[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

EP0587751A1 - Therapeutic compositions for osteoinduction - Google Patents

Therapeutic compositions for osteoinduction

Info

Publication number
EP0587751A1
EP0587751A1 EP92913307A EP92913307A EP0587751A1 EP 0587751 A1 EP0587751 A1 EP 0587751A1 EP 92913307 A EP92913307 A EP 92913307A EP 92913307 A EP92913307 A EP 92913307A EP 0587751 A1 EP0587751 A1 EP 0587751A1
Authority
EP
European Patent Office
Prior art keywords
bmp
vitamin
issued
compound
deluca
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92913307A
Other languages
German (de)
French (fr)
Inventor
Roger Lee Stone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Procter and Gamble Co
Original Assignee
Procter and Gamble Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Procter and Gamble Co filed Critical Procter and Gamble Co
Publication of EP0587751A1 publication Critical patent/EP0587751A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • 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 orphogenetic proteins and a Vitamin D compound, resulting in synergistic bone growth.
  • osteopor ⁇ osis is an abnormal loss of bony tissue often occurring in post- enopausal 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 treat ⁇ ments that go beyond the prevention of bone loss and promote bone formation and/or reverse bone loss.
  • Various other bone morphogenetic proteins/factors, osteoin ⁇ ductive factors, osteogenic factors and other proteins/factors related to bone growth are disclosed in the following publica ⁇ tions: 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 4,294,753, 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 145 155, Sen, published July 10, 1985; World Patent Applica- tion 89/10934, Roos, Burns, Guy and McKnight, published November 16, 1989; World Patent Applications 89/09787 and 89/09788, Oppermann, Kubersampath, Rueger and Ozkaynak, published October 19, 1989; and World Patent Application 88/00205, Wang, Wozney and Rosen, published Janaury 14, 1988.
  • 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 BMPs
  • a pharmaceutically-acceptable carrier BMPs
  • the present invention comprises the administration to a mammal of a combination of a safe and effective amount of a Vitamin D compound amd a safe and effective amount of one or more BMPs or an osteoinducrtive extract comprising one or more BMPs. It has been determined that treatment with a Vitamin D compound, BMP or osteoinductive ex t ract alone increases bone growth. Surpris- ingly, it has been further determined that treatment with a Vitamin D compound, BMP or osteoinductive ex t ract alone increases bone growth. Surpris- ingly, it has been further determined that treatment with a Vitamin D compound, BMP or osteoinductive ex t ract alone increases bone growth. Surpris- ingly, it has been further determined that treatment with a Vitamin D compound, BMP or osteoinductive ex t ract alone increases bone growth. Surpris- ingly, it has been further determined that treatment with a Vitamin D compound, BMP or osteoinductive ex t ract alone increases bone growth. Surpris- ingly
  • Vitamin D compound -m 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, osteoinducrrive extract or Vitamin D compound alone.
  • Subjects in need o-r such treatment suffer from a variety of ailments which may ue treated via this procedure, including but not l imited to, borne fractures (closed and open) , non-union fractures, congenita " defects, as an adjunct in pl astic surgery, in treating oncological resections, all diseases cl assified as osteoporosis, rheum ⁇ etoid arthritis, osteoarthritis, septic arthritis, rickets, rtrganic incorporation of prosthetic joints and dental implants, pe.-iodcntal disease and defects, as well as osteopenic and osteo ⁇ alacic conditions and disease.
  • safe and effective amount means an amount of compound or compicsition sufficient to significantly induce a positive modification, in the condition to be treated, but low enough to avoid seri ⁇ us side effects (at a reasonable benefit/risk ratio) , within the s.rope of sound medical judgment.
  • the safe and effective amount of the expound or composition will vary with the particular condition beir-g treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the tre __tmer._ . the nature of concurrent therapy, the specific compound or composition employed, the particular pharma ⁇ ceutical ly-acceptabl •£ carrier util ized, and l ike factors within the knowledge and expertise of the attending physician.
  • fracture reduction means the restoration of a bone fracture by 3urg-cal or manipulative means to its normal anatomical relation.
  • q.s . means quantity sufficient.
  • re onal treatment
  • systemic treatment includes treating diseases classified as osteoporosis, rheumatoid arthritis, osteo- arthritis, septic arthritis, rickets, and osteopenic conditions and diseases.
  • mineralized tissue means bone and teeth.
  • Vitamin D compound includes Vitamin D, ergocalciferol (Vitamin D 2 ), cholecalciferol (Vitamin D 3 ) 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 D 3 (Sigma, St.
  • the Vitamin D compound is selected from 25-hydroxy Vitamin D 2 , 25-hydroxy Vitamin D 3 , 1,25-dihydroxy Vitamin D 2 , 24,25-dihydroxy Vitamin D 2 , 24,25-dihy ⁇ droxy Vitamin D 3 , and 1,25-dihydroxy Vitamin D 3 , more preferably 1,25-dihydroxy Vitamin D 3 .
  • Additional Vitamin D compounds useful in the present invention are well known to those skilled in the art and include, but are not limited to, those disclosed by the following U.S. Patents, each of which is incorporated herein by reference: U.S. Patent 4,970,203, DeLuca and Kwiecinski, issued November 13, 1990; U.S.
  • Patent 4,313,942 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 4,224,230 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; U.S. Patent 4,201,881, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued May 6, 1980; U.S.
  • Patent 4,196,133 DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued April 1, 1980; U.S. Patent 4,195,027, DeLuca, Hamer, Paaren and Schnoes, issued March 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.
  • Vitamin D compounds useful in the present invention and disclosed by these references include, but are not limited to, hydroxylated 24-homo-vitamin D; cyclopentano-vitamin D; hydroxylated 26-homo vitamin D; 1 a-hydroxyvitamin D; 1-hydroxyvitamin D; 1 ⁇ -hydroxy- vitamin D 2 ; 1 ⁇ ,25-dihydroxy-22Z-dehydroxyvitamin D; 26,26,26,- 27,27-pentafluoro-l ⁇ -hydroxy-27-methoxyvitamin D 3 ; 2 ⁇ -fluoro- vitamin D 3 ; 1,24-dihydroxy-delta 22-vitamin D 3 ; 23,23-difluoro- 25-hydroxy-vitamin D 3 ; l-hydroxy-3,5-cyclovitamin D; 23,23-di- fluoro-1 ⁇ ,25-dihydroxy-vitamin D 3 ; 1,23-dihydroxyvitamin D; hydroxyvitamin D 2 ; 23,23-difluoro-l ⁇ ,25-d
  • Vitamin D compounds useful in the present invention further include those disclosed in The Handbook of Vitamins, L. J. Machlin, Ed., Mercel Dekker, Inc. (1984), incorporated herein by reference.
  • Vitamin D compounds useful in the present invention disclosed by this reference include, bur are not limited to, 1,25-dihydroxy Vitamin D, 3-deoxy-1,25-dihydroxy Vitamin D, 27- ⁇ or-25-hydroxy Vitamin D 3 , 26,27-bis-nor-25-hydroxy Vitamin D 3 24-no.-25-hyd.oxy Vitamin D 3 , 25-hydroxy Vitamin D, 1,25-dihydroxy Vitamin D, l ⁇ -hydroxy Vitamin D 3 and 25-fluoro-la-hydroxy Vitamin D 3 . - 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 ⁇ g, more preferably from about 20 ng to about 10 ⁇ g.
  • 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.
  • doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.
  • 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.
  • a Vitamin D compound is administered in combination with one or more BMPs to generate new bone growth in a mammal.
  • BMPs are preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4,
  • a safe and effective amount of a BMP preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, is dosed in combination with a Vitamin D compound.
  • a preferred dose range for administration of the BMP for systemic treatment is from about 1 pg to about 100 ⁇ g, preferably from about 1 ng to about 10 ⁇ g, more preferably from about 10 ng to about 2.5 ⁇ g.
  • a preferred dose range for the BMP is from about 1 pg to about 100 ⁇ g, more preferably from about 1.5 ⁇ g to about 90 ⁇ g, preferably from about 1.8 ⁇ g to about 75 ⁇ g, more preferably from about 2.0 ⁇ g to about 50 ⁇ g, more preferably still from about 2.2 ⁇ g to about 25 ⁇ g, more preferably from about 2.3 ⁇ g to about 10 ⁇ g, most preferably from about 2.5 ⁇ g to about 5 ⁇ g.
  • the dose range is at least about 2.5 ⁇ g. 56
  • doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.
  • 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.
  • BMP-1 means a peptide encoded by a DNA sequence comprising SEQ ID N0:1.
  • BMP-2 means a peptide encoded by a DNA sequence comprising SEQ ID NO:2.
  • BMP-3 means a peptide encoded by a DNA sequence comprising SEQ ID NO:3.
  • BMP-4" means a peptide encoded by a DNA sequence comprising SEQ ID NO:4.
  • BMP-5" means a peptide encoded by a DNA sequence comprising SEQ ID NO:5.
  • BMP-6 means a peptide encoded by a DNA' sequence comprising SEQ ID NO: 6.
  • BMP-7 means a peptide encoded by a DNA sequence comprising SEQ ID NO: 7.
  • A”, “T”, “G”, and “C” refer to the nucleo ⁇ tides containing adenine, thymine, guanine and cytosine respectively.
  • 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.
  • 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:
  • the acid demineral zed bone particles are deproteinized as follows: Weigh the material following the overnight drying. For each gram of material, add a solution of 30 ml 4M guanidine-HCl, lOmM Tris and l.OmM PMSF pH 6.4 to the bone material in a beaker. 5 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 10 combine with the previously saved solution. The bone particles are now demineralized and deproteinized.
  • 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 carboxymethyl- sepharose (CM-Sepharose) equilibrated with the same buffer.
  • CM-Sepharose carboxymethyl- sepharose
  • the 35 column is washed with 6M urea, 25mM Na acetate, pH 4.6 and activity eluted using a 0 - 0.5M NaCl gradient.
  • Fractions are analyzed for protein concentration and sodium dodecyl sulfate gel electrophoresis. The activity located in the seven fractions before and after the beginning of the major protein peak are 13 - pooled for further purification.
  • the pooled CM-Sepharose fractions are dialyzed three times for 24 hours each against 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, 0.5M NaCl, 25mM Na phosphate, pH 7.4 to 6M urea, 0.5M NaCl, 25mM Na acetate, pH 4.6.
  • a safe and effective amount of osteoinductive extract is dosed in combination with a Vitamin D compound.
  • the osteoinductive extract dosed preferably comprises at least one BMP in an amount from about 1 pg to about 100 ⁇ g, preferably from about 1 ng to about 10 ⁇ g, more preferably from about 10 ng to about 2.5 ⁇ g.
  • the osteoinductive extract dosed preferably comprises at least one BMP in an amount from about 1 pg to about 100 ⁇ g, more preferably from about 1.5 ⁇ g to about 90 ⁇ g, preferably from about 1.8 ⁇ g to about 75 ⁇ g, more preferably from about 2.0 ⁇ g to about 50 ⁇ g, more preferably still from about 2.2 ⁇ g to about 25 ⁇ g, more preferably from about 2.3 ⁇ g to about 10 ⁇ g, most preferably from about 2.5 ⁇ g to about 5 ⁇ g.
  • the dose range is at least about 2.5 ⁇ g.
  • doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.
  • 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 are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.
  • the Vitamin D compound, osteoinductive extract, or BMP may be administered via a pharmaceutically acceptable carrier.
  • pharmaceutically-acceptable carrier means one - 14 - or more compatible solid or liquid filler diluents or encapsu ⁇ lating substances which are suitable for administration to a human or lower animal.
  • compatible means that the components of the pharmaceutical compositions are capable of being commingled with the compound(s) of the subject invention, and with each other in a manner such that there is no interaction which would substantially reduce the pharmaceutical efficacy of the pharmaceutical composition under ordinary usage situations.
  • Pharmaceutically-acceptable carriers must, of course, be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to human or lower animal being treated.
  • substances which can ssr ⁇ e 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 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 formulation
  • wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, tableting agents, stabilizers, antioxidants, and preservatives, can also be present.
  • Other compatible pharmaceutical additives and actives e.g., NSAI drugs; pain killers; muscle relaxants
  • NSAI drugs e.g., NSAI drugs; pain killers; muscle relaxants
  • 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).
  • a pharmaceutically-acceptable carrier to be used in conjunction with the compounds of the present invention is - 15 - determined by the way the compound is to be administered.
  • the preferred modes of administering the compounds of the present invention are by injection, oral administration, topical-oral administration, and nasopharyngeal administration or a combination of modes (i.e., osteoinductive extract via injection and Vitamin D compound via oral administration).
  • the preferred pharmaceutically-acceptable carrier is sterile, physiological saline.
  • 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 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 pharmaceutically- acceptable 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%.
  • compositions of the present invention are described more fully hereinafter.
  • Oral Dose Forms are described more fully hereinafter.
  • the vita ⁇ rn D compound is administered via an oral dose form.
  • oral dosage forms can be used, including such solid forms as tablets, capsules, granules, bulk powders and microcapsules of the drug. These oral forms comprise a safe and effective amount, usually at least about .5%, and preferably from about 1% to about 10% of .ne compound of the present invention. Tablets can be compressed, enteric-coated, sugar-coated or film- coated containing suitable tinders, lubricants, surfactants, dilu ⁇ ents, disintegrating agents, coloring agents, flavoring agents, preservatives, flow-inducing agents, and melting agents.
  • Liquid oral dosage forms include aqueous and nonaqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents, and flavoring agents.
  • Preferred carriers for or ⁇ l administration include gelatin and propylene glycol.
  • Specific examples of pharmaceutically-accept ⁇ able carriers and excipient.5 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.
  • Topical-oral carrier 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 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, espe ⁇ cially 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.
  • 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 0.5% to about 10%), a thickening agent (from about 0.1% to about 5%) a humectant (from about 10% to about 55%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), a coloring agent (from about 0.01% to about 0.5%) and water (from about 2% to about 45%).
  • a dental abrasive from about 10% to about 50%
  • a sur ⁇ factant from about 0.5% to about 10%
  • a thickening agent from about 0.1% to about 5%
  • a humectant from about 10% to about 55%)
  • a flavoring agent from about 0.04% to about 2%
  • a sweetening agent from about 0.1% to about 3%
  • a coloring agent from about 0.01% to
  • Such toothpastes may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.1% to about 13%), and an antiplaque agent (from about 0.1% to about 5%).
  • an anticaries agent from about 0.05% to about 0.3% as fluoride ion
  • an anticalculus agent from about 0.1% to about 13%)
  • an antiplaque agent from about 0.1% to about 5%
  • compositions of the subject invention are mouthwashes and mouthsprays.
  • Components of such mouthwashes and mouthsprays include water (from about 45% to about 95%), ethanol (from about 0% to about 25%), a humectant (from about 0% to about 50%), a surfactant agent (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), and a coloring agent (from about 0.001% to about 0.5%).
  • Such mouthwashes and mouthsprays may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.01% to about 3%), and an antiplaque agent (from about 0.1% to about 5%).
  • an anticaries agent from about 0.05% to about 0.3% as fluoride ion
  • an anticalculus agent from about 0.01% to about 3%
  • an antiplaque agent from about 0.1% to about 5%
  • compositions of the subject invention are dental solutions.
  • Components of such dental solutions generally include water (from about 90% to about 99%), preservative (from about 0.01% to about 0.5%), thickening agent (from about 0% to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from 0% to about 5%) .
  • Topical-oral carrier as used herein, also denotes fibers, strips or tubes which can be impregnated with the active components of the present invention and inserted or implanted into a periodontal pocket.
  • Such compositions of the subject invention can readily be achieved by one of ordinary skill in the art using the teachings disclosed hereinbefore, the following references, incorporated herein by reference, and related well-known technol- ogies: U.S. Patent No. 4,666,897 issued to Golub, McNamara & Ramamurthy on May 19, 1987; European Patent Application No. 244,118 Al in the name of Baker, published on November 4, 1987; European Patent Application No.
  • 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 concen ⁇ tration, 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.
  • 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.
  • the injectable compo ⁇ sition 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.
  • solubilizers are known. Examples of such solubilizers are as follows: urea compounds (e.g., urea; urethan); surfactants (e.g., Tweens; Spans; sodium deoxycholate and Pluronics); cellulosic agents (e.g., carboxymethylcellulose); carbohydrates (e.g., sorbitol; mannitol); B vitamins (e.g., nicotinamide); xanthine derivatives; and alco ⁇ hols (e.g., benzyl alcohol).
  • urea compounds e.g., urea; urethan
  • surfactants e.g., Tweens; Spans; sodium deoxycholate and Pluronics
  • cellulosic agents e.g., carb
  • acids to be used include the following: glucuronic; galacturonic; fumaric; gentisic; acetic; citric and lactobionic.
  • Types of antimicrobial agents that can be used are the following: phenyl mercuric nitrate; thi erosal ; benzethoniu chloride; benzal konium chl oride; phenol ; cresol ; and chlorobutanol .
  • An art-known local anesthetic e.g. , benzyl alcohol ; Novocaine*; l idocaine may al so be included.
  • the osteoinductive extract and the BMP's are administered via an injectable dose form.
  • An injectable composition comprising • the osteoinductive- extract and an oral composition comprising 1,25-dihydroxy Vitami.n D 3 for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques.
  • BMP composition comprising • the osteoinductive- extract and an oral composition comprising 1,25-dihydroxy Vitami.n D 3 for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques.
  • composition 1.25-dihvdroxy Vitamin D, composition
  • 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 ⁇ g 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 Vitamin D 3 are adminis- 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 conven ⁇ tional mixing techniques.
  • 100.00 0.1 cc of the composition is injected into the fracture site at the time of fracture reduction and once daily thereafter until desired repair is achieved.
  • EXAMPLE III A composition for inducing bone growth following reconstruc ⁇ tive surgery is prepared by combining the following components utilizing conventional mixing techniques.
  • EXAMPLE IV A composition for accelerating the healing and providing a stronger bond between natural bone and an artificial prosthesis is prepared by combining the following components utilizing conven- tional mixing techniques.
  • a topical oral carrier composition for periodontal therapy is 0 prepared by combining the following components utilizing conven ⁇ tional mixing techniques.
  • An injectable composition comprising the BMPs 2, 3, 4 and 5 and an oral composition comprising 1,25-dihydroxy Vitamin D 3 for treatment of osteoporosis is prepared by combining the follow i ng components utilizing conventional mixing techn i ques.
  • 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 injection and once daily thereafter.
  • the osteoinductive extract and 1,25-dihydroxy Vitamin D are administered over a 7-day period.
  • non-union fracture means a fracture that has failed to heal normally.
  • Acid demineralized bone particles 90.000 NaCl 0.900 Sterile water for injection q.s.
  • GCATCTCTGT CACACCCGGG GAGAAGATCA TCCTGAACTT CACGTCCCTG GACCTGTACC 114
  • CTGCGGTCTC CTAAAGGTCG ACCATGGTGG CCGGGACCCG CTGTCTTCTA GCGTTGCTGC
  • GAAACCAAAG TCAACTCCTT GGCCATCTGT CAGTGGATAT GGCCAAATCT CATCGAGATA 9
  • ATAGCCACAT CAGAGCTGCC CTTTCCATTG AGCGGAGGAA GAAGCGCTCT ACTGGGGTCT 12
  • CCAGCATGTC AGGATTAGCC GATCGTTACC TCAAGGGAGT GGGAATTGGG CCCAGCTCCG 120
  • ACCAGGCTAC CAGGCCTTCT ACTGCCATGG GGACTGCCCC TTTCCACTGG CTGACCACCT 144
  • CAACTCAACC AACCATGCCA TTGTGCAGAC CCTGGTCAAT TCTGTCAATT CCAGTATCCC 150
  • CTCCTCCCCC AAAAACCCAC CAAAATTAGT TTTAGCTGTA GATCAAGCTA TTTGGGGTGT 2040 TTGTTAGTAA ATAGGGAAAA TAATCTCAAA GGAGTTAAAT GTATTCTTGG CTAAAGGATC 21

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physical Education & Sports Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Rheumatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Procédé de production d'une croissance d'os neuf chez un mammifère consistant à administrer à ce dernier une dose sans danger et efficace d'un composé de vitamine D combinée à une dose sans danger et efficace d'un extrait ostéoinducteur d'au moins une protéine morphogénétique osseuse.Method for producing new bone growth in a mammal comprising administering to the latter a safe and effective dose of a vitamin D compound combined with a safe and effective dose of an osteoinductive extract of at least a bone morphogenetic protein.

Description

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 orphogenetic proteins and a Vitamin D compound, resulting in synergistic bone growth.
BACKGROUND 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- enopausal 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 treat¬ ments that go beyond the prevention of bone loss and promote bone formation and/or reverse bone loss.
(1989) "Bone Morphogenic Proteins and Vitamin D", Nutrition Reviews. Vol. 47, pp. 364-366 concludes that Vitamin D in the diet prevents the loss of the osteoinductive activity of bone matrix.
Turner, R. T., J. Farley, J. J. Vandersteenhoven, S. Epstein, N. H. Bell, and D. J. Baylink, (1988) "Demonstration of Reduced Mitogenic and Osteoinductive Activities in Demineralized Allo- geneic Bone Matrix from Vitamin D-deficient Rats", The Journal of Clinical Investigation. Inc.. Vol. 82, pp. 212-217, discloses the implantation of demineralized bone matrix from Vitamin D-deficient rats into normal rats. The demineralized bone matrix from Vitamin D-deficient rats did not promote osteoinduction as effectively as demineralized bone matrix from normal rats. Sampath, T. K., S. eintraub, and A. H. Reddi, (1984) "Extra¬ cellular 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, osteoin¬ ductive factors, osteogenic factors and other proteins/factors related to bone growth are disclosed in the following publica¬ tions: 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, S estad, 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, 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 Applica¬ tion 145 155, Sen, published July 10, 1985; World Patent Applica- tion 89/10934, Roos, Burns, Guy and McKnight, published November 16, 1989; World Patent Applications 89/09787 and 89/09788, Oppermann, Kubersampath, Rueger and Ozkaynak, published October 19, 1989; and World Patent Application 88/00205, Wang, Wozney and Rosen, published Janaury 14, 1988. OBJECTS OF THE PRESENT INVENTION
It is an object of the present invention to provide a method for generating new bone growth in a mammal.
It is a further object of the present invention to provide a pharmaceutical composition which can be used to generate new bone growth in a mammal .
SUMMARY OF THE INVENTION
The present invention relates to a method of generating new bone growth in mammals comprising administration to a mammal a combination of a safe and effective amount of a Vitamin D compound, and a safe and effective amount of one or more BMPs or osteoinductive extract comprising one or more BMPs.
The present invention further relates to a composition for generating new bone growth in mammals comprising a safe and effective amount of a Vitamin D compound; a safe and effective amount of a BMP or osteoinductive extract comprising one or more
BMPs; and a pharmaceutically-acceptable carrier.
DFTAILFD DESCRIPTION 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 amd a safe and effective amount of one or more BMPs or an osteoinducrtive 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. Surpris- ingly, it has been further determined that treatment with a
Vitamin D compound -m 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, osteoinducrrive extract or Vitamin D compound alone. Subjects in need o-r such treatment suffer from a variety of ailments which may ue treated via this procedure, including but not l imited to, borne fractures (closed and open) , non-union fractures, congenita" defects, as an adjunct in pl astic surgery, in treating oncological resections, all diseases cl assified as osteoporosis, rheumβetoid arthritis, osteoarthritis, septic arthritis, rickets, rtrganic incorporation of prosthetic joints and dental implants, pe.-iodcntal disease and defects, as well as osteopenic and osteoππalacic conditions and disease.
As used herein, "safe and effective amount" means an amount of compound or compicsition sufficient to significantly induce a positive modification, in the condition to be treated, but low enough to avoid seriωus side effects (at a reasonable benefit/risk ratio) , within the s.rope of sound medical judgment. The safe and effective amount of the expound or composition will vary with the particular condition beir-g treated, the age and physical condition of the patient being treated, the severity of the condition, the duration of the tre __tmer._ . the nature of concurrent therapy, the specific compound or composition employed, the particular pharma¬ ceutical ly-acceptabl •£ carrier util ized, and l ike factors within the knowledge and expertise of the attending physician.
As used herein, "fracture reduction" means the restoration of a bone fracture by 3urg-cal or manipulative means to its normal anatomical relation.
As used herein, "BKr* βeans bone morphogenetic protein . As used herein, "q.s . " means quantity sufficient.
As used herein, all percentages are by weight unless other¬ wise specified.
As used herein "re"onal treatment" includes treating bone fractures (closed and :pen) , treating non-union fractures, - 5 - treating congenital defects, as an adjunct treatment to plastic surgery, treating oncological resections, organic incorporation of prosthetic joints, organic incorporation of dental implants, and treatment of periodontal disease and defects. As used herein "systemic treatment" includes treating diseases classified as osteoporosis, rheumatoid arthritis, osteo- arthritis, septic arthritis, rickets, and osteopenic conditions and diseases.
As used herein, all dose ranges for systemic treatment are recited as the dry weight of the actives per kg body weight of the mammal .
As used herein, all dose ranges for regional treatment are recited as the dry weight of the actives per cm2 surface area of mineralized tissue to be treated. As used herein, "mineralized tissue" means bone and teeth.
Vitamin D Compounds
One component involved in the method of the invention is a Vitamin D compound. As used herein, "Vitamin D compound" includes Vitamin D, ergocalciferol (Vitamin D2), cholecalciferol (Vitamin D3) and their biologically active metabolites and precursors. Preferred Vitamin D compounds include, but are not limited to, Vitamin D2 (Sigma, St. Louis, MO), Vitamin D3 (Sigma, St. Louis, MO), 1-o-hydroxy Vitamin D3, 1-β-fluoro Vitamin D3, 3-deoxy-l,25- dihydroxy Vitamin D3, 25-hydroxy-5,6-trans Vitamin D3, 25-hydroxy Vitamin D2, 25-hydroxy Vitamin D3 (Hoffman LaRoche), 1,25-dihy- droxy Vitamin D2, 24,25-dihydroxy Vitamin D2, 24,25-dihydroxy Vitamin D,3 (Hoffman LaRoche), and 1,25-dihydroxy Vitamin D3 (Duphar, Veenendaal, Holland). Preferably, the Vitamin D compound is selected from 25-hydroxy Vitamin 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 D3. Additional Vitamin D compounds useful in the present invention are well known to those skilled in the art and include, but are not limited to, those disclosed by the following U.S. Patents, each of which is incorporated herein by reference: U.S. Patent 4,970,203, DeLuca and Kwiecinski, issued November 13, 1990; U.S. Patent 4,927,815, DeLuca, Kutner, Perlman and Schnoes, issued May 22, 1990; U.S. Patent 4,857,518, DeLuca, Ikekawa and Tanaka, issued August 15, 1989; U.S. Patent 4,851,401, DeLuca, Kutner, Perlman and Schnoes, issued July 25, 1989; U.S. Patent 4,851,400, DeLuca, Ikekawa and Tanaka, issued July 25, 1989; U.S. Patent 4,847,012, DeLuca, Kutner, Perlman, Phelps, Schnoes and Sicinski, issued July 11, 1989; U.S. Patent 4,816,417, Dame, DeLuca and Pierce, issued March 28, 1989; U.S. Patent 4,769,181, DeLuca, Schnoes, Sicinski and Tanaka, issued September 6, 1988; U.S. Patent 4,755,329, DeLuca, Lee and Schnoes, issued July 5, 1988; U.S. Patent 4,719,205, DeLuca, Schnoes, Sicinski and Tanaka, issued January 12, 1988; U.S. Patent 4,719,204, DeLuca, Schnoes, Sicinski and Tanaka, issued January 12, 1988; U.S. Patent 4,717,721, DeLuca, Ikekawa, Ostre and Schnoes, issued January 5, 1988; U.S. Patent 4,689,180, DeLuca, Schnoes, Sicinski and Tanaka, issued August 25, 1987; U.S. Patent 4,619,920, DeLuca, Ikekawa, Kobayashi and Tanaka, issued October 28, 1986; U.S. Patent 4,594,192, DeLuca, Ikekawa, Kobayashi and Tanaka, issued June 10, 1986; U.S. Patent 4,588,716, DeLuca and Schnoes, issued May 13, 1986; U.S. Patent 4,588,528, DeLuca, Ikekawa and Tanaka, issued May 13, 1986; U.S. Patent 4,564,474, DeLuca, Ikekawa, Kobayashi and Tanaka, issued January 14, 1986; U.S. Patent 4,555,364, DeLuca, Lee, Phelps and Schnoes, issued November 26, 1985; U.S. Patent 4,554,106, DeLuca, Lee, Phelps and Schnoes, issued November 19, 1985; U.S. Patent 4,552,698, DeLuca, Ikekawa, Kobayashi and Tanaka, issued November 11, 1985; U.S. Patent 4,512,925, DeLuca, Lee and Schnoes, issued April 23, 1985; U.S. Patent 4,505,906, DeLuca, Schnoes, Sicinski and Tanaka, issued March 19, 1985; U.S. Patent 4,502,991, DeLuca, Ikekawa, Kobayashi and Tanaka, issued March 5, 1985; U.S. Patent 4,500,460, DeLuca, Ikekawa, Kobayashi and Tanaka, issued February 19, 1985; U.S. Patent 4,481,198, Chu, DeLuca, Kabakoff and Schnoes, issued November 6, 1984; U.S. Patent 4,461,766, DeLuca, Hart and Schnoes, issued July 24, 1984; U.S. Patent 4,448,726, DeLuca, Paaren, Schnoes and Smith, issued May 15, 1984; U.S. Patent 4,448,721, DeLuca, Morzycki and Schnoes, issued May 15, 1984; U.S. Patent 4,428,946, DeLuca, Jorgensen and Schnoes, issued January 31, 1984; U.S. Patent 4,411,833, DeLuca, Ikekawa, Kobayashi and Tanaka, issued October 25, 1983; U.S. Patent 4,367,177, DeLuca, Schnoes and Wich an, 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, 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; U.S. Patent 4,201,881, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued May 6, 1980; U.S. Patent 4,196,133, DeLuca, Ikekawa, Kobayashi, Schnoes and Tanaka, issued April 1, 1980; U.S. Patent 4,195,027, DeLuca, Hamer, Paaren and Schnoes, issued March 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 D; 1 a-hydroxyvitamin D; 1-hydroxyvitamin D; 1 α-hydroxy- vitamin D2; 1 α,25-dihydroxy-22Z-dehydroxyvitamin D; 26,26,26,- 27,27-pentafluoro-l α-hydroxy-27-methoxyvitamin D3; 2 α-fluoro- vitamin D3; 1,24-dihydroxy-delta 22-vitamin D3; 23,23-difluoro- 25-hydroxy-vitamin D3; l-hydroxy-3,5-cyclovitamin D; 23,23-di- fluoro-1 α,25-dihydroxy-vitamin D3; 1,23-dihydroxyvitamin D; hydroxyvitamin D2; 23,23-difluoro-l α,25-dihydroxy-vitamin D3; 23,23-difluoro-25-hydroxy-vitamin D3; 26,26,26,27,27,27-hexa- fluoro-1 α,25-dihydroxycholesterol; 23,25-dihydroxyvitamin D3; 26,26,26,27,27,27-hexafluoro-l α,25-dihydroxycholecalciferol; 1 a,25-dihydroxy-2 ?-fluorovitamin D3; 24-fluoro-25-hydroxycholecal- ciferol; 5,6-trans-vitamin D; 1 a-hydroxy-25-keto-27-nor-chole- calciferol; fluorovitamin D; 1 β-hydroxy-2 3-fluorocholecalci- ferol ; 3-deoxy-l α-hydroxycholecalciferol ; 25-hydroxy-26,26,26,- 27,27,27-hexafluorocholecaliferol; __-hydroxy-3,5-cyclovitamin D; 25-hydroxycholecalciferol ; 24,24-difluoro-l o,25-dihydroxychole- calciferol; 25-hydroxycholecalciferol; 25-hydroxycholecalciferol- 26,23-1actone; 24,24-difluoro-lα,25-dihydroxycholecalciferol ; 24,24-difluoro-25-hydroxycholecalciferol; 3,5-cyclovitamin D; and 3-deoxy-α-hydroxycholecalciferol. Additional Vitamin D compounds useful in the present invention further include those disclosed in The Handbook of Vitamins, L. J. Machlin, Ed., Mercel Dekker, Inc. (1984), incorporated herein by reference. Vitamin D compounds useful in the present invention disclosed by this reference, include, bur are not limited to, 1,25-dihydroxy Vitamin D, 3-deoxy-1,25-dihydroxy Vitamin D, 27-πor-25-hydroxy Vitamin D3, 26,27-bis-nor-25-hydroxy Vitamin D3 24-no.-25-hyd.oxy Vitamin D3, 25-hydroxy Vitamin D, 1,25-dihydroxy Vitamin D, lβ-hydroxy Vitamin D3 and 25-fluoro-la-hydroxy Vitamin D3. - 9 -
A safe and effective amount of a Vitamin D compound is dosed in combination with at least one BMP or in combination with an osteoinductive extract comprising at least one BMP.
A preferred dose range for administration of the Vitamin D compound for systemic treatment is from about 1 ng to about 1 mg, preferably from about 10 ng to about 500 μg, more preferably from about 20 ng to about 10 μg.
For purposes of regional treatment, the dose range of the
Vitamin D compound is preferably from about 1 ng to about 1 mg, preferably from about 10 ng to about 500 ng, more preferably from about 10 ng to about 50 ng, most preferably from about 20 ng to about 30 ng.
Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month. Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day. Bone Morphogenetic Proteins
In one embodiment of the present invention, a Vitamin D compound is administered in combination with one or more BMPs to generate new bone growth in a mammal. These BMPs are preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4,
BMP-5, BMP-6 and BMP-7.
A safe and effective amount of a BMP, preferably selected from the group consisting of BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, is dosed in combination with a Vitamin D compound.
A preferred dose range for administration of the BMP for systemic treatment is from about 1 pg to about 100 μg, preferably from about 1 ng to about 10 μg, more preferably from about 10 ng to about 2.5 μg.
For purposes of regional treatment, a preferred dose range for the BMP is from about 1 pg to about 100 μg, more preferably from about 1.5 μg to about 90 μg, preferably from about 1.8 μg to about 75 μg, more preferably from about 2.0 μg to about 50 μg, more preferably still from about 2.2 μg to about 25 μg, more preferably from about 2.3 μg to about 10 μg, most preferably from about 2.5 μg to about 5 μg. Preferably the dose range is at least about 2.5 μg. 56
- 10 -
Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month.
Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day.
As used herein, "BMP-1" means a peptide encoded by a DNA sequence comprising SEQ ID N0:1.
As used herein, "BMP-2" means a peptide encoded by a DNA sequence comprising SEQ ID NO:2. 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, "BMP-5" means a peptide encoded by a DNA sequence comprising SEQ ID NO:5.
As used herein,. "BMP-6" means a peptide encoded by a DNA' sequence comprising SEQ ID NO: 6.
As used herein, "BMP-7" means a peptide encoded by a DNA sequence comprising SEQ ID NO: 7. As used herein, "A", "T", "G", and "C" refer to the nucleo¬ tides containing adenine, thymine, guanine and cytosine respectively. Osteoinductive Extract
Another component of the invention is an osteoinductive extract. As used herein, "osteoinductive extract" means a chemical extract of bone, comprising one or more various bone morphogenetic proteins, including, but not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7, wherein each BMP has a molecular weight of from about 28,000 to about 40,000 daltons. The 28,000 to 40,000 dalton molecular weight range is in reference to the BMP's dimer weight. Preferably, the molecular weight of the dimer is from about 30,000 to about 34,000 daltons. The BMP dimer comprises two monomers, each having a molecular weight of from about 14,000 to about 20,000 daltons, preferably from about 15,000 to about 17,000 daltons.
A preferred method of obtaining the osteoinductive extract 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 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 (U.S.A. Standard Sieve Series, Newark Wire Cloth Co., Newark, N.J.; sieve #40 retains particles greater than 425 μm and sieve #170 retains particles greater than 90 μm), isolate the bone particles in the 90-425um range. Grind any particles greater than 425μm 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 425μm particles until the amount of total recovery is greater than 2/3 of the initial weight of the bone. Store the particles at 4°C until the next step. Weigh the particles isolated thus far. For each gram of particles, add 25 ml of 0.6N 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 HC1 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 times. Swirl, allow to settle, and remove the supernatant. Rinse the bone particles with ethyl ether three times as above. Dry overnight in glass plates. The dried bone particles are referred to as "acid demineralized bone particles". - 12 -
The acid demineral zed bone particles are deproteinized as follows: Weigh the material following the overnight drying. For each gram of material, add a solution of 30 ml 4M guanidine-HCl, lOmM Tris and l.OmM PMSF pH 6.4 to the bone material in a beaker. 5 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 10 combine with the previously saved solution. The bone particles are now demineralized and deproteinized.
Dialyze the saved guanidine-HCl solution against distilled H20 at 4°C using 50 mm dialysis tubing (3500 molecular weight cutoff). Following dialysis, lyopholize the material and resolu- 15 bilize the lyophilized material in 4M Urea-0.05M Tris-O.IM NaCl, pH 7.4. Mix the solubilized material in a conical centrifuge tube with Heparin-Agarose and mix overnight on a rotator at 4°C. Pour the Heparin-Agarose slurry into a column. Wash with 1 column volume 4M urea, 0.05M Tris, 0.1M NaCl, pH 7.4 buffer. Collect the 20 fraction. Wash with 3 column volumes of 4M urea, 0.05M Tris, 0.2M NaCl, pH 7.4 buffer. Step off the material with 3 column volumes of 4M urea, 0.05M Tris, 0.75M NaCl, pH 7.4. Concentrate this sample in a 50 ml Amicon concentrator (Amicon Corp., Danvers, MA) with filter (10,000 molecular weight cut off) to about 4-5ml. 25 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 30 dialyzed against 1M 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 carboxymethyl- sepharose (CM-Sepharose) equilibrated with the same buffer. The 35 column is washed with 6M urea, 25mM Na acetate, pH 4.6 and activity eluted using a 0 - 0.5M NaCl gradient. Fractions are analyzed for protein concentration and sodium dodecyl sulfate gel electrophoresis. The activity located in the seven fractions before and after the beginning of the major protein peak are 13 - pooled for further purification.
The pooled CM-Sepharose fractions are dialyzed three times for 24 hours each against 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, 0.5M NaCl, 25mM Na phosphate, pH 7.4 to 6M urea, 0.5M NaCl, 25mM Na acetate, pH 4.6. Aliquots of each fraction are labeled with 125I and analyzed by SDS gel electrophoresis. Aliquots (100 μl) of each fraction are combined with 400 μl of elution buffer, dialyzed against 1% acetic acid and assayed for activity. Highly purified molecular weight range (Mr) 25-40 kD peptides are assayed in the bone induction assay.
A safe and effective amount of osteoinductive extract is dosed in combination with a Vitamin D compound. For purposes of systemic treatment, the osteoinductive extract dosed preferably comprises at least one BMP in an amount from about 1 pg to about 100 μg, preferably from about 1 ng to about 10 μg, more preferably from about 10 ng to about 2.5 μg.
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 μg, more preferably from about 1.5 μg to about 90 μg, preferably from about 1.8 μg to about 75 μg, more preferably from about 2.0 μg to about 50 μg, more preferably still from about 2.2 μg to about 25 μg, more preferably from about 2.3 μg to about 10 μg, most preferably from about 2.5 μg to about 5 μg. Preferably the dose range is at least about 2.5 μg. Preferably, doses are administered over a 1 day to 6 month period, more preferably from about 1 week to about 1 month. Preferably doses are administered from about once per month to about 5 times per day, more preferably from about once per week to about once per day. Pharmaceutically Acceptable Carrier
The Vitamin D compound, osteoinductive extract, or BMP may be administered via a pharmaceutically acceptable carrier. The term "pharmaceutically-acceptable carrier", as used herein, means one - 14 - or more compatible solid or liquid filler diluents or 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 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 ssr^e 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 carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; vegetable oils such a peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; sugar; alginic acid; pyrogen-free water; isotonic saline; phosphate buffer solutions; cocoa butter (suppository base); emulsifiers, such as the Tweens*; as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, tableting agents, stabilizers, antioxidants, and preservatives, can also be present. Other compatible pharmaceutical additives and actives (e.g., NSAI drugs; pain killers; muscle relaxants) may be included in the pharmaceutically-acceptable carrier for use in the compositions of the present invention. For example, art-known local anesthetics may be included in the pharmaceutically-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 - 15 - determined by the way the compound is to be administered. The preferred modes of administering the compounds of the present invention are by injection, oral administration, topical-oral administration, and nasopharyngeal administration or a combination of modes (i.e., osteoinductive extract via injection and Vitamin D compound via oral administration). If the compound is to be injected, the preferred pharmaceutically-acceptable carrier is sterile, physiological saline. Suitable 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 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 pharmaceutically- acceptable 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. Represen¬ tative pharmaceutical compositions of the present invention are provided in the Examples hereinafter. Pharmaceutically-acceptable carriers suitable for the pre¬ paration of unit dosage forms for oral administration, topical- oral administration, nasopharyngeal administration and injection are well-known in the art. Their selection will depend on secondary considerations like taste, cost, and/or shelf stability, - 16 - which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art. Pharmaceutically-acceptable carriers useful in the compositions of the present invention are described more fully hereinafter. A. Oral Dose Forms:
Preferably, the vitaπrn D compound is administered via an oral dose form. Various oral dosage forms can be used, including such solid forms as tablets, capsules, granules, bulk powders and microcapsules of the drug. These oral forms comprise a safe and effective amount, usually at least about .5%, and preferably from about 1% to about 10% of .ne compound of the present invention. Tablets can be compressed, enteric-coated, sugar-coated or film- coated containing suitable tinders, lubricants, surfactants, dilu¬ ents, disintegrating agents, coloring agents, flavoring agents, preservatives, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous and nonaqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents, and flavoring agents. Preferred carriers for orϊl administration include gelatin and propylene glycol. Specific examples of pharmaceutically-accept¬ able carriers and excipient.5 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 composi¬ tions for making solid oral dosage forms are described in Marshall, "Solid Oral Dosage Forms," Modern Pharmaceutics. Vol. 7. (Banker and Rhodes, editors), 359-427 (1979), incorporated herein by reference. Techniques and compositions for making tablets (compressed, formulas anc molded), capsules (hard and soft gelatin) and pills are described in Remington's Pharmaceutical Sciences (Arthur Osol, ec-.tor), 1553-1593 (1980), incorporated herein by reference. B. Topical-oral Dose Forms
"Topical-oral carrier", as used herein, denotes a carrier for the component of interest which results in a composition which is administered topically to the oral cavity, held therein for a period of time, and then is largely expectorated rather than being 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, espe¬ cially the Vitamin D compound and osteoinductive extract or BMP, used in an oral composition of the subject invention. Preferred topical-oral carriers thus provide the desired characteristics for toothpastes, tooth gels, tooth powders, mouthwashes, mouthsprays, prophylaxis pastes, dental treatment solutions, and the like.. The topical-oral carriers of the subject invention comprise components typically used in such compositions which are well known to a skilled practitioner. Such components include, but are not limited to anticaries agents, antiplaque agents, anticalculus agents, dental abrasives, surfactants, flavoring agents, sweetening agents, binders, humectants, thickening agents, buffering agents, preservatives, coloring agents and pigments, ethanol, and water.
Preferred compositions of the subject invention are in the form of toothpastes. Components of such toothpastes generally include a dental abrasive (from about 10% to about 50%), a sur¬ factant (from about 0.5% to about 10%), a thickening agent (from about 0.1% to about 5%) a humectant (from about 10% to about 55%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), a coloring agent (from about 0.01% to about 0.5%) and water (from about 2% to about 45%). Such toothpastes may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.1% to about 13%), and an antiplaque agent (from about 0.1% to about 5%).
Other preferred compositions of the subject invention are mouthwashes and mouthsprays. Components of such mouthwashes and mouthsprays include water (from about 45% to about 95%), ethanol (from about 0% to about 25%), a humectant (from about 0% to about 50%), a surfactant agent (from about 0.01% to about 7%), a flavoring agent (from about 0.04% to about 2%), a sweetening agent (from about 0.1% to about 3%), and a coloring agent (from about 0.001% to about 0.5%). Such mouthwashes and mouthsprays may also include one or more of an anticaries agent (from about 0.05% to about 0.3% as fluoride ion), an anticalculus agent (from about 0.01% to about 3%), and an antiplaque agent (from about 0.1% to about 5%).
Other preferred compositions of the subject invention are dental solutions. Components of such dental solutions generally include water (from about 90% to about 99%), preservative (from about 0.01% to about 0.5%), thickening agent (from about 0% to about 5%), flavoring agent (from about 0.04% to about 2%), sweetening agent (from about 0.1% to about 3%), and surfactant (from 0% to about 5%) .
"Topical-oral carrier" as used herein, also denotes fibers, strips or tubes which can be impregnated with the active components of the present invention and inserted or implanted into a periodontal pocket. Such compositions of the subject invention can readily be achieved by one of ordinary skill in the art using the teachings disclosed hereinbefore, the following references, incorporated herein by reference, and related well-known technol- ogies: U.S. Patent No. 4,666,897 issued to Golub, McNamara & Ramamurthy on May 19, 1987; European Patent Application No. 244,118 Al in the name of Baker, published on November 4, 1987; European Patent Application No. 286,802 A2 in the name of Kametaka, Miyazaki, Hayashi, Handa & Kameda, published October 19, 1988; Addy, M., L. Rawle, R. Handley, H. Newman & J. Coventry, "The development and in vitro evaluation of acrylic strips and dialysis tubing for local drug delivery", Journal of Periodon¬ tology. Vol. 53 (1982), pp. 693-698; Goodson, J.M., A.D. Haffajee _ S.S. Socransky, "Periodontal therapy by local delivery of tetracycline, Journal of Clinical Periodontology. Vol. 6 (1979), pp. 83-92; Goodson, J., D. Holborow, R. Dunn, P. Hogan & S. Dunham, "Monolithic tetracycline containing fibers for controlled delivery to periodontal pockets", Journal of Periodontology. Vol. 54 (1983), pp. 575-579; Dunn, R., J. Gibson, B. Perkins, J. Goodson & L. Laufe, "Fibrous delivery systems for antimicrobial agents", Polv er Science and Technology. Vol. 32 (1985), pp. 47-59; Dunn, R., J. Gibson, B. Perkins, J. Goodson & L. Laufe, "Fibrous delivery systems for antimicrobial agents", Polymer Material Science Engineering. Vol. 51 (1984), pp. 28-31; Olanoff, L. & J. Anderson, "Controlled release of tetracycline - III: A physiological pharmacokinetic model of the pregnant rat", Journal of Phar acokinetics and Biopharmaceutics. Vol. 8 (1980), pp. 599-620; Elkaya , R., M. Friedman, A. Stabholz, A. Soskolne, M. Sela & L. Golub, "Sustained release device containing minocycline for local treatment of periodontal disease", Journal of Controlled Release. Vol. 7 (1988), pp. 231-236; and Goodson, J., "Multi- center evaluation of tetracycline fiber therapy. I. Experimental Design", Journal of Dental Research. Vol. 68 (1989), p. 197; and references cited therein. C. In.iectable 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 concen¬ tration, and like factors within the specific knowledge and expertise of the attending physician and commensurate with a reasonable benefit/risk ratio associated with the use of any drug compound. In addition, lower dosages will be utilized when only local or minor bone growth is desired, whereas higher dosages will be utilized when general or major bone growth is desired.
Methods and materials for manufacturing injectables can be found in Remington's Pharmaceutical Sciences. 17ed., 1985, Chapter 85, p. 1518, the disclosures of which are incorporated herein by reference in their entirety. Preferably, the injectable compo¬ sition is an aqueous solution.
The aqueous solutions preferably consist of water (preferably from about 80% to about 99.999%), a suitable solubilizer, various types of acids, and an antimicrobial agent. Several solubilizers are known. Examples of such solubilizers are as follows: urea compounds (e.g., urea; urethan); surfactants (e.g., Tweens; Spans; sodium deoxycholate and Pluronics); cellulosic agents (e.g., carboxymethylcellulose); carbohydrates (e.g., sorbitol; mannitol); B vitamins (e.g., nicotinamide); xanthine derivatives; and alco¬ hols (e.g., benzyl alcohol). Examples of acids to be used include the following: glucuronic; galacturonic; fumaric; gentisic; acetic; citric and lactobionic. Types of antimicrobial agents that can be used are the following: phenyl mercuric nitrate; thi erosal ; benzethoniu chloride; benzal konium chl oride; phenol ; cresol ; and chlorobutanol . An art-known local anesthetic (e.g. , benzyl alcohol ; Novocaine*; l idocaine) may al so 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 l imitations 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 Vitami.n D3 for bone fracture repair is prepared by combining the following components utilizing conventional mixing techniques. BMP composition
Percent by Weight Component of Composition
BMP-1 0.04
NaCl 0.90
Sterile water Q.s.
100.00
1.25-dihvdroxy Vitamin D, composition
Percent by Weight
Component of Composition 1,25-dihydroxy Vitamin D3 0.01 Corn starch 18.49 Lactose 63.00 Talc 18.00 Stearic acid 0.50
100.00 0.1 cc of the BMP composition is injected into the fracture site at the time of fracture reduction and once daily thereafter. 100 μg of the 1,25-dihydroxy Vitamin D3 composition is orally administered 24 hours before fracture reduction and once daily thereafter. The BMP and 1,25-dihydroxy Vitamin D3 are adminis- 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 conven¬ tional 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 α.s.
100.00 0.1 cc of the composition is injected into the fracture site at the time of fracture reduction and once daily thereafter until desired repair is achieved.
EXAMPLE III A composition for inducing bone growth following reconstruc¬ tive surgery is prepared by combining the following components utilizing conventional mixing techniques.
Percent by Weight
Component of Composition
BMP-3 0.04
1,25-dihydroxy Vitamin D2 0.01 NaCl 0.90
Sterile water Q.S.
100.00 0.1 cc of the composition per cm2 of surface area of surgically reconstructed bone is deposited directly onto the bone surface.
EXAMPLE IV A composition for accelerating the healing and providing a stronger bond between natural bone and an artificial prosthesis is prepared by combining the following components utilizing conven- tional mixing techniques.
Percent by Weight Component of Composition
BMP-1 0.04
BMP-2 0.04 0.04
BMP-4 0.01
24,25-dihydroxy Vitamin D3 0.90
NaCl q.s. sterile water 100.00
0 1 cc of the composition per cm2 surface area of natural bone proximate to the prosthesis is deposited directly onto the natural bone.
FXAMPLE V
A topical oral carrier composition for periodontal therapy is 0 prepared by combining the following components utilizing conven¬ tional mixing techniques.
Percent by Weight of Composition r.nmponent 0.04
'5 BMP-2
'3 0.90
NaCl
Sterile water °" "
100.00
After the patient is prepared using conventional periodontal surgical therapy 0.1 cc of the composition per exposed tooth is
20 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.
FXAMPLE VI
25
An injectable composition comprising the BMPs 2, 3, 4 and 5 and an oral composition comprising 1,25-dihydroxy Vitamin D3 for treatment of osteoporosis is prepared by combining the following components utilizing conventional mixing techniques.
30 ncton.nriiictive «" -"-. rn oosition
Percent by Weight of Composition Component
0.001
BHP"2 0.001
BMP-3 0.001 5 BMP-4 0.001
BMP'5 0.900
NaCl
Q.S.
Sterile water
100.000 - 23 1.25-dihvdroxy Vitamin Di composition
Percent by Weight
Component of Composition 1,25-dihydroxy Vitamin D3 0.01 Corn starch 18.49 Lactose 63.00 Talc 18.00 Stearic acid 0.50
100.00 1 cc of the BMP composition is injected intravenously once per day. 50 mg of the 1,25-dihydroxy Vitamin D3 composition is orally administered within one hour of the osteoinductive extract injection and once daily thereafter. The osteoinductive extract and 1,25-dihydroxy Vitamin D are administered over a 7-day period.
EXAMPLE VII A composition for inducing bone growth of a non-union fracture is prepared by combining the following components utilizing conventional mixing techniques. As used herein, "non-union fracture" means a fracture that has failed to heal normally.
Percent by Weight
Component of Composition BMP-4 0.004
1,25-dihydroxy vitamin D3 0.01 Acid demineralized bone particles 90.000 NaCl 0.900 Sterile water for injection q.s.
100.000 At the time of fracture reduction, a sufficient quantity of the above composition is deposited directly into the non-union site thereby filling in any bone deficit. _ _ . _ _
- 24 - SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: STONE, ROGER L.
(ii) TITLE OF INVENTION: THERAPEUTIC FORMULAS FOR OSTEOINDUCTION
(iii) NUMBER OF SEQUENCES: 7
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: The Procter & Gamble Company
(B) STREET: 11810 East Miami River Road (C) CITY: Cincinnati
(D) STATE: Ohio
(E) COUNTRY: U.S.A.
(F) ZIP: 45239-8707
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Corstanje, Brahm J.
(B) REGISTRATION NUMBER: 34,804
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513-245-2858
(B) TELEFAX: 513-741-3012 - 25 (2) INFORMATION FOR SEQ ID N0: 1 :
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 2487 base pai rs (B) TYPE : nucl ei c acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GCCGCTTCCC TCGCCGCCGC CCCGCCAGCA TGCCCGGCGT GGCCCGCCTG CCGCTGCTGC
TCGGGCTGCT GCTGCTCCCG CGTCCCGGCC GGCCGCTGGA CTTGGCCGAC TACACCTATG
ACCTGGCGGA GGAGGACGAC TCGGAGCCCC TCAACTACAA AGACCCCTGC AAGGCGGCTG
CCTTTCTTGG GGACATTGCC CTGGACGAAG AGGACCTGAG GGCCTTCCAG GTACAGCAGG
CTGTGGATCT CAGACGGCAC ACAGCTCGTA AGTCCTCCAT CAAAGCTGCA GTTCCAGGAA
ACACTTCTAC CCCCAGCTGC CAGAGCACCA ACGGGCAGCC TCAGAGGGGA GCCTGTGGGA
GATGGAGAGG TAGATCCCGT AGCCGGCGGG CGGCGACGTC CCGACCAGAG CGTGTGTGGC
CCGATGGGGT CATCCCCTTT GTCATTGGGG GAAACTTCAC TGGTAGCCAG AGGGCAGTCT
TCCGGCAGGC CATGAGGCAC TGGGAGAAGC ACACCTGTGT CACCTTCCTG GAGCGCACTG
ACGAGGACAG CTATATTGTG TTCACCTATC GACCTTGCGG GTGCTGCTCC TACGTGGGTC
GCCGCGGCGG GGGCCCCCAG GCCATCTCCA TCGGCAAGAA CTGTGACAAG TTCGGCATTG
TGGTCCACGA GCTGGGCCAC GTCGTCGGCT TCTGGCACGA ACACACTCGG CCAGACCGGG 7
ACCGCCACGT TTCCATCGTT CGTGAGAACA TCCAGCCAGG GCAGGAGTAT AACTTCCTGA 7 AGATGGAGCC TCAGGAGGTG GAGTCCCTGG GGGAGACCTA TGACTTCGAC AGCATCATGC 8
ATTACGCTCG GAACACATTC TCCAGGGGCA TCTTCCTGGA TACCATTGTC CCCAAGTATG 90
AGGTGAACGG GGTGAAACCT CCCATTGGCC AAAGGACACG GCTCAGCAAG GGGGACATTG 96
CCCAAGCCCG CAAGCTTTAC AAGTGCCCAG CCTGTGGAGA GACCCTGCAA GACAGCACAG 102
GCAACTTCTC CTCCCCTGAA TACCCCAATG GCTACTCTGC TCACATGCAC TGCGTGTGGC 108
GCATCTCTGT CACACCCGGG GAGAAGATCA TCCTGAACTT CACGTCCCTG GACCTGTACC 114
GCAGCCGCCT GTGCTGGTAC GACTATGTGG AGGTCCGAGA TGGCTTCTGG AGGAAGGCGC 120
CCCTCCGAGG CCGCTTCTGC GGGTCCAAAC TCCCTGAGCC TATCGTCTCC ACTGACAGCC 126
GCCTCTGGGT TGAATTCCGC AGCAGCAGCA ATTGGGTTGG AAAGGGCTTC TTTGCAGTCT 132
ACGAAGCCAT CTGCGGGGGT GATGTGAAAA AGGACTATGG CCACATTCAA TCGCCCAACT 1380
ACCCAGACGA TTACCGGCCC AGCAAAGTCT GCATCTGGCG GATCCAGGTG TCTGAGGGCT 1440
TCCACGTGGG CCTCACATTC CAGTCCTTTG AGATTGAGCG CCACGACAGC TGTGCCTACG 1500
ACTATCTGGA GGTGCGCGAC GGGCACAGTG AGAGCAGCAC CCTCATCGGG CGCTACTGTG 1560
GCTATGAGAA GCCTGATGAC ATCAAGAGCA CGTCCAGCCG CCTCTGGCTC AAGTTCGTCT 1620
CTGACGGGTC CATTAACAAA GCGGGCTTTG CCGTCAACTT TTTCAAAGAG GTGGACGAGT 1680
GCTCTCGGCC CAACCGCGGG GGCTGTGAGC AGCGGTGCCT CAACACCCTG GGCAGCTACA 1740
AGTGCAGCTG TGACCCCGGG TACGAGCTGG CCCCAGACAA GCGCCGCTGT GAGGCTGCTT 1800
GTGGCGGATT CCTCACCAAG CTCAACGGCT CCATCACCAG CCCGGGCTGG CCCAAGGAGT 1860
ACCCCCCCAA CAAGAACTGC ATCTGGCAGC TGGTGGCCCC CACCCAGTAC CGCATCTCCC 1920
TGCAGTTTGA CTTCTTTGAG ACAGAGGGCA ATGATGTGTG CAAGTACGAC TTCGTGGAGG 1980 TGCGCAGTGG ACTCACAGCT GACTCCAAGC TGCATGGCAA GTTCTGTGGT TCTGAGAAGC 2
CCGAGGTCAT CACCTCCCAG TACAACAACA TGCGCGTGGA GTTCAAGTCC GACAACACCG 2
TGTCCAAAAA GGGCTTCAAG GCCCACTTCT TCTCAGAAAA GAGGCCAGCT CTGCAGCCCC 2
CTCGGGGACG CCCCCACCAG CTCAAATTCC GAGTGCAGAA AAGAAACCGG ACCCCCCAGT 2
GAGGCCTGCC AGGCCTCCCG GACCCCTTGT TACTCAGGAA CCTCACCTTG GACGGAATGG 2
GATGGGGGCT TCGGTGCCCA CCAACCCCCC ACCTCCACTC TGCCATTCCG GCCCACCTCC 2
CTCTGGCCGG ACAGAACTGG TGCTCTCTTC TCCCCACTGT GCCCGTCCGC GGACCGGGGA 2
CCCTTCCCCG TGCCCTACCC CCTCCCATTT TGATGGTGTC TGTGACATTT CCTGTTGTGA 2
AGTAAAAGAG GGACCCCTGC GTCCTGC 2
(2) INFORMATION FOR SEQ ID N0: 2 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 1547 base pai rs
(B) TYPE: nucl eic acid
(C) STRANDEDNESS : doubl e (D) TOPOLOGY : l i near
( i i ) MOLECULE TYPE : cDNA
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :
GGGGACTTCT TGAACTTGCA GGGAGAATAA CTTGCGCACC CCACTTTGCG CCGGTGCCTT
TGCCCCAGCG GAGCCTGCTT CGCCATCTCC GAGCCCCACC GCCCCTCCAC TCCTCGGCCT 1
TGCCCGACAC TGAGACGCTG TTCCCAGCGT GAAAAGAGAG ACTGCGCGGC CGGCACCCGG 1
GAGAAGGAGG AGGCAAAGAA AAGGAACGGA CATTCGGTCC TTGCGCCAGG TCCTTTGACC 2 AGAGTTTTTC CATGTGGACG CTCTTTCAAT GGACGTGTCC CCGCGTGCTT CTTAGACGGA
CTGCGGTCTC CTAAAGGTCG ACCATGGTGG CCGGGACCCG CTGTCTTCTA GCGTTGCTGC
TTCCCCAGGT CCTCCTGGGC GGCGCGGCTG GCCTCGTTCC GGAGCTGGGC CGCAGGAAGT
TCGCGGCGGC GTCGTCGGGC CGCCCCTCAT CCCAGCCCTC TGACGAGGTC CTGAGCGAGT 4
TCGAGTTGCG GCTGCTCAGC ATGTTCGGCC TGAAACAGAG ACCCACCCCC AGCAGGGACG 5
CCGTGGTGCC CCCCTACATG CTAGACCTGT ATCGCAGGCA CTCAGGTCAG CCGGGCTCAC 6
CCGCCCCAGA CCACCGGTTG GAGAGGGCAG CCAGCCGAGC CAACACTGTG CGCAGCTTCC 6
ACCATGAAGA ATCTTTGGAA GAACTACCAG AAACGAGTGG GAAAACAACC CGGAGATTCT 7
TCTTTAATTT AAGTTCTATC CCCACGGAGG AGTTTATCAC CTCAGCAGAG CTTCAGGTTT 7
TCCGAGAACA GATGCAAGAT GCTTTAGGAA ACAATAGCAG TTTCCATCAC CGAATTAATA 8
TTTATGAAAT CATAAAACCT GCAACAGCCA ACTCGAAATT CCCCGTGACC AGACTTTTGG 90
ACACCAGGTT GGTGAATCAG AATGCAAGCA GGTGGGAAAG TTTTGATGTC ACCCCCGCTG 96
TGATGCGGTG GACTGCACAG GGACACGCCA ACCATGGATT CGTGGTGGAA GTGGCCCACT 102
TGGAGGAGAA ACAAGGTGTC TCCAAGAGAC ATGTTAGGAT AAGCAGGTCT TTGCACCAAG 108
ATGAACACAG CTGGTCACAG ATAAGGCCAT TGCTAGTAAC TTTTGGCCAT GATGGAAAAG 114
GGCATCCTCT CCACAAAAGA GAAAAACGTC AAGCCAAACA CAAACAGCGG AAACGCCTTA 120
AGTCCAGCTG TAAGAGACAC CCTTTGTACG TGGACTTCAG TGACGTGGGG TGGAATGACT 126
GGATTGTGGC TCCCCCGGGG TATCACGCCT TTTACTGCCA CGGAGAATGC CCTTTTCCTC 132
TGGCTGATCA TCTGAACTCC ACTAATCATG CCATTGTTCA GACGTTGGTC AACTCTGTTA 1380
ACTCTAAGAT TCCTAAGGCA TGCTGTGTCC CGACAGAACT CAGTGCTATC TCGATGCTGT 1440 ACCTTGACGA GAATGAAAAG GTTGTATTAA AGAACTATCA GGACATGGTT GTGGAGGGTT 1
GTGGGTGTCG CTAGTACAGC AAAATTAAAT ACATAAATAT ATATATA 1
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1774 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AGATCTTGAA AACACCCGGG CCACACACGC CGCGACCTAC AGCTCTTTCT CAGCGTTGGA
GTGGAGACGG CGCCCGCAGC GCCCTGCGCG GGTGAGGTCC GCGCAGCTGC TGGGGAAGAG 1
CCCACCTGTC AGGCTGCGCT GGGTCAGCGC AGCAAGTGGG GCTGGCCGCT ATCTCGCTGC 1
ACCCGGCCGC GTCCCGGGCT CCGTGCGCCC TCGCCCCAGC TGGTTTGGAG TTCAACCCTC 2
GGCTCCGCCG CCGGCTCCTT GCGCCTTCGG AGTGTCCCGC AGCGACGCCG GGAGCCGACG 3
CGCCGCGCGG GTACCTAGCC ATGGCTGGGG CGAGCAGGCT GCTCTTTCTG TGGCTGGGCT 3
GCTTCTGCGT GAGCCTGGCG CAGGGAGAGA GACCGAAGCC ACCTTTCCCG GAGCTCCGCA 4
AAGCTGTGCC AGGTGACCGC ACGGCAGGTG GTGGCCCGGA CTCCGAGCTG CAGCCGCAAG 4
ACAAGGTCTC TGAACACATG CTGCGGCTCT ATGACAGGTA CAGCACGGTC CAGGCGGCCC 5
GGACACCGGG CTCCCTGGAG GGAGGCTCGC AGCCCTGGCG CCCTCGGCTC CTGCGCGAAG 6
GCAACACGGT TCGCAGCTTT CGGGCGGCAG CAGCAGAAAC TCTTGAAAGA AAAGGACTGT 6 ATATCTTCAA TCTGACATCG CTAACCAAGT CTGAAAACAT TTTGTCTGCC ACACTGTATT 7
TCTGTATTGG AGAGCTAGGA AACATCAGCC TGAGTTGTCC AGTGTCTGGA GGATGCTCCC 7
ATCATGCTCA GAGGAAACAC ATTCAGATTG ATCTTTCTGC ATGGACCCTC AAATTCAGCA 8
GAAACCAAAG TCAACTCCTT GGCCATCTGT CAGTGGATAT GGCCAAATCT CATCGAGATA 9
TTATGTCCTG GCTGTCTAAA GATATCACTC AATTCTTGAG GAAGGCCAAA GAAAATGAAG 9
AGTTCCTCAT AGGATTTAAC ATTACGTCCA AGGGACGCCA GCTGCCAAAG AGGAGGTTAC 10
CTTTTCCAGA GCCTTATATC TTGGTATATG CCAATGATGC CGCCATTTCT GAGCCAGAAA 10
GTGTGGTATC AAGCTTACAG GGACACCGGA ATTTTCCCAC TGGAACTGTT CCCAAATGGG . 11
ATAGCCACAT CAGAGCTGCC CTTTCCATTG AGCGGAGGAA GAAGCGCTCT ACTGGGGTCT 12
TGCTGCCTCT GCAGAACAAC GAGCTTCCTG GGGCAGAATA CCAGTATAAA AAGGATGAGG 126
TGTGGGAGGA GAGAAAGCCT TACAAGACCC TTCAGGCTCA GGCCCCTGAA AAGAGTAAGA 132
ATAAAAAGAA ACAGAGAAAG GGGCCTCATC GGAAGAGCCA GACGCTCCAA TTTGATGAGC 138
AGACCCTGAA AAAGGCAAGG AGAAAGCAGT GGATTGAACC TCGGAATTGC GCCAGGAGAT 144
ACCTCAAGGT AGACTTTGCA GATATTGGCT GGAGTGAATG GATTATCTCC CCCAAGTCCT 150
TTGATGCCTA TTATTGCTCT GGAGCATGCC AGTTCCCCAT GCCAAAGTCT TTGAAGCCAT 156
CAAATCATGC TACCATCCAG AGTATAGTGA GAGCTGTGGG GGTCGTTCCT GGGATTCCTG 162
AGCCTTGCTG TGTACCAGAA AAGATGTCCT CACTCAGTAT TTTATTCTTT GATGAAAATA 1680
AGAATGTAGT GCTTAAAGTA TACCCTAACA TGACAGTAGA GTCTTGCGCT TGCAGATAAC 1740
CTGGCAAAGA ACTCATTTGA ATGCTTAATT CAAT 1774
(2) INFORMATION FOR SEQ ID N0:4 : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1751 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGCAGAGGAG GAGGGAGGGA GGGAAGGAGC GCGGAGCCCG GCCCGGAAGC TAGGTGAGTG 6
TGGCATCCGA GCTGAGGGAC GCGAGCCTGA GACGCCGCTG CTGCTCCGGC TGAGTATCTA 12
GCTTGTCTCC CCGATGGGAT TCCCGTCCAA GCTATCTCGA GCCTGCAGCG CCACAGTCCC 18
CGGCCCTCGC CCAGGTTCAC TGCAACCGTT CAGAGGTCCC CAGGAGCTGC TGCTGGCGAG 24
CCCGCTACTG CAGGGACCTA TGGAGCCATT CCGTAGTGCC ATCCCGAGCA ACGCACTGCT 300
GCAGCTTCCC TGAGCCTTTC CAGCAAGTTT GTTCAAGATT GGCTGTCAAG AATCATGGAC 360
TGTTATTATA TGCCTTGTTT TCTGTCAAGA CACCATGATT CCTGGTAACC GAATGCTGAT 420
GGTCGTTTTA TTATGCCAAG TCCTGCTAGG AGGCGCGAGC CATGCTAGTT TGATACCTGA 480
GACGGGGAAG AAAAAAGTCG CCGAGATTCA GGGCCACGCG GGAGGACGCC GCTCAGGGCA 540
GAGCCATGAG CTCCTGCGGG ACTTCGAGGC GACACTTCTG CAGATGTTTG GGCTGCGCCG " 600
CCGCCCGCAG CCTAGCAAGA GTGCCGTCAT TCCGGACTAC ATGCGGGATC TTTACCGGCT 660
TCAGTCTGGG GAGGAGGAGG AAGAGCAGAT CCACAGCACT GGTCTTGAGT ATCCTGAGCG 720
CCCGGCCAGC CGGGCCAACA CCGTGAGGAG CTTCCACCAC GAAGAACATC TGGAGAACAT 780
CCCAGGGACC AGTGAAAACT CTGCTTTTCG TTTCCTCTTT AACCTCAGCA GCATCCCTGA 840 GAACGAGGTG ATCTCCTCTG CAGAGCTTCG GCTCTTCCGG GAGCAGGTGG ACCAGGGCCC 9
TGATTGGGAA AGGGGCTTCC ACCGTATAAA CATTTATGAG GTTATGAAGC CCCCAGCAGA 9
AGTGGTGCCT GGGCACCTCA TCACACGACT ACTGGACACG AGACTGGTCC ACCACAATGT 10
GACACGGTGG GAAACTTTTG ATGTGAGCCC TGCGGTCCTT CGCTGGACCC GGGAGAAGCA 10
GCCAAACTAT GGGCTAGCCA TTGAGGTGAC TCACCTCCAT CAGACTCGGA CCCACCAGGG 114
CCAGCATGTC AGGATTAGCC GATCGTTACC TCAAGGGAGT GGGAATTGGG CCCAGCTCCG 120
GCCCCTCCTG GTCACCTTTG GCCATGATGG CCGGGGCCAT GCCTTGACCC GACGCCGGAG 126
GGCCAAGCGT AGCCCTAAGC ATCACTCACA GCGGGCCAGG AAGAAGAATA AGAACTGCCG 132
GCGCCACTCG CTCTATGTGG ACTTCAGCGA TGTGGGCTGG AATGACTGGA TTGTGGCCCC 138
ACCAGGCTAC CAGGCCTTCT ACTGCCATGG GGACTGCCCC TTTCCACTGG CTGACCACCT 144
CAACTCAACC AACCATGCCA TTGTGCAGAC CCTGGTCAAT TCTGTCAATT CCAGTATCCC 150
CAAAGCCTGT TGTGTGCCCA CTGAACTGAG TGCCATCTCC ATGCTGTACC TGGATGAGTA 156
TGATAAGGTG GTACTGAAAA ATTATCAGGA GATGGTAGTA GAGGGATGTG GGTGCCGCTG 1620
AGATCAGGCA GTCCTTGAGG ATAGACAGAT ATACACACCA CACACACACA CCACATACAC 1680
CACACACACA CGTTCCCATC CACTCACCCA CACACTACAC AGACTGCTTC CTTATAGCTG 1740
GACTTTTATT T 1751
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2153 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTGGTATATT TGTGCCTGCT GGAGGTGGAA TTAACAGTAA GAAGGAGAAA GGGATTGAAT
GGACTTACAG GAAGGATTTC AAGTAAATTC AGGGAAACAC ATTTACTTGA ATAGTACAAC 1
CTAGAGTATT ATTTTACACT AAGACGACAC AAAAGATGTT AAAGTTATCA CCAAGCTGCC 1
GGACAGATAT ATATTCCAAC ACCAAGGTGC AGATCAGCAT AGATCTGTGA TTCAGAAATC 2
AGGATTTGTT TTGGAAAGAG CTCAAGGGTT GAGAAGAACT CAAAAGCAAG TGAAGATTAC 3
TTTGGGAACT ACAGTTTATC AGAAGATCAA CTTTTGCTAA TTCAAATACC AAAGGCCTGA 3
TTATCATAAA TTCATATAGG AATGCATAGG TCATCTGATC AAATAATATT AGCCGTCTTC 4
TGCTACATCA ATGCAGCAAA AACTCTTAAC AACTGTGGAT AATTGGAAAT CTGAGTTTCA 4
GCTTTCTTAG AAATAACTAC TCTTGACATA TTCCAAAATA TTTAAAATAG GACAGGAAAA 5
TCGGTGAGGA TGTTGTGCTC AGAAATGTCA CTGTCATGAA AAATAGGTAA ATTTGTTTTT 6
TCAGCTACTG GGAAACTGTA CCTCCTAGAA CCTTAGGTTT TTTTTTTTTT AAGAGGACAA 6
GAAGGACTAA AAATATCAAC TTTTGCTTTT GGACAAAAAT GCATCTGACT GTATTTTTAC 7
TTAAGGGTAT TGTGGGTTTC CTCTGGAGCT GCTGGGTTCT AGTGGGTTAT GCAAAAGGAG 7
GTTTGGGAGA CAATCATGTT CACTCCAGTT TTATTTATAG AAGACTACGG AACCACGAAA 8
GACGGGAAAT ACAAAGGGAA ATTCTCTCTA TCTTGGGTTT GCCTCACAGA CCCAGACCAT 9
TTTCACCTGG AAAACAAGCG TCCTCTGCAC CTCTCTTTAT GCTGGATCTC TACAATGCCA 9
TGACCAATGA AGAAAATCCT GAAGAGTCGG AGTACTCAGT AAGGGCATCC TTGGCAGAAG 102 AGACCAGAGG GGCAAGAAAG GGATACCCAG CCTCTCCCAA TGGGTATCCT CGTCGCATAC 108
AGTTATCTCG GACGACTCCT CTGACCACCC AGAGTCCTCC TCTAGCCAGC CTCCATGATA 114
CCAACTTTCT GAATGATGCT GACATGGTCA TGAGCTTTGT CAACTTAGTT GAAAGAGACA 120
AGGATTTTTC TCACCAGCGA AGGCATTACA AAGAATTTCG ATTTGATCTT ACCCAAATTC 126
CTCATGGAGA GGCAGTGACA GCAGCTGAAT TCCGGATATA CAAGGACCGG AGCAACAACC 132
GATTTGAAAA TGAAACAATT AAGATTAGCA TATATCAAAT CATCAAGGAA TACACAAATA 1380
GGGATGCAGA TCTGTTCTTG TTAGACACAA GAAAGGCCCA AGCTTTAGAT GTGGGTTGGC 1440
TTGTCTTTGA TATCACTGTG ACCAGCAATC ATTGGGTGAT TAATCCCCAG AATAATTTGG 1500
GCTTACAGCT CTGTGCAGAA ACAGGGGATG GACGCAGTAT CAACGTAAAA TCTGCTGGTC 1560
TTGTGGGAAG ACAGGGACCT CAGTCAAAAC AACCATTCAT GGTGGCCTTC TTCAAGGCGA 1620
GTGAGGTACT TCTTCGATCC GTGAGAGCAG CCAACAAACG AAAAAATCAA AACCGCAATA 1680
AATCCAGCTC TCATCAGGAC TCCTCCAGAA TGTCCAGTGT TGGAGATTAT AACACAAGTG 1740
AGCAAAAACA AGCCTGTAAG AAGCACGAAC TCTATGTGAG CTTCCGGGAT CTGGGATGGC 1800
AGGACTGGAT TATAGCACCA GAAGGATACG CTGCATTTTA TTGTGATGGA GAATGTTCTT 1860
TTCCACTTAA CGCCCATATG AATGCCACCA ACCACGCTAT AGTTCAGACT CTGGTTCATC 1920
TGATGTTTCC TGACCACGTA CCAAAGCCTT GTTGTGCTCC AACCAAATTA AATGCCATCT 1980
CTGTTCTGTA CTTTGATGAC AGCTCCAATG TCATTTTGAA AAAATATAGA AATATGGTAG 2040
TACGCTCATG TGGCTGCCAC TAATATTAAA TAATATTGAT AATAACAAAA AGATCTGTAT 2100
TAAGGTTTAT GGCTGCAATA AAAAGCATAC TTTCAGACAA ACAGAAAAAA AAA 2153
(2) INFORMATION FOR SEQ ID N0:6: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2923 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
CGACCATGAG AGATAAGGAC TGAGGGCCAG GAAGGGGAAG CGAGCCCGCC GAGAGGTGGC
GGGGACTGCT CACGCCAAGG GCCACAGCGG CCGCGCTCCG GCCTCGCTCC GCCGCTCCAC 1
" -TCGCGGG ATCCGCGGGG GCAGCCCGGC CGGGCGGGGA TGCCGGGGCT GGGGCGGAGG 1
GCGCAGTGGC TGTGCTGGTG GTGGGGGCTG CTGTGCAGCT GCTGCGGGCC CCCGCCGCTG 2
CGGCCGCCCT TGCCCGCTGC CGCGGCCGCC GCCGCCGGGG GGCAGCTGCT GGGGGACGGC 30
GGGAGCCCCG GCCGCACGGA GCAGCCGCCG CCGTCGCCGC AGTCCTCCTC GGGCTTCCTG 36
TACCGGCGGC TCAAGACGCA GGAGAAGCGG GAGATGCAGA AGGAGATCTT GTCGGTGCTG 42
GGGCTCCCGC ACCGGCCCCG GCCCCTGCAC GGCCTCCAAC AGCCGCAGCC CCCGGCGCTC 48
CGGCAGCAGG AGGAGCAGCA GCAGCAGCAG CAGCTGCCTC GCGGAGAGCC CCCTCCCGGG 54
CGACTGAAGT CCGCGCCCCT CTTCATGCTG GATCTGTACA ACGCCCTGTC CGCCGACAAC 60
GACGAGGACG GGGCGTCGGA GGGGGAGAGG CAGCAGTCCT GGCCCCACGA AGCAGCCAGC 66
TCGTCCCAGC GTCGGCAGCC GCCCCCGGGC GCCGCGCACC CGCTCAACCG CAAGAGCCTT 72
CTGGCCCCCG GATCTGGCAG CGGCGGCGCG TCCCCACTGA CCAGCGCGCA GGACAGCGCC 78
TTCCTCAACG ACGCGGACAT GGTCATGAGC TTTGTGAACC TGGTGGAGTA CGACAAGGAG 84 TTCTCCCCTC GTCAGCGACA CCACAAAGAG TTCAAGTTCA ACTTATCCCA GATTCCTGAG 9
GGTGAGGTGG TGACGGCTGC AGAATTCCGC ATCTACAAGG ACTGTGTTAT GGGGAGTTTT 9
AAAAACCAAA CTTTTCTTAT CAGCATTTAT CAAGTCTTAC AGGAGCATCA GCACAGAGAC 102
TCTGACCTGT TTTTGTTGGA CACCCGTGTA GTATGGGCCT CAGAAGAAGG CTGGCTGGAA 108
TTTGACATCA CGGCCACTAG CAATCTGTGG GTTGTGACTC CACAGCATAA CATGGGGCTT 114
CAGCTGAGCG TGGTGACAAG GGATGGAGTC CACGTCCACC CCCGAGCCGC AGGCCTGGTG 120
GGCAGAGACG GCCCTTACGA TAAGCAGCCC TTCATGGTGG CTTTCTTCAA AGTGAGTGAG 126
GTCCACGTGC GCACCACCAG GTCAGCCTCC AGCCGGCGCC GACAACAGAG TCGTAATCGC 132
TCTACCCAGT CCCAGGACGT GGCGCGGGTC TCCAGTGCTT CAGATTACAA CAGCAGTGAA 138
TTGAAAACAG CCTGCAGGAA GCATGAGCTG TATGTGAGTT TCCAAGACCT GGGATGGCAG 144
GACTGGATCA TTGCACCCAA GGGCTATGCT GCCAATTACT GTGATGGAGA ATGCTCCTTC 150
CCACTCAACG CACACATGAA TGCAACCAAC CACGCGATTG TGCAGACCTT GGTTCACCTT 1560
ATGAACCCCG AGTATGTCCC CAAACCGTGC TGTGCGCCAA CTAAGCTAAA TGCCATCTCG 1620
GTTCTTTACT TTGATGACAA CTCCAATGTC ATTCTGAAAA AATACAGGAA TATGGTTGTA 1680
AGAGCTTGTG GATGCCACTA ACTCGAAACC AGATGCTGGG GACACACATT CTGCCTTGGA 1740
TTCCTAGATT ACATCTGCCT TAAAAAAACA CGGAAGCACA GTTGGAGGTG GGACGATGAG 1800
ACTTTGAAAC TATCTCATGC CAGTGCCTTA TTACCCAGGA AGATTTTAAA GGACCTCATT 1860
AATAATTTGC TCACTTGGTA AATGACGTGA GTAGTTGTTG GTCTGTAGCA AGCTGAGTTT 1920
GGATGTCTGT AGCATAAGGT CTGGTAACTG CAGAAACATA ACCGTGAAGC TCTTCCTACC 1980
CTCCTCCCCC AAAAACCCAC CAAAATTAGT TTTAGCTGTA GATCAAGCTA TTTGGGGTGT 2040 TTGTTAGTAA ATAGGGAAAA TAATCTCAAA GGAGTTAAAT GTATTCTTGG CTAAAGGATC 21
AGCTGGTTCA GTACTGTCTA TCAAAGGTAG ATTTTACAGA GAACAGAAAT CGGGGAAGTG 21
GGGGGAACGC CTCTGTTCAG TTCATTCCCA GAAGTCCACA GGACGCACAG CCCAGGCCAC 22
AGCCAGGGCT CCACGGGGCG CCCTTGTCTC AGTCATTGCT GTTGTATGTT CGTGCTGGAG 22
TTTTGTTGGT GTGAAAATAC ACTTATTTCA GCCAAAACAT ACCATTTCTA CACCTCAATC 23
CTCCATTTGC TGTACTCTTT GCTAGTACCA AAAGTAGACT GATTACACTG AGGTGAGGCT 24
ACAAGGGGTG TGTAACCGTG TAACACGTGA AGGCAGTGCT CACCTCTTCT TTACCAGAAC 24
GGTTCTTTGA CCAGCACATT AACTTCTGGA CTGCCGGCTC TAGTACCTTT TCAGTAAAGT 25
GGTTCTCTGC CTTTTTACTA TACAGCATAC CACGCCACAG GGTTAGAACC AACGAAGAAA 25
ATAAAATGAG GGTGCCCAGC TTATAAGAAT GGTGTTAGGG GGATGAGCAT GCTGTTTATG 264
AACGGAAATC ATGATTTCCC TGTAGAAAGT GAGGCTCAGA TTAAATTTTA GAATATTTTC 270
TAAATGTCTT TTTCACAATC ATGTGACTGG GAAGGCAATT TCATACTAAA CTGATTAAAT 276
AATACATTTA TAATCTACAA CTGTTTGCAC TTACAGCTTT TTTTGTAAAT ATAAACTATA 282
ATTTATTGTC TATTTTATAT CTGTTTTGCT GTGGCGTTGG GGGGGGGGCC GGGCTTTTGG 288
GGGGGGGGGT TTGTTTGGGG GGTGTCGTGG TGTGGGCGGG CGG 292
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1448 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GTGACCGAGC GGCGCGGACG GCCGCCTGCC CCCTCTGCCA CCTGGGGCGG TGCGGGCCCG
GAGCCCGGAG CCCGGGTAGC GCGTAGAGCC GGCGCGATGC ACGTGCGCTC ACTGCGAGCT 1
GCGGCGCCGC ACAGCTTCGT GGCGCTCTGG GCACCCCTGT TCCTGCTGCG CTCCGCCCTG 1
GCCGACπCA GCCTGGACAA CGAGGTGCAC TCGAGCTTCA TCCACCGGCG CCTCCGCAGC 2
CAGGAGCGGC GGGAGATGCA GCGCGAGATC CTCTCCATTT TGGGCTTGCC CCACCGCCCG 3
CGCCCGCACC TCCAGGGCAA GCACAACTCG GCACCCATGT TCATGCTGGA CCTGTACAAC 36
GCCATGGCGG TGGAGGAGGG CGGCGGGCCC GGCGGCCAGG GCTTCTCCTA CCCCTACAAG • 42
GCCGTCTTCA GTACCCAGGG CCCCCCTCTG GCCAGCCTGC AAGATAGCCA TTTCCTCACC 48
GACGCCGACA TGGTCATGAG CTTCGTCAAC CTCGTGGAAC ATGACAAGGA ATTCTTCCAC 54
CCACGCTACC ACCATCGAGA GTTCCGGTTT GATCTTTCCA AGATCCCAGA AGGGGAAGCT 60
GTCACGGCAG CCGAATTCCG GATCTACAAG GACTACATCC GGGAACGCTT CGACAATGAG 66
ACGTTCCGGA TCAGCGTTTA TCAGGTGCTC CAGGAGCACT TGGGCAGGGA ATCGGATCTC 72
TTCCTGCTCG ACAGCCGTAC CCTCTGGGCC TCGGAGGAGG GCTGGCTGGT GTTTGACATC 78
ACAGCCACCA GCAACCACTG GGTGGTCAAT CCGCGGCACA ACCTGGGCCT GCAGCTCTCG 840
GTGGAGACGC TGGATGGGCA GAGCATCAAC CCCAAGTTGG CGGGCCTGAT TGGGCGGCAC 900
GGGCCCCAGA ACAAGCAGCC CTTCATGGTG GCTTTCTTCA AGGCCACGGA GGTCCACTTC 960
CGCAGCATCC GGTCCACGGG GAGCAAACAG CGCAGCCAGA ACCGCTCCAA GACGCCCAAG 1020
AACCAGGAAG CCCTGCGGAT GGCCAACGTG GCAGAGAACA GCAGCAGCGA CCAGAGGCAG 1080
GCCTGTAAGA AGCACGAGCT GTATGTCAGC TTCCGAGACC TGGGCTGGCA GGACTGGATC 1140 ATCGCGCCTG AAGGCTACGC CGCCTACTAC TGTGAGGGGG AGTGTGCCTT CCCTCTGAAC 12
TCCTACATGA ACGCCACCAA CCACGCCATC GTGCAGACGC TGGTCCACTT CATCAACCCG 12
GAAACGGTGC CCAAGCCCTG CTGTGCGCCC ACGCAGCTCA ATGCCATCTC CGTCCTCTAC 13
TTCGATGACA GCTCCAACGT CATCCTGAAG AAATACAGAA ACATGGTGGT CCGGGCCTGT 13
GGCTGCCACT AGCTCCTCCG AGAATTCAGA CCCTTTGGGG CCAAGTTTTT CTGGATCCTC 14
CATTGCTC 14
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:

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 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 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-α-hydroxy Vitamin D3, l-c.-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 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 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 lmg and the safe and effective amount of the BMP is from about 1 pg to about 100 μg.
10. The method 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 method of Claim 10 wherein the Vitamin D compound is selected from the group consisting of Vitamin D2, Vitamin D3, 1-α- hydroxy Vitamin D3, 1-α-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 method of Claim 11 wherein the Vitamin D compound is 1,25- dihydroxy Vitamin D3.
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.
15. 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 comprisinq 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.
EP92913307A 1991-06-05 1992-05-26 Therapeutic compositions for osteoinduction Withdrawn EP0587751A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US70962191A 1991-06-05 1991-06-05
US709621 1991-06-05
US85611092A 1992-03-27 1992-03-27
US856110 1992-03-27

Publications (1)

Publication Number Publication Date
EP0587751A1 true EP0587751A1 (en) 1994-03-23

Family

ID=27108286

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92913307A Withdrawn EP0587751A1 (en) 1991-06-05 1992-05-26 Therapeutic compositions for osteoinduction

Country Status (13)

Country Link
EP (1) EP0587751A1 (en)
JP (1) JPH06508140A (en)
AU (1) AU667815B2 (en)
BR (1) BR9206110A (en)
CA (1) CA2110410C (en)
CZ (1) CZ282231B6 (en)
IE (1) IE921812A1 (en)
NO (1) NO934381L (en)
NZ (1) NZ243018A (en)
PT (1) PT100567A (en)
RU (1) RU2107512C1 (en)
SK (1) SK136693A3 (en)
WO (1) WO1992021365A1 (en)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5674844A (en) * 1991-03-11 1997-10-07 Creative Biomolecules, Inc. Treatment to prevent loss of and/or increase bone mass in metabolic bone diseases
US7056882B2 (en) 1991-03-11 2006-06-06 Curis, Inc. Treatment to prevent loss of and/or increase bone mass in metabolic bone diseases
JP4235689B2 (en) * 1991-08-30 2009-03-11 ストライカー・コーポレーション Bone morphogenetic protein for treating bone disease
US6071695A (en) * 1992-02-21 2000-06-06 Creative Biomolecules, Inc. Methods and products for identification of modulators of osteogenic protein-1 gene expression
JP3699111B2 (en) * 1993-03-29 2005-09-28 ザイモジェネティクス，インコーポレイティド Composition for stimulating osteoblast growth containing PDGF and vitamin D
CA2191583C (en) * 1994-06-07 2007-03-27 Engin Ozkaynak Methods and compositions for modulating morphogenic protein expression
US7026292B1 (en) 1995-12-12 2006-04-11 Stryker Corporation Compositions and therapeutic methods using morphogenic proteins and stimulatory factors
US6048964A (en) * 1995-12-12 2000-04-11 Stryker Corporation Compositions and therapeutic methods using morphogenic proteins and stimulatory factors
US6492508B1 (en) 1996-06-03 2002-12-10 United States Surgical Corp. A Division Of Tyco Healthcare Group Nucleic acids encoding extracellular matrix proteins
US6281195B1 (en) * 1997-02-07 2001-08-28 Stryker Corporation Matrix-free osteogenic devices, implants and methods of use thereof
RU2336902C2 (en) * 2001-10-31 2008-10-27 Алькон, Инк. Morphogene bone proteins (bmp), bmp receptors and binding bmp proteins and their application for diagnostics and glaucoma treatment
PL2148684T3 (en) * 2007-04-25 2013-06-28 Cytochroma Inc Method of treating vitamin d insufficiency and deficiency
EP2396025A2 (en) * 2009-02-12 2011-12-21 Stryker Corporation Peripheral administration of proteins including tgf-beta superfamily members for systemic treatment of disorders and disease
EP2396026A2 (en) * 2009-02-12 2011-12-21 Stryker Corporation Compositions and methods for minimally-invasive systemic delivery of proteins including tgf- superfamily members
RU2595804C1 (en) * 2015-04-23 2016-08-27 Федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский государственный технический университет имени Гагарина Ю.А." (СГТУ имени Гагарина Ю.А.) Composition for producing dental therapeutic film

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4833125A (en) * 1986-12-05 1989-05-23 The General Hospital Corporation Method of increasing bone mass

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9221365A1 *

Also Published As

Publication number Publication date
RU2107512C1 (en) 1998-03-27
NZ243018A (en) 1995-12-21
CZ262493A3 (en) 1994-07-13
WO1992021365A1 (en) 1992-12-10
BR9206110A (en) 1995-07-11
NO934381L (en) 1994-01-31
CZ282231B6 (en) 1997-06-11
NO934381D0 (en) 1993-12-02
PT100567A (en) 1994-01-31
SK136693A3 (en) 1994-10-05
AU2145492A (en) 1993-01-08
AU667815B2 (en) 1996-04-18
CA2110410A1 (en) 1992-12-10
JPH06508140A (en) 1994-09-14
CA2110410C (en) 1997-04-15
IE921812A1 (en) 1992-12-16

Similar Documents

Publication Publication Date Title
US5693615A (en) Therapeutic compositions for osteoinduction
EP0679097B1 (en) Tgf-beta formulation for inducing bone growth
US5422340A (en) TGF-βformulation for inducing bone growth
Asahina et al. Repair of bone defect in primate mandible using a bone morphogenetic protein (BMP)-hydroxyapatite-collagen composite
Simion et al. Vertical ridge augmentation by means of deproteinized bovine bone block and recombinant human platelet-derived growth factor-BB: a histologic study in a dog model.
Kao et al. The use of biologic mediators and tissue engineering in dentistry.
AU667815B2 (en) Therapeutic compositions for osteoinduction
Barboza et al. Potential of recombinant human bone morphogenetic protein-2 in bone regeneration
CA2417207C (en) Dental products comprising bone growth enhancing peptide
US9089606B2 (en) Brain-derived neurotrophic factor or neurotrophin-4/5 to treat periodontal diseases and pulpal diseases
AU2005328325B2 (en) Activating extraction of demineralized bone matrix
Aghaloo et al. Growth Factors for Site Preparation: Current Science, Indications, and Practice
DE69325913T2 (en) PERIODONTOL TISSUE REGENERATION INDUCED BY MORPHOGEN
Aghaloo et al. Growth Factors for Site Preparation: Current Science, Indications
US20080107706A1 (en) Osteogenic Oligonucleotides and Uses Thereof
Parimala Comparative evaluation of bovine porous bone mineral (Bio-Oss) bone craft with and without platelet rich plasma (PRP) in the treatment of periodontal intrabony defects: a clinical study
GIANNOBILE Advanced reconstructive technologies for periodontal tissue repair
RIDGE SELECTED READINGS IN ORAL AND MAXILLOFACIAL SURGERY

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19931126

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

17Q First examination report despatched

Effective date: 19951208

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19981010