CA2110410C - Therapeutic compositions for osteoinduction - Google Patents

Abstract

A method for generating new bone growth in a mammal comprising administering 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

W092/21365 ~ glaPcr/US92/04356 ~ ,~

THERAPEUTIC COMPOSITIONS FOR OSTEOINDUCTION
t TECHNICAL FlFln The present invention relates to the field of osteoinduction (bone growth). Specific~lly, the present invention relates to novel therapeutic formulations comprising the administration of bone ~ 3 tic proteins and a Vitamin D compound, resulting in o synergistic bone growth.
B.t~ R( OF THE INVENTION
In healthy individuals bone growth generally proceeds nor-mally 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 osteopor-osis. Osteoporosis is an abnormal loss of bony tissue often occurring in post r -~ al 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.
2s Unfortunately, these treatments are merely preventative against the further loss of bone. There is a need in the art for treat-ments that go beyond the prevention of bone loss and promote bone formation and/or reverse bone loss.
(1989) ~Bone ~ ' . ic 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 Allo-geneic 80ne Matrix from Vitamin D-deficient Rats", The Journal of Clinical Investi4ation. Inc.. Vol. 82, pp. 212-217, discloses the _ . _ _ _ . . . _ . _ .

WO 92~21365 PCr/US92/04356 211~1~1Q; - 2 -implantation of deminer~lized bone matrix from Vitamin D-deficient rats i nto normal rats . The demi ner~l i zed bone m~tri x from Vi t~mi n D-deficient rats did not promote osteoinduction as effectively ~s demineralized bone matrix from normal rats.
Sampath, T. K., S. ileintraub, and A. H. Reddi, 1l984) ~Extra-cellular Matrix Proteins Involved in bone Induction are Vitamin D
Dependent~, Biochemical and BioDhvsical Research Communications.
Vol. 124, pp. 829-835, discloses a study involving implantation of demineralized bone matrix from norm~1 r~ts and demineralized bone matrix from rachitic rats wherein the rachitic bone matrix did not induce bone growth whi1e 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.
lS U.S. Patent No. 4,761,471, Urist, assigned to the Regents of the University of California. issued August 2, 1988, discloses a bone ~ , Lic protein composition comprising BMP factor and 8MP 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 Ho. 4,455,256, Urist, assigned to the Regents of the University of California, issued June 19, 1984, discloses a bone . .~:, tic protein h~ving a molecular weight in the range of 1,000 to 100,000 daltons.
\larious other bone ~ ,r, - tic proteins/factors, osteoin-ductive factors, osteogenic factors ~nd other proteins/factors rel~ted to bone growth ~re disclosed in the following public~-tions: U.S. Patent 4,968,590, 1~' ath and Rueger, issued November 6, 1990; U.S. Patent 4,698,328, Neer, Potts and Slovik.
~ssued ~ctober 6, 1987; U.S. Patent 4,877,864, Wang, Wozney and Rosen, issued October 31, 1989; U.S. Patent 4,861,757, Antoni~des, Lynch and Williams, issued August 29, 1989; U.S. P~tent 4,810,691, Seyedin, Thomas, Bentz. Ellingsworth ~nd Armstrong, issued M~rch ` 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, 1lal1~ce, Smestad, McPherson, Piez ~nd Ross, issued December 6, 1988; U.S. P~tent 4,789,732, Urist. issued December 6, 1988; U.S. P~tent 4,774,322, Segedin, Thom~s, Bentz, Ellingsworth and Armstrong, ~ssued September 27, 1988; U.S. P~tent 4,698,328, _ _ _ . _ _ 2 ~ 4 ~ ~ . PCr~US92/04356 Heer and Slovik, issued October 6, 1987; U.S. Patent ~,6Z7,982, 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 Applica-tion 145 ISS, Sen, published July 10, 1985; World Patent Applica-lS tion 89/10934, Roos, Burns, Guy and McKnight, publ ished November 16, 1989; llorld Patent Applications 89/09787 and 89/09788, Oppermann, Kubersampath, Rueger and Ozkaynak, published October 19, 1989; and llorld Patent Application 88/00205, llang, liozney 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 composit~on 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 effect~ve 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 ma~mals comprising a safe and effective amount of a Vitamin D compound; a safe and effective 35 amount of a BMP or osteoinductive extract comprising one or more BMPs; and a pharmaceutically-acceptable carrier.
DETAII Fn l)F~r~TPT10N OF THE INVENTION
The present invention comprises the administration to a mammal of 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 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 ~,UIllL~ dLiull with osteoinductive extract or in UolllL~ill " I with at least one BMP results in a level of new bone growth greater than that achieved through ddllli~ lLldLiull 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 fractions, congenital defects, as an adjunct in plastic surgery, in treating oncological resections, all diseases classified as u~L~ Jol u~is, rheumatoid arth ritis, U~ od l l11 11ti~,, septic arthritis, rickets, orga n ic i ncu, uu, d li~ll I of prosthetic joints and dental implants, periodontal disease and defects, as well as osteopenic and u~Leollldldcic conditions and disease.
As used herein, "safe and effective amount" means an amount of compound or uu" ,~,osiL;,.I~ sufficient to signif cantly induce a positive " l~ldiriudLioll in the condition to be treated, but low enough to avoid serious side effects (ata reasonable benefiVrisk natio), within the scope of sound medical judgment.
2û The safe and effective amount of the compound or co,,,~,u~iLiu,l 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 cûl,,r~ , employed, the particular pharn~ce~' "y ~cceptable can-ier 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 IllLn,ullo~ , protein.
As used herein, "q.s." means quantity sufficient.
As used herein, all p~lu~llldg~ are by weight unless otherwise specif ed.
As used herein "regional treatment" includes treating bone fractures (closed and open), treating non-union Fractures, ~ s 21 104~0 s 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 periodonta~ disease and defects.
S As used herein ~systemic treatment~ includes treating diseases classified as osteoporosis, rheumatoid arthritis. osteo-arthritis, septic arthritis, rickets, and osteopenic conditlons and di seases .
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.
IS As used herein, ~mineralized tissue~ means bone and teeth.
Vitamin O ComDounds 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 Dz), cholecalciferol (Vitamin D,) and their biologically active metabolites and precursorS
Preferred Vitamin D compounds include, but are not limited to, Vitamin D2 (Sigma, St. Louis, liO), Vitamin D3 (Sigm~, St. Louis, MO), 1-~2-hydroxy Vitamin D~ -fluoro Vitamin D3, 3-deoxy-1,25-dihydroxy Vit~min D" 25-hydroxy-5,6-trans Vitamin D3, 25-hydroxy Vitamin D2 . 25-hydroxy Vitamin D, (Hoffman LaRoche), 1.25-dihy-droxy Vitamin D2, 24.25-dihydroxy Vitamin D2, 24,25-dihydroxy V~tamin D3 (Hoffman LaRoche), and 1,25-dihydroxy Vitamin D, (Duph~r, Veenenda~l, Holland). Preferably, the Vitamin D compound is selected from 25-hydroxy V~t~min D2, 25-hydroxy Vitamin D3, 1,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D2, 24,25-dihy-droxy Vitamin D3, and 1,25-dihydroxy Vitamin D3, more preferably 1,25-dihydroxy Vitamin D~. Addition~l Vitamin D compounds useful in the present invention are well known to those skilled in the art and include, but dre not limited to, those disclosed by the following U.S. Patents, 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, ,~
-WO 92121365 PCr/US92~04356 Ikekawa and Tanaka, issued August IS, 1989; U.S. Patent 4,851,401, DeLuca, Kutner, Perlman and 5chnoes, lssued July 25, 1983; U S.
Patent 4,851,400, DeLuca, Ikekawa and Tanaka, issued July 25, 1989; U.S. Patent 4,847,012, DeLuca, Kutner, Perlman, Phelps, S 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 S, 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 S, 1988; U.S. Patent 4,689,180, DeLu~a, Schnoes, Sicinski and Tanaka, issued August 25, 1987; U.S. Patent 4,619,920, DeLuca, Ikekawa, IS 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, lkekawa, Kobayashi and Tanaka, issued March S, 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 11ay IS, 1984; U.S. Patent 4,448,721, DeLuca, Morzycki and Schnoes, issued May IS, 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 liichman, 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 9ZJ2136S ~ PCr~US92/043~i6 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 ilichmann, issued June 2Z, 1982; U.5. Patent 4,313,942, DeLuca, Frank, Paaren and Schnoes, issued February 2, 198Z; U.S. Patent 4,307,231, DeLuca, Paaren, Schnoes, Tanaka and l~lichmann, issued December 22, 1981; U.5. Patent 4,307,025, DeLuca, Ikekawa, Morisaki, Oshida, Schnoes and Tanaka, issued December 22, 1981; U.S. Patent 4,305,880, DeLuca, Ikeka~a, 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, Napol i, Onisko and Schnoes, issued April 21, 1981; U.S. Patent 4,260,804, DeLuca, Esvelt and Schnoes, issued Apri1 1, 1981; U.S. Patent 4,260,549, DeLuca, Hamer, Paaren and Schnoes, issued April 7, 1981; U.5. 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, DeLuc~, 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 35 23, 1980; U.S. Patent 4,223,131, DeLuca, Schnoes and ~lichman, issued September 16, 1980; U.S. Patent 4,217,288, DeLuca, OniskD
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 Hay 13, 1980;
~=

21 1~410 U.S Patent 4,201,881, DeLuca. Ikeka~a, Kobayashi, Schnoes and Tanaka, issued Hay 6, 198û; U.S. Patent 4,196,133, Oe~uca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued April 1, 1980; U.S.
Patent 4,195,027. OeLuca, Hamer, Paaren and Schnoes, issued March 25, 1980; U.S. Patent 4,188,345, DeLuca, Hapol i, Oniski and Schnoes, issued February 12, 1980; and U.S. Patent 3,906,014, DeLuca, Lam and Schnoes, issued September 16, 1975. Additional Vitamin O compounds useful in the present inYention and disclosed by these rerérl - include, but are not limited to, hydroxylated 24-homo-vitamin 0; cyclopeneano-vitamin 0; hydroxylated 26-homo vitamin D; 1 Q-hydro%yvitamin 0; 1-hydroxyvitamin D; 1 l-hydroxy-vitamin 2; I Q,25-dihydroxy-22Z-d-h~J-u~iitamin 0; 26,26,26,-27,27-pentafluoro-1 Q-hydroxy-27-methoxyvitamin 0~; 2 Q-fluoro-vitamin D3; 1,24-dihydroxy-delta 22-vitamin D3; 23,23-difluoro-25-hydroxy-vitamin 0~; 1-hydroxy-3.5-cyclovitamin D; 23.23-di-fluoro-1 Q,25-dihydroxy-vitamin D3; 1,23-dihydroxyvitamin D;
hydroxyvitamin D2; 23,23-difluoro-} Q,25-dihydroxy-vitamin 03;
23,23-difluoro-25-hydroxy-Yitamin D,; 26,26,26,27,27,27-hexa-fluoro-l Q,25-dih~u'~u~r-holesterol; 23,25-dihydroxyvitamin D3;
26,26,26,27,21.27-hexafluoro-1 ,25-dih~u,u~ ,olecalciferol; 1 Q,25-dihydroxy-2 ~-fluorovitamin 0,; 24-fluoro-25-h.~.u~ ,olecal-ciferol; 5,6-trans-vitamin 0; 1 Q-hydroxy-25-keto-27-nor-chole-calciferol; fluorcvitamin D; 1 Q-hydroxy-2 ~-fluorocholecalci-ferol; 3-deoxy-1 ~-hru,u~ lecalciferol; 25-hydroxy-26,26,26,-27.27,27-hexafluorocholecaliferol; ~-hydroxy-3,5-cyclovitamin D;
25-h~u. u~.h~lecalciferol; 24,24-difluoro-1 Q,25-dihjul u~.l,ûle-calciferol; 25~ UluA.r~holec~lciferol; 25-l~d.u~ lecalciferol-26,23-lactone; 24~24-difluoro-lQ~2s-dik~dluAr~llolecalciferol;
24~24-difluoro-2s-hrJlù~r~holecalciferol; 3.5-cyc~ovitamin D; and 3-deoxy-Q-l.~i û~r~hùiecalciferol . Additional Vitamin O compounds useful in the present invention further include those disclosed in The Handbook of Vitamins, L. J. Hachlin, Ed., Hercel Dekker, lnc.
(1984), Vitamin D cûmpormds useful in the present invention disc]osed by this reference, inc]ude, bur are not limited to, 1,25-dihydroxy Yitamin 0, 3-deoxy-1,25-dihydroxy Vit~min D, 27-nor-25-hydroxy Vitamin D3, 26,21-bis-nor-25-hydroxy Vitamin D3 24-nor-25-hydroxy Vitamin 03, 25-hydroxy Vitamin 0, 1,25-dihydroxy Vitamin 0, 1Q-hydroxy Vitamin 03 and 25-fluoro-1Q-hydroxy Vitamin D3.
r ... . . . ......

5 2 1 1 ~ Pcr/US92/04356 A safe and effective amount of a Vitamin D compound ls dosed in combination with at least one ~MP or in combination with an osteoinductive extract comprising at least one BMP.
A preferred dose range for administration of the Vitamin D
S compound for systemic treatment is from about I ng to about I mg, preferably from about 10 ng to about 500 /l9, more preferably from about 20 ng to about 10 ~9.
For purposes of regional treatment, the dose range of the Vitamin D compound is preferably from about I ng to about I mg, lo preferably from about 10 ng to about 500 ng, more preferably from about 10 ng to about S0 ng, most preferably from about 20 ng to about 30 ng.
Preferably, doses are administered over a I 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 S times per day, more preferably from about once per week to about once per day.
Bone l~u..~r. etic Proteins In one: ' ~., t 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-I, BMP-2, BMP-3, BMP-4, BMP-S, BMP-6 and BMP-7.
A safe and effective amount of a BMP, preferably selected from the group consisting of BMP-I, BMP-2, BMP-3, BMP-4, BMP-S, 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 I pg to about 100 19, preferably from about I ng to about 10 1l9, ~ore preferably from about 10 ng to about 2.5 ug.
For purposes of regional treatment, a preferred dose range for the BMP is from about I pg to about 100 119. more preferably from about 1.5 IL9 to about 90 ug, preferably from about 1.8 ~9 to about 75 1~9, more preferably from about 2.0 1~9 to about S0 1l9, more preferably still from about 2.2 1~9 to about 25 ag, more preferably from about 2.3 1~9 to about 10 119, most preferably from about 2.5 !~9 to about 5 1~9. Preferably the dose range is at least ~bout 2 . 5 ,ug .

,, : A PCI-/US92~0435C
2~ 10-Preferably, doses are administered over a 1 day to 6 month period, more preferably from about I week to about I month.
Preferably doses are administered from about once per month to about S times per day, more preferably from about once per week to S about once per day.
As used herein, ~BMP-I~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO~
As used herein, "BMP-2~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:2.
Io As used herein, ~BMP-3~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:3.
As used herein, "BMP-4~ means a peptide encoded by a DNA
sequence comprising SEQ ID NO:4.
As used herein, rBMP-S~ means a peptide encoded by a DNA
IS sequence comprising SEQ ID NO:S.
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, rA~, rT~, ~G~, and ~C~ refer to the nucleo-tides containing adenine, thymine, guanine and cytosine respectively.
Osteoinductive Extract Another component of the invention is ~n osteoinductive extract. As used herein, ~osteoinductive extract~ means a chemical extract of bone, comprising one or more various bone .~ ., tic proteins, including, but not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-S, 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 r~nge 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 ~4,000 to about 20,000 daltons, preferably from about 15,000 to about 17,000 daltons.
A preferred method of obtaining the osteoinductive extract is ~s fol l ows:
Snip the skin at the ankles of a 7-8 week old Long-Evans r~t (Charles River laboratories, ~ilmington, MA). Remove both tibiae , ... . ...... . ..

W0 92/2i365 ~ PCr/US92/04356 and pl~ce in cold water. Rinse the bone with distilled water to remove non-osseous t~ssue (t~ssue other than bone). Allow the bone to air dry. Grind the bones by placing ~n an Osterizer (Oster Co~mercial, Hilwaukee, 1~) blender with water and ice.
llith the blender set at ~liquefy~ speed, cont~nue 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 lo and stir. Add I ml of lmM of phenylmethylsulfonyl fluoride (PM5F). ~lash for I 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.
IS Allow the bone chips to air dry overnight.
~eigh the bone chips following the overnight drying. Using a sieve (U.S.A. Standard Sieve Series, Newark llire Cloth Co., Newark, N.J.; sieve ~40 retains particles greater than ~25 ~m and sieve ~170 retains particles greater thàn 90 ~m), isolate the bone particles in the 90-425um range. Grind any particles greater than 42511m in a MicroMill (Scienceware Bel-Art Products, ~
NJ) for I minute adding dry ice to the bone part~cles to keep the material cold. Repeat the sieving and MicroMill grinding steps of the greater than 4251Jm particles until the amount of total recovery is greater than 2/3 of the initial weight of the bone.
Store the particles at 4C until the next step. I~eigh the particles isolated thus far. For each gram of particles, add 25 ml of 0.6N HCl. Stir vigorously at 4-C for 2 hours. After 2 hours, stop stirring and allow the part~cles 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 S and S.S. Rinse the bone particles with ethanol three times. Swirl, allow to settle, and remove the supernatant. Rinse the bone particles with ethyl ether three ttmes as above. Dry overnight in glass plates. The dried bone parttcles are referred to as ~acid demineralized bone part~cles~.
. _ _ _ _ _ WO 92/21365 2 ~ 1 0 ~ ~ PCr/USs2~043s6 The acid demineral ized bone particles are deproteini2ed as follows: lleigh the material following the overnight drying. For each gram of material, add a solution of 30 ml ~H guanidine-HCl, IOmM Tris and 1.0mM PMSF pH 6.4 to the bone materi~l in a beaker.
S 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-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 H~O at 4C using SO mm dialysis tubing (3SOO molecular weight cutoff). Following dialysis, lyopholize the material and resolu-ls bilize the lyophilized material in 4M Urea-O.OSH Tris-O.lM NaCl, pH 7.4. Mix the solubilized material in a conical centrifuge tube with Heparin-Agarose and mix overnight on a rotator at 4C. Pour the Heparin-Agarose slurry into a column. Iiash with I column volume 4M urea, O.OSM Tris, O.IM NaCl, pH 7.4 buffer. Collect the fraction. Ilash ~ith 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, O.OSM Tris, 0.75M NaCl, pH 7.4. Concentrate this sample in a SO ml Amicon concentrator (Amicon Corp., Danvers, MA) with filter (10,000 molecular weight cut off) to about 4-Sml.
Assay for protein concentration using BCA (bicinchoninic acid) Protein Assay Reagent (Pierce, Rockford, IL) and dialyze (3500 molecular weight cutoff dialysis tubing) in 41i guanidine-O.Ol 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 ~re combined and dialyzed against three changes of 6M urea, 25mM Ha acetate, pH
4.6. The dialysate is loaded onto a column of carboxymethyl-sepharose (CM-Sepharose) equilibrated with the s~me buffer. The 3s column is washed with 6M urea, 25mM Na acetate, pH 4.6 and ~ctivity eluted using a O - O.SM NaCl gradient. Fractions are analyzed for protein concentration and sodium dodecyl sulfate gel ele_Lr~ sis. The activity located in the seven fractions before and after the beginning of the major protein peak are ,,, _ . . . _ _ .

wo 92/21365 2 1 1 ~ 4 1 ~; PcrJUS92/04356 pooled for further pur1f~cation.
The pooled CM-Sepharose fractions are dialyzed three times for 2~ hours each against 1% acetic acid. The dialysate is - lyophilized to dryness and the protein pellet dissolved into 30 ml s 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 abov~ buffer. The column is washed with the above buffer and then eluted with a gradient from 6M urea, 0.5M
llaCl, 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 l2'l and analyzed by SDS gel ele~r" ' .~sis. Aliquots (lO0 pl) of each fraction are co~bined with 400 pl 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 ~t least one BMP in an amount from about 1 pg to about 100 pg, preferably from about 1 ng to about 10 pg, more preferably fro~ about 10 ng to about 2.5 pg.
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 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 lO pg, most preferably from about 2.5 pg to about 5 1~9. Preferably the dose range is at least about 2.5 pg.
Preferably, doses ~re 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 ~bout once per day.
",~": ~licallY AcceDtable Carrier The Vitamin D compound, osteoinductive extract, or BMP may be ~dministered vi~ a pharm~ceutically acceptable carrier. The term '. ~ic~lly-~ccept~ble carrier~, ~s used herein, means one WO 92~21365 ~ 4l0 -14 PCI/USgz/04356 or more compattble solid or liquid filler diluents or encapsu-lating 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 S 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 pharmaceuti-cally-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 ~u~ . thylcellulose, ethyl cel 1 ul ose, cel 1 ul ose acetate; powdered tragacanth; mal t;
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 ~ell as other non-toxic compatible substances used in pharmaceutical formulations.
Wetting agents and lubri~ants 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-accept-able carrier (e.g., benzyl alcohol; Novocaine~; lidocaine).
Additional examples of carriers include collagen, deminer-alized 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 ~dministered. 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 S of modes ~i.e., osteoinductive extract via injection and Vitamin compound via oral administration). If the compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological s~line. Suitable pharmaceutically-acceptable 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 drcps, sprays, mists and pawders.
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 pharmaceutically-acceptable carriers, in tot~l, may comprise from about 0.1% to about gg.g9959% by weight of the pharmaceutical compositions of the present invention, preferably from about 50X to about gg.gg9X, and most preferably from ~bout 75% to about g9.9%.
Specific oral and inject~ble carriers useful in this invention are described in the following U.S. Patents: 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.
RCl!l c:: cllLLLi yc ~ of the present invention are provided in the Examples hereinafter.
Pl,~ ly-acceptable carriers suitable for the preparation of unit dosage forms for oral a.~ ;."., topical-oral ad,.1i11i~L~iu1~, nasopharyngeal A~11~1;1~;~11,11;~ 11l and injection are well-known in the art. Their selection will depend on secondary ~ like taste cost, and/or shelf stability, -' which are not eritical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art. Pl~ liy-acceptab~e earriers useful in the rnmrn~itl~mC of the present invention are deseribed more S fully hereinafter.
A. Oral Dose ~ormc Preferably, the vitamin D eompound is adl.lilu~,clc~ via an oral dose form. Various oral dosage forms can be used, including such solid forms as tablets, capsules, grarlules, bulk powders and ulicLu~a~J~ulc~ of the drug. These oral forms comprise a safe and effective amount, usually ât least about .5%, andpreferab~y from about 1% to about 10% of tbe compound of the present invention. Tablets can be compressed, enteric-coated, sugar-coated or film-coated containing suitable binders, lubricants, surfactants, diluents, ~ lr~ g~
âgents, colormg agents, f~avoring agents, ~JIc~lYali~, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous and solutions, emulsions, ~U*)~ iol~ solutions and/or cll~r~n~inne from non-crrt~ granules, containing suitable solvents, IJlC:~ClV~
clL~Ul~iryillg agents, suspending agents, diluentc, sweeteners, melting agents, coloring agents, and flavoring agents. Preferred earriers for oral a~lluli~lLa~ion inelude gelatin and propylene glyeol. Speeifie examples of ~,1.", 1.,;.,~,.1;. ~lly-acceptable carriers and e%cipients that may be used in r.""",l~l;"~ oral dosage forms eontaining compounds of the present invention are described in U.S.
Patent 3,903,297, Robert, issued September 2, 1975. Techniques and cnmrocition~ for making solid oral dosage forms are described in Marshall, "Solid Oral Dosage Forms," Modern E~ C Vol. 7. (Banker arld Rhodes, editors), 359-427 (1979). Techniques and eompositions for making tablets (~UlllplC;~,d, formulas amd molded), capsules (hard and soft gelatin) and pillsaredescribedinRemin~ton'sPl,..",~ irs~lSciences(ArthurOsol,editor), 1553-1593 (1980).
B. Topieal-oral I:~ose 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 c~lJc.,~ d rather than being .~
..... ..

W092/2136~ 211i~D Pcr/us92Jo4356 swallowed. Such compositions include toothpastes, tooth gels, tooth powders, mouthwashes, mouthsprays, prophyl axi s pastes, dental treatment solutions, biogels or other sustained release - products, and the like.
S 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, espe-cially the Vitamin D compound and oste6inductive 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 lS 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 sur-factant (from about O.SX to about IOX), a thickening agent (from about O.IX to about 5X) a humectant (from about IOX to about 55%), a flavoring agent (from about 0.04X to about 2X), a sweetening agent (from about O.lX to about 3X), a coloring agent (from about 0.01% to about 0.5X) and water (from about 2X to about 45X). 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 O.lX to about 13%), and an antiplaque agent (from about 0.1% to about SX).
Other preferred compositions of the subject invention are mouthwashes and mouthsprays. Components of such mouthwashes and mouthsprays include water (from about 45% to about 95X), ethanol (from about 0% to about 25%), a humectant (from about OX to about 50X), a surfactant agent (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2X), a sweetening agent -(from about 0.1% to about 3%), and a coloring agent (from about 2~ ~4~0 0.001% to about 0.5%). Such mouthwashes and mouthsprays may a1so include one cr more of an anticaries ~gent (from about O.QS% to about 0.3X as fluoride ion), an anticalculus agent (from about O.OIX to about 3%), and an antiplaque agent (from about 0.1% tc S about SX).
Other preferred compositicns of the subiect inventicn are dental soluticns. Components cf such dental soluticns generally include water (from about 90% to about 99X), preserYative (frcm about 0.01% tc abcut 0.5%), thickening agent (from about OX to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from OX to about SX).
"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.
and related well-known technol-ogies: U.S. Patent No. ~,666,897 issued to Golub, McNamara lhy on May 19, 1987; European Patent Application No.
244,118 Al in the name of Baker. published on November 4, 1987;
European Patent Appl ication No. 286,802 A2 in the name cf Kametaka, Miyazaki, Hayashi, Handa ~ I(ameda, published Octcber 19, 1988; Addy, M., L. Rawle, R. Handley, H. Newman ~ J. Ccventry, ~The development and in vitro ev~luation cf acryl ic strips and dialysis tubing fcr local drug delivery~, Jcurnal of Periodon-toloqY, Vol. 53 (1982), pp. 693-698; Goodson, J.M., A.D. Haffajee ~ S.S. Socransky, ~Periodontal therapy by local delivery cf tetracycl ine, Journal of Cl inical PeriodontoloaY, Vol . 6 (1979), pp. 83-92; Goodson, J., D. Holborow, R. Dunn, P. Hogan ~ S.
Dunham, ~Monolithic tetracycline containing fibers for controlled delivery tc periodontal pockets~, Journal of PeriodontclcqY, Vcl.
54 (1983), pp. 575-579; Dunn, R., J. Gibson, B. Perkins, J.
Goodson ~ L. ~aufe, ~Fibrous delivery systems fcr antimicrobial agents~, PolYmer Science and Technoloav. Vol. 32 (1985), pp.
47-S9; Dunn, R., J. Gibson, B. Perkins, J. Goodson ~ L. Laufe, ~Fibrous delivery systems for antimicrobial agents~, Polvmer Material Science Enqineerinq, Vol. Sl (1984), pp. 28-31; Olanoff, L. ~ J. Anderson, 'Controlled rslease of tetr~cycline ~ A
physiological ph~,",~-G~in~tic model of the pregnant r~t-, ~Qy~
of Pharmacokinetics and BioDharmaceutics. Yol. 8 (1980), pp.
599-620; Elkayam, R., H. Friedman, A. Stabhol2, A. Soskolne, H.
Sela ~ L. Golub, ~Sustained release deYice containing minocycline for local treatment of pericdontal disease~, Journal of Ccntrolled ~, Vol. 7 (1988), pp. 231-236; and Goodson, J r 'Multi-center evaluation of tetr~cycline fiber therapy. I. Experimental Design~, Journal of Oent~l Research, Vol. 68 (1989), p. 197; and r~F~I .. ~es cited therein.
C. Iniectable Oose Forms:
The actiYe 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 ~ith the particular condition being treated, the severity of the condition, the duration cf treatment, the specific mixture of compounds employed and its usage concen-tration, and like factors within the specific knowledge and expertise of the ~ttending physician and ~ ~te 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 gro~th is desired.
Hethods and m~terials for manufacturing injectables can be found in Reminqton' s Ph.""~ tical Sciences. 17ed., 1985, Ch~pter 85, p. 1518. Preferably, the injectable compo-sition is an aqueous solution.
The aqueous solutions preferably consist of water (preferably from about 80% to about 99.999X), ~ suitable solubilizer, various types of acids, and an antimicrobial agent. Several solubilizers are known . Exampl es of such sol ubi l i zers are as fol l ows: urea compounds (e.g., urea; urethan); surfactants (e.g., Tweens; Spansi sodium deoxycholate and Pluronics); cellulosic agents (e.g., Cdr~G~j thylcellulose); L.,bu~ tes (e.g., sorbitol; mannitol~;
~ vitamins (e.g., nicotin~mide); xanthine derivatives; and ~lco-hols (e.g., benzyl alcohol). Examples of acids to be used include the following: glucuronic; galacturonic; fumaric; gentisic;
~cetic; citric and l~ctobionic. Types of antimicrobial agents ~' , _ W092/21365 , ~ 4ln PCI~/US92~04356 _ - 20 -that c~n be used are the following: phenylmercur~c n~trate;
th~merosal; benzethon~um chlor~de; benzalkonium chloride; phenol;
cresol; and chlorobutanol . An art-known local anesthet~c (e.g.
benzyl alcohol; ~lovoca~ne~; lidocaine) may also be included.
Preferably the osteo~nductive extract and the BMP s are adm~nistered v~a an ~njectable dose form.
The follow~ng examples further descr~be and demonstrate the preferred . - I im l,s ~l~th~n the scope of the present ~nvent~on.
The examples are g~ven solely for the purpose of ~llustration and are not to be construed as lim~tat~ons of the present ~nvent~on s~nce many variat~ons thereof are poss~ble without depart~ng from ~ts sp~r~t and scope.
EXAMPLE I
An ~njectable composit~on compr~s~ng the osteo~nduct~ve lS extract and an oral composit~on compr~s~ng 1 25-dihydroxy Vitamin D3 for bone fracture repair ~s prepared by comb~n~ng the follow~ng components ut~l~z~ng conventional mix~ng techn~ques.
BMP comDosit~on Percent by ~le~ght Z0ComDonent of ComDosition BMP - I 0 . 04 NaCl o . 90 Ster~le water a.s.
100 . 00 1.25-d~hvdroxY Vjtamin D comDosit~on Percent by ~e~ght ComDonent of ComDosit~on 1 25-d~hydroxy Vitam~n D 0.01 30Corn starch 18 . 49 Lactose 63.00 Tal c 18 . 00 Stear~c ac~d O.S0 100 . 00 0.1 cc of the BMP composit~on ~s ~njected ~nto the fracture site at the time of fracture reduction and once daily thereafter.
100 119 of the 1 25-dihydroxY Vitam~n D3 composit~on is orally adm~n~stered 2~ hours before fracture reduction and once daily - _ thereafter. The BMP and 1 25-dihydroxy Vitamin D, are admin~s-, ~_ .
.

W092/21365 2llaA ~ a Pcr~US92/04356 tered until desired repair is ~chieYed, perferably over a seven day period.

An injectable composition for bone fracture repair is s prepared by combining the following components ut~lizing conven-t i onal mi xi ng techn i ques .
Percent by Weight ComDonent of CQmpQs i t i on BMP - 2 0 . 04 lo25-hydroxy Vitamin D2 .l NaCl 0.09 Sterile water for injection o.s.
100 . 00 0.1 cc of the composition is injected into the fracture site Is at the time of fracture reduction and once daily thereafter until desired repair is achieved.

A composition for inducing bone growth following reconstruc-tive surgery is prepared by combining the following components utilizing conventional mixing techniques.
Percent by ~leight ComDonent of ComDos i t i on BMP-3 0.04 1,25-dihydroxy Vitamin D2 0.01 25NaCl O . 90 Steri 1 e water o . s .
100 .00 0.1 cc of the composition per cm2 of surface area of surgically r, ~ru~ed bone is deposited directly onto the bone 30surface.
EXAMPI F 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-3stional mixing techniques.
Percent by l~leight ComDonent of ComDosition BMP- 1 0 . 04 BHP- 2 0 . 04 WO 92~21365 PCI`/US92/04356 BMP-~ 0.01 24,25-dihydroxy Yitamin D3 O.OI
NaCl 0 . 90 Steri 1 e water q . s .
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 conven-tional mixing techniques.
Percent by ileight ComDonent of ComDosition SBMP-2 0.04 NaCl O . 90 Steri 1 e water a . s .
100 .00 After the patient is prepared using conventional periodontal surgical therapy O.l 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 di sease .
EXAMPLE Vl An injectable composition comprising the BMPs 2, 3, 4 and S
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 comDosition Percent by ~leight ComDQnent of ComDos i t i on BMP-2 O.OOl BMP-3 O.OOl 35BMP-4 O.OOl BMP - S 0 . 001 NaCl O . 9O0 Steri 1 e water a . s .
100.000 WO 92/21365 PCI`/US92/0435 2110~10 6 1.25-dihYdroxY Vitamin D~ comDosit~on Percent by Weiqht CompQnent of ComDosit~on 1, 25 -di hydroxy Vi tami n D, 0 . 01 Corn starch 18.49 Lactose 63 . 00 Tal c 18 . 00 Stearic acid 0.50 100 .00 1 cc of the BMP composition is injected intravenously once per day~ 50 mq 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 ut~lizing conventional mixing techniques. As used herein, ~non-union fracture~ means a fracture that has failed to heal normal ly.
Percent by Weight ComDonent of ComDosition 8MP - 4 0 . 004 251,25-dihydroxy vitamin D3 0.01 Ac~d dem~neral~zed bone particles go.ooo NaCl 0.900 Sterile water for ~njection ~.s.
100 . 000 At the time of fracture reduct~on, a sufficient quant~ty of the above composit~on ~s deposited d~rectly into the non-union site thereby filling in ~ny bone deficit.

6~
PCI /US92/043~i6 211~ 410 SEQUENCE LISTING
(1) 6ENERAL INFORMATION:
(i) APPLICANT: STONE, ROGER L.
(ii) TITLE OF INVENTION: THERAPEUTIC FORMULAS FOR O5TEOINDUCTION
(iii) NUMBER OF SEQUENCE5: 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) SOFT~IARE: Patentln Release ~1.0, Version ~1.25 (vi ) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
( v i i i ) ATTORNEY/AGENT INFORMAT ION:
(A) NAME: Corstanje, Brahm J.
(B) REGISTRATION NUMBER: 34,804 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513-245-2B58 (B) TELEFAX: 513-7~1-3012 1365 ~ 4 1 o Pcr/usg2/o4356 (2) INFORMATION FOR SEQ ID NO:I:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2487 base pairs S (B) TYPE: nucleic acid - (C) STRANDEDNESS: double (D) TOPOLO6Y: l inear ( i i ) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:I:
lSGCC6CTTCCC TCGCCGCCGC CCCGCCAGCA TGCCCGGCGT GGCCCGCCTG CCGCTGCTGC 60 TbbGGI,~b~,l GCTGCTCCCG CGI~CCGGCC GGCCGCTGGA CTTGGCCGAC TACACCTATG 120 ACCTGGCGGA r~qr~ CAC TCGGAGCCCC TCMCTACM AGACCCCTGC MGGCGGCTG 180 C~ I1,I IbG GGACATTGCC CTGGACGMG AGGACCTGAG GGCCTTCCAG GTACAGCAG6 240 35 G~CGCr'~ r~'CCCCAG GCCATCTCCA TCGGCMGM CTGTGACMG TTCGGCATTG 660 TGGTCCACGA GCTGGGCCAC 6lbbll~b6~l TCTGGCACGA ACACACTCGG ccA~ cccct`~ 720 WO 92/21365 PCl'/US92/04356 21ila4lo -26-AGAT6GAGCC TCA66A66T6 GAGTCCCT66 666A6ACCTA T6ACTTC6AC A6CATCAT6C 8~0 S A66T6MCGG GGTGMMCCT CCCATT66CC AA/`rC~r4C6 6CTCA6CM6 66G6ACATT6 960 CCCA!\rCCCG CM6CTTTAC M6T6CCCA6 CCTGTG6A6A 6ACCCT6CM 6ACA6CACA6 1020 6CMCTTCTC CTCCCCT6M TACCCCMT6 6CTACTCT6C TCACAT6CAC T6C6T6T66C ~080 GCAGCC6CCT GTGCTGGTAC GACTATGTGG AGGTCCGAGA TGGCTTCTGG ArCAArrCGC 1200 ' ~ CCCTCCGAGG CCGCTTCTGC GGGTCCMMC TCCCTGAGCC TATCGTCTCC ACTGACA6CC 1260 ACGM6CCAT l.lbCG66Gbl 6ATGTGMAA A66ACTATG6 CCACATTCM TC6CCCMCT 1380 GCTAT6AGM GCCTGATGAC ATCMGA6CA CGTCCAGCCG Cl, 1~ 1 bG(, I 1, MGTTCGTCT 1620 AGT6CA6CT6 T6ACCCC666 TAC6A6CT6G CCCC.4r'\r~A 6CbbCG~lbl 6A66CT6CTT 1800 356T66C6GATT CCTCACCM6 CTCMC66CT CCATCACCA6 CCC6661.1b6 CCCM66AGT 1860 A~CCCCCCAA CM6MCT6C ATCT66CA6C r6blb6CCCC CACCCA6TAC C6CATCTCCC 1920 T6CA6TTT6A CTTCm6AG A~'~r'~G'rr4 ATGATGTGTG CAA6TAC6AC TTC6TGGAGG 1980 WO 92~
21365 PCI~/US92/04356 22il~
TGCGCAGTGG ACTCACAGCT GACTCCMGC TGCATGGCAA li1 l~lbl~bl TCTGAGMGC Z040 s TGTCCAAAM GGGCTTCMG GCCCACTTCT TCTCAGMM GAGGCCAGCT CTGCAGCCCC 2160 GAGGCCTGCC AGGC~1~CCG GACCCCTTGT TACTCAGGM CCTCACCTTG GACGGMTGG 2280 GCCGG ACAGMCTGG Tl.u11,1[,1 1C TCCCCACTGT GCCC61~GC GGACCGGGGA 2400 lS CCCTTCCCCG TGCCCTACCC CCTCCCAm TGATGGTGTC TGTGACATTT CCTGTTGTGA 2460 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(AJ LENGTH: 1547 base pairs (8) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GGGGACTTCT TGMCTTGCA GGGAGMTM CTTGCGCACC CCACTTTGCG CC61,1i,~11 60 TGCCCCAGCG GAGCCTGCTT CGCCATCTCC GAGCCCCACC GCCCCTCCAC ~C~1G66C~1 120 TGCCCGACAC TGAGACGCTG TTCCCAGCGT rNVU~ C ACTGCGCGGC cr~cA~ccrr~ 180 CA'`~'\r'`~CC l\C~r~r~. Mî~^~.Cr'`~ CATTCGGTCC TTGCGCCAGG TCCTTTGACC 240 WO 92/21365 ~ 28 - PCI/US92/04356 AGAGTTTTTC CATGTG6ACG CTCTTTCMT GGACGTGTCC CCGCI, ~bl, 11 CTTAGACGGA 300 CTGCGGTCTC CTMMGGTCG ACCATGGTGG CC~ ACCCG CTGTCTTCTA GCGTTGCTGC 360 S TTCCCCAGGT C~ l b~ l b6GC GGCGCGGCTG GCCTCGTTCC GGAGCTGGGC CGCAGGMGT ~20 TCGCGGCGGC GTCIllbG6GC CGCCCCTCAT CCCAGCCtTC TGACGAGGTC CTGAGCGAGT 480 TCGAGTTGCG GCTGCTCAGC ATGTTCGGCC TGMMCAGAG ACCCACCCCC AGCAGGGACG 5~0 15 ACCATGMGA ATCmGGM GMCTACCAG MMCGAGTGG GMMCMCC CGGAGATTCT 720 TCTTTMTTT MGTTCTATC Crr~rG'''\CC AGTTTATCAC CTCAGCAGAG CTTCAGGTTT 780 mATGMMT CATMMCCT GCMCAGCCA ACTCGMATT CCCCGTGACC AGACTTTTGG 900 ATGMCACAG CTGGTCACAG ATMGGCCAT TGCTAGTMC TTTTGGCCAT GATGGMMG 11~0 35 GGATTGTGGC ICCCCCGGGG TATCACGCCT mACTGCCA CGGAGMTGC CCTTTTCCTC 1320 ACTCTMGAT TCCTMGGCA TGCTGTGTCC CGACAGMCT CAGTGCTATC TCGATGCTGT lU0 WO 92~21365 2 ~ Pcr/US92~043~;6 bTGGbIbl~6 CTAGTACAGC AAMTTMMT ACATMATAT ATATATA I547 S(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: I 774 base pai rs (B) TYPE: nucleic ~cid I0 (C) STRANDEDNESS: double (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE: cDNA
'S
(xi) SEQUENCE DESCRIPTION: SEQ rD NO:3:

GTGGAGACGG C'''CC~CAGC GCCblbCGCG GGT6AG6TCC GCGCAGCTGC TGGGGMGAG 120 CCCACCTGTC AGGCTGCGCT GGGTCAGCGC AGCMGTGGG G~,lbGCCG~,I ATCTCGCTGC I80 25ACCCGGCCGC G I CCCGGGu I CCb ~bbGCCC TCGCCCCAGC TGGTTTGGAG TTCMCCCTC 240 66CII,C6CC6 CCGGCTCCTT GC6CI,llb66 AGTGTCCCGC ACrCACG~G rrq~CC~\~6 300 C~ Ç~GCGÇ GTACCTAGCC ATGGCTGGGG CGAGCAGGCT 6l.~bllll.1b I6G~,IbG6~,l 360 6~11elbCGI GAGCCTGGCG CA''''~ GACCGMGCC ACCTTTCCCG GAGCTCCGCA ~20 35ACMGGTCTC TGMCACATG ~, I bC6GI. 11,1 ATGACAGGTA CAGCACGGTC CArrrrrrcc S~o CC'1'4CC~C' CTCCCTGGAG GGAGGCTCGC AGCCulbGCG CCu~(,GG~,Il, CTGCGCGMG 600 GCMCACGGT TCGCAGCTTT Cl`''CGrr4' CA~'`4'1MC TCTTGAMGA AAAGGACTGT 660 2 ~ 30 PCl'/US92/04356 ATATCTTCM TCTGACATCG CTMCCMGT CTGMMCAT 1 1 ~bTGll,CC ACACTGTATT 720 TTATGTCCTG GCTGTCTMM GATATCACTC MTTCTTGAG C~ CC~^~ GMAATGMG 960 ATAGCCACAT CAGAGCTGCC CTTTCCATTG A~r'`qrr^.^. GMGCGCTCT ACTGGGGTCT 1200 ATMAMGM ACAGAGMMG GGGCCTCATC C~CM~^~CCCA GACGCTCCM TTTGATGAGC 1380 25 AGACCCTGM MMGGCMGG AGMMGCAGT GGATTGMCC TCGGMTTGC GCCAGGAGAT 14~0 AGCCTTGCTG TGTACCAGM MGATGTCCT CACTCAGTAT IIIAII~.III GATGMMTA 1680 35 AGMTGTAGT GCTTMMGTA TACCCTMCA TGACAGTAGA Glt,l I~CGCl TGCA6ATMC 17~0 (2) INFORMATION FOR SEQ ID NO~4:

WO 92/21365 2 1~ PCI~/US92~04356 (i) SEQUENCE CHARACTERISTICS:
(A) LEH6TH: 1751 base pairs (B) TYPE: nucleic ~cid (C) STRANDEDHESS: double S (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
G6CAGAG6A6 6~rr~qrrrq CCCMrt~Gr ~rqr'CCG ~CCrr~CC TA66T6A6TG 60 lS T66CATCCGA GCT6A666AC 6CGAGCCTGA 6AC6CC6CTG ~ILCI~GGC TGAGTATCTA 120 CGGCCC1~6C CCAGGTTCAC TGCMCCGTT CAGA66TCCC CAG6AGCT6C T6CTG6C6A6 240 GCAGCTTCCC T&AGCCTTTC CAGCMGTTT GTTCMGATT G6CTGTCMG MTCATG6AC 360 T6TTATTATA lbCu~ TCT6TCM6A CACCAT6ATT CCTGGTMCC GMTGCTGAT 420 66TCGTTTTA TTATGCCMG TCCTGCTAGG Arrt~r~ACt CATGCTA6TT TGATACCTGA 480 GACCrrr~AE MMMGTCG CCGAGATTCA trrCCACrrG 6rqtrqC~rC GCTCAGG6CA 540 6AGCCATGA6 CTCCT6CGGG ACTTCGAGGC GACACTTCT6 CA6ATGm6 GG~I~C6CCG 600 CC~CC~:C46 CCTAGCMGA 6TGCCGTCAT TCC6GACTAC ATGC66GATC mACCGGCT 660 TCAGTCT66G CArr'~rrlrr MGAGCAGAT CCACAGCACT GGTCTTGAGT ATCCTGAGCG 720 CCCl`rrCACC CrrrtCA~'4 CCGTGAGGA6 CTTCCACCAC GMGMCATC TG6A6MCAT 780 CC~4rrr'~CC AGTGMMCT ~ 1 1 1 11,6 1 1 11,u1 L I I I MCCTCA6CA GCATCCCT6A 840 WO 92/21365 ~ PCr/US92/04356 21104~ 32 GMCGAGGTG ATCTCCTCTG CAGAGCTTCG G~, l b l l cC6G GAGCAGGTGG A~CA'`rr~C 90O

6Ccc~ 6 GTCACCTTTG GCCATGATGG CCGGGGCCAT GCCTTGACCC GACGCCGGAG 1260 GGCCMGCGT AGCCCTMGC ATCACTCACA GCGI GrCACC MGMGMTA AGMCTGCCG 1320 TGATMGGTG GTACTGMM ATTATCAGGA GATGGTAGTA GAGGGATGTG 6blbCC61~1G 1620 (2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENCE CHARACTERISTICS: t (A) LENGTH: 2153 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear WO 92/21365 2 1 1 ~ 4 ~ o PCr/us92/o4356 ( i i ) MOLECULE TYPE: cDNA
S (xi) SEqUENCE DESCRIPTION: SEQ ID HO:5:
CTGGTATATT TGTGCCTGCT GGAGGTGGM TTMCAGTM t:M('''~r'\M GGGATTGMT 60 GGACTTACAG GMGGATTTC M6TMMTTC A~`rl;~lMrl~C ATTTACTTGA ATAGTACMt 120 GCmCTTAG MMTMCTAC TCTTGACATA TTCCMMTA TTTMMTAG GACAGGMAA 540 TCAGCTACTG GGMMCTGTA CCTCCTAGM CCTTAGGTTT l l l l l l l l l l MGAGGACM 660 GMGGACTM MATATCMC l l l I bl, l 111 GGACMAMT GCATCTGACT GTATTTTTAC 720 TTMGGGTAT I b I b6G I I l l; CTCTGGAGCT 6b 1 bbb I I ( I AGTGGGTTAT GCMA~lrr ~C~ 780 GACGGGMMT ACAMrrrAA ATTCTCTCTA ~bl lbGbl I I GCCTCACAGA CCCAGACCAT 900 mCACCTGG AAMCMGCG TCCTCTGCAC CTCTCTTTAT GCTGGATCTC TACMTGCCA 960 WO 92t21365 PCltUS92tO43~6 2110~lp 34 AGACCAGAG6 ~ Ar~AA'` GGATACCCAG CCTCTCCCM TGGGTATCCT CGTCGCATAC 1080 S CCMCTTTCT GMTGATGCT GACATGGTCA TGAGCTTTGT CMCTTAGTT CAl\~CAr'\'~ 1200 GGGATGCAGA I~IGI li;l IG TTAGACACM GMMGGCCCA AGCTTTAGAT GTGGGTTGGC 1440 35 TACGCTCATG TGGCTGCCAC TMTATTMM TMTATT6AT MTMCMM AGATCTGTAT 2~00 (2) INFORMATION FOR SEQ ID HO:6:

Wo 92/21365 2 ~ ) Pr~/us92/o43s6 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2923 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: doubl e S (D) TOPOLOGY: line~r ( i i ) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CGACCATGAG AGATMGGAC TGAGGGCCAG CMrrrC'~AC CGAGCCCGCC GAGAGGTGGC 60 IS GGGGACTGCT CACGCCMGG GCCACAGCGG CCGC6~1~CG GCCTCGCTCC GCCGCTCCAC 120 ^~ICGCGGG ATCCGCGGGG GCAGCCCGGC CCCCCCCrr~ TGCCGG66~T GGr~rrrr~rC I80 GCGCAGTGGC TGTGCTGGTG blb66Gbl,lb CTGTGCA6CT GCTGCGGGCC CCbGCC6~1G 240 CGGCC6CC~1 T6~CC6~1bC rG~GG(~G~ 6~G~C~''C GGCAGCTGCT rrCCC~rG6C 300 CCC.~CCCCG GCCGCACGGA rCAGCrGCC6 CCbl-bCC6C AGTCCTCCTC GGGCTTCCTG 360 25 TACCGGCGGC TCMGACGCA ~r'~r'U\rrrr 6AGATGCAGA AGGAGATCTT GTCGGTGCTG 420 GGG~ CbC ArCr~rrCCCG GCCCCTGCAC GGCCTCCMC .4rrCGCACCC CC~G6C6~1~ 480 cr~r~c4cc~cc /~rr'~rC4rr4 rC4rrq~r4G CAGCTGCCTC r~rr~9r~rCc CC~ Cb66 540 CGACTGMGT CC6C6CCC~1 CTTCATGCTG GATCTGTACA ACGCCCTGTC CGCCGACMC 600 GACGAGGACG GGGCGTCGGA Grrrr~CACr CAGCAGTCCT GGCCCCACGA ArCAGCC9rr 660 35 TCGTCCCAGC GTCGGCAGCC Gr~ r r~r~GCGCACC CGCTCMCCG CMGAGCCTT 720 Clb6CCCCCG GATCTG6CAG ~C"~,rGrG TCCCCACTGA CCAr'Çrr`r~ rr~rcGcc 780 TTCCTCMCG ACGCGGACAT GGTCAT6AGC mGTGMCC TGGTGGAGTA CCAr4,^,r~C 840 WO 92/21365 PCr/US92/04356 2110~ 36-I I L I Ll,l,l, I L GTCA6CGACA CCAr4M'`~ TTCAAGTTCA ACTTATCCCA 6ATTCCTGA6 goo m6ACATCA C66CCACTA6 CMTCT6T66 6TT6T6ACTC CACA6CATM CAT6666CTT 1140 156TCCAC6T6C r,~4~CACCA6 6TCA6CCTCC Arr~rr-rrrC 6ACMCA6A6 TCGTMTCGC 1320 TCTACCCA6T CCCA66AC6T GGCGCGGblL TCCA6T6CTT CA6ATTACM CA6CA6T6M 1380 ACmGMMC TATCTCATGC CAGT6CCTTA TTACCCA66A A6ATTTTMM G6ACCTCATT 1860 LrLl,lLCCcc ANVIACCCAC CMMTTA6T TTTA6CT6TA 6ATCM6CTA 11 IbG6blGl 2040 WO92/2136S 9~lDt ff4 1 0 AGCCAGGGCT CCACGGGGCG CCC1 IG1~r~ AGTCATTGCT GTTGTATGTT CGTGCTGGAG 2280 GGTTCTTTGA CCAGCACATT MCTTCTGGA ~1bCC66~1C TAGTACCTTT TCAGTMMGT 2520 GGTTCTCTGC CTmTACTA TACAGCATAC CACGCCACA~ GGTTAGMCC A/\~qAr \M 2580 MCGGMMTC ATGATTTCCC TGTAGAAAGT GAGGCTCAGA TTMMTmA GMTATTTTC 2700 TMATGTCTT mCACAATC ATGTGACTGG GMGGCMTT TCATACTMA CTGATTMMT 2760 25 MTACATTTA TMTCTACAA CTGTTTGCAC TTACAGCTTT mmGTMMT ATMMCTATA 2820 ATTTATTGTC TATTTTATAT [,IGI 11 Ibbr GlbGCbl Ib6 ''''' '''~C G6Gbl I l Ibb 2880 GG666666br 1 IGI I IbG6G GblGll.blbG IG~bGGCGG6 CGG 2923 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1448 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: doub1e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

WO 92/2136~; PCr/US92/043~i6 2ii0~1~ 38- ~
(xi) SEQUENCE OESCRIPTION: SEQ ID NO:7:
GT6ACCGA6C GGCGCGGACG 6CCGC~16CC CCCTCTGCCA C1,1bGGGCGG lbCG6GCCCG 60 G~rrCG'CCG~ ACAGCTTCGT GGCGCTCTGG GCACCCCTGT 1C~ .CG CTCCGCCCTG 180 CA6GAGCGGC GGGAGATGCA GCGCGAGATC CTCTCCAm TGGGCTTGCC CCACCGCCCG 300 IS GCCATGGCGG TGGAGGAGGG ~rLrGGGrCC GGCGGCCAGG GCTTCTCCTA CCCCTACMG 420 ~CGTCTTCA GTACCCAGGG CCCCCCTCTG GCCAGCCTGC MGATAGCCA TTTCCTCACC 480 CCACGCTACC ACCATCGAGA Gl 1i,CGI,l 1 I GATCTTTCCA AGATCCCAGA AGGGGMGCT 600 1 11,1,1b~.1t,G ACAGCCGTAC C~1~1b~.bCC TCGGAGGAGG GCTGGCTGGT GmGACATC 780 rrr~(~CCCACA ACMGCAGCC CTTCATGGTG GCTTTCTTCA Ar~rCACrr'\ GGTCCACTTC 960 35 CGCAGCATCC GGTCCACGGG CA~r~rqC CGCAGCCAGA ACCGCTCCM GACGCCCMG I020 MrC,qGr~ '` CCCTGCGGAT GGCCMCGTG GCAGAGMCA GCArCACCGA CC4r'lCrC~C 1080 -WO 92/2136S Pc'r/ /04356 ~ 2 1 1 ~ US92 TCCTACATGA A~':fCA~CM CCACGCCATC GTGCAGACGC TGGTCCACTT CATCMCCCG 1260 S GMMCGGT6C CCM6CCCTG l.lGlbC6CCC AC6CAGCTCA AT6CCATCTC CGTCCTCTAC 1320 TTCGAT6ACA GCTCCMC6T CATCCT6MG MMTACAGM ACATGGTGGT CCG66C~,llil 1380 GGCTGCCACT AGCTCCTCCG AGMTTCAGA CCI, I I I b66G CCM6TmT CTGGATCCTC 1440 The invention has been described herein with reference to certain preferred embodiments ~nd examples. Obvious variations may Is appear to those skilled in the art. Therefore, the invention is not to be considered limited thereto but only by the cl~ims which follow.
~IHAT IS CLAIHED IS:

Claims (14)

Claims:

1. 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 BMP selected from the group consisting of 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 accept-able carrier.

2. The composition of Claim 1 wherein the safe and effective amount of the Vitamin D compound is from about 30 ng to about 10 µg and at least about 2.5 µg of the BMP.

3. The composition of Claim 2 wherein the Vitamin D compound is selected from the group consisting of Vitamin D2, Vitamin D3, 1-.alpha.-hydroxy Vitamin D3, 1-.alpha.-fluoro Vitamin D3, 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.

4. The composition of Claim 3 wherein the Vitamin D compound is 1,25-dihydroxy Vitamin D3.

5. The composition of Claim 3 wherein the pharmaceutically-acceptable carrier is an injectable carrier.

6. The composition of Claim 3 wherein the pharmaceutically-acceptable carrier is a topical-oral carrier.

7. The composition of any one of Claims 1-6 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. The use of 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 for the purpose of generating new bone growth in mammals.

9. The use of Claim 8 wherein the safe and effective amount of the Vitamin D compound is from about 1 ng to about 1mg and the safe and effective amount of the BMP is from about 1 pg to about 100 µg.

10. The use of Claim 9 wherein the safe and effective amount of the Vitamin D compound is from about 30 ng to about 10 µg and the safe and effective amount of the BMP is at least about 2.5 µg.

11. The use of Claim 10 wherein the Vitamin D compound is selected from the group consisting of Vitamin D2, Vitamin D3, 1-.alpha.-hydroxy Vitamin D3, 1-.alpha.-fluoro Vitamin D3, 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 use of Claim 11 wherein the Vitamin D compound is 1,25-dihydroxy Vitamin D3.

13. The use of any one of Claims 8-12 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.

14. The use of a composition as claimed in any one of Claims 1-6 for generating new bone growth in mammals.