Inhibitors of C-reactive protein induced inflammation

Details Inhibitors of C-reactive protein induced inflammation Inhibitors of C-reactive protein induced inflammation

2001-06-08

2004-07-20

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S007210, C435S337000, C530S363000

Reexamination Certificate

active

06764826

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to methods and compositions that modulate C-reactive protein. Such modulators are useful for inhibiting C-reactive protein induced vascular inflammation and other inflammatory diseases.
2. Description of Related Art
Inflammatory response plays an important role in the onset, development and evolution of atherosclerotic lesions. Elevated serum levels of C-reactive protein (CRP) are non-specific but sensitive markers of the acute inflammatory response. A number of epidemiological studies have shown that the acute-phase reactant C-reactive protein is an important risk factor for atherosclerosis and ischemic heart disease. Higher levels of C-reactive protein are also related to increased risk of coronary events in patients with stable and unstable angina (Liuzzo et al., 1994). The basic mechanisms of this association are not clear and C-reactive protein can merely be a marker of inflammation, with no specific role in the pathogenesis of atherosclerosis. However, although C-reactive protein is present in atherosclerotic lesions, no previous study has specifically assessed the possible effects of C-reactive protein on vascular cells.
High levels of C-reactive protein are frequently observed in patients with unstable angina and acute myocardial infarction (Liuzzo et al., 1994). Patients with unstable angina levels >3 &mgr;g/mL were associated with increased risk of coronary events (death, myocardial infarct and urgent coronary revascularization) and the association is even stronger for patients with >10 &mgr;g/mL (Liuzzo et al., 1994). This observation has suggested that C-reactive protein is a risk factor for atherosclerosis and ischemic heart disease. These studies have shown that even small increases in the levels of C-reactive protein are associated with higher risk of atherosclerosis and ischemic heart disease in apparently healthy subjects (Ridker et al., 1997; Koeing et al., 1999p; Ridker et al., 2000) and the increased risk is independent of lipid-related and non-lipid-related cardiovascular risk. In patients with stable angina, levels of C-reactive protein >3.6 &mgr;g/mL were associated with a two-fold increase in the risk of coronary events (Haverkate, et al., 1997).
C-reactive protein is an acute phase reactant protein usually present in human serum with a concentration of <1 &mgr;g/mL. However, C-reactive protein levels can increase up to 100 or even 500 times during acute inflammation. This staggering response is mainly regulated by proinflammatory cytokines, in particular interleukin-6, and is largely unaffected by anti-inflammatory drugs and hormones (Kilpatrick et al., 1991). Indeed, in patients with unstable angina with high C-reactive protein levels at discharge, C-reactive protein remains elevated during the follow-up and is associated with high risk of new coronary events, in particular in patients in the upper tertile of C-reactive protein levels (>8.6 &mgr;g/mL) (Biasucci et al., 1999). In a recent large prospective study, patients with unstable angina and C-reactive protein levels of >15 &mgr;g/mL at discharge had a 3-fold higher risk of coronary events during a 90-day follow-up (Ferreiros et al., 1999). These results suggest that the proinflammatory effects of C-reactive protein may contribute to the adverse outcome associated with higher levels of this acute phase reactant protein.
Although there is now strong evidence that C-reactive protein is an independent risk factor for ischemic heart disease (Shah 2000; Ridker et al., 2000), the mechanisms underlying this association are not clear. Since inflammatory responses play an important role in the development and evolution of atherosclerosis and may contribute to its thrombotic complications, C-reactive protein may merely be a marker of inflammatory response. Alternatively, C-reactive protein may have a direct role in the pathogenesis of atherosclerosis (Shah 2000; Lagrand et al., 1999). Due to its ligand binding properties, C-reactive protein plays a part in the innate immunity (opsonization) and in the removal of membrane and nuclear material from necrotic cells. C-reactive protein can also bind to complement factor C1q and factor H and activate the classic pathway of complement activation. In addition, recent studies have shown that C-reactive protein can bind to receptor FC&ggr;RI (with low affinity) and FC&ggr;RII (with high affinity) on leukocytes (Bharadwaj et al., 1999). Interestingly, C-reactive protein is present in atherosclerotic plaques but not in the normal vessel wall (Reynolds et al., 1987) where often colocalize with the terminal complement complex (Torzewski et al., 1998). C-reactive protein can also induce tissue factor expression by human monocytes (Cermak et al., 1993).
SUMMARY OF THE INVENTION
This invention relates generally to methods of screening for modulators of C-reactive protein (CRP). In certain embodiments of the invention, a composition of the modulator may be useful for inhibiting the development of C-reactive protein induced inflammatory diseases, e.g., cardiovascular diseases. The present inventors envision that blocking or lowering C-reactive protein levels may have beneficial effects on the evolution of atherosclerosis and may reduce the risk of coronary events.
In a specific embodiment of the present invention, it is provided a method of screening for modulators of C-reactive protein comprising: obtaining a C-reactive protein; contacting the C-reactive protein with at least a first candidate substance; and assaying for an interaction between the C-reactive protein and the first candidate substance with an assay. The assay may be used to assay for C-reactive protein induction of the expression of an adhesion molecule, a receptor, a signaling molecule, a cytokine or an enzyme. Exemplary adhesion molecules include, but are not limited to, intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM), or E-selectin. A specific example of a cytokine that is contemplated in the present invention is a chemokine, e.g., monocyte chemoattractant protein-1 (MCP-1). It is known that chemokines are small cytokines that are involved in the migration and activation of cells, especially phagocytic cells and lymphocytes. Furthermore, the candidate substance may inhibit or enhance the C-reactive protein induced expression of the adhesion molecule. It can be appreciated that the modulator can modulate either C-reactive protein or a co-factor involved in the function of C-reactive protein. Further, co-factors may be isolated from serum.
In yet another embodiment, the assay endpoint comprises assaying for inducible nitric oxide synthase (iNOS) induction, receptor for advanced glycation endproducts, monocyte chemoattractant protein-1, P-selectin, endothelin-1, endothelin-receptor, interleukin-6 or heme oxygenase-1. One skilled in the art will recognize that a variety of assays may be used to assay these endpoints, such as, FACS, ELISA, Northern blotting and/or Western blotting.
In another specific embodiment, C-reactive protein can be obtained by expressing C-reactive protein in a transgenic cell or an animal; isolating the expressed C-reactive protein; procuring from serum (i.e., human serum); and procuring from cells. Further, the (a transgenic cell) cell comprises a recombinant nucleic acid sequence encoding a C-reactive protein, thus the C-reactive protein is expressed from the recombinant nucleic acid sequence.
A specific embodiment may include contacting the C-reactive protein with a first candidate substance by incubating a cell in a composition comprising C-reactive protein. One particular aspect includes that the C-reactive protein is expressed in the cell before contacting the C-reactive protein with a first candidate substance. Further, the cell is incubated with C-reactive protein and serum. The serum may be human serum. A skilled artisan will recognize that serums from other species may be utilized in the present invention, such as, bovine or guine

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