CA2102808A1 - Targeted delivery of bone growth factors - Google Patents
Targeted delivery of bone growth factorsInfo
- Publication number
- CA2102808A1 CA2102808A1 CA002102808A CA2102808A CA2102808A1 CA 2102808 A1 CA2102808 A1 CA 2102808A1 CA 002102808 A CA002102808 A CA 002102808A CA 2102808 A CA2102808 A CA 2102808A CA 2102808 A1 CA2102808 A1 CA 2102808A1
- Authority
- CA
- Canada
- Prior art keywords
- bone
- targeting molecule
- composition
- growth factor
- tgf
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1841—Transforming growth factor [TGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
- A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- General Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
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- Immunology (AREA)
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- Zoology (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Diabetes (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
Methods and compositions for using conjugates of bone growth factors and targeting molecules in bone repair and augmentation are provided. The bone growth factor and targeting molecule are chemically conjugated to a crosslinker. The crosslinker is preferably a synthetic hydrophilic polymer. The bone growth factor is preferably TGF-.beta., activin, or bone morphogenic protein (BMP).
Targeting molecules preferably have an affinity for bone, such as tetracycline, calcein, bisphosphonate, polyaspartic acid, polyglutamic acid, aminophosphosugars, or estrogen.
Targeting molecules preferably have an affinity for bone, such as tetracycline, calcein, bisphosphonate, polyaspartic acid, polyglutamic acid, aminophosphosugars, or estrogen.
Description
WO92~20371 PCT/US92/03~0 2~ 0~ 0~
TARGETED ~ELIVERY_~BONE GROWTH FACTO~
Pescription Technical Fleld This invention relates to the fields of pro-tein engineering and medical treatment. More par-ticularly, this in~ention relates to conjugates of bone growth factors and targeting molecules and their uses in bone repair and augmentation and the like.
Back~round of the Invention "Transforming grow*h factor-~" (TGF-~) represents a family of proteins which are evolutionarily highly conserved, and which affect a broad spectrum o~
cell types. Two forms, TGF-~1 and TGF-~2, have been identified in platelet releasates and bone. Both -proteins exist as 26 kD disulfide-linked homodimers.
Even though there are 14 amino acid differences in the first 36 amino acids residues of the two forms, their biological acti~ities are similar (Cheifetz et al., Cell (1987) 48:409-415; S. Seyeidin et al., J. Biol~_Chem.
~1987) 262:1946-~94~ }though TGF-~1 and TGF-~2 appar-ently bind to common cell surface receptors, the amino acid sequence homology is only about 70%. TGF-~3 exhibits about 76%;homology with TGF-~l, and about 79%
homo~ogy with TGF-~2.
Additional TGF-~s have also been described.
U.S. Patent No. 4,886,747 describes the identification of TGF-~3 and its nucleotide sequence, and describes a WO~2~20371 PCT/US92/03~0 3 ~ -2-method for recovery of TGF-~3 from recombinant cell cultures. S.B. Jakowlew et al., Molec. Endocrinol.
~1988) 2:1186-llg5 describes TGF-~4 and its nucleotide sequence, identified by cDNA characterization. A.B.
Roberts et al., ~rowth Factors, Vol. 2 ~19g0), pp. 135-147, describes the purification of TGF-~5 from Xenopus-conditioned medium.
TGF-~ was first identified as a factor which permitted anchorage-independent growth o~ primary cell cultures. In vivo, TGF-~ promotes the ~eposition of connective tissue, and induces ell growth for cells of mesenchymal origin. TGF-~1 and TGF-~2 affect the proli~eration and differentiation o~ cells o~ the immune system including macrophages (Wall et al., Proc. Natl.
~cad. Sci. U.S.A. (1987) 84:5788), pre-B cells (Le~ et al., J. Ex~. Med. (19B7) 166:1~90), hematopoietic stem cells (Ohta et al., Nature ~(1987) 329:539; Ishibashi et al~ (1987) 69:1737; Keller et al., J. Ex~. Med.
(1988) in press; Sing et al., Blood (1988) in press), and NK cells (Rook et al., J. Immunol. (1986) ~ 3916).
Bentz et al., U.S. Patent No. 4,806,523, disclosed that TGF-~ may also be administered to suppress inflammatory responses, including immune suppression. McPherson et al., U.S. Patent No. 4,816,442 disclosed that TGF-~ may also be administered to suppress hyperproliferation, for example, cancer and leukemia.
Activins are dimeric proteins structurally similar;to lnhibin, TGF-B1, TGF-~2, and other proteins that ma~eup a family of proteins structurally related to TGF-B1. These proteins exhibit the chromatographic properties of TGF-~s.~ In addition to having homology with respect to the amino acid sequences, activins exhibit conservation of cysteine positions characteristic :
of the TGF-~s. Activins exhibit a molecular weight of 25 ; 35 kD under nonreducing conditions by SDS-PAGE ~and a : ~ :
WO92/20371 PCT/US92/03~0 'J1~ d molecular weight of 14 kD under reducing canditions).
There are two known forms of the activin subunit~, which have been termed ~A or ~B. Homodimeric forms ~AA and ~BB
and a heterodimeric form ~AB have been des~ribed in the literature. Activin subunits have about a 30% homology to TGF-Bl and T~F-B2 chains in terms of their amino a~id sequences. Inhibins are polypeptides which are also structurally related to actiYins. Inhibins are heterodimers of the activin ~A or ~B subunit and a separate ~ subunit. Inhibins exhibit activity essen-tially opposite to activin.
The activin ~A homodimer and ~AB heterodimer have been purified from porcine follicular fluid, and have been shown to stimulate the release of ~ollicle stimulating hormo~e (FSH) from rat pituitary cells in vitro (W. Vale et al., Nature (1986) ~ 776-79). Other reported activities include stimulation of oxytocin release from neurosecretory neurons (P.E. Sawchemko, et al., Nature (1988) 334:~15~17; W. Vale et al., "Recent Progre~s in Hormone Research" (1988) 44:1-34);
stimulation of insulin secretion from pancreatic islets~
~Y. Totsuka et al., Biochem. & BiophYs. Res Comm. (1988) 156:335-39); and stimulation of erythroid and multi-potential progenitor cell colony formation in bone marrow culture (J. Yu et al., Nature (1987) 330:765-67; H.E.
Broxmeyer et~al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85:9052-56). Activin ~A is apparently identical to erythroid differentiation factor (EDF) (M. Murata et al.
Proc. Natl. Acad. Sci. U.S.A. (1988) 85:2434-38).
Despite the fact~that activin is similar in ` amino acid se~uence to TGF-~, activin does not compete with TGF-~ for binding to TGF-~ receptors types I, II, or III present on fibroblasts and epithelial cells.
However, activin has been reported to compete against binding of TGF-~1 to rat pi~tuitary tumor cells (S.
2 1a2 ~ ~ 8 4 Cheifetz et al., J. ~iol. Chem. (1988) 263:17225-Z8).
TGF-~l and TGF-~2 have been reported to induce formation of endochondral bone in VlVO (M.E. Joyce et al~, J. Cell Biol~ ~1990) ~ 2195-2207, H. Bentz, et al. (198g) J.
S Biol. Chem., 264:20805-10)~
A "bone morphogenetic protQin" (BMP) was extracted from demineralized bone using urea or gu~nidine hydrochloride and reprecipitated according to the disclosures in U~S. Patent Nos. 4,294,753 and 4,455,256 to Urist. Urist subsequently reported (Urist, M. R., Clin. Orthop. Rel. Res. ~1982) 162:219) that ion exchange purification of this crude protein mixture yielded an activity which was unadsorbed to carboxymethyl cellulose resin (CMC) at pH 4.8. Urist's reports in Science (1983?
220:680-685 and Proc. Natl. Acad. Sci. U S.A. (1984)~
81:371-375 describe BMPs having molecular weights of 17,500 and 18,500 daltons. Urist's patent publication, EPA Publication No. 0212474, describes BMP fragments of 4,000 to 7,000 daltons obtained by limited proteolysis of BMP.
U.S. Patent No. 4,608,199 describes a bone-derived protein of 30,000-32,000 daltons. The protein is described as being water soluble and having no affinity for ¢oncanavalin A.
TARGETED ~ELIVERY_~BONE GROWTH FACTO~
Pescription Technical Fleld This invention relates to the fields of pro-tein engineering and medical treatment. More par-ticularly, this in~ention relates to conjugates of bone growth factors and targeting molecules and their uses in bone repair and augmentation and the like.
Back~round of the Invention "Transforming grow*h factor-~" (TGF-~) represents a family of proteins which are evolutionarily highly conserved, and which affect a broad spectrum o~
cell types. Two forms, TGF-~1 and TGF-~2, have been identified in platelet releasates and bone. Both -proteins exist as 26 kD disulfide-linked homodimers.
Even though there are 14 amino acid differences in the first 36 amino acids residues of the two forms, their biological acti~ities are similar (Cheifetz et al., Cell (1987) 48:409-415; S. Seyeidin et al., J. Biol~_Chem.
~1987) 262:1946-~94~ }though TGF-~1 and TGF-~2 appar-ently bind to common cell surface receptors, the amino acid sequence homology is only about 70%. TGF-~3 exhibits about 76%;homology with TGF-~l, and about 79%
homo~ogy with TGF-~2.
Additional TGF-~s have also been described.
U.S. Patent No. 4,886,747 describes the identification of TGF-~3 and its nucleotide sequence, and describes a WO~2~20371 PCT/US92/03~0 3 ~ -2-method for recovery of TGF-~3 from recombinant cell cultures. S.B. Jakowlew et al., Molec. Endocrinol.
~1988) 2:1186-llg5 describes TGF-~4 and its nucleotide sequence, identified by cDNA characterization. A.B.
Roberts et al., ~rowth Factors, Vol. 2 ~19g0), pp. 135-147, describes the purification of TGF-~5 from Xenopus-conditioned medium.
TGF-~ was first identified as a factor which permitted anchorage-independent growth o~ primary cell cultures. In vivo, TGF-~ promotes the ~eposition of connective tissue, and induces ell growth for cells of mesenchymal origin. TGF-~1 and TGF-~2 affect the proli~eration and differentiation o~ cells o~ the immune system including macrophages (Wall et al., Proc. Natl.
~cad. Sci. U.S.A. (1987) 84:5788), pre-B cells (Le~ et al., J. Ex~. Med. (19B7) 166:1~90), hematopoietic stem cells (Ohta et al., Nature ~(1987) 329:539; Ishibashi et al~ (1987) 69:1737; Keller et al., J. Ex~. Med.
(1988) in press; Sing et al., Blood (1988) in press), and NK cells (Rook et al., J. Immunol. (1986) ~ 3916).
Bentz et al., U.S. Patent No. 4,806,523, disclosed that TGF-~ may also be administered to suppress inflammatory responses, including immune suppression. McPherson et al., U.S. Patent No. 4,816,442 disclosed that TGF-~ may also be administered to suppress hyperproliferation, for example, cancer and leukemia.
Activins are dimeric proteins structurally similar;to lnhibin, TGF-B1, TGF-~2, and other proteins that ma~eup a family of proteins structurally related to TGF-B1. These proteins exhibit the chromatographic properties of TGF-~s.~ In addition to having homology with respect to the amino acid sequences, activins exhibit conservation of cysteine positions characteristic :
of the TGF-~s. Activins exhibit a molecular weight of 25 ; 35 kD under nonreducing conditions by SDS-PAGE ~and a : ~ :
WO92/20371 PCT/US92/03~0 'J1~ d molecular weight of 14 kD under reducing canditions).
There are two known forms of the activin subunit~, which have been termed ~A or ~B. Homodimeric forms ~AA and ~BB
and a heterodimeric form ~AB have been des~ribed in the literature. Activin subunits have about a 30% homology to TGF-Bl and T~F-B2 chains in terms of their amino a~id sequences. Inhibins are polypeptides which are also structurally related to actiYins. Inhibins are heterodimers of the activin ~A or ~B subunit and a separate ~ subunit. Inhibins exhibit activity essen-tially opposite to activin.
The activin ~A homodimer and ~AB heterodimer have been purified from porcine follicular fluid, and have been shown to stimulate the release of ~ollicle stimulating hormo~e (FSH) from rat pituitary cells in vitro (W. Vale et al., Nature (1986) ~ 776-79). Other reported activities include stimulation of oxytocin release from neurosecretory neurons (P.E. Sawchemko, et al., Nature (1988) 334:~15~17; W. Vale et al., "Recent Progre~s in Hormone Research" (1988) 44:1-34);
stimulation of insulin secretion from pancreatic islets~
~Y. Totsuka et al., Biochem. & BiophYs. Res Comm. (1988) 156:335-39); and stimulation of erythroid and multi-potential progenitor cell colony formation in bone marrow culture (J. Yu et al., Nature (1987) 330:765-67; H.E.
Broxmeyer et~al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85:9052-56). Activin ~A is apparently identical to erythroid differentiation factor (EDF) (M. Murata et al.
Proc. Natl. Acad. Sci. U.S.A. (1988) 85:2434-38).
Despite the fact~that activin is similar in ` amino acid se~uence to TGF-~, activin does not compete with TGF-~ for binding to TGF-~ receptors types I, II, or III present on fibroblasts and epithelial cells.
However, activin has been reported to compete against binding of TGF-~1 to rat pi~tuitary tumor cells (S.
2 1a2 ~ ~ 8 4 Cheifetz et al., J. ~iol. Chem. (1988) 263:17225-Z8).
TGF-~l and TGF-~2 have been reported to induce formation of endochondral bone in VlVO (M.E. Joyce et al~, J. Cell Biol~ ~1990) ~ 2195-2207, H. Bentz, et al. (198g) J.
S Biol. Chem., 264:20805-10)~
A "bone morphogenetic protQin" (BMP) was extracted from demineralized bone using urea or gu~nidine hydrochloride and reprecipitated according to the disclosures in U~S. Patent Nos. 4,294,753 and 4,455,256 to Urist. Urist subsequently reported (Urist, M. R., Clin. Orthop. Rel. Res. ~1982) 162:219) that ion exchange purification of this crude protein mixture yielded an activity which was unadsorbed to carboxymethyl cellulose resin (CMC) at pH 4.8. Urist's reports in Science (1983?
220:680-685 and Proc. Natl. Acad. Sci. U S.A. (1984)~
81:371-375 describe BMPs having molecular weights of 17,500 and 18,500 daltons. Urist's patent publication, EPA Publication No. 0212474, describes BMP fragments of 4,000 to 7,000 daltons obtained by limited proteolysis of BMP.
U.S. Patent No. 4,608,199 describes a bone-derived protein of 30,000-32,000 daltons. The protein is described as being water soluble and having no affinity for ¢oncanavalin A.
5 reports four proteins, designated BMP-l, BMP-2 Class I ("BMP-2"), BMP-3, and BMP-2 Class II
("BMP-4"), that are alleged to have osteogenic activity.
: It is not known! whether t'hese BMPs have oste~genic activity by themselves or require combination with other factors.
J.M. Wozney, in Growth Factor Research, Vol. l (1989):, pp. 267-280, describes three additi~nal BMP
proteins closely~related to BMP-2, and which have been ~ designated BMP-5, BMP-6 and BMP-7.
: 35 Wo 89/09787 and 89/09788 describe a protein .
WO92t20371 PCT/US92/03~0 210~81~
called "OP-1", now known to be BMP-7. The cloning of BMP-7 is described in E. Ozkaynak et al., EMBO Journal (19g0) 2:2085-2093, and the purification of BMP-7 is de~cribed in T.K. Sampath et al., J. Biol. Chem. (1990) 265:131g8-13205.
U.S. Patent No. 4,434,094 to Seyedin and Th~mas reported the partial puri~ication o~ a bone generation-stimulating, bone-derived protein by extraction with chaotropic agents, fractionation on anion and cation exchange columns, and recovery of the activity ~rom a fraction adsorbed to CMC at pH 4.8. This new protein fraction was termed "osteogenia factor" (OF) and was characterized as having a molecular weight ~elow about 30,000 daltons.
There are several re~erences in the art to proteins modified by covalent conjuga~ion to polymers, to alter the solubility, antigenicity and biological clearance of the protein. For ~xample, U.S. P~tent No.
4,261,973 disclosed the conjugation of several allergens to PEG (polyethylene glycol) or PPG (polypropylene gly-col) to reduce the proteins' immunogenicity. U.S. Pate~t ~o. 4,301,144 disclosed the conjugation of hemoglobin with PEG and other polymers to in~rease the protein's oxygen carrying capability. EPO 98,110 disclosed coupling an enzyme or interferon to a polyoxyethylene-polyoxypropylene (POE-POP) block polymer increases the protein's halflife in serum. U.S. Patent No. 4,179,337 disclosed qonjugat.Lng hydrophilic enzymes and însulin to PEG or PPG to reduce immunogenicity.
Davis et al., Lancet (1981) 2:281~83 disclosed the enzyme ~; uricase modified by conjugation with PEG to provide uric acid metabolism in serum having a long halflife and low immunogenicity. Nishida et al., J. Pharm. Pharmacol.
~1984) _ :354-55 disclosed PEG-uricase conjugates administered orally to chickens, demonstrating decreased WOs2/20371 PCT/US92/03~0 2 1 ~ 8 serum levels of uric acid. Inada et al., Biochem.
Biophys. Res. Comm. (1984) 122:845-50 disclosed lipoprotein lipase conjugation with PEG to render it soluble in organic solvents. Takahashi et al., Biochem.
& Biophys. Res. Comm. (1984) 121:261-65 disclosed HRP
conjugated with PEG to render the enzyme soluble in benzene. Abuchowski et al., Cancer Biochem. BiQphvs.
(1984) 7:175-86, disclosed that enzymes such as asparaginase, catalase, uricase, arg4nase, trypsin, superoxide dismutase, adenosine deaminase, phenylalanine ammonia-lyase, and the like, conjugated with PEG exhibit longer half-lives in serum and decreased immunogenicity.
U.S. Patent No. 4,830,847, and European Patent Application 5er. No. Z07,557 disclose diphosphonate-derivatized macromolecules which may be labeled withtechnium-99m Both antibodies and liposomes have been used to direct drugs to target sites ~see, for example, Tyle and Ram, eds., Tarqeted Thera~eutic Svstems, Marcel Dekker, New York, 1990.) Tarqeted delivery of bone growth factors may ~
reduce harmful or~undesirable effects of those molecules, allow the use of lower doses because relatively~higher doses can be delivered to t~e site of interest, and may prolong the effect at ~ the site of interest. In addition, the use of a targeting molecule that influences bone metabolism, lncluding bone resorption and formation, may result in an additive orlsynergistic effeqt.
:
:30 summarY of the Invention~
It~is an object of the invention to provide a composition c~omprising a~bone growth factor and a ` targeting molecule having affinity for a tissue of interest, where~th~e;bone growth factor and targeting molecule are chemically conjugated to a crosslinker. The :~ -:: : ` ~ : :
. . .
WO 92/20371 PCI'/US92/03840 ~ 1 0 2 ~ D8 crosslinker is preferablyi a synthetic hydrophilic polymer. The bone yrowth factor is preferably TGF-~, activin, or bone morphogenic protein ~BMP). Targeting molecules preferably have an affinity for bone, such as tetracycline, calcein, bisphosphonate, polyaspartic acid, polyglutamic a~id, aminophosphosugars, or eætrogen.
It is another object of the invention to pro~ide a method for augmenting bone formation in a sub;ect.
It i5 another object of the invention to provide a method for treating bone loss in a subject.
Modes of Carrvinq Out The Invention A. Definitions The term "bone growth factor" refers to the families of proteins which affect bone formation.
Examples of bone growth faotors include transforming growth factor-beta tTGF-~), activin, and bone morphogenic : 20 factor (BMP).
The term "TGF-~I' refers to beta-type trans-forming growth factors, including TGF-~1, TGF-~2, TGF-~3, TGF-~4, TGF-~5, heterodimers of the TGF-~ polypeptide chains (e.g.~ TGF-~1.2), and ~ragmen~s thereof, synthetic.
peptides, and homologous proteins having substantially : equivalent biological activity in any of the numerous known TGF-~ assays, such as the one described in Methods for Preparation of Med~ia! SuPplements and Substrate for Serum-free Animal Cell Culture (1984) pp. 181-194. Alan R. Liss, Inc. This particular assay determines ability : to induce anchorage-dependent growth in non-neoplastic normal rat kidney~(NRK) fibroblasts by measuring the ~` formation of cell;colonies in soft agar. Other known TGF-~ activity assays include but are not limited to stimulation of osteoblast proliferation, inhibition of W0~2/~0371 PCT~US92/03~0 ~1 02808 -8-keratinocyte proliferation, stimulation of collagen synthesis in a variety of cells, inhibition of mitogen-stimulated T-cell proliferation, and stimulation of chemotactic activity. Preparation of TGF-~l and TGF-~2 is described in U.S~ Patent No. 4,774,322, incorporated herein by reference. Additional TGF-Bs have also been described. U.S. Patent No. 4,886,747 describes the identification of TGF-~3 and its nucleotide sequence, and describes a method for recovery of TGF-~ from recombinant cell cultures. S. B. Jakowlew et al., Molec~ Endocrinol.
(1988) 2:1186-1195, describes TGF-~4 and its nucleotide sequence, ldentified by cDNA characterization. A.B.
Roberts et al., Growth F~ctors, Vol. 2 (1990) pp. 135-147, describes the purification of T5F-B5 from Xenopus-conditioned medium.
The term "TGF-~" as used herein is also intended to include the heterodimer TGF-~2 . 3, disclosed in copending patent application U.S. Ser. No. 614,306, filed 11/16/9o. T~F-~2.3 may be prepared from an extract of bone by pooling side fractions from peaks of column chromatography, subjecting those fractions to reverse phase HPLC and recovering those fraction which migrate more slowly than TGF-~2 by SDS-PAGE, subjecting those slower migrating fractions to FPLC and recovering those that migrate during a~pH 4.6 to 6.7 gradient, subjecting the pH 4.6 to 6.7 eluant to reverse phase HPLC or gel electrophoresis, and recovering substantially pure TGF-~2.3. ~ j ~
The term l'TGF-~" as used herein is also intended to include any other~synthetic molecule whose mechanism of action is mediated through the TGF-~receptor or second messenger~pathway.
Because these proteins are non-species-specific in their activity they maybe used to treat su~jects in general, lncluding sport, pet, and farm animals, and WO92/Z0371 PCr/US92/03~0 2 ~
g humans.
As used herein, "bone morphogenetic proteinl' (BMP) refers to a class of bone-derived proteins capable of inducing bone formation, as measured by activity in an 5 in vivo rat bone formation assay. BMPs include BMP-1, B~P-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, and ~ragments, deletions, additions, substitutions, mutations and modifications thereof which retain the biological c~aracteristics of the na~ural BM~. The BMP may be of human, bovine, or other species origin.
The term "activin" as used herein refers to activin ~AA, activin ~AB, activin ~BB, and ~ragments thereof, synthetic peptides, and proteins having similar acti~ity in a standard cell culture assay where the protein stimulates the release of ~ollicular stimulating hormone (FSH) from rat pituitary cells (Vale, et al., Nature (1986) ~ 776). Briefly, in this assay, anterior pituitaries from adult male Sprague-Dawley rats (200-220 g) are dissociated by collagenase and plated at a concentration of 0.33 X 1o6 cells per well in 24 well tissue culture dishes. The cells are allowed to recover for 72 hr in medium containing 2% fetal bovine serum (FBS). Following the reco~ery period, the cells are washed twice in fresh medium containing 2% FBS. All t eatments are added at thîs time and the cells are incubated for 72 hr. The media i5 then collected and the FSH levels are determined using a radioimmunoassay kit provided~by the NationallHormone and Pituitary program of NlADDK.
` As used hereinj the~term "targeting molecule"
refers to a molecule that binds to the tissue of interest or that influences metabolism of that tissue of interest.
So, for example, bone targeting molecules may include bone-seeking molecules such as tetracycline, calcein, ~5 bisphosphonates, chelators, phosphates, polyaspartic .
21 ~2~û8 -lo-a~id, polyglutamic acid, aminophosphosugars, peptides known to be associaked with the mineral phase of bone such as osteonectin, bone sialoprotein and osteopontin, proteins with bone mineral binding domains, and the like.
- 5 Bone-targeting molecules may also include molecules which affect bone resorption and bone formation ra~es, such as bisphosphonates, and estrogens and other steroids~ These bone-targeting molecules may bring the bone growth factors to the bone ~nd/or result in a synergistic or additive eff~ct on bone resorption or ~ormation.
The term "hydrophilic polymer" as used herein refers to a ~ynthetic or natural polymer having an average molecular weight and composition which renders the polymer essentially water-soluble. Most hydrophilic polymers achi~ve this property by incorporating a sufficient number of oxygen (or less frequently nitrogen) atoms available for ~orming hydrogen bonds in aqueous solution. Hydrophilic polymers used herein will generally be propylene glycol, polyoxyethylene, poly-ethylene glycol, polytrimethylene glycols, polylacticacid, or derivatives thereof. Other suitable polymers include polyoxyethylene-polyoxypropylene block polymers and copolymers. Naturally occurring polymers such as starch, heparin and the like are also included within the scope of this invention. All suitable polymers will be non-toxic and non-inflammatory when administered subcutaneously, and will preferably be essentially non-degradable!in VlVOi over a period of several months.
Presently preferred hydrophilic polymers are synthetic, ` 30 preferably polyethylene~glycols (PEG) having an average molecular weight between about 200 and about 10,000, more preferably between about 600 and about 8,000, and most preferably about 3400.
`~ The PEG is preferably difunctional, i.e., a ` 35 crosslinker between the bone growth factor and the ::;
. ~. .
WO92/20371 PCT/U~92/03840 ~ :I U i' .'~ 0 8 tar~eting molecule, consisting of homobifunctional groups such as in PEG 600 diglycidylether, or of heterofunctional groups. Branched polyfunctional PEG
crosslinkers may also be used. The nature of the functional groups on the crosslinker depends on the reactivities of the molecules to be conjugated.
The term "chemically conjugated" as used herein means attached through a covalent chemical bond. In the practice of the invention, a hydrophilic polymer and a bone growth factor may be ch~mically conjugated by using a linking radical, so that the polymer and the bone growth factor are each bound to the radical, but not directly to each other.
Those of ordinary skill in the art will appre-ciate that polymers such as polyethylene glycol cannotpractically be prepared ha~:ing exa~t molecular weights, and that the term "molecular weight" as used herein refeirs to the average mole~ular wei~ht o~ a number o~
molecules.in any given sample, as commonly used in the art. Thus, a sample of PEG 2000 might contain polymer molecules ranging in weight from, for example, 1,200 to :: 2,500 daltons. Specification of a molecular weight range indicates that the average molecular weight may~be any value between the limits specified, and may include molecules outside those limits. Thus, a molecular weight range of about 800 to about 20,000 indicates an averaqe molecular weight of at least about 800, ranging to about 20 kD.
The term "available lysine residue" as used herein reXers to lysine side chains exposed on the outer surface of the bone growth factor or targeting molecule, which are positioned in a~manner allowing reaction with activated PEG. : In general, 10-100%, and more preferably, 10-50% of available l~ysine residues may be used. The number of available lysine residues may be determined by 7! PCT/US92/03840 Z 10%~~ -12-reaction with sodium 2,4,6-trinitrobenzenesulfonate (TNBS). Bone growth factor genetic variants can be constructed so as to substitute lysine residues at specific sites in the molecule, thus impro~ing the efficiency of the crosslinking. In practice, such substitution will be conservative so as n~t to inter~ere with the receptor binding of growth factor and wi~l be within exterior or more hydrophilic regions of the growth factor.
The term "treat" or "treatment" as used herein refer to repair, prevention, or alleviation of bone defects, especially defects due to loss of bone.
Treatment of bone defects includes augmentation or restoration of lost bone as well as the. prevention of .
further bone loss due to metabolic dise~se, chronic ...
inflammatory processes (e.g., osteoporosis, osteoarthritis, Paget's disease, osteomalacia, osteoha}isteresis, multiple myeloma and other forms of cancer, and age-related loss of bone mass). Compositions of the invention may include additional biologically active factors to aid in healing or regrowth of normal tissue.
The term "tissue of interest" as used herein refers to a desired target~in the body for treatment or or~placement~of~the~bone growth factor. Tissues of interest may~inc~lude~;bone, cartilage, or other tissues or cell types to which`bone growth factors may be targeted.
B. General Methods The;`~composltions of the invention comprise a bone growth~factor chemicalIy conjugated to a targeting molecule. Examples of~;chemistries for conjugating or coupling proteins havè~been~summarized in the art (see, for example, P~.Tyl~e~and B. Ram, eds., Taraeted Therapeutic~Systems,~Marcel Dekker, New York, 1990; and ~........ :
WO92r20371 PCT/US92/03~0 2 1 ~ 8 Means and Feeney, "Chemical Modifications of Proteins, History and Applications," Bioconiuqate Chem. 1:2-12 (1990), hereby incorporated by reference in their entirety).
One such method includes chemical aonjugation of the bone growth factor to a selected synthetic hydrophilic polymer or polymers, which in turn i8 linked to a targ~ting molecule. Thus, for example, a reactive gro~p on the bone growth factor may be con~ugated through a crosslink~r to a polymer, and the targeting molecule may be conjugated to the bone growth facto~-polymer through a second crosslinker, where th~ second cross-inker may be of the same or different speci~s as the first, depending on the reactive groups involved in the targeting molecule~ Examples of reactive groups on proteins include but are not limited to the carboxyl groups of the C-terminus or OL aspartic and glutamic acid; amino groups of N-terminal and lysine residues;
imidazole of histidine and phenolic functions of tyrosine; sulfhydryl groups of cysteine; and guanidine ~groups of arginine.
For ~example, TGF-~ rontains a number of available ami~o, carboxyl, and hydroxy groups which may be used to bind the synthetic hydrophilic polymer. The polymer may be connected using a "linking group", as the native hydroxy~or amino groups in TGF-~ and in the polymer frequently~require activation before they can be linked~ One may thuslemploy compounds such as dicarb-oxylic anhydrides (e.g., glutaric or succinic anhydride) to form a polymer~deri~ative (e.g., succinate), which may then be activated by~e terification with a convPnient leaving group, for Pxample,: N-hydroxysuccinimide, N,N'-disuccinimidyl oxalate, N,N'-disuccinimidyl carbonate, and the like. Presently preferred dicarbox-ylic anhydrides~`that are used to form polymer-glutarate ~: ' WO92/20371 PCT/US92/0384~
~1~2808 compositions include glutaric anhydride, adipic anhy-dride, ~,8-naphthalene dicarboxylic anhydride, and 1,4,5,8-naphthalenetetracarboxylic dianhydride. The polymer thus activated is then allowed to react with the TGF-~, forming a TGF-~-polymer conjugate.
TGF-~ is difficult to dissol~e in solutions of appropriate pH for coupling to hydrophilic polym~rs. In a pres~ntly preferred mekh~d, the TGF-~ is lyophilized in the absence of a carrier protein, and dissolved in a mild acid, preferably about 10 mM HCl. The solution is neutralized by adding a strong baæe, preferably NaOH (1 N
solution) in bu~fered saline, additionally including a solubilizing amoun~ of ~MSO. The final solution preferably contains about 50% DMSO or CH3CN to solubil-ize the TGF-~ and to prevent aggregation.
In;one embodiment, monomethylpolyethylene glycol (mPEG) ~mw 5,000) is~reacted with glutaric anhydride to form mP~G qlutarate. The glutarate derivative is then reacted with N-hy~roxysuccinimide to form a succinim~idyl monomethylpolyethylene glycol glutarate. The succinimidyl~ester is then capablerof reacting with ~ree amino groups present on a bone growth faotor (lysine residues) to form a bone growth factor -PEG conjugate, which may then be further conjugated to a targeting molecule. Other p~lymers may be substituted for the mPEG, as described above. Similarly, the coupling reaction may be carried out using any known method for derivatizing proteins and synthetic polymers.
The activated~mPEG may be replaced, in whole or in part, by bifunctional~activated PEG (i.e., non-methylated PEG which is then activated at each end), - ~ thus providing a crosslinked or partially crosslinked bone~growth factor composition. The character of the compos~tion may be adjusted as desired, by varying the amount of bifunctional PEG included during the pro~ess.
.
` :
`:
WO92/20371 PCT/~S92/03~0 2~a2~8 The resulting bone growth factor-targeting molecule conjugates may be purified by standard techniques, pre~erably by reverse-phase HPLC ~RP-HPLC), size-exclusion HPLC ~SEC-HPLC; e.g., tetrahydrogel-HPLC) or ion-excha~ge chromatography. RP-HPLC i~ preferably performed using a C1~ column using 90% acetonitrile or 90% isopropanol with 0.1% trifluoroaceti~ acid (TFA) as the B solvent. For SEC-HPLC, a preferred runnîng buffer can be 5 mM sodium acetat~ at pH 5.5.
Whether a homobifunctional group or a heterobifunctional group is used depends on the nature of the reactive groups on the bone growth fac~ox ~nd the targeting molecule. For example, homobifunctional reagents may have the gen~ral formula R-PEG-R, where R is glycidylether(e.g., to form diglycidyl-PEG 600), succinimidyl succinate, p-nitrophenylcarbonate (e.g., to form bis(p-nitrophenylcarbonate) PEG3400, imidazoylcarbonyl, and the like. These and similar crosslinkers are available from Sigma, Union Carbide, and Polyscience. Shorter crosslinkers such as caxbodiimide, not containing the PEG bridging part, have well described chemistxies in the art and are available commercially ~e.g., Pierce).
The bone growth factor-targeting compositions of the invèntion are preferably administered by parenteral routes, intravenous injection, intranasal or bronchial aerosol, and the like. Sustained release de~ices may be surgicallylimplanted under the skin or within the peritoneal cavity.
Pharmaceutical formulations of the invention which include a bone~growth factor and a targeting molecule for administration will generally include an osteogenically effective amount of the bone growth factor to promote bone growth, in addition to a pharma-ceutlcally acceptable excipient. Suitable excipients WOg2/2~371 PCT/US92/03X40 2;1l~2~08 include most carriers approved for parenteral adminis-tration, including water, saline, Ringer's solution, Hank's solution, and solutions of glucose, lactose, dextrose, ethanol, glycerol, albumin, and the like.
These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives. The bone growth factor-targeting composition may also be delivered in a slow release form from a suitable carriex.
A presently preferred vehicle comprises about 1 mg/ml serum albumin (species-speci~ic) in phosphate-buf-fered saline (PBS) or isotonic citrate buffer. A
thorough discussion o~ suitable vehicles for parenteral administration may be found in E.W. Martin, "Remington's ~harmaceutical Sciences" (Mack Pub. Co., current edltion sections relating to the excipient vehicles and formu-lating being incorporated herein by reference to disclose such). Such formulations ~re generally known to those skilled in the art and are administered systemically to - 20 provide systemic treatment.
The "ef~ective amount" of bone growth factor ~-targeting composition necessary to effect treatment will depend upon the growth factor, the nature and severity o~
the condition to be treated, the age and general health of the subject, the specific activity of the composition prepared, and other factors which may be determined by the practitioner of ordinary skill in the art. As a general, gu~de,~ ho~ever,~an effective amount of bone growth factor-targeting molecule conjugate for treating a bone loss or defect will range from about 0.1 ~g/kg to about 25 ~g/kg. ~ ~ ~
The bone growth~factor - targeting compositions of the invention may be ~formulated as solutions or suspensions, or may ~e lyophilized for later reconstitution~ ~
WO92J20371 PCT/US92/03~0 C. Exam~les The examples presented below are pro~ided a~ a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the inventio~ in any way.
ExamPle_1 (TGF-~-PEG-Tetracycline co~jugate Preparation) Tetracycline ~2 ~mol; Aldrich Chem. Co.) i5 dissolved in 1 ~mol bis-epoxy-P~G (Polyscience) and reacted at 90C under nitrogen for 24 hr with gentle stirring. The resulting tetracycline-PEG conjugate is then reacted with TGF-~2 by adding 100 ~g TGF-~2 dissolved in 0.0~ M sodium borate, 0.02~ SDS, 50%
acetonitrile, pH g.0 to 52 ~l of the tetracycline-PEG
conjugate. The mixture is incubated at 37 C for 48 hr.
The resulting TGF-~-PEG-tetracycline conjugates are then - ~ purified by molecular sieve chromatography~and C1~-RP-HPLC.
, ExamPle II
(Conjugation of TGF-~2 to 3-amino-1-hydroxypropylidene-l,l-bisphosphonates IADP)~via bi~-epoxy PEG 600?.
~yophilized TGF-~is dissolved in 50%
water/acetonitri-e (final concentration 200-500 ~g/ml).
The pH is adjusted to 7-8 with phosphate or borate buffer.~ A~ 0! maIar excess of bis-epoxy~PEG 600 or tetra-epoxy PEG 1700 (Polyscience) is added and the mixture is reacted;for 15-20 hours at pH 7.0-B.0 at 40C.
Amicon ultrafiltration~may~be used to remove unattached PEG. The pH is raised to 9.5 with 1% deoxycholate, pH
9.5, 0.0l N NaOH, or 0.~1 M borate buffer. The 3-amino 1-hydroxypropylidene 1,1-bisphosphonate (APD, CIBA-GEIGY) i`s dissolved in 1% deoxycholate, pH 9.5, at 2 mg/ml. The : , .
~ , r~, ~. ~r ~ ~
2i~Q8 bisphosphonate is then added in a 5-200 molar excess and stirred overnight at 40C. The reaction mixture is acidified and filtered through a 0.45 ~m low protein binding syringe filter prior to chromatography.
Purification is performed by gel filtration, ion exchange (Mono-S) and reverse-phase HPLC.
Exam~le III
~Conjugation of TGF-~ to 3-amino-1-hydroxypropylidene-1,1-bisphosphonate ~APD) carbodiimide) Lyophilized TGF-~ ~200 ~g) is dissolved in 0.1 M NES (2-(N-Morpholino) ethanesul~onic acid) buffer, 50%
acetonitrile, at pH 4.7. Sulfo-NHS (N-hydroxysulfosuccinimide, Pierce) is dissolved in aqueous 50% acetonitrile and 2-10 ~moles are added to the TGF-~
solution. The water soluble l-ethyl-3-~3-dimethylaminopropyl)carbodiimide HCL (EDC, Pierce) is dissolved in 0.1 MES buffer, pH 4.7~ and added in 2-50 molar amounts. Finally, the bisphosphonake (ADP, C}BA-GEIGY) dissolved in 0.1 N HCl ~5 mg/100 ~1) is added in 2-5Q ~molar amounts. The pH is adjusted to 4.7-5.5 wit~h 0.01 N NaOH.~ The mixture is reacted overnight at room temperature. The TGF-~/APD conjugate is purified by ion-exchange (Mono-S) chromatography and reverse-phase HPLC.
Example IV
(Conjugation of TGF-~ to Tetracycline (Einhorn Reaction)) Tetracycline;dissolved in methanol is reacted with 38% formaldehyde. TGF-~ dissolved in a 1:5 solution of water:methanol is added dropwise. The reaction mixture is sti~rred gent~y for 2 hr at room temperature.
The resulting TGF-~-N-lysinemethyltetracycline can be separated from other reaction products by size-exclusion and RP-HP~C. (Bernardelli, et al. Liebiqs Ann. Chem.
706:243-249 (1967)).
; .
WO92/20371 PCT/US92/03~0 2 1 (~ 9-Example V
(Conjugation of TGF-~ to Tetracycline) Alkoxyalkyltetracycline~ can be obtained by condensing a tetracycline with an aldehyde in alcohol.
For example, tetracycline, mono-hydroxy-methoxy ~EG-2000 ~MeOoPEG 2000-OH), and formalin are added in equimolar amounts to a round bottom ~lask ~itted with a reflux condenser. The mixture is dissolved in tert butyl alcohol by heating to approximately 80C ~oil bath). The temperature is increased to 115C and held for 50 houræ.
This reaction is conducted under nitrogen. The system is then attached to a water aspirator to remove water from the reaction mixture, after which the mixture is allowed to cool to room temperature. Ethanol is added to dissolve the tert butyl alcohol and the miXture is f~ltered. The remaining wine-red precipitate is ~urther purified by acetone extrac~ion. The so-ob~ained intermediate can be further purified by ion exchange and siz~ exclusion chromatography. After activation, the tetracycline-~EG conjugate can then be coupled to the TGF-~, preferably in an organic solvent ~uch as methylene chloride, dimethylformamide, D~SO, and the like.
~;
.
~ 30 .: ~
~ ' ~
:
:
("BMP-4"), that are alleged to have osteogenic activity.
: It is not known! whether t'hese BMPs have oste~genic activity by themselves or require combination with other factors.
J.M. Wozney, in Growth Factor Research, Vol. l (1989):, pp. 267-280, describes three additi~nal BMP
proteins closely~related to BMP-2, and which have been ~ designated BMP-5, BMP-6 and BMP-7.
: 35 Wo 89/09787 and 89/09788 describe a protein .
WO92t20371 PCT/US92/03~0 210~81~
called "OP-1", now known to be BMP-7. The cloning of BMP-7 is described in E. Ozkaynak et al., EMBO Journal (19g0) 2:2085-2093, and the purification of BMP-7 is de~cribed in T.K. Sampath et al., J. Biol. Chem. (1990) 265:131g8-13205.
U.S. Patent No. 4,434,094 to Seyedin and Th~mas reported the partial puri~ication o~ a bone generation-stimulating, bone-derived protein by extraction with chaotropic agents, fractionation on anion and cation exchange columns, and recovery of the activity ~rom a fraction adsorbed to CMC at pH 4.8. This new protein fraction was termed "osteogenia factor" (OF) and was characterized as having a molecular weight ~elow about 30,000 daltons.
There are several re~erences in the art to proteins modified by covalent conjuga~ion to polymers, to alter the solubility, antigenicity and biological clearance of the protein. For ~xample, U.S. P~tent No.
4,261,973 disclosed the conjugation of several allergens to PEG (polyethylene glycol) or PPG (polypropylene gly-col) to reduce the proteins' immunogenicity. U.S. Pate~t ~o. 4,301,144 disclosed the conjugation of hemoglobin with PEG and other polymers to in~rease the protein's oxygen carrying capability. EPO 98,110 disclosed coupling an enzyme or interferon to a polyoxyethylene-polyoxypropylene (POE-POP) block polymer increases the protein's halflife in serum. U.S. Patent No. 4,179,337 disclosed qonjugat.Lng hydrophilic enzymes and însulin to PEG or PPG to reduce immunogenicity.
Davis et al., Lancet (1981) 2:281~83 disclosed the enzyme ~; uricase modified by conjugation with PEG to provide uric acid metabolism in serum having a long halflife and low immunogenicity. Nishida et al., J. Pharm. Pharmacol.
~1984) _ :354-55 disclosed PEG-uricase conjugates administered orally to chickens, demonstrating decreased WOs2/20371 PCT/US92/03~0 2 1 ~ 8 serum levels of uric acid. Inada et al., Biochem.
Biophys. Res. Comm. (1984) 122:845-50 disclosed lipoprotein lipase conjugation with PEG to render it soluble in organic solvents. Takahashi et al., Biochem.
& Biophys. Res. Comm. (1984) 121:261-65 disclosed HRP
conjugated with PEG to render the enzyme soluble in benzene. Abuchowski et al., Cancer Biochem. BiQphvs.
(1984) 7:175-86, disclosed that enzymes such as asparaginase, catalase, uricase, arg4nase, trypsin, superoxide dismutase, adenosine deaminase, phenylalanine ammonia-lyase, and the like, conjugated with PEG exhibit longer half-lives in serum and decreased immunogenicity.
U.S. Patent No. 4,830,847, and European Patent Application 5er. No. Z07,557 disclose diphosphonate-derivatized macromolecules which may be labeled withtechnium-99m Both antibodies and liposomes have been used to direct drugs to target sites ~see, for example, Tyle and Ram, eds., Tarqeted Thera~eutic Svstems, Marcel Dekker, New York, 1990.) Tarqeted delivery of bone growth factors may ~
reduce harmful or~undesirable effects of those molecules, allow the use of lower doses because relatively~higher doses can be delivered to t~e site of interest, and may prolong the effect at ~ the site of interest. In addition, the use of a targeting molecule that influences bone metabolism, lncluding bone resorption and formation, may result in an additive orlsynergistic effeqt.
:
:30 summarY of the Invention~
It~is an object of the invention to provide a composition c~omprising a~bone growth factor and a ` targeting molecule having affinity for a tissue of interest, where~th~e;bone growth factor and targeting molecule are chemically conjugated to a crosslinker. The :~ -:: : ` ~ : :
. . .
WO 92/20371 PCI'/US92/03840 ~ 1 0 2 ~ D8 crosslinker is preferablyi a synthetic hydrophilic polymer. The bone yrowth factor is preferably TGF-~, activin, or bone morphogenic protein ~BMP). Targeting molecules preferably have an affinity for bone, such as tetracycline, calcein, bisphosphonate, polyaspartic acid, polyglutamic a~id, aminophosphosugars, or eætrogen.
It is another object of the invention to pro~ide a method for augmenting bone formation in a sub;ect.
It i5 another object of the invention to provide a method for treating bone loss in a subject.
Modes of Carrvinq Out The Invention A. Definitions The term "bone growth factor" refers to the families of proteins which affect bone formation.
Examples of bone growth faotors include transforming growth factor-beta tTGF-~), activin, and bone morphogenic : 20 factor (BMP).
The term "TGF-~I' refers to beta-type trans-forming growth factors, including TGF-~1, TGF-~2, TGF-~3, TGF-~4, TGF-~5, heterodimers of the TGF-~ polypeptide chains (e.g.~ TGF-~1.2), and ~ragmen~s thereof, synthetic.
peptides, and homologous proteins having substantially : equivalent biological activity in any of the numerous known TGF-~ assays, such as the one described in Methods for Preparation of Med~ia! SuPplements and Substrate for Serum-free Animal Cell Culture (1984) pp. 181-194. Alan R. Liss, Inc. This particular assay determines ability : to induce anchorage-dependent growth in non-neoplastic normal rat kidney~(NRK) fibroblasts by measuring the ~` formation of cell;colonies in soft agar. Other known TGF-~ activity assays include but are not limited to stimulation of osteoblast proliferation, inhibition of W0~2/~0371 PCT~US92/03~0 ~1 02808 -8-keratinocyte proliferation, stimulation of collagen synthesis in a variety of cells, inhibition of mitogen-stimulated T-cell proliferation, and stimulation of chemotactic activity. Preparation of TGF-~l and TGF-~2 is described in U.S~ Patent No. 4,774,322, incorporated herein by reference. Additional TGF-Bs have also been described. U.S. Patent No. 4,886,747 describes the identification of TGF-~3 and its nucleotide sequence, and describes a method for recovery of TGF-~ from recombinant cell cultures. S. B. Jakowlew et al., Molec~ Endocrinol.
(1988) 2:1186-1195, describes TGF-~4 and its nucleotide sequence, ldentified by cDNA characterization. A.B.
Roberts et al., Growth F~ctors, Vol. 2 (1990) pp. 135-147, describes the purification of T5F-B5 from Xenopus-conditioned medium.
The term "TGF-~" as used herein is also intended to include the heterodimer TGF-~2 . 3, disclosed in copending patent application U.S. Ser. No. 614,306, filed 11/16/9o. T~F-~2.3 may be prepared from an extract of bone by pooling side fractions from peaks of column chromatography, subjecting those fractions to reverse phase HPLC and recovering those fraction which migrate more slowly than TGF-~2 by SDS-PAGE, subjecting those slower migrating fractions to FPLC and recovering those that migrate during a~pH 4.6 to 6.7 gradient, subjecting the pH 4.6 to 6.7 eluant to reverse phase HPLC or gel electrophoresis, and recovering substantially pure TGF-~2.3. ~ j ~
The term l'TGF-~" as used herein is also intended to include any other~synthetic molecule whose mechanism of action is mediated through the TGF-~receptor or second messenger~pathway.
Because these proteins are non-species-specific in their activity they maybe used to treat su~jects in general, lncluding sport, pet, and farm animals, and WO92/Z0371 PCr/US92/03~0 2 ~
g humans.
As used herein, "bone morphogenetic proteinl' (BMP) refers to a class of bone-derived proteins capable of inducing bone formation, as measured by activity in an 5 in vivo rat bone formation assay. BMPs include BMP-1, B~P-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, and ~ragments, deletions, additions, substitutions, mutations and modifications thereof which retain the biological c~aracteristics of the na~ural BM~. The BMP may be of human, bovine, or other species origin.
The term "activin" as used herein refers to activin ~AA, activin ~AB, activin ~BB, and ~ragments thereof, synthetic peptides, and proteins having similar acti~ity in a standard cell culture assay where the protein stimulates the release of ~ollicular stimulating hormone (FSH) from rat pituitary cells (Vale, et al., Nature (1986) ~ 776). Briefly, in this assay, anterior pituitaries from adult male Sprague-Dawley rats (200-220 g) are dissociated by collagenase and plated at a concentration of 0.33 X 1o6 cells per well in 24 well tissue culture dishes. The cells are allowed to recover for 72 hr in medium containing 2% fetal bovine serum (FBS). Following the reco~ery period, the cells are washed twice in fresh medium containing 2% FBS. All t eatments are added at thîs time and the cells are incubated for 72 hr. The media i5 then collected and the FSH levels are determined using a radioimmunoassay kit provided~by the NationallHormone and Pituitary program of NlADDK.
` As used hereinj the~term "targeting molecule"
refers to a molecule that binds to the tissue of interest or that influences metabolism of that tissue of interest.
So, for example, bone targeting molecules may include bone-seeking molecules such as tetracycline, calcein, ~5 bisphosphonates, chelators, phosphates, polyaspartic .
21 ~2~û8 -lo-a~id, polyglutamic acid, aminophosphosugars, peptides known to be associaked with the mineral phase of bone such as osteonectin, bone sialoprotein and osteopontin, proteins with bone mineral binding domains, and the like.
- 5 Bone-targeting molecules may also include molecules which affect bone resorption and bone formation ra~es, such as bisphosphonates, and estrogens and other steroids~ These bone-targeting molecules may bring the bone growth factors to the bone ~nd/or result in a synergistic or additive eff~ct on bone resorption or ~ormation.
The term "hydrophilic polymer" as used herein refers to a ~ynthetic or natural polymer having an average molecular weight and composition which renders the polymer essentially water-soluble. Most hydrophilic polymers achi~ve this property by incorporating a sufficient number of oxygen (or less frequently nitrogen) atoms available for ~orming hydrogen bonds in aqueous solution. Hydrophilic polymers used herein will generally be propylene glycol, polyoxyethylene, poly-ethylene glycol, polytrimethylene glycols, polylacticacid, or derivatives thereof. Other suitable polymers include polyoxyethylene-polyoxypropylene block polymers and copolymers. Naturally occurring polymers such as starch, heparin and the like are also included within the scope of this invention. All suitable polymers will be non-toxic and non-inflammatory when administered subcutaneously, and will preferably be essentially non-degradable!in VlVOi over a period of several months.
Presently preferred hydrophilic polymers are synthetic, ` 30 preferably polyethylene~glycols (PEG) having an average molecular weight between about 200 and about 10,000, more preferably between about 600 and about 8,000, and most preferably about 3400.
`~ The PEG is preferably difunctional, i.e., a ` 35 crosslinker between the bone growth factor and the ::;
. ~. .
WO92/20371 PCT/U~92/03840 ~ :I U i' .'~ 0 8 tar~eting molecule, consisting of homobifunctional groups such as in PEG 600 diglycidylether, or of heterofunctional groups. Branched polyfunctional PEG
crosslinkers may also be used. The nature of the functional groups on the crosslinker depends on the reactivities of the molecules to be conjugated.
The term "chemically conjugated" as used herein means attached through a covalent chemical bond. In the practice of the invention, a hydrophilic polymer and a bone growth factor may be ch~mically conjugated by using a linking radical, so that the polymer and the bone growth factor are each bound to the radical, but not directly to each other.
Those of ordinary skill in the art will appre-ciate that polymers such as polyethylene glycol cannotpractically be prepared ha~:ing exa~t molecular weights, and that the term "molecular weight" as used herein refeirs to the average mole~ular wei~ht o~ a number o~
molecules.in any given sample, as commonly used in the art. Thus, a sample of PEG 2000 might contain polymer molecules ranging in weight from, for example, 1,200 to :: 2,500 daltons. Specification of a molecular weight range indicates that the average molecular weight may~be any value between the limits specified, and may include molecules outside those limits. Thus, a molecular weight range of about 800 to about 20,000 indicates an averaqe molecular weight of at least about 800, ranging to about 20 kD.
The term "available lysine residue" as used herein reXers to lysine side chains exposed on the outer surface of the bone growth factor or targeting molecule, which are positioned in a~manner allowing reaction with activated PEG. : In general, 10-100%, and more preferably, 10-50% of available l~ysine residues may be used. The number of available lysine residues may be determined by 7! PCT/US92/03840 Z 10%~~ -12-reaction with sodium 2,4,6-trinitrobenzenesulfonate (TNBS). Bone growth factor genetic variants can be constructed so as to substitute lysine residues at specific sites in the molecule, thus impro~ing the efficiency of the crosslinking. In practice, such substitution will be conservative so as n~t to inter~ere with the receptor binding of growth factor and wi~l be within exterior or more hydrophilic regions of the growth factor.
The term "treat" or "treatment" as used herein refer to repair, prevention, or alleviation of bone defects, especially defects due to loss of bone.
Treatment of bone defects includes augmentation or restoration of lost bone as well as the. prevention of .
further bone loss due to metabolic dise~se, chronic ...
inflammatory processes (e.g., osteoporosis, osteoarthritis, Paget's disease, osteomalacia, osteoha}isteresis, multiple myeloma and other forms of cancer, and age-related loss of bone mass). Compositions of the invention may include additional biologically active factors to aid in healing or regrowth of normal tissue.
The term "tissue of interest" as used herein refers to a desired target~in the body for treatment or or~placement~of~the~bone growth factor. Tissues of interest may~inc~lude~;bone, cartilage, or other tissues or cell types to which`bone growth factors may be targeted.
B. General Methods The;`~composltions of the invention comprise a bone growth~factor chemicalIy conjugated to a targeting molecule. Examples of~;chemistries for conjugating or coupling proteins havè~been~summarized in the art (see, for example, P~.Tyl~e~and B. Ram, eds., Taraeted Therapeutic~Systems,~Marcel Dekker, New York, 1990; and ~........ :
WO92r20371 PCT/US92/03~0 2 1 ~ 8 Means and Feeney, "Chemical Modifications of Proteins, History and Applications," Bioconiuqate Chem. 1:2-12 (1990), hereby incorporated by reference in their entirety).
One such method includes chemical aonjugation of the bone growth factor to a selected synthetic hydrophilic polymer or polymers, which in turn i8 linked to a targ~ting molecule. Thus, for example, a reactive gro~p on the bone growth factor may be con~ugated through a crosslink~r to a polymer, and the targeting molecule may be conjugated to the bone growth facto~-polymer through a second crosslinker, where th~ second cross-inker may be of the same or different speci~s as the first, depending on the reactive groups involved in the targeting molecule~ Examples of reactive groups on proteins include but are not limited to the carboxyl groups of the C-terminus or OL aspartic and glutamic acid; amino groups of N-terminal and lysine residues;
imidazole of histidine and phenolic functions of tyrosine; sulfhydryl groups of cysteine; and guanidine ~groups of arginine.
For ~example, TGF-~ rontains a number of available ami~o, carboxyl, and hydroxy groups which may be used to bind the synthetic hydrophilic polymer. The polymer may be connected using a "linking group", as the native hydroxy~or amino groups in TGF-~ and in the polymer frequently~require activation before they can be linked~ One may thuslemploy compounds such as dicarb-oxylic anhydrides (e.g., glutaric or succinic anhydride) to form a polymer~deri~ative (e.g., succinate), which may then be activated by~e terification with a convPnient leaving group, for Pxample,: N-hydroxysuccinimide, N,N'-disuccinimidyl oxalate, N,N'-disuccinimidyl carbonate, and the like. Presently preferred dicarbox-ylic anhydrides~`that are used to form polymer-glutarate ~: ' WO92/20371 PCT/US92/0384~
~1~2808 compositions include glutaric anhydride, adipic anhy-dride, ~,8-naphthalene dicarboxylic anhydride, and 1,4,5,8-naphthalenetetracarboxylic dianhydride. The polymer thus activated is then allowed to react with the TGF-~, forming a TGF-~-polymer conjugate.
TGF-~ is difficult to dissol~e in solutions of appropriate pH for coupling to hydrophilic polym~rs. In a pres~ntly preferred mekh~d, the TGF-~ is lyophilized in the absence of a carrier protein, and dissolved in a mild acid, preferably about 10 mM HCl. The solution is neutralized by adding a strong baæe, preferably NaOH (1 N
solution) in bu~fered saline, additionally including a solubilizing amoun~ of ~MSO. The final solution preferably contains about 50% DMSO or CH3CN to solubil-ize the TGF-~ and to prevent aggregation.
In;one embodiment, monomethylpolyethylene glycol (mPEG) ~mw 5,000) is~reacted with glutaric anhydride to form mP~G qlutarate. The glutarate derivative is then reacted with N-hy~roxysuccinimide to form a succinim~idyl monomethylpolyethylene glycol glutarate. The succinimidyl~ester is then capablerof reacting with ~ree amino groups present on a bone growth faotor (lysine residues) to form a bone growth factor -PEG conjugate, which may then be further conjugated to a targeting molecule. Other p~lymers may be substituted for the mPEG, as described above. Similarly, the coupling reaction may be carried out using any known method for derivatizing proteins and synthetic polymers.
The activated~mPEG may be replaced, in whole or in part, by bifunctional~activated PEG (i.e., non-methylated PEG which is then activated at each end), - ~ thus providing a crosslinked or partially crosslinked bone~growth factor composition. The character of the compos~tion may be adjusted as desired, by varying the amount of bifunctional PEG included during the pro~ess.
.
` :
`:
WO92/20371 PCT/~S92/03~0 2~a2~8 The resulting bone growth factor-targeting molecule conjugates may be purified by standard techniques, pre~erably by reverse-phase HPLC ~RP-HPLC), size-exclusion HPLC ~SEC-HPLC; e.g., tetrahydrogel-HPLC) or ion-excha~ge chromatography. RP-HPLC i~ preferably performed using a C1~ column using 90% acetonitrile or 90% isopropanol with 0.1% trifluoroaceti~ acid (TFA) as the B solvent. For SEC-HPLC, a preferred runnîng buffer can be 5 mM sodium acetat~ at pH 5.5.
Whether a homobifunctional group or a heterobifunctional group is used depends on the nature of the reactive groups on the bone growth fac~ox ~nd the targeting molecule. For example, homobifunctional reagents may have the gen~ral formula R-PEG-R, where R is glycidylether(e.g., to form diglycidyl-PEG 600), succinimidyl succinate, p-nitrophenylcarbonate (e.g., to form bis(p-nitrophenylcarbonate) PEG3400, imidazoylcarbonyl, and the like. These and similar crosslinkers are available from Sigma, Union Carbide, and Polyscience. Shorter crosslinkers such as caxbodiimide, not containing the PEG bridging part, have well described chemistxies in the art and are available commercially ~e.g., Pierce).
The bone growth factor-targeting compositions of the invèntion are preferably administered by parenteral routes, intravenous injection, intranasal or bronchial aerosol, and the like. Sustained release de~ices may be surgicallylimplanted under the skin or within the peritoneal cavity.
Pharmaceutical formulations of the invention which include a bone~growth factor and a targeting molecule for administration will generally include an osteogenically effective amount of the bone growth factor to promote bone growth, in addition to a pharma-ceutlcally acceptable excipient. Suitable excipients WOg2/2~371 PCT/US92/03X40 2;1l~2~08 include most carriers approved for parenteral adminis-tration, including water, saline, Ringer's solution, Hank's solution, and solutions of glucose, lactose, dextrose, ethanol, glycerol, albumin, and the like.
These compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives. The bone growth factor-targeting composition may also be delivered in a slow release form from a suitable carriex.
A presently preferred vehicle comprises about 1 mg/ml serum albumin (species-speci~ic) in phosphate-buf-fered saline (PBS) or isotonic citrate buffer. A
thorough discussion o~ suitable vehicles for parenteral administration may be found in E.W. Martin, "Remington's ~harmaceutical Sciences" (Mack Pub. Co., current edltion sections relating to the excipient vehicles and formu-lating being incorporated herein by reference to disclose such). Such formulations ~re generally known to those skilled in the art and are administered systemically to - 20 provide systemic treatment.
The "ef~ective amount" of bone growth factor ~-targeting composition necessary to effect treatment will depend upon the growth factor, the nature and severity o~
the condition to be treated, the age and general health of the subject, the specific activity of the composition prepared, and other factors which may be determined by the practitioner of ordinary skill in the art. As a general, gu~de,~ ho~ever,~an effective amount of bone growth factor-targeting molecule conjugate for treating a bone loss or defect will range from about 0.1 ~g/kg to about 25 ~g/kg. ~ ~ ~
The bone growth~factor - targeting compositions of the invention may be ~formulated as solutions or suspensions, or may ~e lyophilized for later reconstitution~ ~
WO92J20371 PCT/US92/03~0 C. Exam~les The examples presented below are pro~ided a~ a further guide to the practitioner of ordinary skill in the art, and are not to be construed as limiting the inventio~ in any way.
ExamPle_1 (TGF-~-PEG-Tetracycline co~jugate Preparation) Tetracycline ~2 ~mol; Aldrich Chem. Co.) i5 dissolved in 1 ~mol bis-epoxy-P~G (Polyscience) and reacted at 90C under nitrogen for 24 hr with gentle stirring. The resulting tetracycline-PEG conjugate is then reacted with TGF-~2 by adding 100 ~g TGF-~2 dissolved in 0.0~ M sodium borate, 0.02~ SDS, 50%
acetonitrile, pH g.0 to 52 ~l of the tetracycline-PEG
conjugate. The mixture is incubated at 37 C for 48 hr.
The resulting TGF-~-PEG-tetracycline conjugates are then - ~ purified by molecular sieve chromatography~and C1~-RP-HPLC.
, ExamPle II
(Conjugation of TGF-~2 to 3-amino-1-hydroxypropylidene-l,l-bisphosphonates IADP)~via bi~-epoxy PEG 600?.
~yophilized TGF-~is dissolved in 50%
water/acetonitri-e (final concentration 200-500 ~g/ml).
The pH is adjusted to 7-8 with phosphate or borate buffer.~ A~ 0! maIar excess of bis-epoxy~PEG 600 or tetra-epoxy PEG 1700 (Polyscience) is added and the mixture is reacted;for 15-20 hours at pH 7.0-B.0 at 40C.
Amicon ultrafiltration~may~be used to remove unattached PEG. The pH is raised to 9.5 with 1% deoxycholate, pH
9.5, 0.0l N NaOH, or 0.~1 M borate buffer. The 3-amino 1-hydroxypropylidene 1,1-bisphosphonate (APD, CIBA-GEIGY) i`s dissolved in 1% deoxycholate, pH 9.5, at 2 mg/ml. The : , .
~ , r~, ~. ~r ~ ~
2i~Q8 bisphosphonate is then added in a 5-200 molar excess and stirred overnight at 40C. The reaction mixture is acidified and filtered through a 0.45 ~m low protein binding syringe filter prior to chromatography.
Purification is performed by gel filtration, ion exchange (Mono-S) and reverse-phase HPLC.
Exam~le III
~Conjugation of TGF-~ to 3-amino-1-hydroxypropylidene-1,1-bisphosphonate ~APD) carbodiimide) Lyophilized TGF-~ ~200 ~g) is dissolved in 0.1 M NES (2-(N-Morpholino) ethanesul~onic acid) buffer, 50%
acetonitrile, at pH 4.7. Sulfo-NHS (N-hydroxysulfosuccinimide, Pierce) is dissolved in aqueous 50% acetonitrile and 2-10 ~moles are added to the TGF-~
solution. The water soluble l-ethyl-3-~3-dimethylaminopropyl)carbodiimide HCL (EDC, Pierce) is dissolved in 0.1 MES buffer, pH 4.7~ and added in 2-50 molar amounts. Finally, the bisphosphonake (ADP, C}BA-GEIGY) dissolved in 0.1 N HCl ~5 mg/100 ~1) is added in 2-5Q ~molar amounts. The pH is adjusted to 4.7-5.5 wit~h 0.01 N NaOH.~ The mixture is reacted overnight at room temperature. The TGF-~/APD conjugate is purified by ion-exchange (Mono-S) chromatography and reverse-phase HPLC.
Example IV
(Conjugation of TGF-~ to Tetracycline (Einhorn Reaction)) Tetracycline;dissolved in methanol is reacted with 38% formaldehyde. TGF-~ dissolved in a 1:5 solution of water:methanol is added dropwise. The reaction mixture is sti~rred gent~y for 2 hr at room temperature.
The resulting TGF-~-N-lysinemethyltetracycline can be separated from other reaction products by size-exclusion and RP-HP~C. (Bernardelli, et al. Liebiqs Ann. Chem.
706:243-249 (1967)).
; .
WO92/20371 PCT/US92/03~0 2 1 (~ 9-Example V
(Conjugation of TGF-~ to Tetracycline) Alkoxyalkyltetracycline~ can be obtained by condensing a tetracycline with an aldehyde in alcohol.
For example, tetracycline, mono-hydroxy-methoxy ~EG-2000 ~MeOoPEG 2000-OH), and formalin are added in equimolar amounts to a round bottom ~lask ~itted with a reflux condenser. The mixture is dissolved in tert butyl alcohol by heating to approximately 80C ~oil bath). The temperature is increased to 115C and held for 50 houræ.
This reaction is conducted under nitrogen. The system is then attached to a water aspirator to remove water from the reaction mixture, after which the mixture is allowed to cool to room temperature. Ethanol is added to dissolve the tert butyl alcohol and the miXture is f~ltered. The remaining wine-red precipitate is ~urther purified by acetone extrac~ion. The so-ob~ained intermediate can be further purified by ion exchange and siz~ exclusion chromatography. After activation, the tetracycline-~EG conjugate can then be coupled to the TGF-~, preferably in an organic solvent ~uch as methylene chloride, dimethylformamide, D~SO, and the like.
~;
.
~ 30 .: ~
~ ' ~
:
:
Claims (46)
1. A composition comprising a bone growth factor and a targeting molecule having affinity for a tissue of interest, wherein said bone growth factor and targeting molecule are chemically conjugated to a crosslinker.
2. The composition of claim 1, wherein the bone growth factor is TGF-.beta..
3. The composition of claim 1, wherein the bone growth factor is an activin.
4. The composition of claim 1, wherein the bone growth factor is a bone morphogenic protein (BMP).
5. The composition of claim 1, wherein the crosslinker is a synthetic hydrophilic polymer.
6. The composition of claim 1, wherein the crosslinker is polyfunctional.
7. The composition of claim 5, wherein said polymer is polyethylene glycol.
8. The composition of claim 7, wherein said polyethylene glycol has a molecular weight of about 200 to about 10,000.
9. The composition of claim 5, wherein said synthetic hydrophilic polymer is bound to an available lysine residue on said bone growth factor.
10. The composition of claim 5, wherein said growth factor has a plurality of available lysine residues and molecules of said synthetic hydrophilic polymer are bound to 10-100% of said available lysine residues.
11. The composition of claim 5, wherein said growth factor has a plurality of available lysine residues and molecules of said synthetic hydrophilic polymer are bound to 10-50% of said available lysine residues.
12. The composition of claim 1, wherein said targeting molecule has an affinity for bone.
13. The composition of claim 1, wherein said targeting molecule is a protein.
14. The composition of claim 1, wherein said targeting molecule is tetracycline.
15. The composition of claim 1, wherein said targeting molecule is calcein.
16. The composition of claim 1, wherein said targeting molecule is a bisphosphonate.
17. The composition of claim 1, wherein said targeting molecule is polyaspartic acid.
18. The composition of claim 1, wherein said targeting molecule is polyglutamic acid.
19. The targeting molecule of claim 1, wherein said targeting molecule is an aminophosphosugar.
20. The targeting molecule of claim 1, wherein said targeting molecule is a steroid.
21. The targeting molecule of claim 1, wherein said targeting molecule is an estrogen.
22. A method for augmenting the formation of mature bone comprising administering an effective amount of the composition of claim 1 in a pharmaceutically acceptable carrier.
23. A method for treating bone loss in a subject, comprising administering the composition of claim 1 to the subject.
24. The method of claim 23, wherein said bone growth factor is TGF-.beta..
25. The method of claim 23, wherein said bone growth factor is an activin.
26. The method of claim 23, wherein said bone growth factor is a bone morphogenic protein (BMP).
27. The composition of claim 23, wherein said targeting molecule has an affinity for bone.
28. The method of claim 23, wherein said targeting molecule is tetracycline.
29. The method of claim 23, wherein said targeting molecule is a calcein.
30. The method of claim 23, wherein said targeting molecule is a bisphosphonate.
31. The method of claim 23, wherein said targeting molecule is polyaspartic acid.
32. The method of claim 23, wherein said targeting molecule is polyglutamic acid.
33. The method of claim 23, wherein said targeting molecule is an aminophosphosugar.
34. The method of claim 23, wherein said targeting molecule is an estrogen.
35. The method of claim 23, wherein the subject is human.
36. The method of claim 23, wherein the bone loss is osteoporosis-related.
37. The method of claim 23, wherein the bone loss is osteomalacia-related.
38. The method of claim 23, wherein the bone loss is osteohalisteresis-related.
39. The method of claim 23, wherein the bone loss is age-related.
40. The method of claim 23, wherein the bone.
loss is related to steroid therapy.
loss is related to steroid therapy.
41. The method of claim 23, wherein the bone loss is osteoarthritis-related.
42. The method of claim 23, wherein the bone loss is related to Paget's disease.
43. The method of claim 23, wherein the bone loss is related to cancer.
44. The method of claim 40, wherein the cancer in multiple myeloma.
45. The method of claim 23, wherein the treatment is prophylactic.
46. The method of claim 23, wherein the composition contains a sustained-release vehicle.
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| US69846791A | 1991-05-10 | 1991-05-10 | |
| US698,467 | 1991-05-10 |
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| CA2102808A1 true CA2102808A1 (en) | 1992-11-11 |
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| CA002102808A Abandoned CA2102808A1 (en) | 1991-05-10 | 1992-05-07 | Targeted delivery of bone growth factors |
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| JP (1) | JPH07500315A (en) |
| AT (1) | ATE156365T1 (en) |
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Families Citing this family (62)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5766897A (en) * | 1990-06-21 | 1998-06-16 | Incyte Pharmaceuticals, Inc. | Cysteine-pegylated proteins |
| US5674844A (en) * | 1991-03-11 | 1997-10-07 | Creative Biomolecules, Inc. | Treatment to prevent loss of and/or increase bone mass in metabolic bone diseases |
| US7056882B2 (en) | 1991-03-11 | 2006-06-06 | Curis, Inc. | Treatment to prevent loss of and/or increase bone mass in metabolic bone diseases |
| CA2116559C (en) * | 1991-08-30 | 2005-05-10 | Thangavel Kuberasampath | Treatment to prevent loss of and/or increase bone mass in metabolic bone diseases |
| EP0612348B1 (en) | 1991-11-04 | 2003-04-23 | Genetics Institute, LLC | Recombinant bone morphogenetic protein heterodimers, compositions and methods of use |
| IL106159A0 (en) * | 1992-06-30 | 1993-10-20 | Dow Chemical Co | Targeted delivery of growth factors for bone regeneration |
| EP2272959A1 (en) | 1993-05-12 | 2011-01-12 | Genetics Institute, LLC | BMP-11 compositions |
| DE4420157B4 (en) * | 1993-08-10 | 2013-04-25 | Biopharm Gesellschaft Zur Biotechnologischen Entwicklung Von Pharmaka Mbh | New growth / differentiation factor of the TGF-β family |
| JP3871713B2 (en) | 1995-05-10 | 2007-01-24 | 協和醗酵工業株式会社 | Novel toxin complex |
| US5776193A (en) | 1995-10-16 | 1998-07-07 | Orquest, Inc. | Bone grafting matrix |
| US6902584B2 (en) | 1995-10-16 | 2005-06-07 | Depuy Spine, Inc. | Bone grafting matrix |
| JPH09143093A (en) * | 1995-11-17 | 1997-06-03 | Hoechst Japan Ltd | Cartilage/bone-inductive restoring material |
| DE69740089D1 (en) | 1996-03-22 | 2011-02-10 | Stryker Corp | Method for improved functional recovery of motor coordination, language or sensation after trauma or ischemia of the CNS |
| US6492508B1 (en) | 1996-06-03 | 2002-12-10 | United States Surgical Corp. A Division Of Tyco Healthcare Group | Nucleic acids encoding extracellular matrix proteins |
| EP0981376A1 (en) | 1997-02-13 | 2000-03-01 | Bone Care International, Inc. | Targeted therapeutic delivery of vitamin d compounds |
| WO1998035703A2 (en) * | 1997-02-14 | 1998-08-20 | The Salk Institute For Biological Studies | Methods and compositions for delivery of therapeutic agents to bone tissue employing conjugates of negatively charged peptide oligomers with therapeutic agents |
| CA2281493A1 (en) * | 1997-02-20 | 1998-08-27 | David S. Dime | Site-specific drug delivery |
| US7033765B1 (en) | 1997-02-20 | 2006-04-25 | Toronto Research Chemicals, Inc. | Site-specific drug delivery |
| US8586781B2 (en) | 1998-04-02 | 2013-11-19 | Mbc Pharma, Inc. | Bone targeted therapeutics and methods of making and using the same |
| US6663870B2 (en) * | 1998-12-07 | 2003-12-16 | Zymogenetics, Inc. | Methods for promoting growth of bone using zvegf3 |
| US6458763B1 (en) * | 1999-09-17 | 2002-10-01 | Depuy Orthopeadics | Bone sialoprotein-based compositions for enhancing connective tissue repair |
| WO2002016557A2 (en) | 2000-08-21 | 2002-02-28 | Rice University | Tissue engineering scaffolds promoting matrix protein production |
| US6436386B1 (en) * | 2000-11-14 | 2002-08-20 | Shearwater Corporation | Hydroxyapatite-targeting poly (ethylene glycol) and related polymers |
| US7829074B2 (en) | 2001-10-18 | 2010-11-09 | Nektar Therapeutics | Hydroxypatite-targeting poly(ethylene glycol) and related polymers |
| TW200526779A (en) | 2001-02-08 | 2005-08-16 | Wyeth Corp | Modified and stabilized GDF propeptides and uses thereof |
| EP1383510B1 (en) * | 2001-04-10 | 2012-10-17 | MBC Pharma, Inc. | Bisphosphonate conjugates and methods of making and using the same |
| WO2004074345A2 (en) * | 2003-02-19 | 2004-09-02 | Pharmacia Corporation | Carbonate esters of polyethylene glycol activated by means of oxalate esters |
| WO2004089345A1 (en) * | 2003-04-03 | 2004-10-21 | Semafore Pharmaceuticals Inc. | Bone targeting of biodegradable drug-containing nanoparticles |
| EP2374471A1 (en) | 2003-09-12 | 2011-10-12 | Wyeth LLC | Injectable hardenable calcium phosphate pastes for delivery of osteogenic proteins |
| ES2887039T3 (en) * | 2004-04-21 | 2021-12-21 | Alexion Pharma Inc | Conjugates for bone delivery and method of using them to target proteins to bone |
| US20070081984A1 (en) | 2005-10-11 | 2007-04-12 | Shunji Tomatsu | Compositions and methods for treating hypophosphatasia |
| TWI299257B (en) | 2005-05-16 | 2008-08-01 | Tung Hai Biotechnology Corp | γ-POLYGLUTAMIC ACID (γ-PGA, H FORM), γ-POLYGLUTAMATES AND γ-POLYGLUTAMATE HYDROGEL FOR USE AS NUTRITION SUPPLEMENTS IN DIETARY PRODUCTS |
| JP2008542388A (en) * | 2005-06-01 | 2008-11-27 | アルザ・コーポレーシヨン | High molecular conjugate of bone morphogenic protein-7 |
| US20100047258A1 (en) * | 2006-08-02 | 2010-02-25 | Dong Wang | Drug Carriers, Their Synthesis, and Methods of Use Thereof |
| WO2008075968A1 (en) * | 2006-12-20 | 2008-06-26 | Ge Healthcare As | Contrast agents |
| AU2008317383B2 (en) * | 2007-10-23 | 2013-11-14 | Nektar Therapeutics | Hydroxyapatite-targeting multiarm polymers and conjugates made therefrom |
| WO2009075258A1 (en) * | 2007-12-10 | 2009-06-18 | Cosmed Pharmaceutical Co., Ltd. | Transdermally absorptive preparation |
| EP2488208B1 (en) | 2009-10-14 | 2018-11-21 | The Governors of the University of Alberta | Compounds having peptides conjugated to bone targeting moieties and methods of making and using thereof |
| SG185079A1 (en) | 2010-04-30 | 2012-12-28 | Alexion Pharma Internat Sarl | Methods, compositions, and kits for the treatment of matrix mineralization disorders |
| CA2823066A1 (en) | 2010-12-27 | 2012-07-05 | Alexion Pharma International Sarl | Compositions comprising natriuretic peptides and methods of use thereof |
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Family Cites Families (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4179337A (en) | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
| GB1578348A (en) | 1976-08-17 | 1980-11-05 | Pharmacia Ab | Products and a method for the therapeutic suppression of reaginic antibodies responsible for common allergic |
| JPS6023084B2 (en) | 1979-07-11 | 1985-06-05 | 味の素株式会社 | blood substitute |
| US4455256A (en) | 1981-05-05 | 1984-06-19 | The Regents Of The University Of California | Bone morphogenetic protein |
| US4294753A (en) | 1980-08-04 | 1981-10-13 | The Regents Of The University Of California | Bone morphogenetic protein process |
| US4609546A (en) | 1982-06-24 | 1986-09-02 | Japan Chemical Research Co., Ltd. | Long-acting composition |
| US4434094A (en) | 1983-04-12 | 1984-02-28 | Collagen Corporation | Partially purified osteogenic factor and process for preparing same from demineralized bone |
| US4863899A (en) * | 1983-05-09 | 1989-09-05 | Todaro George J | Biologically active polypeptides |
| US4795804A (en) | 1983-08-16 | 1989-01-03 | The Regents Of The University Of California | Bone morphogenetic agents |
| ATE128715T1 (en) | 1984-07-16 | 1995-10-15 | Celtrix Pharma | POLYPEPTIDE-INDUCING FACTORS IN BONE AND CARTILAGE. |
| US4830847A (en) | 1985-06-28 | 1989-05-16 | The Procter & Gamble Company | Diphosphonate-derivatized macromolecules |
| US4806523A (en) | 1985-08-06 | 1989-02-21 | Collagen Corporation | Method of treating inflammation |
| IL83003A (en) | 1986-07-01 | 1995-07-31 | Genetics Inst | Osteoinductive factors |
| IN165717B (en) * | 1986-08-07 | 1989-12-23 | Battelle Memorial Institute | |
| EP1225225A3 (en) | 1988-04-08 | 2003-01-08 | Stryker Corporation | Osteogenic devices |
| US5162430A (en) | 1988-11-21 | 1992-11-10 | Collagen Corporation | Collagen-polymer conjugates |
| US5118667A (en) * | 1991-05-03 | 1992-06-02 | Celtrix Pharmaceuticals, Inc. | Bone growth factors and inhibitors of bone resorption for promoting bone formation |
-
1992
- 1992-05-07 AU AU19947/92A patent/AU662155B2/en not_active Ceased
- 1992-05-07 CA CA002102808A patent/CA2102808A1/en not_active Abandoned
- 1992-05-07 JP JP5500099A patent/JPH07500315A/en active Pending
- 1992-05-07 WO PCT/US1992/003840 patent/WO1992020371A1/en active Application Filing
- 1992-05-08 ES ES92304142T patent/ES2104827T3/en not_active Expired - Lifetime
- 1992-05-08 DE DE69221368T patent/DE69221368T2/en not_active Expired - Fee Related
- 1992-05-08 AT AT92304142T patent/ATE156365T1/en not_active IP Right Cessation
- 1992-05-08 EP EP92304142A patent/EP0512844B1/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU662155B2 (en) | 1995-08-24 |
| ATE156365T1 (en) | 1997-08-15 |
| JPH07500315A (en) | 1995-01-12 |
| DE69221368T2 (en) | 1997-12-18 |
| DE69221368D1 (en) | 1997-09-11 |
| EP0512844A1 (en) | 1992-11-11 |
| AU1994792A (en) | 1992-12-30 |
| ES2104827T3 (en) | 1997-10-16 |
| WO1992020371A1 (en) | 1992-11-26 |
| EP0512844B1 (en) | 1997-08-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |