HK1016470A - Methods and uses of connective tissue growth factor as an induction agent - Google Patents

Description

Method and use of connective tissue growth factor as inducer

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The information disclosed in this specification was made, in part, with government support under the fund number GM37223 awarded by the national institutes of health. The government may have certain rights in the invention disclosed in this specification. 1. Statement of related application

This application is related to and is a continuation-in-part application filed on 2.6.1995 under serial No. 08/459,717 entitled "connective tissue growth factor", application No. 08/459,717 is a continuation-in-part application described in 08/386,680 filed on 10.2.1995 under the same title, application No. 08/386,680 is a divisional application filed on 4.12.1993 and now entitled to the application described in serial No. 08/167,628 of U.S. patent No. 5,408,040, and patent No. 5,408,040 is a continuation-in-part application filed on 30.8.1991 under the currently-disclaimed serial No. 07/752,427. 2. Field of the invention

The invention relates generally to the field of growth factors, and more particularly to Connective Tissue Growth Factor (CTGF) and methods of use thereof. 3. Background of the invention

A. Role of growth factors in bone and cartilage formation

Bone and cartilage formation. In all multicellular organisms derived from a single fertilized egg, the formation of tissues and organs requires the differentiation of various specialized cells derived from undifferentiated stem cells. As the process of embryogenesis progresses, more highly specialized cell types and more complex structures are formed. However, no specific information is currently available on the identification of specific factors or the mechanisms by which these factors exert their effect on bone and cartilage formation in vertebrates, including humans.

There are two common types of bone formation in the mammalian system: intramembranous ossification and endochondral ossification. Bone formation of the skull is an example of intramembranous ossification. At that part of the skull, mesenchymal cells from the neural crest interact with the extracellular matrix of cranial epithelial cells and form bone. See Hall, science of the united states (amer. sci), 1988, p.181, 76174. Mesenchymal cells aggregate into small islands of cells and differentiate into osteoblasts and capillaries. Osteoblasts secrete a special type of extracellular matrix, which (osteoid) binds calcium salts.

Endochondral ossification is the process of osteogenesis of long bones (upper and lower limbs) as well as vertebrae and ribs that form the central axis skeleton. See hall. In this process, bone formation occurs through an intermediate stage of cartilage tissue. In mammals, long bones form in certain mesenchymal cells in embryonic limb buds. These cells form chondrocytes and secrete a cartilage matrix. Other mesenchymal cells around form the perichondrium (eventually, periosteum). In some cases, chondrocytes adjacent to the area where chondrocytes are proliferating and forming differentiate into hypertrophic chondrocytes.

Hypertrophic chondrocytes produce a different type of matrix and alter the differentiation direction of the tissue to form long bony growths. The structure of the long bone growth part is arranged into a plurality of cell columns, including a cell hypertrophy area, a proliferation area, a ossification area and a blood vessel forming area. See Hall, supra; gilbert, "transcriptional regulation of gene expression", DEVELOPMENTAL biology (DEVELOPMENT, BIOIOGY), 5 th edition. Sinaur Assoc, page 387-390 (1994). This results in the transformation of cells from chondrocytes to gradually evolve osteoblasts which form bone containing inorganic salts.

Endochondral ossification, which occurs in mammals from infant growth to adult growth, is an active, ongoing ossification process. Mesenchymal cells differentiate into chondrocytes, their proliferation and replacement by osteoblasts are dependent on growth factors, including the TGF- β family, and inorganic salinization of the matrix. Tuan, 1984, J.Imidaho.J. (J.Exp.Zool.) 1, pages 1-13 (1984); syfetad and Caplan, 1984, developmental biology (Devel. biol.)104, Vol.348 and page 386.

With respect to connective tissue, it can be seen that all skeletal components in mammals are derived from a single stem cell capable of differentiating into various types of specialized cells including muscle, cartilage, bone and tendon. These cells also appear to be capable of differentiating into adipose tissue.

Related fields relating to growth factors and bone and cartilage formation. Prior to the advent of this invention, it was generally known that growth factors included a class of secreted polypeptides that stimulated target cells to proliferate, differentiate and constitute developing tissues. In general, the activity of a growth factor depends on its ability to bind to specific receptors and activate signaling within the cell. Some examples of growth factors that have been studied in detail include Platelet Derived Growth Factor (PDGF), insulin-like growth factor (IGF-1), transforming growth factor beta family (TGF- β), transforming growth factor alpha family (TGF- α), Epithelial Growth Factor (EGF), and Fibroblast Growth Factor (FGF).

Effects of TGF-. beta.on chondrocyte growth, differentiation and chondrogenesis. TGF-. beta.plays a role in cartilage formation. As previously reported, TGF-. beta.1 and TGF-. beta.2 increased chondrogenesis in rat embryonic mesenchymal cells (Seyedin, et al, 1987, J.biol.chem., 262, 1946-1947), and both isoforms induced chondroblast formation from cultured mouse muscle mesenchymal cells. Seyedin et al, 1986, J.Biol.chem.261, 5693-5695. The TGF-beta is applied to the chondroprotein (prechondroid) tissues of mouse embryos to increase the differentiation of mesenchymal cells, the generation of proteoglycan and the proliferation of chondroblasts. Centrella, et al, 1994, Endocrine Reviews, Vol.15, pp.27-38; thorp and Jakowlew, 1994, Bone (Bone)15, pp.59-64.

Using in situ hybridization techniques, reduced levels of TGF-beta 3 are seen in growth plates of animals with chondrocyte differentiation arrested and three distinct diseases. In organ cultures of bovine articular cartilage, collagen type II and proteoglycans are increased when TGF-. beta.is used, as measured by the same method (Id.). Morales and Roberts, 1988, journal of Bioand chemistry (J.biol.chem.)263 page 12828-12831. In contrast, TGF-. beta.has been shown to decrease the expression of cartilage-specific collagen types II and X, the synthesis of chondrocyte proteoglycans, and the activity of alkaline phosphatase in cultured chondrocytes. "effects of TGF-. beta.on bone", "Clinical Applications of TGF-. beta." (Clinical Applications of TGF-. beta.), 1991, Wiley Chichester, Ciba Foundation Symposium 157 pp.137-. Chondrocyte differentiation in rabbit growth plates is inhibited by TGF- β, whereas growth plate chondrocyte mitosis is increased. Kato et al, 1988, Proc. Natl. Acad. Sci. USA 85 vol 9552-9556.

In addition, high concentrations of TGF-beta 1 or TGF-beta 2 added to osteoinductive models favor cartilage formation over bone formation when smaller doses are used. Mundy, supra. The accumulation of these apparently contradictory data has hampered research into determining the role TGF- β plays in cartilage formation.

Bone morphogenetic proteins and bone formation. A family of proteins called bone morphogenetic proteins (BMPs are capable of inducing ectopic bone formation in certain species of mammals, all of these proteins, except BMP-1, which encodes a metalloprotease, have structures associated with TGF-. beta.however, if BMPs are involved in the regulation of bone formation during normal embryogenesis, it is not known which of these are responsible.

BMP's, which are factors inducing ectopic bone formation outside the bone, were originally isolated from demineralized bone. Three polypeptides were initially identified as BMP-1, BMP-2A, and BMP-3. Celese, et al, 1990, proceedings of the national academy of sciences of the United states (Proc, Natl.Acad.Sci.USA)87, Vol.9843-; kubler and Urist, 1990, "clinical orthopedics and related research (Clin. orthopedics and Rel. Res.)258, page 279-294. The latter two BMPs are members of the TGF- β superfamily. Subsequently, 5 more closely related members of the BMP protein group have been identified and cloned. BMP-5, BMP-6 and BMP-7 are most similar to vgr/60A, whereas BMP-2 and BMP-4 are most similar to Decapentaplegic. Both Vga/60A and Decapentaplegic are Drosophila genes that control the formation of dorsal/ventral medial axis patterns (patterren). Hoffman, 1992, molecular reproduction and development (mol. reproand Dev.)32 vol.173-178.

In situ hybridization at certain specific stages during development has limited the gene expression of the BMP to the bone-forming regions in the limb bud, suggesting that BMP is physiologically significant. BMP is able to induce the transition from a fibrogenic pattern to a chondrosterioembryogenic (chondrosteroplastic) pattern in the mesenchyme of the postembryonic adventitia. Kubler and Urist, supra. Several series of data suggest that BMPs may act synergistically with TGF-. beta.s to initiate osteoinductive chains in vivo. TGF-. beta.1 was able to enhance the formation of ectopic bone induced by most BMPs in the subcutaneous tissues of mice. BMP-6 (also known as VGR-1) is expressed in hypertrophic cartilage at the same time and site as TGF-. beta.s are expressed, and is also involved in the expression of type X collagen. See Celeste, et al, supra.

Addition of TGF- β 2 to bone graft that has been treated with BMP-2 or BMP-3 increases osteoinductive activity and increases the cartilage to bone ratio when compared to treatment with either factor alone. Bentz, et al, 1991, Matrix (Matrix)11 vol 269-275. However, the synergistic effect between TGF-. beta.s and these proteins is not ubiquitous. TGF-. beta.1 has been shown to directly reduce BMP-2 expression in embryonic rat calvarial bone cultures. Harris, et al, 1994, journal of bone and mineral research (J.bone and mineral Res.)9 Vol.855-863. Since BMP-2 apparently plays an important role in osteoblast differentiation, it can be suggested that TGF- β 1 might act as a switch to monitor the differentiation fate of chondroblasts or osteoblast precursors.

Other factors were found to be expressed in developing tissues. Cyr61 is a growth regulatory factor that is found expressed in developing mouse embryos and extraembryonic tissues. O' Brien and Lau, 1992, Cell Growth differentiation (Cell Growth and Differ), 3 vol.645 page 654. Cyr61 was related to but not identical to CTGF, and the specific activity of Cyr61 was not known prior to the present invention.

B. Role of growth factor in wound healing

Platelet derived growth factor and wound healing. PDGF is a dimeric molecule consisting of an a chain and a B chain. These chains form heterodimers among homodimers and all dimer molecules isolated so far are biologically active. Regarding the activity of this factor, POGF has been described as a positively charged, thermostable protein found in the alpha granules of circulating platelets. The molecule is further described as a mitogen and chemotactic agent towards connective tissue cells such as fibroblasts and smooth muscle cells.

Due to its biological activity and local secretion during wound healing, PDGF has been identified as a growth factor involved in wound healing and in pathological states exhibiting hyperproliferation of connective tissue, including atherosclerosis and fibrotic diseases.

It has been speculated that growth factors other than PDGF may play a role in the normal development, growth and repair of human tissues.

TGF-beta and wound healing. The formation of neogenetic and regenerative tissues requires the coordinated action of various genes that can produce regulatory and structural molecules involved in cell growth and the organization of the tissue-forming organisms. With respect to osteoinduction, it appears that TGF-. beta.acts as a central regulatory molecule in this process. TGF-. beta.is secreted by platelets, macrophages and neutrophils that are present at the initial stage of the repair process. TGF-beta may act as a growth-stimulating factor for mesenchymal cells and a growth-inhibiting factor for endothelial and epithelial cells. It has been proposed that the growth stimulatory effect of TGF-. beta.appears to be mediated by an indirect mechanism involving the induction of other growth factor genes such as PDGF.

The activities possessed by several members of the TGF- β superfamily may be useful in the treatment of cell proliferative disorders, such as cancer. In particular, TGF-. beta.has been demonstrated to be a potent growth inhibitory factor for many cell types. (Massague, 1987, Cell 49, 437), MIS has been shown to inhibit the growth of human endometrial cancer in nude mice (Donano, et al, 1981, Ann. Surg., 194, 472), and this inhibition has been shown to inhibit tumor growth in the ovary and testis (Matzuk, et al, 1992, Nature, 360, 313).

Many members of the TGF- β family are also important regulators of tissue repair. TGF-. beta.has been shown to significantly affect collagen formation and the predisposition to a significant angiogenic response in neonatal mice (Roberts et al, 1986, Proc. Natl. Acad. Sci. USA 83, 4167). Bone Morphogenic Proteins (BMPs) can induce new bone formation and are effective in treating bone fractures and other skeletal defects (Glowacki et al, 1981, Lancet (Lancet), pp.959, Ferguson et al, 1988, St.Clin. Orthopaediation Res. 227 pp.265, Johnson et al, 1988, St.Clin. Orthopted Relat. Res. 230, pp.257).

C. Connective tissue growth factor

A previously unknown growth factor related to PDGF and designated Connective Tissue Growth Factor (CTGF) has been reported in a related patent. See U.S. Pat. No. 5,408,040. CTGF is a cysteine-rich mitogenic polypeptide that is selectively induced in fibroblasts following activation with TGF- β. Igarashi et al, 1993, molecular biology and cells (mol.biol.cell)4 vol 637-645.

CTGF is a member of a family of polypeptides including the serum-inducible gene product Cef10(Simmons et al, 1989, proceedings of the national academy of sciences USA (Proc. Natl. Acad. Sci. USA)86 vol 1178-1182), Cyr61 (O' Brien et al, 1990, molecular Cell biology 10 vol 3569-233), fisp12/IGM1(Ryseck et al, 1993, Cell Growth and differentiation (Cell Growth & Differ) 2 vol 225-233), and the chicken-transformed gene, nov (Joliot et al, 1992, molecular Cell biology 12 vol 10-21 (1992), CTGF and a fruit gene product, twisted progut embryo formation (twg) (twasted tissue homology (Dev. Biol., Masson et al., 1994), developmental process of cells and developmental Genes: Maselop 9.

As reported in that patent, CTGF is the product of a unique gene. CTGF has mitogenic activity as also reported in U.S. patent No. 5,408,040. The net result of this mitogenic activity in vivo is the growth of the target tissue. CTGF also has chemotactic activity, which refers to the chemically induced movement of cells caused by interaction with certain specific molecules.

Although this molecule is antigenically related to PDGF, it is almost unclear whether there is polypeptide sequence homology between CTGF and PDGF. anti-PDGF antibodies have high affinity for the non-reduced forms of PDGF isomers and CTGF molecules, but one-tenth of the affinity for the reduced forms of these polypeptides lacking biological activity.

Yet another protein identified as "connective tissue growth factor-2" or "CTGF-2" has also been reported. See PCT application No. PCT/US94/07736 (International publication No. WO 96/01896). According to the PCT application, CTGF-2 may also be used to enhance the repair of connective and scaffold tissues. Although identified as connective tissue growth factor, CTGF-2 is not germane to CTGF of the present invention. Specifically, the CTGF family includes three distinct proteomes: CTGF/Fisp12, cyr61 and nov. The proteins of the present invention are patented as belonging to the first protein group, in contrast to CTGF-2 belonging to the cyr61 group. PCT application No. PCT/US94/07736 is located at 4(at 4).

Prior to the present invention, although various PDGF-associated growth factors including CTGF have been identified, such factors have not been demonstrated to be effective inducers of matrix production, including bone and/or cartilage tissue induction. 4. Summary of the invention

The subject matter of the present invention provides novel methods and compositions for treating diseases, disorders, or disorders requiring stromal and/or connective tissue production, including bone and/or cartilage production. The present subject matter is also directed to the treatment of diseases, disorders, or conditions requiring the promotion of wound healing.

More specifically, the compositions of the present invention comprise CTGF and/or fragments and/or derivatives thereof (hereinafter collectively referred to as CTGF), alone or in combination with other growth factors. CTGF for use in the composition of the present invention may be obtained by isolation from natural resources, synthetic production, or production using genetic recombinant bioengineering techniques.

In one aspect of the invention, the methods of the invention comprise treating a disease, disorder or condition requiring bone or cartilage tissue-induced production with an effective amount of CTGF, alone or in combination with one or more other compounds. In a preferred embodiment of the method, such additional compound is a growth factor.

In another aspect of the present invention, the methods of the present invention comprise treating a disease, disorder or condition requiring the promotion of wound healing with an effective amount of CTGF alone or in combination with one or more compounds, and more preferably one or more growth factors.

In a preferred embodiment of the invention, the composition comprising CTGF is applied directly to a site where bone or cartilage induced production is desired, to induce the formation of such bone or cartilage. In another embodiment, the composition is formulated for targeted administration, or the composition is designed for release of a new composition at a corresponding site (e.g., a wound requiring cartilage formation). In each case, the CTGF-containing composition is suitably formulated for use in a patient in need thereof. 5. Definition of

In the specification, the term "CTGF" shall mean: (1) a protein encoded by the amino acid sequence depicted in figure 1C, (2) a protein having CTGF activity encoded by the amino acid sequence depicted in figure 1C in which one or more amino acids have been inserted, deleted, mutated, substituted or otherwise altered ("derivative"), and the nucleotide sequence encoding the protein hybridizes under stringent conditions to the nucleotide sequence depicted in figure 1C, or (3) a partial fragment of CTGF or a derivative thereof.

In the present specification, the term "induce" as used herein shall mean to produce, form, cause to produce, or cause to form.

In the present context, an "inducing agent" shall mean an agent comprising a protein or other biological substance that causes a particular end result to occur or form (e.g., the development of connective tissue).

In the present specification, the term "polynucleotide" refers to DNA, cDNA and/or RNA flanking the CTGF structural gene encoding untranslated sequences. For example, a polynucleotide of the present invention includes 5 'regulatory nucleotide sequences and 3' untranslated sequences related to the structural gene of CTGF. A polynucleotide of the present invention comprising 5 'and 3' untranslated regions is illustrated in FIG. 1C. The 5' regulatory region including the promoter is illustrated in FIG. 1B. A more detailed description of polynucleotides of interest in the present invention can be found in U.S. Pat. No. 5,408,040.

In the present specification, "stringent conditions" are used herein to refer to those hybridization conditions (1) using low ionic strength and high temperature for elution, for example, 50 ℃, 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulfate; (2) denaturing agents such as formamide are used during hybridization, such as formamide at 42 ℃, 50% (v/v) formamide containing 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer ph6.5, and 750mM sodium chloride, 75mM sodium citrate; or (3) using 50% formamide, 5 times SSC (0.75M sodium chloride, 0.075M sodium pyrophosphate, 5 times Denhardt's solution, sonicated salmon sperm DNA (50g/ml), 0.1% sodium lauryl sulfate, and 10% dextran sulfate at 42 deg.C while rinsing with 0.2 times SSC and 0.1% sodium lauryl sulfate at 42 deg.C.

In the present specification, the "recombinant expression vector" refers to a plasmid, virus or other vector known in the art, which has been manipulated by insertion or integration of CTGF gene sequences.

In the present specification, "therapeutically effective" refers to an amount of CTGF effective to induce bone or cartilage formation or wound healing. 6. Brief description of the drawings

FIG. 1A shows the structural composition of the CTGF gene. Exons are represented as box regions, and the solid box regions in the gene are open reading frames.

FIG. 1B compares the nucleotide sequences of the CTGF promoter and the fisp12 promoter. Identical nucleotides are indicated by asterisks. TATA boxes and other consensus sequences are indicated and shaded. The transcription start site is at position number + 1.

FIG. 1C shows all the nucleotides and the deduced amino acid sequence of the CTGF structural gene as well as the 5 'and 3' untranslated sequences.

Fig. 2 shows experimental in situ hybridization results for CTGF transcript expression in the long bone growth plates of neonatal mice. In situ hybridization experiments were performed using antisense CTGF RNA probes as described below. The CTGF gene expression of the chondrocytes in the proliferation region was strongly positive, indicating that CTGF is expressed at the site of cartilage growth.

FIG. 3 shows the expression of CTGF gene during embryogenesis in transgenic mice constructed from a fusion gene constructed from the CTGF promoter and the structural gene of beta-galactosidase. The gene introduced into the embryonic line expresses beta-galactosidase at the site of CTGF expression and can be detected by immunohistochemical methods, in which the expressed part of the developing transgenic mouse is added to a substrate X-gal capable of depositing a blue color at the active site of beta-galactosidase. Panel A is a mouse embryo from day 12 of this transgenic mouse. The blue stained area is the area destined to form the Meckel's cartilage, the first piece of cartilage to be formed. Panel B is a photograph of the hind limb and paw demonstrating staining in the long bone ends and paw in the growth area of the metatarsal.

FIG. 4 provides evidence of bone and cartilage induced production in cultures of C3H10T1/2 mouse embryonic stem cells. C3H10T1/2 cells were cultured as described in methods. Cells were not treated (panel A), treated with 5-azacytidine (panel B), treated with 50ng/ml CTGF (panel C) or treated with 5-azacytidine followed by CTGF (panel D).

FIG. 5 illustrates Northern blot analysis of expression of CTGF gene in callus columns transplanted to bone regeneration sites.

FIG. 6 provides evidence that CTGF is expressed in human osteoblasts responsive to TGF- β.

Fig. 7A-7D illustrate the results of a cartilage formation assay.

Figure 7A provides the results of the chondrogenesis assay for the control culture.

FIG. 7B provides the results of chondrogenesis assays for cultures to which 5ng/ml TGF-. beta.1 was added.

FIG. 7C provides the results of chondrogenesis assays for cultures supplemented with 5ng/ml TGF-. beta.1 and 10ng cholera toxin.

FIG. 7D provides the results of chondrogenesis assays for cultures supplemented with 5ng/ml TGF-. beta.1, 10ng/ml cholera toxin, and 5ng/ml CTGF.

Fig. 8A is a Scatchard plot reflecting CTGF binding to NRK cells.

Fig. 8B is a Scatchard plot reflecting the binding of CTGF to rat chondroblasts. 7. Detailed description of the invention

7.1 Process for the preparation of CTGF

A nucleic acid sequence encoding CTGF. According to the present invention, the nucleotide sequence encoding CTGF or functional equivalents thereof may be used to prepare recombinant DNA molecules which direct the expression of the protein or functional equivalents thereof in a suitable host cell. Alternatively, nucleotide sequences that hybridize under stringent conditions to portions of the CTGF sequence may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, and the like. In yet another approach, DNA molecules encoding CTGF may be isolated by a hybridization process that includes antibody screening of expression libraries to detect common structural features.

Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences may also be isolated and used in the practice of the present invention for the cloning and expression of CTGF. These DNA sequences include those that are capable of hybridizing to human CTGF sequences under stringent conditions.

Altered DNA sequences which may be used according to the invention include deletions, insertions or substitutions of different nucleotide residues which result in a sequence encoding the same or a functionally equivalent gene product. The gene product itself may contain deletions, insertions or substitutions of amino acid residues in the CTGF sequence, which changes are silent and thus produce a functionally equivalent protein. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids having an uncharged polar head group and having a similar hydrophilicity value include the following amino acids: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

The DNA sequences of the present invention may be engineered to alter the sequence of the protein to obtain a variety of products including, but not limited to, such changes that alter the processing and expression of the gene product. For example, mutations can be introduced using techniques well known in the art, such as site-directed mutagenesis, e.g., insertion of new restriction sites. For example, in certain expression systems such as yeast, the host cell may hyperglycosylate the gene product. When these expression systems are used, it may be preferable to alter the coding sequence of CTGF to eliminate any N-linked glycosylation sites.

The CTGF sequence may be linked to a heterologous sequence to encode a fusion protein. For example, for polypeptide library screening, it is useful to encode a chimeric CTGF protein that expresses a heterologous epitope recognized by commercially available antibodies. The fusion protein may also be designed to contain a cleavage site between the CTGF sequence and a heterologous protein sequence (e.g., a sequence encoding a growth factor associated with PDGF) to allow cleavage of CTGF from the heterologous moiety.

The coding sequence for CTGF may also be synthesized in whole or in part using chemical methods well known in the art. See, e.g., Caruthers et al, 1980, nucleic acids research Association series (nucleic acids Res. Symp. Ser.) volume 7, page 215-233; crea and Horn, 1980, Nucleic Acids research (Nucleic Acids Res.)9 Vol.10, page 2331; matteucci and Caruthers, 1980, Tetrahedron Letters, 21, volume 719; and Chow and Kempe, 1981, Nucleic Acids research (Nucleic Acids Res.)9, Vol.12, 2807 and 2817. Alternatively, the protein itself may be prepared by chemical methods for synthesizing all or part of the CTGF amino acid sequence. For example, peptides can be synthesized using solid phase techniques, separated from the resin, and then purified using preparative high performance liquid chromatography. See, e.g., Creighton, 1983, "protein Structures And Molecular Principles", W.H.Freeman And CO., p.Y.50-60. The composition of the synthetic peptide can be confirmed by amino acid analysis or sequencing. See, e.g., the Edman degradation process, see Creighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and CO., p.Y.34-49.

More detailed descriptions of nucleic acid sequences encompassed by the present invention and methods for identifying such sequences can be found in U.S. Pat. No. 5,408,040, which is incorporated herein by reference.

Expression of CTGF. For the expression of biologically active CTGF, the nucleotide sequence encoding the protein or functional equivalent as described above is inserted into a suitable expression vector, i.e. a vector containing the elements necessary for the transcription and translation of the inserted coding sequence.

More specifically, methods well known to those skilled in the art can be used to construct expression vectors containing CTGF sequences and appropriate transcription/translation regulatory signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, e.g., techniques described in the following, Maniatis et al, 1989, Molecular Cloning: a laboratory Manual, Cold spring harbor laboratory, N.Y., and Ausubel et al, 1989, methods of modern Molecular Biology (Current Protocols in Molecular Biology), Greene publishing associates and Wiley Interscience, N.Y.

A variety of host expression vector systems may be used to express the CTGF coding sequence. These systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing CTGF coding sequences; yeasts, including Pichia pastoris and Hansenula polymorpha, transformed with a recombinant expression vector containing the CTGF coding sequence; insect cell systems transfected with recombinant viral expression vectors (e.g., baculoviruses) containing CTGF coding sequences; plant cell systems transfected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the CTGF coding sequence; or animal cell systems transfected with recombinant viral expression vectors (e.g., adenovirus, poxvirus, human tumor cells (e.g., HT-1080)), including cell lines engineered to contain multiple copies, stably amplified on double minichromosomes (CHO/dhfr), or unstably amplified CTGF DNA (e.g., mouse cell lines). As used herein, the term "host expression vector system" and, more broadly, "host cell" is understood to include a host cell or any progeny of a host expression vector system. It is further understood that although not all progeny may be identical to the parent cell, because of the mutations that may occur during replication, such progeny are not included within the scope of the present invention.

The expression elements of these systems vary in their strength and specificity. Depending on the host/vector system used, any transcription and translation element that can be used, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning in a bacterial system, an inducible promoter such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage lambda, etc. can be used; when cloning in an insect cell system, a promoter such as a baculovirus polyhedrin promoter can be used; when cloned in a plant cell system, promoters from the genome of plant cells (e.g., heat shock promoters; promoters of RUBISCO small subunits; promoters of chlorophyll a/b binding proteins) or from plant viruses (e.g., 35S RNA promoter of CaMV; TMV capsid protein promoter) may be used; when cloned in mammalian cell systems, promoters from mammalian cell genomes (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; poxvirus 7.5K promoter) may be used; when cell lines containing multiple copies of CTGF DNA are prepared, SV40-, BPV-and EBV-based vectors with appropriate selection markers can be used.

In bacterial systems, many expression vectors have been selected for their advantages, depending on the use of the CTGF to be expressed. For example, suitable vectors for expression in bacteria include T₇Vectors based thereon, as described by Rosenberg et al, 1987, Gene 56, pp.125. By way of further example, when large amounts of CTGF expression are required for peptide librariesIn screening, vectors directing the expression of high yields of protein that are easy to purify may be required. These vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, 1983, EMBOJ.2 Vol.1791) in which the CTGF coding sequence is ligated in-frame with the Lac Z coding region into the vector, resulting in the production of a hybrid AS-Lac Z protein; pIN vector (Inouye)&Inouye, 1985, Nudic Acids Res.13, 3101 and 3109; van Heeke&Schuster, 1989, journal of biochemistry and chemistry (J.biol.chem.)264, 5503 and 5509, etc. pGEX vectors can also be used to express foreign polypeptides such as CTGF with glutathione S-transferase (GST). In summary, these fusion proteins are soluble and can be easily purified from lysed cells, adsorbed to glutathione-agarose beads during purification, and subsequently eluted in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site so that the cloned polypeptide of interest can be cleaved from the GST moiety.

More generally, when the host is a prokaryotic cell, competent cells capable of accepting DNA can be prepared from cells treated as follows: cells are harvested after logarithmic growth and subsequently treated with magnesium chloride or rubidium chloride using the calcium chloride method, or another method well known in the art.

Where the host is a eukaryotic cell, a variety of methods of DNA transfer may be employed. They include transfection with calcium phosphate-precipitated DNA, conventional mechanical methods including microinjection, plasmid insertion encapsulated in liposomes, or the use of viral vectors. Eukaryotic cells can also be co-transformed with a DNA sequence encoding a polypeptide of the invention and another exogenous DNA encoding a selectable phenotype, such as a herpes simplex thymidine kinase gene. Another approach is to use eukaryotic viral vectors, such as simian virus 40(SV40) or bovine papilloma virus, to transiently transfect or transform eukaryotic cells and express proteins. See, Eukaryotic Viral Vectors (Eukaryotic Viral Vectors), 1992, Cold spring harbor laboratory, Gluzman, Ed.). Eukaryotic host cells include yeast, mammalian cells, insect cells and plant cells.

In yeast, a number of vectors containing constitutive or inducible promoters may be used. For a review, see methods of modern Molecular Biology (Current Protocols in Molecular Biology), Vol.2, 1988, Ausubel et al, eds., Greene publishing. Assoc. & Wiley Interscience, Chapter 13; grant et al, 1987, Methods in Enzymology, Wu & Grossman eds, Acad.Press, N.Y., Vol.153, page 516-; glover, 1986, "DNA Cloning", volume II, IRL Press, Wash, d.c., chapter 3; bitter, 1987, heterologous gene Expression in Yeast (heterologous Gene Expression in Yeast), Methods in Enzymology (Methods in Enzymology), Berger & Kimmel, Acad.Press, N.Y., volume 673 684; and "Molecular Biology of Yeast" (The Molecular Biology of The Yeast Saccharomyces), 1982, Stratan et al, eds., Cold Spring Harbor Press, volumes I and II. For example, many shuttle vectors have been reported for expressing foreign genes in yeast. Heinemann et al, 1989, Nature 340, Vol.205; rose et al, 1987, Gene (Gene) volume 60, page 237.

In the case of plant expression vectors, expression of the CTGF coding sequence may be initiated by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al, 1984, Nature 310, 511-514), or the capsid promoter of TMV (Takamatsu et al, 1987, EMBO J.6, 307-311) may be used. Alternatively, plant promoters may be used, such as the promoter of the RUBISCO small subunit (Coruzzi et al, 1984, EMBOJ.3, pp. 1671-; or heat shock promoters such as soybean hsp17.5-E or hsp17.3-B (Gurley et al, 1986, molecular cell biology 6 vol. 559-565). These constructs can be introduced into plant cells by Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and the like. For a review of these techniques, see, e.g., Weissbach & Weissbach, 1988, Methods for Plant molecular biology (Methods for Plant molecular biology) Academic Press, N.Y.section VIII, pp.421-; grierson & Corey, 1988, Plant Molecular Biology, 2 nd edition, Blackie, London, chapters 7-9.

In insect systems, another expression system may be used to express CTGF. In one system, baculovirus is used as a vector for expressing foreign genes. The virus is then grown in insect cells. The coding sequence for CTGF may be cloned into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of a baculovirus promoter. These recombinant viruses are then used to infect insect cells in which the inserted gene is capable of being expressed. See, e.g., Smith et al, 1983, journal of virology (J.Virol.)46, page 584; smith, U.S. patent No. 4,215,051.

In mammalian host cells, a number of viral-based expression systems can be used. In the case where an adenovirus is used as the expression vector, the CTGF coding sequence may be ligated to the adenovirus's transcription/translation regulatory complex, e.g., the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the genome of an adenovirus by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., E)₁Or E₃Region) will produce a recombinant virus that will survive and express CTGF in the infected host. See, e.g., Logan&Shenk, 1984, Proc. Natl. Acad. Sci (USA) 81, 3655, 3659. Alternatively, the poxvirus 7.5K promoter may be used. See, e.g., Mackett et al, 1982, Proc. Natl. Acad. Sci (USA) 79, pages 7415-7419; mackett et al, 1984, J.Virol., 49, pp.857-864; panicali et al, 1982, Proc. Natl. Acad. Sci. 79 Vol. 4927 4931.

In another embodiment, CTGF sequences may be expressed in human tumor cells such as HT-1080 that have been stably transfected with calcium phosphate precipitates and neomycin resistance genes. In yet another embodiment, expression may be carried out using a pMSXND expression vector or similar expression vectors in a variety of mammalian cells, including COS, BHK293 and CHO cells. Lee and Nathans, 1988, journal of biology and chemistry (J.biol.chem.)263, page 3521.

Specific initiation signals are also required for efficient translation of the inserted CTGF coding sequence. These signals include the ATG initiation codon and adjacent sequences. In case the complete CTGF gene, including its own initiation codon and adjacent sequences, is inserted into an appropriate expression vector, no additional translational control signals are required. However, in the case where only a portion of the CTGF coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Further, the initiation codon must be coordinated with the reading frame of the CTGF coding sequence to ensure translation of the complete insert. These exogenous translational regulatory signals and initiation codons can be of various origins, both naturally occurring and synthetic. The efficiency of expression can be enhanced by incorporating appropriate transcription enhancer elements, transcription terminators, and the like. See, e.g., Bitter et al, 1987, Methods in enzymology 153 (Methods in Enzymol.) page 516-544.

In addition, a host cell line may be selected which is capable of regulating the expression of the inserted sequence or modifying or processing the gene product in the particular manner desired. Modification (e.g., glycosylation) and processing (e.g., cleavage) of such protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. Appropriate cell lines or host systems may be selected to ensure proper modification and processing of the foreign protein expressed. To achieve this, eukaryotic host cells containing the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, 293, WI38, HT-1080, and the like.

For long-term, high-yield expression of recombinant proteins, stable expression is preferred. For example, a cell line stably expressing CTGF may be obtained using bioengineering. Instead of using an expression vector containing a viral origin of replication, a host cell can be transformed with CTGF DNA and a selectable marker under the control of appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Following the introduction of the exogenous DNA, the engineered cells can be grown in an enrichment medium for 1-2 days and then transferred to a selection medium. The selectable marker in the recombinant plasmid provides resistance to selection and allows for stable integration of the plasmid into the cell chromosome, which can then be cloned and divided into integration sites in cell lines after cell growth.

A number of selection systems can be employed, including but not limited to herpes simplex virus thymidine kinase (Wigler, et al, 1977, Cell 11, page 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska)&Szybalski, 1962, proceedings of the national academy of sciences of the united states of america (proc.natl.acad.sci. (USA))48, pp.2026), and adenine phosphoribosyltransferase (Lowy, et al, 1980, Cell (Cell)22, pp.817). Genes can be used for tk respectively^-、hgprt^-Or aprt^-A cell.

Furthermore, antimetabolite resistance can be used as the basis for several options, the dhfr gene which provides methotrexate resistance (Wigler, et al 1980, Proc. Natl. Acad. Sci. (USA) 77 pp. 3567; O' Hare, et al 1981, Proc. Natl. Acad. Sci. (USA))78 pp. 1527); the gpt gene which confers mycophenolic acid resistance (Mullgan & Berg, 1981, Proc. Natl. Acad. Sci. (USA))78, page 2072; the neo gene which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al, 1981, J. Mol. biol. 150, page 1); and the hygro Gene which confers hygromycin resistance (Santerre, et al, 1984, Gene 147, Vol. 30, Gene). Recently, alternative genes have been described, trpB, which enables cells to use indole instead of tryptophan, hisD, which enables cells to use histidinol instead of histidine (Hartman & Mullingan, 1988, Proc. Natl. Acad. Sci. (USA)) 85-volume 8047, and ODC (ornithine decarboxylase) (McConlogue, 1987, see; "modern molecular biology Communications in molecular biology", Cold spring harbor laboratory), which provides resistance to the ornithine decarboxylase inhibitor 2- (difluoromethyl) -DL-ornithine (DFMO).

The polypeptides expressed in the host cells of the present invention may be isolated and purified by any conventional method, such as, for example, preparative chromatographic separation and immunological separation, such as those involving the use of monoclonal or polyclonal antibodies.

7.2 identification of transfectants or transformants expressing CTGF

Host cells containing a coding sequence and expressing a biologically active gene product can be identified by at least four general methods: (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of a function of a "marker" gene; (c) assessing the level of transcription based on the determination of CTGF transcript mRNA expression in the host cell; and (d) detecting the gene product according to an assay or determination of its biological activity.

In the first method, the presence of the CTGF coding sequence inserted into the expression vector is detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences homologous to the CTGF coding sequence, or to a partial sequence or its derivatives, respectively.

In the second approach, recombinant expression vector/host systems are identified and selected for the presence or absence of certain "marker" gene functions (e.g., antibiotic resistance, methotrexate resistance, transformation phenotype, formation of inclusion bodies in baculoviruses, etc.). For example, in a preferred embodiment, the CTGF coding sequence is inserted into the anti-neomycin marker gene sequence of the vector, and then recombinants containing the CTGF coding sequence are identified as lacking the function of the marker gene. Alternatively, the marker gene and the CTGF sequence may be placed in tandem under the control of the same or different promoter that controls expression of the CTGF coding sequence. Expression of the marker gene in response to induction or selection indicates expression of the CTGF coding sequence.

In a third method, the transcriptional activity of the CTGF coding region may be assessed by hybridization assays. For example, RNA can be extracted and subjected to Northern blot analysis using a probe homologous to the CTGF coding sequence or a specific portion thereof. Alternatively, all of the nucleic acid of the host cell may be extracted and then hybridized with such probes.

The fourth method involves detection of biological or immunological activity of the CTGF gene product. A number of assays may be used to detect CTGF activity, including, but not limited to, those described in U.S. patent No. 5,408,040.

7.3 therapeutic indications

The methods, compounds and formulations of the invention are each directed to the treatment of disorders, diseases or disorders associated with insufficient proliferation of connective tissue in bone, cartilage or other organs such as skin and muscle, and to the treatment of disorders, diseases or disorders requiring the formation of bone or cartilage.

Such diseases, disorders or conditions include the repair of cartilage or bone defects following various traumatic injuries or diseases including arthritis, osteoporosis and other bone diseases, scar hyperplasia, burns, and vascular hyperplasia. Since the disease is caused by an adverse growth response of fibroblasts, stem cells, chondrocytes, osteoblasts or fibroblasts at the site of injury, it is beneficial to add a bioactive agent that stimulates the growth of these cells.

Another important application of CTGF may be in culture systems for expansion of stem or chondrocytes isolated from an individual prior to re-transplantation. In a similar process, CTGF is added to stem cells or chondrocytes used as grafts to facilitate stimulation of expansion and differentiation of these cells at the site of transplantation. CTGF may also be added to grafts containing cartilage or bone to help stimulate growth.

Another therapeutic indication is directed to the use of CTGF to promote wound healing in a patient in need thereof. Platelet Derived Growth Factor (PDGF) and other growth factors, such as CTGF, are required for the normal healing process of skin wounds. CTGF polypeptide therapy of the invention is of value in the healing of skin wounds or where it is desirable to promote normal healing mechanisms, such as burns. An important benefit of using CTGF protein to promote wound healing is the high proportion of cysteine residues in the molecule. CTGF, or a functional fragment thereof, is more stable and less susceptible to degradation by proteases than PDGF and other growth factors involved in wound healing.

Preferably, the agents of the invention are TGF- β in combination with CTGF, however, other members of the TGF- β family may also be effective in promoting wound healing by inducing CTGF. The compositions of the present invention help to heal wounds, in part, by promoting connective tissue growth. The purified CTGF and TGF- β are combined with a pharmaceutically acceptable carrier substance, such as an inert gum or liquid, to prepare a composition.

Therapeutic indications of interest in connection with wound healing include prospective wounds (e.g., wounds resulting from surgery), as well as unintended wounds (e.g., wounds resulting from trauma).

7.4. Pharmaceutical formulations and routes of administration

The molecules of the invention are administered to a patient in need thereof, either alone or in a pharmaceutical composition comprised of one or more molecules in admixture with suitable carriers or excipients, to treat or alleviate a wide variety of conditions. Alternatively, because CTGF is produced by endothelial cells and fibroblasts both present at the site of bone or cartilage formation and injury, agents that stimulate CTGF secretion may be added to the composition for promoting bone or cartilage induction or wound healing. Preferably, the agent of the invention is transforming growth factor beta. The compositions of the present invention help to heal wounds, in part by promoting the proliferation of connective tissue. In another embodiment, CTGF may be used in combination with a protein or compound believed to promote connective tissue formation.

Whether a composition includes CTGF alone or CTGF and other agents as active ingredients, such compositions are prepared by combining purified CTGF and TGF- β with a pharmaceutically acceptable carrier material, such as an inert gum or liquid.

A therapeutically effective dose further refers to a dose of the compound sufficient to cause symptomatic relief. Techniques for the preparation and administration of compounds of this application can be found in the "Remington pharmaceutical sciences", MackPublishing Co., Easton, Pa., latest edition.

7.4.1. Route of administration

Suitable routes of administration may include, for example, oral, rectal, transmucosal or enteral administration; parenteral administration, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

Alternatively, the compounds may be administered to the patient topically rather than systemically, e.g., by direct injection into the site in need of CTGF, often in a depot or sustained release formulation.

Still further, the patient may be administered using a targeted drug delivery system, e.g., in the form of liposomes coated with a specific antibody, e.g., to target cartilage. Such liposomes will target and be selectively absorbed by the diseased tissue.

7.4.2. Composition/formulation

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping (enrering) or lyophilizing processes.

Thus, pharmaceutical compositions prepared for use according to the present invention may be formulated by conventional methods using one or more physiologically acceptable carriers comprising excipients and adjuvants which facilitate processing of the active molecule into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.

For injection, the agent of the present invention can be prepared as an aqueous solution, preferably using a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal application, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds are readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. These carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral administration to a patient to be treated. Pharmaceutical preparations for oral use can employ solid excipients, if necessary after addition of suitable adjuvants, optionally with or without grinding of the resulting mixture, and processing of the mixture of granules to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If necessary, a lysing agent, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may also be added.

The dragee cores may be coated with a suitable coating. To this end, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polycarbonyl vinyl gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to identify or identify different combinations of active compound doses.

Pharmaceutical preparations for oral use include push-fit capsules consisting of gelatin, as well as soft, closed capsules consisting of gelatin and a forming agent such as glycerol or sorbitol. Push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may also be added. All preparations for oral administration should be formulated in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds suitable for use according to the present invention may conveniently be delivered in the form of an aerosol spray presentation from a pressurised vessel or nebuliser, together with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be measured by a valve delivering a metered dose of aerosol. Capsules of gelatin or other capsules and bullets containing powders of the compounds and suitable powder bases such as lactose or starch for use in an inhaler or insufflator may be prepared.

These molecules may be formulated to be suitable for parenteral administration by injection, e.g., bolus injection or continuous infusion. Formulations for injection containing an added preservative may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers. The compositions may take such forms as suspensions, solutions or emulsions in which an oil or water acts as a carrier, and may contain formulatory (formulating) agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical preparations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Alternatively, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may also optionally contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be mixed with a suitable carrier, for example, sterile pyrogen-free water, in powder form prior to use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., preparations containing conventional suppository bases such as cocoa butter buffers or other glycerides.

In addition to the above formulations, the compounds may also be formulated as a depot preparation. Such long acting formulations may be used by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For example, the compounds may be prepared by co-formulation with suitable polymeric or hydrophobic materials (such as an acceptable emulsion in oil) or ion exchange resins, or slightly soluble derivatives, such as a slightly soluble salt.

A pharmaceutically acceptable carrier for hydrophobic molecules useful in the present invention is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, an organic polymer miscible with water, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution made up of 3% w/v benzyl alcohol, 8% w/v non-polar surfactant polyoxyethylene sorbitol fatty acid ester 80 and 65% w/v polyethylene glycol 300 in absolute ethanol. The VPD co-solvent system (VPD: 5W) was obtained by diluting VPD with 5% aqueous dextrose 1: 1. This co-solubilising system solubilises hydrophobic compounds well and is itself of low toxicity to systemic administration. Of course, the proportions of the various parts of the co-solvent system can vary widely without destroying its solubility and toxicity characteristics. Further, the components of the co-solution may vary: for example, other low toxicity non-polar surfactants may be substituted for the polyoxyethylene sorbitol fatty acid ester 80; the molecular weight of the polyethylene glycol can vary; other biocompatible polymers may be substituted for the polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides instead of dextrose.

Alternatively, other delivery systems for hydrophobic molecules may be used. Liposomes and emulsions are well known examples of carriers or vehicles for hydrophobic drugs. Certain organic solvents such as dimethyl sulfoxide, although more toxic, may also be employed. In addition, compounds, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent, can be carried by sustained release systems. A wide variety of sustained release materials have been developed and are well known to those skilled in the art. Sustained release capsules may release compounds for weeks to more than 100 days, depending on their chemical nature. Depending on the chemical nature and biological stability of the therapeutic agent, additional strategies for maintaining protein stability may also be employed.

The pharmaceutical compositions may also include suitable solid or gel carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

7.4.3 effective dose

Pharmaceutical compositions suitable for use in the present invention include those containing an effective amount of the active ingredient to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent or alleviate the symptoms presented by the subject. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the details provided herein.

For any compound used in the methods of the invention, its effective amount can be preliminarily assessed from cell culture assays. For example, a dose can be formulated in animal models to obtain the IC measured for inclusion in cell culture₅₀(i.e., the concentration at which half of the maximal CTGF activity is obtained for the compound tested). Such information can be used to more accurately determine effective dosages for humans.

A therapeutically effective amount is one that causes the patient to experience remission or survivalDose of the molecule for extended periods. Toxicity and therapeutic efficacy of these molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. E.g. for determining LD₅₀(dose of 50% subject death) and ED₅₀(a therapeutically effective dose in 50% of the subjects). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD₅₀And ED₅₀The ratio of (a) to (b). Molecules that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a range of dosage for application to humans. Preferably, the dose of these molecules includes ED₅₀Within a circulating concentration range with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration. The exact formulation, route of administration and dosage can be selected by each physician in accordance with the patient's condition. See, e.g., Fingl et al, 1975, pharmaceutical bases of therapy (The pharmaceutical Basis of Therapeutics), Chapter I.

The dose and dosing interval are adjusted for each patient so that the plasma concentration of the active moiety is sufficient to maintain the induction effect of CTGF, or the lowest effective concentration (MEC). MEC will vary for each compound but can be estimated from in vitro data; for example, the concentration necessary to achieve 50-90% CTGF activity to induce bone growth is determined using the assay described herein. The dosage necessary to achieve the MEC will depend on the identity and route of administration of each individual. However, plasma concentrations can be determined using HPLC or biological assays.

Dosing intervals may also be measured using MEC values. The compound should be administered by a method that maintains plasma concentrations above the MEC for a period of 10-90%, preferably 30-90%, most preferably 50-90%.

In the case of topical application or selective absorption, the local effective concentration of the drug may not be correlated with plasma concentration.

The amount of the composition used will, of course, depend on the subject, the weight of the subject, the severity of the disease, the mode of administration and the judgment of the prescribing physician.

7.4.4 packaging

If desired, the compositions may be presented in a pack or dispenser containing one or more units of the dosage form containing the active ingredient. For example, the package may comprise a metal or plastic foil, such as a blister pack. The packaging or dispensing device may be accompanied by instructions for administration. Pharmaceutical compositions containing a compound of the invention formulated in a suitable pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the indication of therapy. Indications indicated on the label include treatment of disorders or diseases requiring cartilage or bone induction and wound healing, or the like.

7.5. Identification of Compounds that induce CTGF production in cartilage

Identification of promoter elements of the CTGF gene, specifically TGF- β response/regulatory element (T β RE) (5'-GTGTCAACCCTC-3'; nucleotides-157 and-145) provides a means for screening methods to identify compounds or compositions that affect CTGF expression. More specifically, methods for identifying compositions that enhance the activity of CTGF and thus can be used to enhance bone, tissue and cartilage induction include: (1) incubating components such as, but not limited to, oligonucleotides comprising the composition and TGF- β reactive element of CTGF, wherein said incubating is under conditions sufficient to allow interaction of the components; and (2) determining the effect of the composition on CTGF expression. Preferably, the promoter regions used in the screening assays described herein include nucleotides-823 to +74, although less extensive regions that include TGF- β response elements may also be used in the methods disclosed herein (e.g., -162 to-128, or-154 to-145). In other assays, nucleotides in this region, including the T.beta.RE, are coupled to a receptor gene such as luciferase and transfected into mammalian cells to obtain cell lines which carry the constructed gene and which exhibit activity when incubated with TGF-beta. These candidate drugs, oligonucleotides, and compounds that modify activation function are readily detected in cellular assays. 8. Examples of the invention

The connective tissue growth factor gene is not only expressed in fibroblasts, but also selectively induced by TGF- β in connective tissue cells of mesenchymal origin (e.g., fibroblasts, smooth muscle cells, chondrocytes, osteoblasts, astrocytes, etc.). The gene was expressed in connective tissue cells constituting a skeletal component of vertebrates, which suggests that CTGF plays a role in the formation of cartilage, bone, ligament, and muscle in vertebrates. The results of the following examples demonstrate that CTGF modulates the induction, differentiation and growth of cartilage and bone forming cells in vertebrates, including humans. Specifically, these results confirm that: (1) CTGF transcripts were present in the long bone growth plates of adult rats and neonatal mice; (2) the CTGF gene is expressed at the cartilage induction and growth part of an embryonic mouse; (3) CTGF receptors are present on rat chondrocytes; (4) the CTGF gene is expressed at the bone regeneration part after the injury of the adult rabbit; (5) the CTGF protein can induce the differentiation of pluripotent mouse embryonic stem cells into chondrocytes and osteoblasts; (6) cultured human osteoblasts secrete CTGF.

8.1. Biological experiments

The method comprises the following steps: mitogenic and anchorage-independent growth assays. Mitogenic assays were performed in monolayer cultures using 48-well plates and NRK fibroblasts as target cells, as previously described by Grotendorst et al, 1991, J.CellPhysiolo, Vol.149, page 235-243. Anchorage-independent growth experiments were carried out essentially as described by Guadagno and Assoian, 1991, journal of cell biology (J.CellBiol.)115 vol 1572-1575.

The method comprises the following steps: mRNA induction assay of extracellular matrix proteins. NRK rat fibroblasts were cultured to confluence in Dulbecco's modified eagle medium containing 5% fetal bovine serum, and then serum-starved for 24 hours in DMEM containing 1% bovine serum albumin. Growth factors were added to the cell culture medium and total cellular RNA was extracted after 24 hours and subjected to northern blot analysis as described by Igarashi et al, 1993, molecular biology and cells (mol. biol. cell)4, page 637-645.

To ensure that equal amounts of total RNA were added to each lane of the gel, a₂₆₀/A₂₈₀RNA was quantitated by alignment and then stained with ethidium bromide to ensure equal transfer by comparing ribosomal 28S and 18S RNA in each lane. Additional controls were performed using actin cDNA probes. Random primer labeling kit (Boehringer Mannheim, Indianapolis, IN) was used³²P-dCTP labels the double-stranded cDNA fragment used as a probe. The CTGF probe was derived from a 1.1kb human cDNA fragment containing the open reading frame of the CTGF transcript. The TGF-. beta.1 probe is a 1.0kb NarI fragment of a 2.0kb human TGF-. beta.1 cDNA (G.I.Bell, H.H. institute of medicine, university of Chicago). The α I-type 1 human collagen probe is derived from a 1.5kb open reading frame fragment (ATCC No. 61323) at the 3' end. The α 5 fusin probe is derived from a cDNA insert of a portion of human cDNA containing the open reading frame, obtained from r.associates at miami university. The human fibronectin probe was a 0.9kb EcoR1/Hind III fragment from a 2.2kb cDNA clone supplied by F.Woessner (also university of Miami) containing the 3' region of the open reading frame. Human actin probes used as control RNA probes were purchased from Oncor, Co. (Gaithersberg, MD).

8.2 localization of CTGF transcript in neonatal mice

Experiments were performed to determine whether CTGF transcripts were present in the long bone growth plates of neonatal mice according to Fava et al 1990, Blood 76 (Blood) 1946-page 1955.

The method comprises the following steps: and (4) in situ hybridization. Tissue specimens were quickly placed in 4.0% paraformaldehyde for 1.5 hours, then quickly frozen and embedded. The sections were cut to 5 μm and placed on TESPA-coated slides (Oncor, Gathersburg, Md.). CTGF mRNA was hybridised in situ using standard methods. Briefly, slides with specimens were dehydrated by gradient ethanol, treated with 20 μ g/ml proteinase K in 50mM Tris-HCl pH7.4, 5mM EDTA, and then re-fixed in 4.0% paraformaldehyde and soaked in 0.1M triethanolamine and 1ml acetic anhydride prior to dehydration in continuous gradient ethanol. Using the riboprobe kit (Promega, Madiso)n, WI) using T7 and SP6 promoters, respectively, to construct sense and antisense CTGF RNA probes. The specific activity of the probe was 1X 108 cpm/. mu.g RNA. Hybridization was performed overnight at 54 ℃ under a coverslip in 50% deionized formamide. 10% dextran sulfate, 50mM DTT, 0.3M NaCl, 0.01M Tris pH7.5, 5mM EDTA, 10mM Na₂HPO₄0.02% ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.2mg/ml yeast tRNA and riboprobe (5X 104 cpm/. mu.l). The slide was washed in 250ml of 5 XSSC, 10mM β -mercaptoethanol at 50 ℃ for 30 minutes, in 2 XSSC, 100mM β -mercaptoethanol, 50% formamide at 65 ℃ for 20 minutes, and in TEN buffer (1M Tris, 0.5M EDTA, 5M NaCl) for 10 minutes, 3 times in succession. The second TEN rinse included 10 μ g rnase a. The final two washes were performed in 2 XSSC at 65 ℃ for 15 minutes each. After re-dehydration with a gradient of 0.3M ammonium acetate, the slides were immersed in developing emulsion (Ilford K-5, Polyscience) and incubated at4 ℃ for 8 days. Slides were then developed and sectioned for counterstaining in Mayer's hematoxylin and eosin.

And (6) obtaining the result. These findings indicate that the CTGF gene is expressed in the proliferative region of the growth plate. This zone contains chondrocytes that actively proliferate to increase bone length. CTGF expression at this site is consistent with its function to function as a growth factor for chondrocytes.

Expression of CTGF gene at cartilage induction and growth sites in embryonic mice.

To confirm the role of CTGFs in cartilage induction and growth, the expression of CTGF gene in a mouse embryo at a site where cartilage and bone are to be formed but have not yet been formed has been studied. To achieve the goal of this study, a transgenic mouse line was established containing a transgene comprising a human CTGF promoter element that regulates expression of a bacterial galactosidase gene. Cells expressing this gene are readily identified by X-gal staining, which forms a blue precipitate at the enzyme active site. In this way we stained embryos of transgenic mice to locate CTGF gene expression. As shown in panel a of fig. 4, there is no cartilage or bone formation, indicating that CTGF is expressed before bone formation and may act as an inducer of cartilage and bone. The results further confirm that the expression of the transgene is consistent with the expression detected by CTGF probe in situ hybridization.

These studies also demonstrated gene expression in the growth plate of long bone, the precortial region of cartilage and Meckel's cartilage, the latter being the first piece of cartilage formed during mammalian development. These regions are called pre-chondrogenic mesenchyme and can be identified by cell condensation. The CTGF gene is expressed at these sites but not in nearby tissues. Further, CTGF was expressed at these sites the day before cell condensation, which occurred the day before actual chondrogenesis. These results demonstrate that CTGF is present prior to the formation of cartilage or cells with a true chondrocyte phenotype and is consistent with the role of CTGF in inducing cartilage phenotype in undifferentiated stem cells.

Importantly, these studies demonstrated that CTGF is expressed in embryos at sites where bone formation occurs via intramembranous, or endochondral, pathways, suggesting that CTGF can function as a signal for chondrogenesis either from undifferentiated mesenchymal stem cells forming the limbs bone, or from neural crest cells forming cartilage in Meckel's cartilage and long bones of the skull

Localization of CTGF receptors on rat chondrocytes

In order for cells to react with polypeptide factors such as CTGF, they must express cognate receptors for the specific polypeptide factor on the cell surface.

And (4) carrying out balance test. Equilibrium binding assays were performed on confluent monolayers of NRK-49F rat fibroblasts and primary rat articular chondrocytes to determine the number and affinity of CTGF receptors on these cells. And iodinated recombinant human ctgf (rhctgf) at various concentrations for 4 hours at low temperature. Nonspecific binding was determined by inclusion of a 200-fold molar excess of unlabeled ligand. Representative Scatchard plots are shown in fig. 8A (associated with equilibrium binding assays using NRK cells) and fig. 8B (associated with equilibrium binding assays using rat chondroblasts).

And (4) performing a competition test. Several types of cells were tested for CTGF receptor expression, including normal rat kidney fibroblasts, mouse fibroblasts, mink lung epithelial cells and rat articular chondrocytes. By passing¹²⁵Iodination labels CTGF, and radiolabeled CTGF is used in competitive binding assays to determine CTGF receptors on various types of cells. As shown in table 1 below, only NRK fibroblasts and rat articular chondrocytes express high-affinity receptors for CTGF. Mouse fibroblasts hardly express high affinity receptors, and no binding phenomenon was detected on the surface of mink lung epithelial cells.

TABLE 1

Binding characteristics of rhCTGF to various cells

High affinity, low affinity cell types KD (pM) site/cell KD (nM) site/cell NRK 13-232200 350011-2.2126,000-195,000 chondrocytes 213500-48001.0150,000 NIH3T 35-104801.8102.000 MLEC were not assayed

These data indicate that chondrocytes express CTGF and receptors for CTGF and are therefore able to respond to CTGF as a growth-stimulating factor.

Activity of CTGF in inducing differentiation of pluripotent mouse embryonic stem cell lines into chondrocytes and osteoblasts.

CTGF was evaluated for its ability to induce a chondrocyte and osteocyte phenotype in cultured undifferentiated stem cells. Specifically, the C3H10T1/2 cell line was used to assess this biological activity. These cells are a standard and well-established cell line for these types of studies. C3H10T1/2 can be maintained in an undifferentiated state in culture, and then induced to differentiate into skeletal muscle cells, chondrocytes, osteoblasts and adipocytes. Cells treated with CTGF formed chondrocyte colonies and cartilage nodules. Cells treated with 5-azacytidine overnight followed by CTGF overnight differentiated into osteoblasts and osteoid bodies. Differentiation of these cultured cells into muscle and fat cells can be blocked by the presence of CTGF.

More specifically, cells were treated with 5-azacytidine overnight, followed by incubation for 10-14 days, to allow cell differentiation to occur. The effect of 5-azacytidine and CTGF on these cells was then compared, both alone and in combination. Control cultures that were not treated with either agent maintained the cells in an undifferentiated state with monolayer growth. As shown in FIG. 4, the cells treated with 5-azacytidine alone differentiated mainly into skeletal muscle cells (myotubules) and adipocytes. Chondrocytes were not found in the cultured cells. CTGF treated cells (50ng/ml) resulted in the formation of cartilage nodules for 10 days. These nodules were not found under any other conditions. None of the cultured cells treated with FGF, PDGF, EGF or TGF- β induced the production of these nodules, suggesting that CTGF alone is capable of inducing chondrogenesis in undifferentiated mesenchymal stem cells.

Treatment with 5-azacytidine (overnight) followed by CTGF (50ng/ml) for 10 days had a significant effect on the cultured cells. First, the absence of skeletal muscle tubules was not observed, confirming that CTGF was able to prevent cell differentiation into skeletal muscle cells. Second, when there are some cartilage nodules, most of the nodules appear to be osteoid (bone). Thus CTGF is able to induce undifferentiated mesenchymal cells to form chondrocytes and osteoblasts. These results indicate that the factor can be used to stimulate cartilage and bone differentiation at the site of need.

8.6 CTGF gene expression at the site of bone regeneration following injury in adult rabbits.

An experimental model is established to detect the expression of various regulatory and matrix protein genes during the process of injury repair. In this model, a meshed nylon post was implanted into the ilium of the pelvis of a male New Zealand white rabbit (10kg) that had been anesthetized with ether. A 1.1cm diameter hole is drilled in the ilium of the pelvis with a bone drill and the cylinder is then inserted into the hole. The nylon column is fixed by using part of the tissues of the nearby muscular system and ligaments. Two nylon columns were implanted per transplant in 20 animals.

Animals were sacrificed on days 9, 14, 21, 24, 28, 31, 35, 42, and 56, respectively, after implantation of the nylon column. The nylon column was removed and the tissue on the outside of the nylon column was carefully and completely separated. The nylon column was cut open and the tissue contained within the column was collected.

Total RNA was extracted from tissues obtained from 6 to 18 nylon columns (pooled from 1 to 3 animals) by guanidinium isothiocyanate extraction (Chomcaynski and Sachi, 1987, analytical Biochemistry 162: 156. sup. 159) and cesium chloride centrifugation (Chirgwin et al, 1979, Biochemistry 18: 5294. sup. 5299). The amount of RNA recovered in the tissues collected on different days was between 100 and 300. mu.g. Total RNA was electrophoresed on agarose/formalin gels and then transferred to nitrocellulose membranes. Equal amounts of RNA were transferred to nitrocellulose membranes as judged by staining for ribosomal RNA present in each sample. The CTGF probe is a 900 base pair fragment representing the open reading frame of the CTGF cDNA. The kit was labeled with random primed DNA (Boehringer Mannheim biochemicals Indianapolis, IN, supra., [ solution ]³²p]dCTP labeled Probe, 1X 10⁶cpm/ml of these probes were used to perform hybridization assays. Autoradiography was performed using X-ray film and intensity at-70 ℃ for 24-72 hours.

Blood clotting at the repair site is followed by inflammation, followed by proliferation of connective tissue, which proliferates through certain repair chains. In bones implanted with nylon posts, the dense connective tissue formed is at least similar if not identical to those formed in soft tissue implants.

As shown in fig. 5, the gene expression of CTGF was evident from days 14 to 42. This was 4 days before the first appearance of the histological appearance of the bone in the nylon cavity and was consistent with the time course of bone formation in the cavity. However, as also shown in FIG. 5, some changes in morphology occurred in the connective tissue region approximately 17-18 days after implantation. These areas then began to form bone by day 20-21 after implantation, suggesting that this is a functional model for studying bone regeneration.

The expression of CTGF mRNA in the lumen was slightly advanced and then coincided with the formation and growth of a bone-forming region in the lumen, indicating that CTGF was expressed at the site of bone regeneration in mammals.

8.7 promotion of human osteoblast formation by Using CTGF

And (5) culturing the cells. Human osteoblasts were cultured from a graft of human bone. Containing 10% CO at 37 ℃₂And an environment of 90% air; the cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Calf Serum (FCS).

Western blot analysis. CTGF products in conditioned medium were analyzed by SDS-PAGE on a 12% acrylamide gel, followed by electrotransfer to nitrocellulose membrane. The hybridization membranes were incubated for 1 hour in Tris-buffered saline (100mM sodium chloride, 50mM Tris-HCl pH7.4) containing 2% skim powdered milk (TBS-milk) before overnight incubation with 2g/ml of chicken anti-human CTGF IgY diluted in TBS-milk. The hybridization membrane was washed 5 times in TBS-milk for 5 minutes each, and then incubated with alkaline phosphatase-crosslinked affinity-purified rabbit anti-chicken IgY (1: 1,000 dilution, organic Teknika-Cappel, WestChester, Pa.) in TBS-milk for 90 minutes. The hybridization membrane was washed 3 times with TBS-milk followed by 2 times in TBS before antigen detection with commercial alkaline phosphatase substrate kit (Sigma.St.Louis.MO).

And (6) obtaining the result. In an operation for removing a bone tumor or joint replacement, human osteoblasts are obtained from a donor after surgical removal of bone. Osteoblasts were cultured from bone and identified with standards. The cells were cultured in complete medium containing 10% fetal bovine serum to confluence, and then the medium was changed to serum-free medium, and the cells were stopped from growing by overnight culture. Some cultured cells were treated with TGF-. beta.and compared to non-treated cultured cells. Osteoblasts treated with TGF-beta are stimulated to secrete CTGF, which is detected using a specific anti-CTGF antibody. As shown in fig. 5, the medium was collected and CTGF production and secretion was analyzed by immunopurification of CTGF with an antibody specific to CTGF, and CTGF was detected and quantified by Western blotting using the same antibody. TGF- β induces human osteoblasts to produce CTGF as observed with fibroblasts, smooth muscle cells and chondrocytes. Untreated control cells were unable to synthesize detectable amounts of CTGF.

As demonstrated by this experiment, osteoblasts react with TGF- β similarly to other connective tissue cells with respect to CTGF production.

8.8. Transgenic rabbit model

All experimental studies in mice were performed in accordance with the principles and methods outlined in the "guidelines for the protection and use of experimental animals". The establishment of transgenic animal models was carried out at the university of miami transgenic mouse core facility using standard techniques. Briefly, the gene to be injected (transgene) is linearized by restriction enzyme digestion, and the DNA fragments are separated by low-melting agarose gel electrophoresis and purified by GENECLEAN.

Transgenic mice were prepared by injecting linear DNA into a pronucleus of-100-300 recently fertilized mouse egg cells. Hogan et al, 1986, manipulation of mouse embryos, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. These post-injection surviving ova were transferred into the oviduct of pseudopregnant mice (male mice that had been mated with vasectomized sperm). Rat tail biopsies were taken from young mice one to three weeks after birth and genomic DNA was analyzed by Southern blot to detect the presence of the transferred gene. Transgenic mouse lines were established by crossing transgenic positive mice with control mice. As a result of these experiments, two independent transgenic mouse lines expressing β -galactosidase under the control of the CTGF promoter were established. Both mouse lines showed similar expression patterns.

8.9. Chondrogenesis test

CTGF and TGF-. beta.were detected in chondrogenesis assays as described by Seydin et al, 1983, journal of cell Biology (J.cell Biology)97, pp.1950-53. Briefly, muscle tissue was isolated from limbs of a 19-20 day old Sprague-Dawley embryo and cut into pieces, and primary culture cells of embryonic muscle were obtained from cells that grew from these pieces of muscle tissue. For chondrogenesis experiments, cells were trypsinized and embedded in agarose, followed by addition of medium containing no factors (FIG. 7A), TGF- β alone (FIG. 7B), TGF- β and cholera toxin (FIG. 7C) or TGF- β, cholera toxin and CTGF (FIG. 7D). For each experiment, the medium was changed 1 time every 2-3 days, and then stained with toluidine blue after 21 days of culture, as described in Horwitz and Dorfman, 1970, J.Cell Biol., 45, 434-438.

As shown in fig. 7A-7D, significant chondrocyte growth was observed at the culture sites where CTGF was added to the medium, indicating that CTGF stimulated chondrocyte growth and the production of connective tissue matrix.

The scope of the invention is not intended to be limited to the specific embodiments illustrated as one aspect of the invention, and functionally equivalent methods are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. It is intended that the appended claims cover such modifications.

All references cited in the specification of the present invention are incorporated herein by reference in their entirety. Biological preservation

The sequence of CTGF in the present invention was deposited in the gene bank at 26.7.1990, Los Alamos National Laboratory, Los Alamos, New Mexico, 87545, usa and given accession number M36965. The collection of CTGF sequences is merely illustrative and applicants should not be asked to acknowledge that such collection is necessary to protect the claimed subject matter.

This is possible for all specified countries and is legal according to the laws of the specified countries, and it is necessary for an independent expert to obtain a sample of deposited microorganisms after the invention has been granted, which complies with the relevant patent regulations, such as EPC rule 28(4), United kingdom patent rule 17(3) in 1982, Australian code (Regulation)3, 25(3) and generally similar applicable regulations for any other specified country.

Claims (19)

1. A pharmaceutical composition containing CTGF.

2. A method for inducing bone formation comprising administering to a patient in need thereof a composition comprising CTGF and a pharmaceutically acceptable carrier.

3. The method of claim 2, wherein the composition further comprises a second growth factor.

4. The method of claim 3, wherein the second growth factor is TGF- β.

5. The method of claim 2, wherein the composition further comprises at least one collagen.

6. The method of claim 2 wherein the patient is suffering from a disease that affects bone formation.

7. The method of claim 6, wherein the disease is selected from the group consisting of osteoporosis, osteoarthritis and osteochondritis.

8. A method for inducing tissue formation comprising administering to a patient in need thereof a composition comprising CTGF and a pharmaceutically acceptable carrier.

9. The method of claim 8, wherein the composition further comprises a second growth factor.

10. The method of claim 8, wherein the second growth factor is TGF- β.

11. The method of claim 8, wherein the composition further comprises at least one collagen.

12. A method for inducing chondrogenesis comprising administering to a patient in need thereof a composition comprising CTGF and a pharmaceutically acceptable carrier.

13. The method of claim 12, wherein the composition further comprises a second growth factor.

14. The method of claim 13, wherein the second growth factor is TGF- β.

15. The method of claim 12, wherein the composition further comprises at least one collagen.

16. A method for inducing wound healing comprising administering to a patient in need thereof a composition comprising CTGF and a pharmaceutically acceptable carrier.

17. The method of claim 16, wherein the composition further comprises a second growth factor.

18. The method of claim 16, wherein the second growth factor is TGF- β.

19. The method of claim 16, wherein the composition further comprises at least one collagen.