JP2001522813A - Novel IGF-I compositions and uses thereof - Google Patents

Abstract

  (57) [Summary] Provided is a highly concentrated, low salt, biologically active syrup of IGF-I or a variant thereof and a method of preparing the same. This novel syrup form of IGF-I has a concentration of at least about 250 mg / ml, a density of about 1.0 g / ml to about 1.2 g / ml, and about 13,000 as measured at room temperature (23 ° C.). It has a viscosity of IGF-I from centipoise (cps) to about 19,000 cps. The IGF-I syrup may have a solubility in the solution by precipitating or partitioning the IGF-I from the solution, preferably by adjusting the solution pH to concentrate the IGF-I in the solution, or following the use of a solubility enhancer. It is prepared by removing the accelerator. The precipitated syrup is useful as a means of storing IGF-I in a stable form and as a means of preparing a composition comprising biologically active IGF-I. Pharmaceutical compositions and kits comprising the concentrated IGF-I syrup are provided. Precipitated IGF-I syrups, IGF-I reconstituted from IGF-I syrups, pharmaceutical compositions, and kits are useful in IGF-I treatment for IGF-I responsive conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to novel compositions of IGF-I and variants thereof.

BACKGROUND OF THE INVENTION Insulin-like growth factor I (IGF-I) is a 70 amino acid polypeptide hormone with insulin-like and mitogenic growth biological activity (Rin
derknecht (1978) J. Biol. Chem. 253: 2770;
Rinderknecht (1978) FEBS Lett. 89: 283).
This hormone enhances cell growth and / or survival in various tissues, including the musculoskeletal, liver, kidney, intestinal, nervous system tissues, heart and lungs. Administration of IGF-I has been indicated for treatment of a variety of conditions in humans and animals.

[0003] Various formulations of IGF-I have been made. For example, U.S. Pat.
126,324, 5,324,639, 5,324,660, 5,
374,620 and 5,650,496; International Publication No. WO 94/15.
584 and WO 96/40776; "High and Low Load"
Formation of IGF-I in Multivesicul
co-pending application entitled "Ar Liposomes," Ser. No. 08 / 925,531, filed Sep. 8, 1997; "Injectable."
No. 60 / 080,008,19, co-pending application entitled "Formation Containing Succinate".
Filed on April 3, 1998; and "Method for P, filed concurrently with the present application.
producing Sustained-Release Formula
See co-pending application entitled "on," U.S. Patent Application No. _.

When IGF-I was expressed as a heterologous protein, inclusion bodies containing IGF-I were formed. For example, European Patent Nos. EP123,228, EP128,
733, EP 135,094, EP 230,869 and EP 288,451. When incorporated into such inclusion bodies, IGF-I is generally misfolded and is a biologically inactive form, and is reduced to an active solubilized form, refolded, and refolded. Must be solubilized. 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 /
See 02807.

[0005] Another method of making highly concentrated forms of proteins is to use protein stabilizers and precipitants, such as salts, and / or various manipulations of solution conditions such as pH, temperature, ionic strength, and the like. Includes the use of other techniques known in the art. These methods often result in unwanted protein purification. Because these are
It is biologically inert, relatively dilute, and / or contains pharmaceutically undesired salts or other drugs.

SUMMARY OF THE INVENTION There is provided a highly concentrated, low salt content, biologically active form of IGF-I or a variant thereof, and a method of preparing the same. IG obtained according to the method of the invention
This new form of FI has a viscous "syrup" consistency.
This syrup has at least about 250 mg / m 2 as measured at room temperature (23 ° C.).
l, a density of about 1.0 g / ml to about 1.2 g / ml, and about 13,000 g / ml.
It has a viscosity of IGF-I from 0 centipoise (cps) to about 19,000 cps. When reconstituted from syrup form, IGF-I is biologically active without requiring refolding. IGF-I syrup can be prepared by, for example,
Is prepared by precipitating IGF-I from solution by adjusting the pH of the solution or by removing the dissolution promoter.

[0007] Highly concentrated IGF-I syrups are useful as a means of storing IGF-I in stable form and as a means of preparing compositions containing biologically active IGF-I. Thus, IGF-I syrup, or IGF-I reconstituted from this syrup, can be used to form compositions such as pharmaceutical preparations, such as, for example, sustained release formulations and delivery devices, with other substances. Can be taken in.
Pharmaceutical compositions containing the reconstituted IGF-I syrup are provided. Also provided is a kit containing this highly concentrated syrup form and IGF-I in a separate pharmaceutically acceptable biological buffer. IGF-I syrup, IGF-I reconstituted from syrup, pharmaceutical compositions and kits comprise IGF-I
Useful in treating IGF-I associated with an I-responsive condition.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel form of IGF-I or a variant thereof and a method for its preparation. This highly concentrated, low salt, biologically active form of IGF-I or a variant thereof has the consistency of a viscous "syrup" and is referred to herein as a syrup. Disclosed is a method of preparing a novel form of IGF-I or a variant thereof. The IGF-I syrup is prepared by precipitating IGF-I from the solution, preferably by adjusting the pH of the solution appropriately, or by adding a suitable solubilizing agent to concentrate the IGF-I in the solution. Subsequently, it is prepared by removing the solubilizer. The resulting syrup form of IGF-I or a variant thereof provides a means for packaging more than the amount of IGF-I in a given volume. These by-products are the basis for the disclosed compositions and other methods useful for treating IGF-I associated with an IGF-I responsive condition.

[0009] As used herein, the term "IGF-I" refers to 70 amino acids and about 7,6 amino acids.
Insulin-like growth factor I, a single-chain peptide with a molecular weight of 00 Daltons
(IGF-I). IGF-I stimulates mitotic and proliferative processes associated with cell development.

[0010] The following description of IGF-I syrup form, its preparation, and its use
Refers to FI, but the compositions and methods of the present invention may comprise IGF-I as defined below.
Includes both I and IGF-I variants.

By “highly concentrated” as measured at room temperature (23 ° C.), at least about 250 mg / ml, such as at least about 300 mg / ml, or at least about 350 mg / ml, or at least about 425 mg / ml Or about 45
IGF-I concentrations from 0 mg / ml to 500 mg / ml are contemplated. At these concentrations and temperatures, the syrup has a density from about 1.0 g / ml to about 1.2 g / ml, more preferably about 1.1 g / ml, and from about 13,000 cps to about 19,000 cps. , Preferably from about 14,000 cps to about 18,000 cps
ps, more preferably from about 15,000 cps to about 17,000 cps
And even more preferably from about 15,500 cps to about 16,500 cps
And even more preferably has a viscosity of about 16,000 cps. This is a viscosity substantially lower than the viscosity of IGF-I formed in the inclusion body.
In one embodiment, the syrup has about 350 mg / syrup as measured at room temperature.
It has a concentration of 1.0 ml, a density of about 1.07 g / ml, and a viscosity of about 15,700 cps IGF-I. By "low salt content" is intended an amount of salt that is not sufficient to cause precipitation of the protein. "Biologically active" is intended to mean that IGF-I or a variant thereof, when reconstituted from its syrup form in solution form, is biologically active without having to refold. Is done.

[0012] This highly concentrated IGF-I syrup is prepared according to the method of the present invention.
It is obtained by precipitating I or a variant thereof. This syrup form of IGF-I is free-flowing and apparently milky in white, which can be obtained, for example, by precipitation using ammonium sulfate or IGF-I prepared by "salting out".
This is a feature that distinguishes this from the salting out form of GF-I. As a result of the high solubility of ammonium sulfate (3.9 M in water at 0 ° C.), a preferred high ionic strength solution for IGF-I precipitation can be easily achieved. For example, Voet and Voet (1995)
Biochemistry (John Wiley and Sons, New Y
ork), pp. 79-81. This method results in the precipitation of an apparent white salt protein complex, has a thick paste consistency, and has a substantially higher viscosity than the IGF-I syrup of the present invention. Such precipitated salt protein complexes are unacceptable for rapid and easy recovery of low salt IGF-I. To recover low salt-containing IGF-I, the precipitate must be re-solubilized (and thereby less concentrated) to remove salts from the protein solution.

[0013] The preparation of the highly concentrated IGF-I syrup of the present invention is preferably performed according to the method of the present invention. These methods include removing the solubilizing agent to make IGF-I syrup after manipulating the solution pH or adding a solubilizing agent to enhance the solubility of IGF-I. Both of these methods involve precipitation of IGF-I,
It allows for a highly concentrated, low salt content syrup that can be easily reconstituted to recover a solution of biologically active protein.

[0014] The first of these methods is based on the observation of the abnormal solubility properties of IGF-I. IGF-I is very soluble below pH 5.0, where 50-200
A concentration of mg / ml can be obtained. However, a sharp decrease in solubility was observed at pH 5
. Observed between 0 and pH 5.5. Above pH 5.5, the solubility of IGF-I is less than 10 mg / ml (see FIG. 1).

[0015] This method of preparing a highly concentrated IGF-I syrup comprises the steps of:
Reducing the solubility of IGF-I so as to precipitate from a buffer solution containing GF-I. Precipitation is achieved by adjusting the pH of IGF-I with buffer solution to a pH above about pH 5.0, as disclosed below.

[0016] Thus, IGF-I is adjusted in a suitable buffer solution whose important properties are the initial pH preferred for the solubility of IGF-I. The buffer solution is adjusted to the desired initial pH
Can be any buffer that provides Many suitable buffers are available in the art, including succinate buffers, phosphate buffers, citrate buffers, acetate buffers, acetates such as sodium or potassium acetate. Examples include but are not limited to buffers. In one embodiment of the invention, the buffer is acetic acid. Any buffer can be used as long as the initial pH promotes the solubility of IGF-I.

The buffer solution containing IGF-I has an initial pH below about pH 5.0, preferably from about pH 2.0 to about pH 5.0, and more preferably about pH 3.0.
To about pH 4.5, and even more preferably from about pH 3.5 to about pH 4.0.
It is. The initial concentration of IGF-I in this low pH buffer solution determines the amount of highly concentrated IGF-I syrup that can be obtained by subsequent pH adjustment. Therefore,
Higher initial concentrations of IGF-I produce higher amounts of precipitated IGF-I syrup. Since the solubility of IGF-I decreases rapidly at solution pH above about pH 5.0, an initial solution pH in the range below about 5.0 is preferably
It maximizes the initial concentration of IGF-I in the buffer solution and therefore maximizes the yield of precipitated syrup. Regardless of the initial concentration of IGF-I,
The concentration of -I syrup is at least about 250 mg / ml, as described above.

To obtain this highly concentrated IGF-I syrup, the initial pH of the buffer containing IGF-I is adjusted up to a final pH higher than about pH 5.0, and preferably about 5 From about 0.0 to a pH greater than about pH 9.0, more preferably about p.
H5.0 to a pH greater than about pH 8.0, even more preferably about pH 5.0.
From about pH 5.5 to about pH 7.0, even more preferably from about pH 5.5 to about pH 7.0.
To a pH of 6.5, and most preferably from about pH 5.5 to about pH 6.0.
It is adjusted to. As the pH increases, IGF-I above the solubility limit at high pH conditions forms a viscous syrup and precipitates. The pH of the buffer solution can be adjusted by standard titration methods well known in the art, such as adding sodium hydroxide. Alternatively, dialysis of the initial buffer solution containing IGF-I against any suitable buffer solution having a desired final pH above pH 5.0, as disclosed above. Can be adjusted by Such a buffer, for example,
Includes inorganic (eg, phosphate) and organic (eg, acetate) buffers. In one embodiment of the present invention, an initial IGF-I buffer solution having a pH of 5.0 or less is dialyzed at pH 6.0 against a sodium citrate buffer.

For example, if a solution containing 100 mg / ml IGF-I at an initial pH of 4.0 is adjusted to a final pH of 6.0, only 8.0 mg / ml IGF-I will remain in solution. The remaining 92 mg / ml precipitate forms a highly concentrated IGF-I syrup. IGF-I syrup can be separated from the buffered solution by decanting or aspirating the solution. This syrup has a concentration of IGF-I of at least about 250 mg / ml, as disclosed above.

[0020] This highly concentrated IGF-I syrup exhibits a precipitated form of IGF-I. For some proteins, precipitation results from irreversible denaturation and / or agglutination leading to inactivation of the protein. In the case of the precipitated IGF-I of the present invention, the precipitation reaction is reversible. Thus, the IGF-I syrup can be reconstituted and, when compared to the biological activity of IGF-I not precipitated by the method of the invention, the recovered soluble IGF-I will have a total biological activity Hold. Laminating the buffer solution on a known aliquot of syrup allows reconstitution of IGF-I. Any suitable buffer solution can be used for reconstitution, as long as the buffer capacity maintains the solution pH in a range that allows for the solubility of IGF-I. IG
FI solubility is a function of pH, with greater amounts of soluble IGF-I when the pH solution is below about pH 5.0 than can be recovered when the solution pH is above about 5.0. Can be recovered from the concentrated IGF-I syrup using a given volume of buffer solution.

[0021] The highly concentrated IGF-I syrup of the present invention can also be prepared with a suitable solubilizer or so-called solubility enhancer. For purposes of the present invention, "solubility enhancer" refers to a compound that contains a guanidinium group and is capable of enhancing the solubility of IGF-I or a variant of IGF-I. Examples of such solubilizers include the amino acid arginine, as well as amino acid analogs of arginine, which retain the ability to enhance the solubility of IGF-I above pH 5.5. Such analogs include, without limitation, dipeptides and tripeptides, including arginine. Other suitable solubilizers include, but are not limited to, guanidine carbaniedin (carb
aniedine), guanidine acetate, guanidineamine, guanidine carbonate, guanidine 1-cyano, guanidine 1,3-diphenyl, guanidine 1,3-
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-guanidinoethyl) -5
-Such as isoquinoline sulfonamide, guaninobutyric acid, guanidinopropionic acid and the like, for example, Sigma Chemical Company, St. Lo
guanidine-containing compounds commercially available from uis, Missouri. Of these compounds, arginine, guanidine hydrochloride, agmatine, 4-guanidinobenzoic acid, guanidoacetic acid, and guanidinosuccinic acid are preferred.

By “enhancing solubility” of IGF-I, the presence of a guanidinium-containing compound in the presence of a guanidinium-containing compound in a solution that is the same component but lacking the guanidinium-containing compound
H 5.5 or more, pH 6.0 or more, pH 7.0 or more, pH 8.0 or more, or pH
PH 5.5 and above, pH 6 and above compared to the amount of IGF-I that can be dissolved above 9.0
. It is contemplated that the amount of IGF-I that can be dissolved in a solution of 0 or more, pH 7.0 or more, pH 8.0 or more, or pH 9.0 or more is increased. The ability of a guanidine-containing compound to enhance the solubility of IGF-I can be determined using methods well known in the art. Generally, the concentration of the solubilizer added to the solution is from about 10 mM to about 1 M
And preferably from about 15 mM to about 500 mM, and more preferably, for example, "Compositions Providing for Inc.
The concentration of the compound arginine is in the range of about 20 mM to 200 mM, as disclosed in co-pending application U.S. Patent Application No. _ filed concurrently with the present application entitled "raised IGF-I Solubility".

In this manner, the addition of a solubility enhancer to the solution allows for the preparation of a highly concentrated IGF-I solution. The solubility enhancer is then removed from the IGF-I solution by dialysis or diafiltration. Removal of the solubility enhancer results in precipitation of IGF-I in a 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, IG
FI is biologically active without having to be refolded.

IGF-I prepared in a highly concentrated form according to the method of the present invention can be from any animal species, including but not limited to birds, dogs, cows, pigs, horses and humans . Preferably, the concentrated form is mammalian IGF-I
When used to treat a responsive disorder, IGF-I is from a mammalian species,
And more preferably, it is from a mammal of the same species as the mammal undergoing treatment for such a disorder. It is recognized that IGF-I can be made by recombinant methods using a coding sequence corresponding to IGF-I from the animal species of interest. Such recombination methods are discussed in more detail below.

[0025] Biologically active variants of IGF-I are also included in the methods of the invention. Such variants should retain IGF-I activity, particularly the ability to bind to the IGF-I receptor site. IGF-I activity can be measured using a standard IGF-I bioassay. Representative assays are known radioreceptor assays using placental membranes, such as those described in US Pat. No. 5,324,639; Hall et al. (1974) J. Clin. Endocr, which is incorporated herein by reference.
inol. and Metab. 39: 973-976; and Marshal
I, et al., (1974) J. Am. Clin. Endocrinol. and Metab
. 39: 283-292), BALB / c in a dose dependent manner.
Bioassays that measure the ability of molecules to enhance tritiated thymidine incorporation into 3T3 fibroblast DNA (see, eg, Tamura et al., (1989) J. Am.
Biol. Chem. 262: 5616-5621).
Preferably, the variant has at least the same activity as the native molecule.

Suitable biologically active variants can be IGF-I fragments, analogs, and derivatives. Intact IG by "IGF-I fragment"
Proteins consisting only of part of the FI sequence and structure are contemplated, and IG
It may be a C-terminal deletion or an N-terminal deletion of FI. By "analog" is intended an analog of either IGF-I or an IGF-I fragment comprising the native IGF-I sequence and structure with one or more amino acid substitutions, insertions or deletions. Peptides having one or more peptoids (peptidomimetics) are also included in the term analog (see International Application No. WO 91/04282).
. By "derivative" is meant IGF-I, an IGF-I fragment, or an analog thereof, such as glycosylation, phosphorylation, or the addition of other foreign moieties (as long as IGF-I activity is retained). Any suitable modifications are contemplated. Methods for making IGF-I fragments, analogs, and derivatives are available in the art. In general, U.S. Patent Nos. 4,738,921, 5,158,875, and 5,077, which are incorporated herein by reference.
, 276; International Publication Nos. WO85 / 00831, WO92 / 04363,
See WO 87/01038, and WO 89/05822; and European Patent Nos. EP1355094, EP123228, and EP128733.

The IGF-I variant is generally at least 70%, preferably at least 80%
%, More preferably from about 90% to 95% or more, and most preferably about 98% or more with the amino acid sequence of the reference IGF-I molecule. Variants differ by as few as 10, as few as 5, as few as 4,3,2, or only 1 amino acid residue. By "sequence identity", when a specified contiguous segment of the amino acid sequence of a variant is aligned with and compared to the amino acid sequence of the reference molecule, the same amino acid residue is found in the IGF-I variant and the reference IGF- It is intended to be found in the I molecule. Methods for determining identity between sequences are well-known in the art. For example, the ALIGN program (Dayhoff (1978) At
las of Protein Sequence and Structur
e5: Addendum 3 (National Biomedical Research)
Foundation, Washington, D.C. C. ) And Wiscon
sin Sequence Analysis Package, Versio
n8 (Genetics Computer Group, Madison,
See programs (available from Wisconsin) (eg, GAP programs). For the purpose of optimal alignment of the two sequences, contiguous segments of the amino acid sequence of the variant may have additional or deleted amino acid residues with respect to the amino acid sequence of the reference molecule. The contiguous segment used to compare to the reference amino acid sequence contains at least 20 contiguous nucleotides and can be 30, 40, 50, 100 or more nucleotides. Corrections for increased sequence homology associated with inclusion of gaps in the amino acid sequence of the variant can be made by assigning gap penalties. Methods for sequence alignment are well-known in the art.

When considering the percentage of amino acid sequence identity, the positions of some amino acid residues may differ from the result of conservative amino acid substitutions, which do not affect the nature of the protein function. In these examples, the percent sequence identity may be adjusted upwards to consider similar to conservatively substituted amino acids. Such adjustments are well-known in the art. For example, Meyers and Miller (1988) C
Omputer Applic. Biol. Sci. 4: 11-17.

The art provides substantial guidance involved in preparing and using such IGF-I variants, as discussed further below. A fragment of IGF-I generally comprises at least about 10 contiguous amino acid residues of a full-length molecule,
Preferably, it comprises about 15 to 25 contiguous amino acid residues of the full length molecule, and most preferably, about 20 to 50 or more contiguous amino acid residues of full length IGF-I.
In preparing IGF-I variants, those skilled in the art will appreciate that modifications to the native protein nucleotide or amino acid sequence can be made according to the methods disclosed in the present invention to prepare highly enriched forms of the IGF-I variants. Can easily be determined to produce variants that allow These are generally conservative amino acid substitutions that preserve the charge on the substituted residue (eg, aspartic acid versus glutamic acid).

Some IGF-I variants are known in the art and their IG
Examples of the modified FI include, for example, Proc. Natl. Acad. Sci. US
A 83 (1986): 4904-4907; Biochem. Biophys
. Res. Commun. 149 (1987): 398-404; Biol
. Chem. 263 (1988): 6233-6239; Biochem. Bi
ophys. Res. Commun. 165 (1989): 766-771; F
orsbert et al., (1990) Biochem. J. 271: 357-363
U.S. Patent Nos. 4,876,242 and 5,077,276; and those described in International Publication Nos. WO 87/01038 and WO 89/05822. Representative variants include those with the deletion of the mature molecule Glu-3, variants truncated to 5 amino acids from the N-terminus, the first 3
Variants having two truncations (des (1-3) -IGF-I, des-IGF-I
, TIGF-I, or brain IGF), and variants containing the first 17 amino acids of the B chain of human insulin instead of the first 16 amino acids of human IGF-I.

According to the method of the present invention, IGF-I used to make a highly concentrated syrup form of IGF-I may be in its substantially purified form, native form, recombinantly. It can be in a produced or chemically synthesized form. For example, IGF-I can be isolated directly from blood, such as serum or plasma, by known methods. See, for example, Phillips (1980) New Eng. J M
ed. 302: 371-380; Svoboda et al., (1980) Bioche.
mystery 19: 790-797; Cornell and Boughtdad.
y (1982) Prep. Biochem. 12:57; Cornell and Boughtday (1984) Prep. Biochem. 14: 123; European Patent No. EP123,228; and US Patent No. 4,769,361. Alternatively, IGF-I can be chemically synthesized by any of several techniques known to those skilled in the peptide art. For example, for solid phase peptide synthesis techniques, see Li et al., (1983) Proc. Natl. Ac
ad. Sci. USA 80: 2216-2220, Stewart and Yo
ung (1984) Solid Phase Peptide Syntheses
is (Pierce Chemical Company, Rockford,
Illinois), and Barany and Merrifield (19
80) The Peptides: Analysis, Synthesis, B
Iology, Gross and Meienhofer, eds., Volume 2 (Acade
mic Press, New York, 1980), 3-254 ,; and for classical liquid phase synthesis, see Bodansky (1984) Princip.
Less of Peptide Synthesis (Springer-Ve
rlag, Berlin); and edited by Gross and Meienhofer, (1980) The Peptides: Analysis, Syntheses.
is, Biology, Volume 1 (Academic Press, New Yo
rk). IGF-I can also be prepared chemically by methods of simultaneous synthesis of multiple peptides. For example, Houghten (1985) Proc
. Natl. Acad. Sci. USA 82: 5131-5135; and U.S. Patent No. 4,631,211. These references are incorporated herein by reference.

Genetic manipulation by recombinant DNA technology may be the most efficient way to produce IGF-I. Human DNA sequences encoding IGF-I are known and can be introduced into host cells for expression. IGF-I is produced by recombinant DNA technology.
E. FIG. coli, yeast, insect, and mammalian cells. Secretion I
GF-I can be made by adding a signal sequence to the DNA sequence encoding IGF-I. In addition, the DNA sequence encoding IGF-I
It can be manipulated to make I fragments, IGF-I analogs, or IGF-I derivatives. Such recombinant DNA technology is generally available in the art. For example, International Publication No. WO96 / 07424 (here, recombinant human IG
(FI protein is produced in yeast).

[0033] As a method for preparing this highly concentrated IGF-I syrup has been provided, the syrup itself has several uses as disclosed in the present invention. First, the syrup provides a means to enclose high concentrations of IGF-I in a small volume space. Thus, this syrup provides a means for easy storage of IGF-I. When stored in a suitable container, an aliquot of the syrup can be reconstituted by using a suitable buffer solution to recover the soluble IGF-I that retains its biological activity. The storage of IGF-I syrup is preferably between about 2 ° C and about 10 ° C.
C., more preferably at a temperature of about 2C to about 8C, most preferably about 4C. Storage in this manner provides a shelf life of 18-24 months or more. In addition, IGF
-I syrup is formulated with a protein stabilizer to maintain its activity. Such stabilizers are known in the art and include, for example, polyhydroxylated compounds such as simple salts, buffer salts, glycerol, mannitol, sucrose, and polyethylene glycol, and surfactants. See, for example, International Publication No. WO 92/11844.

[0034] Containers containing the highly concentrated IGF-I syrup may be packaged in kit form for subsequent preparation of a pharmaceutical composition useful in IGF-I therapy. In addition, such kits include appropriate buffers for reconstituting IGF-I in a more highly concentrated syrup form. The resulting reconstituted IGF-I may be utilized in formulating a pharmaceutical composition as outlined below. The pharmaceutical composition is administered at a known concentration of IGF-I such that administration at a therapeutically effective dose promotes the desired therapeutic response, particularly with respect to the IGF-I responsive state being treated. Prescribed. A "desired therapeutic response" is intended to ameliorate the condition, or a symptom associated with the condition.

The syrup form of IGF-I is useful as a means of preparing a composition comprising IGF-I. In this approach, the IGF-I syrup may be directly incorporated into one or more substances to form a composition comprising the biologically active IGF-I in a highly concentrated form. Alternatively, an aliquot of IGF-I syrup is
-I can serve as a source for I, and then incorporated into one or more substances to form a composition comprising the biologically active IGF-I in its reconstituted state obtain. Of particular interest are, for example, pharmaceutical compositions and compositions comprising IGF-I in an encapsulated form that are useful in the formulation of sustained release pharmaceutical compositions.

[0036] The highly concentrated IGF-I syrup of the present invention is administered to an IGF-I treatment directed to any IGF-I responsive condition to the treatment site being treated for such condition. It is useful if Therefore, I in this syrup form
GF-I, or IGF-I reconstituted from IGF-I syrup, is incorporated into one or more substances to form a pharmaceutical composition that is then placed in contact with the treatment site. Administration of a therapeutically effective amount of such a composition promotes the desired therapeutic response, particularly with respect to IGF-I responsive conditions undergoing IGF-I treatment.
The preferred form of IGF-I, ie, syrup or reconstituted, depends on the preferred method of delivering the pharmaceutical composition, as outlined below.

Suitable methods of delivering a pharmaceutical composition containing the concentrated IGF-I syrup include gelled formulations, viscous solutions, sustained release formulations, implantable delivery systems such as pumps, and the like. But not limited to these. Such a delivery system allows for controlled and focused delivery of IGF-I to the treatment site. The exact formulation employed will depend on the type of application desired. For example, gel-like formulations can be utilized for topical or incisional wound healing, while viscous, water-soluble formulations require a more fluid formulation with a high moisture content. Can be used for those applications that do.

The highly concentrated IGF-I syrup of the present invention is used in a gel formulation,
A controlled delivery system can be provided. "Controlled delivery" contemplates sufficient drug release to maintain therapeutic levels for an extended period of up to several months. The concentrated IGF-I syrups of the invention can be utilized in formulations that increase the residence time of growth factors and provide sustained release dosage forms. This is a significant advantage as it allows less application of the formulation to the treatment site, thereby allowing injury to the wound or site and less of its cellular components. For example, U.S. Patent Nos. 5,705,485, 5,457,093, and 3,932.
See 4,013, and 5,071,644.

The gel formulations of the present invention include those that contain 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 to prolong the presence of IGF-I in treated mammals (generally more than one day). Sustained release pharmaceutical compositions generally provide the pharmaceutical composition in a polymer, preferably a hydrophilic polymer, for sustained release of the drug. Methods for preparing many sustained release formulations are known in the art, and are described in Remington's P
Harmaceutical Sciences (18th edition; Mack Pub)
. Co. : Eaton, Pennsylvania, 1990), which is incorporated herein by reference. Generally, IGF-I can be encapsulated in a semi-permeable matrix of solid hydrophobic polymer. This matrix can be formed into a film or microcapsules. Examples of such matrices include polyesters, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Siddam et al. (1983) Biopolymers 22:54).
7-556)), poly-actide (U.S. Pat.
73,919 and EP 58,481), polylactic acid polyglycolic acid (PL
GA) hydrogels (eg, Langer et al. (1981) J. Biomed. M
ater. Res. 15: 167-277; Langer (1982) Chem.
. Tech. 12: 98-105), non-degradable vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as Lupron Depot ^™ ), and poly-D-(-)-3-hydroxybutyric acid (EP 133). 988), but not limited thereto. Suitable microcapsules may also include hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylic acid microcapsules prepared by coacervation techniques or interfacial polymerization. In addition, microemulsion or colloid drug delivery systems, such as liposomes and albumin microspheres, can also be used. Remi
ngton's Pharmaceutical Sciences (18th edition; Mack Pub. Co .: Eaton, Pennsylvania, 199)
See 0).

Such sustained release pharmaceutical compositions are described, for example, in US Pat. No. 4,178,361.
No. 4,404,183; 4,343,789; 5,614,487
No. 5,422,116; No. 4,309,405; No. 4,248,858
No. 4,524,060; 4,973,470; 4,539,199
No. 4,309,406; No. 4,309,404; No. 4,248,857
No. 4,248,856, all of which are incorporated herein by reference. One such sustained release composition, in which a polypeptide such as IGF-I is entrapped in biodegradable microparticles, is described in "Methods for Making Sustained Release Formulations", which were filed concurrently with the specification. It is described in the entitled U.S. Patent Application No .-- application, which is incorporated herein by reference in its entirety.

[0042] In one embodiment of the invention, the pharmaceutical composition comprising the highly concentrated IGF-I syrup is administered via topical drug delivery. This topical application of the drug provides a concentrated delivery of the drug, which reaches a tissue level that is not available through other routes of administration. Because drug release occurs through a time-dependent process of diffusion, delivery to its target site depends on the delivery system utilized,
It is maintained for days to weeks or more. In addition, local delivery
Reduces systemic drug exposure, thereby limiting systemic side effects. It also allows for the delivery of drugs that would otherwise be difficult or impossible to deliver via oral or intravenous routes due to solubility or formulation issues. Topical administration also offers the potential for the use of otherwise non-administrable drugs due to the dosage range that reaches the toxic limit by conventional routes of administration.

Means for local drug delivery include balloon catheter delivery systems, intravascular polymer-coated stents, facilitated diffusion, polymer endoluminal pavement (
paving), and controlled release matrices. For example, Eccl
eston et al. (1995) International Cardiolo
gy Monitor 1: 33-40-41, and Slepian (199
6) Intervention Cardiol. 1: 103-116 (hereby incorporated by reference).

In a preferred embodiment, the highly concentrated IGF-I syrup is an osmotically driven DU of ALZA (Palo Alto, California)
Used in implantable pumps, such as the ROS ^™ implantable pump. Such pumps are surgical implants that provide drug delivery in various doses in a continuous, steady manner over several months (eg, 6-12 months). In order for them to be implanted, these pumps must be small to be practical. Reloading the pump most often requires further surgery.
Thus, the material loaded into them allows to keep them implanted for a maximum amount of time, and to minimize the number of surgeries needed to implant another device In order to be highly concentrated. I of the present invention
GF-I syrups provide a means to encapsulate high concentrations of IGF-I in small volumes, typical of such implant devices.

Alternatively, an aliquot of the highly concentrated IGF-I syrup may be incorporated into a liquid injection for parenteral delivery. In this embodiment, an aliquot of the syrup is a pharmaceutically acceptable buffer having a buffering capacity to maintain the pH of the solution in a range that allows for dissolution of IGF-I, preferably below pH 5.0. Using the solution, it is reconstituted as described above. Then, the reconstituted IGF-
I is incorporated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier as described below. Preferably, the carrier is an advantageous carrier for parenteral delivery, and is preferably isotonic with the blood of the recipient. Such carriers include, but are not limited to, water, saline, Ringer's solution, and grape solution. Other carriers are described below.

The pharmaceutical composition comprising the reconstituted IGF-I, in a unit dose, and
In an injectable or insoluble form, such as a solution, suspension, or emulsion;
Should be prescribed. It can also be in the form of a lyophilized powder, which can be converted into a solution, suspension, or emulsion before administration. Pharmaceutical compositions containing reconstituted IGF-I are preferably sterilized by membrane filtration and stored in single or multiple dose containers, such as sealed vials or ampoules.

[0047] Any pharmaceutical composition comprising IGF-I, as concentrated above, or IGF-I reconstituted from IGF-I syrup, modulates therapeutic treatment with IGF-I. Other components may be included. Such components include any IGF-I binding protein, IGF-I receptor, and acid labile subunit of the IGF-I binding complex. IGFBP-3 may enhance the stimulatory effect of IGF-I on proteoglycan synthesis (Chevalier et al. (1996)
) British J .; Rheumat. 35: 515-522).
. In addition, acid labile glycoproteins have also been shown to associate with the protein complex formed by IGF-I and its binding proteins. Thus, a therapeutically effective pharmaceutical composition is such that the acid labile glycoprotein and the IGF-I binding protein facilitate the desired positive response in the IGF-I responsive disorder being treated. If it is proven, they may be included. IGF-
The amount of IGFBP administered with I can be determined according to the molar ratio between IGF-I and IGFBP. This molar ratio may range from about 0.5: 1 to about 3: 1 and preferably may range from about 1: 1 (see US Pat. No. 5,187,151). Alternatively, the pharmaceutical composition may include an agent that disrupts the binding of IGF-I to IGFBP, and that comprises an IG present at the affected physiological site.
It may be effective to increase the amount of FI to a therapeutically effective level. In addition to these components, pharmaceutical compositions that include IGF-I may include one or more protease inhibitors. An exemplary protease inhibitor is sodium pentosan polysulfate (PPS), a polysulfated polysaccharide. This protease inhibitor provides low doses of IG
Effective in the treatment of osteoarthritis in combination with FI (intra-articular injection of 1 μg of IGF-I three times a week) (Rogachefsky et al. (1993) Oste
arthritis and Cartilage 1: 105-114).
Such protease inhibitors may be administered by other routes, such as intramuscularly, during the administration of an effective dose of IGF-I.

A pharmaceutical composition according to the invention further comprises one or more other therapeutic agents effective in the treatment of other disorders in the individual, the biochemical effect of the additional therapeutic agent being I
May be included as long as they do not interfere with the efficacy of the intended effect of GF-I treatment. Examples of such agents include, but are not limited to, antibiotics, anti-inflammatory agents, and the like.

[0049] A pharmaceutically acceptable carrier should be mixed with IGF-I and other components used in preparing the pharmaceutical composition. "Pharmaceutically acceptable carrier" intends a carrier commonly used in the art to facilitate storage, administration, and / or healing effects of a therapeutic ingredient. The carrier may also reduce any unwanted side effects of IGF-I. A suitable carrier should be stable, ie, incapable of reacting with the other components of the formulation. that is,
The dosages and concentrations used for treatment should not produce significant local or systemic adverse side effects in the recipient. Such carriers are generally known in the art. Suitable carriers for the present invention are those that conventionally employ large, stable macromolecules, such as: albumin, gelatin, collagen, polysaccharides, monosaccharides, polyvinylpyrrolidone, polylactic acid, Polyglycolic acid, polymerized amino acids, non-volatile oil, ethyl oleate, liposome, glucose, sucrose, lactose, mannose, glucose, dextran, cellulose, mannitol, sorbitol, polyethylene glycol (PEG) and the like.
Sustained-release carriers, such as hyaluronic acid, may also be suitable. Prisel
l et al. (1992) Int. J. Pharmaceu. 85: 51-56, and U.S. Patent No. 5,166,331. Inclusion with hyaluronic acid and other polymers may have additional beneficial effects in osteoarthritis, an IGF-I responsive disorder. Bragantini (1987) Clin. Trials
J. 24 (4): 333-340; Dougados et al. (1993) Oste.
arthritis and Cartilage 1: 97-103; and Lusseir et al. Rheum. 23: 1579-1585;
See in particular (incorporated herein by reference). Other acceptable components in the composition include water, saline, phosphoric acid, citric acid, succinic acid,
Buffers that enhance isotonicity, such as, but not limited to, acetic acid and other organic acids or their salts.

[0050] Preferred pharmaceutical compositions incorporate a buffer having reduced local pain and irritation caused by infusion of the IGF-I composition. Such buffers include, but are not limited to, low phosphate buffers and succinate buffers. For example, in International Publication No. WO94 / 15584, 50 mmol / L
PH 5.5 to 6.5, using a phosphate buffer present in a content of less than
A GF-I solution has been described and reported to produce reduced pain upon infusion.
As another example, a pharmaceutical composition is described in “Inje, filed April 3, 1998.
table Formation Containing Succin
ate ", as in the formulation disclosed in co-pending application, U.S. Patent Application No. 60 / 080,008, which has a pH range from about 4.0 to about 7.5; And the succinic acid ranges from 0.5 mM to 100 mM, preferably less than about 50 mM.

The pharmaceutical composition may further comprise what is called a solubilizer or a so-called solubilizer. Compounds containing a guanidinium group (most preferably arginine)
Are suitable solubilizers for IGF-I described above.

[0052] Methods of formulating pharmaceutical compositions are generally known in the art. Remingt
on's Pharmaceutical Sciences (18th edition; Ma
ck Pub. Co. : Eaton, Pennsylvania, 1990) (
A pharmaceutically acceptable carrier, which is incorporated herein by reference),
A thorough discussion can be found regarding the formulation and selection of stabilizers and isomolytes.

Pharmaceutical compositions comprising concentrated IGF-I syrup, or IGF-I reconstituted from IGF-I syrup, are useful in therapy directed to treatment of IGF-I responsive conditions. . By "IGF-I responsive condition" is intended any condition that responds to IGF-I either positively or negatively for a short or long term. Such an IGF-I responsive condition can be a normal condition. For example, mammals can receive IGF-I treatment to increase normal muscle mass, where increased muscle mass is desirable, as in athletes. In contrast, the IGF-I responsive condition can be a chronic, abnormal condition, and thus occurs more or less continuously, or it is acute,
Subsequent wounds to the site, such as joint or bone wounds, occur.

Symptoms responsive to IGF-I include, but are not limited to, acute or chronic symptoms; hyperglycemic disorders, including all types of diabetes; chronic lung disease Acute and chronic kidney failure; acute and chronic liver failure; cirrhosis; inflammatory responses such as rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, and inflammatory bowel disease; short intestine; including the heart, liver, or brain; Or ischemic injury, such as that resulting from renal necrosis; immunological disorders, such as reduced immune tolerance or immunodeficiency including chemotherapy-induced tissue damage; organ rejection after transplantation; Diseases and dysfunctions of the heart structure or function, such as heart condition, cardiomyopathy, beats, and congestive heart failure; growth retardation; osteoporosis; wound healing; bone damage; ocular symptoms; infertility; Neurodegenerative disorders, such as neuronal disorders, multiple sclerosis, muscular dystrophy, diabetic neuropathy, paralytic peripheral neuropathy, Parkinson's disease, Alzheimer's disease, and sequelae of traumatic spinal cord injury; and osteoarthritis And articular cartilage disorders such as injury involving trauma. Any IGF-I responsive disorder is
-I syrup or reconstituted IGF-I obtained from IGF-I syrup of the present invention
May benefit from administration of a pharmaceutical composition comprising

“Treatment” refers to the treatment of existing normal conditions that are enhanced by IGF-I treatment,
Therapeutic treatment of existing abnormal conditions of IGf-I responsiveness and preventive or preventive procedures to be performed before the abnormal disorder occurs.

A pharmaceutical composition comprising IGF-I syrup or reconstituted IGF-I obtained therefrom can be used in the treatment of any animal for an IGF-I responsive condition. Exemplary animals include cats, dogs, horses, cows, sheep, pigs,
And more preferably includes but is not limited to humans.

[0057] The highly concentrated syrupy IGF-I disclosed in the present invention may be IG
A further use of FI as an essentially water-free preparation has been found. Thus, the method of the present invention provides a means for preparing IGF-I compositions that are essentially water free.

[0058] IGF-I syrup is useful for preparing IGF-I in dry powder form because of its high concentration and essentially no water. This syrup-like IGF-I can be dried more efficiently in less time as little water should be recovered from the IGF-I syrup during lyophilization when compared to the solution formulation. The resulting dry powdered IGF-I is more densely packed (eg, more IGF-I per unit volume) than other lyophilized IGF-I, and its preparation has a lower salt content .

Because of the inherent lack of water, this syrup of IGF-I has found use in other processes that require the removal of water from protein compositions. For example, this IGF-I syrup is available from Johnson et al. (1996) Nature Med.
ICINE 2: P using a low temperature process described by 794-799
IGF was added to LGA (poly (D, L-lactide-co-glycolide)) microspheres.
-I can be used to encapsulate. See also the low temperature process described in US Pat. No. 5,019,400.

The following examples are provided by way of illustration and not by way of limitation.

Experiments IGF-I for use in these experiments was isolated from yeast strain Pichia pa
storis, which are produced recombinantly, and are essentially US Pat.
Purified as described in 4,639, 5,324,660, and 5,650,496 and International Publication No. WO 96/40776.

Example 1 IGF-I Solubility as a Function of pH After isolation, the solubility of recombinant human IGF-I (rhIGF-I) was determined by dialysis. Saturated rhIGF-I solutions were used at higher concentrations (eg, 100 mg / ml in pH 4.0 buffer) for conditions where rhIGF-I was less soluble.
I can be made by dialysis. In this example, rhIGF-I (pH
4.0, 100 mg / ml) was dialyzed (3,000 molecular weight cut-off tubes) against three conversions of 20 volumes of pH 6.0 buffer.

As rhIGF-I above the solubility limit was distributed, two phases formed. That is, a solution phase containing a precipitated rhIGF-I layer and a saturated solution of rhIGF-I. A sample of the solution phase rhIGF-I was removed and filtered through a 0.22 μm 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 extinction coefficient.

FIG. 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 of 10
less than mg / ml. When the tonicifying salt is removed from the buffer solution, the solubility at pH 6.0 is only 3.0 mg / ml.
It is.

Although IGF-I has an isoelectric point near pH 8.7, the solubility profile of rhIGF-I shows that this protein is less soluble at solution pH well below its isoelectric point solution pH. To indicate that

Example 2 Preparation of IGF-I Syrup by Manipulation of Solution pH rhIGF-I described in Example 1 was precipitated as follows. 13mg
/ Ml of bulk rhIGF-I was added to the initial buffer solution at pH 4.0 with 74
mg / ml and then spectral pore tubing 1000
10 mM sodium citrate / 140 mM pH 6.0 using MWCO
Dialyzed against sodium chloride buffer. The concentration of rhIGF-I remaining in the solution is 10
. It was 6 mg / ml. The concentration of rhIGF-I in the first and last solution was
Measured spectrophotometrically at 276 nm in the UV region. The buffer solution was decanted and the precipitated polypeptide regained the form of a milky, viscous syrup.
The concentration of rhIGF-I in the precipitated syrup was determined to be about 350 mg / ml syrup.

The density of the rhIGF-I was determined by weight at room temperature (23 ° C.). 1
0 milliliters (10 ml) of rhIGF-I syrup was prepared for volumetric determination and its weight was determined on a Mettler AE240. rhI
The weight of a 10 ml sample of GF-I syrup was determined to be 10.7 grams. Therefore, the density of this rhIGF-I syrup was determined to be 1.07 g / ml.

The viscosity of the rhIGF-I syrup was measured using a Canon Instruments LV200
It was determined using a zero-speed viscometer. The instrument was calibrated using a viscosity standard provided by the manufacturer. All measurements were performed at room temperature (23 ° C.). This rhIGF
The viscosity of the -I syrup was determined to be about 15,700 centipoise (cps).

Example 3 Preparation of IGF-I Syrup by Removal of Solubility Enhancer Other compounds containing arginine and guanidinium groups have been shown to dramatically increase the solubility of IGF-I ( See FIG. 2). In this example, arginine can be used as a solubility enhancer to prepare a high concentration rhIGF-I solution, from which the high concentration rhIGF-I solution is removed and the rhIGF-I of the present invention is removed. Syrup precipitates.

For example, 100 mg / ml rhIGF-I in 10 mM sodium citrate, 120 mM arginine, pH 6.0 was added to 10 mM sodium citrate, 140 mM
Dialysis was performed at 4 ° C. against mM sodium chloride, pH 6.0. Under these conditions,
rhIGF-I only dissolves up to about 10 mg / ml. First 100mg
/ Ml, 90 mg / ml precipitate to form a milky syrup and 1
0 mg / ml remains in the solution. The solution portion of rhIGF-I can be decanted and the rhIGF-I syrup can be recovered. This rhIGF-I syrup retains its biological activity.

Example 4: Stability of IGF-I syrup Stability studies were performed using rhIGF-I in a highly concentrated, low salt content, biologically active syrup. . rhIGF-I was prepared by the method of Example 2. The precipitated rhIGF-I syrup was recovered. The buffer was overlaid on the syrup to allow some rhIGF-I to be reconstituted. This buffer did not contain any agents that are normally used to promote protein refolding. A sample of this supernatant was removed at time 0 (T = 0 weeks) and the reconstituted rhIGF-I was purified by SDS-PAGE (Laemm
Li et al. (1970) Nature 227: 680-685), RP-HPLC
(Kunitani et al. (1986) J. Chromatogr. 359: 391).
-402), and bioassays (Lopaczynski et al. (1993) R
(Evaluation Peptides 48: 207-216).

The syrup material was then stored at 4 ° C. After two and four weeks, fresh buffer was overlaid on the syrup. This reconstituted material is again converted to SDS
-Analyzed by PAGE, RP-HPLC, and bioassay. The results of this study are shown in Table 1.

[0073] These results indicate that rhIGF-I can be prepared by this method to form a viscous syrup and then reconstituted. These data also indicate that the reconstituted material retains its purity and activity.

[0074]

[Table 1] Example 5 Preparation of IGF-I in Precipitated Form Containing Salt 100 ml of rhIGF-I at 13.2 mg / ml in 0.1 M acetic acid (pH approx.
To 5), ammonium sulfate was added at 4 ° C until 35% saturation. The ammonium sulfate was dissolved by stirring, and the solution turned "milky" white, indicating that the rhIGF-I had precipitated from solution. The milky suspension could be stirred at 4 ° C. overnight and the ammonium sulfate-precipitated rhIGF-I was recovered by centrifugation. The precipitated rhIGF-I was denser than the liquid and settled to the bottom of the centrifuge tube during centrifugation. The precipitated rhIGF-I was recovered from the pellet by decanting the supernatant. The recovered ammonium sulphate-precipitated rhIGF-I was different from the IGF-I composition of the present invention: it was white in appearance (indicating the presence of real salts) and was free-flowing. It had a thicker paste than some syrups.

Example 6: Low Temperature PLGA Encapsulation Process Requiring Lyophilization of Syrup The rhIGF-I syrup of Example 2 or Example 3 was lyophilized for encapsulation and in methylene chloride. And PLGA. The suspension so obtained is combined with liquid nitrogen and essentially Johnson et al. (1996) Nature.
e Spray onto PLGA microspheres produced and recovered as described in Medicine 2: 799-799.

Example 7: Low Temperature Process that does not Require Lyophilization of Syrup The rhIGF-I syrup described in Example 2 or 3 is filtered to substantially remove all water, and then It can be mixed with ethanol and then added to PLGA dissolved in methylene chloride. Alternatively, the ethanol is in methylene chloride. Ethanol mixed with this syrup extract does not bind to water. This ethanol is miscible with the methylene chloride and therefore partitions into the methylene chloride phase containing the PLGA polymer, and is thus available from Johnson et al. (1996) Nature Me.
The cold process described in dicine 2: 795-799 actually facilitates encapsulation of the entire rhIGF-I in syrup.

All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, it will be understood that certain changes and modifications may be practiced within the scope of the appended claims. it is obvious.

[Brief description of the drawings]

FIG. 1 shows rhIGF-I solubility as a function of pH.

FIG. 2 shows rhIGF-I solubility as a function of arginine concentration, or the concentration of some other compounds (compounds with and without guanidino groups).

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF Term (reference) 4C076 AA09 AA94 AA97 CC30 DD48 EE36 FF15 4C084 AA02 BA20 BA23 CA62 DB34 DB58 MA01 MA05 MA28 NA13 NA14 ZB221 ZC031 4H045 AA10 BA20 CA40 DA38 EA30 FA74 GA05

Claims (27)

[Claims] 1. A highly concentrated IGF-I composition, wherein the composition comprises:
A composition comprising biologically active IGF-I or a variant thereof at a concentration of at least about 250 mg / ml. 2. The IGF-I composition of claim 1, wherein the composition is a low salt syrup. 3. The IGF-I composition of claim 2, wherein the syrup comprises IGF-I at a concentration from about 250 mg / ml to about 500 mg / ml. 4. The IGF-I composition of claim 2, wherein said syrup comprises IGI-F at a concentration of about 350 mg / ml, and said syrup comprises about 1.07 g / ml. ml, and a viscosity of about 15,700 cps,
Composition. 5. A method for preparing a highly enriched form of biologically active IGF-I or a variant thereof, comprising: a) the IGF-I or a variant thereof. Preparing a solution comprising: wherein the solution has an initial pH of about pH 5.0 or less; and b) adjusting the pH of the solution to a final pH of about pH 5.5 or more. Process,
Wherein said adjusting comprises causing precipitation of said IGF-I or variant thereof to form a syrup containing said IGF-I or variant thereof at a concentration of at least about 250 mg / ml; and c) Separating the syrup from the solution. 6. The method of claim 5, wherein the syrup comprises:
A method wherein the syrup is a low salt syrup comprising IGF-I at a concentration of about 250 mg / ml to about 500 mg / ml. 7. The method of claim 5, wherein the syrup comprises:
A method wherein the syrup is a low salt content syrup comprising IGF-I at a concentration of about 350 mg / ml, and the syrup has a density of about 1.07 g / ml and a viscosity of about 15,700 cps. 8. A highly concentrated form of biologically active IGF-I prepared according to the method of claim 5. 9. A method for preparing a highly enriched form of biologically active IGF-I or a variant thereof, comprising: a) in the presence of a solubility enhancer Preparing a high concentration solution of IGF-I or a variant thereof; b) removing the enhancer from the solution, wherein the removal of the enhancer is at a concentration of at least about 250 mg / ml. Causing precipitation of the IGF-I or a variant thereof to form a syrup containing the IGF-I or a variant thereof; and c) separating the syrup from the solution. . 10. The method of claim 9, wherein said solubility enhancer is arginine or an arginine analog. 11. The method of claim 9, wherein said solubility enhancer is guanidine hydrochloride. 12. A highly concentrated form of biologically active IGF-I prepared according to the method of claim 9. 13. A kit for reconstituting a pharmaceutical composition comprising biologically active IGF-I or a variant thereof, wherein said kit comprises one or more container means. Including a compartmentalized carrier to receive in a secure restraint, where:
One of the containers contains a highly concentrated form of biologically active IGF-I or a variant thereof, wherein the IGF-I or variant thereof has at least about 25
0 mg / ml and another said container means has a pH of about pH 5.0 or less.
A kit comprising a pharmaceutically acceptable buffered solution having H. 14. A method for preparing a composition comprising biologically active IGF-I, the method comprising the following steps: a) Advanced method of biologically active IGF-I or a variant thereof. Wherein the IGF-I or a variant thereof is present at a concentration of at least about 250 mg / ml; and b) at least to form the composition Incorporating a highly enriched form of said biologically active IGF-I or a variant thereof in one substance. 15. The method of claim 14, wherein the highly concentrated form of the biologically active IGF-I or a variant thereof is about 250 mg.
A syrup comprising a low salt content syrup comprising a concentration of IGF-I from / ml to about 500 mg / ml. 16. A composition prepared according to the method of claim 15, which is a pharmaceutical composition. 17. The pharmaceutical composition according to claim 16, which is a sustained release formulation. 18. The pharmaceutical composition according to claim 16, which is a gel formulation. 19. The composition prepared according to the method of claim 15, wherein the composition is PLGA microparticles made at cryogenic temperatures. 20. The microparticle of claim 19, wherein said microparticle is a lyophilized form of said syrup. 21. A method of treating an IGF-I responsive condition in a mammal, said method comprising a highly enriched form of biologically active IGF-I or a variant thereof. Administering a pharmaceutical composition to said mammal, wherein said IGF-I or variant thereof is present at a concentration of at least about 250 mg / ml. 22. The method of claim 21, wherein the highly concentrated form of the biologically active IGF-I or a variant thereof is about 250 mg / mg.
A method wherein the syrup is a low salt content syrup comprising a concentration of IGF-I from ml to about 500 mg / ml. 23. The method of claim 21, wherein said pharmaceutical composition is administered as a sustained release formulation. 24. The method of claim 21, wherein said pharmaceutical composition is administered as a gel formulation. 25. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is administered as an implant.
The method described in. 26. The method of claim 21, wherein the pharmaceutical composition is a therapeutic site to be treated for the IGF-I responsive condition and is micro-sized for long-term delivery. A method administered in a pump. 27. The method of claim 26, wherein the pump is osmotically driven.