In Vivo Responsiveness to Ezetimibe Correlates with Niemann-Pick C1 Like-1 (NPC1L1) Binding Affinity: Comparison of Multiple Species NPC1L1 Orthologs □ S Brian E. Hawes, Kim A. O’Neill, Xiaorui Yao, James H. Crona, Harry R. Davis Jr., Michael P. Graziano, and Scott W. Altmann Department of Cardiovascular/Metabolic Disease, Schering Plough Research Institute, Kenilworth, New Jersey Received June 12, 2006; accepted September 27, 2006 ABSTRACT Ezetimibe is the first in class 2-azetidinone that decreases plasma cholesterol by blocking intestinal cholesterol absorp- tion. Ezetimibe effectively reduces plasma cholesterol in sev- eral species including human, monkey, dog, hamster, rat, and mouse, but the potency ranges widely. One potential factor responsible for this variation in responsiveness is diversity in ezetimibe metabolism. After oral administration, ezetimibe is glucuronidated. Both ezetimibe and the glucuronide lower plasma cholesterol; however, the glucuronide exhibits greater potency. Recent identification of Niemann-Pick C1 Like-1 (NPC1L1) as the molecular target of ezetimibe enables direct binding studies to be performed. Here, we report the cloning of NPC1L1 derived from multiple species and assess amino acid sequence homology among human, monkey, dog, hamster, rat, and mouse. The rank order of affinity of glucuronidated ezetimibe for NPC1L1 in each species correlates with the rank order of in vivo activity with monkey  dog  hamster and rat  mouse. Ezetimibe analogs that bind to NPC1L1 exhibit in vivo cholesterol-lowering activity, whereas compounds that do not bind NPC1L1 are inactive. Specific structural components of ezetimibe are identified as critical for binding to NPC1L1. The results demonstrate that small variations in ezetimibe structure or in NPC1L1 amino acid sequence can profoundly influence ezetimibe/NPC1L1 interaction and consequently in vivo activ- ity. The results demonstrate that the ability of compounds to bind to NPC1L1 is the major determinant of in vivo responsive- ness. Hypercholesterolemia is linked to cardiovascular disease, myocardial infarction, and stroke. Blood cholesterol levels are regulated by several components, including de novo syn- thesis, dietary cholesterol absorption, and biliary clearance and excretion. Alteration of the rate of any of these processes can drastically affect whole-body cholesterol levels. Several pharmaceutical therapeutics have been developed that in- hibit cholesterol synthesis. These agents, collectively re- ferred to as statins, inhibit the enzyme 3-hydroxymethyl- glutaryl coenzyme A reductase to effectively reduce blood cholesterol levels and represent the standard of care for treatment of dyslipidemia. A new class of cholesterol-lower- ing therapeutics, called 2-azetidinones, decreases plasma cholesterol levels by blocking intestinal absorption of choles- terol. Ezetimibe (Zetia; Merck/Schering-Plough, Kenilworth, NJ), the first-in-class representative of the 2-azetidinones, blocks both dietary and biliary cholesterol absorption in the Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.027896. □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: NPC1L1, Niemann-Pick C1 Like-1; MES, 4-morpholineethanesulfonic acid; SR-BI, scavenger receptor class B, type I; PCR, polymerase chain reaction; HEK, human embryonic kidney; RACE, rapid amplification of cDNA ends; SCH, Schering Plough compound number; SCH60663, 1-O-[4-[trans-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4- fluorophenyl)propyl]-2-azetidinyl]phenyl]--D-glucuronic acid; SCH58235, 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4- fluorophenyl)propyl)]-4(S)-(4-hydroxyphenyl)-2-azetidinone; SCH61159, 1-O-[4-[trans-(2- S,3R)-1-(4- fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4- fluorophenyl)propyl]-2-azetidinyl]phenyl-L]-3-O-(-D-glucopyranosyl)--D-glucopyranose; SCH604813, (R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4- hydroxyphenyl)-1-(4-iodophenyl)-2- azetidinone; SCH58832, trans-1-(4-flu- oropheny)-3-[[2-(4-fluoropheny)-2-oxoethyl]thio]-4-(4-hydroxyphenyl)-2-azetidinone; SCH60179, 1-O-[4-[trans-(3R,4S)-1-(4-methoxyphenyl)-2- oxo-3-(3-phenylpropyl)-4-azetidinyl]phenyl]-2,3,4,6-tetra-O-(phenylmethyl)--D-glucopyranose; SCH50032, rel-(3R,4S)-4-(4- fluorophenyl-L)-1-(4- mehtoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone; SCH354909, 1-O-[4-[1-[4-[3-[[3-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3- yl)-1-oxopropyl]amino]-1-propynyl]phenyl]-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-oxo-4(S)-azetidinyl]phenyl]--D-glucopyranuronic acid; SCH610396, 1- O-[4-[1-[4-[3-[[3-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- S-indacen-3-yl)-1-oxopropyl]amino]-1-propynyl]- phenyl]-3(R)-[3(S)-hydroxy-3-(4- fluorophenyl)propyl]-2-oxo-4(S)-azetidinyl]phenyl]--D-glucopyranuronic acid, methyl ester. 0026-895X/07/7101-19 –29$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 1 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 27896/3159254 Mol Pharmacol 71:19–29, 2007 Printed in U.S.A. 19 http://molpharm.aspetjournals.org/content/suppl/2006/10/02/mol.106.027896.DC1.html Supplemental material to this article can be found at: at ASPET Journals on January 11, 2016 molpharm.aspetjournals.org Downloaded from