Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-09-29
2003-01-21
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S329000, C530S399000
Reexamination Certificate
active
06509318
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to agents that inhibit the activity of TGF-&bgr; for uses such as scar tissue inhibition during wound healing, and more particularly to small peptides with potent anti-transforming growth factor &bgr; activity and useful in countering the effect of increased TGF-&bgr; activity seen in many pathological conditions.
2. Description of Related Art
Increased levels of transforming growth factor &bgr; (TGF-&bgr;) activity are involved in a large number of pathologic conditions, including, but not limited to, the following: (i) fibrosis, scarring, and adhesion during wound healing; (ii) fibrotic diseases of the lungs, liver, kidneys; (iii) atherosclerosis and arteriosclerosis; (iv) certain types of cancer including cancer of the prostate, neuroendocrine tumors of the digestive system, cancer of the cervix, glioblastomas, and gastric cancer; (v) angiopathy, vasculopathy, nephropathy; (vi) systemic sclerosis; (vii) viral infection such as hepatitis C and HIV; and (viii) immunological disorders and deficiencies.
For example, members of the TGF-&bgr; family are among the peptides known to have a number of biological activities related to tumorigenesis (including angiogenesis) and metastasis. TGF-&bgr; inhibits the proliferation of many cell types including capillary endothelial cells and smooth muscle cells. TGF&bgr; downregulates integrin expression (&agr;1&bgr;1, &agr;2&bgr;1, &agr;v&bgr;3 involved in endothelial cell migration). Integrins are involved in the migration of all cells, including metastatic ones. TGF-&bgr; downregulates matrix metalloproteinase expression needed for both angiogenesis and metastasis. TGF-&bgr; induces plasminogen activator inhibitor, which inhibits a proteinase cascade needed for angiogenesis and metastasis. TGF-&bgr; induces normal cells to inhibit transformed cells.
Transforming growth factor-&bgr;s were originally named for their ability to transform normal fibroblasts to cells capable of anchorage-independent growth. The effects of TGF-&bgr;s on cells are generally classified as proliferative and non-proliferative. As originally established with the first experiments on fibroblasts, TGF-&bgr;s are bona fide growth factors. Two important cell types in which proliferation is enhanced by TGF-&bgr; are osteoblasts and Schwann cells of the peripheral nervous system. However, in many cells, TGF-&bgr;s are potent inhibitors of cell proliferation. This negative growth control may be the regulatory mechanism that checks regeneration of certain tissues and may play a role in the initiation of carcinogenesis.
The most important non-proliferative function of TGF-&bgr;s are in enhancing the formation of extracellular matrices. Although this is achieved primarily through the increased transcription of both collagen and fibronectin, the inhibition of the proteases from degrading the matrix also contributes to its stability. Degradation of the extracellular matrix is inhibited by the decrease in the secretion of the proteases themselves and the simultaneous increase in the levels of protease inhibitors.
Because of the wide applicability of TGF-&bgr;s in clinical therapies, they have been the focus of much research. Although much of the research involved in vitro uses, recent in vivo studies have confirmed some of the more promising in vitro effects.
The natural members of the transforming growth factor-&bgr; family range upwards of 25 KDa molecular weight. Clinical uses of growth factors, including TGF-&bgr;s, may be limited because of their size, which can cause immune responses. For example, human TGF &bgr;-1 is a 25,000 dalton homodimeric protein. In addition to possible adverse immunological responses, large proteins are not often the best candidates for drugs because of the difficulties in administration and delivery.
Because of the involvement of TGF-&bgr; in a large number of serious pathological conditions, there is considerable interest in developing inhibitors of TGF-&bgr;. Many of the proposals for TGF-&bgr; inhibitors have involved antibodies.
For example, U.S. Pat. No. 5,662,904, issued Sep. 2, 1997, inventors Ferguson et al., describe a composition for use in treating wounds to inhibit scar tissue formation. An exemplary such composition has growth factor neutralizing antibody, such as antibodies to TGF &bgr;-1, TGF &bgr;-2, and PDGF.
U.S. Pat. No. 5,972,335, issued Oct. 26, 1999, inventors Ferguson et al., disclose compositions comprising at least two antibodies for use in promoting wound healing or fibrotic disorders, where the first antibody is specific for a single epitope on TGF &bgr;-1 and the second antibody is specific for a single epitope on TGF &bgr;-2.
U.S. Pat. No. 6,062,460, issued May 9, 2000, inventor Ferguson, discloses the use of soluble betaglycan (otherwise known as TGF-&bgr; receptor III) for use in promoting wound healing of fibrotic disorders with reduced scarring.
U.S. Pat. No. 5,958,411, issued Sep. 28, 1999, inventors Logan and Baird, disclose methods for treating a CNS pathology by administering neutralizing anti-TGF-&bgr; antibodies.
However, antibodies and receptors are large proteins. Because of the amounts of TGF &bgr;-1 in serum of subjects with diseases such as prostate cancer (which are as high as 35 ng/ml), very large quantities of “neutralizing” proteins, such as antibodies, would need to be administered. This poses a difficult problem and adverse effects can be anticipated. Furthermore, while the amount of growth factor in the extracellular fluid is constantly increasing because of secretion from diseased cells, the number of cells and receptors is not likely to increase at the same rate. Therefore, a method that would target the receptor should mean that smaller amounts of a receptor inhibitor can be administered.
BRIEF SUMMARY OF THE INVENTION
In one aspect of the present invention, a family of small peptides have been found to be inhibitory to TGF &bgr;-1 and which preferably have the primary structure of Formula I.
AA
1
-AA
2
-AA
3
-Pro-D-Glu-Ala FORMULA I
The N-terminal amino acid residue designated AA
1
may include a blocking group, for example may be acylated, and preferably is leucine, phenylalanine, &agr;-aminoisobutric acid (Aib), N-methylalanine (NmeA), N-methylisoleucine, or isoleucine; AA
2
is the same or a different amino acid residue as described for AA
1
; and AA
3
is alanine or N-methylalanine. The C-terminal alanine may also include a blocking group, for example, may be amidated or esterified. Three particularly preferred such inhibitors are Glu-Ile-Ala-Pro-D-Glu-Ala (sometimes hereinafter “CITA-I”), Phe-Ile-Ala-Pro-D-Glu-Ala (“CITA-II”), and Leu-Phe-Ala-Pro-D-Glu-Ala (“CITA-III”). These three particularly preferred embodiments markedly reverse the inhibition of DNA synthesis caused by TGF &bgr;-1.
Because the TGF-&bgr; inhibitors of the present invention are small peptides, they are easily synthesized, obtained in highly pure form, and can be readily delivered in a wide variety of modalities. For example, the Formula I peptides may be applied topically to wounds so as to prevent adhesions and scarring, they may be delivered by injection to affect the behavior of tumors, fibrotic tissues, sites of inflammation, and to modulate the behavior of cells of the immune system.
DETAILED DESCRIPTION OF THE INVENTION
In our copending application Ser. No. 09/113,696, filed Jul. 10, 1998, inventors Bhatnagar, Qian, and Gough, of common assignment herewith, we describe a series of small peptides we called “cytomodulin analogs” which were found active as TGF-&bgr; agonists. One of these was L-I-A-P-(D-Glu)-A. These cytomodulin analogs were suggested as having utility in surgery as agents which promote wound healing and regeneration.
In the subject application, the earlier L-I-A-P-(D-Glu)-A, which we had studied for its activity as a TGF-&bgr; agonist, together with now other,
Bhatnagar Rajendra S.
Gough Craig A.
Qian Jing Jing
Bozicevic Field & Francis LLP
Francis Carol L.
Gupta Anish
Low Christopher S. F.
The Regents of the University of California
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