WO1998030234A1 - Compositions favorisant l'osteogenese et la chondrogenese - Google Patents

Compositions favorisant l'osteogenese et la chondrogenese Download PDF

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WO1998030234A1
WO1998030234A1 PCT/JP1998/000112 JP9800112W WO9830234A1 WO 1998030234 A1 WO1998030234 A1 WO 1998030234A1 JP 9800112 W JP9800112 W JP 9800112W WO 9830234 A1 WO9830234 A1 WO 9830234A1
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cells
shh
bmp
bone
rhbmp
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PCT/JP1998/000112
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Masahiro Iwamoto
Tomonao Aikawa
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Masahiro Iwamoto
Tomonao Aikawa
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Priority to AU54955/98A priority Critical patent/AU5495598A/en
Publication of WO1998030234A1 publication Critical patent/WO1998030234A1/fr

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    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention concerns a drug composition characterized by containing a bone mo ⁇ hogenesis factor; and specifically, concerns a composition for osteogenesis and chondrogenesis.
  • Bone morphogenetic proteins are members of the TGF- ⁇ superfamily of proteins which are known to have activity in the growth and differentiation of cells and tissue. Many BMPs are active protein that cause undifferentiated mesenchymal cells of subcutaneous tissue or muscular tissue to differentiate into chondroblasts or osteoblasts, thereby stimulating osteogenesis or chondrogenesis.
  • the genes that code for various BMPs have been isolated, including the genes encoding BMPs 1 through 18, GDFs 1-14, MP52, BIP, and HP269. See also: Science 242, 1528-1534 (1988); Proc. Natl. Acad. Sci. USA 87, 2220-2224 (1990); Progress in Growth Factor Research 1, 267-280 (1989); National Publication [Tokuhyo] No.
  • the hedgehog (“HH”) family is a group of proteins that has received attention as critical factors in mo ⁇ hogenesis. At present, there at least four types of hedgehog that are present among vertebrates, the sonic hedgehog (Shh), the desert hedgehog (Dhh), the Indian hedgehog (Ihh) and the tiggy-winkle hedgehog (twhh).
  • Shh protein is of particular interest among vertebrates in terms of its function as a neural tube and somite ventralizing factor, and as a factor in determining the antero-posterior axis of limb buds.
  • Bone and cartilage tissues are formed via a continuous process wherein bone and cartilage precursor cells or bone and cartilage invades into the region in which formation is to occur, followed by the growth and differentiation of the bone or cartilage precursor cells or bone and cartilage cells, along with the synthesis of bone and cartilage matrix. This process depends on the size of the region to be generated, but in general, a lengthy period of a few weeks to a few months is required. Consequently, the discovery of a drug that facilitates osteogenesis and chondrogenesis and can rapidly induce the formation of bone and cartilage tissue is strongly desired in order to aid in the recovery of persons needing repair or induction of bone and cartilage tissue.
  • the present invention comprises a drug composition, characterized by containing BMP and hedgehog protein; and specifically, comprises a drug composition used for osteogenesis and chondrogenesis.
  • This composition exhibits much higher levels of osteogenesis and chondrogenesis performance in comparison to conventional drugs for osteogenesis and chondrogenesis containing BMP alone.
  • Figure 1 is a diagram showing the effect of the introduction of Shh-N and BMP-2/-4 genes into osteoblasts and the effect of the products of the introduced genes on osteoblast differentiation based on APase activity in Example 1.
  • Figure 2 is a diagram showing the effect of Shh-N-CM and BMP-2/-4 on osteoblast induction based on APase activity in Example 2.
  • Figure 3 is a diagram showing the effect of Shh-N-CM and rhBMP-2 on osteoblast differentiation based on APase activity in Example 3.
  • Figure 3 A represents the change over time
  • Figure 3B represents the Shh-N concentration dependence
  • Figure 3C represents the rhBMP-2 concentration dependence.
  • Figure 4 is a diagram showing the effect (concentration dependence) of recombinant murine ("rm”) Shh-N and rhBMP-2 on osteoblast differentiation based on APase activity in Example 4.
  • Figure 5 is a diagram showing the effect of Shh-N-CM and BMP-2/-4-CM on the differentiation of RMD-1 cells into chondrocytes in Example 5 using the amount of 35 S- sulfuric acid inco ⁇ orated into proteoglycan as an indicator.
  • Figure 6 is a diagram showing the influence of the Shh-N-CM treatment time on the differentiation of RMD-1 cells into chondrocytes in Example 6 using the amount of 35 S- sulfuric acid inco ⁇ orated into proteoglycan as an indicator.
  • Figure 7 is a diagram showing the effect of rmShh-N and rhBMP-2 on the differentiation of RMD-1 cells into chondrocytes in Example 7 (concentration dependence of rmShh-N), using the amount of 35 S-sulfuric acid inco ⁇ orated into proteoglycan as an indicator.
  • bone mo ⁇ hogenesis proteins any substance can be used without particular restrictions, provided that it is a protein that acts on undifferentiated mesenchymal cells, causing these cells to differentiate into chondrocytes and osteoblasts.
  • BMPs bone mo ⁇ hogenesis proteins
  • human BMP manufactured by recombinant DNA technologies is desirable from the standpoint of producing large quantities of good quality material that is clinically safe from an immunological standpoint, etc.
  • a transformant (cells or microorganism) containing recombinant DNA including the base sequence coding for the human bone mo ⁇ hogenesis protein is cultured, and the recombinant human bone mo ⁇ hogenesis protein produced by these transformants is isolated and purified to prepare the recombinant human bone mo ⁇ hogenesis protein (rhBMP).
  • rhBMPs human bone mo ⁇ hogenesis proteins
  • rhBMP-2 rhBMP-3, rhBMP-4 (also referred to as rhBMP-2B), rhBMP-5, rhBMP- 6, rhBMP-7 (rhOP-1), rhBMP-8, rhBMP-9, rhBMP-12, rhBMP-13, rhBMP-15, rhBMP-16, rhBMP-17, rhBMP-18, rhGDF-1, rhGDF-3, rhGDF-5, rhGDF-6, rhGDF-7, rhGDF-8, rhGDF-9, rhGDF-10, rhGDF-11, rhGDF-12, rhGDF-14.
  • BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7 disclosed in United States Patents 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publication WO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP- 10, disclosed in United States Patent 5,637,480; BMP-11, disclosed in United States Patent 5,639,638, or BMP-12 or BMP-13, disclosed in United States Patent 5,658,882, BMP-15, disclosed United States Patent 5,635,372 and BMP- 16, disclosed in co-pending patent application serial number 08/715,202.
  • compositions which may also be useful include Vgr-2, and any of the growth and differentiation factors [GDFs], including those described in PCT applications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others.
  • GDFs growth and differentiation factors
  • Also useful in the present invention may be BIP, disclosed in WO94/01557; HP00269, disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCT application WO93/16099. The disclosures of all of these applications are hereby inco ⁇ orated herein by reference.
  • Also useful in the present invention are heterodimers of the above and modified proteins or partial deletion products thereof. These proteins can be used individually or in mixtures of two or more, and rhBMP-2 is preferred.
  • the BMP proteins that are used are preferably expressed by mammalian cells (for example, CHO cells), bacteria (for example, E. coli), and yeast cells.
  • mammalian cells for example, CHO cells
  • bacteria for example, E. coli
  • yeast cells for example, rhBMP-2
  • rhBMP-2 is an example of an rhBMP protein for which large-scale production and purification methods have been established
  • the other rhBMP proteins can also be used after manufacturing and purifying them in the same manner (Progress in Growth Factor Research 1, 267-280 (1989)).
  • the known purified rhBMP-2 is a dimeric protein with a molecular weight of about 30,000. Each of the monomers has a high-mannose sugar chain at residue Asn 56 (Abstract Sixth Interactive Symposium of the Protein Society, San Diego CA (1992)).
  • the amount of BMP in the bone and cartilage forming compositions of the present invention can be any concentration at which bone and cartilage induction is manifested, but preferably, when rhBMP-2 is used, the amount is preferably 0.1 ⁇ g/mL or more, with 1 - 1000 ⁇ g/mL being more preferred and 1-300 ⁇ g/mL being additionally desirable.
  • Shh has similar biological activities in terms of their capacity for double limb induction (Science 273, 613-622 (1996)), and that the activity of Shh resides entirely in the N-terminus domain (Shh-N) ((Curr. Biol. 5, 791- 796 (1995), Cell 81, 313-316 (1995)).
  • Shh, Ihh, or the N-terminus domain Shh-N which is the active region of Shh, can be used as the hedgehog protein in the present invention.
  • the hedgehog proteins that have been manufactured by recombinant DNA technologies are preferred from the standpoint of obtaining large quantities of good quality raw material.
  • a transformant containing recombinant DNA that includes the base sequence encoding the hedgehog protein is cultured, and the recombinant hedgehog protein produced by these transformants is isolated and purified to prepare a crude recombinant hedgehog protein.
  • Methods whereby physiologically active recombinant hedgehog protein is produced in large quantities using E. coli have been documented by numerous researchers (Curr. Biol. 5, 791-796 (1995), Nature 375, 322-25 (1995)).
  • the Shh and Ihh genes are highly conserved across all species ranging from chickens to humans (Cell 75, 1401-1416 (1993), Science 273, 613-622 (1996), Cell 75, 1417-1430 (1993), Gene Bank Accession Numbers L38517 and L38518), and so the animal types are unlimited for hedgehog proteins.
  • the molecular weight of the protein is small in comparison to Shh and Ihh, and thus the active N-terminus domain Shh-N of Shh, which is that has similar physiological activity, is preferred.
  • the amount of hedgehog protein contained in the compositions of the present invention can be any concentration at which bone and cartilage induction is manifested, but when Shh-N is used, the amount is preferably 2 ng/mL or greater, with 5 ng/mL-500 ⁇ g/mL being more preferred, and 10 ng/mL to 10 ⁇ g/mL being additionally desirable.
  • the bone and cartilage forming composition of the present invention can be administered systemically or locally as a solution that contains BMP and HH, or the proteins can be held on an appropriate support, and can be administered to the regions where osteogenesis or chondrogenesis is desired.
  • the cartilage forming composition of the present invention can contain components other than those indicated above. Specific examples of these optional components include bio-absorbable supports, stabilizers, preservatives, solubilizers, pH adjusters and thickeners.
  • supplements that are effective in osteogenesis and chondrogenesis can also be included, examples of which include fibronectin and osteonectin.
  • the bone and cartilage forming composition of the present invention is administered by any method to the area at which osteogenesis or chondrogenesis is needed.
  • the composition can be administered by injection, when in the form of a paste or clay, the composition can be administered by implanting, or when held on an appropriate support, the composition can be administered by hardening and molding the material into the desired shape, and then implanting the material as a solid.
  • the therapeutic method includes administering the composition topically, systemically, or locally as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or other connective tissue or other tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than the compositions which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the compositions may be administered using a carrier or matrix which may provide slow release of BMP and/or HH protein, as well as proper presentation and appropriate environment for cellular infiltration. Such matrices may be formed of materials presently in use for other implanted medical applications.
  • compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid and polyanhydrides.
  • Other potential .materials are biodegradable and biologically well defined, such as bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • the carrier is preferably a viscous gel, such as a collagen or polymer.
  • Carriers and matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
  • the bone and cartilage forming composition of the present invention can be used in organisms following methods known in the relevant fields, and to this end, appropriate use can be made depending on the application, region to be treated, and condition of the patient.
  • the bone and cartilage forming compositions of the present invention do not have to be used individually, and these substances can also be used in combination with other known implants.
  • the composition can be used in combination with a collagen film, or a polylactic acid film or Goretex film employing the GTR method. These materials are biocompatible films used for localization and immobilization of the composition.
  • the proteins of the present invention may be delivered through the use of appropriate vectors in gene therapy applications.
  • the vectors encoding both BMP and HH may be transfected into the cells of a patient ex vivo, and the cells may be reintroduced into a patient.
  • the vectors may be introduced into a patient in vivo through targeted transfection.
  • the efficacy of the drug composition of the present invention with respect to osteogenesis and chondrogenesis can be confirmed in the manner described below.
  • the activity with respect to osteogenesis can be evaluated 1) by investigating the degree of osteoblast differentiation and propagation in C3H10T1/2 cells (Growth Factors 9, 57-71 (1993)), an undifferentiated mesenchymal cell line that has been reported to have a capacity for differentiation into osteoblasts, and 2) by investigating the differentiation and manifestation of functionality in primary cultured osteoblasts isolated from the skulls of chicken embryos. In both cases, variation in alkaline phosphatase activity (APase) of the cells is a marker for differentiation, and is used as an indicator.
  • APase alkaline phosphatase activity
  • Activity with respect to chondrogenesis can be evaluated by investigating chondrocyte differentiation in RMD cells, a undifferentiated rat mesenchymal cell line having a capacity for differentiation into chondrocytes (J. Bone Miner. Res. 11, 544-553 (1996).
  • the degree of differentiation into chondrocytes can be measured using the inco ⁇ oration of 35 S-sulfuric acid into the extracellular matrix via the synthesis of cartilaginous macromolecular proteoglycan (GAG) (J. Bone Miner. Res. 11, 544-553 (1996).
  • GAG cartilaginous macromolecular proteoglycan
  • accumulation of cartilaginous macromolecular proteoglycan can be easily determined by staining the cells with Alcian blue at pH 1.0.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the composition, e.g. amount of bone or other tissue weight desired to be formed, the site of bone or tissue damage, the condition of the damaged bone tissue, the size of a wound, type of damaged tissue, the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and the types of BMP proteins in the composition.
  • systemic or injectable administration will be initiated at a dose which is minimally effective, and the dose will be increased over a preselected time course until a positive effect is observed.
  • IGF I insulin like growth factor I
  • Progress can be monitored by periodic assessment of bone or tissue growth and/or repair. The progress can be monitored, for example, x-rays, histomo ⁇ hometric determinations and tetracycline labeling.
  • Shh cDNA was recovered by the RT-PCR method from the total RNA of stage 25 chicken embryos. Next, the region coding for the 200 amino acids at the N-terminus was amplified by PCR while inserting a stop codon at the 200th residue. The resulting DNA fragment was subjected to terminal blunting, and after ligating a Cla I adaptor, the fragment was subcloned into the chicken retrovirus vector RCAS (A) (J. Virol. 61, 3004-3012 (1987)).
  • This viral DNA was then transfected using calcium phosphate into chicken embryo fibroblast (CEF) cells that were prepared from 11 -day virus-free eggs (M egg line, Nisseiken, Yamanashi) (Mol. Cell. Biol.7, 2745-2752 (1987)).
  • CEF cells were cultured in medium 199 (GIBCO, Gaithersburg MD, USA) containing 10% fetal bovine calf serum (FBS; Cell Culture Laboratories OH, USA).
  • the culture supernatant from the CEF cells transfected with the viral DNA contained Shh-N recombinant virus and chicken Shh-N protein.
  • the viral particles present in the culture supernatant that was collected were concentrated by ultracentrifugation according to the method of Cepko et al. (Mol. Cell. Biol. 13, 2604-2613 (1993)), and were subjected to infectivity experiments.
  • the culture supernatant containing the chicken N-Shh protein (Shh-N-CM) was then used in a virus-free mouse or rat cell culture system.
  • Culture supernatant from CEF cells containing the viral vector alone (RCAS-CM) was used as a control. Preparation of mouse recombinant N-Shh (rmShh-N)
  • Mouse Shh cDNA containing the entire length of the coding sequence was obtained by RT-PCR from the total RNA of 10-day mouse embryos.
  • the 73-651 base sequence region of the mouse Shh cDNA sequence based on the recording of MacMahon (Gene Bank Accession Number X76290, Cell 75, 1417-1430 (1993)) was amplified using a primer pair containing the Sac I site. At this time, a stop codon was inserted after the base 851 of the sequence.
  • the resulting DNA fragment was then digested with Sac I, and was subcloned into the pQE30 plasmid using the QIA Express Kit (QIA Express Kit, Qiagen, GmbH, Germany). The plasmid was then transformed into E.
  • Human BMP-2 cDNA containing the entire coding sequence was cloned from the total RNA of human osteosarcoma cells using the RT-PCR method. This DNA fragment was sub-cloned into sub-group A and B RCAS vectors (RCAS(A), RCAS(B)) (J. Virol. 61, 3004-3012, (1987)) using the same method that was used in constructing the chicken N-Shh recombinant virus as described above.
  • hBMP-2-CM The culture supernatant containing the hBMP-2 protein (hBMP-2-CM) was then used in a virus-free mouse or rat cell culture system.
  • Mouse BMP-4 cDNA containing the entire coding sequence was cloned out of the total RNA of 16-day mouse embryos using the RT-PCR method. This DNA fragment was used, and was subcloned into subgroup A and B RCAS vectors (RCAS(A), RCAS(B)) (J. Virol. 61, 3004-3012, (1987)) using the same method that was used in constructing the chicken N-Shh recombinant virus as described above. The culture supernatant from CEF cells transfected with the viral DNA was then concentrated and was used in infectivity testing. Culture supernatant containing mBMP-4 protein (mBMP-4-CM) was then used in a virus-free mouse or rat cell culture system.
  • mBMP-4-CM mBMP-4 protein
  • Chicken embryo undifferentiated fibroblast (CEF) cells were isolated from 11-day virus-free embryos, and were cultured in Medium 199 (GIBCO, Gaithersburg MD) containing 10% fetal bovine serum (FBS, Cell Culture Laboratories, OH USA).
  • the mouse fibroblast ceil strain C3H10T1/2 Clone 8 was received from the Riken Cell Bank, and was subcultured with Eagle's Minimal Essential Medium (MEM, Nissui, Tokyo) containing 10% FBS).
  • the rat undifferentiated fibroblast strain RMD-1 was subcultured in Ham's F-12 medium containing 10% FBS.
  • the chondrocyte differentiation tests for the RMD-1 cells were carried out by culturing in Ham F-12 containing 5% FBS.
  • the Shh-N and BMP genes were introduced into osteoblast cells using the recombinant viruses, and the effects of the products of the introduced genes on osteoblast differentiation were investigated.
  • Primary culture osteoblast cells were isolated from the skulls of 18-day virus-free chickens according to the method of Gerstenfeld et al. (Dev. Biol. 122, 49-60 (1987)). Culturing was carried out with alphaMEM [sic] containing 10% FBS.
  • the cells were introduced into culture plates, and after 2 h, chicken Shh-N recombinant virus (Shh-N-RCAS(A)), human BMP-2 recombinant virus (BMP-2-RCAS(B)) and mouse BMP- 4 recombinant virus (BMP-4-RCAS(B) were added to the media.
  • Cells were infected with individual viruses, or were double-infected with the different subcloned Shh virus and BMP virus. Controls were produced that consisted of a group that was left uninfected (none) and a groups that were infected with vector alone (RCAS(A), RCAS(B)). After 6 days, the cells were harvested using trypsin.
  • the remainder of the cells were introduced into 48-well plates (Corning NY, USA) at 30,000 cells/well, and culturing was continued for 7 days.
  • the culture medium (CM) was changed every 3 days. After completion of culturing, the APase activity was measured according to the method of Pacifici et al. (Exp. Cell Res. 195, 38-46 (1991)).
  • cells were lysed by sonication in 0.2% Triton X-100 and 0.9% NaCl, and colorimetric determination was carried out based on the paranitrophenol produced when the supernatant was added to a reaction liquid containing paranitrophenol-2-phosphoric acid as the substrate.
  • the amount of DNA from the cells was measured by a fluorescent dye method (Anal. Biochem. 122, 338-344, Exp. Cell Res. 195, 38-46 (1991)).
  • the APase activity is expressed in the figures with 1 unit representing the activity that generates 1 nmole of paranitrophenol in 1 min from 1 ⁇ g DNA.
  • the results are shown in Figure 1. Only 1 unit ⁇ g DNA or less of APase activity was detected in the None, -RCAS(A) and RCAS(B) groups.
  • the cells that had been infected with Shh-N-RCAS(A), BMP-2-RCAS(B) or BMP-4-RCAS (B) showed an increase in APase activity of 2-3 times in comparison to the control groups.
  • Shh-N-CM and BMP-2/-4-CM were investigated.
  • C3H10T1/2 cells were introduced into 48-well plates (Corning, NY USA) at 30,000 cells per well, and after 24 h, Shh-N-CM, hBMP-2-CM and mBMP-4-CM were added to a concentration of 25%. Culturing was carried out for 7 days. The culture liquid and CM were changed every 3 days, and after culturing, the APase activity was measured following the same method as in Example 1.
  • C3H10T1/2 cells were introduced into 48-well plates (Corning NY, USA) at 30,000 cells per well, and after 24 h, Shh-N-CM was added at a concentration of 25%, recombinant human BMP-2 (rhBMP- 2, obtained from Yamanouchi Seiyaku) was added in the amount of 1 ⁇ g/mL, or both were added, and culturing was carried out for 7 days.
  • the APase activity was measured by the same method as in Practical Example 1 on the 2nd, 4th and 7th days of culturing. The results are shown in Figure 3A.
  • C3H10T1/2 cells were introduced into a 48 well plate (Corning, NY USA) at 30,000 cells per well, and after 24 h, rmShh-N was added in concentrations ranging from 10 ng/mL to 10 ⁇ g/mL in the presence and absence of 1 ⁇ g/mL of rhBMP-2, and culturing was carried out for 7 days. After completion of culturing, the APase activity was measured by the same method as in Example 1.
  • Shh-N stimulated APase activity in accordance with concentration in the same manner as Shh-N- CM, and clear synergistic effects were seen in the presence of BMP-2.
  • RMD-1 cells were introduced at 400,000 cells/well into 24-well plates (Iwaki Garasu, Chiba) coated with type I collagen, and HAM F-12 medium containing 5% FBS was added. After 24 h, Shh-N-CM, hBMP-2-CM and mBMP-4-CM were added individually or in conjunction to a concentration of 25%, and culturing was carried out. The medium was changed after 48 h, and culturing was then continued without adding CM.
  • the amount of proteoglycan synthesis was measured by the inco ⁇ oration of 35 S-sulfuric acid into the cetyl pyrimidine chloride [sic; probable error for "cetyl pyridinium chloride”] precipitate fraction (J. Bone Miner. Res. 11, 544-553, (1996)). Specifically, 1.5 ⁇ Ci of 35 S-sulfuric acid was added per well, and labeling was carried out by incubation for 6 h. The amount of 35 S- sulfuric acid inco ⁇ orated into the proteoglycan of the cell layer and the amount contained in the culture supernatant were then measured.
  • RMD-1 cells were introduced at 400,000 cells/well into 24-well plates (Iwaki Garasu, Chiba) coated with type I collagen, and HAM F-12 medium containing 5% FBS was added. After 24 h, culturing was canied out while treating with 25% Shh-N-CM for 2 days, 4 days or 6 days in the presence and absence of 200 ng/mL of rhBMP-2.
  • the synthesis of proteoglycan which is an indicator of chondrocyte differentiation, was measured by the inco ⁇ oration of 35 S-sulfuric acid into the cetyl pyridinium chloride precipitate fraction.
  • RMD-1 cells were introduced at 400,000 cells/well into 24- well plates (Iwaki Garasu, Chiba) coated with type I collagen, and HAM F-12 medium containing 5% FBS was added. After 24 h, concentrations of rmShh-N ranging from 10 ng/mL to 10 ⁇ g/mL were added in the presence or absence of 200 ng/mL of rhBMP-2, and culturing was carried out. After 48 h, the culture medium was changed, whereupon culturing was continued for an additional 7 days without adding recombinant protein.
  • proteoglycan which is an indication of chondrocyte differentiation, was measured based on the inco ⁇ oration of 35 S-sulfuric acid into the cetyl pyridinium chloride precipitate fraction.
  • rmShh-N increased proteoglycan synthesis in accordance with increasing concentration, and remarkably high levels of proteoglycan synthesis were seen in the presence of BMP-2. ( Figure 5)
  • the implants of the present invention can rapidly induce osteogenesis and chondrogenesis in vivo, and can form (regenerate) bone and cartilage tissue. Consequently, the compositions can be used in patients in accordance with methods known in the relevant fields in order to repair various types of bone and cartilage loss resulting from injury, illness or congenital condition.
  • the transplant of the present invention can be used in a number of fields, for example, for repairing areas deficient in bone or cartilage resulting from accident, disease, congenital condition or various types of surgery, for accelerating the healing of various types of bone fractures, for forming bone around implants such as artificial teeth roots, for -accelerating the fixation of artificial implants, for accelerating spine fusions and for regenerating or filling-in bone or cartilage in the field of orthopedic medicine, for example, in leg elongations.
  • the invention can also be used in the field of plastic surgery in order to fill in bone or cartilage, or in the field of dentistry for repairing cavities, for regenerating alveolar bones, for increasing bone mass to repair cementum and for use with implants.

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Abstract

Composition médicamenteuse caractérisée par le fait qu'elle contient des protéines morphogéniques osseuses (BMP) et des protéines du hérisson et, plus particulièrement, composition médicamenteuse favorisant l'ostéogenèse et la chondrogenèse et exerçant des effets d'ostéogenèse et de chondrogenèse supérieurs à ceux qu'on obtient avec des médicaments classiques servant à développer les os et les cartilages et contenant les protéines morphogéniques osseuses (BMP) seules.
PCT/JP1998/000112 1997-01-14 1998-01-14 Compositions favorisant l'osteogenese et la chondrogenese WO1998030234A1 (fr)

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AU54955/98A AU5495598A (en) 1997-01-14 1998-01-14 Compositions for osteogenesis and chondrogenesis

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JP9004936A JPH10194987A (ja) 1997-01-14 1997-01-14 骨・軟骨形成用組成物
JP9/4936 1997-01-14

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EP1002547A1 (fr) * 1998-09-01 2000-05-24 F. Hoffmann-La Roche Ag Complex ionique hydrosoluble de la protein Hedgehog et son utilisation pharmaceutique
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Cited By (21)

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US6767888B1 (en) 1997-06-27 2004-07-27 Curis, Inc. Neuroprotective methods and reagents
US7144997B2 (en) 1997-07-24 2006-12-05 Curis, Inc. Vertebrate embryonic patterning-inducing proteins, compositions and uses related therto
US7138492B2 (en) 1997-07-24 2006-11-21 Curis, Inc. Method of treating dopaminergic and GABA-nergic disorders
US6639051B2 (en) 1997-10-20 2003-10-28 Curis, Inc. Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto
EP0978285A1 (fr) * 1998-08-07 2000-02-09 F. Hoffmann-La Roche Ag Compositions pharmaceutiques contenant des protéines Hedgehog, et leur utilisation
US6207718B1 (en) 1998-08-07 2001-03-27 Ontogeny, Inc. Pharmaceutical compositions containing hedgehog protein
AU761341B2 (en) * 1998-08-07 2003-06-05 Curis, Inc. Pharmaceutical composition of hedgehog proteins and use thereof
EP0987028A1 (fr) * 1998-08-07 2000-03-22 F. Hoffmann-La Roche Ag Compositions pharmaceutiques comprennant des hedgehog-proteines et leur utilisation
CN1325118C (zh) * 1998-09-01 2007-07-11 霍夫曼-拉罗奇有限公司 离子复合体形式的水溶性药物组合物及其应用
US8519098B2 (en) 1998-09-01 2013-08-27 Hoffmann-La Roche Inc. Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof
SG85670A1 (en) * 1998-09-01 2002-01-15 Hoffmann La Roche Water-soluble pharmaceutical composition in an ionic complex and the use thereof
US6867182B2 (en) 1998-09-01 2005-03-15 Hoffmann-La Roche Inc. Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof
EP1002547A1 (fr) * 1998-09-01 2000-05-24 F. Hoffmann-La Roche Ag Complex ionique hydrosoluble de la protein Hedgehog et son utilisation pharmaceutique
US6884770B1 (en) 1998-11-06 2005-04-26 Curis, Inc. Methods and compositions for treating or preventing peripheral neuropathies
US6890897B1 (en) 1999-01-28 2005-05-10 Scil Technology Gmbh Use of a melanoma inhibiting activity factor (MIA) for cartilage and bone repair
EP1025871A1 (fr) * 1999-01-28 2000-08-09 F. Hoffmann-La Roche Ag Utilisation d'un facteur à activité inhibitrice du mélanome pour la réparation de cartilage et des os
WO2000044401A1 (fr) * 1999-01-28 2000-08-03 Hoffmann La Roche Utilisation d'un facteur d'activite inhibitrice de melanome (mia) pour la reparation de cartilages et d'os
AU779472B2 (en) * 1999-07-28 2005-01-27 Interface Biotech A/S In vitro repair of bone and/or cartilage defects
WO2001006949A3 (fr) * 1999-07-28 2001-05-10 Interface Biotech As Reparation in vivo de defauts osseux et/ou du cartilage
WO2001006949A2 (fr) * 1999-07-28 2001-02-01 Interface Biotech A/S Reparation in vivo de defauts osseux et/ou du cartilage
WO2003011343A1 (fr) * 2001-07-27 2003-02-13 National Institute Of Advanced Industrial Science And Technology Technique de regeneration de tissus osseux/cartilagineux par un transfert du gene de facteur de transcription

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JPH10194987A (ja) 1998-07-28

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