Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-03-22
2003-11-25
Wessendorf, T. D. (Department: 1639)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S023000, C530S327000, C530S300000
Reexamination Certificate
active
06653285
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to peptide inhibitors of glycosaminoglycans. This invention also relates to formulations, uses and methods of identifying such inhibitors.
BACKGROUND OF THE INVENTION
The extracellular matrix (ECM) is a dynamic assemblage of interacting molecules that regulate cell functions and interactions in response to stimulation. One class of extracellular matrix macromolecules, the glycosaminoglycans, are molecules known to be involved in a wide array of both normal and abnormal biological processes, including cell migration, differentiation, proliferation, immune response and cytoskeletal organization.
The glycosaminoglycan hyaluronan (HA) is a repeating disaccharide of [GlcNAc&bgr;1-4GlcUA&bgr;1-3]
n
that exists in vivo as a high molecular weight linear polysaccharide. HA is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid, and is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with HA creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. HA plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole,
Cell Biol. Extracell. Matrix,
Hay (ed), Plenum Press, New York, 1384-1386 (1991); Bertrand et al.
Int. J. Cancer
52:1-6 (1992); Knudson et al,
FASEB J.
7:1233-1241 (1993)). HA levels have been shown to correlate with tumor aggressiveness (Ozello et al.,
Cancer Res.
20:600-604 (1960); Takeuchi et al.,
Cancer Res.
36:2133-2139 (1976); Kimata et al.,
Cancer Res.
43:1347-1354 (1983)), and can be indicative of the invasive properties of tumor cells. M. M. Knupfer et al.,
Anticancer Res
18:353-6 (1998).
HA is also involved in immune response, and thus may mediate this response in both normal and abnormal biological reactions. Increased binding of HA to one of its receptors, CD44, has been shown to mediate the primary adhesion (“rolling”) of lymphocytes to vascular endothelial cells under conditions of physiologic shear stress, and this interaction mediates activated T cell extravasation into an inflamed site in vivo in mice. H. C. DeGrendele, et al.,
J. Exp. Med.
183:1119-1130 (1996); H. D. DeGrendele,et al.,
Science
278:672-675 (1997). H. C. DeGrendele et al.,
J. Immunol.
159:2549-2553 (1997). Alterations in levels of HA and other glycosaminoglycans have also been associated with unwanted immune responses, and may be involved in diseases and disorders such as rheumatoid arthritis, atopic dermatitis, psoriasis, multiple sclerosis, transplantation rejection. For example, HA and other glycosaminoglycans display are altered in autoimiune disorders such as arthritis, and decreased levels of both hyaluronic acid and chondroitin 6-sulfate have been found in the diseased synovial fluid of both adults with rheumatoid arthritis (A. Bensouyad et al.,
Ann Rheum Dis
49:301-7 (1990)) and children with juvenile rheumatoid arthritis (P. F. Spelling et al.
Clin Exp Rheumatol
9:195-9 (1991)).
Dendritic cells (DC) play essential roles in the induction of cellular immune responses to a variety of relevant antigens. DC are known to play critical roles in the induction of cellular immune responses against a wide variety of antigens of relevance, including chemical haptens, foreign proteins, infectious microbes, and tumor-associated antigens (Steinman, “The dendritic cell system and its role in immunogenicity.”
Ann. Rev. Immunol.
9:271 (1991); Stingl et al., “The Epidermis: An Immunologic Microenvironment In Dermatology in General Medicine.” T. B. Fitzpatrick, ed. McGraw Hill and Co., New York, p. 172 (1993)). Interaction between HA, expressed on endothelial cells, and CD44, expressed on activated dendritic cells as well as T cells, and granulocytes, is believed to mediate homing of such leukocytes to their target sites.
Glycosaminoglycans, and particularly HA, are also known to mediate other cellular interactions that involve binding and entry into a cell. For example, HA is involved in infection of mammalian cells by the Human Immunodeficiency Virus (HIV), since HIV is known to bind to HA upon infection. Both HA and monoclonal antibodies to its receptor CD44 were found to inhibit HIV infection of monocytes by monocytotropic HIV. M. C. Levesque and B. F. Haynes,
J. Immunol
156:1557-65 (1996). HA is also involved in mammalian zygote formation by mediating binding of the oocyte and the sperm. Data suggests that HA in the cumulus matrix may act to prime the fertilizing sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida. HA is thought to mediate this interaction by binding to the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiate the acrosome reaction. K. Sabeur et al.,
Zygote
6:103-11 (1998). HA mediates sperm motility by enhancing phosphorylation of proteins including HA binding protein. S. Ranganathan et al.,
Cell Mol Biol Res
41:467-76 (1995).
The role of glycosaminoglycans, and particularly hyaluronic acid, in such varying physiological processes make them attractive targets for therapeutic agents. Unfortunately, glycosaminoglycans have been found to be nearly non-antigenic, and very few antibodies that recognize glycosaminoglycans have been isolated. Due to this lack of antigenicity, it has been technically difficult to develop inhibitors or probes of glycosaminoglycans. Thus, there is a need in the art for inhibitors of glycosaminoglycan-mediated processes, and in particular for inhibitors of hyaluronic acid-mediated processes. There is also a need for a method of identifying effective glycosaminoglycan inhibitors in a systematic, reproducible manner.
SUMMARY OF THE INVENTION
The present invention provides peptides with a specific affinity for glycosaminoglycan molecules. These peptides exhibit any number of functions, including but not limited to inhibitors of glycosaminoglycan-mediated processes, enhancers of glycosaminoglycan-mediated processes, and as molecular probes to identify the presence of a specific glycosaminoglycan. Peptides of the invention are administered in order to inhibit, alter the interaction of, or otherwise affect the activity or function of any glycosaminoglycan, including hyaluronic acid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, heparin, keratan sulfate, keratosulfate, chitin, chitosan 1, and chitosan 2. These isolated peptides are formulated and administered by injection for the treatment or prevention of diseases involving viral infection, inflammatory diseases, cancer, infections, etc. Moreover, these peptides are formulated and administered for the stimulation of normal biological responses, such as wound healing, angiogenesis, etc. The peptides of the invention cab be labeled directly or indirectly and as such are useful in in vitro, ex vivo or in vivo probes to determine the presence of a particular glycosaminoglycan in a biological sample and/or patient.
In a preferred embodiment, the invention provides isolated and substantially purified peptides which specifically bind to and as such inhibit or otherwise affect the activity of HA. The isolated peptide inhibitors are characterized by aliphatic or polar aliphatic residues at positions 4, 5 and 6 and/or 9, 10 and 11.
An aspect of the invention is a composition comprising a carrier material and an active ingredient. The active ingredient is characterized by all or any of (1) specifically and selectively binding to a glycosaminoglycan which is preferably hyaluronic acid; (2) inhibiting the normal function of or altering the normal interactions of or otherwise affecting the normal activity of a glycosaminoglycan; (3) having an amino acid sequence corresponding to any of SEQ ID NOS:1, 2, 3, 4 or 5; or (4) having sufficient homology with any of SEQ ID NOS:1, 2, 3, 4 or 5 so as to present a structure characterized by (1) or (2) above.
Mohamadzadeh Mansour
Mummert Mark E.
Takashima Akira
Bozicevic Field & Francis LLP
Francis Carol L.
The University of Texas System
Wessendorf T. D.
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