Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2001-08-02
2004-12-07
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S007240, C435S007720, C435S007920
Reexamination Certificate
active
06828091
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is in the field of assays useful for the screening of chemical compounds able to serve as immunosuppressive agents.
The present invention arises from the discovery of a method for the identification of agents that selectively induce apoptosis in activated T lymphocytes. Compounds of this nature are useful as therapeutically effective immunosuppressive agents. In particular, the invention relates to the use of primary T cell cultures to identify compounds that directly or indirectly activate the caspase cascade.
Also taught are methods for using the immunosuppressive agents and pharmaceutical compositions for their use.
2. Related Art
Immunopathological Diseases: The immune system is a remarkably evolved defense system in vertebrates for protection against pathogenic microorganisms. The same immune system can also lead to various pathological conditions. For example, the immune system can cause rejection of grafts during transplantation (Rosenberg, A. and S. Singer,
Annu. Rev. Immunol.
10:333 (1992)). Also, graft versus host disease (GvHD) develops when a graft containing immunocompetent T cells recognize and react with the recipient's cells (Woo, S.-B., et al.,
Crit. Rev. Oral. Biol. Med.
8:201 (1997)). Mechanisms of self-tolerance normally protect an individual from self-reactive T lymphocytes. However, should these mechanisms fail, an inappropriate immune response occurs leading to what is known as autoimmunity. Rheumatoid arthritis is a well-known example of autoimmunity. In this degenerative condition, auto-reactive T cells destroy the tissue around the joints causing inflammation and tissue destruction.
Transplantation or Graft Rejection: A problem arises during kidney, cardiac, lung or liver transplants and skin grafts when the host immune system recognizes the transplant graft as foreign tissue and develops immune reactivity that ends in rejection of the transplanted/grafted tissue. Several attempts are being made to induce immunological tolerance across the major histocompatibility complex (MHC) barriers. This is generally achieved by three mechanisms:
1) clonal deletion of the activated antigen/MHC reactive lymphocytes:
2) clonal anergy and suppression on the other hand by antibody mediated blockade of the gene expression; or
3) Suppression of one subset of the T cells (Th1) and expansion of the other (Th2) in situations of cardiac allografts studies were also partly successful (Bach. F., et al.,
Nat. Med.
3:196-204 (1997); Sayegh, M H., et al.,
J. Exp. Med.
181:1869-1874 (1995).
Graft-Versus-Host Disease (GvHD): GvHD is the most important complication of bone marrow transplantation (BMT) (Ferrara, J. and H. Deeg,
N. Engl. J. Med.
324:667 (1991)). When competent T cells are transferred from a donor to a recipient who is incapable of rejecting them, the grafted cells survive, start recognizing the host antigens and develop immune reactivity towards them. Instead of the normal transplantation reaction of host versus graft, the reverse is seen in this case. Research indicates that increased donor T cell and monocyte/macrophage expansion and inflammatory cytokines are responsible for this syndrome (Via, C., et al,
J. Immunol.
157:5387 (1996): Krenger, W., et al,
Transplantation
64:553 (1997); Hattori, K., et al.,
Blood
91:4051 (1998); Mori, T., et al.,
Blood
92:101 (1998)).
Autoimmune Diseases: Among this group of diseases is rheumatoid arthritis which is a chronic inflammatory disease of the joints, characterized by infiltration of T lymphocytes into the synovial fluid and eventual destruction of the cartilage and bones in the affected joints. Several studies have suggested that the infiltrating T lymphocytes are activated and cause neighboring tissue destruction. Other autoimmune diseases due to autoreactive T lymphocytes include multiple sclerosis, insulin-dependent diabetes mellitus, lupus, and muscular dystrophy (Liblau, R., et al.,
Immunol. Today
16:34 (1995)).
Immunosuppressive Agents: Current immunosuppressive treatments result in generalized immunosuppression and leave the patient prone to various infections. These therapies are also aimed at slowing down the proliferation of activated T cells and thereby due to lack of specificity, effect the growth of all normal dividing cells and result in side effects and toxicity. The primary methods of treatment for immunopathological disorders such as graft or transplantation rejection. GvHD and rheumatoid arthritis are corticosteroid and immunosuppressive agents. Current immunosuppressive drugs like cyclosporin A (CsA) and FK506 work by blocking a calcium dependent protein phosphatase calcineurin (Cn), but they often have unwanted side effects such as cancer, kidney failure, and diabetes. Progress is being made in enhancing the effectiveness of each of these agents.
Despite reduced side effects from immunosuppression, certain tissue transplantations still result in morbidity and mortality. Because of the frequent occurrence of corticosteroid related side effects in transplant patients, alternative therapeutic agents are desirable for these and other related disorders. One such therapeutic agent is methotrexate (MTX), a folate antagonist first developed for malignancies (Farber, S., et al.,
Advances in Cancer Res.
2-73 (1956)) and subsequently used as an anti-inflammatory and/or immunosuppressive drug. MTX is now the most commonly used treatment for rheumatoid arthritis (Weinblatt, M., et al.,
N. Engl. J. Med.
312:818-822(1985); Williams, B., et al.,
Arthritis Rheum.
28:721-730 (1985)).
Apoptosis: A normal checkpoint in the life of cells in multicellular organisms is the process of apoptosis (see, e.g., Evan and Littlewood,
Science
281:1317-1322 (1998)). Apoptosis is the highly conserved mechanism by which cells commit suicide. Characteristics of the process include an execution phase that includes loss of cell volume, plasma membrane blebbing and chromatin condensation, followed by packing of the cellular contents into membrane-enclosed vesicles called apoptotic bodies that are rapidly phagocytosed by neighboring cells. Apoptosis differs from necrosis, which is cell death resulting from physical injury.
Since autoimmune diseases and certain degenerative diseases also involve the proliferation of abnormal cells, therapeutic treatment for these diseases could also involve the enhancement of the apoptotic process through the administration of appropriate drugs.
It is pertinent, therefore, to inquire into the mechanism of apoptosis in order to develop a method for the identification of compounds for the treatment of autoimmune diseases. It has been found that a group of proteases are a key element in apoptosis (see e.g. Thornberry,
Chemistry and Biology
5:R97-R103 (1998); Thornberry,
British Med. Bull.
53:478-490 (1996)). Genetic studies in the nematode
Caenorhabditis elegans
revealed that apoptotic cell death involves at least 14 genes, two of which are the pro-apoptotic (death-promoting) ced (for cell death abnormal) genes, ced-3 and ced-4. CED-3 is homologous to interleukin 1&bgr;-converting enzyme (ICE), a cysteine protease, which is now called caspase-1. When these data were ultimately applied to mammals, and upon further extensive investigation, it was found that the mammalian apoptosis system appears to involve a cascade of caspases, or a system that behaves like a cascade of caspases. At present, the caspase family of cysteine proteases comprises 10 different members, and more may be discovered in the future. All known caspases are synthesized as zymogens that require cleavage at an aspartyl residue prior to forming the active enzyme. Thus, caspases are capable of activating other caspases, in the manner of an amplifying cascade.
The caspase cascade can be involved in disease processes in two major aspects. Excessive activity of the caspase cascade can lead to excessive apoptosis and organ failure. Among the diseases that could result from this excessive activity are myocardial infarction, congestive heart fail
Green Douglas R.
Kasibhatla Shailaja
Tseng Ben
Chan Christina
Cytovia, Inc.
Huynh Phuong
Sterne Kessler Goldstein & Fox P.L.L.C.
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