Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-06-03
2002-03-05
Romeo, David S. (Department: 1647)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S484000, C424S426000, C530S350000
Reexamination Certificate
active
06352972
ABSTRACT:
BACKGROUND OF THE INVENTION
The Transforming Growth Factor beta (TGF&bgr;) superfamily is a large group of cytokines that exert profound influences on the physiology of wound healing. Their mode of action in wound healing includes the modulation of stem cell populations, as well as their expression of specific genes that encode matrix proteins, cellular receptors, matrix proteinases and proteinase inhibitors. Numerous animal studies have demonstrated the efficacy of exogenous TGF&bgr; in promoting wound healing, which lead to the first clinical applications in the repair of bone, surgical wound healing and in the treatment of diabetic ulcers and burns. Moreover, a single systemic dose of TGF&bgr;
1
, given prior to injury (surgery), has been demonstrated to enhance tissue repair and wound healing, suggesting that a single dose, administered systemically before surgery, may improve patient recovery rates.
Clinical studies using TGF&bgr;
1
as a therapeutic agent have been hampered by its limited availability. TGF&bgr;
1
is usually purified from either human platelets, bone or soft tissues such as placenta and kidney. It is estimated that approximately one ton of bone is required to purify enough TGF&bgr;
1
for a single therapeutic treatment. Small amounts of TGF&bgr;
1
have been isolated as a recombinant protein which was processed and secreted by transfected mammalian cells into conditioned growth medium. However, the small amounts of TGF&bgr; obtained and the high cost of production do not make this a method of production commercially viable.
The potential utility of TGF&bgr;
1
as a clinical agent to promote wound healing is complicated by TGF&bgr;
1
's potent chemo-attractant and its macrophage and fibroblast activation properties. At elevated levels of TGF&bgr;
1
such as occur in chronic fibrotic disorders, especially when local inflammation persists, macrophages and fibroblasts accumulate at the site of the disease. Elevated plasma levels of TGF&bgr;
1
has been shown to correlate with a high incidence of hepatic fibrosis, and has also been associated with glomerulosclerosis and pulmonary fibrosis. Therefore, delivery to and activation of TGF&bgr;
1
at the site of a wound is desirable for prolonged treatment with TGF&bgr;
1
.
Three distinct TGF&bgr; polypeptides have been identified and are designated TGF&bgr;
1
, TGF&bgr;
2
and TGF&bgr;
3
. The TGF&bgr; proteins are expressed as precursor molecules of 380, 442 and 410 amino acids, respectively. These inactive latent TGF&bgr; proteins are activated by proteinases such as plasmin, latent TGF&bgr;
1
binding protein (LTBP) and thrombospondin. The mature form of the proteins are dimers of identical polypeptide chains of 112 amino acids in length. The amino acid sequence of the TGF&bgr;
1
, TGF&bgr;
2
and TGF&bgr;3 polypeptides shows 70 to 80% homology and the sequence conserved in the mature polypeptides includes 9 cysteine residues which determine the inter- and intra-polypeptide disulfide bridge formation in the mature proteins.
LTBP and a 60 kD TGF&bgr;
1
binding protein appear to mediate the binding of TGF&bgr;
1
to the extracellular matrix. The close association of TGF&bgr;
1
with the extracellular matrix possibly maintains the elevated growth factor concentration within the local environment of the healing wound.
A subset of the TGF&bgr; proteins are the bone morphogentic proteins (BMP). In postnatal life mammals continuously remodel their skeleton and retain the ability to initiate repair in response to injury and trauma. The cellular and molecular basis for this bone morphogenesis and repair has been attributed to the BMP's and their responding inducible osteogenic precursor cells. Bone marrow has cells with osteogenic potential and consists of determined osteogenic precursor cells that are committed to osteogenesis. Determined osteogenic precursor cells can differentiate into bone without an exogenous signal. Inducible osteogenic precursor cells requires a molecular signal for initiating the differentiation program. BMP's effects include: chemotaxis of monocytes and mesenchymal cells, mitosis of mesenchymal progenitor cells; initiation of differentiation; inhibition of proliferation of differentiated cells; promotion of osteoblast phenotype; maintenance of bone; and cartilage phenotype and binding to extracellular matrix.
The BMP's include at least 9 different proteins, named: BMP-2 (also known as BMP-2A); BMP-3 (also known as osteogenin); BMP-4 (also known as BMP-2B); BMP-5; BMP-6; osteogenic protein-1 (OP-1, also known as BMP-7); osteogenic protein-2 (OP-2, also known as BMP-8); BMP-3b and GDF-10 (also known as BMP-10). There are several subgroups among the BMP's as follows: BMP-2 and 4; BMP-5 to 8; and BMP-3 and GDF-10 share physical properties. All BMP's are expressed as a protein of about 300 to about 400 amino acid residues. The proteins are subsequently processed to a mature region having between about 100 to about 200 amino acids at a consensus sequence of Arg-X-X-Arg SEQ ID NO: 49.
The use of TGF&bgr; or BMP-based medical therapies require the availability of large quantities of pharmaceutical grade TGF&bgr; and BMP that is free of transmittable hazards omnipresent in products extracted from animal, in particular human, sources. Therefore, it is desirable to develop a means for preparing large quantities of the mature TGF&bgr; and BMP. It is also desirable that the protein(s) is (are) made from a source which eliminates the possibility of hazardous material contaminating the final product. It is also desirable that the protein is engineered to target specific sites where wound healing is desired.
SUMMARY OF THE INVENTION
The present invention is directed at a bone morphogenetic fusion protein, a method of preparation of the bone morphogenetic fusion protein and methods of using the bone morphogenetic fusion protein.
The bone morphogenetic fusion protein comprises a purification tag and a bone morphogenetic active fragment.
The method of preparation bone morphogenetic fusion protein comprises purifying and renaturing bone morphogenetic protein to provide an active bone morphogenetic fusion protein preparation.
Methods of use of the bone morphogenetic fusion protein include methods to promote bone growth at the site of a fracture and as a replacement for bone in performing bone grafts.
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Sampath et al. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J. Biol. Ch Oct. 1992.*
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Bowie et al. Deciphering the message in protein sequences: tolerance to amino acid substitutions. Science, (Mar. 16, 1990) 247 (4948) 1306-10.*
Kingsley D M. The TGF-superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes and Development, (Jan. 1994) 8 (2) 133-46.*
Shah M; Foreman D M; Ferguson M W. Neutralization of TGF-1 and TGF-2 or exogenous addition of TGF-3 to cutaneous rat wounds reduces scarring. J. Cell Sci., (Mar. 1995) 108 (Pt 3) 985-1002.*
Reddi A. H. Role of morphogenetic proteins in skeletal tissue engineering and regeneration. Nat Biotechnol, (Mar. 1998) 16 (3) 247-52.*
Reddi A. H. Bone morphogenetic proteins: an unconventional ap
Hall Frederick L.
Han Bo
Nimni Marcel E.
Shors Edwin C.
Wu Lingtau
Oppenheimer Wolff & Donnelly LLP
Romeo David S.
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