AU667815B2 - Therapeutic compositions for osteoinduction - Google Patents

Description

WO 92/21365 PCT/US92/04356 THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION TECHNICAL FIELD 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.

BACKGROUND OF THE INVENTION 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 Nutrition Reviews, Vol. 47, pp. 364-366 concludes that Vitamin D in the diet prevents the loss of the osteoinductive activity of bone matrix.

STurner, R. J. Farley, J. J. Vandersteenhoven, S. Epstein, N. H. Bell, and D. J. Baylink, (1988) "Demonstration of Reduced Mitogenic and Osteoinductive Activities in Demineralized Allogeneic Bcne Matrix from Vitamin D-deficient Rats", The Journal of Clinical Investigation, Inc., Vol. 82, pp. 212-217, discloses the WO 92/21365 PCT/US92/04356 -2implantation 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. 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, i 1 -Y~ SUMMARY OF THE INVENTION The present invention relates to a method of generating new bone growth in a mammal in need of such treatment comprising administrating to the mammal a safe and effective amount of a Vitamin D compound in combination with a safe and effective amount of a BMP selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7.

The present invention further relates to a composition for generating new bone growth in a mammal in need of such treatment comprising: a. a safe and effective amount of a Vitamin D compound; b. a safe and effective amount of a bone morphogenetic protein (BMP) selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7; and c. a safe and effective amount of a pharmaceutically acceptable carrier.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives or components or integers.

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 i i S* 3a 1 WO 92/21365 PCr/US92/04356 -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, Dasch and Seyedin, published October 11, 1989; European Patent Application 145 155, Sen, published July 10, 1985; World Patent Application 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 OFTHE INVENTIDL- The present invention_ phe administration to a mammal ination of a safe and effective amount of a

';I

WO 92/21365 PCT/US92/04356 4 Vitamin D compound amd 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. Surprisingly, it has been further determined that treatment with a Vitamin D 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, osteoinduc=ive extract or Vitamin D compound alone.

Subjects in need of such treatment suffer from a variety of ailments which may se treated via this procedure, including but not limited to, borne fractures (closed and open), non-union fractures, congenita" defects, as an adjunct in plastic surgery, in treating oncolog-,cal resections, all diseases classified as osteoporosis, rheumnetoid arthritis, osteoarthritis, septic arthritis, rickets, nrganic incorporation of prosthetic joints and dental implants, per-iod:ntal disease and defects, as well as osteopenic and osteonmalacic conditions and disease.

As used herein. "safe and effective amount" means an amount of compound or comptcsition sufficient to significantly induce a positive modification in the condition to be treated, but low enough to avoid serius side effects (at a reasonable benefit/risk ratio), within the s;=ope of sound medical judgment. The safe and effective amount of -he compound or composition will vary with the particular condition beir- treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the treamer.t the nature of concurrent therapy, the specific compound or comgosition employed, the particular pharmaceutically-acceptable carrier utilized, and like factors within the knowledge and exmertise 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, "BMF' means bone morphogenetic protein.

As used herein, means quantity sufficient.

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

As used herein "re;:snal treatment" includes treating bone fractures (closed and :pen), treating non-union fractures, 4i WO 92/21365 PCT/US92/04356 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 cm 2 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 D2), cholecalciferol (Vitamin 03) and their biologically active metabolites and precursors.

Preferred Vitamin D compounds include, but are not limited to, Vitamin D 2 (Sigma, St. Louis, MO), Vitamin 03 (Sigma, St. Louis, MO), 1-a-hydroxy Vitamin 03, 1-a-fluoro Vitamin D3, 3-deoxy-1,25dihydroxy Vitamin D3, 25-hydroxy-5,6-trans Vitamin 03, Vitamin D2, 25-hydroxy Vitamin D3 (Hoffman LaRoche), 1,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D0, 24,25-dihydroxy Vitamin D3 (Hoffman LaRoche), and 1,25-dihydroxy Vitamin D 3 (Duphar, Veenendaal, Holland). Preferably, the Vitamin D compound is selected from 25-hydroxy Vitamin 02, 25-hydroxy Vitamin D3, 1,25-dihydroxy Vitamin D 2 24,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D3, and 1,25-dihydroxy Vitamin D3, 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 i 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, WO 92/21365 PCT/US92/04356 -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 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 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 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 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, I kekawa, 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, WO 92/21365 PCT/US92/04356 20 WO 92/21365 PCT/US92/04356 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 15, 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; WO 92/21365 PCI'/US92/04356 -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, Hamner, Paaren and Schnoes, issued March 25, 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 0; 1 a-hydroxyvitamin 0; 1-hydroxyvitamin D; 1 a-hydroxyvitamin 02; I a,25-dihydroxy-22Z-dehydroxyvitamin D; 26,26,26,- 27,27-pentafluoro-1 a-hydroxy-27-methoxyvitamin 03; 2 o-fluorovitamin 03; 1,24-dihydroxy-delta 22-vitamin D 3 23,23-difluoro- 25-hydroxy-vitamin 03; 1-hydroxy-3,5-cyclovitamin 0; 23,23-difluoro-1 a,25-dihydroxy-vitamin 03; 1,23-dihydroxyvitamin 0; hydroxyvitamin D2; 23,23-difluoro-1 c,25-dihydroxy-vitamin 03; 23,23-difluoro-25-hydroxy-vitamin D3; 26,26,26,27,27,27-hexafluoro-1 a,25-dihydroxycholesterol; 23,25-dihydroxyvitamin 03; 26,26,26,27,27,27-hexafluoro-1 &,25-dihydroxycholecalciferol; 1 a,25-dihydroxy-2 fi-fluorovitamin 03; 24-fluoro-25-hydroxycholecalciferol; 5,6-trans-vitamin 0; 1 a-hydroxy-25-keto-27-nor-cholecalciferol; fluorovitamin D; 1 ct-hydroxy-2 R-fluorocholecalciferol; 3-deoxy-1 a-hydroxycholecalciferol; 25-hydroxy-26,26,26,- 27,27,27-hexafluorocholecal iferol of-hydroxy-3,5-cyclovitamin 0; 24,24-difluoro-1 calciferol; 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 0 compounds useful in the present invention further include those disclosed in The Handbook of Vitamins, L. J. Machlim, 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 0, 3-deoxy-1,25-dihydroxy Vitamin 0, 27-nor-25-hydroxy Vitamin D31 26,27-bis-nor-25-hydroxy Vitamin 03- 24-nor-25-hydroxy Vitamin D31 Vitamin D, 1,25-dihydroxy Vitamin D, le-hydroxy Vitamin 03 and 25-fluoro-lo-hydroxy Vitamin D 3 -1 I I I WO 92/21365 PC/lUS92/04356 -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 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-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-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 Mg, 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 jig, more preferably from about 1.5 pg to about 90 pg, preferably from about 1.8 Mg to about 75 ig, more preferably from about 2.0 pg to about 50 ig, more preferably still from about 2.2 Mg to about 25 uq, more preferably from about 2.3 ig to about 10 pg, most preferably from about 2.5 jg to about 5 g. Preferably the dose range is at least about 2.5 pg.

WO 92/21365 PCT/US92/04356 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 sequence comprising SEQ ID NO:1.

As used herein, "BMP-2" means sequence comprising SEQ ID NO:2.

As used herein, "BMP-3" means sequence comprising SEQ ID NO:3.

As used herein, "BMP-4' means sequence comprising SEQ ID NO:4.

As used herein, "BMP-5" means sequence comprising SEQ ID As used herein,. "BMP-6" means sequence comprising SEQ ID NO: 6.

As used herein, "BMP-7" means sequence compr'sing SEQ ID NO: 7.

As used herein, a peptide encoded by a DNA a peptide encoded a peptide encoded a peptide encoded a peptide encoded a peptide encoded by a DNA by a DNA by a DNA by a DNA by a DNA a peptide encoded by a DNA and refer to the nucleotides containing adenine, thymine, guanine and cytosine respectively.

Osteoinductive Extract Another component of the inven 'Io 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 is 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 WO 92/21365 PCT/US92/04356 -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, WI) 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 phenylmethylsulfonyl 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 Standard Sieve Series, Newark Wire Cloth Co., Newark, 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 425upm particles until the amount of total recovery is greater than 2/3 of the initial weight of the bone.

Store the particles at 4oC until the next step. Weigh the particles isolated thus far. For each gram of particles, add ml of 0.6N HC1. Stir vigorously at 4'C for 2 hours. After 2 hours, stop stirring and allow the particles to settle. Decant the HC1. Add fresh 0.6N HC1 and stir again for 2 hours. Decant the HC1 and add fresh 0.6N HC1 a third time and stir for two hours. Decant the HCI and rinse with distilled water. Using litmus paper, check the pH of the water for the presence of HC1.

Continue rinsing with distilled water until the pH is between about 5 and 5.5. Rinse the bone particles with ethanol three Stimes. 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'.

F WO 92/21365 WO 92/21365 PC/US92/04356 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-HC1, lOmM Tris and 1.OmM PMSF pH 6.4 to the bone material in a beaker.

Extract for 16 hours at 4* with vigorous stirring. Following the 16 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-HC1 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 H 0 at 4oC using 50 mm dialysis tubing (3500 molecular weight cutoff). Following dialysis, lyopholize the material and resolubilize the lyophilized material in 4M Urea-0.05M Tris-O.1M NaCl, pH 7.4. Mix the solubilized material in a conical centrifuge tube with Heparin-Agarose and mix overnight on a rotator at 40C. 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 4-5m1.

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 NaCI 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 WO 92/21365 92/21365 PCT/US92/04356 13 pooled for further purification.

The pooled CM-Sepharose fractions are dialyzed three times for 24 hours each against 1% 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, NaC1, 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 1251 and analyzed by SDS gel electrophoresis. Aliquots (100 il) of each fraction are combined with 400 Al of elution buffer, dialyzed against 1% acetic acid and assayed for activity. Highly purified molecular weight range

M

r) 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 preferably from about 1 ng to about 10 g, more preferably from about 10 ng to about 2.5 jg.

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 Mg, more preferably from about 1.5 Ig to about 90 ig, preferably from about 1.8 Ig to about 75 Mg, more preferably from about 2.0 Mg to about 50 Ag, more preferably still from about 2.2 ig to about 25 ig, more preferably from about 2.3 ig to about 10 Mg, most preferably from about 2.5 Mg to about pg. Preferably the dose range is at least about 2.5 Mg.

Preferably, doses are administered over a 1 day to 6 month period, more preferably from about I 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 WO 92/21365 PCT/US92/0435 6 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 5 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 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 benzyl alcohol; Novocainee; 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 r a--l I r I I I-- WO 92/21365 PCT/US92/04356 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 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, WO 92/21365 PCT/US92/04356 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 vitam-n 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 a: least about and preferably from about 1% to about 10% of t.ne compound of the present invention.

Tablets can be compressed, enteric-coated, sugar-coated or filmcoated containing suitable :inders, 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, sol.tions 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. Specifi: 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 Dral 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 anc molded), capsules (hard and soft gelatin) and pills are described in Remington's Pharmaceutical Sciences (Arthur Osol, edctor), 1553-1593 (1980), incorporated herein by reference.

B. Topical-oral Dose Formns "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 L C: -i 1~ r I- i 1~1 1 I "I DETAILED DESCRIPTION OF THE INVENTN----- The present invention the administration to a mammal m ination of a safe and effective amount of a WO 92/21365

S.

S92/21365 PCT/US92/04356 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 a surfactant (from about 0.5% to about a thickening agent (from about 0.1% to about a humectant (from about 10% to about a flavoring agent (from about 0.04% to about a sweetening agent (from about 0.1% to about a coloring agent (from about 0.01% to about and water (from about 2% to about 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 and an antiplaque agent (from about 0.1% to about Other preferred compositions of the subject invention are mouthwashes and mouthsprays. Components of such mouthwashes and mouthsprays include water (from about 45% to about ethanol (from about 0% to about a humectant (from about 0% to about a surfactant agent (from about 0.01% to about a flavoring agent (from about 0.041 to about a sweetening agent (from about 0.1% to about and a coloring agent (from about ~Y~i i:: WO 92/21365 PCT/US92/04356 18

.T

0.001% to about 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 and an antiplaque agent (from about 0.1% to about Other preferred compositions of the subject invention are dental solutions. Components of such dental solutions generally include water (from about 90% to about preservative (from about 0.01% to about thickening agent (from about 0% to about flavoring agent (from about 0.04% to about sweetening agent (from about 0.1% to about and surfactant (from 0% to about "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 technologies: 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, A.D. Haffajee S.S. Socransky, "Periodontal therapy by local delivery of tetracycline, Journal of Clinical Periodontoloqy, Vol. 6 (1979), pp. 83-92; Goodson, 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, J. Gibson, B. Perkins, J.

Goodson L. Laufe, "Fibrous delivery systems for antimicrobial agents", Polymer Science and TechnoloaY, Vol. 32 (1985), pp.

47-59; Dunn, J. Gibson, 8. Perkins, J. Goodson L. Laufe, "Fibrous delivery systems for antimicrobial agents", Polymer Material Science Engineering, Vol. 51 (1984), pp. 28-31; Olanoff, WO 92/21365 PCT/US92/04356 -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, 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, "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 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 Sare known. Examples of such solubilizers are as follows: urea compounds urea; urethan); surfactants Tweens; Spans; sodium deoxycholate and Pluronics); cellulosic agents carboxymethylcellulose); carbohydrates sorbitol; mannitol); B vitamins nicotinamide); xanthine derivatives; and alcohols benzyl alcohol). Examples of acids to be used include the following: glucuronic; galacturonic; fumaric; gentisic; acetic; citric and lactobionic. Types of antimicrobial agents -LP- i i i CI~- WO 92/21365 PCT/US92/04356 20 that can be used are the following: phenylmercuric nitrate; thimerosal; benzethonium chloride; benzalkonium chloride; phenol; cresol; and chlorobutanol. An art-known local anesthetic 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

D

3 for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques.

BMP composition Component

BMP-I

NaCl Sterile water Percent by Weight of Composition 0.04 0.90 100.00 100.00 1.25-dihydroxy Vitamin D, composition Percent by Weight Component of Composition 1,25-dihydroxy Vitamin D 3 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 pg of the 1,25-dihydroxy Vitamin D 3 composition is orally administered 24 hours before fracture reduction and once daily thereafter. The BMP and 1,25-dihydroxy Vitamir D 3 are adminis-

A

WO 92/21365 WO92/21365 PCT/US92/04356 21 tered until desired repair is achieved, perferably over a seven day period.

EXAMPLE II An injectable composition for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques.

Percent by Weight Component of Composition BMP-2 0.04 25-hydroxy Vitamin D2 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 reconstructive 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 q.s.

100.00 0.1 cc of the composition per cm 2 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 conventional mixing techniques.

Percent by Weight Component of Composition BMP-1 0.04 BMP-2 0.04 WO 92/21365 P /US92/4356 PCT/US92/04356

A-,

22 BMP-4 24,25-dihydroxy Vitamin D 3 NaCl 0.04 0.01 0.90 Sterile water q.s.

100.00 0.1 cc of the composition per cm 2 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.

Component BMP-2 NaCl Percent by Weight of Composition 0.04 0.90 Sterile water Q.s.

100.00 After the patient is prepared using conventional periodontal 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 and an oral composition comprising 1,25-dihydroxy Vitamin D 3 for treatment of osteoporosis is prepared by combining the following components utilizing conventional mixing techniques.

osteoinductive extract composition Component BMP-2 BMP-3 BMP-4 NaCl Sterile water Percent by Weight of Composition 0.001 0.001 0.001 0.001 0.900 00.000 100.000

I

J" W 92/21365 PCT/US92/04356 23 125-dihydroxy Vitamin D, composition Percent by Weight Component of Composition 1,25-dihydroxy Vitamin D 3 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 D 3 composition is orally administered within one hour of the osteoinductive extract inj..tion 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 03 0.01 Acid demineralized bone particles 90.000 NaCl 0.900 Sterile water for injection 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.

1 PCT/US92/04356 -24 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: STONE, ROGER L.

(ii) TITLE OF INVENTION: THERAPEUTIC FORIULAS FOR OSTEOINDUCTION (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: The Procter Gamble Company STREET: 11810 East Miami River Road CITY: Cincinnati STATE: Ohio COUNTRY: U.S.A.

ZIP: 45239-8707 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

S(viii) ATTORNEY/AGENT INFORMATION: 1 NAME: Corstanje, Brahm J.

REGISTRATION NUMBER: 34,804 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 513-245-2858 TELEFAX: 513-741-3012 I WO 92/21365 PCr/US92/04356 25 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 2487 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GCCGCTTCCC TCGCCGCCGC CCCGCCAGCA TGCCCGGCGT

TCGGGCTGCT

ACCTGGCGGA

CCTTTCTTGG

CTGTGGATCT

ACACTTCTAC

GATGGAGAGG

CCGATGGGGT

TCCGGCAGGC

ACGAGGACAG

GCCGCGGCGG

TGGTCCACGA

ACCGCCACGT

GCTGCTCCCG

GGAGGACGAC

GGACATTGCC

CAGACGGCAC

CCC CAGCTGC

TAGATCCCGT

CATCCCCTTT

CATGAGGCAC

CTATATTGTG

GGGCCCCCAG

GCTGGGCCAC

TTCCATCGTT

CGTCCCGGCC

TCGGAGCCCC

CTGGACGMAG

ACAGCTCGTA

CAGAGCACCA

AGCCGGCGGG

GTCATTGGGG

TGGGAGAAGC

TTCACCTATC

GCCATCTCCA

GTCGTCGGCT

CGTGAGAACA

GGCCGCTGGA

TCAACTACAA

AGGACCIGAG

AGTCCTCCAT

ACGGGCAGCC

CGGCGACGTC

GAAACTTCAC

ACACCTGTGT

GACCTTGCGG

TCGGCAAGA.A

TCTGGCACGA

TCCAGCCAGG

GGCCCGCCTG

CTTGGCCGAC

AGACCCCTGC

GGCCTTCCAG

CAAAGCTGCA

TCAGAGGGGA

CCGACCAGAG

TGGTAGCCAG

CACCTTCCTG

GTGCTGCTCC

CTGTGACAAG

ACACACTCGG

GCAGGAGTAT

CCGCTGCTGC

TACACCTATG

AAGGCGGCTG

GTACAGCAGG

GTTCCAGGAA

GCCTGTGGGA

CGTGTGTGGC

AGGGCAGTCT

GAGCGCACTG

TACGTGGGTC

TTCGGCATTG

CCAGACCGGG

AACTTCCTGA

WO 92/21365 PCT/US92/04356

AGATGGAGCC

ATTACGCTCG

AGGTGAACGG

CCCMAGCCCG

GCAACTTCTC

GCATCTCTGT

GCAGCCGCCT

CCCTC CGAGG

GCCTCTGGGT

ACGMAGCCAT

ACCCAGACGA

TCCACGTGGG

ACTATCTGGA

GCTATGAGMA

CTGACGGGTC

GCTCTCGGCC

AGTGCAGCTG

GTGGCGGATT

ACCCCCCCAA

TCAGGAGGTG

GAACACATTC

GGTBAAACCT

CMAGCTTTAC

C TC C CCTGAA

CACACCCGGG

GTGCTGGTAC

CCGCTTCTGC

TGAATTCCGC

CTGCGGGGGT

TTACCGGCCC

CCTCACATTC

GGTGCGCGAC

GCCTGATGAC

CATTAACAAA

CMACCGCGGG

TGACCCCGGG

CCTCACCAAG

CAAGAACTGC

GAGTCCCTGG

TCCAGGGGCA

CCCATTGGCC

AAGTGCCCAG

TACCCCAATG

GAGAAGATCA

GACTATGTGG

GGGTCCAAAC

AGCAGCAGCA

GATGTGAAAA

AGCAAAGTCT

CAGTCCTTTG

GGGCACAGTG

ATCAAGAGCA

GCGGGCTTTG

GGCTGTGAGC

TACGAGCTGG

CTCAACGGCT

ATCTGGCAGC

26

GGGAGACCTA

TCTTCCTGGA

AAAGGACACG

CCTGTGGAGA

GCTACTCTGC

TCCTGAACTT

AGGTCCGAGA

TCCCTGAGCC

ATTGGGTTGG

AGGACTATGG

GCATCTGGCG

AGATTGAGCG

AGAGCAGCAC

CGTCCAGCCG

CCGTCMACTT

AGCGGTGCCT

CC CCAGACAA

CCATCACCAG

TGGTGGCCCC

TGACTTCGAC

TACCATTGTC

GCTCAGCAAG

GACCCTGCMA

TCACATGCAC

CACGTCCCTG

TGGCTTCTGG

TATCGTCTCC

AAAGGGCTTC

CCACATTCMA

GATCCAGGTG

CCACGACAGC

CCTCATCGGG

CCTCTGGCTC

TTTCAAAGAG

CMCACCCTG

GCGCCGCTGT

CCCGGGCTGG

CACCCAGTAC

AGCATCATGC

CCCAAGTATG

GGGGACATTG

GACAGCACAG

TGCGTGTGGC

GACCTGTAC C

AGGAAGGCGC

ACTGACAGCC

TTTGCAGTCT

TCGCCCAACT

TCTGAGGGCT

TGTGCCTACG

CGCTACTGTG

AAGTTCGTCT

GTGGACGAGT

G.GCAGCTACA

GAGGCTGCTT

CCCAAGGAGT

CGCATCTCCC

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 TGCAGTTTGA CTTCTTTGAG ACAGAGGGCA ATGATGTGTG CMAGTACGAC TTCGTGGAGG

-I

WO 92/2 1365 PCT/US92/04356 27 TGCGCAGTGG ACTCACAGCT GACTCCAAGC TGCATGGCAA CCGAGGTCAT CACCTCCCAG TACAACAACA TGCGCGTGGA TGTCCAAAAA GGGCTTCAAG GCCCACTTCT TCTCAGAAAA CTCGGGGACG CCCCCACCAG CTCAAATTCC GAGTGCAGAA GAGGCCTGCC AGGCCTCCCG GACCCCTTGT TACTCAGGAA GATGGGGGCT TCGGTGCCCA CCAACCCCCC ACCTCCACTC CTCTGGCCGG ACAGAACTGG TGCTCTCTTC TCCCCACTGT CCCTTCCCCG TGCCCTACCC CCTCCCATTT TGATGGTGTC AGTAAA.AGAG GGACCCCTGC GTCCTGC INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1547 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

GTTCTGTGGT

GTTCAAGTCC

GAGGCCAGCT

AAGAAACCGG

CCTCACCTTG

TGCCATTCCG

GCCCGTCCGC

TGTGACATTT

TCTGAGAAGC

GACMACACCG

CTGCAGCCCC

ACCCCCCAGT

GACGGMATGG

GCCCACCTCC

GGACCGGGGA

CCTGTTGTGA

2040 2100 2160 2220 2280 2340 2400 2460 2487 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GGGGACTTCT TGMACTTGCA GGGAGAATAA CTTGCGCACC CCACTTTGCG CCGGTGCCTT TGCCCCAGCG GAGCCTGCTT CGCCATCTCC GAGCCCCACC GCCCCTCCAC TCCTCGGCCT TGCCCGACAC TGAGACGCTG TTCCCAGCGT GAAAAGAGAG ACTGCGCGGC CGGCACCCGG GAGAAGGAGG AGGCMAAGAA MAGGAACGGA CATTCGGTCC TTGCGCCAGG TCCTTTGACC WO 92/21365 PCT/US92/04356 28

AGAGTTTTTC

CTGCGGTCTC

TTCCCCAGGT

TCGCGGCGGC

TCGAGTTGCG

CCGTGGTGCC

CCGCCCCAGA

ACCATGAAGA

TCTTTAATTT

TCCGAGAACA

TTTATGAAAT

ACACCAGGTT

TGATGCGGTG

TGGAGGAGAA

ATGMACACAG

GGCATCCTCT

AGTCCAGCTG

GGATTGTGGC

TGGCTGATCA

ACTCTAAGAT

CATGTGGACG

CTMAAGGTCG

CCTCCTGGGC

GTCGTCGGGC

GCTGCTCAGC

CCCCTACATG

CCACCGGTTG

ATCTTTGGAA

AAGTTCTATC

GATGCAAGAT

CATAAAACCT

GGTGMATCAG

GACTGCACAG

ACAAGGTGTC

CTGGTCACAG

CCACAAAAGA

TAAGAGACAC

TCC CCC GGGG

TCTGAACTCC

TCCTAAGGCA

CTCTTTCMAT

ACCATGGTGG

GGCGCGGCTG

C GC C C CCAT

ATGTTCGGCC

CTAGACCTGT

GAGAGGGCAG

GAACTACCAG

CCCACGGAGG

GCTTTAGGAA

GCAACAGCCA

AATGCAAGCA

GGACACGCCA

TCCAAGAGAC

ATAAGGC CAT

GMAAACGTC

CCTTTGTACG

TATCACGCCT

ACTAATCATG

TGCTGTGTCC

GGACGTGTCC

CCGGGACCCG

GCCTCGTTCC

CCCAGCCCTC

TGMAACAGAG

ATCGCAGGCA

CCAGCCGAGC

AAACGAGTGG

AGTTTATCAC

ACAATAGCAG

ACTCGAAATT

GGTGGGMAAG

ACCATGGATT

ATGTTAGGAT

TGCTAGTAAC

AAGCCAAACA

TGGACTTCAG

TTTACTGCCA

CCATTGTTCA

CGACAGAACT

CCGCGTGCTT

CTGTCTTCTA

GGAGCTGGGC

TGACGAGGTC

AC CCAC CCCC

CTCAGGTCAG

CAACACTGTG

GAAAACAACC

CTCAGCAGAG

TTTCCATCAC

CCCCGTGACC

TTTTGATGTC

CGTGGTGGAA

MAGCAGGTCT

TTTTGGCCAT

CAAACAGCGG

TGACGTGGGG

CGGAGMATGC

GACGTTGGTC

CAGTGCTATC

CTTAGACGGA

GCGTTGCTGC

CGCAGGAAGT

CTGAGCGAGT

AGCAGGGACG

CCGGGCTCAC

CGCAGCTTCC

CGGAGATTCT

CTTCAGGTTT

CGAATTMATA

AGACTTTTGG

ACCCCCGCTG

GTGGCCCACT

TTGCACCAAG

GATGGAAMAG

,AAACGCCTTA

TGGAATGACT

CCTTTTCCTC

AACTCTGTTA

TCGATGCTGT

300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 11V 1200 1260 1320 1380 1440 WO 92/21365 PCT/US92/ 04356 29 ACCTTGACGA GAATGAAAAG GTTGTATTMA AGAACTATCA GGACATGGTT GTGGAGGGTT GTGGGTGTCG CTAGTACAGC AAAATTAAAT ACATAAATAT ATATATA INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1774 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 1500 1547 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGATCTTGAA AACACCCGGG CCACACACGC CGCGACCTAC

GTGGAGACGG

CCCACCTGTC

ACCCGGCCGC

GGCTCCGCCG

CGCCGCGCGG

GCTTCTGCGT

AAGCTGTGCC

ACAAGGTCTC

GGACACCGGG

GCAACACGGT

CGCCCGCAGC

AGGCTGCGCT

GTCCCGGGCT

CCGGCTCCTT

GTACCTAGCC

GAGCCTGGCG

AGGTGACCGC

TGAACACATG

CTCCCTGGAG

TCGCAGCTTT

GCCCTGCGCG

GGGTCAGCGC

CC GTGCGC CC

GCGCCTTCGG

ATGGCTGGGG

CAGGGAGAGA

ACGGCAGGTG

CTGCGGCTCT

GGAGGCTCGC

CGGGCGGCAG

GGTGAGGTCC

AGCAAGTGGG

TCGCCCCAGC

AGTGTCCCGC

CGAGCAGGCT

GACCGAAGCC

GTGGCCCGGA

ATGACAGGTA

AGCCCTGGCG

CAGCAGAAAC

AGCTCTTTCT CAGCGTTGGA GCGCAGCTGC TGGGGAAGAG GCTGGCCGCT ATCTCGCTGC TGGTTTGGAG TTCMACCCTC AGCGACGCCG GGAGCCGACG GCTCTTTCTG TGGCTGGGCT ACCTTTCCCG GAGCTCCGCA CTCCGAGCTG CAGCCGCAAG CAGCACGGTC CAGGCGGCCC CCCTCGGCTC CTGCGCGAAG TCTTGAAAGA AAAGGACTGT 120 180 240 300 360 420 480 540 600 660 7 WO 92/21365 PCr/US92/04356 30 ATATCTTCMA TCTGACATCG CTAACCAAGT CTGAAAACAT

TCTGTATTGG

ATCATGCTCA

GAAACCAAAG

TTATGTCCTG

AGTTCCT CAT

CTTTTCCAGA

GTGTGGTATC

ATAGCCACAT

TGCTGCCTCT

TGTGGGAGGA

ATAAAAAGAA

AGACCCTGAA

ACCTCAAGGT

TTGATGCCTA

CAMATCATGC

AGCCTTGCTG

AGAATGTAGT

AGAGCTAGGA

GAGGAAACAC

TCAACTCCTT

GCTGTCTAAA

AGGATTTAAC

GCCTTATATC

AAGCTTACAG

CAGAGCTGCC

GCAGAACAAC

GAGMAAGCCT

ACAGAGAAAG

AAAGGCAAGG

AGACTTTGCA

TTATTGCTCT

TACCATCCAG

TGTACCAGAA

GCTTAAAGTA

AACATCAGCC

ATTCAGATTG

GGCCATCTGT

GATATCACTC

ATTACGTCCA

TTGGTATATG

GGACACCGGA

CTTTCCATTG

GAGCTTCCTG

TACAAGACCC

GGGCCTCATC

AGAAAGCAGT

GATATTGGCT

GGAGCATGCC

AGTATAGTGA

AAGATGTCCT

TACCCTAACA

TGAGTTGTCC

ATCTTTCTGC

CAGTGGATAT

AATTCTTGAG

AGGGACGCCA

CCAA.TGATGC

ATTTTCCCAC

AGCGGAGGAA

GGGCAGMATA

TTCAGGCTCA

GGAAGAGCCA

GGATTGAACC

GGAGTGMATG

AGTTC CC CAT

GAGCTGTGGG

CACTCAGTAT

TGACAGTAGA

CAAT

TTTGTCTGCC

AGTGTCTGGA

ATGGACCCTC

GGCCAAATCT

GAAGGCCAAA

GCTGCCAAAG

CGCCATTTCT

TGGMACTGTT

GMGCGCTCT

CCAGTATAAA

GGCCCCTGAA

GACGCTCCMA

TCGGAATTGC

GATTATCTCC

GCCAAAGTCT

GGTCGTTCCT

TTTATTCTTT

GTCTTGCGCT

ACACTGTATT

GGATGCTCCC

AAATTCAGCA

CATCGAGATA

GAAAATGAAG

AGGAGGTTAC

GAGCCAGAAA

CCCAAATGGG

ACTGGGGTCT

AGGATGAGG

AAGAGTAAGA

TTTGATGAGC

GCCAGGAGAT

CCCAAGTCCT

TTGAAGC CAT

GGGATTCCTG

GATGMAAATA

TGCAGATAAC

720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1774 CTGGCAAAGA ACTCATTTGA ATGCTTMATT INFORMATION FOR SEQ ID NO:4: WO 92/21365 WO 9221365PCTr/US92/04356 31 SEQUENCE CHARACTERISTICS: LENGTH: 1751 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GGCAGAGGAG GAGGGAGGGA GGGAAGGAGC GCGGAGCCCG

TGGCATCCGA

GCTTGTCTCC

CGGCCCTCGC

CCCGCTACTG

GCAGCTTCCC

TGTTATTATA

GGTCGTTTTA

GACGGGGMAG

GAGCCATGAG

CCGCCCGCAG

35 TCAGTCTGGG

CCCGGCCAGC

CCCAGGGACC

GCTGAGGGAC

CCGATGGGAT

CCAGGTTCAC

CAGGGACCTA

TGAGCCTTTC

TGCCTTGTTT

TTATGCCAAG

AAAAAAGTCG

CTCCTGCGGG

CCTAGCAAGA

GAGGAGGAGG

CGGGCCAACA

AGTGAAAACT

GCGAGCCTGA

TCCCGTCCAA

TGCAACCGTT

TGGAGCCATT

CAGCAAGTTT

TCTGTCAAGA

TCCTGCTAGG

CCGAGATTCA

ACTTCGAGGC

GTGCCGTCAT

AAGAGCAGAT

CCGTGAGGAG

CTGCTTTTCG

GACGCCGCTG

GCTATCTCGA

CAGAGGTCCC

CC GTAGTGC C

GTTCAAGATT

CACCATGATT

AGGCGCGAGC

GGGCCACGCG

GACACTTCTG

TCCGGACTAC

CCACAGCACT

CTTCCACCAC

TTTCCTCTTT

GCCCGGAAGC

CTGCTCCGGC

GCCTGCAGCG

CAGGAGCTGC

ATCCCGAGCA

GGCTGTCMAG

CCTGGTAACC

CATGCTAGTT

GGAGGACGCC

CAGATGTTTG

ATGCGGGATC

GGTCTTGAGT

GAAGAACATC

AACCTCAGCA

TAGGTGAGTG

TGAGTATCTA

C CACAGT CC C

TGCTGGCGAG

ACGCACTGCT

AATCATGGAC

GAATGCTGAT

TGATACCTGA

GCTCAGGGCA

GGCTGCGCCG

TTTACCGGCT

ATCCTGAGCG

TGGAGAACAT

GCATCCCTGA

.120 180 240 300 360 420 480 540 600 660 720 780 840 4

I

I

I

WO 92/21365 PFU9/45 PC-r/US92/04356 32 GMCGAGGTG ATCTCCTCTG CAGAGCTTCG GCTC TGATTGGGMA AGGGGCTTCC ACCGTATAAA CATT AGTGGTGCCT GGGCACCTCA TCACACGACT ACTG GACACGGTGG GAAACTTTTG ATGTGAGCCC TGCG GCCAAACTAT GGGCTAGCCA TTGAGGTGAC TCAC CCAGCATGTC AGGATTAGCC GATCGTTACC TCAA GCCCCTCCTG GTCACCTTTG GCCATGATGG CCGG GGCCAAGCGT AGCCCTAAGC ATCACTCACA GCGG GCGCCACTCG CTCTATGTGG ACTTCAGCGA TGTG ACCAGGCTAC CAGGCCTTCT ACTGCCATGG GGAC CAACTCAACC MACCATGCCA TTGTGCAGAC CCTG CAAAGCCTGT TGTGTGCCCA CTGAACTGAG TGCC TGATAAGGTG GTACTGAAAA ATTATCAGGA GATG AGATCAGGCA GTCCTTGAGG ATAGACAGAT ATAC CACACACACA CGTTCCCATC CACTCACCCA CACA GACTTTTATT T INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2153 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear

TTCCGG

TATGAG

GACACG

GTCCTT

CTC CAT

GGGAGT

GGC CAT

GCCAGG

GGCTGG

TGCCCC

GTCAAT

ATCTCC

LGTAGTA

ACACCA

CTACAC

GAGCAGGTGG

GTTATGAAGC

AGACTGGTCC

CGCTGGACCC

CAGACTCGGA

GGGAATTGGG

GCCTTGACCC

AAGMAGMTA

AATGACTGGA

TT-TCCACTGG

TCTGTCA.ATT

ATGCTGTACC

GAGGGATGTG

CACACACACA

AGACTGCTTC

ACCAGGGCCC

CCCCAGCAGA

ACCACAATGT

GGGAGAAGCA

CCCACCAGGG

CCCAGCTCCG

GACGCCGGAG

AGAACTGCCG

TTGTGG CCCC

CTGACCACCT

C CAGTATCC C

TGGATGAGTA

GGTGCCGCTG

CCACATACAC

CTTATAGCTG

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1751 WO 92/21365 PCT/US92/94356 33 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CTGGTATATT TGTGCCTGCT GGAGGTGGMA TTMACAGTMA GGACTTACAG GAAGGATTTC MAGTAAATTC CTAGAGTATT ATTTTACACT AAGACGACAC GGACAGATAT ATATTCCMAC ACCAAGGTGC AGGATTTGTT TTGGM.AGAG CTCAAGGGTT TTTGGGAACT ACAGTTTATC AGAAGATCMA TTATCATMAA TTCATATAGG MATGCATAGG TGCTACATCA ATGCAGCAAA AACTCTTAAC GCTTTCTTAG AMATAACTAC TCTTGACATA TCGGTGAGGA TGTTGTGCTC AGAAATGTCA TCAGCTACTG GGAAACTGTA CCTCCTAGAA GAAGGACTAA AAATATCAAC TTTTGCTTTT TTAAGGGTAT TGTGGGTTTC CTCTGGAGCT GTTTGGGAGA CMATCATGTT CACTCCAGTT GACGGGAAAT ACAAAGGGAA ATTCTCTCTA TTTCACCTGG AAAACAAGCG TCCTCTGCAC TGACCAATGA AGAAAATCCT GAAGAGTCGG

AGGGA.AACAC

AMAGATGTT

AGATCAGCAT

GAGMAGAACT

CTTTTGCTAA

TCATCTGATC

AACTGTGGAT

TTCCAkAATA

CTGTCATGAA

CCTTAGGTTT

GGACAAMAAT

GCTGGGTTCT

TTATTTATAG

TCTTGGGTTT

CTCTCTTTAT

AGTACTCAG T

GAAGGAGAMA

ATTTACTTGA

AAAGTTATCA

AGATCTGTGA

CAAAAGCAAG

TTCAMATACC

AAATATATT

MATTGGAAAT

TTTMAAATAG

AAATAGGTAA

TTTTTTTTTT

GCATCTGACT

AGTGGGTTAT

AiAGACTACGG

GCCTCACAGA

GCTGGATCTC

AAGGGCATCC

GGGATTGMAT

ATAGTACMAC

C CAAGCTGC C

TTCAGAAATC

TGAAGATTAC

AAAGGCCTGA

AGCCGTCTTC

CTGAGTTTCA

GACAGGAA

ATTTGTTTTT

MAGAGGACAA

GTATTTTTAC

GCMAAGGAG

AACCACGAAA

CCCAGAC CAT

TACMATGCCA

TTGGCAGAAG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 WO 92/21365 PCF/US92/04356 34

AGACCAGAGG

AGTTATCTCG

CCAACTTTCT

AGGATTTTTC

CTCATGGAGA

GATTTGAAAA

GGGATGCAGA

TTGTCTTTGA

GCTTACAGCT

TTGTGGGAAG

GTGAGGTACT

AATCCAGCTC

AGCAAAAACA

AGGACTGGAT

TTCCACTTAA

TGATGTTTCC

CTGTTCTGTA

TACGCTCATG

GGCAAGAAAG

GACGACTCCT

GAATGATGCT

TCACCAGCGA

GGCAGTGACA

TGAAACAATT

TCTGTTCTTG

TATCACTGTG

CTGTGCAGAA

ACAGGGACCT

TCTTCGATCC

TCATCAGGAC

AGCCTGTAAG

TATAGCACCA

CGCCCATATG

TGACCACGTA

CTTTGATGAC

TGGCTGCCAC

GGATACCCAG

CTGACCACCC

GACATGGTCA

AGGCATTACA

GCAGCTGAAT

AAGATTAGCA

TTAGACACAA

ACGAG CAATC

ACAGGGGATG

C AG T CAAAC

GTGAGAGCAG

TCCTCCAGAA

AAGCACGMAC

GAAGGATACG

AATGCCACCA

CCAAAGCCTT

AGCTCCMATG

TMJTATTAAA,

CCTCTCCCAA

AGAGTCCTCC

TGAGCTTTGT

AAGAATTTCG

TCCGGATATA

TATATCAAAT

GMAAGGCCCA

ATTGGGTGAT

GACGCAGTAT

AACCATTCAT

CCAACAAACG

TGTCCAGTGT

TCTATGTGAG

CTGCATTTTA

ACCACGCTAT

GTTGTGCTCC

TCATTTTGAA

TAATATTGAT

TTTCAGACAA

TGGGTATCCT

TCTAGCCAGC

CMACTTAGTT

ATTTGATCTT

CAAGGACCGG

CATCMAGGMA

AGCTTTAGAT

TAATCC C AG

CAACGTAA

GGTGGCCTTC

AAAAAATCAA

TGGAGATTAT

CTTCCGGGAT

TTGTGATGGA

AGTTCAGACTr

AACCAAATTA

AAAATATAGA

AATAACAA

ACAGAAAA

CGTCGCATAC

CTCCA'YGATA

GAAAGAGACA

ACCCAAATTC

AGCAACAACC

TACACAAATA

GTGGGTTGGC

AATAATTTGG

TCTGCTGGTC

TTCAAGGCGA

MACCGCMATA

AACACAAGTG

CTGGGATGGC

GPATGTTCTT

CTGGTTCATC

AATGCCATCT

MATATGGTAG

AGATCTGTAT

A

1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2153

I

TMAGGTTTAT GGCTGCAATA AMAAGCATAC INFORMATION FOR SEQ ID HO:6: WO 92/21365 WO 9221365PC]'/US92/04356 35 SEQUENCE CHARACTERISTICS: LENGTH: 2923 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGACCATGAG AGATAAGGAC TGAGGGCCAG GAAGGGGAAG GGGGACTGCT CACGCCAAGG "-TCGCGGG ATCCGCGGGG GCGCAGTGGC TGTGCTGGTG CGGCCGCCCT TGCCCGCTGC GGGAGCCCCG GCCGCACGGA TACCGGCGGC TCAAGACGCA GGGCTCCCGC ACCGGCCCCG CGGCAGCAGG AGGAGCAGCA CGACTGMAGT CCGCGCCCCT GACGAGGACG GGGCGTCGGA TCGTCCCAGC GTCGGCAGCC CTGGCCCCCG GATCTGGCAG TTCCTCAACG ACGCGGACAT

GCCACAGCGG

GCAGCCCGGC

GTGGGGGCTG

CGCGGCCGCC

GCAGCCGCCG

GGAGAAGCGG

GCCCCTGCAC

GCAGCAGCAG

CTTCATGCTG

GGGGGAGAGG

GCCCCCGGGC

CGGCGGCGCG

GGTCATGAGC

CCGCGCTCCG

CGGGCGGGGA

CTGTGCAGCT

GCCGCCGGGG

CCGTCGCCGC

GAGATGCAGA

GGCCTCCAAC

CAGCTGCCTC

GATCTGTACA

CAGCAGTCCT

GCCGCGCACC

TCCCCACTGA

TTTGTGAACC

C GAG CCCGC C

GCCTCGCTCC

TGCCGGGGCT

GCTGCGGGCC

GGCAGCTGCT

AGTCCTCCTC

AGGAGATCTT

AGCCGCAGCC

GCGGAGAGCC

ACGCCCTGTC

GGCCCCACGA

CGCTCAACCG

CCAGCGCGCA

TGGTGGAGTA

GAGAGGTGGC

GCCGCTCCAC

GGGGCGGAGG

CCC GCC GCTG

GGGGGACGGC

GGGCTTCCTG

GTCGGTGCTG

CCC GGC GCTC

CCCTCCCGGG

CGCCGACAAC

AGCAGCCAGC

CAAGAGCCTT

GGACAGCGCC

CGACAAGGAG

A WO 92/21365 PCI/US92/04356 36 TTCTCCCCTC GTCAGCGACA CCACAAAGAG TTCMAGTTCA ACTTATCCCA

GGTGAGGTGG

AAAAACCAAA

TCTGACCTGT

TTTGACATCA

CAGCTGAGCG

GGCAGAGAC G

GTCCACGTGC

TCTACCCAGT

TTGAAAMCAG

GACTGGATCA

C CACTCAACG

ATGAACCCCG

GTTCTTTACT

AGAGCTTGTG

TTCCTAGATT

ACTTTGAAAC

AATAATTTGC

GGATGTCTGT

CTCCTCCCCC

TGACGGCTGC

CTTTTCTTAT

TTTTGTTGGA

C GGCC AC TAG

TGGTGACMAG

GCCCTTACGA

GCACCACCAG

CCCAGGACGT

CCTGCAGGAA

TTGCACCCAA

CACACATGAA

AGTATGTCCC

TTGATGACAA

GATGCCACTA

ACATCTGCCT

TATCTCATGC

TCACTTGGTA

AGCATMAGGT

AAAACCCAC

AGAATTCCGC

CAGCATTTAT

CACCCGTGTA

CAATCTGTGG

GGATGGA'TC

TAAGCAGCCC

GTCAGCCTCC

GGCGCGGGTC

GCATGAGCTG

GGGCTATGCT

TGCMACCAAC

CAAACCGTGC

CTCCAATGTC

ACTCGAAACC

TAAAAAAACA

CAGTGCCTTA

AATGACGTGA

CTCGTAACTG

CAAAATTAGT

ATCTACAAGG

CAAGTCTTAC

GTATGGGCCT

GTTGTGACTC

CACGTCCACC

TTCATGGTGG

AGCCGGCGCC

TCCAGTGCTT

TATGTGf4IGTT

GCCAATTACT

CACGCGA'TTG

TGTGCGCCMA

ATTCTGAAAA

AGATGCTGGG

CGGAAGCACA

TTACCCAGGA

GTAGTTGTTG

CAGAIICATA

TTTAGCTGTA

ACTGTGTTAT

AGGAGCATCA

CAGAAGAAGG

CACAGCATAA

C CC GAGC CG C

CTTTCTTCAA

GACAACAGAG

CAGATTACMA

TCCAAGACCT

GTGATGGAGA

TGCAGACCTT

C TMG CTAMA

AATACAGGAA

GACACACATT

GTTGGAGGTG

AGATTTTAAA

GTCTGTAGCA

ACCGTGMAGC

GATCAAGCTA

GATTC CIGAG

GGGGAGTTTT

GCACAGAGAC

CTGGCTGGAA

CATGGGGCTT

AGGCCTGGTG

AGTGAGTGAG

TCGTAkATCGC

CAGCAGTGAA

GGGATGGCAG

ATGCTCCTTC

GGTTCACCTT

TGCCATCTCG

TATGGTTGTA

CTGCCTTGGA

GGACGATGAG

GGACCTCATT

AGCTGAGTTT

TCTTCCTACC

TTTGGGGTGT

960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 WO 92/21365 WO 9221365PCr/US92/04356 37 TTGTTAGTAA ATAGGGAAAA TAATCTCAAA AGCTGGTTCA 'GTACTGTCTA TCAAAGGTAG GGGGGAACGC CTCTGTTCAG TTCATTCCCA AGCCAGGGCT CCACGGGGCG CCCTTGTCTC TTTTGTTGGT GTGMAAATAC ACTTATTTCA CTCCATTTGC TGTACTCTTT GCTAGTACCA ACAAGGGGTG TGTAACCGTG TAACACGTGA GGTTCTTTGA CCAGCACATT AACTTCTGGA GGTTCTCTGC CTTTTTACTA TACAGCATAC ATAAAATGAG GGTGCCCAGC TTATAAGAAT MACGGAAATC ATGATTTCCC TGTAGAAAGT TAAATGTCTT TTTCACAATC ATGTGACTGG AATACATTTA TMATCTACAA CTGTTTGCAC ATTTATTGTC TATTTTATAT CTGTTTTGCT GGGGGGGGGT TTGTTTGGGG GGTGTCGTGG

GGAGTTAMAT

ATTTTACAGA

GAAGTCCACA

AGTCATTGCT

GCCAMAACAT

AAAGTAGACT

AGGCAGTGCT

CTGCCGGCTC

CACGCCACAG

GGTGTTAGGG

GAGGCTCAGA

GAAGGCAATT

TTACAGCTTT

GTGGCGTTGG

TGTGGGCGGG

GTATTCTTGG

GAACAGAAAT

GGACGCACAG

GTTGTATGTT

ACCATTTCTA

GATTACACTG

CACCTCTTCT

TAGTAC CTTT

GGTTAGMACC

GGATGAGCAT

TTAAATTTTA

TCATACTAAA

TTTTGTMAAT

GGGGGGGGCC

CGG

CTAAAGGATC

CGGGGAAGTG

CCCAGGCCAtC

CGTGCTGGAG

CACCTCAATC

AGGTGAGGCT

TTACCAGAAC

TCAGTAAAGT

AACGAAGAAA

GCTGTTTATG

GX',ATATTTTC

CTGATTMAAT

ATAAACTATA

GGGCTTTTGG

2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2923 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 1448 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) M4OLECULE TYPE; cDiRA WO 92/21365 WO 92/ 1365PCr/US92/04356 38 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTGACCGAGC GGCGCGGACG GCCGCCTGCC CCCTCTGCCA V GAGCCCGGAG

GCGGCGCCGC

U GCCGACTTCA 1 U CAGGAGCGGC

CGCCCGCACC

GCCATGGCGG

"CGTCTTCA

GACGCCGACA

CCACGCTACC

GTCACGGCAG

ACGTTCCGGA

TTCCTGCTCG

ACAGCCACCA

GTGGAGACGC

GGGCCCCAGA

CGCAGCATCC

AACCAGGAAG

GCCTGTAAGA

CCC GGGTAG C

ACAGCTTCGT

GCCTGGACAA

GGGAGATGCA

TCCAGGGCAA

TGGAGGAGGG

G TA C CCAG GG

TGGTCATGAG

ACCATCGAGA

CCGAATTCCG

TCAGCGTTTA

ACAGCCGTAC

GCAACCACTG

TGGATGGGCA

ACAAGCAGCC

GGTCCACGGG

CCCTGCGGAT

AGCACGAGCT

GCGTAGAGCC

GGCGCTCTGG

CGAGGTGCAC

GCGCGAGATC

GCACAACTCG

CGGCGGGCCC

CCCCCCTCTG

CTTCGTCAAC

GTTCCGGTTT

GATCTACMAG

TCAGGTGCTC

CCTCTGGGCC

GGTGGTCAAT

GAG CATCAAC

CTTCATGGTG

GAGCAAACAG

GGCCAACGTG

GTATGTCAGC

GGCGCGATGC

GCACCCCTGT

TCGAGCTTCA

CTCTCCATTT

GCACCCATGT

GGCGGCCAGG

GCCAGCCTGC

CTCGTGGAAC

GATCTTTCCA

GACTACATCC

CAGGAGCACT

TCGGAGGAGG

CCGCGGCACA

CCCAAGTTGG

GCTTTCTTCA

CGCAGCCAGA

GCAGAGAACA

TTCCGAGACC

CCTGGGGCGG

ACGTGCGCTC

TCCTGCTGCG

TCCACCGGCG

TGGGCTTGCC

TCATGCTGGA

GCTTCTCCTA

MAGATAGCCA

ATGACAAGGA

AGATCCCAGA

GGGAACGCTT

TGGGCAGGGA

GCTGGCTGGT

ACCTGGGCCT

CGGGCCTGAT

AGGCCACGGA

ACCGCTCCAA

GCAGCAGCGA

TGGGCTGGC~i

TGCGGGCCCG

ACTGCGAGCT

C TC CGC C CTG

CCTCCGCAGC

CCACCGCCCG

CCTGTACAAC

CCC CTACAAG

TTTCCTCACC

ATTCTTCCAC

AGGGGAAGCT

CGACMATGAG

ATCGGATCTC

GTTTGACATC

GCAGCTCTCG

TGGGCGGCAC

GGTCCACTTC

GACGCCCAAG

CCAGAGGCAG

GGACTGGATC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140

Claims (4)

1. A composition for generating new bone growth in a mammal in need of such treatment comprising: a. a safe and effective amount of Vitamin D compound; b. a safe and effective amount of agBMP selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7; and c. a safe and effective amount of a pharmaceutically acceptable carrier.

2. The composition of Claim 2 wherein the safe and effective amount of the Vitamin D compound is from about 30 ng to about 10 pg and at least about 2.5 pg of the BMP.

3. The composition of Claim 2 wherein the Vitamin D compound is selected from the group consisting of Vitamin D,, Vitamin D3, 1-a-hydroxy Vitamin D3, 1-a-fluoro Vitamin D3, 3-deoxy-1,25-dihydroxy Vitamin D3, 25-hydroxy-5,6-trans Vitamin 03, 25-hydroxy Vitamin D 2

25-hydroxy Vitamin D3, 1,25-dihydroxy Vitamin D 2 24,25-dihydroxy Vitamin D 2 24,25-dihydroxy Vitamin D 3 and 1,25-dihydroxy Vitamin 03. 4. The composition of Claim 3 wherein the Vitamin D compound is 1,25-dihydroxy Vitamin 03. The composition of Claim 4 wherein the pharmaceutically-acceptable carrier is an injectable carrier. 6. The composition of Claim 4 wherein the pharmaceutically-acceptable carrier is a topical-oral carrier. 7. The composition of any of Claims 2-7 wherein the BMP is a component of an osteoinductive extract, the osteoinductive extract comprising one or more BMP's selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. 8. A method of generating new bone growth in a mammal in need of such treatment comprising administrating to the mammal a safe and effective amount of a Vitamin D compound in combination I I WO 92/21365 PTU9/45 PC]r/US92/04356 ATCGCGCCTG TCCTACATGA GAAACGGTGC TTCGATGACA GGCTGCCACT CATTGCTC AAGGCTACGC ACGCCACCMA CCAAGCCCTG GCTCCA.ACGT AGCTCCTCCG CGCCTACTAC CCACGCCATC CTGTGCGCCC CATCCTGAAG AGAATTCAGA 39 TGTGAGGGGG GTGCAGACGC ACGCAGCTCA AAATACAGAA CCCTTTGGGG AGTGTGCCTT TGGTCCACTT ATGCCATCTC ACATGGTGGT CCAAGTTTTT CCCTCTGAAC CATCAACCCG CGTCCTCTAC CCGGGCCTGT CTGGATCCTC 1200 1260 1320 1380 1440 1448 The invention has been described herein with reference to certain preferred embodiments and examples. Obvious variations may appear to those skilled in the art. Therefore, the invention is not to be considered limited thereto but only by the claims which follow. WHAT IS CLAIMED IS: :i WO 92/21365 PCT/US92/04356 41 with a safe and effective amount of a BMP selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. 9. The method of. Claim 8 wherein the safe and effective amount of the Vitamin D compound is from about 1 ng to about rr'g and the safe and effective amount of the BMP is from about 1 pg to about 100 Ag. The method of Claim 9 wherein the safe and effective amount of the Vitamin D compound is from about 30 ng to about j.g and the safe and effective amount of the BMP is at least about pg. 11. The method .of Claim 10 wherein the Vitamin D compound is selected from the group consisting of Vitamin Vitamin D3, l1-a- hydroxy Vitamin 03, 1-c-fluoro Vitamin 03, 3-deoxy-1,25-dihydroxy Vitamin D3, 25-hydroxy-5,6-trans Vitamin D3, 25-hydroxy Vitamin D2, 25-hydroxy Vitamin D3, 1,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D3, and 1,25-dihydroxy Vitamin D3. 12. The method of Claim 11 wherein the Vitamin D compound is 1,25- dihydroxy Vitamin 03. 13. The method of Claim 11 wherein the administration of the Vitamin D compound and the BMP is by injection. 14. The method of Claim 11 wherein the administration of the Vitamin D compound and the BMP is topical-oral. The method of Claim 11 wherein the administration of the Vitamin D compound is oral and the administration of the BMP is by injection. 16. The method of any of Claims 8-15 wherein the BMP is a component of an osteoinductive extract, the osteoinductive extract comorisinQ one or more BMP's selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7. I -n e I; 17. A composition according to claim 1 substantially as herein described with reference to any one of the Examples. DATED: 9 February, 1996 THE PROCTER GAMBLE COMPANY By their Patent Attorneys PHILLIPS ORMONDE FITZPATRICK *42 I 42 :t u I S1 4 HI