Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-03-31
2002-01-15
Wang, Andrew (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S366000, C435S375000, C536S023100, C536S024310, C536S024500
Reexamination Certificate
active
06339066
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to therapies, diagnostics, and research reagents for disease states which respond to modulation of the expression of the &bgr;
I
, &bgr;
II
, &ggr;, &dgr;, &egr;, &zgr; or &eegr; isoform of protein kinase C. In particular, preferred embodiments, this invention relates to sequence-specific antisense phosphorothioate oligonucleotides comprising nucleosides joined by intersugar linkages, wherein the intersugar linkages are substantially pure (i.e. substantially all Sp or Rp) chiral phosphorothioate linkages which are specifically hybridizable with nucleic acids relating to the &bgr;
I
, &bgr;
II
, &ggr;, &dgr;, &egr;, &zgr; and &eegr; isoforms of protein kinase C. These oligonucleotides modulate the expression of the &bgr;
I
, &bgr;
II
, &ggr;, &dgr;, &egr;, &zgr; and &eegr; isoforms of protein kinase C, and are especially well suited as diagnostics, therapeutics and research reagents.
1. Protein Kinase C Isoforms
The phosphorylation of proteins plays a key role in the transduction of extracellular signals into cells. Kinases, the enzymes which effect such phosphorylations, are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation. One of the major signal transduction pathways involves the enzyme protein kinase C (PKC), which is known to have many critical influences on cell proliferation and differentiation. PKC is activated by diacylglycerols (DAGs), which are metabolites released in signal transduction.
PKC is not a single enzyme, but a family of enzymes. At the present time at least seven isoforms (isozymes) of PKC have been identified. Isoforms &agr;, &bgr;, and &ggr; have been purified to homogeneity and isoforms &dgr;, &egr;, &zgr; and &eegr; have been identified by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka,
Nature,
334:661, 1988, for a review) and may serve different physiological functions. For example, PKC-&ggr; seems to be expressed only in the central nervous system. As another example, PKC-&eegr; has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than, for example, in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain (Osada et al.,
J. Biol. Chem.
265:22434, 1990). As a third example, PKC-&agr; and -&bgr; are expressed in most tissues, but have different patterns of expression in different cell types. Both PKC-&agr; and PKC-&bgr; are expressed in, and have been purified from, human epidermis; however, while PKC-&agr; has been detected mainly in keratinocytes of the basal layers of the epidermis, PKC-&bgr; is found mainly in the middle layers of the epidermis and Langerhans cells. It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. For example, in psoriatic lesions there is an alteration in the ratio between PKC-&agr; and PKC-&bgr;, with preferential loss of PKC-&bgr; compared to normal skin (Hegemann, L. and G. Mahrle,
Pharmacology of the Skin
, H. Mukhtar, ed., pp. 357-368, CRC Press, Boca Raton, Fla., 1992).
Interest in PKC was stimulated by the finding that PKC is a major cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells (Gescher et al.,
Anti
-
Cancer Drug Design
4:93, 1989). Phorbols capable of tumor production can mimic the effect of DAG in activating PKC, suggesting that these tumor promoters act through PKC and that activation of PKC is at least partially responsible for the resulting tumorigenesis (Parker et al.,
Science
233:853, 1986).
Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestinal tract convert lipids to DAG, thus activating PKC and altering cell proliferation. This may explain the correlation between high dietary fat and colon cancer (Weinstein,
Cancer Res
. (Suppl.) 51:5080s, 1991). It has also been demonstrated that a greater proportion of the PKC in the colonic mucosa of patients with colorectal cancer is in an activated state compared to that of patients without cancer (Sakanoue et al.,
Int. J. Cancer
48:803, 1991).
Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice. A mutant form of PKC induces highly malignant tumor cells with increased metastatic potential. Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiation-induced transformation in vivo (Endo et al.,
Cancer Research
51:1613, 1991; Borek et al.,
Proc. Natl. Acad. Sci
. (
USA
) 88:1953, 1991). A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs (Gescher, A. and Dale, I. L.,
Anti
-
Cancer Drug Design,
4:93, 1989).
Experiments also indicate that PKC plays an important role in the pathophysiology of hyperproliferative skin disorders such as psoriasis and skin cancer. Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells. Various studies indicate a role for PKC in causing these symptoms. PKC stimulation in cultured keratinocytes can be shown to cause hyperproliferation. Inflammation can be induced by phorbol esters and is regulated by PKC. DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis (Hegemann, L. and G. Mahrle,
Pharmacology of the Skin
, H. Mukhtar, ed., pp. 357-368, CRC Press, Boca Raton, Fla., 1992).
The oligonucleotides of the invention are useful in the therapeutic treatment of diseases associated with PKC isoforms. Such diseases include, but are not limited to, hyperproliferative and inflammatory conditions including psoriasis, tumors and cancers, for example glioblastoma, bladder cancer, breast cancer, lung cancer and colon cancer.
Although numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara,
Trends in Pharm. Sci.
8:162, 1987, for a review), few have been found which inhibit PKC specifically. While the quinoline sulfonamide derivatives such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC at micromolar concentrations, they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are potent in vitro inhibitors of PKC. However, these drugs exhibit only limited selectivity among different protein kinases (Gescher,
Anti
-
Cancer Drug Design
4:93, 1989). Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.
There is also a desire to inhibit specific PKC isozymes, both as a research tool and in diagnosis and treatment of diseases which may be associated with particular isozymes. Godson et al. (
J. Biol. Chem.
268:11946, 1993) disclose the stable transfection of antisense PKC-&agr; cDNA in cytomegalovirus promotor-based expression vectors to specifically decrease expression of PKC-&agr; protein by approximately 70%. It was demonstrated that this inhibition caused a loss of phospholipase A
2
-mediated arachidonic acid release in response to the phorbol ester PMA. Attempts b
Bennett C. Frank
Cook Phillip Dan
Dean Nicholas M.
Hoke Glenn
ISIS Pharmaceuticals Inc.
Shibuya Mark L.
Wang Andrew
Woodcock & Washburn LLP
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