MXPA99003229A - Use of a combination of an osteoinductive protein and a dorsalizing factor for cartilage induction - Google Patents

Abstract

A composition of an osteoinductive protein and a dorsalizing factor at a ratio of 10:1 to 1:10 is a useful pharmaceutical agent for chondrogenesis.

Description

DORSALIZATION FACTOR FOR CARTILAGE INDUCTION Description of the invention: The present invention relates to a method and composition for the induction of chondrocytes from mesenchymal stem cells using a combination of an osteoinductive protein with a dorsalation factor. Osteoinductive proteins are proteins that induce the differentiation of mesenchymal stem cells into chondrocytes and osteocytes and are, for example, hedgehog proteins (sonic (Shh), Indian (Ihh), desert (Dhh), Kinto et al., FEBS Letters 404 (1997) 319-323), or morphogenetic proteins »osseous. Bone morphogenetic proteins (BMPs) are molecules that are responsible for the formation of bones, cartilage, tendons and other tissues present in bones. The only inductive activities of these proteins during their presence in bone suggest that they are important regulators of bone repair processes and may be involved in the normal preservation of bone tissue. Many of these proteins are known and can be divided into several sub-families (Reddi, A.H., Cytokine &Growth Factor Revie s ("Journal of Cytokines and Growth Factor") 8 (1997) 11-20). Said BMPs are, for example, BMP-2 to BMP-14 and GDF-1 to GDF-14. BMPs are pleiotropic regulators, and therefore regulate the sequential cascade in multiple steps of bone and cartilage formation such as qui iotaxis, mitosis and differentiation. Especially, BMP-2, BMP-4, BMP-5, BMP-7, initiate chondrogenesis and osteogenesis. ref. 29815 However, BMP-2, BMP-4, BMP-5 and BMP-7 are not able to induce chondrogenesis alone without the simultaneous induction of osteogenesis. Thus, the pharmaceutical application of BMP-2, BMP-4, BMP-5 and BMP-7, in connection with endochondral bone formation will always result in chondrocytic and osteocytic induction. In connection with cartilage tissue repair, however, it would be advantageous to achieve the initiation of chondrogenesis without the simultaneous induction of osteogenesis. Antagonists of bone morphogenetic protein are substances that inhibit the osteogenic properties of BMP, and are, for example, noguine, cordin or follistatin. Noguina is an antagonist of the growth factor and of a bone morphogenetic protein (BMP), which is described p. ex. in US Patent No. 5,670,481. The noguina is expressed in the Spemann organizer and appears to be a mediator of the effects of the Spemann organizer, namely the neural induction and the dorsalization of the mesoderm. Given that noguina is expressed in the notocordium and cephalic mesoderm, it seems that noguina influences either the dorsal-ventral model or the antero-posterior model of the neural plate. Since noguina is expressed on the crest of the branchial neural arch, it appears that noguina influences either the cartilage deposit of the cells of the neural crest and also influences the growth and remodeling of the last branchial arch. The noguina is expressed in the neural crest of the tail fin, and since the neural crest is necessary for the growth of the fin, noguina can act as a growth factor for the epidermis or mesenchyme. Noguina can bind to BMP-4 with a high affinity (Piccollo et al., Cell 86 (1996) 12141-12145) and is an antagonist of BMPs (Re'em-Kalma et al., Proc. Nati. Acad.

Sci. USA 92 (1995) 12141-12145; Sasai et al., Nature 376 (1995) 333-336; Fainsod et al., Mechanisms of Development ("Mechanisms of Development") 63 (1997) 39-50). Cordina is a segregated polypeptide and a potent dorsalizing and neural inducing factor (US Patent No. 5,679,783, Sasai et al., Cell 79 (1994) 779-790). The cordina is expressed in the Spemann organizer. The overexpression of cordina induces the notocordic and neural tissue, but not the mesoderm. However, the cordina can modify the specification of the mesoderm. The expression of cordin overlaps with noguine and can act in a similar way to noguine. The cordina acts antagonistically to BMP-4 (Fainsod et al., EMBO 13 (1994) 5015-5025) and can bind to BMP-4 with high affinity (Picollo et al., Cell 86 (1996) 589-598). Follistatin is a segregated polypeptide and is expressed in the Spemann organizer and acts as a dorsalizing and neural inducing factor, and is originally described as an activin binding protein and an inhibitor of activin function (Na amura et al., Science 247 (1990) 836-838; Hemmati-Brivanlou et al., Cell 79 (1994) 169-179). Follistatin can also bind BMPs (Fainsod et al., Mechanisms of Development) 63 (1997) 39-50). In the case of cartilage defects, only limited success has been achieved. The standard procedures are shaving of fibrillated articular cartilage, debridement, spongiolization, diamond abrasion, osteotomies, microfracture and cartilage grafting techniques such as perichondrial autografts, periosteal autografts. A simple and effective treatment does not yet exist (see review by Heath and Margari, Biotechnology and Bioengineering (50) (1996) 430-437). New therapies could include chondrocyte transplantation, growth factor therapy, cell therapy (US Patent No. 5,486,359), gene therapy, new biomaterials. Normally they are not yet available. The invention provides a method for cartilage repair, employing a composition of an osteogenic factor and an antagonist of bone morphogenetic protein.

Surprisingly it was found that osteoinductive (osteogenic) proteins, preferably morphogenetic proteins from bone 2, 4, 5 and 7 or hedgehog protein are not completely inhibited in vivo by BMP antagonists such as noguine, cordin and follistatin, when they are present in a ratio of 10: 1 to 1:10 (BMP: noguina). In this, the initiation of osteogenesis is however inhibited on a large scale while chondrogenesis is hardly affected. For an effect in accordance with the present invention it is essential that the BMP antagonist possess a high binding affinity for BMP-2, BMP-4, BMP-5 and BMP-7.

By "oso-inducing protein" is meant preferably an osteogenic protein that induces BPM-dependent osteogenesis based on mesenchymal stem cells. The BMPs are therefore upregulated, which ultimately leads to the formation of chondrocytes. This induction of BPM-dependent osteogenesis can be achieved, for example, through the same BPM or through substances that induce the expression of BMP in cells, e.g. ex. the hedgehog proteins. The ability of a substance to induce BMP-dependent osteogenesis can be determined in a simple manner. For this, for example, mesenchymal cells, p. ex. C3H10T1 / 2 cells, are cultured with and without the potentially osteoinductive factor. The controls and the treated cells are measured to detect the activity of osteocalcin and alkaline phosphatase. Osteocalcin can be determined by commercially available ELISA analysis, e.g. ex. from Dako Co. Alkaline phosphatase can be measured photometrically using a suitable colorimetric substrate, e.g. ex. p-nitrophenyl phosphate. An increased activity of osteocalcin and / or alkaline phosphatase is considered as osteoinduction. Alternatively, overregulation of osteocalcin and alkaline phosphatase is measured by RT-PCR using the appropriate primers for osteocalcin and alkaline phosphatase. By "dorsalization factor", according to the invention, is meant a substance, preferably a protein, which likewise an antagonist of a bone morphogenetic protein, inhibits the osteoinductive property of the BPM, preferably the BPM- 2 or hedgehog. Such inhibition can be determined, for example, in an assay with a bone marrow stromal cell line (Zimmermann et al., Cell 86 (1996) 599-606). A dorsalization factor has the property of promoting the formation of dorsal structures in Xenopus. The dorsalizing activity can be determined by means of a shell test of an Xe-nopus animal. The animal shell test Xenopus (Ruiz and Altaba, Essential Developmental Biology, A Practical Approach ("Essential Development of Biology, a Practical Approach"), IRL Press, 1993, IRL Press, pp. 147-152; Lamb et al. ., Science 262 (1993) 713-718) is used to determine neural induction. After treatment, the animal caps are stained for the presence of neural markers > p. ex. NCAM, and the absence of mesodermal markers, p. ex. muscle actin These factors are, for example, noguine, cordin and follistatin (Fainsod et al., "Mechanisms of Development") 63 (1997) 39-50 BMP antagonists are described, for example, by Re. 'em-Kalma et al., Proc. Nati, Acad. Sci. USA 92 (1995) 12141-12145; Sasai et al., Nature 376 (1995) 333-336; and Fainsod et al., Mechanisms of Development (" Mechanisms of Development ") 63 (1997) 39-50 In a later preferred version of the invention, the osteogenic protein and the dorsalization factor can be introduced into cells via gene therapy, for this method the genes encoding the osteogenic protein and the dorsalization factor can be introduced into a vector, preferably under the control of the same promoter, or in separate vectors The invention thus describes a method for the preparation of a pharmaceutical composition for the treatment of a patient in need of repair of a cartilage, characterized in that an expression vector capable of expression of an osteoinductive protein, and a dorsalization factor, or a combination of a vector capable of the expression of an osteoinductive protein and a vector capable of the expression of a dorsalization factor, is employed as an essential component of said composition. For an efficient expression of the osteogenic factor and the dorsalization factor, it is necessary to use potent promoters in the vectors. These promoters are p. ex. , promoters PGK or CMV. Preferably, the expression vector consists of this potent promoter, the mRNA of the total length of the chosen gene, e.g. ex. , BPM-2, BPM-4, BPM-5, BPM-7, Shh, Ihh, or Dhh, noguina, cordina, or follistatin, an artificial intron and a poly-A site. For the application, the DNA is either lyophilized in collagen sponges, or it is applied with any other support, preferably hyaluronic acid or alginate. The pharmaceutical formulation according to the invention may also include an appropriate matrix, for example, for the delivery and / or support of the composition and / or to provide a surface for cartilage formation. The matrix can provide the slow release of the osteoinductive protein and the antagonist of the bone morphogenetic protein. Preferably the composition includes a matrix which is biocompatible and / or biodegradable. Potential matrices for the compositions contain, for example, hyaluronic acid, alginate, calcium sulfate, tricalcium phosphate, hydroxylapatite, polylactic acid, polyanhydrides, or collagen. The dosage regimen will be determined by the physician attending the patient, taking into consideration several facts that modify the action of the formulation of the invention. Factors that may modify the action of the formulation include, the amount of cartilage to be formed, site of application, condition of the lesion, age of the patient, sex and diet, the severity of any infection, duration of administration and other clinical factors. The dosage can vary with the type of matrix used in the reconstitution of the cartilage. Another object of the invention is a method for the preparation of a pharmaceutical composition containing a bone morphogenetic protein and a bone morphogenetic protein antagonist, in a ratio of 10: 1 to 1:10, by combining said protein and said antagonist, and the use of said pharmaceutical composition for chondrogenesis in vivo, especially for the treatment of a patient suffering from defects in the cartilage and therefore needs a cartilage repair. The following examples and references are provided for a better understanding of the present invention, the true purpose of which is described in the appended claims. It is understood that modifications can be made to the described methods without departing from the spirit of the invention. EXAMPLE 1 Micromass Assay of the Mouse Leg Primordia of mouse Ell (NMRI) embryo legs are isolated using microdissection scissors and watchmaker's forceps under sterile conditions. A cell suspension is obtained by rinsing and incubating in calcium and magnesium-free saline solution, then incubating for 20 minutes in 1% trypsin solution at 37 ° C, followed by disintegration by repeated aspiration with a 1 ml pipette. The cells are adjusted to 2 × 10 7 per ml in Ham medium and five aliquots of 10 μl are introduced into sterile Petri dishes. After 2 hours of culture, 2 ml of Ham's medium alone, or Ham's medium is added together with the test compound, which consists of: (a) the BMP antagonist, noguine, cordin or follistatin alone, ( b) BMP-2, BMP-4, BMP-5, BMP-7 or Shh, and (c) combinations of BMP-2, BMP-4, BMP-5, BMP-7 or Shh, and the antagonist in various doses from less than 10 ng / ml to approximately 100 μg / ml. The medium is not changed during a period of five days of cultivation. The cultures are then fixed and stained with Alcian blue or crystal violet, washed with running water and dried (Steele and Copping, Post-implantation Mammalian Embryos, A Practical Approach ("Post-implantation of mammalian embryos, a practical approach"), IRL Press 1990,229-232). Differentiation is determined after dyeing, with Alcian blue for chondrogenesis and with crystal violet for fibroblast-like cells. In addition, quantitative RT-PCR analyzes are performed to determine cartilage expression (eg collagen II, MIA, collagen X) and bone markers (eg osteocalcin, phospha-alkaline rate, osteopontin). Example 2 Culture of chondrocytes For articular chondrocytes isolated from mice of 2 months of age (NMRI), the tibial plate of the knee joint is surgically removed, and the articular tissue 1 is carefully released from adhering contaminating tissues, including the fibrocartilage layer opposite the synovial cavity. The tissue is comminuted with a scalpel, incubated for 16 hours in a mixture of 0.25% trypsin and 0.1% crude collagenase in Hank's buffered saline. Cells are grown for nine days in Dulbecco's glucose-rich and serum-free modified medium (DMEM), in the form of a monolayer or as agarose cultures (three-dimensional matrix) (d'Angelo and Pacifici, J. Bone and Min. Res. 12 (1997) 1368-1377). The cells are treated with (a) the BMP antagonist alone, (b) the induction or modification factor alone, and (c) combinations of the induction or modification factor and the antagonist, in different doses, from a dose lower than 10 ng / ml up to approx. 100 μg / ml. After nine days of treatment, quantitative RT-PCR analyzes are performed to determine the expression of early cartilage proliferative markers (eg, collagen II, MIA), late markers of mature cartilage (eg, collagen X) ), and the fibrous tissue marker (eg, collagen I). Example 3 Mouse bioassay, to determine the induction of cartilage, bone, tendon and ligament. Similar to the rat ectopic implant test of Sampath and Reddi, a mouse ectopic implant test was performed using non-consanguineous NMRI mice or consanguineous C3H mice, 2 months of age (Sampath and Reddi, Proc. Natio. Acad. Sci. USA 80 (1983) 6591-6595, International Application No. WO 95/16035). The (a), BMP antagonist alone, (b), induction factor or modification alone, and (c), combinations of the induction or modification factor and the antagonist, in the appropriate buffer, are lyophilized. ex. 0.1% trifluoroacetic acid for BMP proteins. For the combinations of factors, buffers are chosen according to stability. Any suitable support can be used, p. ex. a type I collagen matrix, collagen-heparin mixture, gelatin capsules, hyaluronic acid or other functionally equivalent device, based on biocompatibility, biodegradability, stability, and mechanical properties. The implants fit intramuscularly in the muscle of the hind quarter of the mouse for 7 and 14 days. After 7 and 14 days the mice are sacrificed by cervical dislocation. Implants are isolated and processed using standard histological techniques (see Theory and Practice of Histological Techniques, eds. Bancroft and Stevens, Churchill Livingstone 1996). Sections of paraffin (8 μm) or glycolmethacrylate (1 μm) are stained with toluidine blue, Alcian blue, von Kossa, Movat or hematoxylin / eosin to visualize and quantify the amount of tendon, ligament, cartilage and bone tissue induced in each implant. Positive implant control groups (eg, BMP-2, GDF-5) and negative (eg, simulator device) are compared with experimental implants. To determine the quality of cartilage and / or induced bone, gene expression can be studied by in situ hybridization of RNA and by quantitative RT-PCR assay for cartilage and bone markers, as described above. EXAMPLE 4 Repair Model of Articular Cartilage at Full Thickness A lack model of the total thickness of the articular cartilage in the femoral-patellar joint of adult rabbits is used to determine the capacity of the BMP antagonist combinations of the induction factor. or modification and support, to affect the repair of cartilage and bone. Adult rabbits are anesthetized and prepared for sterile surgery. A hole up to 4 4 mm is made through the articular cartilage and into the subchondral bone below, in the patellar or patellar canal of the knee joint. The orifice is left either empty, either filled with the appropriate support, or filled with a support containing the BMP antagonist and the factor of induction or modification of each factor, alone. The animals are allowed to move freely for four weeks. After this time the animals are sacrificed without causing suffering and the place of lack of articular cartilage / subchondral bone, is evaluated histologically to determine the tissue argument, quantity and quality of the repair. EXAMPLE 5 Repair Model of Articular Cartilage to Partial Thickness A model of articular cartilage deficiency to partial thickness in the femoral-patellar joint of adult rabbits is used to determine the capacity of the BMP antagonist combinations of the factor of induction or modification, and of the support, to affect the repair of cartilage and bone. Adult rabbits are anesthetized and prepared for sterile surgery. An orifice of up to 4 x 4 mm is made through the articular cartilage within the patellar or patellar canal of the knee joint, leaving the subchondral bone below. The orifice is left either empty, or filled with the appropriate support, or filled with a support containing the BMP antagonist and the induction or modification factor, or with each factor alone. The animals are allowed to move freely for four weeks. After this time, the animals are sacrificed without causing suffering and the place of lack of articular cartilage is evaluated histologically to determine the tissue argument, quantity and quality of the repair. Example 6 Bioassay with rabbits to determine the induction of cartilage, bone, tendon and ligament, by using DNA Similar to the ectopic implant test in rats of Sampath and Reddi, an ectopic implant test on mouse is performed, employing p . ex. NMRI non-consanguineous mice or consanguineous C3H mice, 2 months of age (Sampath and Reddi, Proc. Nati. Acad. Sci. USA 80 (1983) 6591-6595; International Application No. WO 95/16035). Expression vectors for: (a) dorsalization factor, alone, (b) osteoinduction factor, alone, and (c) combinations of both factors, are lyophilized in the appropriate buffer, p. ex. , TE buffer (Fang et al., Proc. Nati, Acad. Sci. USA 93 (1996) 5773-5758). Any suitable support, e.g. ex. , a type I collagen matrix, collagen-heparin mixture, gelatin capsules, hyaluronic acid, or other functionally eguivalent device, based on biocompatibility, biodegradability, stability and mechanical properties. The implants are performed intramuscularly in the muscle of the hind quarter of the mouse for 7 and 14 days. After said time, the mice are sacrificed by cervical dislocation. The implants are isolated and processed using standard histological techniques (see Theory and Practice of Histological! Techniques ("Theory and Practice of Histological Techniques"), ed., Bancroft and Stevens, Churchill Livingstone, 1996). Sections of paraffin (8 μm) or glycolmethacrylate (l μm) are stained with toluidine blue, Alcian blue, von Kossa, Msvat or hematoxylin / eosin to visualize and quantify the amount of tendon, ligament, cartilage or bone tissue induced in each implant . Positive implant control groups (eg, BMP-2, GDF-5) and negative (eg, simulator device) are compared with experimental implants. To determine the quality of cartilage and / or induced bone, gene expression can be studied by in situ hybridization of RNA and a quantitative RT-PCR assay for cartilage and bone markers, as described above. List of references d'Angelo and Pacifici, J. Bone and Min. Res. 12 (1997) 1368-1377 Fainsod et al., EMBO 13 (1994) 5015-5025 Fainsod et al., Mechanisms of Development ("Mechanisms of Development "), 63 (1997) 39-50 Fang et al., Proc. Nati Acad. Sci. USA 93 (1996) 5753-5758 Heath and Margari, Biotechnology and Bioengineering ("Biotechnology and Bioengineering") 50 (1996) 430-437 Hemmati-Brivanlou et al. , Cell ("Cell") 79 (1994) 169-179 Kinto et al., FEBS Letters 404 (1997) 319-323 Lamb et al., Science ("Science") 262 (1993) 713-718 Nakamura et al. , Science ("Science") 247 (1990) 836-838 Piccollo et al., Cell ("Cell") 86 (1996) 12141-12145 Piccollo et al., Cell ("Cell") 86 (1996) 589-598 Re'em-Kalma et al., Proc. Nati Acad. Sci USA 92 (1995) 1214. 1-12145 Reddi, A.H., Cytokine & Grs th Factor Reviews ("Cytokine Magazines &Growth Factor"), 8 (1997) 11-20 Ruiz i Altaba, Essential Developmental Biology, A Practical Approach, ("Essential Development of Biology, a Practical Approach"), IRL Press, 1993, pp. 147-152. Sampath and Reddi, Proc. Nati Acad. Sci. USA 80 (1983) 6591-6595 Sasai et al., Cell ("Cell") 79 (1994) 779-790 Sasai et al., Nature ("Nature") 376 (1995) 333-336 Steele and Copping, Postimplantation Mammalian Embryos, A Practical Approach ("Postimplantation of mammalian embryos, a practical approach"), IRL Press 1990, 229-232 Theory and Practice of Histological Techniques ("Theory and practice of histological techniques"), ed. Bancroft and Stevens, Churchill Livingstone 1996 US Patent 5,486,359 US Patent 5,670,481 US Patent 5,679,783 WO 95/16035 Zim ermann et al., Cell ("Cell") 86 (1996) 599-606 It is noted that with In relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (13)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A pharmaceutical composition, characterized in that it contains a combination of an osteoinductive protein and a dorsalization factor, in a ratio of 10: 1 to 1:10.

2. A pharmaceutical composition as claimed in claim 1, characterized in that the osteoinductive protein is BMP-2, BMP-4, BMP-5, BMP-7, or a hedgehog protein.

3. A pharmaceutical composition as claimed in claim 1 or 2, characterized in that the dorsalization factor is noguine, cordina or follistatin.

4. A pharmaceutical composition as claimed in claims 1 to 3, characterized in that the composition includes a biocompatible matrix.

5. A pharmaceutical composition as claimed in claims 1 to 4, characterized in that the biocompatible matrix is hyaluronic acid, alginate or collagen.

6. A method for the preparation of a pharmaceutical composition containing an osteoinductive protein and a dorsalization factor, characterized in that the ratio of the composition and said factor is 10: 1 to 1:10, by combining said protein and said dorsalization factor.

7. A method as claimed in claim 6, characterized in that the osteoinductive factor is BMP-2, BMP-4, BMP-5, BMP-7, or a hedgehog protein.

8. A method as claimed in claim 6 or 7 characterized in that the dorsalization factor is noguine, cordina or follistatin.

9. A method as claimed in claims 6 to 8, characterized in that the osteoin-ductor.a protein and the dorsalizing factor are combined with a bipcompatible matrix.

10. A method as claimed in claim 9, characterized in that the biocompatible matrix is hyaluronic acid, collagen or alginate.

11. The use of a combination of an osteoinductive protein and a dorsalization factor, for the treatment of a patient to whom it is necessary to carry out the repair of a cartilage.

12. A pharmaceutical composition, characterized in that it contains an expression vector for an osteoinductive protein and a dorsalization factor or a combination of a vector for the expression of an osteoinductive protein with a vector capable of the expression of a dorsalization factor.

13. A method for the preparation of a pharmaceutical composition for the treatment of a patient to whom it is necessary to carry out the repair of a cartilage, characterized in that the expression vector capable of the expression of an osteoinductive protein is used, and a dorsalization factor , or a combination of a vector capable of the expression of an osteoinductive protein and a layer vector of the expression of a dorsalization factor, as an essential component of said composition.