A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC. Free cholesterol (FC) has an important role as a component of cell membranes and a starting material for bile acid synthesis. However, cholesteryl ester (CE) is inactive when it is stored. In the progression of arteriosclerosis, CE accumulates in macrophages and smooth muscle cells and leads to the formation of foam cells. Determination of CE in cells or various tissues is of great importance in the fundamental research into atherosclerosis and the development of anti-atherosclerotic drugs.Several methods of enzymatic determination for FC and total cholesterol (TC) have been published (1-5). Measurement of TC has measured FC resulting from the decomposition of CE by cholesterol esterase, and FC was contained in the native sample. To measure CE, individual TC and FC are measured, and FC is then subtracted from TC (indirect assay). Measuring CE by indirect assay is difficult in a sample solution with a low ratio of CE to FC.This report describes a direct assay of CE by enzymatic determination without measuring TC and FC. In the first step, FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is decomposed into water and oxygen by catalase. At the second step, CE is measured by enzymatic determination using a colorimetric method or a fluorometric method. CE is hydrolyzed to FC by cholesterol esterase. FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Thus, the enzymatic method for assaying FC is based on the measurement of hydrogen peroxide by way of peroxidase-coupled oxidation of hydrogen-sensitive probes. The hydrogen peroxide reacts with 4-aminoantipyrine or Amplex Red in the presence of peroxide to form a pigment or fluorescent products. The enzymatic reactions involved in the assay are as follows:CE content in human plasma, rat liver, and cultured cells was measured using this method, and its usefulness was evaluated.

No abstract

No abstract

[2][3][4]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide hydrochloride (K-604, 2) has been identified as an aqueous-soluble potent inhibitor of human acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also known as SOAT)-1 that exhibits 229-fold selectivity for human ACAT-1 over human ACAT-2. In our molecular design, the insertion of a piperazine unit in place of a 6methylene chain in the linker between the head (pyridylacetamide) and tail (benzimidazole) moieties led to a marked enhancement of the aqueous solubility (up to 19 mg/mL at pH 1.2) and a significant improvement of the oral absorption (the C max of 2 was 1100-fold higher than that of 1 in fasted dogs) compared with those of the previously selected compound, 1. After ensuring the pharmacological effects and safety, we designated 2 as a clinical candidate, named K-604. Considering the therapeutic results of ACAT inhibitors in past clinical trials, we believe that K-604 will be useful for the treatment of incurable diseases involving ACAT-1 overexpression.

No abstract