IE921812A1 - Therapeutic compositions for osteoinduction - Google Patents

Abstract

A method for generating new bone growth in a mammal comprising administrating to the mammal a safe and effective amount of a Vitamin D compound in combination with a safe and effective amount of osteoinductive extract or at least one BMP.

Description

THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION ABSTRACT A method for generating new bone growth in a mammal comprising administrating to the mammal a safe and effective amount of a Vitamin D compound in combination with a safe and effective amount of osteoinductive extract or at least one BMP.

PATENTS ACT, 1964 COMPLETE SPECIFICATION THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION THE PROCTER & GAMBLE COMPANY, a Company organised under the laws of the State of Ohio, United States of America, of One Procter & Gamble Plaza, Cincinnati, Ohio 45202, United States of America. -1 IE 921812 4407R THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION The present invention relates to the field of osteoinduction (bone growth). Specifically, the present invention relates to novel therapeutic formulations comprising the administration of bone morphogenetic proteins and a Vitamin D compound, resulting in synergistic bone growth In healthy individuals bone growth generally proceeds normally and fractures heal without the need for pharmacologic intervention. Nonetheless, in certain instances bones may be weakened or may fail to heal properly. For example, healing may proceed slowly in the elderly and in patients undergoing treatment with corticosteroids, such as transplant patients and those being treated for chronic lung disease. Another example is osteoporosis. Osteoporosis is an abnormal loss of bony tissue often occurring in post-menopausal woman and elderly men. The disorder increases the risks of small fractures occurring in the bones, particularly the spine. At present, osteoporosis is treated mainly by supplements of calcium, vitamin D, estrogen, or calcitonin, a hormone which controls the body's use of calcium.

Unfortunately, these treatments are merely preventative against the further loss of bone. There is a need in the art for treatments that go beyond the prevention of bone loss and promote bone formation and/or reverse bone loss. (1989) Bone Morphogenic Proteins and Vitamin D, Nutrition Reviews, Vol. 47, pp. 364-366 concludes that Vitamin D in the diet prevents the loss of the osteoinductive activity of bone matrix.

Turner, R. T., J. Farley, J. J. Vandersteenhoven, S. Epstein, N. H. Bell, and D. J. Baylink, (1988) Demonstration of Reduced Mitogenic and Osteoinductive Activities in Demineralized Allo35 geneic Bone Matrix from Vitamin D-deficient Rats, The Journal of Clinical Investigation, Inc., Vol. 82, pp. 212-217, discloses the - 2 implantation of demineralized bone matrix from Vitamin D-deficient rats into normal rats. The demineralized bone matrix from Vitamin D-deficient rats did not promote osteoinduction as effectively as demineralized bone matrix from normal rats.

Sampath, T. K., S. Weintraub, and A. H. Reddi, (1984) Extracellular Matrix Proteins Involved in bone Induction are Vitamin D Dependent, Biochemical and Biophysical Research Communications. Vol. 124, pp. 829-835, discloses a study involving implantation of demineralized bone matrix from normal rats and demineralized bone matrix from rachitic rats wherein the rachitic bone matrix did not induce bone growth while the normal bone matrix did. The study concluded that these results demonstrate that Vitamin D is necessary to produce bone inductive proteins in the bone matrix of a living rat.

U.S. Patent No. 4,761,471, Urist, assigned to the Regents of the University of California, issued August 2, 1988, discloses a bone morphogenetic protein composition comprising BMP factor and BMP associated protein having a molecular weight of 34,000 daltons. Use of such factors and compositions to induce bone formation in mammals is also disclosed.

U.S. Patent No. 4,455,256, Urist, assigned to the Regents of the University of California, issued June 19, 1984, discloses a bone morphogenetic protein having a molecular weight in the range of 1,000 to 100,000 daltons.

Various other bone morphogenetic proteins/factors, osteoinductive factors, osteogenic factors and other proteins/factors related to bone growth are disclosed in the following publications: U.S. Patent 4,968,590, Kubersampath and Rueger, issued November 6, 1990; U.S. Patent 4,698,328, Neer, Potts and Slovik, issued October 6, 1987; U.S. Patent 4,877,864, Wang, Wozney and Rosen, issued October 31, 1989; U.S. Patent 4,861,757, Antoniades, Lynch and Williams, issued August 29, 1989; U.S. Patent 4,810,691, Seyedin, Thomas, Bentz, Ellingsworth and Armstrong, issued March 7, 1989; U.S. Patent 4,804,744, Sen, issued February 14, 1989; U.S. Patent 4,795,804, Urist, issued January 3, 1989; U.S. Patent 4,789,663, Wallace, Smestad, McPherson, Piez and Ross, issued December 6, 1988; U.S. Patent 4,789,732, Urist, issued December 6, 1988; U.S. Patent 4,774,322, Seyedin, Thomas, Bentz, Ellingsworth and Armstrong, issued September 27, 1988; U.S. Patent 4,698,328, - 3 Neer and Slovik, issued October 6, 1987; U.S. Patent 4,627,982, Seydin and Thomas, issued December 9, 1986; U.S. Patent 4,619,989, Urist, issued October 28, 1986; U.S. Patent 4,596,574, Urist, issued June 24, 1986; U.S. Patent 4,563,489, Urist, issued January 7, 1986; U.S. Patent 4,563,350, Nathan, Seyedin and Bentz, issued January 7, 1986; U.S. Patent 4,526,909, Urist, issued July 2, 1985; U.S. Patent 4,434,894, Seyedin and Thomas, issued February 23, 1984; U.S. Patent 4,294,753, Urist, issued October 13, 1981; European Patent Application 349 048, Bab, Muhlrad, Gazit and Shteyer, published January 3, 1990; European Patent Application 309 241, Chu, Nathan and Seyedin, published March 29, 1989; European Patent Application 336 760, Bentz, Nathan, Rosen, Oasch and Seyedin, published October 11, 1989; European Patent Application 145 155, Sen, published July 10, 1985; World Patent Applica15 tion 89/10934, Roos, Burns, Guy and McKnight, published November 16, 1989; World Patent Applications 89/09787 and 89/09788, Oppermann, Kubersampath, Rueger and Ozkaynak, published October 19, 1989; and World Patent Application 88/00205, Wang, Wozney and Rosen, published Janaury 14, 1988.

OBJECTS OF THE PRESENT INVENTION It is an object of the present invention to provide a method for generating new bone growth in a mammal.

It is a further object of the present invention to provide a pharmaceutical composition which can be used to generate new bone growth in a mammal.

SUMMARY OF THE INVENTION The present invention relates to a method of generating new bone growth in mammals comprising administration to a mammal a combination of a safe and effective amount of a Vitamin D compound, and a safe and effective amount of one or more BMPs or osteoinductive extract comprising one or more BMPs.

The present invention further relates to a composition for generating new bone growth in mammals comprising a safe and effective amount of a Vitamin D compound; a safe and effective amount of a BMP or osteoinductive extract comprising one or more BMPs; and a pharmaceutically-acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION The present invention comprises the administration to a mammal of a combination of a safe and effective amount of a - 4 Vitamin D compound and a safe and effective amount of one or more BMPs or an osteoinductive extract comprising one or more BMPs. It has been determined that treatment with a Vitamin D compound, BMP or osteoinductive extract alone increases bone growth. Surpris5 ingly, it has been further determined that treatment with a Vitamin 0 compound in combination with osteoinductive extract or in combination with at least one BMP results in a level of new bone growth greater than that achieved through administration of the BMP, osteoinductive extract or Vitamin D compound alone.

Subjects in need of such treatment suffer from a variety of ailments which may be treated via this procedure, including but not limited to, bone fractures (closed and open), non-union fractures, congenital defects, as an adjunct in plastic surgery, in treating oncological resections, all diseases classified as osteoporosis, rheumetoid arthritis, osteoarthritis, septic arthritis, rickets, organic incorporation of prosthetic joints and dental implants, periodontal disease and defects, as well as osteopenic and osteomalacic conditions and disease.

As used herein, safe and effective amount means an amount of compound or composition sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The safe and effective amount of the compound or composition will vary with the particular condition being treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the specific compound or composition employed, the particular pharmaceutically-acceptable carrier utilized, and like factors within the knowledge and expertise of the attending physician.

As used herein, fracture reduction means the restoration of a bone fracture by surgical or manipulative means to its normal anatomical relation.

As used herein, BMP means bone morphogenetic protein.

As used herein, q.s. means quantity sufficient.

As used herein, all percentages are by weight unless otherwise specified.

As used herein regional treatment includes treating bone fractures (closed and open), treating non-union fractures, - 5 treating congenital defects, as an adjunct treatment to plastic surgery, treating oncological resections, organic incorporation of prosthetic joints, organic incorporation of dental implants, and treatment of periodontal disease and defects.

As used herein systemic treatment includes treating diseases classified as osteoporosis, rheumatoid arthritis, osteoarthritis, septic arthritis, rickets, and osteopenic conditions and diseases.

As used herein, all dose ranges for systemic treatment are recited as the dry weight of the actives per kg body weight of the mammal.

As used herein, all dose ranges for regional treatment are recited as the dry weight of the actives per cm2 surface area of mineralized tissue to be treated.

As used herein, mineralized tissue means bone and teeth.

Vitamin D Compounds One component involved in the method of the invention is a Vitamin D compound. As used herein, Vitamin D compound includes Vitamin D, ergocalciferol (Vitamin 02), cholecalciferol (Vitamin D3) and their biologically active metabolites and precursors.

Preferred Vitamin D compounds include, but are not limited to, Vitamin 02 (Sigma, St. Louis, MO), Vitamin D3 (Sigma, St. Louis, MO), Ι-α-hydroxy Vitamin D3, Ι-α-fluoro Vitamin D3, 3-deoxy-l,25dihydroxy Vitamin D3, 25-hydroxy-5,6-trans Vitamin D3, 25-hydroxy Vitamin D2, 25-hydroxy Vitamin D3 (Hoffman LaRoche), 1,25-dihydroxy Vitamin O2, 24,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D3 (Hoffman LaRoche), and 1,25-dihydroxy Vitamin 03 (Duphar, Veenendaal, Holland). Preferably, the Vitamin D compound is selected from 25-hydroxy Vitamin D2, 25-hydroxy Vitamin 03, 1,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D3, and 1,25-dihydroxy Vitamin 03, more preferably 1,25-dihydroxy Vitamin D3. Additional Vitamin D compounds useful in the present invention are well known to those skilled in the art and include, but are not limited to, those disclosed by the following U.S. Patents, each of which is incorporated herein by reference: U.S. Patent 4,970,203, DeLuca and Kwiecinski, issued November 13, 1990; U.S. Patent 4,927,815, DeLuca, Kutner, Perlman and Schnoes, issued May 22, 1990; U.S. Patent 4,857,518, DeLuca, - 6 Ikekawa and Tanaka, issued August 15, 1989; U.S. Patent 4,851,401, DeLuca, Kutner, Perlman and Schnoes, issued July 25, 1989; U.S. Patent 4,851,400, DeLuca, Ikekawa and Tanaka, issued July 25, 1989; U.S. Patent 4,847,012, DeLuca, Kutner, Perlman, Phelps, Schnoes and Sicinski, issued July 11, 1989; U.S. Patent 4,816,417, Dame, DeLuca and Pierce, issued March 28, 1989; U.S. Patent 4,769,181, DeLuca, Schnoes, Sicinski and Tanaka, issued September 6, 1988; U.S. Patent 4,755,329, DeLuca, Lee and Schnoes, issued July 5, 1988; U.S. Patent 4,719,205, DeLuca, Schnoes, Sicinski and Tanaka, issued January 12, 1988; U.S. Patent 4,719,204, DeLuca, Schnoes, Sicinski and Tanaka, issued January 12, 1988; U.S. Patent 4,717,721, DeLuca, Ikekawa, Ostrem and Schnoes, issued January 5, 1988; U.S. Patent 4,689,180, DeLuca, Schnoes, Sicinski and Tanaka, issued August 25, 1987; U.S. Patent 4,619,920, DeLuca, Ikekawa, Kobayashi and Tanaka, issued October 28, 1986; U.S. Patent 4,594,192, DeLuca, Ikekawa, Kobayashi and Tanaka, issued June 10, 1986; U.S. Patent 4,588,716, DeLuca and Schnoes, issued May 13, 1986; U.S. Patent 4,588,528, DeLuca, Ikekawa and Tanaka, issued May 13, 1986; U.S. Patent 4,564,474, DeLuca, Ikekawa, Kobayashi and Tanaka, issued January 14, 1986; U.S. Patent 4,555,364, DeLuca, Lee, Phelps and Schnoes, issued November 26, 1985; U.S. Patent 4,554,106, DeLuca, Lee, Phelps and Schnoes, issued November 19, 1985; U.S. Patent 4,552,698, DeLuca, Ikekawa, Kobayashi and Tanaka, issued November 11, 1985; U.S. Patent 4,512,925, DeLuca, Lee and Schnoes, issued April 23, 1985; U.S. Patent 4,505,906, DeLuca, Schnoes, Sicinski and Tanaka, issued March 19, 1985; U.S. Patent 4,502,991, DeLuca, Ikekawa, Kobayashi and Tanaka, issued March 5, 1985; U.S. Patent 4,500,460, DeLuca, Ikekawa, Kobayashi and Tanaka, issued February 19, 1985; U.S. Patent 4,481,198, Chu, DeLuca, Kabakoff and Schnoes, issued November 6, 1984; U.S. Patent 4,461,766, DeLuca, Hart and Schnoes, issued July 24, 1984; U.S. Patent 4,448,726, DeLuca, Paaren, Schnoes and Smith, issued May 15, 1984; U.S. Patent 4,448,721, DeLuca, Morzycki and Schnoes, issued May 15, 1984; U.S. Patent 4,428,946, DeLuca, Jorgensen and Schnoes, issued January 31, 1984; U.S. Patent 4,411,833, DeLuca, Ikekawa, Kobayashi and Tanaka, issued October 25, 1983; U.S. Patent 4,367,177, DeLuca, Schnoes and Wichman, issued January 4, 1983; U.S. Patent 4,358,406, DeLuca, Ikekawa, Kobayashi and Tanaka, issued November 9, 1982; U.S. Patent 4,338,312, DeLuca, - 7 Jorgensen and Schnoes, issued July 6, 1982; U.S. Patent 4,338,250, DeLuca, Hamer, Paaren and Schnoes, issued July 6, 1982; U.S. Patent 4,336,193, DeLuca, Fivizzani, Paaren, Schnoes and Wichmann, issued June 22, 1982; U.S. Patent 4,313,942, DeLuca, Frank, Paaren and Schnoes, issued February 2, 1982; U.S. Patent 4,307,231, DeLuca, Paaren, Schnoes, Tanaka and Wichmann, issued December 22, 1981; U.S. Patent 4,307,025, DeLuca, Ikekawa, Morisaki, Oshida, Schnoes and Tanaka, issued December 22, 1981; U.S. Patent 4,305,880, DeLuca, Ikekawa, Kobayashi and Tanaka, issued December , 1981; U.S. Patent 4,297,289, DeLuca, Fivizzani, Paaren and Schnoes, issued October 27, 1981; U.S. Patent 4,292,250, DeLuca, Levan and Schnoes, issued September 29, 1981; U.S. Patent 4,265,822, DeLuca, Hamer, Paaren and Schnoes, issued May 5, 1981; U.S. Patent 4,264,513, DeLuca, Fivizzani, Napoli and Schnoes, issued April 28, 1981; U.S. Patent 4,263,214, DeLuca, Napoli, Onisko and Schnoes, issued April 21, 1981; U.S. Patent 4,260,804, DeLuca, Esvelt and Schnoes, issued April 7, 1981; U.S. Patent 4,260,549, DeLuca, Hamer, Paaren and Schnoes, issued April 7, 1981; U.S. Patent 4,254,045, DeLuca, Ikekawa, Morisaki, Oshida and Tanaka, issued March 3, 1981; U.S. Reissue Patent 30,538, DeLuca, Lam and Schnoes, issued March 3, 1981; U.S. Patent 4,248,791, DeLuca, Ikekawa, Kobayashi and Tanaka, issued February 3, 1981; U.S. Patent 4,234,495, DeLuca, Hamer, Paaren and Schnoes, issued November 18, 1980; U.S. Patent 4,230,627, DeLuca, Napoli, Onisko and Schnoes, issued October 28, 1980; U.S. Patent 4,229,359, Alper, DeLuca, Schnoes and Tanaka, issued October 21, 1980; U.S. Patent 4,229,358, DeLuca, Napoli, Onisko and Schnoes, issued October 21, 1980; U.S. Patent 4,229,357, DeLuca, Napoli, Onisko and Schnoes, issued October 21, 1980; U.S. Patent 4,226,788, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued October 7, 1980; U.S. Patent 4,226,787, DeLuca, Napoli, Onisko and Schnoes, issued October 7, 1980; U.S. Patent 4,224,231, Alper, DeLuca, Schnoes and Tanaka, issued September 23, 1980; U.S. Patent 4,224,230, DeLuca, Napoli, Onisko and Schnoes, issued September 23, 1980; U.S. Patent 4,223,131, DeLuca, Schnoes and Wichman, issued September 16, 1980; U.S. Patent 4,217,288, DeLuca, Onisko and Schnoes, issued August 12, 1980; U.S. Patent 4,209,634, DeLuca, Esvelt and Schnoes, issued June 24, 1980; U.S. Patent 4,202,829, DeLuca, Hamer, Paaren and Schnoes, issued May 13, 1980; - 8 U.S. Patent 4,201,881, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued May 6, 1980; U.S. Patent 4,196,133, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued April 1, 1980; U.S. Patent 4,195,027, DeLuca, Hamer, Paaren and Schnoes, issued March , 1980; U.S. Patent 4,188,345, DeLuca, Napoli, Oniski and Schnoes, issued February 12, 1980; and U.S. Patent 3,906,014, DeLuca, Lam and Schnoes, issued September 16, 1975. Additional Vitamin D compounds useful in the present invention and disclosed by these references include, but are not limited to, hydroxylated 24-homo-vitamin D; cyclopentano-vitamin D; hydroxylated 26-homo vitamin D; 1 α-hydroxyvitamin D; 1-hydroxyvitamin D; 1 o-hydroxyvitamin D2; 1 a,25-dihydroxy-22Z-dehydroxyvitamin D; 26,26,26,27.27- pentafluoro-l a-hydroxy-27-methoxyvitamin D3; 2 a-fluorovitamin D3; 1,24-dihydroxy-delta 22-vitamin D3; 23,23-difluoro15 25-hydroxy-vitamin D3; l-hydroxy-3,5-cyclovitamin D; 23,23-difluoro-1 e,25-dihydroxy-vitamin D3; 1,23-dihydroxyvitamin D; hydroxyvitamin D2; 23,23-difluoro-l a,25-dihydroxy-vitamin D3; 23.23- difluoro-25-hydroxy-vitamin D3; 26,26,26,27,27,27-hexafluoro-1 a,25-dihydroxycholesterol; 23,25-dihydroxyvitamin D3; 26,26,26,27,27,27-hexafluoro-l a,25-dihydroxycholecalciferol; 1 a,25-dihydroxy-2 /l-fluorovitamin D3; 24-fluoro-25-hydroxycholecalciferol; 5,6-trans-vitamin D; 1 a-hydroxy-25-keto-27-nor-cholecalciferol; fluorovitamin D; 1 a-hydroxy-2 /J-fluorocholecalciferol; 3-deoxy-l α-hydroxychol ecalciferol; 25-hydroxy-26,26,26,25 27,27,27-hexafluorocholecaliferol; a-hydroxy-3,5-cyclovitamin D; -hydroxycholecalciferol; 24,24-difluoro-l a,25-dihydroxycholecalciferol ; 25-hydroxycholecalciferol; 25-hydroxycholecalciferol 26.23- lactone; 24,24-difluoro-la,25-dihydroxycholecalciferol; 24.24- difluoro-25-hydroxycholecalciferol; 3,5-cyclovitamin D; and 3-deoxy-a-hydroxycholecalciferol. Additional Vitamin D compounds useful in the present invention further include those disclosed in The Handbook of Vitamins. L. J. Maehl in, Ed., Mercel Dekker, Inc. (1984), incorporated herein by reference. Vitamin D compounds useful in the present invention disclosed by this reference, include, bur are not limited to, 1,25-dihydroxy Vitamin D, 3-deoxy-1,25-dihydroxy Vitamin D, 27-nor-25-hydroxy Vitamin D3, 26.27- bis-nor-25-hydroxy Vitamin D3 24-nor-25-hydroxy Vitamin D3, 25-hydroxy Vitamin D, 1,25-dihydroxy Vitamin D, Ια-hydroxy Vitamin D3 and 25-fluoro-Ια-hydroxy Vitamin D3. - 9 A safe and effective amount of a Vitamin D compound is dosed in combination with at least one BMP or in combination with an osteoinductive extract comprising at least one BMP.

A preferred dose range for administration of the Vitamin D 5 compound for systemic treatment is from about 1 ng to about 1 mg, preferably from about 10 ng to about 500 pg, more preferably from about 20 ng to about 10 pg.

For purposes of regional treatment, the dose range of the Vitamin D compound is preferably from about 1 ng to about 1 mg, preferably from about 10 ng to about 500 ng, more preferably from about 10 ng to about 50 ng, most preferably from about 20 ng to about 30 ng.

Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.

Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day.

Bone Morphogenetic Proteins In one embodiment of the present invention, a Vitamin D compound is administered in combination with one or more BMPs to generate new bone growth in a mammal. These BMPs are preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7.

A safe and effective amount of a BMP, preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, is dosed in combination with a Vitamin D compound.

A preferred dose range for administration of the BMP for systemic treatment is from about 1 pg to about 100 pg, preferably from about 1 ng to about 10 pg, more preferably from about 10 ng to about 2.5 pg.

For purposes of regional treatment, a preferred dose range for the BMP is from about 1 pg to about 100 pg, more preferably from about 1.5 pg to about 90 pg, preferably from about 1.8 pg to about 75 pg, more preferably from about 2.0 pg to about 50 pg, more preferably still from about 2.2 pg to about 25 pg, more preferably from about 2.3 pg to about 10 pg, most preferably from about 2.5 pg to about 5 pg. Preferably the dose range is at least about 2.5 pg. - 10 Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month. Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day.

As used herein, BMP-1 means a peptide encoded by a DNA sequence comprising SEQ ID N0:l. As used herein, BMP-2 means a peptide encoded by a DNA sequence comprising SEQ ID N0:2. As used herein, BMP-3 means a peptide encoded by a DNA sequence comprising SEQ ID N0:3. As used herein, BMP-4 means a peptide encoded by a DNA sequence comprising SEQ ID NO:4. As used herein, BMP-5 means a peptide encoded by a DNA sequence comprising SEQ ID N0:5. As used herein, BMP-6 means a peptide encoded by a DNA sequence comprising SEQ ID NO: 6. As used herein, BMP-7 means a peptide encoded by a DNA sequence comprising SEQ ID NO: 7.

As used herein, A, T, G, and C refer to the nucleotides containing adenine, thymine, guanine and cytosine respectively.

Osteoinductive Extract Another component of the invention is an osteoinductive extract. As used herein, osteoinductive extract means a chemical extract of bone, comprising one or more various bone morphogenetic proteins, including, but not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, wherein each BMP has a molecular weight of from about 28,000 to about 40,000 daltons.

The 28,000 to 40,000 dalton molecular weight range is in reference to the BMP's dimer weight. Preferably, the molecular weight of the dimer is from about 30,000 to about 34,000 daltons. The BMP dimer comprises two monomers, each having a molecular weight of from about 14,000 to about 20,000 daltons, preferably from about 15,000 to about 17,000 daltons.

A preferred method of obtaining the osteoinductive extract 1s as follows: Snip the skin at the ankles of a 7-8 week old Long-Evans rat (Charles River laboratories, Wilmington, MA). Remove both tibiae - 11 and place in cold water. Rinse the bone with distilled water to remove non-osseous tissue (tissue other than bone). Allow the bone to air dry. Grind the bones by placing in an Osterizer (Oster Commercial, Milwaukee, HI) blender with water and ice.

With the blender set at liquefy speed, continue to add bone.

Allow the blended material to settle for a few minutes. Decant the liquid layer. Place the solid layer on a stirring plate and add distilled water to wash. Continue washing until the distilled water washes clear. Once the distilled water is clear, add ice and stir. Add 1 ml of ImM of phenyl methyl sul fonyl fluoride (PMSF). Wash for 1 hour adding ice frequently. Repeat with a second water wash. Place the sample in an ice water bath on a stirring plate. Defat with absolute ethanol, then defat twice with ethyl ether. Spread bone material onto glass petri dishes.

Allow the bone chips to air dry overnight.

Weigh the bone chips following the overnight drying. Using a sieve (U.S.A. Standard Sieve Series, Newark Wire Cloth Co., Newark, N.J.; sieve #40 retains particles greater than 425 pm and sieve #170 retains particles greater than 90 pm), isolate the bone particles in the 90-425um range. Grind any particles greater than 425pm in a MicroMill (Scienceware Bel-Art Products, Pequannock, NJ) for 1 minute adding dry ice to the bone particles to keep the material cold. Repeat the sieving and MicroMill grinding steps of the greater than 425pm particles until the amount of total recovery is greater than 2/3 of the initial weight of the bone.

Store the particles at 4°C until the next step. Weigh the particles isolated thus far. For each gram of particles, add 25 ml of 0.6N HCl. Stir vigorously at 4C for 2 hours. After 2 hours, stop stirring and allow the particles to settle. Decant the HCl. Add fresh 0.6N HCl and stir again for 2 hours. Decant the HCl and add fresh 0.6N HCl a third time and stir for two hours. Decant the HCl and rinse with distilled water. Using litmus paper, check the pH of the water for the presence of HCl. Continue rinsing with distilled water until the pH is between about 5 and 5.5. Rinse the bone particles with ethanol three times. Swirl, allow to settle, and remove the supernatant. Rinse the bone particles with ethyl ether three times as above. Dry overnight in glass plates. The dried bone particles are referred to as acid demineralized bone particles. - 12 The acid demineralized bone particles are deproteinized as follows: Weigh the material following the overnight drying. For each gram of material, add a solution of 30 ml 4M guanidine-HCl, lOmM Tris and l.OmM PMSF pH 6.4 to the bone material in a beaker.

Extract for 16 hours at 4’ with vigorous stirring. Following the hour extraction, cease stirring and allow the material to settle. Pour off the guanidine solution and save. Extract the material a second time for 6-7 hours using fresh guanidine-HCl solution. Following the extraction, pour off the solution and combine with the previously saved solution. The bone particles are now demineralized and deproteinized.

Dialyze the saved guanidine-HCl solution against distilled H20 at 4°C using 50 mm dialysis tubing (3500 molecular weight cutoff). Following dialysis, lyopholize the material and resolu15 bilize the lyophilized material in 4M Urea-0.05M Tris-0.1M NaCl, pH 7.4. Mix the solubilized material in a conical centrifuge tube with Heparin-Agarose and mix overnight on a rotator at 4°C. Pour the Heparin-Agarose slurry into a column. Wash with 1 column volume 4M urea, 0.05M Tris, 0.1M NaCl, pH 7.4 buffer. Collect the fraction. Wash with 3 column volumes of 4M urea, 0.05M Tris, 0.2M NaCl, pH 7.4 buffer. Step off the material with 3 column volumes of 4M urea, 0.05M Tris, 0.75M NaCl, pH 7.4. Concentrate this sample in a 50 ml Amicon concentrator (Amicon Corp., Danvers, MA) with filter (10,000 molecular weight cut off) to about 4-5ml.

Assay for protein concentration using BCA (bicinchoninic acid) Protein Assay Reagent (Pierce, Rockford, IL) and dialyze (3500 molecular weight cutoff dialysis tubing) in 4M guanidine-0.01 M Tris pH 7.4. Load material on Sephacryl S-200 column and collect fractions. The fractions containing the major protein peak are dialyzed against IM acetic acid and assayed for activity.

Active fractions from the gel filtration are combined and dialyzed against three changes of 6M urea, 25mM Na acetate, pH 4.6. The dialysate is loaded onto a column of carboxymethylsepharose (CM-Sepharose) equilibrated with the same buffer. The column is washed with 6M urea, 25mM Na acetate, pH 4.6 and activity eluted using a 0 - 0.5M NaCl gradient. Fractions are analyzed for protein concentration and sodium dodecyl sulfate gel electrophoresis. The activity located in the seven fractions before and after the beginning of the major protein peak are - 13 pooled for further purification.

The pooled CM-Sepharose fractions are dialyzed three times for 24 hours each against IX acetic acid. The dialysate is lyophilized to dryness and the protein pellet dissolved into 30 ml of 6M urea, 0.5M NaCl, 25mM Na phosphate, pH 7.4. The sample is applied on a column of chelating Sepharose charged with zinc and equilibrated with the above buffer. The column is washed with the above buffer and then eluted with a gradient from 6M urea, 0.5M NaCl, 25mM Na phosphate, pH 7.4 to 6M urea, 0.5M NaCl, 25mM Na acetate, pH 4.6. Aliquots of each fraction are labeled with 12SI and analyzed by SDS gel electrophoresis. Aliquots (100 /il) of each fraction are combined with 400 /il of elution buffer, dialyzed against 1% acetic acid and assayed for activity. Highly purified molecular weight range (Mr) 25-40 kD peptides are assayed in the bone induction assay.

A safe and effective amount of osteoinductive extract is dosed in combination with a Vitamin D compound. For purposes of systemic treatment, the osteoinductive extract dosed preferably comprises at least one BMP in an amount from about 1 pg to about 100 /xg, preferably from about 1 ng to about 10 μg, more preferably from about 10 ng to about 2.5 Mg.

For purposes of regional treatment, the osteoinductive extract dosed preferably comprises at least one BMP in an amount from about 1 pg to about 100 /ig, more preferably from about 1.5 Mg to about 90 M9, preferably from about 1.8 M9 to about 75 /ig, more preferably from about 2.0 MS to about 50 μg, more preferably still from about 2.2 μg to about 25 μg, more preferably from about 2.3 Mg to about 10 μg, most preferably from about 2.5 μg to about 5 Mg. Preferably the dose range is at least about 2.5 μg.

Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month. Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day.

Pharmaceutically Acceptable Carrier The Vitamin D compound, osteoinductive extract, or BMP may be administered via a pharmaceutically acceptable carrier. The term pharmaceutically-acceptable carrier, as used herein, means one - 14 or more compatible solid or liquid filler diluents or encapsulating substances which are suitable for administration to a human or lower animal. The term compatible, as used herein, means that the components of the pharmaceutical compositions are capable of being commingled with the compound(s) of the subject invention, and with each other in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the pharmaceutical composition under ordinary usage situations. Pharmaceutically-acceptable carriers must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to human or lower animal being treated.

Some examples of substances which can serve as pharmaceutically-acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils such a peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; sugar; alginic acid; pyrogen-free water; isotonic saline; phosphate buffer solutions; cocoa butter (suppository base); emulsifiers, such as the Tweens®; as well as other non-toxic compatible substances used in pharmaceutical formulations.

Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, tableting agents, stabilizers, antioxidants, and preservatives, can also be present. Other compatible pharmaceutical additives and actives (e.g., NSAI drugs; pain killers; muscle relaxants) may be included in the pharmaceutically-acceptable carrier for use in the compositions of the present invention. For example, art-known local anesthetics may be included in the pharmaceutically-acceptable carrier (e.g., benzyl alcohol; Novocaine®; lidocaine).

Additional examples of carriers include collagen, demineralized bone particles, ceramic and metallic implant materials, collagen membrane and bone grafts (isogenic or allogenic).

The choice of a pharmaceutically-acceptable carrier to be used in conjunction with the compounds of the present invention is determined by the way the compound is to be administered. The preferred modes of administering the compounds of the present invention are by injection, oral administration, topical-oral administration, and nasopharyngeal administration or a combination of modes (i.e., osteoinductive extract via injection and Vitamin D compound via oral administration). If the compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological saline. Suitable pharmaceuticallyacceptable carriers for oral administration include those suited for tablets, and capsules. Suitable pharmaceutically-acceptable carriers for topical-oral administration include those suited for pastes, gels, and liquids. Suitable pharmaceutically-acceptable carriers for nasopharyngeal administration include those suited for drops, sprays, mists and powders.

A separate pharmaceutically-acceptable carrier may be used in conjunction with each active component of the present invention or a single pharmaceutically-acceptable carrier may be employed in conjunction with a mixture of the active components of the present invention. In either case, the pharmaceutically-acceptable carrier is used at a concentration sufficient to provide a practical size to dosage relationship. The pharmaceuticallyacceptable carriers, in total, may comprise from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions of the present invention, preferably from about 50% to about 99.999%, and most preferably from about 75% to about 99.9%.

Specific oral and injectable carriers useful in this invention are described in the following U.S. Patents, all incorporated by reference herein: U.S. Patent No. 4,401,663, Buckwalter, et al, issued August 30, 1983; U.S. Patent No. 4,424,205, LaHann, et al, issued January 31, 1984; U.S. Patent No. 4,443,473, Buckwalter, et al, issued April 12, 1984; U.S. Patent No. 4,493,848, LaHann, et al, issued January 15, 1984. Representative pharmaceutical compositions of the present invention are provided in the Examples hereinafter.

Pharmaceutically-acceptable carriers suitable for the preparation of unit dosage forms for oral administration, topicaloral administration, nasopharyngeal administration and injection are well-known in the art. Their selection will depend on secondary considerations like taste, cost, and/or shelf stability, - 16 which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art. Pharmaceutically-acceptable carriers useful in the compositions of the present invention are described more fully hereinafter.

A. Oral Dose Forms: Preferably, the vitamin D compound is administered via an oral dose form. Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules, bulk powders and microcapsules of the drug. These oral forms comprise a safe and effective amount, usually at least about .5%, and preferably from about 1% to about 10% of the compound of the present invention. Tablets can be compressed, enteric-coated, sugar-coated or filmcoated containing suitable binders, lubricants, surfactants, diluents, disintegrating agents, coloring agents, flavoring agents, preservatives, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous and nonaqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents, and flavoring agents.

Preferred carriers for oral administration include gelatin and propylene glycol. Specific examples of pharmaceutically-acceptable carriers and excipients that may be used in formulating oral dosage forms containing compounds of the present invention are described in U.S. Patent 3,903,297, Robert, issued September 2, 1975, incorporated by reference herein. Techniques and compositions for making solid oral dosage forms are described in Marshall, Solid Oral Dosage Forms, Modern Pharmaceutics. Vol. 7. (Banker and Rhodes, editors), 359-427 (1979), incorporated herein by reference. Techniques and compositions for making tablets (compressed, formulas and molded), capsules (hard and soft gelatin) and pills are described in Remington's Pharmaceutical Sciences (Arthur Osol, editor), 1553-1593 (1980), incorporated herein by reference.

B. Topical-oral Dose Forms Topical-oral carrier, as used herein, denotes a carrier for the component of interest which results in a composition which is administered topically to the oral cavity, held therein for a period of time, and then is largely expectorated rather than being - 17 swallowed. Such compositions include toothpastes, tooth gels, tooth powders, mouthwashes, mouthsprays, prophylaxis pastes, dental treatment solutions, biogels or other sustained release products, and the like.

Components of the topical-oral carrier are suitable for administration to the oral cavity of a human or lower animal and are compatible with one another and the other components, especially the Vitamin D compound and osteoinductive extract or BMP, used in an oral composition of the subject invention. Preferred topical-oral carriers thus provide the desired characteristics for toothpastes, tooth gels, tooth powders, mouthwashes, mouthsprays, prophylaxis pastes, dental treatment solutions, and the like. The topical-oral carriers of the subject invention comprise components typically used in such compositions which are well known to a skilled practitioner. Such components include, but are not limited to anticaries agents, antiplaque agents, anticalculus agents, dental abrasives, surfactants, flavoring agents, sweetening agents, binders, humectants, thickening agents, buffering agents, preservatives, coloring agents and pigments, ethanol, and water.

Preferred compositions of the subject invention are in the form of toothpastes. Components of such toothpastes generally include a dental abrasive (from about 10% to about 50%), a surfactant (from about 0.5% to about 10%), a thickening agent (from about 0.1% to about 5%) a humectant (from about 10% to about 55%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), a coloring agent (from about 0.01% to about 0.5%) and water (from about 2% to about 45%). Such toothpastes may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.1% to about 13%), and an antiplaque agent (from about 0.1% to about 5%).

Other preferred compositions of the subject invention are mouthwashes and mouthsprays. Components of such mouthwashes and mouthsprays include water (from about 45% to about 95%), ethanol (from about 0% to about 25%), a humectant (from about 0% to about 50%), a surfactant agent (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), and a coloring agent (from about - 18 0.001% to about 0.5%). Such mouthwashes and mouthsprays may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.01% to about 3%), and an antiplaque agent (from about 0.1% to about 5%).

Other preferred compositions of the subject invention are dental solutions. Components of such dental solutions generally include water (from about 90% to about 99%), preservative (from about 0.01% to about 0.5%), thickening agent (from about 0% to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from 0% to about 5%).

Topical-oral carrier as used herein, also denotes fibers, strips or tubes which can be impregnated with the active components of the present invention and inserted or implanted into a periodontal pocket. Such compositions of the subject invention can readily be achieved by one of ordinary skill in the art using the teachings disclosed hereinbefore, the following references, incorporated herein by reference, and related well-known technol20 ogies: U.S. Patent No. 4,666,897 issued to Golub, McNamara & Ramamurthy on May 19, 1987; European Patent Application No. 244,118 Al in the name of Baker, published on November 4, 1987; European Patent Application No. 286,802 A2 in the name of Kametaka, Miyazaki, Hayashi, Handa & Kameda, published October 19, 1988; Addy, M., L. Rawle, R. Handley, H. Newman & J. Coventry, The development and in vitro evaluation of acrylic strips and dialysis tubing for local drug delivery, Journal of Periodontology, Vol. 53 (1982), pp. 693-698; Goodson, J.M., A.D. Haffajee & S.S. Socransky, Periodontal therapy by local delivery of tetracycline, Journal of Clinical Periodontology. Vol. 6 (1979), pp. 83-92; Goodson, J., D. Holborow, R. Dunn, P. Hogan & S. Dunham, Monolithic tetracycline containing fibers for controlled delivery to periodontal pockets, Journal of Periodontology, Vol. 54 (1983), pp. 575-579; Dunn, R., J. Gibson, B. Perkins, J.

Goodson & L. Laufe, Fibrous delivery systems for antimicrobial agents, Polymer Science and Technology, Vol. 32 (1985), pp. 47-59; Dunn, R., J. Gibson, B. Perkins, J. Goodson & L. Laufe, Fibrous delivery systems for antimicrobial agents, Polymer Material Science Engineering, Vol. 51 (1984), pp. 28-31; Olanoff, - 19 L. & J. Anderson, Controlled release of tetracycline - III: A physiological pharmacokinetic model of the pregnant rat, Journal of Pharmacokinetics and Biopharmaceutics. Vol. 8 (1980), pp. 599-620; Elkayam, R., M. Friedman, A. Stabholz, A. Soskolne, M.

Sela & L. Golub, Sustained release device containing minocycline for local treatment of periodontal disease, Journal of Controlled Release. Vol. 7 (1988), pp. 231-236; and Goodson, J., Multicenter evaluation of tetracycline fiber therapy. I. Experimental Design, Journal of Dental Research. Vol. 68 (1989), p. 197; and references cited therein.

C. Injectable Dose Forms: The active components of the present invention are also useful when injected. The dosage of the active components of the present invention which is both safe and effective to provide bone growth activity will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the specific mixture of compounds employed and its usage concentration, and like factors within the specific knowledge and expertise of the attending physician and commensurate with a reasonable benefit/risk ratio associated with the use of any drug compound. In addition, lower dosages will be utilized when only local or minor bone growth is desired, whereas higher dosages will be utilized when general or major bone growth is desired.

Methods and materials for manufacturing injectables can be found in Remington's Pharmaceutical Sciences. 17ed., 1985, Chapter 85, p. 1518, the disclosures of which are incorporated herein by reference in their entirety. Preferably, the injectable composition is an aqueous solution.

The aqueous solutions preferably consist of water (preferably from about 80% to about 99.999%), a suitable solubilizer, various types of acids, and an antimicrobial agent. Several solubilizers are known. Examples of such solubilizers are as follows: urea compounds (e.g., urea; urethan); surfactants (e.g., Tweens; Spans; sodium deoxycholate and Pluronics); cellulosic agents (e.g., carboxymethylcellulose); carbohydrates (e.g., sorbitol; mannitol); B vitamins (e.g., nicotinamide); xanthine derivatives; and alcohols (e.g., benzyl alcohol). Examples of acids to be used include the following: glucuronic; galacturonic; fumaric; gentisic; acetic; citric and lactobionic. Types of antimicrobial agents - 20 that can be used are the following: phenylmercuric nitrate; thimerosal; benzethonium chloride; benzalkonium chloride; phenol; cresol; and chlorobutanol. An art-known local anesthetic (e.g., benzyl alcohol; Novocaine·; lidocaine) may also be included.

Preferably, the osteoinductive extract and the BMP's are administered via an injectable dose form.

The following examples further describe and demonstrate the preferred embodiments within the scope of the present invention. The examples are given solely for the purpose of illustration, and are not to be construed as limitations of the present invention since many variations thereof are possible without departing from its spirit and scope.

EXAMPLE I An injectable composition comprising the osteoinductive extract and an oral composition comprising 1,25-dihydroxy Vitamin D3 for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques.

BMP composition Percent by Weight Component of Composition BMP-1 0.04 NaCl 0.90 Sterile water q.s.. . 100.00 1,25-dihydroxy Vitamin 0, composition Component Percent by Weight of Composition 1,25-dihydroxy Vitamin D3 0.01 Corn starch 18.49 Lactose 63.00 Talc 18.00 Stearic acid 0.50 100.00 0.1 cc of the BMP composition is injected into the fracture site at the time of fracture reduction and once daily thereafter. 100 /ig of the 1,25-dihydroxy Vitamin D3 composition is orally administered 24 hours before fracture reduction and once daily thereafter. The BMP and 1,25-dihydroxy Vitamin 03 are adminisIE 921812 - 21 10 tered until desired repair is achieved, perferably over a seven day period. EXAMPLE^! An injectable composition for bone fracture repair is prepared by combining the following components utilizing conven- tional mixing techniques. Percent by Weight Component of Composition BMP-2 0.04 25-hydroxy Vitamin 02 0.01 NaCl 0.09 Sterile water for injection q.s. 100.00 0.1 cc of the composition is injected into the fracture site at the time of fracture reduction and once daily thereafter until desired repair is achieved. EXAMPLE III A composition for inducing bone growth following reconstruc- tive surgery is prepared by combining the following components utilizing conventional mixing techniques. Percent by Weight Component of Composition BMP-3 0.04 1,25-dihydroxy Vitamin D2 0.01 NaCl 0.90 Sterile water a.s. 100.00 0.1 cc of the composition per cm2 of surface area of surgically reconstructed bone is deposited directly onto the bone surface. EXAMPLE IV A composition for accelerating the healing and providing a stronger bond between natural bone and an artificial prosthesis is prepared by combining the following components utilizing conven- tional mixing techniques. Percent by Weight Component of Composition BMP-1 0.04 BMP-2 0.04 BMP-4 0.04 24,25-dihydroxy Vitamin D3 0.01 NaCl 0.90 Sterile water 100.00 0.1 cc of the composition per cm2 surface area of natural bone proximate to the prosthesis is deposited directly onto the natural bone.

EXAMPLE V A topical oral carrier composition for periodontal therapy is prepared by combining the following components utilizing conventional mixing techniques.

Percent by Weight ComDonent of Composition 15 BMP-2 0.04 NaCl 0.90 Sterile water - 100.00 After the patient is prepared using conventional periodontal 20 surgical therapy 0.1 cc of the composition per exposed tooth is deposited into the surgery site. Soft tissue flaps are then sutured to close the surgical site. This treatment is useful for restoring alveolar and supporting bone in the periodontium lost by disease.

EXAMPLE VI An injectable composition comprising the BMPs 2, 3, 4 and 5 and an oral composition comprising 1,25-dihydroxy Vitamin D3 for treatment of osteoporosis is prepared by combining the following components utilizing conventional mixing techniques. osteoinductive extract composition Percent by Weight Component of Composition BMP-2 0.001 BMP-3 0.001 BMP-4 0.001 BMP-5 0.001 NaCl 0.900 Sterile water . 100.000 1,25-dihvdroxy Vitamin 0» composition Percent by Weight of Composition Component 1,25-dihydroxy Vitamin D3 0.01 Corn starch 18.49 Lactose 63.00 Talc 18.00 Stearic acid 0.50 100.00 1 cc of the BMP composition is injected intravenously once per day. 50 mg of the 1,25-dihydroxy Vitamin D3 composition is orally administered within one hour of the osteoinductive extract injection and once daily thereafter. The osteoinductive extract and 1,25-dihydroxy Vitamin D3 are administered over a 7-day period.

EXAMPLE VII A composition for inducing bone growth of a non-union fracture is prepared by combining the following components utilizing conventional mixing techniques. As used herein, non-union fracture means a fracture that has failed to heal normally.

Percent by Weight Component of Composition BMP-4 0.004 1,25-dihydroxy vitamin D3 0.01 Acid demineralized bone particles 90.000 NaCl 0.900 Sterile water for injection Q.S. 100.000 At the time of fracture reduction, a sufficient quantity of the above composition is deposited directly into the non-union site thereby filling in any bone deficit. - 24 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: STONE, ROGER L. (ii) TITLE OF INVENTION: THERAPEUTIC FORMULAS FOR OSTEOINDUCTION (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: The Procter & Gamble Company (B) STREET: 11810 East Miami River Road (C) CITY: Cincinnati (D) STATE: Ohio (E) COUNTRY: U.S.A.

(F) ZIP: 45239-8707 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Corstanje, Brahm J.

(B) REGISTRATION NUMBER: 34,804 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 513-245-2858 (B) TELEFAX: 513-741-3012 - 25 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2487 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GCCGCTTCCC TCGCCGCCGC CCCGCCAGCA TGCCCGGCGT GGCCCGCCTG CCGCTGCTGC 60 TCGGGCTGCT GCTGCTCCCG CGTCCCGGCC GGCCGCTGGA CTTGGCCGAC TACACCTATG 120 ACCTGGCGGA GGAGGACGAC TCGGAGCCCC TCAACTACAA AGACCCCTGC AAGGCGGCTG 180 20 CCTTTCTTGG GGACATTGCC CTGGACGAAG AGGACCTGAG GGCCTTCCAG GTACAGCAGG 240 CTGTGGATCT CAGACGGCAC ACAGCTCGTA AGTCCTCCAT CAAAGCTGCA GTTCCAGGAA 300 25 ACACTTCTAC CCCCAGCTGC CAGAGCACCA ACGGGCAGCC TCAGAGGGGA GCCTGTGGGA 360 GATGGAGAGG TAGATCCCGT AGCCGGCGGG CGGCGACGTC CCGACCAGAG CGTGTGTGGC 420 CCGATGGGGT CATCCCCTTT GTCATTGGGG GAAACTTCAC TGGTAGCCAG AGGGCAGTCT 480 30 TCCGGCAGGC CATGAGGCAC TGGGAGAAGC ACACCTGTGT CACCTTCCTG GAGCGCACTG 540 ACGAGGACAG CTATATTGTG TTCACCTATC GACCTTGCGG GTGCTGCTCC TACGTGGGTC 600 35 GCCGCGGCGG GGGCCCCCAG GCCATCTCCA TCGGCAAGAA CTGTGACAAG TTCGGCATTG 660 TGGTCCACGA GCTGGGCCAC GTCGTCGGCT TCTGGCACGA ACACACTCGG CCAGACCGGG 720 ACCGCCACGT TTCCATCGTT CGTGAGAACA TCCAGCCAGG GCAGGAGTAT AACTTCCTGA 780 - 26 AGATGGAGCC TCAGGAGGTG GAGTCCCTGG GGGAGACCTA TGACTTCGAC AGCATCATGC ATTACGCTCG GAACACATTC TCCAGGGGCA TCTTCCTGGA TACCATTGTC CCCAAGTATG AGGTGAACGG GGTGAAACCT CCCATTGGCC AAAGGACACG GCTCAGCAAG GGGGACATTG CCCAAGCCCG CAAGCTTTAC AAGTGCCCAG CCTGTGGAGA GACCCTGCAA GACAGCACAG GCAACTTCTC CTCCCCTGAA TACCCCAATG GCTACTCTGC TCACATGCAC TGCGTGTGGC GCATCTCTGT CACACCCGGG GAGAAGATCA TCCTGAACTT CACGTCCCTG GACCTGTACC GCAGCCGCCT GTGCTGGTAC GACTATGTGG AGGTCCGAGA TGGCTTCTGG AGGAAGGCGC CCCTCCGAGG CCGCTTCTGC GGGTCCAAAC TCCCTGAGCC TATCGTCTCC ACTGACAGCC GCCTCTGGGT TGAATTCCGC AGCAGCAGCA ATTGGGTTGG AAAGGGCTTC TTTGCAGTCT ACGAAGCCAT CTGCGGGGGT GATGTGAAAA AGGACTATGG CCACATTCAA TCGCCCAACT ACCCAGACGA TTACCGGCCC AGCAAAGTCT GCATCTGGCG GATCCAGGTG TCTGAGGGCT TCCACGTGGG CCTCACATTC CAGTCCTTTG AGATTGAGCG CCACGACAGC TGTGCCTACG ACTATCTGGA GGTGCGCGAC GGGCACAGTG AGAGCAGCAC CCTCATCGGG CGCTACTGTG GCTATGAGAA GCCTGATGAC ATCAAGAGCA CGTCCAGCCG CCTCTGGCTC AAGTTCGTCT CTGACGGGTC CATTAACAAA GCGGGCTTTG CCGTCAACTT TTTCAAAGAG GTGGACGAGT 30 GCTCTCGGCC CAACCGCGGG GGCTGTGAGC AGCGGTGCCT CAACACCCTG GGCAGCTACA AGTGCAGCTG TGACCCCGGG TACGAGCTGG CCCCAGACAA GCGCCGCTGT GAGGCTGCTT GTGGCGGATT CCTCACCAAG CTCAACGGCT CCATCACCAG CCCGGGCTGG CCCAAGGAGT ACCCCCCCAA CAAGAACTGC ATCTGGCAGC TGGTGGCCCC CACCCAGTAC CGCATCTCCC TGCAGTTTGA CTTCTTTGAG ACAGAGGGCA ATGATGTGTG CAAGTACGAC TTCGTGGAGG - 27 T6CGCAGTGG ACTCACAGCT GACTCCAAGC TGCATGGCAA GTTCTGTGGT TCTGAGAAGC CCGAGGTCAT CACCTCCCAG TACAACAACA TGCGCGTGGA GTTCAAGTCC GACAACACCG TGTCCAAAAA GGGCTTCAAG GCCCACTTCT TCTCAGAAAA GAGGCCAGCT CTGCAGCCCC CTCGGGGACG CCCCCACCAG CTCAAATTCC GAGTGCAGAA AAGAAACCGG ACCCCCCAGT GAGGCCTGCC AGGCCTCCCG GACCCCTTGT TACTCAGGAA CCTCACCTTG GACGGAATGG GATGGGGGCT TCGGTGCCCA CCAACCCCCC ACCTCCACTC TGCCATTCCG GCCCACCTCC C1CTGGCCGG ACAGAACTGG TGCTCTCTTC TCCCCACTGT GCCCGTCCGC GGACCGGGGA CCCTTCCCCG TGCCCTACCC CCTCCCATTT TGATGGTGTC TGTGACATTT CCTGTTGTGA AGTAAAAGAG GGACCCCTGC GTCCTGC (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1547 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: GGGGACTTCT TGAACTTGCA GGGAGAATAA CTTGCGCACC CCACTTTGCG CCGGTGCCTT TGCCCCAGCG GAGCCTGCTT CGCCATCTCC GAGCCCCACC GCCCCTCCAC TCCTCGGCCT TGCCCGACAC TGAGACGCTG TTCCCAGCGT GAAAAGAGAG ACTGCGCGGC CGGCACCCGG GAGAAGGAGG AGGCAAAGAA AAGGAACGGA CATTCGGTCC TTGCGCCAGG TCCTTTGACC AGAGTTTTTC CATGTGGACG CTCTTTCAAT GGACGTGTCC CCGCGTGCTT CTTAGACGGA 300 CTGCGGTCTC CTAAAGGTCG ACCATGGTGG CCGGGACCCG CTGTCTTCTA GCGTTGCTGC 360 5 TTCCCCAGGT CCTCCTGGGC GGCGCGGCTG GCCTCGTTCC GGAGCTGGGC CGCAGGAAGT 420 TCGCGGCGGC GTCGTCGGGC CGCCCCTCAT CCCAGCCCTC TGACGAGGTC CTGAGCGAGT 480 TCGAGTTGCG GCTGCTCAGC ATGTTCGGCC TGAAACAGAG ACCCACCCCC AGCAGGGACG 540 10 CCGTGGTGCC CCCCTACATG CTAGACCTGT ATCGCAGGCA CTCAGGTCAG CCGGGCTCAC 600 CCGCCCCAGA CCACCGGTTG GAGAGGGCAG CCAGCCGAGC CAACACTGTG CGCAGCTTCC 660 15 ACCATGAAGA ATCTTTGGAA GAACTACCAG AAACGAGTGG GAAAACAACC CGGAGATTCT 720 TCTTTAATTT AAGTTCTATC CCCACGGAGG AGTTTATCAC CTCAGCAGAG CTTCAGGTTT 780 TCCGAGAACA GATGCAAGAT GCTTTAGGAA ACAATAGCAG TTTCCATCAC CGAATTAATA 840 20 TTTATGAAAT CATAAAACCT GCAACAGCCA ACTCGAAATT CCCCGTGACC AGACTTTTGG 900 ACACCAGGTT GGTGAATCAG AATGCAAGCA GfTGGGAAAG TTTTGATGTC ACCCCCGCTG 960 25 TGATGCGGTG GACTGCACAG GGACACGCCA ACCATGGATT CGTGGTGGAA GTGGCCCACT 1020 TGGAGGAGAA ACAAGGTGTC TCCAAGAGAC ATGTTAGGAT AAGCAGGTCT TTGCACCAAG 1080 ATGAACACAG CTGGTCACAG ATAAGGCCAT TGCTAGTAAC TTTTGGCCAT GATGGAAAAG 1140 30 GGCATCCTCT CCACAAAAGA GAAAAACGTC AAGCCAAACA CAAACAGCGG AAACGCCTTA 1200 AGTCCAGCTG TAAGAGACAC CCTTTGTACG TGGACTTCAG TGACGTGGGG TGGAATGACT 1260 35 GGATTGTGGC TCCCCCGGGG TATCACGCCT TTTACTGCCA CGGAGAATGC CCTTTTCCTC 1320 TGGCTGATCA TCTGAACTCC ACTAATCATG CCATTGTTCA GACGTTGGTC AACTCTGTTA 1380 ACTCTAAGAT TCCTAAGGCA TGCTGTGTCC CGACAGAACT CAGTGCTATC TCGATGCTGT 1440 - 29 ACCTTGACGA GAATGAAAAG GTTGTATTAA AGAACTATCA GGACATGGTT GTGGAGGGTT 1500 GTGGGTGTCG CTAGTACAGC AAAATTAAAT ACATAAATAT ΛΤΑΤΑΤΑ 1547 (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1774 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: AGATCTTGAA AACACCCGGG CCACACACGC CGCGACCTAC AGCTCTTTCT CAGCGTTGGA 60 GTGGAGACGG CGCCCGCAGC GCCCTGCGCG GGTGAGGTCC GCGCAGCTGC TGGGGAAGAG 120 CCCACCTGTC AGGCTGCGCT GGGTCAGCGC AGCAAGTGGG GCTGGCCGCT ATCTCGCTGC 180 ACCCGGCCGC GTCCCGGGCT CCGTGCGCCC TCGCCCCAGC TGGTTTGGAG TTCAACCCTC 240 GGCTCCGCCG CCGGCTCCTT GCGCCTTCGG AGTGTCCCGC AGCGACGCCG GGAGCCGACG 300 CGCCGCGCGG GTACCTAGCC ATGGCTGGGG CGAGCAGGCT GCTCTTTCTG TGGCTGGGCT 360 GCTTCTGCGT GAGCCTGGCG CAGGGAGAGA GACCGAAGCC ACCTTTCCCG GAGCTCCGCA 420 AAGCTGTGCC AGGTGACCGC ACGGCAGGTG GTGGCCCGGA CTCCGAGCTG CAGCCGCAAG 480 ACAAGGTCTC TGAACACATG CTGCGGCTCT ATGACAGGTA CAGCACGGTC CAGGCGGCCC 540 GGACACCGGG CTCCCTGGAG GGAGGCTCGC AGCCCTGGCG CCCTCGGCTC CTGCGCGAAG 600 GCAACACGGT TCGCAGCTTT CGGGCGGCAG CAGCAGAAAC TCTTGAAAGA AAAGGACTGT 660 ATATCTTCAA TCTGACATCG CTAACCAAGT CTGAAAACAT TTTGTCTGCC ACACTGTATT TCTGTATTGG AGAGCTAGGA AACATCAGCC TGAGTTGTCC AGTGTCTGGA GGATGCTCCC 5 ATCATGCTCA GAGGAAACAC ATTCAGATTG ATCTTTCTGC ATGGACCCTC AAATTCAGCA GAAACCAAAG TCAACTCCTT GGCCATCTGT CAGTGGATAT GGCCAAATCT CATCGAGATA TTATGTCCTG GCTGTCTAAA GATATCACTC AATTCTTGAG GAAGGCCAAA GAAAATGAAG 10 AGTTCCTCAT AGGATTTAAC ATTACGTCCA AGGGACGCCA GCTGCCAAAG AGGAGGTTAC CTTTTCCAGA GCCTTATATC TTGGTATATG CCAATGATGC CGCCATTTCT GAGCCAGAAA 15 GTGTGGTATC AAGCTTACAG GGACACCGGA ATTTTCCCAC TGGAACTGTT CCCAAATGGG ATAGCCACAT CAGAGCTGCC CTTTCCATTG AGCGGAGGAA GAAGCGCTCT ACTGGGGTCT TGCTGCCTCT GCAGAACAAC GAGCTTCCTG GGGCAGAATA CCAGTATAAA AAGGATGAGG 20 TGTGGGAGGA GAGAAAGCCT TACAAGACCC TTCAGGCTCA GGCCCCTGAA AAGAGTAAGA ATAAAAAGAA ACAGAGAAAG GGGCCTCATC GGAAGAGCCA GACGCTCCAA TTTGATGAGC 25 AGACCCTGAA AAAGGCAAGG AGAAAGCAGT GGATTGAACC TCGGAATTGC GCCAGGAGAT ACCTCAAGGT AGACTTTGCA GATATTGGCT GGAGTGAATG GATTATCTCC CCCAAGTCCT TTGATGCCTA TTATTGCTCT GGAGCATGCC AGTTCCCCAT GCCAAAGTCT TTGAAGCCAT 30 CAAATCATGC TACCATCCAG AGTATAGTGA GAGCTGTGGG GGTCGTTCCT GGGATTCCTG AGCCTTGCTG TGTACCAGAA AAGATGTCCT CACTCAGTAT TTTATTCTTT GATGAAAATA 35 AGAATGTAGT CTGGCAAAGA GCTTAAAGTA ACTCATTTGA TACCCTAACA ATGCTTAATT TGACAGTAGA CAAT GTCTTGCGCT TGCAGATAAC (2) INFORMATION FOR SEQ ID N0:4: - 31 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1751 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: GGCAGAGGAG GAGGGAGGGA GGGAAGGAGC GCGGAGCCCG GCCCGGAAGC TAGGTGAGTG 60 15 TGGCATCCGA GCTGAGGGAC GCGAGCCTGA GACGCCGCTG CTGCTCCGGC TGAGTATCTA 120 GCTTGTCTCC CCGATGGGAT TCCCGTCCAA GCTATCTCGA GCCTGCAGCG CCACAGTCCC 180 CGGCCCTCGC CCAGGTTCAC TGCAACCGTT CAGAGGTCCC CAGGAGCTGC TGCTGGCGAG 240 20 CCCGCTACTG CAGGGACCTA TGGAGCCATT CCGTAGTGCC ATCCCGAGCA ACGCACTGCT 300 GCAGCTTCCC TGAGCCTTTC CAGCAAGTTT GTTCAAGATT GGCTGTCAAG AATCATGGAC 360 25 TGTTATTATA TGCCTTGTTT TCTGTCAAGA CACCATGATT CCTGGTAACC GAATGCTGAT 420 GGTCGTTTTA TTATGCCAAG TCCTGCTAGG AGGCGCGAGC CATGCTAGTT TGATACCTGA 480 GACGGGGAAG AAAAAAGTCG CCGAGATTCA GGGCCACGCG GSAGGACGCC GCTCAGGGCA 540 30 GAGCCATGAG CTCCTGCGGG ACTTCGAGGC GACACTTCTG CAGATGTTTG GGCTGCGCCG 600 CCGCCCGCAG CCTAGCAAGA GTGCCGTCAT TCCGGACTAC ATGCGGGATC TTTACCGGCT 660 35 TCAGTCTGGG GAGGAGGAGG AAGAGCAGAT CCACAGCACT GGTCTTGAGT ATCCTGAGCG 720 CCCGGCCAGC CGGGCCAACA CCGTGAGGAG CTTCCACCAC GAAGAACATC TGGAGAACAT 780 CCCAGGGACC AGTGAAAACT CTGCTTTTCG TTTCCTCTTT AACCTCAGCA GCATCCCTGA 840 GAACGAGGTG ATCTCCTCTG CAGAGCTTCG GCTCTTCCGG GAGCAGGTGG ACCAGGGCCC 900 TGATTGGGAA AGGGGCTTCC ACCGTATAAA CATTTATGAG GTTATGAAGC CCCCAGCAGA 960 5 AGTGGTGCCT GGGCACCTCA TCACACGACT ACTGGACACG AGACTGGTCC ACCACAATGT 1020 GACACGGTGG GAAACTTTTG ATGTGAGCCC TGCGGTCCTT CGCTGGACCC GGGAGAAGCA 1080 GCCAAACTAT GGGCTAGCCA TTGAGGTGAC TCACCTCCAT CAGACTCGGA CCCACCAGGG 1140 10 CCAGCATGTC AGGATTAGCC GATCGTTACC TCAAGGGAGT GGGAATTGGG CCCAGCTCCG 1200 GCCCCTCCTG GTCACCTTTG GCCATGATGG CCGGGGCCAT GCCTTGACCC GACGCCGGAG 1260 15 GGCCAAGCGT AGCCCTAAGC ATCACTCACA GCGGGCCAGG AAGAAGAATA AGAACTGCCG 1320 GCGCCACTCG CTCTATGTGG ACTTCAGCGA TGTGGGCTGG AATGACTGGA TTGTGGCCCC 1380 ACCAGGCTAC CAGGCCTTCT ACTGCCATGG GGACTGCCCC TTTCCACTGG CTGACCACCT 1440 20 CAACTCAACC AACCATGCCA TTGTGCAGAC CCTGGTCAAT TCTGTCAATT CCAGTATCCC 1500 CAAAGCCTGT TGTGTGCCCA CTGAACTGAG TGCCATCTCC ATGCTGTACC TGGATGAGTA 1560 25 TGATAAGGTG GTACTGAAAA ATTATCAGGA GATGGTAGTA GAGGGATGTG GGTGCCGCTG 1620 AGATCAGGCA GTCCTTGAGG ATAGACAGAT ATACACACCA CACACACACA CCACATACAC 1680 CACACACACA CGTTCCCATC CACTCACCCA CACACTACAC AGACTGCTTC CTTATAGCTG 1740 GACTTTTATT T 1751 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2153 base pairs (B) TYRE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - 33 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTGGTATATT TGTGCCTGCT GGAGGTGGAA TTAACAGTAA GAAGGAGAAA GGGATTGAAT GGACTTACAG GAAGGATTTC AAGTAAATTC AGGGAAACAC ATTTACTTGA ATAGTACAAC CTAGAGTATT ATTTTACACT AAGACGACAC AAAAGATGTT AAAGTTATCA CCAAGCTGCC GGACAGATAT ATATTCCAAC ACCAAGGTGC AGATCAGCAT AGATCTGTGA TTCAGAAATC AGGATTTGTT TTGGAAAGAG CTCAAGGGTT GAGAAGAACT CAAAAGCAAG TGAAGATTAC TTTGGGAACT ACAGTTTATC AGAAGATCAA CTTTTGCTAA TTCAAATACC AAAGGCCTGA TTATCATAAA TTCATATAGG AATGCATAGG TCATCTGATC AAATAATATT AGCCGTCTTC TGCTACATCA ATGCAGCAAA AACTCTTAAC AACTGTGGAT AATTGGAAAT CTGAGTTTCA GCTTTCTTAG AAATAACTAC TCTTGACATA TTCCAAAATA TTTAAAATAG GACAGGAAAA TCGGTGAGGA TGTTGTGCTC AGAAATGTCA CTGTCATGAA AAATAGGTAA ATTTGTTTTT TCAGCTACTG GGAAACTGTA CCTCCTAGAA CCTTAGGTTT TTTTTTTTTT AAGAGGACAA GAAGGACTAA AAATATCAAC TTTTGCTTTT GGACAAAAAT GCATCTGACT GTATTTTTAC 30 TTAAGGGTAT TGTGGGTTTC CTCTGGAGCT GCTGGGTTCT AGTGGGTTAT GCAAAAGGAG GTTTGGGAGA CAATCATGTT CACTCCAGTT TTATTTATAG AAGACTACGG AACCACGAAA GACGGGAAAT ACAAAGGGAA ATTCTCTCTA TCTTGGGTTT GCCTCACAGA CCCAGACCAT TTTCACCTGG AAAACAAGCG TCCTCTGCAC CTCTCTTTAT GCTGGATCTC TACAATGCCA TGACCAATGA AGAAAATCCT GAAGAGTCGG AGTACTCAGT AAGGGCATCC TTGGCAGAAG AGACCAGAGG AGTTATCTCG CCAACTTTCT AGGATTTTTC CTCATGGAGA GATTTGAAAA GGGATGCAGA TTGTCTTTGA GCTTACAGCT TTGTGGGAAG GTGAGGTACT AATCCAGCTC AGCAAAAACA AGGACTGGAT TTCCACTTAA TGATGTTTCC CTGTTCTGTA TACGCTCATG TAAGGTTTAT GGCAAGAAAG GGATACCCAG GACGACTCCT CTGACCACCC GAATGATGCT GACATGGTCA TCACCAGCGA AGGCATTACA GGCAGTGACA GCAGCTGAAT TGAAACAATT AAGATTAGCA TCTGTTCTTG TTAGACACAA TATCACTGTG ACCAGCAATC CTGTGCAGAA ACAGGGGATG ACAGGGACCT CAGTCAAAAC TCTTCGATCC GTGAGAGCAG TCATCAGGAC TCCTCCAGAA AGCCTGTAAG AAGCACGAAC TATAGCACCA GAAGGATACG CGCCCATATG AATGCCACCA TGACCACGTA CCAAAGCCTT CTTTGATGAC AGCTCCAATG TGGCTGCCAC TAATATTAAA GGCTGCAATA AAAAGCATAC - 34 CCTCTCCCAA TGGGTATCCT AGAGTCCTCC TCTAGCCAGC TGAGCTTTGT CAACTTAGTT AAGAATTTCG ATTTGATCTT TCCGGATATA CAAGGACCGG TATATCAAAT CATCAAGGAA GAAAGGCCCA AGCTTTAGAT ATTGGGTGAT TAATCCCCAG GACGCAGTAT CAACGTAAAA AACCATTCAT GGTGGCCTTC CCAACAAACG AAAAAATCAA TGTCCAGTGT TGGAGATTAT TCTATGTGAG CTTCCGGGAT CTGCATTTTA TTGTGATGGA ACCACGCTAT AGTTCAGACT GTTGTGCTCC AACCAAATTA TCATTTTGAA AAAATATAGA TAATATTGAT AATAACAAAA TTTCAGACAA ACAGAAAAAA ' CGTCGCATAC : CTCCATGATA GAAAGAGACA ACCCAAATTC AGCAACAACC TACACAAATA GTGGGTTGGC AATAATTTGG TCTGCTGGTC TTCAAGGCGA AACCGCAATA AACACAAGTG CTGGGATGGC GAATGTTCTT CTGGTTCATC AATGCCATCT AATATGGTAG AGATCTGTAT AAA (2) INFORMATION FOR SEQ ID NO:6: - 35 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2923 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: CGACCATGAG AGATAAGGAC TGAGGGCCAG GAAGGGGAAG CGAGCCCGCC GAGAGGTGGC GGGGACTGCT CACGCCAAGG GCCACAGCGG CCGCGCTCCG GCCTCGCTCC GCCGCTCCAC GCCTCGCGGG ATCCGCGGGG GCAGCCCGGC CGGGCGGGGA TGCCGGGGCT GGGGCGGAGG GCGCAGTGGC TGTGCTGGTG GTGGGGGCTG CTGTGCAGCT GCTGCGGGCC CCCGCCGCTG CGGCCGCCCT TGCCCGCTGC CGCGGCCGCC GCCGCCGGGG GGCAGCTGCT GGGGGACGGC GGGAGCCCCG GCCGCACGGA GCAGCCGCCG CCGTCGCCGC AGTCCTCCTC GGGCTTCCTG TACCGGCGGC TCAAGACGCA GGAGAAGCGG GAGATGCAGA AGGAGATCTT GTCGGTGCTG GGGCTCCCGC ACCGGCCCCG GCCCCTGCAC GGCCTCCAAC AGCCGCAGCC CCCGGCGCTC CGGCAGCAGG AGGAGCAGCA GCAGCAGCAG CAGCTGCCTC GCGGAGAGCC CCCTCCCGGG 30 CGACTGAAGT CCGCGCCCCT CTTCATGCTG GATCTGTACA ACGCCCTGTC CGCCGACAAC GACGAGGACG GGGCGTCGGA GGGGGAGAGG CAGCAGTCCT GGCCCCACGA AGCAGCCAGC TCGTCCCAGC GTCGGCAGCC GCCCCCGGGC GCCGCGCACC CGCTCAACCG CAAGAGCCTT CTGGCCCCCG GATCTGGCAG CGGCGGCGCG TCCCCACTGA CCAGCGCGCA GGACAGCGCC TTCCTCAACG ACGCGGACAT GGTCATGAGC TTTGTGAACC TGGTGGAGTA CGACAAGGAG - 36 TTCTCCCCTC GTCAGCGACA CCACAAAGAG TTCAAGTTCA ACT7ATCCCA GATTCCTGAG GGTGAGGTGG TGACGGCTGC AGAATTCCGC ATCTACAAGG ACTGTGTTAT GGGGAGTTTT AAAAACCAAA CTTTTCTTAT CAGCATTTAT CAAGTCTTAC AGGAGCATCA GCACAGAGAC TCTGACCTGT TTTTGTTGGA CACCCGTGTA GTATGGGCCT CAGAAGAAGG CTGGCTGGAA TTTGACATCA CGGCCACTAG CAATCTGTGG GTTGTGACTC CACAGCATAA CATGGGGCTT CAGCTGAGCG TGGTGACAAG GGATGGAGTC CACGTCCACC CCCGAGCCGC AGGCCTGGTG GGCAGAGACG GCCCTTACGA TAAGCAGCCC TTCATGGTGG CTTTCTTCAA AGTGAGTGAG GTCCACGTGC GCACCACCAG GTCAGCCTCC AGCCGGCGCC GACAACAGAG TCGTAATCGC TCTACCCAGT CCCAGGACGT GGCGCGGGTC TCCAGTGCTT CAGATTACAA CAGCAGTGAA TTGAAAACAG CCTGCAGGAA GCATGAGCTG TATGTGAGTT TCCAAGACCT GGGATGGCAG GACTGGATCA TTGCACCCAA GGGCTATGCT GCCAATTACT GTGATGGAGA ATGCTCCTTC CCACTCAACG CACACATGAA TGCAACCAAC CACGCGATTG TGCAGACCTT GGTTCACCTT ATGAACCCCG AGTATGTCCC CAAACCGTGC TGTGCGCCAA CTAAGCTAAA TGCCATCTCG GTTCTTTACT TTGATGACAA CTCCAATGTC ATTCTGAAAA AATACAGGAA TATGGTTGTA AGAGCTTGTG GATGCCACTA ACTCGAAACC AGATGCTGGG GACACACATT CTGCCTTGGA 30 TTCCTAGATT ACATCTGCCT TAAAAAAACA CGGAAGCACA GTTGGAGGTG GGACGATGAG ACTTTGAAAC TATCTCATGC CAGTGCCTTA TTACCCAGGA AGATTTTAAA GGACCTCATT AATAATTTGC TCACTTGGTA AATGACGTGA GTAGTTGTTG GTCTGTAGCA AGCTGAGTTT GGATGTCTGT AGCATAAGGT CTGGTAACTG CAGAAACATA ACCGTGAAGC TCTTCCTACC CTCCTCCCCC AAAAACCCAC CAAAATTAGT TTTAGCTGTA GATCAAGCTA TTTGGGGTGT TTGTTAGTAA - 37 - 2100 ATAGGGAAAA TAATCTCAAA GGAGTTAAAT GTATTCTTGG CTAAAGGATC AGCTGGTTCA GTACTGTCTA TCAAAGGTAG ATTTTACAGA GAACAGAAAT CGGGGAAGTG 2160 5 GGGGGAACGC CTCTGTTCAG TTCATTCCCA GAAGTCCACA GGACGCACAG CCCAGGCCAC 2220 AGCCAGGGCT CCACGGGGCG CCCTTGTCTC AGTCATTGCT GTTGTATGTT CGTGCTGGAG 2280 TTTTGTTGGT GTGAAAATAC ACTTATTTCA GCCAAAACAT ACCATTTCTA CACCTCAATC 2340 10 CTCCATTTGC TGTACTCTTT GCTAGTACCA AAAGTAGACT GATTACACTG AGGTGAGGCT 2400 ACAAGGGGTG TGTAACCGTG TAACACGTGA AGGCAGTGCT CACCTCTTCT TTACCAGAAC 2460 15 GGTTCTTTGA CCAGCACATT AACTTCTGGA CTGCCGGCTC TAGTACCTTT TCAGTAAAGT 2520 GGTTCTCTGC CTTTTTACTA TACAGCATAC CACGCCACAG GGTTAGAACC AACGAAGAAA 2580 ATAAAATGAG GGTGCCCAGC TTATAAGAAT GGTGTTAGGG GGATGAGCAT GCTGTTTATG 2640 20 AACGGAAATC ATGATTTCCC TGTAGAAAGT GAGGCTCAGA TTAAATTTTA GAATATTTTC 2700 TAAATGTCTT TTTCACAATC ATGTGACTGG GAAGGCAATT TCATACTAAA CTGATTAAAT 2760 25 AATACATTTA TAATCTACAA CTGTTTGCAC TTACAGCTTT TTTTGTAAAT ATAAACTATA 2820 ATTTATTGTC TATTTTATAT CTGTTTTGCT GTGGCGTTGG GGGGGGGGCC GGGCTTTTGG 2880 GGGGGGGGGT TTGTTTGGGG GGTGTCGTGG TGTGGGCGGG CGG 2923 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1448 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA - 38 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTGACCGAGC GGCGCGGACG GCCGCCTGCC CCCTCTGCCA CCTGGGGCGG TGCGGGCCCG 60 5 GAGCCCGGAG CCCGGGTAGC GCGTAGAGCC GGCGCGATGC ACGTGCGCTC ACTGCGAGCT 120 GCGGCGCCGC ACAGCTTCGT GGCGCTCTGG GCACCCCTGT TCCTGCTGCG CTCCGCCCTG 180 GCCGACTTCA GCCTGGACAA CGAGGTGCAC TCGAGCTTCA TCCACCGGCG CCTCCGCAGC 240 10 CAGGAGCGGC GGGAGATGCA GCGCGAGATC CTCTCCATTT TGGGCTTGCC CCACCGCCCG 300 CGCCCGCACC TCCAGGGCAA GCACAACTCG GCACCCATGT TCATGCTGGA CCTGTACAAC 360 15 GCCATGGCGG TGGAGGAGGG CGGCGGGCCC GGCGGCCAGG GCTTCTCCTA CCCCTACAAG 420 GCCGTCTTCA GTACCCAGGG CCCCCCTCTG GCCAGCCTGC AAGATAGCCA TTTCCTCACC 480 GACGCCGACA TGGTCATGAG CTTCGTCAAC CTCGTGGAAC ATGACAAGGA ATTCTTCCAC 540 20 CCACGCTACC ACCATCGAGA GTTCCGGTTT GATCTTTCCA AGATCCCAGA AGGGGAAGCT 600 GTCACGGCAG CCGAATTCCG GATCTACAAG GACTACATCC GGGAACGCTT CGACAATGAG 660 25 ACGTTCCGGA TCAGCGTTTA TCAGGTGCTC CAGGAGCACT TGGGCAGGGA ATCGGATCTC 720 TTCCTGCTCG ACAGCCGTAC CCTCTGGGCC TCGGAGGAGG GCTGGCTGGT GTTTGACATC 780 ACAGCCACCA GCAACCACTG GGTGGTCAAT CCGCGGCACA ACCTGGGCCT GCAGCTCTCG 840 30 GTGGAGACGC TGGATGGGCA GAGCATCAAC CCCAAGTTGG CGGGCCTGAT TGGGCGGCAC 900 GGGCCCCAGA ACAAGCAGCC CTTCATGGTG GCTTTCTTCA AGGCCACGGA GGTCCACTTC 960 35 CGCAGCATCC GGTCCACGGG GAGCAAACAG CGCAGCCAGA ACCGCTCCAA GACGCCCAAG 1020 AACCAGGAAG CCCTGCGGAT GGCCAACGTG GCAGAGAACA GCAGCAGCGA CCAGAGGCAG 1080 GCCTGTAAGA AGCACGAGCT GTATGTCAGC TTCCGAGACC TGGGCTGGCA GGACTGGATC 1140 IE 921812 - 39 ATCGCGCCTG AAGGCTACGC CGCCTACTAC TGTGAGGGGG AGTGTGCCTT CCCTCTGAAC 1200 TCCTACATGA ACGCCACCAA CCACGCCATC GTGCAGACGC TGGTCCACTT CATCAACCCG 1260 GAAACGGTGC CCAAGCCCTG CTGTGCGCCC ACGCAGCTCA ATGCCATCTC CGTCCTCTAC 1320 TTCGATGACA GCTCCAACGT CATCCTGAAG AAATACAGAA ACATGGTGGT CCGGGCCTGT 1380

Claims (4)

1. CATTGCTC 1448 The invention has been described herein with reference to certain preferred embodiments and examples. Obvious variations may 15 appear to those skilled in the art. Therefore, the invention is not to be considered limited thereto but only by the claims which follow. 2.

2. 3.

3. 4.

4. A composition for generating new bone growth in a mammal in need of such treatment characterized in that it comprises: a. a safe and effective amount, preferably from 1 ng to 1 mg, more preferably from 30 ng to 10 pg, of a Vitamin D compound, preferably the Vitamin D compound is selected from Vitamin D z , Vitamin D 3 , Ι-α-hydroxy Vitamin D 3 , 1-a-fluoro Vitamin D 3 , 3-deoxy-l,25-dihydroxy Vitamin D 3 , 25-hydroxy-5,6-trans Vitamin D 3 , 25-hydroxy Vitamin D z , 25-hydroxy Vitamin 0 3 , 1,25-dihydroxy Vitamin 0 z , 24.25- dihydroxy Vitamin D 2 , 24,25-dihydroxy Vitamin-D 3 , and 1.25- dihydroxy Vitamin 0 3 ; more preferably 1,25-dihydroxy Vitamin D 3 ; ί b. a safe and effective amount, preferably at least 2.5 pg, of a BMP selected from BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7; and c. a safe and effective amount of a pharmaceutically-acceptable carrier. The composition of Claim 1 wherein the pharmaceuticallyacceptable carrier is an injectable carrier. The composition of Claim 1 wherein the pharmaceuticallyacceptable carrier is a topical-oral carrier. The composition of any of Claims 1-3 wherein the BMP is a component of an osteoinductive extract, the osteoinductive extract comprising one or more BMP's selected t from BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. A composition according to Claim 1, substantially as hereinbefore described and exemplified.