Abstract
Ezetimibe is the first in class 2-azetidinone that decreases plasma cholesterol by blocking intestinal cholesterol absorption. Ezetimibe effectively reduces plasma cholesterol in several 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 activity. The results demonstrate that the ability of compounds to bind to NPC1L1 is the major determinant of in vivo responsiveness.
Footnotes
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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]-β-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-(2S,3R)-1-(4-fluorophenyl)-4-oxo-3-[3(S)-hydroxy-3-(4-fluorophenyl)propyl]-2-azetidinyl]phenyl-l]-3-O-(β-d-glucopyranosyl)-β-d-glucopyranose; SCH604813, (R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl)-1-(4-iodophenyl)-2-azetidinone; SCH58832, trans-1-(4-fluoropheny)-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)-β-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]-β-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]-β-d-glucopyranuronic acid, methyl ester.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Received June 12, 2006.
- Accepted September 27, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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