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    • 专利摘要:
    A new pharmaceutical composition for insulin-like growth factor / insulin-like growth factor binding protein complexes is disclosed. Insulin-like growth factor / insulin-like growth factor binding protein complex, preferably recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex, optionally adds excipients and buffering salts without the addition of osmotic control salts To prepare. Also disclosed is a lyophilized composition containing an insulin like growth factor / insulin like growth factor binding protein complex.
    公开号:KR20010052500A
    申请号:KR1020007013631
    申请日:1999-05-28
    公开日:2001-06-25
    发明作者:데이비드 패스모어；스테펜 단코；야수쉬 오가와
    申请人:셀트릭스 파마슈티칼스, 인크.；
    IPC主号:
    专利说明:

    Pharmaceutical formulations for IGF / IGFBP}
    Growth factors are polypeptides that cause a wide range of biological responses in specific target cell populations, such as DNA synthesis, cell division, and expression of specific genomes. Many different growth factor families include metastatic growth factor beta (TGF-βs), epidermal growth factor and metastatic growth factor alpha (the TGF-αs), platelet-derived growth factors (PDGFs), and fibroblast growth factor family FGFs. And insulin-like growth factor groups (IGFs) including insulin-like growth factor-I and insulin-like growth factor-II.
    Insulin-like growth factor-I and insulin-like growth factor-II (IGFs) are similar in structure to the amino acid sequence and each polypeptide has a molecular weight of about 7.5 kDa. Insulin-like growth factor-I is involved in the main effects of growth hormone and is an important substance involved in postnatal growth. Insulin-like growth factor-I is also associated with the action of a variety of other growth factors, and treatment of these growth factors in cells increases insulin-like growth factor-I production. In contrast, insulin-like growth factor-II is believed to play an important role in prenatal growth. As the name of both insulin-like growth factor-I and insulin-like growth factor-II shows, it is a substance that has insulin-like activity and causes division of neural tissue cells.
    Most all insulin-like growth factors form a non-covalent complex consisting of insulin-like growth factor-I, insulin-like binding protein-3, and a broader range of protein subunits called acid-degradable subunits (ALS). And therefore, little free insulin-like growth factor-I is found. The ternary complex consists of three components, each with the same molar concentration. Although there are some reports that insulin-like growth factor binding protein-3 can bind to acid-degradable subunits in mice without insulin-like growth factor (Lee et al., Endocrinology 136: 4982-4989, 1995), acid-degradable subunits Does not bind directly to insulin-like growth factor, but only by binding to insulin-like growth factor / insulin-like growth factor binding protein-3 complex (Baxter et al., J. Biol. Chem 264 (20): 11843-11848, 1989 ). The molecular weight of the tri-element complex of insulin-like growth factor / insulin-like growth factor binding protein-3 / acid degradable subunit is about 150 kDa. This three-element complex is "a reservoir or buffer of insulin-like growth factor-I and insulin-like growth factor-II that blocks rapid changes in free insulin-like growth factor concentrations" (Blum et al. (1991), "Insulin in Plasma". Clinical indicators indicating the level of similar growth factor binding protein-3 "in MODERN CONCEPTS OF INSULINE-LIKE GROWTH FACTORS, pp. 381-393, EM Spencer, ed., Elsevier, New York). There is no excess of unbound insulin-like growth factor binding protein-3 in the circulatory system, while there is a significant amount of excess free acid-degradable subunit (Baxter, J. Clin. Endocrinol. Metab. 67: 265-272, 1988).
    The complex of insulin-like growth factor-I and insulin-like growth factor binding protein-3 ("binary complex" or "insulin-like growth factor-I / insulin-like growth factor binding protein-3") is an uncombined insulin-like growth factor- Physically and chemically different from I. Binary complexes are about five times larger than uncombined insulin-like growth factor-I and differ in overall isoelectric point and overall hydrophobicity. Because of these differences, binary complexes act significantly differently from insulin-like growth factor-I.
    Because of the wide range of activity of insulin-like growth factor-I, insulin-like growth factor-I has been developed as a treatment for a variety of diseases, including myotrophic lateral sclerosis and diabetes, commonly known as Lou Gehrig's disease. Unfortunately, administration of insulin-like growth factor-I may cause hypoglycemia, edema that can cause Bell stroke and tunnel wrist syndrome and various harmful diseases, hypophosphatemia in the blood, and hypernatremia in the blood with high sodium concentration. It involves many side effects, including. Administration of insulin-like growth factor-I in the form of a complex of insulin-like growth factor-I and insulin-like growth factor binding protein-3 can reduce or eliminate these side effects (Adams et al., 1996, Prog. Growth Factor Res). 6: 2-4).
    Dosing in the form of an insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex is preferred, but in most formulations this complex, like other proteins, has very limited storage stability (expiration date). Many insulin-like growth factor-I compositions have been developed which have been intentionally stabilized by combining insulin-like growth factor-I alone or with other proteins such as growth hormones, but insulin-like growth factor-I / insulin-like growth factor due to the poor stability of the protein. The stability of compositions such as binding protein-3 is not satisfactory. In the compositions of the binary complex the protein must be frozen at very low temperatures, for example -70 ° C. Freezers, especially cryogenic freezers, must maintain -70 ° C and are very expensive equipment that is not suitable for use other than experimental equipment. Thus, the fact that such binary composite compositions can be stored at or at higher temperatures in common refrigerators is critical to the commercialization of insulin-like growth factor-I / insulin-like growth factor binding proteins.
    Various compositions for insulin-like growth factor, in particular insulin-like growth factor-I, have been published. For example, U.S. Pat. Insulin-like growth factor-I compositions containing a preservative such as benzyl alcohol or phenol are disclosed. WO 97/07816 discloses a liquid insulin-like growth factor-I composition containing insulin-like growth factor-I and mannitol in a buffer. However, because there are substantial physical and chemical differences between insulin-like growth factor-I and insulin-like growth factor / insulin-like growth factor binding protein-3, the insulin-like growth factor-I composition is suitable for insulin-like growth factor binding protein-3. There is no reasonable prediction.
    Note that insulin-like growth factor binding protein-3 is the most common insulin-like growth factor binding protein ("IGFBPs"), but at least five different distinct insulin-like growth factor binding proteins have been identified in various tissues and body fluids. Is the point. Although these proteins bind to insulin-like growth factors, they each come from different genomes and have distinctly different amino acid sequences. Unlike insulin-like growth factor binding protein-3, circulating insulin-like growth factor binding proteins are not saturated with insulin-like growth factor. Insulin-like growth factor binding protein-3 is the only insulin-like growth factor binding protein that forms a 150 kDa ternary complex with the insulin-like growth factor, acid-degradable subunit. However, some of the other insulin-like growth factor binding proteins have been proposed to be used in combination with insulin-like growth factor-I as a medicament.
    However, despite the advantages of administering insulin-like growth factor-I in the form of a complex with insulin-like growth factor binding protein-3, little is known about the compositions suitable for their pharmaceutical use. Bagi et al. (J. Bone Mineral Res. 9 (8): 1301-1311,1994) reported the use of insulin-like growth factor-I / insulin-like growth factor binding protein-3 in mice with ovarian depletion. Announced. Insulin-like growth factor-I / insulin-like growth factor binding protein-3 complexes were prepared in conventional phosphate buffered saline (PBS). Celtrix Pharmaceuticals has released an insulin-like growth factor-I / insulin-like growth factor-binding protein-3 composition prepared using acetate buffer pH 5.5 containing 105 mM sodium chloride as an osmolality salt. have. However, this composition is not preferable as a commercial pharmaceutical composition because the product cannot be lyophilized.
    Lyophilization, drying while freezing under certain conditions, is a commonly used method for long term preservation of proteins. Lyophilized proteins do not degrade, aggregate, oxidize, or otherwise decompose during the lyophilized state. Lyophilized proteins are usually used to restore the original protein form with water and optionally contain preservatives that inhibit the growth of bacteria such as benzyl alcohol. Unfortunately, various preservatives, such as, for example, benzyl alcohol, are not recommended to be easily mixed with protein or are currently recommended for medications that are currently administered for at least 24 hours or longer, and such recommendations are recommended for medications sold in the United States. It is a requirement.
    Lyophilized pharmaceutical products that are commercially acceptable must be made in the form of acceptable lyo cakes (lumps of lyophilized products). Preferably, the lyocake should have a smooth surface and a uniform appearance. The lyophilized protein itself is difficult to make in the form of an acceptable lyocake so that an appropriate excipient must be added. Typically, carbohydrates such as mannitol, sorbitol, and sugar are used as excipients in lyophilized pharmaceuticals. Buffers are also commonly added, especially in medical compositions consisting of proteins such as growth factors or cytokines. For example, if the medicament is in the liquid state before lyophilization and after reconstitution, the buffer is typically used to control the pH of the composition since the protein is typically particularly sensitive to rapid changes or extreme pH.
    Therefore, there is a need for a pharmaceutically acceptable composition preparation technique capable of providing high stability to a medicament consisting of insulin-like growth factor-I / insulin-like growth factor binding protein-3.
    The present invention relates generally to protein therapeutic composition, and more particularly to a pharmaceutical composition comprising a complex of insulin-like growth factor-I and insulin-like growth factor binding protein-3.
    We have made a new composition of insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex that provides long term stability of the insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex. The composition of the present invention is a pharmaceutically acceptable composition.
    We find the surprising fact that pharmaceutical compositions of insulin-like growth factor-I / insulin-like growth factor binding protein-3 complexes containing very low osmoticity-controlling salts are more stable than salt-added compositions. It was. In addition, the inventors have found surprising and unexpected finding that insulin-like growth factor-I / insulin-like growth factor binding protein-3 complexes can be prepared that further increase stability without the addition of pH buffer salts.
    In a more surprising and unexpected finding, the inventors found that insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex compositions containing high concentrations of protein and low osmoticity salts and no pH buffer salts were added. It is the fact that it has stability.
    One embodiment of the present invention shows that the composition of the present invention consists of an insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex, an excipient, and a pH buffer salt. In the composition of this example, no osmotic control salt is added.
    In another embodiment of the invention, the composition of the invention comprises only insulin-like growth factor-I / insulin-like growth factor binding protein-3 complexes and excipients. No osmotic salts or pH buffering salts are added to the compositions of this example. The composition of this example is particularly useful because it allows the preparation of a pharmaceutical composition containing very high concentrations of protein.
    The composition of the present invention may be a liquid composition or a lyophilized composition. They may also optionally contain nonionic surfactants. Liquid formulations may optionally contain a preservative to reduce bacterial growth or to eliminate bacteria.
    The appended amino acid sequence listing shows the amino acid sequence of a variant of mature insulin like growth factor binding protein-3 (Ala 5 ).
    The inventors have discovered a number of surprising and unexpected facts that allow the production of commercial, pharmaceutically acceptable, stable insulin-like growth factor / insulin-like growth factor binding protein compositions. The inventors found surprising and unexpected facts that the stability of the insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex is increased even after removing the added osmotic control salt. Also, more unexpectedly, the inventors found that the stability of the insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex is increased even after the pH buffer salt is removed. The inventors surprising the fact that the composition without the addition of pH buffer salts contained low levels of osmotic salts and increased stability when benzyl alcohol was commonly used as a drug preservative, which often promotes aggregation of proteins. Found. The compositions of the present invention contain less osmotic control salts, and with or without the addition of a pH buffer, make high concentrations of insulin-like growth factor-I / insulin-like growth factor binding protein complex compositions without substantial loss of protein. Can be. "Insulin-like growth factor" or "IGFs" of the present invention include, but are not limited to, insulin-like growth factor-I and insulin-like growth factor-II. Insulin-like growth factors are polypeptides having a molecular weight of about 7.5 kDa. Insulin-like growth factors of the present invention include naturally occurring insulin-like growth factor-I or insulin-like growth factor-II, analogs or variants thereof. Variants are substituted with tyrosine, phenylalanine, tryptophan, for example, wherein one, two or more tyrosine residues of insulin-like growth factor-I, 24, 31 or 60 residues are non-aromatic residues, 49, 50, 51, 53 , Variants similar to amino acid residues 55 and 56, eg, residues 49-51 modified with threonine-serine-isoleucine or residues 55-56 modified with tyrosine-glutamine and insulin-like growth factor-I Or insulin-like growth factor-II fused with other amino acid sequences. Insulin-like growth factors can be obtained from natural sources and prepared by recombinant methods.
    As used herein, the term "insulin-like growth factor binding protein" or "IGFBPs" refers to insulin-like growth factor-binding protein-1, insulin-like growth factor-binding protein-2, insulin-like, either from natural sources or from recombinant sources. Insulin-like growth factor-binding protein group consisting of growth factor-binding protein-3, insulin-like growth factor-binding protein-4, insulin-like growth factor-binding protein-5 and insulin-like growth factor-binding protein-6, but not limited thereto.
    "Insulin-like growth factor binding protein-3" or "IGFBP-3" is a member of the insulin-like growth factor binding protein family. In humans, a mature protein consists of at least two naturally occurring allelic proteins, 264 amino acids, of which the fifth amino acid residue is glycine or alanine (Gly 5 insulin-like growth factor binding protein-3 and Ala). 5 insulin-like growth factor-binding protein-3, respectively). When proteins are produced in cells of humans and other stray animals, the proteins undergo post-translational modifications up to three distinct sites where three nitrogen-binding glycosylations have occurred. If the protein is produced in bacteria, the protein is not glycosylated. Insulin-like growth factor-binding protein-3 also includes variants of the protein, for example, variants in which the position of the amino acid where glycosylation normally occurs is bound to another amino acid. Variants of the sequence are represented by X # Y, X is a single letter representing the amino acid code of the amino acid residue of the native protein, # represents the residue number of the mature protein sequence, and Y represents the amino acid with the residue replaced. In particular aspartate, N89D; N109D; N172D; N89D, N109; N89D, DN172D; N109D, N172D and N89D, N109D, N172D variants or N89X; N109X; N172X; N89X, N109X; N89X, N172X; N109X, N173X; And N89X, N109X, N172X variants, and the like. Other variants include alterations at positions 116 and 135, so that the natural sequence of aspartate is glutamate (i.e. D116E, D135E and D116E, D135E) or other amino acids (i.e., D116X, D135X and D116X, D135X) and insulin Variants of the local localizing sequence (NLS) located in the nucleus of similar growth factor binding protein-3 (ie, K228E, R230G and K228E, R230G and / or 215, 216 and / or 231 residues). Again variants of insulin-like growth factor binding protein-3 include one or more variants, ie variants of insulin-like growth factor binding protein-3, as well as hydrolysis-resistant variants as well as gene sequence variants located in the nucleus. Insulin-like growth factor-binding protein-3 is produced by purification of natural raw materials or recombinant prokaryotic host cells or eukaryotic host cells, and is preferably produced by recombination with naturally occurring allelic modified proteins.
    "Excipient" is a term that refers to an additive that is added to the lyocake to increase the volume as a pharmaceutically acceptable ingredient. Excipients that can be used include dextrose, ribose, simple sugars such as fructose, mannitol, alcohol sugars such as inositol and sorbitol, carbohydrates such as trehaloses, sugars, and lactose, and starch, dextran, chitosan, Include, but are not limited to, alluronates, proteins and glycogens such as gelatin and serum albumin, synthetic monomers and polymers. The excipients used in the present invention preferably also function as osmotic control salts (additives that make liquid compositions into normal serum and isotonic solution in humans).
    As used herein, the term "osmotic control salt" is a salt that is added to make the composition normal to human serum and isotonic solution. Osmotic pressure-controlling salts consider stability in dosing in humans and generally contain sodium chloride, calcium chloride, potassium chloride, and the like. Pharmaceutically acceptable osmotic control salts can generally be found in the United States Pharmacopieal, United States Pharmacopieal Covention, Inc., Rockville, MD, 1995.
    "Preservatives" are bacterial growth inhibitors, fungicides, fungal growth inhibitors and compounds that kill fungi, and are added herein to inhibit or eliminate the growth of bacteria or other contaminating microorganisms in the composition. Preservatives should be pharmaceutically acceptable and of general stability when administered to humans. Preservatives which may be added to the compositions of the present invention are, for example, benzyl alcohol, phenol, benzalkonium chloride, meta-cresol, thimerazole, chlorobutanol, methylparaben, propylparaben and the like. Medically acceptable preservatives can generally be found in the United States Pharmacopoeia (Id). 0.9-1% (v / v) benzyl alcohol is a preservative preferably used in liquid compositions and reconstituted lyophilized compositions.
    As used herein, the term "nonionic surfactant" is a compound that reduces the surface tension of water. Surfactants are useful in many ways, including preservatives that reduce the formation of aggregates of proteins, thereby reducing the protein bound to storage and dosing devices or to facilitate redissolution of the protein during reconstitution of the lyophilized composition. Very useful as Surfactants useful in the present invention should not cause denaturation of the protein. Examples of the surfactants acceptable in the present invention include polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), dodecyl poly (oxyethylene glycol ether) 23 (Brij 35) and octylphenol poly (ethylene glycol ether) 10 (Triton X-100), but is not limited to such. In general, nonionic surfactants that can be used as components in the compositions of the present invention can be found in the US Pharmacopoeia.
    In the present invention, the term "stable" as used in connection with a particular composition means that the composition satisfies the minimum acceptable criteria after storage at a particular time and in a particular condition. This means that aggregates for one year when stored at normal storage conditions, i.e., lyophilized composition, at about 20 ° C., or refrigerated, frozen or at 20 ° C. in a liquid state, are subjected to size exclusion chromatography. The composition of the composition, as determined by graphimetry, is determined by measuring the ratio of the peak area of the substance that does not belong to the main peak of the insulin-like growth factor / insulin-like growth factor binding protein to the total peak area. By insulin-like growth factor / insulin-like growth factor binding protein preferably increases by a percentage of less than 30, more preferably less than 15. A stable composition is also characterized by separation by reverse phase HPLC (substance is separated by reverse phase-HPLC, and the degradation rate is the ratio between the total peak area and the area between peaks other than the main peak of insulin-like growth factor and insulin-like growth factor binding protein. Obtained by measurement), where the degradation over a year in normal storage conditions is increased by a percentage, preferably less than 10, more preferably less than 5.
    Such insulin-like growth factors include wild-type insulin-like growth factor-I, most preferably recombinant human insulin-like growth factor-I and insulin-like growth factors, variants prepared by other methods known in the art. Preferably, recombinant human insulin-like growth factor-I is prepared by recombinant methods, using the techniques disclosed in WO 94/04076 and WO 96/40722. The insulin-like growth factor binding protein described above includes recombinant naturally-occurring human insulin-like growth factor binding protein-3, and glycine of naturally occurring allelic mutagens, particularly naturally-type human insulin-like growth factor binding protein-3. Allelic variant genomes of and alanines and variants of human insulin-like growth factor binding protein-3, eg, variants 89, 109, 116, 135, 172, 228 and 230.
    Formation of the insulin-like growth factor / insulin-like growth factor binding protein complex is preferably achieved by simply mixing the insulin-like growth factor and the insulin-like growth factor binding protein. Insulin-like growth factor-I and insulin-like growth factor binding protein-3 rapidly form complexes without further manipulation. Preferably, the complex may be further purified after complex formation. Such purification can be accomplished by several techniques known in the art.
    Preferably, the insulin-like growth factor / insulin-like growth factor binding protein complexes used in the composition contain less than 5% degradation products and less than 15% aggregates.
    Typically, complexes are prepared using insulin-like growth factors and insulin-like growth factor binding proteins in aqueous solutions containing pH buffer salts and osmotic pressure-controlling salts such as, for example, sodium chloride. To prepare the compositions of the present invention, the dissolved salts and optionally included pH buffer salts must be removed from the solution. Such removal is accomplished by buffer exchange techniques known in the art, including but not limited to filtration, dialysis, reverse osmosis and other ultrafiltration techniques, and desalination by size exclusion chromatogaphy. no. The protein solution can be replaced directly in the composition, but is preferably replaced with pure water. If the protein solution is replaced with pure water, the other components of the composition are added to the water / protein solution and mixed thoroughly. For example, the components of the composition, such as excipients, are added in the form of dried chemicals (in the form of most excipients and some surfactants supplied by the manufacturer) or liquid concentrates.
    No osmotic control salt is added to the composition of the present invention. This is because it is almost impossible to completely remove the salt from the buffer added during the preparation and purification of insulin-like growth factor and insulin-like growth factor binding protein, especially when the composition is prepared in a commercial process. This is because there can be no composition at all. However, the concentration of osmotic salts in the compositions of the present invention is low, preferably less than 12.5 mM, more preferably less than 2.5 mM, most preferably less than 1 mM.
    In one preferred embodiment, the insulin-like growth factor / insulin-like growth factor binding protein complex is formulated in pH buffer, a solution containing a buffer salt that buffers the pH change. The pH of this pH buffer solution is preferably about 5.0 to 7.0, more preferably 5.5 to 6.5. To the insulin-like growth factor / insulin-like growth factor binding protein complex solution is added a pH buffered salt concentration solution with the desired pH, or after adding a dry pH buffer salt, the insulin-like growth factor / insulin-like growth factor binding protein complex is added to the water. It can also be buffer exchanged. Alternatively, insulin-like growth factor / insulin-like growth factor binding proteins may also be buffer exchanged directly into pH buffer. Preferably, the insulin-like growth factor / insulin-like growth factor binding protein is buffer exchanged directly in pH buffer. Buffer salts are some pharmaceutically acceptable buffer salts such as sodium phosphate, potassium phosphate, sodium acetate, sodium citrate and sodium succinate. Preferred buffer salts are sodium citrate and sodium succinate, more preferably sodium succinate. In stability validation experiments, Applicants found that a composition containing a complex consisting of insulin-like growth factor-I / insulin-like growth factor binding protein-3 and a pH buffer without the osmolality salt was more stable than the composition containing the osmotic salt. I found that. More surprisingly, the Applicant has found that a composition containing succinate buffer at pH 5.5 is more stable than a composition containing citric acid and acetate buffer.
    In another embodiment of the invention, the insulin-like growth factor / insulin-like growth factor binding protein complex is buffered and exchanged into pure water. If necessary, excipients and additives are added to the insulin-like growth factor / insulin-like growth factor binding protein solution to make it into human serum and isotonic solution. Preferably, the excipient is mannitol, sorbitol, sugar, inositol, lactose, dextrose or a mixture of excipients. In one preferred embodiment, the excipient is mannitol and sugar, the excipient adds 6% (w / v) of the total amount, and the preferred ratio of mannitol to sugar is 3: 2, i.e. 3.6% (w / v) Mannitol and about 2.4% (w / v) sugar. In addition, compositions that are reconstituted with a relatively small amount of water after storage and lyophilization can be considered to produce isotonic compositions with increased protein concentrations. For example, the insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex is made up of 1.5% mannitol, 1% sugar in 50 mg / ml protein, and after lyophilization to a volume of 0.5 times the original volume It is prepared to be reconstituted to a composition of 100 mg / ml that is isotonic with human serum. Neither osmotic salts nor buffer salts are added to the composition of this example. Applicants have added a pharmaceutical composition with mannitol, sugar and insulin-like growth factor-I / insulin-like growth factor binding protein-3 complex without the addition of pH buffer or osmotic control salt, to which pH buffer and osmotic salt are added. It was found to be more stable than the composition. Applicants have also found that the compositions of these examples are particularly useful because they contain proteins at the highest possible concentrations (see Example 5).
    The composition of the present invention is stored as a liquid composition or in lyophilized state. Liquid compositions are preferred to be frozen for long term storage. Frozen liquid compositions are stored in cryogenic freezers below about −70 ° C., non-defrost freezers below −20 ° C., or defrost freezers in the range of 5 ° C. to −15 ° C. Preferably, the liquid composition is preferably stored in a cryogenic freezer, but it is also possible to store it in a non-defrost freezer or defrost freezer.
    The lyophilized composition is first prepared in liquid state, then frozen and lyophilized. Lyophilization processes are well known to those skilled in the art and involve sublimation of water from compositions frozen under certain conditions. The lyophilized composition is stored at about 20 ° C., at refrigeration or room temperature. The lyophilized composition is restored to a form for use by adding an aqueous solution to the redissolved composition. Solutions containing preservatives or excipients may also be used, but preferably, the solution used to restore the protein may be water (e.g., the U.S. Pharmacopoeia WFI, or water for injection) or water that inhibits the growth of bacteria (e.g., For example, the United States Pharmacopoeia (WFI) containing 0.9% benzyl alcohol is suitable. Water or water that inhibits the growth of bacteria is suitable as a solution used for protein restoration. Phenol (preferably about 0.2 to 0.3%), m-cresol (preferably about 0.25 to 0.3%), thimerosal (preferably about 0.25 to 0.3%), methylparaben (preferably about 0.25 to 0.3%), propyl Other preservatives, including parabens (preferably about 0.25-0.3%), chlorobutanol (preferably about 0.5%) and the like, can be added to the solution used for the restoration of the protein.
    Example 1: Composition of pH Buffer
    Frozen recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3 is dissolved in 1 ml of 50 mM acetate in 10 mg ratio, the pH is adjusted to 5.5, and 105 mM sodium chloride is dissolved in 3 ml of sample. One sample was dialyzed with 500 ml of three changes and replaced with 20 mM sodium citrate, 3% mannitol, 2% sugar, and a pH of 5.5. The second sample was dialyzed with 500 ml of three substituents and replaced with 20 mM sodium succinate, 3% mannitol and 2% sugar, with a pH of 5.5. After dialysis the sample was readjusted to 10 mg / ml. The samples were placed on the shelf of the lyophilizer and allowed to equilibrate at 18 ° C. for about 10 minutes and then the temperature was lowered to 5 ° C. for about 18 minutes. After equilibration at 5 ° C, the temperature was rapidly lowered to -15 ° C and held for about 12 minutes, then lowered to -35 ° C at which time the pressure of the freeze dryer was reduced to 200 to 300 millitorr and the sample was lyophilized for 4 hours. I was. The temperature was increased to 20 ° C. for at least 6 hours in vacuo and then held at 20 ° C. for 28 hours in vacuo.
    Recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3, frozen in two recombinant samples as a control sample, was dissolved in 1 ml of 50 mM sodium acetate at a rate of 10 mg, dissolved in 105 mM sodium chloride, and adjusted to pH 5.5. Incubate at 37 ° C. for 10 days after the addition. On day 10, samples were visually inspected and analyzed by reverse phase high performance liquid chromatography (HPLC) and size exclusion chromatography. Reversed phase HPLC was performed using a Vydac 4.6 X 250 mm C 18 column with a bead size of 5 μm and injected with 5% acetonitrile, followed by separation of 26 to 34% concentration with 0.1% trifluoroacetic acid (TFA). The material to be analyzed was eluted for at least 40 minutes. Size exclusion chromatography analysis used a Pharmacia Superdex 75 HR 10/30 column, 50 mM potassium phosphate, 0.5 M sodium chloride, and at pH 7.0, operated at a flow rate of 0.5 ml / min. Frozen recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3 was dissolved in 1 ml of 50 mM acetate in a 10 mg ratio, pH 5.5 and dissolved in 105 mM sodium chloride, maintained at -80 ° C during the experiment. The sample was dissolved and used as a control sample.
    Reverse phase-HPLC analysis measures the amount of insulin-like growth factor-I and insulin-like growth factor binding protein-3 degradation, the peak of total matter and the major peak of insulin-like growth factor-I and insulin-like growth factor binding protein-3. It is expressed by the ratio of the substance extreme value of. Citric acid and succinic acid buffers were nearly equivalent in this experiment and also equivalent to the control group. As a control, acetate buffer caused 3.6% degradation, while citric acid and succinic acid produced 3.5% and 4.1% degradation, respectively.
    Size exclusion chromatography analysis showed significant differences between the three samples. Size exclusion chromatography analysis compares the ratio of the substance extremes of the major peaks of insulin-like growth factor-I and insulin-like growth factor binding protein-3 to the total material of this sample, indicating the coagulant percentage of the coagulant. Formation was measured. The control formed the highest level of 6% coagulum, while citric acid formed 5% of the coagulum. Succinic acid was surprisingly much better than the other compositions, showing only 2.5% coagulum after 10 days experiments at 37 ° C.
    Example 2: pH Optimization for Compositions Containing pH Buffers
    Three 5 ml samples of human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 dissolved at a rate of 10 mg per ml of 5 mM acetate were 20 mM sodium succinate buffer containing 3% mannitol and 2% sugar. Dialysis at pH4.5, 5.0, 5.5, 6.0 or 6.5 for 24 hours against 500 ml of substituents. After dialysis, the protein content was examined by measuring OD 276 and the concentration was necessarily adjusted to make the sample 10 mg / ml. The pH of each sample was examined to maintain within 0.1 pH points of the intended pH, the sample was filtered under sterile conditions and lyophilized according to Example 1. The pH of the sample was inspected after recombination, the sample was filtered again in a sterile state, and each sample was allowed to stand for 10 days at 5 ° C and 37 ° C. The stability of the composition was analyzed by reverse phase HPLC and size exclusion chromatography.
    Size exclusion chromatography analysis indicates that the increase in coagulant is associated with an increase in pH. Buffers with a pH of 4.5 form 3.5% of the coagulum, buffers with a pH of 5 give 4% of the coagulum, buffers with a pH of 5.5 form 4.2% of the coagulum, while pHs of 6 and 6.5 The buffer formed 5.3% and 7.2% of coagulum, respectively.
    Reversed phase HPLC analysis showed the opposite trend, with increasing degradation generally lowering the pH. The highest degradation of 6.2% occurred at pH 4.5, while the degradation of 4%, 3.8%, 3.5% and 4.2% occurred at pH 5, 5.5, 6 and 6.5, respectively.
    Based on these results, pH 5.5 was chosen as the optimal pH for the composition containing the pH buffer.
    Example 3: Optimization of pH Buffer Concentration for Compositions Containing pH Buffer
    5 ml sample of human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 was mixed with 3% mannitol (w / v), 2% sugar (w / v) and various concentrations of 0.5mM, 10mM and 20mM. Dialysis with a solution containing 40 mM sodium succinate. After dialysis, the protein content was examined by measuring OD 276 , and the concentration was necessarily adjusted to 10 mg / ml of the sample. The pH of each sample was examined to maintain within 0.1 pH point of the intended pH, and the sample was sterile filtered and then lyophilized according to Example 1. The pH of the sample was inspected after recombination, the sample was sterilely filtered again, and each sample was divided into 5 ° C and 37 ° C for 10 days. The stability of the composition was analyzed by reverse phase HPLC and size exclusion chromatography.
    Size exclusion chromatography analysis indicates a direct correlation between coagulant and succinic acid buffer concentrations. 40 mM succinic acid formed 5.5% of coagulum, while samples of 20mM, 10mM, 5mM and 0mM formed 4.5%, 3.6%, 3.2% and 2.4% of coagulum, respectively.
    Reversed phase HPLC analysis indicated that all samples, except for 5 mM samples that caused 7.5% degradation, resulted in 3.5-4% degradation and are at the same level in degradation. This "spike" in degradation may be due to salts that are optimal for processes or enzymes involved in insulin-like growth factor binding protein-3 degradation.
    These results indicate that the lyophilized composition without further pH buffer is more stable than the composition with pH buffer.
    Example 4 Composition Containing High Concentrations of Insulin-like Growth Factor-I / Insulin-like Growth Factor Binding Protein-3
    30 ml of the sample of 10 mg / ml of recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3 was completely dissolved in a composition solution of 3.6% (w / v) mannitol and 2.4% (w / v) sugar. After dialysis, Amicon Centricon 10 Adjust the concentration so that the initial concentration of the ultrafiltered solution is 10 mg / ml, 20 mg / ml and 40 mg / ml using a centrifugal ultrafiltration device, and then check the concentration with OD 276 , and then add the composition solution. The concentration was adjusted. Samples of 10 mg / ml, 20 mg / ml and 40 mg / ml of recombinant human insulin-like growth factor-I / insulin-like growth factor binding protein-3 in the composition solution were lyophilized according to the method of Example 1, followed by water or 0.9% The benzyl alcohol was added to restore the original protein in the form of water, and filtered aseptically. Samples were placed at 37 ° C. for 7 days and then analyzed for stability by reverse phase HPLC and size exclusion chromatography. The analysis results are shown in Table 1.
    Reversed phase HPLC analysis showed that increasing protein concentration had little effect on proteolysis, and addition of benzyl alcohol had no effect on degradation.
    Size exclusion chromatography analysis indicates that increasing protein concentration increases coagulant levels in the sample. Interestingly, the addition of benzyl alcohol, which generally increases the coagulant of the protein, is particularly free of osmotic control salts or pH buffers in insulin-like growth factor-I / insulin-like growth factor binding protein-3 complexes. There was no substantial increase in protein coagulant in the mannitol / sugar composition.
    Table 1.
    Sample, digestion, coagulation body
    10 mg / ml 1.4% 1.2%
    10 mg / ml + benzyl alcohol 1.2% 1.2%
    20 mg / ml 1.9% 2.1%
    20 mg / ml + Benzyl Alcohol 1.7% 2.1%
    40 mg / ml 1.9% 4.2%
    40 mg / ml + benzyl alcohol 1.3% 6.1%
    Experiments were performed using human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 at a concentration of 75 mg / ml. The protein was first dialyzed extensively in 3.6% mannitol, 2.4% sugar solution, and then the concentration was adjusted using a stirred cell ultrafiltration device with a 10 kDa blocking filtration device. The solution was filtered aseptically and lyophilized by the method described above and then restored to the original protein form. The concentration of the protein restored to the original protein form was 75 mg / ml and analyzed by OD 276 . Samples with or without 0.9% benzyl alcohol were incubated at 37 ° C. for 7 days and then analyzed by reverse phase HPLC and size exclusion chromatography. The results are shown in Table 2 below.
    Extremely high protein concentrations increased the coagulum of the protein. Interestingly, benzyl alcohol does not affect coagulation or degradation at low protein concentrations, but benzyl alcohol addition enhances coagulation but does not affect degradation at very high protein concentrations.
    Table 2.
    Sample coagulation digest
    75 mg / ml 15% 3.4%
    75 mg / ml + benzyl alcohol 27% 3.0%
    Example 5 Compositions Containing Very High Concentrations of Insulin-like Growth Factor-I / Insulin-like Growth Factor Binding Protein-3
    Human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 contains four different compositions: (1) 3.6% mannitol, 2.4% sugar; (2) 1.5% mannitol, 1% sugar; ( 3) 0.525% mannitol, 0.35% sugar; Or (4) complete dialysis in one of the waters. After dialysis, the solution was adjusted to 50 mg / ml protein using a stirred cell concentration controller.
    Samples of 1 ml of composition 1, 2 ml of composition 2, 4 ml of composition 3, and 10 ml of composition 4 were transferred to a glass bottle and lyophilized by the method of Example 1. Different compositions were adjusted to obtain yields named human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 at concentrations of 50 mg / ml, 100 mg / ml, 200 mg / ml and 500 mg / ml, respectively (final concentration). Was exactly 49 mg / ml, 96 mg / ml, 187 mg / ml, 495 mg / ml). The total weight of the sample was reconstituted to 1 g each, and the protein concentration of the sample excluding composition 4 which could not be analyzed by forming a highly viscous syrup was measured by OD 276 . Purity was analyzed by size exclusion chromatography using Brij 35 as standard and in this assay 98.57% purity using lyophilized human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 as a sample. Got. The results are shown in Table 3.
    Table 3.
    Composition Nominal Concentration Expected Concentration Concentration Determination
    1 50 mg / ml 49 mg / ml 50 mg / ml 98.29%
    2 100 mg / ml 96 mg / ml 96 mg / ml 98.31%
    3 200 mg / ml 187 mg / ml 186 mg / ml 97.57%
    4 500 mg / ml 495 mg / ml Not measurable
    pH values were measured before lyophilization and after restoration to the original protein form. The results are shown in Table 4.
    Table 4.
    Composition Named Concentration pH Before Lyophilization pH After Reconstitution
    1 50 mg / ml Not measured 6.88
    2 100 mg / ml 6.91 6.87
    3 200 mg / ml 6.62 6.80
    4 500 mg / ml 6.60 Not measured
    Osmotic pressure was also measured using an osmometer before lyophilization and after restoring to the original protein form. Experimental normal osmolarity levels range from 280 to 295 mOsm / kg in blood, plasma and serum (Merck Manual of Diagnosis and Therapy, R. Berkow ed., 16th edition, 2581, 1992). The results are shown in Table 5.
    Table 5.
    Composition Named Concentration Predicted Osmotic Pressure Measured Osmotic Pressure
    1 50 mg / ml 304 mOsm / kg 309.5 mOsm / kg
    2 100 mg / ml 293 mOsm / kg 297 mOsm / kg
    3 200 mg / ml 294 mOsm / kg 290 mOsm / kg
    In a second experiment to determine the stability of the lyophilized protein of such a composition, human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 was dialyzed against composition 1, 2 or 3, followed by Example 1 Lyophilized according to the method of. After lyophilization, the sample is restored to the original protein form at concentrations of (a) 50 mg / ml, 100 mg / ml and 200 mg / ml ("Time 0"), or (b) each month at 22 ° C or 37 ° C. After 50mg / ml, 100mg / ml or 200mg / ml was restored to the original protein form of the nominal concentration. The purity of the sample is analyzed by the method of Example 1 using SEC or reverse phase HPLC. Human recombinant insulin-like growth factor-I / insulin-like growth factor binding protein-3 was stored at −80 ° C. as the control. The results of size exclusion chromatography or reverse phase HPLC analysis are shown in Tables 6 and 7, respectively. Results of size exclusion chromatography or reversed phase HPLC analysis show that compositions 1, 2 and 3 are stable under these conditions, but compositions with low concentrations of excipients, such as composition 3, are des (glycine-alanine) insulin-like growth factor-I. The stability is slightly lower due to formation.
    Table 6
    Composition Name Concentration SEC Purity (%) SEC Purity (%) SEC Purity (%)
    Time 0 1 mo. 22 ° C. 1 mo. 37 ℃
    Control group 10 mg / ml 96.7 97.5 * 97.5 *
    1 50 mg / ml Not measured 97.9 97.5
    2 100 mg / ml 98.4 96.5 97.8
    3 200 mg / ml 98.3 98.3 97.3
    Control samples were stored at −80 ° C. during the incubation period.
    TABLE 7
    Composition Nominal Concentration HPLC Purity (%) HPLC Purity (%) HPLC Purity (%)
    Time 0 1mo. 22 ° C. 1mo. 37 ℃
    Control group 10 mg / ml 99.3 99.6 * 99.6 *
    1 50 mg / ml Not measured 99.5 99.6
    2 100 mg / ml 99.4 99.4 99.6
    3 200 mg / ml 99.4 99.3 99.4
    Control samples were stored at −80 ° C. during the incubation period.
    The patents, patent applications and documents cited herein are incorporated by reference in their entirety.
    The present invention has been described in detail through direct description and examples. Equivalents and findings of the present invention will be apparent to those skilled in the art, all of which are within the scope of the present invention.
    <110> CELTRIX PHARMACEUTICALS, INC.
    <120> PHARMACEUTICAL FORMULATIONS FOR IGF / IGFBP
    <130> WO99 / 62536
    <150> US 09 / 089,062
    <151> 1998-06-01
    <160> 1
    <170> KOPATIN 1.5
    <210> 1
    <211> 264
    <212> PRT
    <213> Homo sapiens
    <400> 1
    Gly Ala Ser Ser Ala Gly Leu Gly Pro Val Val Arg Cys Glu Pro Cys
    1 5 10 15
    Asp Ala Arg Ala Leu Ala Gln Cys Ala Pro Pro Pro Ala Val Cys Ala
    20 25 30
    Glu Leu Val Arg Glu Pro Gly Cys Gly Cys Cys Leu Thr Cys Ala Leu
    35 40 45
    Ser Glu Gly Gln Pro Cys Gly Ile Tyr Thr Glu Arg Cys Gly Ser Gly
    50 55 60
    Leu Arg Cys Gln Pro Ser Pro Asp Glu Ala Arg Pro Leu Gln Ala Leu
    65 70 75 80
    Leu Asp Gly Arg Gly Leu Cys Val Asn Ala Ser Ala Val Ser Arg Leu
    85 90 95
    Arg Ala Tyr Leu Leu Pro Ala Pro Pro Ala Pro Gly Asn Ala Ser Glu
    100 105 110
    Ser Glu Glu Asp Arg Ser Ala Gly Ser Val Glu Ser Pro Ser Val Ser
    115 120 125
    Ser Thr His Arg Val Ser Asp Pro Lys Phe His Pro Leu His Ser Lys
    130 135 140
    Ile Ile Ile Ile Lys Lys Gly His Ala Lys Asp Ser Gln Arg Tyr Lys
    145 150 155 160
    Val Asp Tyr Glu Ser Gln Ser Thr Asp Thr Gln Asn Phe Ser Ser Glu
    165 170 175
    Ser Lys Arg Glu Thr Glu Tyr Gly Pro Cys Arg Arg Glu Met Glu Asp
    180 185 190
    Thr Leu Asn His Leu Lys Phe Leu Asn Val Leu Ser Pro Arg Gly Val
    195 200 205
    His Ile Pro Asn Cys Asp Lys Lys Gly Phe Tyr Lys Lys Lys Gln Cys
    210 215 220
    Arg Pro Ser Lys Gly Arg Lys Arg Gly Phe Cys Trp Cys Val Asp Lys
    225 230 235 240
    Tyr Gly Gln Pro Leu Pro Gly Tyr Thr Thr Lys Gly Lys Glu Asp Val
    245 250 255
    His Cys Tyr Ser Met Gln Ser Lys
    260
    权利要求:
    Claims (37)
    [1" claim-type="Currently amended] A pharmaceutical composition of an insulin-like growth factor and an insulin-like growth factor-binding protein complex, comprising a complex of an insulin-like growth factor and an insulin-like growth factor-binding protein, an excipient and a pH buffering salt, and no osmotic control salt added.
    [2" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the pH buffer salt comprises sodium succinate.
    [3" claim-type="Currently amended] The pharmaceutical composition of claim 2, wherein the concentration of the pH buffer salt is less than about 40 millimolar (mM).
    [4" claim-type="Currently amended] 4. A pharmaceutical composition according to claim 3 wherein the concentration of said pH buffer salt is less than about 20 mM.
    [5" claim-type="Currently amended] The pharmaceutical composition according to claim 2, wherein the concentration of the pH buffer salt is less than about 10 mM.
    [6" claim-type="Currently amended] The pharmaceutical composition of claim 1, wherein the pH of the composition is about 5.5 to 6.5.
    [7" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the excipient comprises mannitol.
    [8" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the excipient comprises sorbitol.
    [9" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the excipient comprises sugar.
    [10" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the excipient comprises mannitol and sorbitol.
    [11" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the content of the excipient is about 6% (w / v).
    [12" claim-type="Currently amended] The pharmaceutical composition of claim 11 wherein the mannitol is about 3.6% (w / v) and the sugar is about 2.4% (w / v).
    [13" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the composition further comprises a nonionic surfactant.
    [14" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the insulin-like growth factor binding protein is insulin-like growth factor binding protein-3.
    [15" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the insulin-like growth factor is insulin-like growth factor-I.
    [16" claim-type="Currently amended] The pharmaceutical composition according to claim 15, wherein the insulin-like growth factor binding protein is insulin-like growth factor binding protein-3.
    [17" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the composition further comprises a preservative.
    [18" claim-type="Currently amended] The pharmaceutical composition according to claim 17, wherein the preservative is benzyl alcohol.
    [19" claim-type="Currently amended] The method of claim 10 wherein the insulin-like growth factor / insulin-like growth factor binding protein complex is 50 milligrams per milliliter (mg / ml), the mannitol is 1.5% (w / v) and the sugar is 1% (w / and v) pharmaceutical composition.
    [20" claim-type="Currently amended] The method of claim 10, wherein the insulin-like growth factor / insulin-like growth factor binding protein complex is 100mg / ml, the mannitol is 3% (w / v) and the sugar is 2% (w / v) characterized in that Pharmaceutical composition.
    [21" claim-type="Currently amended] Insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP), characterized in that it contains an insulin-like growth factor / insulin-like growth factor binding protein complex and excipients and does not contain osmotic control salts and pH buffering salts. ) Pharmaceutical composition of the complex.
    [22" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the excipient is mannitol.
    [23" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the excipient is sorbitol.
    [24" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the excipient is sugar.
    [25" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the excipient contains mannitol and sugar.
    [26" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the content of the excipient is about 6%.
    [27" claim-type="Currently amended] The pharmaceutical composition of claim 26, wherein the mannitol is about 3.6% (w / v) and the sugar is about 2.4% (w / v).
    [28" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the composition further comprises a nonionic surfactant.
    [29" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the insulin-like growth factor is insulin-like growth factor-I.
    [30" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the insulin-like growth factor binding protein is insulin-like growth factor binding protein-3.
    [31" claim-type="Currently amended] The pharmaceutical composition according to claim 29, wherein the insulin-like growth factor binding protein is insulin-like growth factor binding protein-3.
    [32" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the composition further comprises a preservative.
    [33" claim-type="Currently amended] The pharmaceutical composition according to claim 32, wherein the preservative is benzyl alcohol.
    [34" claim-type="Currently amended] The pharmaceutical composition according to claim 1, wherein the composition is lyophilized.
    [35" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the composition is lyophilized.
    [36" claim-type="Currently amended] Insulin-like growth factor, insulin-like growth factor-binding protein, a mixture containing excipients and pH buffer salts and no osmolality salt is prepared, and the mixture is lyophilized to produce insulin-like growth factor, insulin-like growth factor-binding protein, A lyophilized pharmaceutical composition of an insulin-like growth factor and insulin-like growth factor binding protein complex prepared by making a lyophilized formulation of an excipient and a pH buffer salt.
    [37" claim-type="Currently amended] Lyophilized formulations of insulin-like growth factor, insulin-like growth factor binding protein and excipients are prepared by preparing a mixture containing insulin-like growth factor, insulin-like growth factor binding protein and excipients, but without osmotic control salt, and lyophilizing the mixture. A lyophilized pharmaceutical composition of an insulin-like growth factor and insulin-like growth factor binding protein complex prepared by the preparation.
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    同族专利:
    公开号 | 公开日
    US6436897B2|2002-08-20|
    US20020004478A1|2002-01-10|
    CA2330925A1|1999-12-09|
    AU4326799A|1999-12-20|
    JP2002516871A|2002-06-11|
    DE69918690D1|2004-08-19|
    WO1999062536A2|1999-12-09|
    BR9910863A|2002-06-11|
    WO1999062536A3|2000-03-30|
    DE69918690T2|2005-08-18|
    ES2221456T3|2004-12-16|
    AT270898T|2004-07-15|
    CN1315868A|2001-10-03|
    EP1082133B1|2004-07-14|
    US20030087806A1|2003-05-08|
    IL139969D0|2002-02-10|
    CN1201816C|2005-05-18|
    HK1040637A1|2006-01-06|
    EP1082133A2|2001-03-14|
    CA2330925C|2010-02-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-06-01|Priority to US09/089,062
    1998-06-01|Priority to US09/089,062
    1999-05-28|Application filed by 셀트릭스 파마슈티칼스, 인크.
    1999-05-28|Priority to PCT/US1999/012173
    2001-06-25|Publication of KR20010052500A
    优先权:
    申请号 | 申请日 | 专利标题
    US09/089,062|1998-06-01|
    US09/089,062|US6436897B2|1998-06-01|1998-06-01|Pharmaceutical formulations for IGF/IGFBP|
    PCT/US1999/012173|WO1999062536A2|1998-06-01|1999-05-28|Pharmaceutical formulations for igf/igfbp|
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