WO1995028950A1 - Administration of platelet-derived growth factor and bone seeking drugs for osteoporosis and bone regeneration - Google Patents
Administration of platelet-derived growth factor and bone seeking drugs for osteoporosis and bone regeneration Download PDFInfo
- Publication number
- WO1995028950A1 WO1995028950A1 PCT/US1995/005047 US9505047W WO9528950A1 WO 1995028950 A1 WO1995028950 A1 WO 1995028950A1 US 9505047 W US9505047 W US 9505047W WO 9528950 A1 WO9528950 A1 WO 9528950A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- bone
- pdgf
- mammal
- growth factor
- platelet
- Prior art date
Links
Classifications
-
- 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
- A61K38/1858—Platelet-derived growth factor [PDGF]
Definitions
- the invention relates to pharmaceutical formulations useful for treating bone loss.
- Bone loss in mammals commonly results from a number of conditions including, for example, osteoporosis, traumatic injuries, rheumatoid arthritis, malignancies, and periodontal diseases.
- Osteoporosis is a disease process in which bone mass is progressively lost in almost all sites in the skeletal system.
- Post-menopausal osteoporosis (Type I) dramatically increases the rate at which bone mass decreases in women. Prior to menopause, women over the age of 35 lose cortical bone mass at a rate of 0.3 - 0.5% per year.
- Age-related osteoporosis (Type II) affects almost all aging women, and it can affect men as well.
- osteoporosis Currently available treatments for osteoporosis, such as estrogen or calcitonin therapy, are of limited efficacy. While these therapies may prevent the loss of bone mass due to Type I osteoporosis, the current treatments fail to reverse the effects of the disease, and they fail to halt Type II osteoporosis. Furthermore, estrogen therapy is not acceptable for women with a history of, or predisposition for, thromboembolic disorders, diabetes, cancer, or liver disorders. Estrogen replacement therapy also is not an acceptable treatment for men with osteoporosis.
- Growth factors also referred to as tissue growth promoting factors, are polypeptides which interact with specific cell surface receptors in defined target cells, resulting in specific cell effects. Growth factors are often multifunctional, and they may either stimulate or inhibit cell proliferation. They may also affect differentiated cell function. Examples of growth factors include, but are not limited to, platelet-derived growth factors (PDGFs) (R. Ross et al. (1986) Cell 46:155-169), insulin-like growth factors (IGFs) (V.R. Sara and K. Hall (1990) Physiol. Rev. 70:591-614) fibroblast growth factors (FGFs) (D. Gospodarowicz et al. (1987)
- PDGFs platelet-derived growth factors
- IGFs insulin-like growth factors
- FGFs fibroblast growth factors
- EGFs epidermal growth factors
- NGFs nerve growth factors
- TGFs transforming growth factors
- BMPs bone morphogenetic proteins
- BMPs which are part of the TGF-,9 superfamily, are characterized by their unique ability to induce osteoblastic differentiation.
- growth factors such as PDGF, TGF-3, IGF-I, IGF-II, basic (b)-FGF, and bone morphogenetic proteins are cationic at physiological pH.
- Tissue growth promoting factors have been shown to promote bone formation in vitro and in vivo.
- the combinations of PDGF and IGF-I, and PDGF and IGF-II promote bone formation or regeneration (See, for example, S. E. Lynch et al. (1991) J. Periodontol . 62:458-467; and U.S. Patents 4,861,757 and 5,019,559).
- bisphosphonate drugs are known to be direct inhibitors of osteoclastic bone resorption (H. Fleisch (1983) in: Bone and Mineral Research Annual 1:319-357).
- bisphosphonates include etidronate, clodronate, tiludronate, pamidronate, alendronate, and risedronate.
- the invention features a method for treating bone loss, which includes systemically administering to a mammal PDGF which is not covalently bonded to any other component of the composition.
- the composition used in this method also includes an anti-resorptive agent.
- the invention also features a pharmaceutical formulation for treating bone loss, including PDGF, a bone-targeting anionic compound having at least one negative charge at pH 6.8, and a physiologically acceptable excipient, wherein the PDGF is not covalently bonded to any other compound in the formulation.
- the association of PDGF with the bone-targeting anionic compound is sufficiently strong that the two components remain associated in vivo for a period of time sufficient for delivery of PDGF to bone by the anionic compound.
- the fact that PDGF and the anionic compound do not have to be covalently bonded greatly reduces the cost of producing the bone-targeting pharmaceutical formulation, as simply mixing the two components in solution is all that is required.
- This pharmaceutical formulation may contain in addition another growth-promoting factor, such as IGF-I, IGF-II, TGF-3, aFGF, bFGF, EGF, NGF, or a bone morphogenetic protein.
- the invention also features a pharmaceutical composition consisting of PDGF and an anti-resorptive agent; this composition may also contain another growth- promoting factor.
- the non-covalent association of PDGF and the anionic compound involves at least in part an ionic attraction between the negatively charged anionic compound and PDGF, which is positively charged at pH 6.8.
- the positive charge on PDGF can be increased by addition of one or more cationic groups such as amino groups, or by point mutations which substitute positively charged amino acids such as lysine for neutral or negatively-charged amino acids; these modifications can be carried out by standard techniques.
- PDGF and the other growth factors useful in the invention may be derived from natural sources, produced by recombinant DNA technology, or chemically synthesized.
- the growth factor is purified and deemed to be at least 90% pure, by weight, as determined by techniques such as gel electrophoresis and amino acid sequence analysis.
- PDGF includes all forms of biologically active PDGF including all isoforms, homodimeric and hete ⁇ odimeric forms, and analogs.
- the bone-targeting anionic compound can serve as the bone-targeting anionic compound, including sulfonates, phosphonates, and dicarboxylates (e.g.. oxalic acids) .
- the anionic compound has at least 1 negative charge at pH 6.8. More preferably, the anionic compound has at least 2 negative charges at pH 6.8. Still more preferably, the anionic compound is a bisphosphonate of the formula:
- R x is hydrogen, fluorine, chlorine, hydroxyl, or methyl
- R 2 is hydrogen, fluorine, chlorine, methyl, S- C 6 H 5 -C1, (CH 2 ) 2 NH 3 , (CH 2 ) 3 NH 3 , (CH 2 ) 2 N(CH 3 )C 5 H U , CH 2 -pyridine, or NHC 7 H 14 .
- bisphosphonates include, but are not limited to, etidronate, clodronate, tiludronate, pamidronate, alendronate, and risedronate.
- at least one negative charge of the anionic compound interacts with PDGF, and at least one negative charge interacts with calcium on the bone.
- the anti-resorptive agent is a phosphonate, and more preferably, it is a bisphosphonate.
- Methods for preparing anti-resorptives are well-known in the art.
- the formulation of the invention is administered to a mammal in an amount between about 0.001 and 50 mg/kg of the body weight of the mammal (preferably a human), more preferably in an amount between about 0.01 and 10.0 mg/kg of the body weight of the mammal.
- PDGF treatment of bone increases bone mineral density compared with saline treatment of bone.
- Applicants have also found that the combination of PDGF and an anionic compound, such as a bisphosphonate, has a greater effect on both whole skeleton bone mineral density and spinal bone mineral density (than either compound alone does) .
- the formulations of this invention can be used to treat or prevent osteoporosis in a mammal.
- PBS phosphate buffered saline
- DXA dual energy X-ray absorptio etry
- PDGF can be produced by standard recombinant DNA techniques and purified by conventional chromatography. These techniques are well known to those skilled in the art. 0.285 ml of 2 M Tris-hydrochloride and 3.41 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) were added to 36.3 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) to yield a 40 ml solution with a final pH of 7.1. From this solution, six 6-ml portions for subsequent administration were prepared and stored at -20°C.
- Alendronate (0.015 g) was dissolved in 10 ml of water containing 10 ⁇ l of 10 M sodium hydroxide. To this solution was added 30 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1), yielding a final solution with a pH of 7.1. From this solution, six 6-ml portions for subsequent administration were prepared and stored at - 20°C.
- Alendronate (0.015 g) was added to 36.3 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) along with 0.10 ml PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) and 0.20 ml of 10 M NaOH.
- PBS 20 mM phosphate, 150 mM NaCl, pH 7.1
- 0.275 ml of 2 M Tris-hydrochloride 0.275 ml of 2 M Tris-hydrochloride and 0.002 ml glacial acetic acid to yield a 40 ml solution with a final pH of 7.04. From this solution, six 6-ml portions for subsequent administration were prepared and stored at -20°C.
- Alendronate (0.015 g) was added to 3.63 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) along with 0.10 ml PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) and 0.020 ml of 10 M NaOH. To this solution was added 26.25 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) to yield a 40 ml solution with a final pH of 7.12. From this solution, six 6-ml portions for subsequent administration were prepared and stored at - 20°C.
- Ovariectomized rats were injected 3 times per week. Bone mineral was measured by DXA scanning at baseline before treatment and then at 2.5 weeks after the start of treatment. a Different from PBS, p ⁇ 0.05; b different from PBS, p ⁇ 0.001; different from PDGF, p ⁇ 0.001; d different from alendronate, p ⁇ 0.01; e different from PDGF, p ⁇ 0.01. The numbers in parentheses are the number of observations.
- Bone mineral density in the group treated with the complex of alendronate and low dose PDGF was increased over baseline 25.0% more than the corresponding change from baseline in alendronate-treated animals.
- Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 300.0% more than the corresponding change from baseline in PBS-treated animals.
- Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 100.0% more than the corresponding change from baseline in PDGF-treated animals.
- Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 50.0% more than the corresponding change from baseline in alendronate-treated animals.
- results presented above indicate that the addition of PDGF to alendronate increased bone density (hence bone formation activity) in long bones as well.
- results presented above also provide evidence for the dose-dependence of the effect of the PDGF added to alendronate. Stimulation of spinal bone growth by the combination of PDGF plus alendronate
- Ovariectomized rats were injected 3 times per week. Bone mineral was measured by DXA scanning at baseline before treatment and then at 2.5 weeks after the start of treatment.
- a Different from PBS p ⁇ 0.05; b different from PBS, p ⁇ 0.001; c different from alendronate, p ⁇ 0.001; d different from PDGF, p ⁇ 0.01; e different from alendronate, p ⁇ 0.05; f different from alendronate plus low dose PDGF, p ⁇ 0.05.
- the numbers in parentheses are the number of observations.
- Applicants have found that 2.5 weeks of treatment with either PDGF or the combination of PDGF and alendronate increased spinal bone mineral density compared with PBS-treated animals. Mineral density in PDGF-treated animals, alendronate-treated animals, alendronate plus low-dose PDGF-treated animals, and alendronate plus high-dose PDGF-treated animals was significantly increased over baseline compared with the corresponding change from baseline in PBS-treated control animals. Applicants have also found that treatment with the complex of PDGF and alendronate increased spinal bone mineral density compared with animals treated with alendronate or PDGF alone. Mineral density in low-dose PDGF plus alendronate-treated animals was increased over baseline 100.0% more than the corresponding change from baseline in alendronate-treated animals.
- compositions of the invention may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; intranasally; topically; or transdermally.
- compositions of this invention may also be formulated for implants.
- the compositions of this invention may be supplied as at least two components, such that the components are either mixed before use or used sequentially, but close enough in time to be therapeutically effective.
- a composition of the invention may be conveniently administered in unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences ((1980) Mack Pub. Co., Easton, PA).
- Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like.
- polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like.
- biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxethylene- polyoxypropylene copolymers may be useful excipients to control the release of a compound of the invention.
- Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes
- Formulations for inhalation administration may contain excipients, for example, lactose.
- Inhalation formulas may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
- Compositions for parenteral administration may also include glycocholate for buccal administration, or citric acid for vaginal administration.
- compositions described herein in a physiologically acceptable mixture will vary depending on a number of factors, including the dosage of the compound to be administered, the chemical characteristics of the compositions employed, and the route of administration.
- the compositions of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v for parenteral administration. Typical dose ranges are from about 0.001 g PDGF/kg to about 50 g PDGF/kg of body weight per day, given in 1-4 divided doses.
- the preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of bone loss, the overall health status of the particular patient, the relative biological efficacy of the composition selected, the formulation of the excipients, and its route of administration. Other embodiments are within the following claims. What is claimed is:
Abstract
The invention features pharmaceutical formulations useful for treating bone loss. The formulation may consist of a simple mixture of platelet-derived growth factor (PDGF) and a bone-targeting anionic compound of at least one negative charge at pH 6.8. The invention also features a composition containing PDGF and an anti-resorptive agent. The aforementioned compositions may also contain a second growth factor such as IGF-I, IGF-II, NGF, TGF, EGF, aFGF, bFGF, or a bone morphogenetic protein. The invention also features methods of treating bone of a mammal by systemically administering PDGF or the above-mentioned pharmaceutical formulations in an amount sufficient to enhance bone formation or regenerate or maintain bone.
Description
ADMINISTRATION OF PLATELET-DERIVED GROWTH FACTOR AND BONE SEEKING DRUGS FOR OSTEOPOROSIS AND BONE REGENERATION
Background of the Invention
The invention relates to pharmaceutical formulations useful for treating bone loss.
Bone loss in mammals commonly results from a number of conditions including, for example, osteoporosis, traumatic injuries, rheumatoid arthritis, malignancies, and periodontal diseases. Hence, it is desirable in medicine and dentistry to provide a therapeutic agent that will either prevent or treat bone loss in a mammal, e.g. a human patient. Osteoporosis is a disease process in which bone mass is progressively lost in almost all sites in the skeletal system. Post-menopausal osteoporosis (Type I) dramatically increases the rate at which bone mass decreases in women. Prior to menopause, women over the age of 35 lose cortical bone mass at a rate of 0.3 - 0.5% per year. However, after menopause, that rate increases to 2 - 3%, and the loss rate for trabecular bone mass reaches 5 - 8%. Age-related osteoporosis (Type II) affects almost all aging women, and it can affect men as well.
Currently available treatments for osteoporosis, such as estrogen or calcitonin therapy, are of limited efficacy. While these therapies may prevent the loss of bone mass due to Type I osteoporosis, the current treatments fail to reverse the effects of the disease, and they fail to halt Type II osteoporosis. Furthermore, estrogen therapy is not acceptable for women with a history of, or predisposition for, thromboembolic
disorders, diabetes, cancer, or liver disorders. Estrogen replacement therapy also is not an acceptable treatment for men with osteoporosis.
Growth factors, also referred to as tissue growth promoting factors, are polypeptides which interact with specific cell surface receptors in defined target cells, resulting in specific cell effects. Growth factors are often multifunctional, and they may either stimulate or inhibit cell proliferation. They may also affect differentiated cell function. Examples of growth factors include, but are not limited to, platelet-derived growth factors (PDGFs) (R. Ross et al. (1986) Cell 46:155-169), insulin-like growth factors (IGFs) (V.R. Sara and K. Hall (1990) Physiol. Rev. 70:591-614) fibroblast growth factors (FGFs) (D. Gospodarowicz et al. (1987)
Endocrinol. Rev. 8:95-114), epidermal growth factors (EGFs) (L.C. Read and C. George-Nascimento (1989-1990) Biotechnology Therapeutics 1:237-272), nerve growth factors (NGFs) (H. Theonen and Y.A. Barde (1980) Physiol. Rev. 60:1284-1335), transforming growth factors (TGFs) (J. assagu§ (1990) Annu. Rev. Cell Biol. 6:597-641) and bone morphogenetic proteins (BMPs) (A.J. Celeste et al. (1990) Proc. Natl. Aσad. Sci., USA 87:9843-9847). BMPs, which are part of the TGF-,9 superfamily, are characterized by their unique ability to induce osteoblastic differentiation. In general, growth factors such as PDGF, TGF-3, IGF-I, IGF-II, basic (b)-FGF, and bone morphogenetic proteins are cationic at physiological pH. Tissue growth promoting factors have been shown to promote bone formation in vitro and in vivo. For example, the combinations of PDGF and IGF-I, and PDGF and IGF-II promote bone formation or regeneration (See, for example, S. E. Lynch et al. (1991) J. Periodontol . 62:458-467; and U.S. Patents 4,861,757 and 5,019,559).
Other data indicate that IGF-I alone may be sufficient to increase bone growth (Tobias et al. (1992) Endocrinology 131:2387-2392; and Spencer et al. (1991) Bone 12:21-26). The growth factors TGF-/3 and bFGF have also been shown to increase new bone formation (M. Noda and J.J. Camilliere
(1989) Endocrinology 124:2991-2994; M.E. Joyce et al.
(1990) J. Cell Biol. 110:2195-2207; H. Mayahara et al. (1993) Growth Factors 9:73-80; J.S. Wang and P. Aspenberg (1993) Acta Orthop. Scand. 64:557-561). The BMPs, when applied within mesenchymal tissues, also increase new bone formation [A.J. Celeste et al. (1990) Proc. Natl. Acad. Sci., USA 87:9843-9847).
Members of the phosphonate class of drugs currently are under investigation for the treatment of osteoporosis (S.E. Papapoulos et al. (1992) Bone 13:S41- S40) . The bisphosphonate drugs are known to be direct inhibitors of osteoclastic bone resorption (H. Fleisch (1983) in: Bone and Mineral Research Annual 1:319-357). Examples of bisphosphonates include etidronate, clodronate, tiludronate, pamidronate, alendronate, and risedronate.
Summary of the Invention Applicants have discovered that systemic administration of PDGF increases bone mineral density in mammals, without the PDGF being covalently bonded to a bone targeting compound. Accordingly, the invention features a method for treating bone loss, which includes systemically administering to a mammal PDGF which is not covalently bonded to any other component of the composition. In preferred embodiments, the composition used in this method also includes an anti-resorptive agent.
The invention also features a pharmaceutical formulation for treating bone loss, including PDGF, a
bone-targeting anionic compound having at least one negative charge at pH 6.8, and a physiologically acceptable excipient, wherein the PDGF is not covalently bonded to any other compound in the formulation. In preferred embodiments, the association of PDGF with the bone-targeting anionic compound is sufficiently strong that the two components remain associated in vivo for a period of time sufficient for delivery of PDGF to bone by the anionic compound. The fact that PDGF and the anionic compound do not have to be covalently bonded greatly reduces the cost of producing the bone-targeting pharmaceutical formulation, as simply mixing the two components in solution is all that is required. This pharmaceutical formulation may contain in addition another growth-promoting factor, such as IGF-I, IGF-II, TGF-3, aFGF, bFGF, EGF, NGF, or a bone morphogenetic protein.
The invention also features a pharmaceutical composition consisting of PDGF and an anti-resorptive agent; this composition may also contain another growth- promoting factor.
In preferred embodiments of the invention which features PDGF and an anionic compound, the non-covalent association of PDGF and the anionic compound involves at least in part an ionic attraction between the negatively charged anionic compound and PDGF, which is positively charged at pH 6.8. The positive charge on PDGF can be increased by addition of one or more cationic groups such as amino groups, or by point mutations which substitute positively charged amino acids such as lysine for neutral or negatively-charged amino acids; these modifications can be carried out by standard techniques.
PDGF and the other growth factors useful in the invention may be derived from natural sources, produced by recombinant DNA technology, or chemically synthesized.
Preferably, the growth factor is purified and deemed to be at least 90% pure, by weight, as determined by techniques such as gel electrophoresis and amino acid sequence analysis. PDGF includes all forms of biologically active PDGF including all isoforms, homodimeric and hete^odimeric forms, and analogs.
Many compounds can serve as the bone-targeting anionic compound, including sulfonates, phosphonates, and dicarboxylates (e.g.. oxalic acids) . Preferably, the anionic compound has at least 1 negative charge at pH 6.8. More preferably, the anionic compound has at least 2 negative charges at pH 6.8. Still more preferably, the anionic compound is a bisphosphonate of the formula:
wherein: Rx is hydrogen, fluorine, chlorine, hydroxyl, or methyl; and
R2 is hydrogen, fluorine, chlorine, methyl, S- C6H5-C1, (CH2)2NH3, (CH2)3NH3, (CH2)2N(CH3)C5HU, CH2-pyridine, or NHC7H14.
Examples of bisphosphonates include, but are not limited to, etidronate, clodronate, tiludronate, pamidronate, alendronate, and risedronate. Without limiting the scope of the invention as set forth in the claims, at least one negative charge of the anionic compound interacts with PDGF, and at least one negative charge interacts with calcium on the bone.
Preferably, the anti-resorptive agent is a phosphonate, and more preferably, it is a bisphosphonate.
Methods for preparing anti-resorptives are well-known in the art.
Preferably, the formulation of the invention is administered to a mammal in an amount between about 0.001 and 50 mg/kg of the body weight of the mammal (preferably a human), more preferably in an amount between about 0.01 and 10.0 mg/kg of the body weight of the mammal.
Applicants have found that PDGF treatment of bone increases bone mineral density compared with saline treatment of bone. Applicants have also found that the combination of PDGF and an anionic compound, such as a bisphosphonate, has a greater effect on both whole skeleton bone mineral density and spinal bone mineral density (than either compound alone does) . The formulations of this invention can be used to treat or prevent osteoporosis in a mammal.
Abbreviations used herein are:
PBS = phosphate buffered saline
DXA = dual energy X-ray absorptio etry
Other features and advantages of the invention will be apparent from the following description thereof, and from the claims. The following examples are presented to more clearly set forth the invention without imposing any limits on the scope of the invention as set forth in the claims.
Detailed Description
Preparation of PDGF for Injection
PDGF can be produced by standard recombinant DNA techniques and purified by conventional chromatography. These techniques are well known to those skilled in the art. 0.285 ml of 2 M Tris-hydrochloride and 3.41 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) were added to
36.3 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) to yield a 40 ml solution with a final pH of 7.1. From this solution, six 6-ml portions for subsequent administration were prepared and stored at -20°C.
Preparation of Alendronate (a form of bisphosphonate) for Injection
Alendronate (0.015 g) was dissolved in 10 ml of water containing 10 μl of 10 M sodium hydroxide. To this solution was added 30 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1), yielding a final solution with a pH of 7.1. From this solution, six 6-ml portions for subsequent administration were prepared and stored at - 20°C.
Preparation of the Mixture of PDGF (high dose) and Alendronate for Injection
Alendronate (0.015 g) was added to 36.3 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) along with 0.10 ml PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) and 0.20 ml of 10 M NaOH. To this solution were added 3.31 ml PBS (20 mM phosphate, 150 mM NaCl, pH 7.1), 0.275 ml of 2 M Tris-hydrochloride and 0.002 ml glacial acetic acid to yield a 40 ml solution with a final pH of 7.04. From this solution, six 6-ml portions for subsequent administration were prepared and stored at -20°C.
Preparation of the Mixture of PDGF (low dose) and Alendronate for Injection
Alendronate (0.015 g) was added to 3.63 ml of 50 mM sodium acetate buffer (pH 5.00) containing PDGF (11.03 mg/ml) along with 0.10 ml PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) and 0.020 ml of 10 M NaOH. To this solution
was added 26.25 ml of PBS (20 mM phosphate, 150 mM NaCl, pH 7.1) to yield a 40 ml solution with a final pH of 7.12. From this solution, six 6-ml portions for subsequent administration were prepared and stored at - 20°C.
Stimulation of Whole Skeleton Bone Growth by the Combination of PDGF plus Alendronate
Six-month-old female Sprague-Dawley rats were obtained. Animals were ovariectomized, and baseline bone mineral measurements were obtained by DXA scanning according to the method of B. H. Mitlak et al. ((1991) J. Bone Min. Res , 6:1317-1321). Animals were injected 3 times per week via tail-vein injection with 200 μl of PBS (vehicle) , alendronate alone, PDGF alone, or alendronate plus PDGF in two doses (high and low) . At 2.5 weeks after the start of injections, bone measurements were obtained by DXA scanning. The results are given in Table 1. Data are given as means ± standard errors; the numbers in parentheses are the number of observations.
Table 1: Whole Skeleton Bone Mineral Density (g/cm2)
Group Baseline After 2.5 Weeks of Change froa Baseline
(Before Treatβent) Treataent to 2.5 Weeks
PBS 0.160 ± 0.002 (8) 0.164 ± 0.003 (8) 0.003 ± 0.001 (8)
PDGF 0.159 ± 0.002 (8) 0.165 ± 0.003 (8) 0.006 ± 0.001 (8)
Alendronate 0.157 ± 0.001 (8) 0.165 1 0.001 (8) 0.008 ± 0.001 (8)b
Alendronate plus 0.158 i 0.002 (8) 0.168 t 0.001 (8) 0.010 ± 0.001 (8)b'e PDGF (low dose)
Alendronate plus 0.161 i 0.002 (9) 0.173 t 0.003 (9)a 0.012 ± 0.001 PDGF (high dose) (0)b.c.d
Ovariectomized rats were injected 3 times per week. Bone mineral was measured by DXA scanning at baseline before treatment and then at 2.5 weeks after the start of treatment. aDifferent from PBS, p < 0.05; bdifferent from PBS, p < 0.001; different from PDGF, p < 0.001; ddifferent from alendronate, p < 0.01; edifferent from PDGF, p < 0.01. The numbers in parentheses are the number of observations.
It is readily apparent that 2.5 weeks of treatment with the combination of PDGF and alendronate increased whole skeleton bone mineral density compared with PBS-, PDGF-, and alendronate-treated animals. The analysis which follows demonstrates that PDGF and alendronate produced a greater effect on bone density than either PDGF or alendronate alone. Bone mineral density in the group treated with the complex of alendronate and low dose PDGF was increased over baseline 233.3% more than the corresponding change from baseline in PBS-treated animals. Bone mineral density in the group treated with the complex of alendronate and low dose PDGF was increased over baseline 66.7% more than the corresponding change from baseline in PDGF-treated animals. Bone
mineral density in the group treated with the complex of alendronate and low dose PDGF was increased over baseline 25.0% more than the corresponding change from baseline in alendronate-treated animals. Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 300.0% more than the corresponding change from baseline in PBS-treated animals. Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 100.0% more than the corresponding change from baseline in PDGF-treated animals. Bone mineral density in the group treated with the complex of alendronate and high dose PDGF was increased over baseline 50.0% more than the corresponding change from baseline in alendronate-treated animals.
Applicants have found that the combination of PDGF and alendronate increased bone mineral density compared with alendronate alone in the whole skeleton. Measurement of the whole skeleton is mainly reflective of cortical long bone. Even though the bisphosphonates are being studied for the treatment or prevention of osteoporosis, other data suggest that the bisphosphonates may be more effective in spinal bone than in long bone- (E. B. Silberstein and W. Schnur (1992) J". Nucl . Wed. 33:1-5; M. R. McClung and A. J. Yates (1993) J. Bone Min . Res. 8 (Suppl. 1):S141A). The results presented above indicate that the addition of PDGF to alendronate increased bone density (hence bone formation activity) in long bones as well. The results presented above also provide evidence for the dose-dependence of the effect of the PDGF added to alendronate.
Stimulation of spinal bone growth by the combination of PDGF plus alendronate
Six-month-old female Sprague-Dawley rats were obtained. Animals were ovariectomized, and baseline spinal bone mineral measurements were obtained by DXA scanning according to the method of B.H. Mitlak et al. ((1991) J. Bone Min . Res . 6:1317-1321). Animals were injected three times per week via tail-vein injection with 200 μl of PBS (vehicle) , PDGF alone, alendronate alone, or the combination of PDGF and alendronate. At 2.5 weeks after the start of injections, bone measurements were obtained by DXA scanning. The results are given in Table 2. Data are given as means ± standard errors; the numbers in parentheses are the number of observations.
TABLE 2 : Spinal Bone Mineral Density (g/cm2)
Group Baseline After 2.5 Weeks of Change free Baseline to Treat-aent 2.5 Weeks
PBS 0.252 ± '0.006 (8) 0.248 t 0.005 (8) -0.004 t 0.002 (8)
PDGF 0.250 ± 0.005 (8) 0.263 ± 0.005 (8) 0.013 ± 0.002 (8)b'e
Alendronate 0.250 ± 0.005 (8) 0.257 ± 0.004 (8) 0.007 ± 0.002 (8)b
Alendronate plus PDGF 0.249 ± 0.006 (8) 0.263 ± 0.006 (8) 0.014 t 0.002 (8)b'e (low dose)
Alendronate plus PDGF 0.251 ± 0.005 (9) 0.271 ± 0.005 (9)a 0.020 ± 0.002 (high dose) (θ)b,c,df f
Ovariectomized rats were injected 3 times per week. Bone mineral was measured by DXA scanning at baseline before treatment and then at 2.5 weeks after the start of treatment. aDifferent from PBS, p < 0.05; bdifferent from PBS, p < 0.001; cdifferent from alendronate, p < 0.001; ddifferent from PDGF, p < 0.01; edifferent from alendronate, p < 0.05; fdifferent from alendronate plus
low dose PDGF, p < 0.05. The numbers in parentheses are the number of observations.
Applicants have found that 2.5 weeks of treatment with either PDGF or the combination of PDGF and alendronate increased spinal bone mineral density compared with PBS-treated animals. Mineral density in PDGF-treated animals, alendronate-treated animals, alendronate plus low-dose PDGF-treated animals, and alendronate plus high-dose PDGF-treated animals was significantly increased over baseline compared with the corresponding change from baseline in PBS-treated control animals. Applicants have also found that treatment with the complex of PDGF and alendronate increased spinal bone mineral density compared with animals treated with alendronate or PDGF alone. Mineral density in low-dose PDGF plus alendronate-treated animals was increased over baseline 100.0% more than the corresponding change from baseline in alendronate-treated animals. Mineral density in low-dose PDGF and alendronate-treated animals was increased over baseline 7.7% more than the corresponding change from baseline in PDGF-treated animals. Mineral density in high-dose PDGF plus alendronate-treated animals was increased over baseline 185.7% more than the .corresponding change from baseline in alendronate-treated animals. Mineral density in high- dose PDGF plus alendronate-treated animals was increased over baseline 53.8% more than the corresponding change from baseline in PDGF-treated animals. The results presented above provide evidence for the efficacy of PDGF treatment and for the efficacy of treatment with PDGF delivered in combination with alendronate. These data also show the dose-;dependence of the PDGF effect in the presence of alendronate, since the change in bone mineral density over baseline in alendronate plus high dose PDGF-
treated animals was significantly higher (42.9%) than the corresponding change from baseline in alendronate plus low dose PDGF-treated animals.
Use The compositions of the invention may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; intranasally; topically; or transdermally. The compositions of this invention may also be formulated for implants. The compositions of this invention may be supplied as at least two components, such that the components are either mixed before use or used sequentially, but close enough in time to be therapeutically effective.
A composition of the invention may be conveniently administered in unit dosage form, and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences ((1980) Mack Pub. Co., Easton, PA).
Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphtalenes, and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxethylene- polyoxypropylene copolymers may be useful excipients to control the release of a compound of the invention. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes, Formulations for inhalation administration may contain excipients, for example, lactose. Inhalation formulas may be aqueous solutions containing, for
example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or they may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Compositions for parenteral administration may also include glycocholate for buccal administration, or citric acid for vaginal administration.
The concentration of a composition described herein in a physiologically acceptable mixture will vary depending on a number of factors, including the dosage of the compound to be administered, the chemical characteristics of the compositions employed, and the route of administration. In general terms, the compositions of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v for parenteral administration. Typical dose ranges are from about 0.001 g PDGF/kg to about 50 g PDGF/kg of body weight per day, given in 1-4 divided doses. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of bone loss, the overall health status of the particular patient, the relative biological efficacy of the composition selected, the formulation of the excipients, and its route of administration. Other embodiments are within the following claims. What is claimed is:
Claims
1. A pharmaceutical formulation effective for treating bone in a mammal, comprising a) platelet-derived growth factor (PDGF) ; b) a physiologically acceptable excipient; and c) a bone-targeting anionic compound having at least 1 negative charge at pH 6.8, wherein said PDGF is not covalently bonded to the excipient or the bone targeting anionic compound.
2. The formulation of claim 1, wherein the association of said platelet-derived growth factor with said anionic compound is sufficiently strong that they remain associated in vivo for a period of time sufficient for delivery of a bone therapeutically effective amount of said platelet-derived growth factor to bone by said anionic compound.
3. The formulation of claim 2, wherein said anionic compound has at least 2 negative charges at pH 6.8.
4. The formulation of claim 2, wherein said anionic compound is selected from the group consisting of sulfonates, phosphonates, and dicarboxylate.
5. The formulation of claim 4, wherein said phosphonate is a bisphosphonate.
6. The formulation of claim 2, further comprising a compound selected from the group consisting of IGF-I,
IGF-II, aFGF, bFGF, NGFs, EGFs, TGFs, and bone morphogenetic proteins.
7. The formulation of claim 6, wherein said compound is IGF-I.
8. A method of treating a bone of a mammal, said method comprising systemically administering to said mammal a therapeutically effective amount of a composition comprising PDGF which is not covalently bonded to any other component of said composition.
9. The method of claim 8, wherein said composition further comprises an anti-resorptive agent.
10. The method of claim 8, wherein said mammal suffers from osteoporosis.
11. The method of claim 8, wherein said composition is administered to said mammal in an amount between about 0.001 and 50 mg PDGF/kg of the body weight of said mammal.
12. The method of claim 11, wherein said formulation is administered to said mammal in an amount between about 0.01 and 10.0 mg PDGF/kg of the body weight of said mammal.
13. A composition comprising platelet-derived growth factor and an anti-resorptive agent, wherein the association of said platelet-derived growth factor with said anti-resorptive agent is noncovalent.
14. The composition of claim 13, wherein said anti-resorptive agent is a phosphonate.
15. The composition of claim 14, wherein said phosphonate is a bisphosphonate. - 17 -
16. The composition of claim 13, further comprising a compound selected from the group consisting of IGF-I, IGF-II, aFGF, bFGF, NGFs, EGFs, TGFs, and bone morphogenetic proteins.
17. The composition of claim 16, wherein said compound is IGF-I.
18. A method of treating a bone of a mammal, said method comprising administering to said mammal a therapeutically effective amount of the composition of claim 13.
19. The method of claim 18, wherein administration of platelet-derived growth factor and administration of anti-resorptive agent occur close enough in time to be therapeutically effective.
20. The method of claim 18, wherein administration of platelet-derived growth factor and administration of anti-resorptive agent occur close enough in time to act synergistically.
21. The method of claim 18, wherein said platelet-derived growth factor and said anti-resorptive agent are administered to said mammal in an amount between about 0.001 and 50 mg PDGF/kg of the body weight of said mammal.
22. The method of claim 21, wherein said platelet-derived growth factor and said anti-resorptive agent are administered to said mammal in an amount between about 0.01 and 10.0 mg PDGF/kg of the body weight of said mammal.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU22973/95A AU2297395A (en) | 1994-04-20 | 1995-04-18 | Administration of platelet-derived growth factor and bone seeking drugs for osteoporosis and bone regeneration |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US23011494A | 1994-04-20 | 1994-04-20 | |
US08/230,114 | 1994-04-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995028950A1 true WO1995028950A1 (en) | 1995-11-02 |
Family
ID=22864006
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/005047 WO1995028950A1 (en) | 1994-04-20 | 1995-04-18 | Administration of platelet-derived growth factor and bone seeking drugs for osteoporosis and bone regeneration |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2297395A (en) |
WO (1) | WO1995028950A1 (en) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000071148A2 (en) * | 1999-05-26 | 2000-11-30 | The Brigham And Women's Hospital, Inc. | Therapeutic uses of agents that modulate the activity of alpha-smooth muscle actin |
WO2001032197A2 (en) * | 1999-11-02 | 2001-05-10 | Eli Lilly And Company | Methods of using lp8, a pdgf-related protein, to treat musculoskeletal disorders |
US6710025B1 (en) | 1999-05-26 | 2004-03-23 | The Brigham And Women's Hospital, Inc. | Treatment of damaged tissue using agents that modulate the activity of alpha-smooth muscle actin |
AU2005295919B2 (en) * | 2004-10-14 | 2009-03-26 | Biomimetic Therapeutics, Inc. | Platelet-derived growth factor compositions and methods of use thereof |
US8257737B2 (en) * | 2001-08-09 | 2012-09-04 | Giuseppe Intini | Tissue implants and methods for making and using same |
US8399409B2 (en) | 2006-11-03 | 2013-03-19 | Biomimetic Therapeutics Inc. | Compositions and methods for arthrodetic procedures |
WO2014087037A1 (en) * | 2012-12-03 | 2014-06-12 | Servicio Andaluz De Salud | Use of the growth factor derived from platelets or a selective agonist of the at2 receptor for the treatment of vascular calcifications |
US8870954B2 (en) | 2008-09-09 | 2014-10-28 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries |
US9161967B2 (en) | 2006-06-30 | 2015-10-20 | Biomimetic Therapeutics, Llc | Compositions and methods for treating the vertebral column |
US9642891B2 (en) | 2006-06-30 | 2017-05-09 | Biomimetic Therapeutics, Llc | Compositions and methods for treating rotator cuff injuries |
US10071182B2 (en) | 2014-10-14 | 2018-09-11 | Samuel E. Lynch | Methods for treating wounds |
US10258566B2 (en) | 2004-10-14 | 2019-04-16 | Biomimetic Therapeutics, Llc | Compositions and methods for treating bone |
US11235030B2 (en) | 2010-02-22 | 2022-02-01 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods for the treatment of tendinopathies |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4861757A (en) * | 1986-11-14 | 1989-08-29 | Institute Of Molecular Biology | Wound healing and bone regeneration using PDGF and IGF-I |
US5118667A (en) * | 1991-05-03 | 1992-06-02 | Celtrix Pharmaceuticals, Inc. | Bone growth factors and inhibitors of bone resorption for promoting bone formation |
US5158934A (en) * | 1989-09-01 | 1992-10-27 | Genentech, Inc. | Method of inducing bone growth using TGF-β |
-
1995
- 1995-04-18 AU AU22973/95A patent/AU2297395A/en not_active Abandoned
- 1995-04-18 WO PCT/US1995/005047 patent/WO1995028950A1/en active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4861757A (en) * | 1986-11-14 | 1989-08-29 | Institute Of Molecular Biology | Wound healing and bone regeneration using PDGF and IGF-I |
US5158934A (en) * | 1989-09-01 | 1992-10-27 | Genentech, Inc. | Method of inducing bone growth using TGF-β |
US5118667A (en) * | 1991-05-03 | 1992-06-02 | Celtrix Pharmaceuticals, Inc. | Bone growth factors and inhibitors of bone resorption for promoting bone formation |
Non-Patent Citations (2)
Title |
---|
JOURNAL OF PERIODONTOLOGY, Volume 62, Number 11, issued November 1991, S.E. LYNCH et al., "Effects of the Platelet-Derived Growth Factor/Insulin-Like Growth Factor-I Combination on Bone Regeneration Around Titanium Dental Implants. Results of a Pilot Study in Beagle Dogs", pages 710-716. * |
JOURNAL OF PERIODONTOLOGY, Volume 62, Number 7, issued July 1991, S.E. LYNCH et al., "The Effects of Short-Term Application of a Combination of Platelet-Derived and Insullin Like Growth Factors on Periodontal Wound Healing", pages 458-467. * |
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000071148A3 (en) * | 1999-05-26 | 2001-09-27 | Brigham & Womens Hospital | Therapeutic uses of agents that modulate the activity of alpha-smooth muscle actin |
US6710025B1 (en) | 1999-05-26 | 2004-03-23 | The Brigham And Women's Hospital, Inc. | Treatment of damaged tissue using agents that modulate the activity of alpha-smooth muscle actin |
WO2000071148A2 (en) * | 1999-05-26 | 2000-11-30 | The Brigham And Women's Hospital, Inc. | Therapeutic uses of agents that modulate the activity of alpha-smooth muscle actin |
WO2001032197A2 (en) * | 1999-11-02 | 2001-05-10 | Eli Lilly And Company | Methods of using lp8, a pdgf-related protein, to treat musculoskeletal disorders |
WO2001032197A3 (en) * | 1999-11-02 | 2002-03-07 | Lilly Co Eli | Methods of using lp8, a pdgf-related protein, to treat musculoskeletal disorders |
US8257737B2 (en) * | 2001-08-09 | 2012-09-04 | Giuseppe Intini | Tissue implants and methods for making and using same |
US10258566B2 (en) | 2004-10-14 | 2019-04-16 | Biomimetic Therapeutics, Llc | Compositions and methods for treating bone |
AU2005295919B2 (en) * | 2004-10-14 | 2009-03-26 | Biomimetic Therapeutics, Inc. | Platelet-derived growth factor compositions and methods of use thereof |
US11571497B2 (en) | 2004-10-14 | 2023-02-07 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods of use thereof |
US11364325B2 (en) | 2004-10-14 | 2022-06-21 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods of use thereof |
US11318230B2 (en) | 2004-10-14 | 2022-05-03 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods of use thereof |
US9545377B2 (en) | 2004-10-14 | 2017-01-17 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods of use thereof |
US9161967B2 (en) | 2006-06-30 | 2015-10-20 | Biomimetic Therapeutics, Llc | Compositions and methods for treating the vertebral column |
US9642891B2 (en) | 2006-06-30 | 2017-05-09 | Biomimetic Therapeutics, Llc | Compositions and methods for treating rotator cuff injuries |
US10456450B2 (en) | 2006-06-30 | 2019-10-29 | Biomimetic Therapeutics, Llc | Compositions and methods for treating rotator cuff injuries |
US11058801B2 (en) | 2006-06-30 | 2021-07-13 | Biomimetic Therapeutics, Llc | Compositions and methods for treating the vertebral column |
US8399409B2 (en) | 2006-11-03 | 2013-03-19 | Biomimetic Therapeutics Inc. | Compositions and methods for arthrodetic procedures |
US11135341B2 (en) | 2008-09-09 | 2021-10-05 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor composition and methods for the treatment of tendon and ligament injuries |
US8870954B2 (en) | 2008-09-09 | 2014-10-28 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods for the treatment of tendon and ligament injuries |
US11235030B2 (en) | 2010-02-22 | 2022-02-01 | Biomimetic Therapeutics, Llc | Platelet-derived growth factor compositions and methods for the treatment of tendinopathies |
WO2014087037A1 (en) * | 2012-12-03 | 2014-06-12 | Servicio Andaluz De Salud | Use of the growth factor derived from platelets or a selective agonist of the at2 receptor for the treatment of vascular calcifications |
US10071182B2 (en) | 2014-10-14 | 2018-09-11 | Samuel E. Lynch | Methods for treating wounds |
Also Published As
Publication number | Publication date |
---|---|
AU2297395A (en) | 1995-11-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2831132B2 (en) | Bone growth factors and bone resorption inhibitors for promoting bone formation | |
EP0527787B1 (en) | Method of predisposing mammals to accelerated tissue repair | |
EP0436469B1 (en) | Composition and method for the treatment of osteoporosis in mammals | |
US9333237B2 (en) | Bone morphogenic protein binding peptide | |
US9855368B2 (en) | Bone morphogenic protein binding peptide | |
US9610320B2 (en) | Surgical applications for BMP binding protein | |
CA2589930C (en) | Activating extraction of demineralized bone matrix | |
WO1995028950A1 (en) | Administration of platelet-derived growth factor and bone seeking drugs for osteoporosis and bone regeneration | |
JPH09510209A (en) | Use of fibroblast growth factor to stimulate bone growth | |
AU2007338627A1 (en) | Surgical applications for BMP binding protein | |
EP0499242B1 (en) | Stimulation of endosteal bone formation with a cell growth factor | |
WO1993010810A1 (en) | Bone regeneration | |
EP0672420A1 (en) | Wound remedy |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NO NZ PL PT RO RU SD SE SI SK TJ TT UA UZ VN |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
NENP | Non-entry into the national phase |
Ref country code: CA |
|
122 | Ep: pct application non-entry in european phase |