Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-01
2002-09-10
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007720, C435S007800, C435S006120, C435S069100, C435S091100, C435S091200, C435S325000, C435S375000, C435S244000
Reexamination Certificate
active
06448019
ABSTRACT:
BACKGROUND OF THE INVENTION
Vascular diseases are the major cause of morbidity and mortality in the United States. Despite decades of intensive research, the mechanisms responsible for these diseases are poorly understood.
The low incidence of vascular diseases in pre-menopausal women and the rapid increase in vascular diseases, including cerebrovascular and ischemic heart disease, in women following the menopause are well recognized. It is now recognized that the hormone estrogen plays a significant role in preventing atherosclerotic vascular disease. Most attempts to explain the effects of estrogen on the development of vascular disease in women focus on indirect effects of estrogen on known risk factors, such as lipid and/or carbohydrate metabolism, counterbalancing of androgen-mediated effects, or indirect effects of sex hormones on the thrombotic milieu.
Estrogen replacement therapy has been rather successful in reducing the incidence of vascular disease in post-menopausal women. It can reduce the incidence of coronary artery disease by as much as 30-50 percent. However, estrogen replacement therapy has a number of significant drawbacks, including an increased risk of endometrial cancer, an increased risk of breast cancer in women, and side effects such as endometrial bleeding and breast tenderness. In addition, though estrogen therapy may theoretically have beneficial effects for vascular disease in men as well as women, attempts to examine this possibility have been limited by adverse effects observed with currently available agents and the possibility of feminization that such therapy harbors.
There is a need for more selective therapies which have some or all of the vasoprotective benefits of estrogen therapy, but have fewer undesirable side-effects.
SUMMARY OF THE INVENTION
The present invention relates to screening methods which can be used to identify agents which inhibit vascular smooth muscle cell activation and/or proliferation or enhance vascular endothelial cell activation and/or proliferation or activate estrogen responsive genes in vascular cells. Such agents are potentially useful for treatment or prevention of vascular disease and are referred to as “vasoprotective agents.” Preferred vasoprotective agents are relatively vasospecific, i.e., their effect on one or more types of vascular cells is more pronounced than their effect on other cell types. Treatment with such vasospecific agents will generally be associated with fewer undesirable side-effects than treatment with estrogen. Vascular disease can result from overproliferation of vascular smooth muscle cells. This proliferation can be inhibited by activation and/or proliferation of vascular endothelial cells. For example, a preferred agent would inhibit the proliferation of vascular smooth muscle cells and/or increase proliferation of vascular endothelial cells associated with the development of atherosclerosis, but not have any significant effect on cells of the reproductive system, e.g., breast cells or uterine cells.
The methods of the invention permit identification of agents which inhibit vascular smooth muscle cell proliferation directly by altering the proliferation rate of vascular smooth muscle cells. The methods of the invention also permit identification of agents which inhibit vascular smooth muscle cell proliferation more indirectly by enhancing the proliferation or activation of vascular endothelial cells, which can, in vivo, exert an inhibitory effect on the proliferation of vascular smooth muscle cells and can also promote vascular stability through an increased rate or extent of re-endothelialization.
The term “vasoprotective agent” refers to agents which have the potential to reduce vascular disease in patients because they inhibit, directly or indirectly, unwanted proliferation of vascular smooth muscle cells and/or enhance, directly or indirectly, endothelial cell growth or regrowth at sites of vascular injury.
The methods of the invention are screening assays in which candidate agents are examined to identify vasoprotective agents. One type of screening assay involves examining the effect of a candidate agent on cell proliferation and/or cell activation. Another type of screening assay involves examining the effect of a candidate agent on the expression of a gene which is responsive to estrogen (“an estrogen responsive gene”). Both screening assays involve the use of vascular cells and non-vascular cells. The use of both cell types is important because the cellular milieu is very likely to influence the effect of a candidate agent on cell proliferation, cell activation, and the expression of an given estrogen receptor responsive gene.
The term “estrogen responsive gene” refers to a gene whose expression can be affected by the presence of an estrogen.
The methods of the invention can be used to identify agents which exert their effect through a known estrogen receptor, e.g., estrogen receptor &agr; or estrogen receptor &bgr;, or through an as yet unidentified estrogen receptor (“estrogen receptor-dependent agent”). Moreover, the methods of the invention can be used to identify agents which exert their effect through a receptor other than an estrogen receptor. These other receptors may recognize the same DNA binding site (recognition element) as an estrogen receptor or may bind to a recognition element which is distinct from an estrogen receptor recognition element.
The term “estrogen receptor” means a protein which specifically binds an estrogen (e.g., 17&bgr;-estradiol or E2). Estrogen binding can be measured using any standard estrogen binding assay, e.g., the assay described by Gibson et al. (
Endocrinology,
29:2000, 1991). The term encompasses, but is not limited to, both the well-known estrogen receptor &agr; (Green et al.,
Nature
320:134,1986) and the more recently described estrogen receptor &bgr; (Kuiper et al.,
Proc. Nat'l Acad. Sci. USA
93:5925, 1996; Genbank Accession Number U57439).
The term “estrogen receptor-dependent agent” refers to an agent the effect of which on a cell depends on the presence of an estrogen receptor (e.g., estrogen receptor &agr; or estrogen receptor &bgr; or some other estrogen receptor) in the cell. Accordingly, the term includes agents which have one effect on a cell when an estrogen receptor is expressed by the cell and a different effect or no effect on the same cell when an estrogen receptor is not expressed by the cell. The agent may, of course, exert other effects which are not mediated via the estrogen receptor. Tamoxifen, 17&bgr;-estradiol, and ICI 182,780 are all examples of estrogen receptor-dependent agents.
In selecting a vasoprotective agent, among the undesirable side-effects to be avoided or minimized are activating or growth promoting effects on: (a) cancerous cells or pre-cancerous cells; (b) uterine cells or tissue; (c) breast cells or tissue; and (d) non-vascular, non-reproductive cells. Of course, it may not be possible to completely avoid all or even some of these undesirable side-effects in selecting a vasoprotective agent. A vasoprotective agent can be therapeutically beneficial even if its use is associated to some extent with some or all of these side effects.
In addition to identifying vasoprotective agents which avoid or minimize undesirable side effects, it is desirable to identify vasoprotective agents which are osteoprotective. The term “osteoprotective agent” refers to agents which have the potential to reduce osteoporosis in patients because they increase the activation and/or proliferation of osteoclasts, which are responsible for bone formation, or because they inhibit activation and/or proliferation of osteoclasts, which are responsible for bone absorbtion.
Some of the methods which can be used to identify vasoprotective agents involve measuring cell activation and/or proliferation. Cell proliferation can be measured by measuring cell number, DNA content, or
3
H-thymidine uptake. Cell activation can be monitored by measuring the expression of genes that are up regulated by cell activation. For example
Karas Richard H.
Mendelsohn Michael E.
Clark & Elbing LLP
Kemmerer Elizabeth
New England Medical Center Hospitals Inc.
Prasad Sarada C
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