In a major advance in understanding the intestinal pathway for cholesterol absorption and the mechanism of action for Zetia (ezetimibe), scientists at Schering-Plough Research Institute (SPRI) have identified and characterized a long sought protein critical to intestinal cholesterol absorption. In an article published in the Feb. 20 issue of the journal Science, Schering-Plough scientists report on the identification of the protein, named NPC1L1, as playing an essential role in the ezetimibe-sensitive cholesterol absorption pathway.

Cholesterol levels in the blood are largely controlled through two sources in the body: the liver, which synthesizes (produces) cholesterol, and the intestine, where cholesterol is absorbed into the blood stream.

ýýýBy demonstrating the function of the NPC1L1 protein, scientists at SPRI have made a significant advance toward deciphering the cholesterol absorption pathway in the intestine, which has eluded scientists for some time,ýýý said Cecil Pickett, president, SPRI. ýýýThis discovery reflects the successful integration of new technologies, including genomics and bioinformatics, into discovery research and scientific excellence by a team of SPRI scientists representing a variety of disciplines.ýýý

ýýýWhile research in the past few decades has contributed much to our understanding about the production of cholesterol in the liver, this finding represents an important new discovery which helps explain how the body regulates cholesterol absorption in the second critical pathway-the intestine,ýýý said Christie Ballantyne, director of the Center for Cardiovascular Disease Prevention and professor of medicine at Baylor College of Medicine/The Methodist DeBakey Heart Center in Houston.

Led by Michael Graziano, senior director, Cardiovascular/Metabolic Discovery Research, and senior author on the research paper, Schering-Plough scientists identified NPC1L1 after years of studying specific intestinal cells, called enterocytes, which are known to absorb cholesterol. These cells comprise a small percentage of the total number of cells in the intestine, and have not previously been studied in great detail.

Scientists began by compiling two ýýýlibrariesýýý of more than 16,000 segments of nucleotide sequences (ESTs, or expressed sequence tags) of the genes that are present in enterocytes. The bioinformatics team, led by Nicholas Murgolo, senior principal scientist, Discovery Technologies and Bioinformatics, SPRI, used proprietary capabilities to explore publicly available genomics databases. The team focused their search on genes encoding proteins whose predicted structures suggested cell surface expression and potential interaction with cholesterol, two key characteristics that a protein involved in cholesterol absorption would likely possess. The scientists characterized and determined the function of a previously identified gene known as Niemann-Pick C1-Like 1 (NPC1L1) gene, whose name derives from its similarity to another gene that is mutated in individuals with the rare disorder Niemann-Pick disease. NPC1L1 has no role in Niemann-Pick disease.

Schering-Plough molecular biologists led by Scott Altmann, associate principal scientist, Cardiovascular/Metabolic Discovery Research, cloned the NPC1L1 gene and characterized its expression. Employing immunohistochemistry they showed that the NPC1L1 protein is specifically located on the brush border membranes of jejunal enterocytes, the side of the cell that comes in direct contact with the contents of the small intestine. The jejunum is the specific region of the small intestine where the majority of cholesterol absorption occurs.

To confirm the role of the newly identified protein in cholesterol absorption, scientists led by coauthor Harry ýýýChipýýý Davis, distinguished research fellow, Cardiovascular/Metabolic Discovery Research, studied ýýýgene knockoutýýý mice that had been genetically engineered to lack the NPC1L1 protein. Davis and his colleagues found that the knockout mice absorbed 70 percent less cholesterol from their diets than did normal mice, demonstrating that NPC1L1 is a critical component in the cholesterol absorption pathway. Administration of Zetia had no effect in the knockout mice, suggesting that the compound works by blocking NPC1L1. In addition, when Zetia was administered to mice that were not NPC1L1 deficient, the percentage reduction in cholesterol absorption was similar to the NPC1L1 deficient mice. While further research is needed, these findings suggest that Zetia interacts with NPC1L1 to reduce cholesterol absorption. Scientists are beginning to identify other proteins in the pathway that orchestrate cholesterol absorption from the intestine.

ýýýWith further research, we will continue to better understand the potential benefits of targeting both sources of cholesterol through dual inhibition which decreases cholesterol production in the liver as well as blocks cholesterol absorption in the intestinal pathway,ýýý said Dr Ballantyne.