos. 5,324,639, 5,324,660, and 5,650,496 and International Publication No. WO 96/40776.
  • Example 1 Solubility of IGF-I as a Function of pH Following isolation, the solubility of recombinant human IGF-I (rhIGF-I) was determined by dialysis.
  • a saturated rhIGF-I solution can be created by dialyzing rhIGF-I at high concentration (e.g., 100 mg/ml in pH 4.0 buffer) against conditions where rhIGF-I is less soluble.
  • rhIGF-I pH 4.0, 100 mg/ml
  • rhIGF-I pH 4.0, 100 mg/ml
  • rhIGF-I As rhIGF-I above the solubility limit partitioned, two phases formed, a layer of precipitated rhIGF-I and a solution phase containing a saturated solution of rhIGF-I. A sample of the solution phase rhIGF-I was removed and filtered through a 0.22 ⁇ m filter to remove any insoluble material. The concentration of the filtered rhIGF-I solution was then determined by UV spectroscopy using the known IGF-I absorption coefficient.
  • Figure 1 shows rhIGF-I solubility as a function of pH.
  • rhIGF-I remains very soluble below pH 5.0.
  • a sharp decrease in solubility occurs between pH 5.0 and pH 5.5, with solubility above pH 5.5 being less than 10 mg/ml.
  • solubility at pH 6.0 is only 3.0 mg/ml.
  • IGF-I has an isoelectric point around pH 8.7
  • solubility profile for rhIGF-I indicates the protein becomes much less soluble at solution pH well below that of its isoelectric point.
  • Example 2 Preparation of IGF-I Syrup by Manipulation of Solution pH rhIGF-I as described in Example 1 was precipitated as follows. Bulk rhIGF-I at a concentration of 13 mg/ml was concentrated to 74 mg/ml in an initial buffer solution at pH 4.0 and then dialyzed in a 10 mM sodium citrate/140 mM sodium chloride buffer at pH 6.0 using spectra por tubing 1000 MWCO. The concentration of rhIGF-I remaining in solution was 10.6 mg/ml. Concentration of rhIGF-I in the initial and final solutions was measured spectrophotometrically in the uv region at 276 nm. The buffer solution was decanted off and the precipitated polypeptide recovered in the form of an opalescent viscous syrup. Concentration of rhIGF-I in the precipitated syrup form was determined at about 350 mg/ml of syrup.
  • the density of the rhIGF-I syrup was determined by weight at ambient temperature (23°C). Ten milliliters (10 ml) of rhIGF-I syrup was prepared volumetrically and its weight determined on a Mettler AE240. The weight of the 10 ml sample of rhIGF-I syrup was determined to be 10.7 grams. Therefore, the density of the rhIGF-I syrup was determined to be 1.07 g/ml.
  • the viscosity of the rhIGF-I syrup was determined with a Cannon Instruments LV2000 Rotary Viscometer. The instrument was calibrated with a viscosity standard provided by the manufacturer. All measurements were performed at ambient temperature (23°C). The viscosity of the rhIGF-I syrup was determined to be approximately 15,700 centipoise (cps).
  • Example 3 Preparation of IGF-I Syrup by Removal of Solubility Enhancer Arginine and other compounds containing a guanidinium group have been shown to dramatically increase the solubility of IGF-I (see Figure 2).
  • arginine is used as a solubility enhancer to prepare a high concentration rhIGF-I solution from which the solubility enhancer is removed to precipitate the rhIGF-I syrup of the present invention.
  • rhIGF-I at 100 mg/ml in 10 mM sodium citrate, 120 mM arginine, pH 6.0 is dialyzed against 10 mM sodium citrate, 140 mM sodium chloride, pH 6.0 at 4°C.
  • rhIGF-I is only soluble to about 10 mg/ml. Of the original 100 mg/ml, 90 mg/ml precipitates to form an opalescent syrup and 10 mg/ml remains in solution. The soluble portion of the rhIGF-I can be decanted off and the rhIGF-I syrup recovered. This rhIGF-I syrup retains its biological activity.
  • Example 4 Stability of IGF-I Syrup A stability study was conducted with the highly concentrated, low salt- containing, biologically active syrup form of rhIGF-I.
  • Example 5 Preparation of Salt-Containing Precipitated Form of IGF-I
  • rhIGF-I To 100 ml of rhIGF-I at 13.2 mg/ml in 0.1 M acetic acid (approximately pH 3.5), ammonium sulfate was added to 35% saturation at 4°C. The ammonium sulfate was dissolved with stirring, and the solution became "milky" white indicating that the rhIGF-I had precipitated from solution. The milky suspension was allowed to stir at 4°C overnight, and the ammonium sulfate-precipitated rhIGF-I was recovered by centrifugation.
  • the precipitated rhIGF-I being more dense than the liquid, sedimented to the bottom of the centrifuge tube during centrifugation.
  • the precipitated rhIGF-I was recovered by decanting the supernatant from the pellet.
  • the recovered ammonium sulfate-precipitated rhIGF-I was unlike the IGF-I composition of the present invention; it was white in appearance (indicating the presence of substantial salt) and had the consistency of a thick paste rather than a flowable syrup.
  • the rhIGF-I syrup of Example 2 or Example 3 is lyophilized and mixed with the PLGA in methylene chloride for encapsulation.
  • the suspension so obtained is sprayed into liquid nitrogen and PLGA microspheres produced and recovered essentially as described by Johnson et al. (1996) Nature Medicine 2:795- 799.
  • Example 7 Cryogenic Process Not Requiring Syrup Lyophilization
  • the rhIGF-I syrup of Example 2 or 3 is filtered to remove substantially all water, is then mixed with a small quantity of ethanol, and is then added to PLGA dissolved in methylene chloride. Alternatively, the ethanol is present in methylene chloride. Ethanol mixed with the syrup extracts unbound water. This ethanol, being miscible with methylene chloride, partitions into the methylene chloride phase containing the PLGA polymer, thus facilitating practically the entire rhIGF-I in the syrup form to be encapsulated by the cryogenic process described by Johnson et al. (1996) Nature Medicine 2:795-799.

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Abstract

L'invention concerne un sirop biologiquement actif hautement concentré à faible teneur en sel, à base d'IGF-I ou une variante de ce sirop. L'invention concerne également les procédés de préparation de ce sirop. Ce nouveau sirop d'IGF-I présente une concentration d'IGF-I d'au moins 250 mg/ml, une densité variant entre 1,0 g/ml et 1,2 g/ml, et une viscosité située entre 13 000 centipoises (cps) et 19 000 cps environ, telle que mesurée à température ambiante (23 °C). On prépare ce sirop d'IGF-I en faisant précipiter ou en divisant l'IGF-I de la solution, de préférence en ajustant le pH de cette solution ou en utilisant un agent facilitant la solubilité afin de concentrer l'IGF-I dans la solution, avant d'éliminer ledit agent facilitant la solubilité. Le sirop précipité peut être utilisé en tant que moyen permettant de stocker ledit IGF-I dans une forme stable et de préparer des compositions renfermant l'IGF-I biologiquement actif. L'invention concerne également des compositions pharmaceutiques et des kits comprenant ce sirop concentré d'IGF-I. L'invention concerne enfin le sirop précipité d'IGF-I, l'IGF-I reconstitué à partir de ce sirop précipité d'IGF-I, et des compositions pharmaceutiques et des kits utiles à une thérapie utilisant l'IGF-I pour traiter des dysfonctionnements réagissant à l'IGF-I.
PCT/US1998/023672 1997-11-07 1998-11-06 Nouvelle composition d'igf-i et son utilisation WO1999024062A1 (fr)

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Cited By (6)

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WO1999051262A2 (fr) * 1998-04-03 1999-10-14 Chiron Corporation Utilisation d'igf1 pour traiter des maladies du cartilage articulaire
EP1284748A1 (fr) * 2000-05-03 2003-02-26 GroPep Limited Traitement de tissus conjonctifs endommages
US6573238B2 (en) 1997-11-07 2003-06-03 Chiron Corporation Method for producing sustained-release formulations
US6641840B2 (en) 2000-08-30 2003-11-04 Pfizer Inc. Sustained release formulations for growth hormone secretagogues
WO2008035230A2 (fr) * 2006-09-18 2008-03-27 Novozymes Gropep Limited Compositions protéiques liquides
KR101614983B1 (ko) 2009-11-17 2016-04-22 입센 파마 에스.에이.에스 Ｈｇｈ 및 ｒｈｉｇｆ―１의 조합물을 위한 제제

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110152188A1 (en) * 2009-12-23 2011-06-23 Hanns-Christian Mahler Pharmaceutical compositions of igf/i proteins

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EP0297860A1 (fr) * 1987-07-01 1989-01-04 Genentech, Inc. Compositions thérapeutiques et méthode pour la prévention de dépôts de fibrine et d'adhésions
EP0440989A1 (fr) * 1990-01-05 1991-08-14 Fujisawa Pharmaceutical Co., Ltd. Méthode pour la préparation d'une composition sèche du "insulin-like growth factor" (IGF-I)

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Publication number Priority date Publication date Assignee Title
EP0297860A1 (fr) * 1987-07-01 1989-01-04 Genentech, Inc. Compositions thérapeutiques et méthode pour la prévention de dépôts de fibrine et d'adhésions
EP0440989A1 (fr) * 1990-01-05 1991-08-14 Fujisawa Pharmaceutical Co., Ltd. Méthode pour la préparation d'une composition sèche du "insulin-like growth factor" (IGF-I)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6573238B2 (en) 1997-11-07 2003-06-03 Chiron Corporation Method for producing sustained-release formulations
WO1999051262A2 (fr) * 1998-04-03 1999-10-14 Chiron Corporation Utilisation d'igf1 pour traiter des maladies du cartilage articulaire
WO1999051262A3 (fr) * 1998-04-03 1999-12-09 Chiron Corp Utilisation d'igf1 pour traiter des maladies du cartilage articulaire
US7141545B2 (en) 1998-04-03 2006-11-28 Novartis Vaccines And Diagnostics, Inc. Compositions and methods for treating articular cartilage disorders
EP1284748A1 (fr) * 2000-05-03 2003-02-26 GroPep Limited Traitement de tissus conjonctifs endommages
EP1284748A4 (fr) * 2000-05-03 2005-02-16 Gropep Ltd Traitement de tissus conjonctifs endommages
US6641840B2 (en) 2000-08-30 2003-11-04 Pfizer Inc. Sustained release formulations for growth hormone secretagogues
WO2008035230A2 (fr) * 2006-09-18 2008-03-27 Novozymes Gropep Limited Compositions protéiques liquides
WO2008035230A3 (fr) * 2006-09-18 2008-06-05 Novozymes Gropep Ltd Compositions protéiques liquides
KR101614983B1 (ko) 2009-11-17 2016-04-22 입센 파마 에스.에이.에스 Ｈｇｈ 및 ｒｈｉｇｆ―１의 조합물을 위한 제제

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