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WO1994023740A1 - Conjugues du facteur de croissance et d'inhibiteur de la resorption osseuse - Google Patents

Conjugues du facteur de croissance et d'inhibiteur de la resorption osseuse Download PDF

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Publication number
WO1994023740A1
WO1994023740A1 PCT/US1994/001662 US9401662W WO9423740A1 WO 1994023740 A1 WO1994023740 A1 WO 1994023740A1 US 9401662 W US9401662 W US 9401662W WO 9423740 A1 WO9423740 A1 WO 9423740A1
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Prior art keywords
growth factor
tgf
bone
peg
hydrophilic polymer
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Johanna Bentz
David M. Rosen
Cedo Bagi
Robert Brommage
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Celtrix Pharmaceuticals Inc
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Celtrix Pharmaceuticals Inc
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Priority to AU65863/94A priority Critical patent/AU6586394A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • 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]

Definitions

  • This invention relates to the use of transforming growth factors in treatment of systemic disease, and, particularly, to the use of molecules in any of the "TGF-0 Superfa ily" of proteins, such as the beta-type transforming growth factors ( ⁇ TGF-3s”) , the bone morphogenetic proteins (“BMPs”) , the activins, and the inhibins, for promoting bone formation in vivo, particularly for treatment of osteopenic bone diseases.
  • proteins such as the beta-type transforming growth factors ( ⁇ TGF-3s”) , the bone morphogenetic proteins (“BMPs”) , the activins, and the inhibins.
  • a targeting molecule having an affinity for the targeted tissue such as a monoclonal antibody that binds cell surface receptors of targeted cell types, is conjugated to a molecule of the agent.
  • the antibody-agent conjugate circulates until it reaches a cell of the target type, whereupon the antibody binds the cell surface receptor, localizing the antibody- agent at the targeted site.
  • TGF-3 The family of peptides known as TGF-3 can regulate both cell growth and cell differentiation. Depending upon the particular cell type, the peptides of the TGF-j8 family can stimulate or inhibit cell proliferation. Almost all tissues from all species of animals which have been examined contain TGF- ⁇ s of some type.
  • compositions for treating bone loss that include a bone growth factor, such as TGF-/3, activin, or bone morphogenic protein ("BMP") , chemically conjugated (preferably via a crosslinker) to a targeting molecule having an affinity for bone, such as tetracycline, calcein, bisphosphonate, polyaspartic acid, polyglutamic acid, aminophosphosugars, or estrogen.
  • BMP bone morphogenic protein
  • Preferred crosslinkers, according to the '467 application include PEG having average molecular weights between about 200 and about 10,000 da1tons.
  • EP 0 98,110 describes increasing the half-life of an enzyme or an interferon by coupling with a polyoxyethylene-polyoxypropylene (“POE-POP") block polymer.
  • POE-POP polyoxyethylene-polyoxypropylene
  • a conjugate of a recombinant TGF-/3 and a hydrophilic polymer such as a polyethylene glycol (“PEG”), administered to an animal in vivo can be substantially more effective for stimulating bone formation than unmodified TGF-3; and, in particular, a PEG-TGF-jS conjugate can be effective for stimulating bone formation in vivo when administered at dose levels at which unmodified TGF-S alone is ineffective.
  • the present invention also offers in vivo combination therapy for stimulating new bone formation through the combined administration of the PEG-TGF-
  • the invention features a method for treating a systemic disease condition by administering to the animal an effective amount of a conjugate of a growth factor and a hydrophilic polymer.
  • the growth factor is a bone growth factor and the systemic disease condition is treated by stimulating bone formation.
  • a "bone growth factor”, as that term is used herein, includes any of the TGF-/3 family of growth factors, and includes activin and bone morphogenetic proteins ("BMP") .
  • the bone growth factor is a TGF-3, such as TGF-02, and more preferably is a recombinant TGF-j8, such as recombinant TGF-j82.
  • TGF-3 includes TGF-01, TGF-j-,2, TGF-03, TGF-04 and TGF-/S5, and heterodimers thereof; and, more broadly, "TGF-0" means and includes any molecule that competes with binding of the native form of TGF-/3 for any of the cell surface TGF-3- binding proteins, including any of the TGF-/S receptors types I through IX that have to date been characterized.
  • hydrophilic polymer is a synthetic or natural polymer having an average molecular weight and composition that render the polymer essentially water soluble. Most hydrophilic polymers have this property by virtue of their having a sufficient number of oxygen atoms (less frequently nitrogen atoms) available for forming hydrogen bonds in aqueous solution. Hydrophilic polymers generally used herein include PPG, PEG, POE, polytrimethylene glycols, polylactic acid, and derivatives thereof. Particularly suitable hydrophilic polymers include a polyethylene glycol —(CH 2 CH 2 0) n — (“PEG”), a polypropylene glycol
  • PPG poly(CH 2 CH 2 CH 2 0) n —
  • preferred hydrophilic polymers include, for example, PEGs or PPGs having a molecular weight between 200 and 100,000, such as, for example, PEG 5000, PEG 1700, or PEG 35,000.
  • the hydrophilic polymer is a PEG and the growth factor is a TGF-32
  • 1 - 14 molecules of the hydrophilic polymer, and more preferably 1 - 7 molecules of the hydrophilic polymer are attached to each TGF-
  • Other suitable polymers include POE-POP block polymers and copolymers.
  • a "conjugate" of a hydrophilic polymer and a growth factor is a composition in which the hydrophilic polymer is attached to the growth factor via a covalent bond.
  • inhibitor of bone resorption refers to prevention of bone loss, especially the inhibition of removal of existing bone either from the mineral phase and/or the organic matrix phase, through direct or indirect inhibition of osteoclast formation or activity.
  • inhibitor of bone resorption refers to agents that prevent bone loss by the direct or indirect inhibition of osteoclast formation or activity.
  • the invention features a composition for stimulating bone deposition, including a conjugate of a growth factor and a hydrophilic polymer.
  • the invention features a composition for stimulating bone deposition, including a conjugate of a growth factor and a hydrophilic polymer and an inhibitor of bone resorption.
  • the growth factor may be brought into more effective proximity to the tissue site on which the growth factor is effective, when conjugated with a hydrophilic polymer according to the invention. This may result from a more or less specific affinity of the hydrophilic polymer (or of the conjugate) for the tissue.
  • PEG-TGF-32 conjugates according to the invention may effectively localize to bone following systemic administration.
  • TGF-/32 on both osteoblasts and osteoclasts may be enhanced or prolonged; and an in vivo inhibition of osteoclast activity by TGF-j82 (as can be observed in vitro) can be effected, resulting in a net increase in bone.
  • preferred hydrophilic polymers may (either in and of themselves or when conjugated with a bone growth factor) have a specific affinity for bone tissue.
  • the pharmacokinetics of the growth factor may be altered by conjugation with a hydrophilic polymer according to the invention.
  • a differential effect of the bone growth factor (such as TGF- ⁇ 2) on osteoblasts may result from conjugation with the hydrophilic polymer (such as PEG) , resulting in a net increase in bone.
  • Osteoblasts and osteoclasts may have different receptors for TGF-0, for example, binding different portions of a particular TGF-
  • the invention features a method for stimulating bone formation in an animal by administering to the animal a PEG-TGF-/32 conjugate in which the PEG has a molecular weight between about 5 kd and about 100 kd, and preferably about 35 kd, and in which the higher molecular weight PEG is present with the TGF-/32 homodimer in a molar ratio in the range about 1:1 to about 3:1.
  • the preferred molar ratio for a given combination of hydrophilic polymer and growth factor depends among other variables upon the molecular size of the hydrophilic polymer.
  • TGF- ⁇ s for example, have a molecular size (dimer) about 26,000; preferred conjugates of hydrophilic polymers with TGF-3s have a molecular size at least about 55,000 or 60,000; such molecular sizes can be obtained by conjugation of PEG 5000 and a TGF- ⁇ at molar ratios at least about 6:1, or by conjugation of PEG 35,000 and a TGF-0 at a molar ratio of at least about 1:1.
  • a lower molar ratio of higher molecular size PEGs in PEG-TGF-/3 conjugates can be more effective than a higher molar ratio of lower molecular size PEGs.
  • the activity is significantly reduced or lost at molar ratios of PEG 5000 to TGF-02 greater than 7:1.
  • the hydrophilic polymer — growth factor conjugate can be administered in conjunction with a bone resorption inhibitor.
  • the conjugate is administered in conjunction with a bone resorption inhibitor such as, for example, an estrogen, a bisphosphonate or a calcitonin.
  • Administration of the conjugate can commence either prior to, at the same time as, or following administration of the bone resorption inhibitor.
  • the conjugate and bone resorption inhibitor can be administered at least partly concurrently. Where administration is to be simultaneous, the conjugate and the bone resorption inhibitor may or may not be combined in a single composition. Drugs which prevent bone loss and/or add back lost bone are often first tested in the ovariectomized rat.
  • inhibitors of bone resorption include estrogens such as estradiol, tamoxifen, bisphosphonates, calcitonins, or other small peptides or molecules that may inhibit bone resorption.
  • estrogens such as estradiol, tamoxifen, bisphosphonates, calcitonins, or other small peptides or molecules that may inhibit bone resorption.
  • echistatin which includes the arginine- glycine-aspartate (RGD) sequence which is recognized by some cell surface adhesion receptors and apparently disrupts osteoclast interactions (Fisher et al. (1993) Endocrinology 132: 1411-13) .
  • RGD arginine- glycine-aspartate
  • Another example of a bone resorption factor is OPF or osteoclastpoietic factor (PCT Publication WO 93/01827 published 4 February 1993) .
  • Bisphosphonates include, but are not limited to, pamidronate, alendronate, residronate and tiludronate.
  • hydrophilic polymer — growth factor conjugates There follow illustrative protocols for making hydrophilic polymer — growth factor conjugates according to the invention, showing by way of example protocols for production of PEG-TGF-j82 conjugates.
  • Other hydrophilic polymers than polyethylene glycols, and other growth factors than TGF-j ⁇ s can be coupled to make the conjugates of the invention. Adjustment of steps and particular parameters given in the protocols can be made as appropriate for the particular materials and according to the custom of the particular laboratory, all without undue experimentation and within the ordinary skill of the art.
  • the growth factor can be conjugated at a reactive group to the selected hydrophilic polymer or polymers.
  • Reactive groups on the growth factor include, but are not limited to, carboxyl groups of the polypeptide C-terminus or of aspartic acid or glutamic acid residues, amino groups of the polypeptide N-terminus or of lysine residues, imidazole functions of histidine residues and phenolic functions of tyrosine residues, sulfhydryl groups of residues, and guanidine groups of arginine residues.
  • the molar ratio of hydrophilic polymer molecules to growth factor molecules can be controlled by selection of specific conjugation chemistries (for example, by reacting the polymer predominantly with primary amine substituents on lysine residues, of which there are a fixed number on any given growth factor molecule) and, as will be appreciated, by control of reaction conditions such as temperature or pH.
  • the bone growth factor TGF-,3 contains a number of available amino, carboxyl, and hydroxy groups that may be used to bind the hydrophilic polymer.
  • the hydrophilic polymer may be connected using a "linking group", as the native hydroxy or amino groups in TGF-3 and in the hydrophilic polymer frequently require activation before they can be linked.
  • a compound such as a dicarboxylic anhydride e.g., glutaric or succinic anhydride
  • a polymer derivative polymer glutarate or polymer succinate, e .g. , using PEG and succinic anhydride, a PEG-succinate
  • a convenient leaving group e.g. , N-hydroxysuccinimide, N,N , -disuccinimidyl oxalate, N,N'-disuccinimidyl carbonate, or the like.
  • the activated polymer is then allowed to react with the bone growth factor, to form the hydrophilic polymer — bone growth factor conjugate.
  • Preferred dicarboxylic anhydrides for use in forming polymer-glutarate compositions include glutaric anhydride, adipic anhydride, 1,8-naphthalene dicarboxylic anhydride, and 1,4,5,8- naphthalenetetracarboxylic dianhydride.
  • Suitable crosslinkers have chemistries well known in the art, and they are commercially available.
  • the resulting hydrophilic polymer — growth factor conjugate can be purified using a standard technique, such as by reverse-phase high performance liquid chromatography ("RP-HPLC"), size-exclusion HPLC ("SEC-HPLC”; e .g. , tetrahydrogel-HPLC) , or ion- exchange chromatography.
  • RP-HPLC reverse-phase high performance liquid chromatography
  • SEC-HPLC size-exclusion HPLC
  • e tetrahydrogel-HPLC e etrahydrogel-HPLC
  • ion- exchange chromatography ion- exchange chromatography.
  • RP-HPLC is preferably performed using a C18 column using gradient elution with 80 - 100% acetonitrile, ethanol or isopropanol with 0.1% trifluoroacetic acid (“TFA”) as the eluting solvent.
  • a preferred running buffer can be 5 mM sodium acetate at pH 5.5, preferably including an organic
  • a PEG— GF-/3 conjugate can be made according to the following protocols, which make specific reference to formation of rTGF-32(PEG 5000) 6 from PEG 5000 and rTGF-/32 and to formation of rTGF-02(PEG 35,000) ⁇ from PEG 35,000 and rTGF-02.
  • TGF-3 is difficult to dissolve in solutions of appropriate pH for coupling to hydrophilic polymers.
  • the TGF-3 is lyophilized in the absence of a carrier protein or is held in solution in acid/organic solvent.
  • TGF-3 is dissolved in a mild acid, preferably about 10 mM HC1, in the presence of a disaggregating reagent such as an organic solvent
  • the solution is then neutralized by adding a base, preferably NaOH (1 N solution) in buffered saline (e.g., phosphate buffered saline) .
  • buffered saline e.g., phosphate buffered saline
  • the final solution preferably contains about 40 - 50% DMSO or CH 3 CN to solubilize the TGF- ⁇ and to prevent aggregation, thus preserving TGF-jS activity.
  • rhTGF-fl2.PEG 5000.., In a preferred protocol, the activated PEG
  • succinimydyl ester made, for example, as follows. Monomethylpolyethylene glycol, average olecular weight about 5,000 daltons, (“mPEG 5000") is reacted with glutaric anhydride to form mPEG glutarate. The glutarate derivative is then reacted with N-hydroxysuccinimide to form a succinimydyl mPEG glutarate. The succinimydyl ester is then capable of reacting with free amino groups (lysine residues) on the TGF-j ⁇ to form a TGF-/5 — PEG conjugate.
  • mPEG 5000 Monomethylpolyethylene glycol, average olecular weight about 5,000 daltons
  • rhTGF-/_.2(PEG 5000) 6 was made in a 5 mg batch from recombinant human TGF-32 (rhTGF-32) and methoxypolyethylene glycol succinimydyl succinate 5000 (M-S-PEG 5000).
  • rhTGF-32 recombinant human TGF-32
  • M-S-PEG 5000 methoxypolyethylene glycol succinimydyl succinate 5000
  • M-S-PEG 5000 (Sigma, lot 11H8040) (8 ⁇ mol; 40 mg) , stored dry at - 20 °C, was added (either directly or dissolved in acetonitrile) at a molar ratio of 40:1, to the rhTGF-/32 mixture, and permitted to react for 2 hours at room temperature.
  • the resulting mixture was then diluted at least 1:3 with 0.1% TFA, the pH was adjusted to between pH 2 and pH 4 using 1% TFA, and the mixture was fractionated by C18-RP-HPLC (Vydac 218TP510, 1 x 25 cm).
  • Solvent A was 0.1% TFA and solvent B was 90% acetonitrile in A.
  • unreacted TGF-/32 eluted first at 41% solvent B followed by TGF-0 with one PEG 5000 molecule attached.
  • TGF-jS2 having increasingly higher amounts of PEG 500 eluted from the column.
  • rhTGF-02(PEG 35,000) ⁇ _ 3 from recombinant human TGF-j82 (rhTGF-32) and bis-polyethylene glycol succinimydyl carbonate 35,000 (BSC-PEG 35,000).
  • rhTGF-32 recombinant human TGF-j82
  • BSC-PEG 35,000 bis-polyethylene glycol succinimydyl carbonate 35,000
  • PG 1.0 ml HPLC-grade water, 1.0 ml lOx PBS and 100 ⁇ l 0.1 N NaOH to make about 8.7 ml total volume.
  • BSC- PEG 35,000 provided by Milton Harris, University of Alabama, Huntsville, AL, was added either (A) in a 5:1 mol ratio (20 mg) or (B) in a 2:1 mol ratio (8 mg) , to the rhTGF-32 mixture, to yield a protein concentration 0.268 mg/ml, 36.3% organic solvent, 10.3% PG, at pH 7.2.
  • the mixture of rhTGF-02 and BSC-PEG 35,000 was permitted to react for 90 min. at room temperature, pH 7.2.
  • the resulting mixture was then diluted at least 1:3 with 0.1% TFA, the pH was adjusted to between pH 2 and pH 4 using 1% TFA, and the TGF-jS2(PEG 35,000) was purified by C18-RP-HPLC according to the purification protocol referenced above.
  • the resulting HPLC fractions were analyzed under non-reduced and reduced conditions on SDS-PAGE, 5-15% gradient.
  • the protein concentration of pooled purified samples was determined at OD 280 nm, and aliquots were prepared for use as described above.
  • compositions containing hydrophilic polymer—growth factor conjugate are preferably administered by parenteral routes, intravenous injection, intranasal or bronchial aerosol, and the like.
  • the compositions can be employed in sustained release vehicles, such as from a slow-release carrier, or from a sustained release device that may be surgically implanted subdermally or within the peritoneal cavity.
  • compositions for administration of the hydrophilic polymer — bone growth factor generally include an osteogenically effective amount of the bone growth factor in addition to a pharmaceutically acceptable excipient.
  • Suitable excipients include most carriers approved for parenteral administration, including water, saline, Ringer's solution, Hank's solution, as well as solutions of lactose, dextrose, ethanol, glycerol, albumin, and the like.
  • the compositions may optionally include stabilizers, antioxidants, antimicrobials, preservatives, buffering agents, surfactants, and other accessory additives, such as propylene glycol.
  • a preferred mode of administration includes about 10-50% propylene glycol. The more preferred mode includes about 40% propylene glycol.
  • saline or phosphate- buffered saline can be a preferred vehicle for parenteral administration of rTGF-3—PEG.
  • PBS phosphate- buffered saline
  • hydrophilic polymer — growth factor compositions of the invention may be formulated as solutions or suspensions, or they may be lyophilized for later reconstitution.
  • hydrophilic polymer — growth factor conjugate that is, an amount sufficient to effect treatment in the subject, will depend upon the particular growth factor and the particular hydrophilic polymer used in the conjugate and the number of polymer molecules attached to each growth factor molecule in the conjugate, as well as the nature and severity of the condition to be treated, the age and general health of the subject, the specific activity of the composition, and other factors that may be determined by the practitioner of ordinary skill in the art of treating bone disease.
  • doses of conjugate in the range 0.001 to 10 ⁇ g/kg body weight, and more preferably in the range 0.001 to l ⁇ g/kg body weight, and most preferably in the range 0.01 to 0.1 ⁇ g/kg body weight, should be effective.
  • weights or volumes given /kg refer to /kg of body weight.
  • hydrophilic polymer — bone growth factor conjugates of the invention are substantially more effective for stimulating bone formation in vivo than the bone growth factor alone, effective systemic dosages of the conjugate according to the invention are much lower and may be less frequently administered than are effective dosages of the corresponding unconjugated growth factor.
  • the number of molecules of the conjugate that need be administered systemically to achieve the desired treatment effect can be at least 10- to 100-fold lower than the number of molecules of unmodified TGF-32 that must be administered to achieve the desired treatment effect.
  • conjugate compositions according to the invention that have relatively lower activities as determined by an in vitro assay can be effective for treatment in vivo.
  • An effective dose for estrogen is about 1 ⁇ g/kg to about 1 mg/kg of body weight.
  • An effective dose for bisphosphonates is quite variable but generally between about 0.05 ⁇ g/kg to about 15 mg/kg of body weight.
  • An effective dose for calcitonin is about 0.05 IU (International Units or Medical Research Council Units)/kg to about 2.5 IU/kg of body weight.
  • the combination of PEG-TGF-/3 and an inhibitor of bone resorption is useful for treating bone fractures, defects, and disorders which result in weakened bones such as osteoporosis (including postmenopausal, age-related and idiopathic) , osteoarthritis, Paget's disease, osteohalisteresis, osteomalacia, bone loss resulting from multiple myeloma and other forms of cancer, and bone loss resulting from side effects of other medical treatment (such as steroids) .
  • osteoporosis including postmenopausal, age-related and idiopathic
  • osteoarthritis including postmenopausal, age-related and idiopathic
  • Paget's disease osteohalisteresis
  • osteomalacia bone loss resulting from multiple myeloma and other forms of cancer
  • side effects of other medical treatment such as steroids
  • hydrophilic polymer — growth factor conjugates according to the invention can be administered for treatment of diseases where bone loss occurs, such as, for example, osteoporosis.
  • mice were divided into five treatment groups of six mice each.
  • Mice in the first group were treated with mouse seru albumin (MSA) , a vehicle control; those in the second and third groups were treated with rTGF-j ⁇ 2 at doses of 20 ⁇ g and 0.1 ⁇ g per mouse (760 ⁇ g and 3.8 ⁇ g per kg body weight) , respectively; and those in the fourth and fifth groups were treated with rTGF-/32(PEG 5000) 6 at doses of 0.1 ⁇ g and 0.02 ⁇ g per mouse (3.8 ⁇ g and 0.76 ⁇ g per kg body weight), respectively.
  • MSA mouse seru albumin
  • the body weight index is 26.4 g, the average weight of the animals at the start of the study.
  • Treatments were performed daily for eleven days by subcutaneous injections of 100 ⁇ l each in the tailbase. Demeclocycline was used as a fluorochrome label on day 1, and calcein was used on days 6 and 10 for histomorphometric analyses. A variety of analyses were performed. Body weights of the animals were measured daily.
  • hematology white cell counts, red cell counts, packed cell volume (PCV) , hemoglobin, platelet counts, differential blood cell counts
  • thymic cellularity histology (liver, kidney, spleen)
  • bone histomorphometry femur epiphysis
  • mice treated with rTGF-/32(PEG 5000) 6 in daily doses of 3.8 ⁇ g/kg body weight showed increases in indices of bone formation in all measured parameters, with close to normal values for indices of bone resorption, resulting in an overall increase in cancellous bone mass (increases in percent trabecular area, trabecular thickness, trabecular number, decreases in trabecular separation) .
  • bone formation in mice treated with unmodified rTGF- ⁇ in the same daily doses of 3.8 ⁇ g/kg body weight was not significantly different from bone formation in controls.
  • mice were treated by subcutaneous injection with rTGF-/32 (two different doses), with rTGF-02(PEG 5000) 8 or rTGF-32(PEG 5000) 6 or rTGF-/32(PEG 5000) 4 (two different doses for each molar ratio), with rTGF-02(PEG 35,000),. 3 (two different doses) , or with a vehicle control.
  • rTGF-/32 two different doses
  • rTGF-02(PEG 5000) 8 or rTGF-32(PEG 5000) 6 or rTGF-/32(PEG 5000) 4 two different doses for each molar ratio
  • rTGF-02(PEG 35,000) three
  • a variety of analyses were used to determine the effects of the treatments on bone cell morphology.
  • mice 8 week-old-male C3H mice were divided into treatment groups. Mice in the first group were treated with MSA-PEG 5000, as a control; those in the second group were treated with 3 ⁇ g r- TGF-/32 admixed with PEG 5000 (uncomplexed) ; those in the third and fourth groups were treated with rTGF-32 at doses of 15 ⁇ g and 3 ⁇ g per mouse, respectively; those in the fifth, sixth and seventh groups were treated with 3 ⁇ g per mouse rTGF-jS2(PEG 5000) at molar ratios (PEG: rTGF-02) of 8:1, 6:1, and 4:1, respectively; those in the ninth, tenth and eleventh groups were treated with 0.6 ⁇ g per mouse rTGF-32(PEG 5000) at molar ratios 8:1, 6:1, and 4:1, respectively; and those in the eighth and twelfth groups were treated with rTGF-32(PEG 35,000) w
  • mice treated with rTGF-02(PEG 5000) 8 gained weight slightly over the study period and showed no significant changes in hematologic, bone marrow or thymic analysis.
  • mice treated with rTGF- / 32(PEG 5000) 6 showed significant increases in RBC, hemoglobin and % PCV
  • mice treated at 0.6 ⁇ g with rTGF-32(PEG 5000) 4 or rTGF-02(PEG 35,000) ⁇ _ 3 showed no significant changes in hematologic or bone marrow or thymus analysis.
  • mice treated with rTGF- / 82(PEG 5000) 6 even at the lower dose weight loss was only moderate (about 3.3%) in mice treated at 0.6 ⁇ g with rTGF-/32(PEG 5000) 4 ; and mice treated at
  • OVX rats were treated by subcutaneous injections of PEG-rTGF-
  • OVX rats were ovariectomized at 12 weeks of age and then lost bone until the start of the study when the rats were 95 weeks old.
  • the OVX rats were given a dietary intake restricted to minimize the body weight gain that normally follows ovariectomy.
  • OVX rats had their spinal bone mineral density (BMD) measured by DEXA. Based on spine BMD values, the OVX rats were then randomly assigned among the study groups described below, with reasonable attempts made to have mean BMD values similar for all groups. The rats were given subcutaneous injections (0.1 ml/rat) for 8 weeks
  • estradiol (10 ⁇ g/kg) and/or
  • PEG-rTGF-32 (1 ⁇ g/kg) .
  • PEG-rTGF-/32 was given every day for 3 weeks, then 3 times a week for 4 weeks, and then every day during the last week.
  • the PEG-rTGF-j ⁇ 2 vehicle was 40% propylene glycol with ⁇ 2% ethanol in
  • the tibia lengths and projected areas are presented in Table 4.
  • the tibia bone mineral content, bone mineral density and bone mineral apparent density data are given in Table 5.
  • the bone mineral densities for tibial cortical bone, metaphysis and epiphysis are given in Table 6.
  • Bone (mg/cm 2 . (mg/cm 3 .
  • Table 7 indicates that the estradiol dose was sufficient to normalize the uterine size, but PEG- rTGF-02 had little if any effect.
  • This purpose of this study is to: a) test the ability of rTGF-32 (PEG 5000) 6 to promote bone formation in osteopenic rat skeleton after bilateral ovariectomy; b) compare effects on bone of three different doses of PEG-TGF-/32 when injected daily
  • One hundred and thirty five female, Sprague- 5 Dawley rats are obtained at 90 days of age and housed for one week to acclimate. After acclimation, 23 randomly chosen animals are sham-operated; whereas, the remaining 112 females are bilaterally ovariectomized by the dorsal approach under 10 ketamine/xylazine anesthesia. Eight weeks post sham surgery or ovariectomy, animals are randomly divided in eight experimental groups and are treated as shown:
  • mice from groups 1 and 2 serve as baseline controls and show bone status in sham and OVX groups before treatment with PEG-TGF-32.
  • Declomycin 25 mg/kg is administered at day 7 to animals scheduled to be sacrificed on day 14 and on day 21 to animals scheduled to be sacrificed on day 28.
  • Body weights are monitored for all animals weekly. Animals are necropsied by exsanguination from the vena cava under deep anesthesia with ketamine/xylazine.
  • Livers from the Group 3 mice killed on day 28 are processed for histopathology.
  • Both femurs, tibias, lumbar vertebral bodies, and mandibles are collected, fixed in ethanol and processed for various histomorphometric analyses.
  • Distal femoral metaphyses from right femurs are embedded undecalcified for static and dynamic histomorphometry of cancellous bone.
  • cortical bone histomorphometry is performed on cross-sections from right tibias at the tibio-fibular junction.
  • the proximal portion of the same tibias is demineralized, embedded in paraffin, cut in thin longitudinal sections (4-5 ⁇ m) .
  • Left tibias which have been cleaned of soft tissues are used for determination of wet, dry and ash weight. Also, if necessary, calcium and phosphorus are determined from ash. Left femurs and mandibles are stored undecalcified at -70°C for eventual additional measurements.

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  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

l'invention se rapporte à un procédé de stimulation de la formation osseuse chez un animal qui consiste à administrer à l'animal une dose efficace d'un conjugué d'un facteur de croissance et d'un polymère hydrophile. De même une composition permettant de traiter la maladie ostéopénique des os comprend un conjugué d'un facteur de croissance et d'un polymère hydrophile. Les conjugués préférés comprennent, par exemple, un TGF-β comme facteur de croissance, et un polyéthylène glycol comme polymère hydrophile. Les facteurs de croissance conjugués aux polymères hydrophiles, selon l'invention, peuvent stimuler la formation osseuse à des niveaux de dosage inférieurs auxquels le facteur de croissance, non modifié, est inefficace; et les facteurs de croissance conjugués aux polymères hydrophiles, selon l'invention, favorisent une augmentation nette de la formation osseuse à des niveaux de dosage supérieurs auxquels le facteur de croissance, non modifié, provoque une réduction nette de la masse osseuse, dûe à la stimulation de la résorption osseuse et la formation osseuse par le facteur de croissance. Un inhibiteur de résorption osseuse est éventuellement ajouté afin de diminuer la résorption osseuse et par conséquent de stimuler la formation osseuse.
PCT/US1994/001662 1993-04-22 1994-02-16 Conjugues du facteur de croissance et d'inhibiteur de la resorption osseuse Ceased WO1994023740A1 (fr)

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AU65863/94A AU6586394A (en) 1993-04-22 1994-02-16 Conjugates of growth factor and bone resorption inhibitor

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US5150893A 1993-04-22 1993-04-22
US08/051,508 1993-04-22
US8473293A 1993-06-28 1993-06-28
US08/084,732 1993-06-28

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WO1996028475A1 (fr) * 1995-03-10 1996-09-19 Nakamura, Toshikazu Facteur de croissance des hepatocytes modifie a l'aide de polyethylene glycol
WO1996039430A1 (fr) * 1995-06-06 1996-12-12 Hall Frederick L Proteines de fusion du tgf-beta et leur utilisation dans la cicatrisation de lesions
WO1998008517A3 (fr) * 1996-08-26 1998-05-07 Takeda Chemical Industries Ltd Composition pharmaceutique contenant une substance favorisant l'osteogenese et un polyethylene glycol
US6649168B2 (en) 1999-03-17 2003-11-18 Novartis Ag Pharmaceutical compositions comprising TGF-beta
US7635592B2 (en) 2000-08-21 2009-12-22 Rice University Tissue engineering scaffolds promoting matrix protein production
EP2196538A1 (fr) * 2000-02-10 2010-06-16 Mountain View Pharmaceuticals, Inc. Conjugués préparés avec des protéines sans agrégats
US7927589B2 (en) 1998-08-06 2011-04-19 Mountain View Pharmaceuticals, Inc. PEG-urate oxidase conjugates and use thereof
US10139399B2 (en) 2009-06-25 2018-11-27 Horizon Pharma Rheumatology Llc Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during PEGylated uricase therapy
US10160958B2 (en) 2005-04-11 2018-12-25 Horizon Pharma Rheumatology Llc Variant forms of urate oxidase and use thereof
US12269875B2 (en) 2023-08-03 2025-04-08 Jeff R. Peterson Gout flare prevention methods using IL-1BETA blockers
US12465631B2 (en) 2019-01-30 2025-11-11 Horizon Therapeutics Usa, Inc. Reducing immunogenicity to pegloticase

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US6190695B1 (en) 1919-04-09 2001-02-20 Takeda Chemical Industries, Ltd. Pharmaceutical composition containing osteogenesis-promoting substance
US5977310A (en) * 1995-03-10 1999-11-02 Toshikazu Nakamura And Sumitomo Pharmaceuticals Co., Ltd. Peg-modified HGF
WO1996028475A1 (fr) * 1995-03-10 1996-09-19 Nakamura, Toshikazu Facteur de croissance des hepatocytes modifie a l'aide de polyethylene glycol
WO1996039430A1 (fr) * 1995-06-06 1996-12-12 Hall Frederick L Proteines de fusion du tgf-beta et leur utilisation dans la cicatrisation de lesions
US5800811A (en) * 1995-06-06 1998-09-01 Hall; Frederick L. Artificial skin prepared from coclagen matrix containing transforming growth factor-β having a collagen binding site
WO1998008517A3 (fr) * 1996-08-26 1998-05-07 Takeda Chemical Industries Ltd Composition pharmaceutique contenant une substance favorisant l'osteogenese et un polyethylene glycol
US8618267B2 (en) 1998-08-06 2013-12-31 Mountain View Pharmaceuticals, Inc. PEG-urate oxidase conjugates and use thereof
US9885024B2 (en) 1998-08-06 2018-02-06 Duke University PEG-urate oxidase conjugates and use thereof
US8921064B2 (en) 1998-08-06 2014-12-30 Mountain View Pharmaceuticals, Inc. Method for purifying urate oxidase tetramers and octamers
US7927589B2 (en) 1998-08-06 2011-04-19 Mountain View Pharmaceuticals, Inc. PEG-urate oxidase conjugates and use thereof
US8067553B2 (en) 1998-08-06 2011-11-29 Mountain View Pharmaceuticals, Inc. PEG-urate oxidase conjugates and use thereof
US6649168B2 (en) 1999-03-17 2003-11-18 Novartis Ag Pharmaceutical compositions comprising TGF-beta
EP2196538A1 (fr) * 2000-02-10 2010-06-16 Mountain View Pharmaceuticals, Inc. Conjugués préparés avec des protéines sans agrégats
US7635592B2 (en) 2000-08-21 2009-12-22 Rice University Tissue engineering scaffolds promoting matrix protein production
US11345899B2 (en) 2005-04-11 2022-05-31 Horizon Therapeutics Usa, Inc. Variant forms of urate oxidase and use thereof
US10160958B2 (en) 2005-04-11 2018-12-25 Horizon Pharma Rheumatology Llc Variant forms of urate oxidase and use thereof
US10731139B2 (en) 2005-04-11 2020-08-04 Horizon Pharma Rheumatology Llc Variant forms of urate oxidase and use thereof
US11781119B2 (en) 2005-04-11 2023-10-10 Horizon Therapeutics Usa, Inc. Variant forms of urate oxidase and use thereof
US10823727B2 (en) 2009-06-25 2020-11-03 Horizon Pharma Rheumatology Llc Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy
US10139399B2 (en) 2009-06-25 2018-11-27 Horizon Pharma Rheumatology Llc Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during PEGylated uricase therapy
US11598767B2 (en) 2009-06-25 2023-03-07 Horizon Therapeutics Usa, Inc. Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy
US11639927B2 (en) 2009-06-25 2023-05-02 Horizon Therapeutics Usa, Inc. Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during PEGylated uricase therapy
US11982670B2 (en) 2009-06-25 2024-05-14 Horizon Therapeutics Usa, Inc. Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy
US12188927B2 (en) 2009-06-25 2025-01-07 Horizon Therapeutics Usa, Inc. Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during PEGylated uricase therapy
US12465631B2 (en) 2019-01-30 2025-11-11 Horizon Therapeutics Usa, Inc. Reducing immunogenicity to pegloticase
US12269875B2 (en) 2023-08-03 2025-04-08 Jeff R. Peterson Gout flare prevention methods using IL-1BETA blockers

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