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Pharmacologic method of lowering cholesterol productionRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, In Vivo Diagnosis Or In Vivo Testing, Testing Efficacy Or Toxicity Of A Compound Or Composition (e.g., Drug, Vaccine, Etc.)Pharmacologic method of lowering cholesterol production description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070274917, Pharmacologic method of lowering cholesterol production. Brief Patent Description - Full Patent Description - Patent Application Claims
CLAIM OF PRIORITY [0001] This application claims priority from U.S. Provisional Patent Application No. 60/561,585, filed Apr. 13, 2004, the entire teachings of which are incorporated by reference. FIELD OF INVENTION [0003] The present invention relates to methods and compounds that modulate cholesterol levels as well as screening methods used to identify such compounds. BACKGROUND OF THE INVENTION [0004] Cholesterol is an essential constituent of all animal cell membranes. Cholesterol reduces the permeability of the membrane, increases the membrane's mechanical strength and helps to organize the membrane constituents laterally into domains. In the cell, cholesterol's abundance is tightly controlled through biosynthetic, storage, ingestion, and transfer pathways. Through these pathways, cells have the ability to adjust their cholesterol level to their needs. [0005] Unfortunately, in some instances, in vivo cholesterol levels are not properly adjusted. Cholesterol disorders, specifically high serum levels of cholesterol and the mishandling of cholesterol in cells of arterial walls, may cause disease and death in humans by contributing to the formation of atherosclerotic plaques in arteries throughout the body. In fact, cholesterol disorders in the United States contribute to such a high number of health issues that the National Heart, Lung, and Blood Institute launched the National Cholesterol Education Program in 1985. The goal of the National Cholesterol Education Program is to contribute to reducing illness and death from coronary heart disease in the United States by reducing the percent of Americans with high blood cholesterol. The program plans to do this by raising awareness of the link between high cholesterol levels and coronary heart disease. [0006] Many of the illnesses triggered by cholesterol abnormalities are addressed through both increased education and drug treatment. Current drugs used for treatment of cholesterol related disorders are compounds called statins, which inhibit cholesterol biosynthesis by blocking the production of the cholesterol precursor, mevalonic acid. However, mevalonic acid is used by the body to synthesize many other important cellular constituents. Unfortunately, because of the numerous cellular uses for mevalonic acid, statins, which are largely non-specific, often have side effects because they suppress a variety of metabolic functions other than cholesterol biosynthesis. [0007] Accordingly, there is need to find other therapeutic agents to combat cholesterol disorders as well as methods for identifying such compounds. SUMMARY OF THE INVENTION [0008] One embodiment of the present invention provides a method of screening compounds for cholesterol modulating activity. The screening method involves contacting one or more test agents with one or more cells and determining or measuring whether the one or more test agents has an effect on cholesterol activity, cholesterol concentration or both in a membrane of the one or more cells. The screening method can also involve contacting the one or more cells with a lytic compound, so that the lytic compound causes perforation or lysis of the membrane of the one or more cells when the cholesterol activity, cholesterol concentration or both of the membrane of the one or more cells reaches a level at or above a threshold cholesterol level. In certain screening methods, the cholesterol content of the membrane can be increased or decreased by contacting the one or more cells with a cholesterol modulating compound. [0009] In one embodiment, the invention includes a method of screening compounds for cholesterol modulating activity by contacting test agents with cells and determining whether the test agents has an effect on cholesterol activity, cholesterol concentration or both in the cell membrane. [0010] In a further embodiment, determining whether the one or more test agents has an effect on cholesterol activity, cholesterol concentration or both in the cell membrane includes contacting the cells with a lytic compound that causes perforation or lysis of the membrane of the cells when the cholesterol activity, cholesterol concentration or both of the membrane of the cells reaches a level at or above a threshold cholesterol level. [0011] In yet another embodiment, the cholesterol content of the membrane may be either increased or decreased by contacting the cells with a cholesterol modulating compound. In some embodiments, this will be performed prior to, simultaneous with or subsequent to contact with the test agents. [0012] In another embodiment, the invention includes a method of identifying a compound that modulates cholesterol activity by identifying test agents that modulate the cholesterol activity in a cell membrane, where the test agents has been identified by a method in any one of the preceding paragraphs. [0013] In a further embodiment, the invention comprises a method of manufacturing a compound that modulates cholesterol activity by synthesizing or isolating therapeutic agents identified by the methods of identifying a compound that modulates cholesterol activity. [0014] In yet a further embodiment, the invention includes a method of modulating the cholesterol level of a cell by contacting cells with an effective amount of octanol, ceramide, diglyceride, lysophosphosphatidyl choline, or a combination thereof, thereby increasing or decreasing the cholesterol level of the cells. In certain cases, the cells will be in vivo, while in other cases, the cells will be in vitro. [0015] A specific embodiment of the invention includes a kit for determining the effect of a test agent on the cholesterol activity, cholesterol concentration and/or both in a cell membrane. The kit may includes the instructions for carrying out the method of any one of the previous paragraphs and one or more needed reagents. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 shows the screening of lysis of red blood cells using amphotericin B by turbidity. Human red blood cells were either freshly drawn or, taken from an outdated blood bank source and then washed and suspended in physiological buffer. The cholesterol content of washed human red blood cells was varied with methyl-.beta.-cyclodextrin/methyl-.beta.-cyclodextrin-cholesterol complexes. The cells were distributed into a 96-well plate and incubated for a few minutes with saline (.largecircle.), 0.2 .mu.mol/well octanol (.gradient.) or 1 .mu.g/well lysophosphatidylcholine (.quadrature.). Amphotericin B (20 .mu.g/well) was added and the plate incubated for 1 hour at room temperature. As shown in FIG. 1, the variation of endogenous cholesterol content by methyl-.beta.-cyclodextrin/ methyl-.beta.-cyclodextrin-cholesterol complexes, results in cells that will not be lysed by amphotericin B unless a test agent increases the cholesterol concentration, cholesterol activity and/or both in the cell membrane. In FIG. 1, turbidity (signifying intact cells) was determined in a plate-reading photometer as optical density (OD) at 500 nm; loss of OD signifies lysis. In FIG. 1, results are plotted versus total human red blood cell cholesterol, assessed with Amplex.TM. Red, Molecular Probes, Eugene, Oreg. The cholesterol content of unmodified red blood cells was 0.55 .mu.g/.mu.l cells, close to the threshold of the control curve. [0017] FIG. 2 shows a visual screen of red blood cell lysis caused by amphotericin B. The experiment was as in FIG. 1. Cells with modified cholesterol were placed in the conical wells of a 96-well plate, briefly pre-incubated with the two test compounds, and then treated with amphotericin B (20 .mu.g/well) for one hour at room temperature. The plate was photographed after the cells settled. Row A: saline control. Cells depleted of cholesterol (wells 1-6) resisted lysis (seen as a loss of the central button). Row B: n-octanol (0.2 .mu.mol/well). Even cholesterol depleted cells were lysed by amphotericin B in the presence of octanol but not in controls without amphotericin B (not shown). Row C: Lysophosphatidylcholine (1 .mu.g/well). Even enriched cells resisted amphotericin B lysis. [0018] FIG. 3 shows the screening of red blood cell lysis using cholesterol oxidase. The cholesterol content of washed human red cells was varied with methyl-.beta.-cyclodextrin-cholesterol complexes. The cells were distributed into a 96-well plate and incubated with saline (.largecircle.), 0.2 .mu.mol/well octanol (.gradient.) or 1 .mu.g/well lysophosphatidylcholine (.quadrature.). Cholesterol oxidase (2 IU/ml) was added and the plate incubated for 1 h at 37.degree. C. Turbidity (signifying intact cells) was determined in a plate-reading photometer as O.D. at 500 nm. Loss of turbidity reflects cell lysis. [0019] FIG. 4 depicts the effect of octanol on fibroblast cholesterol. Replicate flasks of fibroblasts were incubated for 24 h in growth medium with octanol. The cells were then assayed for cholesterol and protein. [0020] FIG. 5 shows the effect of ceramide and diglyceride on the oxidation of red cell membrane cholesterol by cholesterol oxidase. The red cell membranes were pretreated with ceramide or diglycerides and then treated with cholesterol oxidase for a hour. An increase in oxidation following cholesterol oxidase treatment signifies an increase in active cellular cholesterol. Continue reading about Pharmacologic method of lowering cholesterol production... Full patent description for Pharmacologic method of lowering cholesterol production Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Pharmacologic method of lowering cholesterol production patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. 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