European Heart Journal Supplements (2001) 3 (Supplement E), E6-EIO
`
`Ezetimibe: a selective inhibitor of cholesterol
`absorption
`
`A. L. Catapano
`
`Dipartimento di Scienze Farmacologiche, Universita degli Studi di Milano, Milano, Italy
`
`is a novel selective cholesterol absorption
`Ezetimibe
`inhibitor that effectively blocks intestinal absorption of
`dietary and biliary cholesterol. Ezetimibe undergoes glu(cid:173)
`curonidation to a single metabolite and is localized in the
`intestinal wall, where it prevents cholesterol absorption.
`Enterohepatic recirculation of ezetimibe and the glucuro(cid:173)
`nide ensures repeated delivery to the site of action and
`limits peripheral exposure. Ezetimibe does not affect the
`absorption of fat-soluble vitamins or triglycerides. Results
`from pre-clinical studies in various animal models have
`shown the lipid-lowering and anti-atherosclerotic properties
`of ezetimibe as a single agent, and a synergistic effect when
`combined with a statin. In cholesterol-fed rhesus mon(cid:173)
`keys, ezetimibe reduced both plasma cholesterol (ED50=
`0·0005 mg. kg- 1
`
`• day - 1) and
`low-density
`lipoprotein
`cholesterol levels in a dose-dependent manner. In apo E
`
`knockout mice, ezetimibe reduced serum cholesterol more
`than 50% and decreased carotid (97%) and aortic (47- 87%)
`atherosclerosis. Ezetimibe inhibited the rise of plasma choles(cid:173)
`terol in cholesterol-fed dogs (ED50 =0·007 mg.kg- 1.day- 1
`).
`Co-administration of ezetimibe and lovastatin in chow-fed
`dogs synergistically reduced plasma cholesterol to levels
`lower than those achieved with either agent alone (P<0·05).
`These results suggest that ezetimibe combined with a statin
`may similarly reduce plasma cholesterol levels in patients
`with hypercholesterolaemia.
`(Eur Heart J Supplements 2001; 3 (Suppl E): E6-EIO)
`© 2001 The European Society of Cardiology
`
`Key Words: Cholesterol absorption inhibitor, ezetimibe,
`pre-clinical studies, atherosclerosis, hypercholesterolaemia,
`statins.
`
`Introduction
`
`Ezetimibe (SCH 58235) (Fig. 1) is a highly potent and
`selective cholesterol absorption inhibitor that prevents
`absorption of cholesterol from dietary and biliary
`sources by preventing transport of cholesterol through
`the intestinal wa111•.21. Ezetimibe prevents intestinal
`absorption of cholesterol without affecting absorption
`of triglycerides, fatty acids, bile acids or fat-soluble
`vitaminsPl, unlike pancreatic lipase
`inhibitors (e.g.
`orlistat), which affect the absorption of triglyceridesl4
`5l
`•
`and may decrease the absorption of fat-soluble vita(cid:173)
`mins161. Furthermore, ezetimibe also differs from the
`bile acid sequestrants (e.g. cholestyramine) which inter(cid:173)
`fere with absorption of vitamins A, D, E and K,
`taurocholate and bile acidsp,s1.
`Ezetimibe undergoes phase II metabolism191, yielding
`a glucuronide that appears to be pharmacodynamica1ly
`active. Fo11owing intraduodenal delivery to rodents,
`ezetimibe is rapidly and extensively glucuronidated in
`the intestinal wall, is absorbed into the portal plasma
`and passes through the liver into the bile within
`minutesl2l. Once glucuronidated, ezetimibe circulates
`
`Correspondence: Professor Alberico L. Catapano, PhD,
`Dipartimento di Scienze Farmacologiche, Universita degli Studi di
`Milano,Via G. Balzaretti, 9, Milano, 20133 Italy.
`
`enterohepatically, repeatedly returning the drug back to
`the primary site of action (the intestine), thus limiting
`peripheral exposure. This recirculation may explain the
`long duration of action of ezetimibe.
`Ezetimibe localizes in the intestinal wa11, mainly as the
`phenolic glucuronide (SCH 60663)121. Comparative
`studies suggest that the glucuronide is a more potent
`inhibitor of cholesterol absorption than ezetimibel21, and
`the localization of the glucuronide at the intestinal villi
`may explain its apparent increase in potency compared
`with ezetimibe.
`
`OH
`
`OH
`
`F
`
`"Q F
`
`Figure 1 Structure of ezetimibe.
`
`1520-765X/01/0E0006+05 $35.00/0
`
`© 2001 The European Society of Cardiology
`
`1 of 5
`
`PENN EX. 2175
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`Ezetimibe inhibits cholesterol absorption E7
`
`Figure 2 Autoradiographic analysis of intestinal wall after intravenous administration of 3H-ezetimibe in
`bile-cannulated rats. Three distinct villi are shown. The dark background is the intestinal lumen. 3H-ezetimibe
`concentrates on the surface of the enterocytes at the tips of the villi (dark-field microscopy, original magnification
`250 x ). (Reproduced with permission121.)
`
`Specific localization of radiolabelled ezetimibe to the
`intestinal villi, its site of action, has been reported in
`bile-cannulated rats intravenously injected with 3H(cid:173)
`ezetimibel21. Autoradiographic analysis of cross-sections
`of the intestines of these rats shows that ezetimibe is
`located throughout the intestinal villi and is concen(cid:173)
`trated in the tips of the intestinal villi (Fig. 2).
`This paper reviews data from several pre-clinical
`studies in various animal models. The first series of
`studies evaluated the lipid-lowering effects of ezetimibe
`in cholesterol-fed rhesus monkeys, as well as the effects
`of an ezetimibe analogue on postprandial chylo(cid:173)
`micron cholesterol content11°1. Another study in apo E
`knockout mice examined the lipid-lowering effects of
`ezetimibe, as well as its effects on atherosclerosis in this
`animal mode11• •1. Finally, data from two studies in
`dogs are reviewed1121. The first study was designed
`to determine whether ezetimibe would decrease
`plasma cholesterol levels in cholesterol-fed dogs, and
`the second evaluated whether the combination of
`ezetimibe with lovastatin would decrease plasma total
`cholesterol levels in animals fed a cholesterol-free chow
`diet.
`
`Effects of ezetimibe in cholesterol-fed
`monkeys
`
`A series of studies was conducted to determine the
`effects of ezetimibe on plasma lipids and lipoprotein
`composition in monkeys with diet-induced hypercholes(cid:173)
`terolaemial10J. All monkeys were fed a chow-based,
`high-fat, high-cholesterol (i.e. 'western') diet containing
`0·25% cholesterol and 22·5% fat ( 15% coconut oil and
`7·5% olive oil).
`In one series of studies, groups of five rhesus monkeys
`received either the western diet alone or with ezetimibe
`(0·3, I, 3 or IO µg. kg-•. day- 1) admixed in the diet.
`After 3 weeks, plasma cholesterol was significantly
`(P<0·05) lower in ezetimibe-treated monkeys compared
`with control animals at all doses tested. Ezetimibe
`inhibited the expected increase in both plasma total
`cholesterol and
`low-density
`lipoprotein cholesterol
`(ED 50=
`(LDL-C)
`in
`a dose-dependent
`fashion
`0·5 µg. kg-•. day-•, or 0·0005 mg. kg-•. day- 1).
`Ezetimibe did not affect plasma high-density lipoprotein
`cholesterol (HDL-C) or triglyceride levelsl' 01.
`
`Eur Heart J Supplements, Vol. 3 (Suppl E) June 2001
`
`2 of 5
`
`PENN EX. 2175
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`EB A. L. Catapano
`
`In another experiment in rhesus monkeys, a crossover
`design was used to evaluate the duration of action
`of ezetimibe and the time course of the reversal of
`pre-established hypercholesterolaemia. Control animals
`and ezetimibe-treated animals (I 00 µg . kg - 1
`• day- 1
`admixed in the diet; five per group) were fed their
`respective diets for 20 days. Monkeys receiving the
`cholesterol-containing diet without drug had a marked
`increase in cholesterol concentration over the 20-day
`period, but there was a complete inhibition of the
`in animals receiving
`the diet with
`dietary effect
`ezetimibe. Treatments were switched at 20 days, i.e. the
`monkeys that had received the cholesterol diet with
`ezetimibe then received the diet without the drug, and
`those that had received the cholesterol diet only were
`given the diet with ezetimibe. Despite a daily intake of
`375 mg of dietary cholesterol for 20 days, plasma chol(cid:173)
`esterol remained unchanged from baseline levels for
`3 days after discontinuation of ezetimibe. Withdrawal of
`ezetimibe eventually did reverse the cholesterol-lowering
`effect, with significant increases in plasma cholesterol
`levels seen IO days after cessation of the drug. Ezetimibe
`had a rapid onset of action after administration and a
`reversal of effect after withdrawal[ 101.
`A single 10 mg . kg - 1 dose of an analogue of
`ezetimibe (SCH 48461)[ 11 was given to a small group of
`cynomolgus monkeys to evaluate postprandial chylo(cid:173)
`micron cholesterol content. The animals were fasted for
`20 h and then fed the high-fat, high-cholesterol diet in
`one single meal with or without the ezetimibe analogue.
`Blood samples were collected 5 h later. Chylomicrons
`and chylomicron remnants were isolated and analyzed
`for cholesteryl ester, free cholesterol and triglyceride
`content. A single dose of the ezetimibe analogue
`decreased postprandial chylomicra cholesterol content
`by 69% (P<0·05), with no significant effect on. the
`triglyceride contentP 01.
`
`Effects of ezetimibe in apo E knockout
`mice
`
`The apo E knockout (KO) mouse is a widely used model
`to study the development and progression of athero(cid:173)
`sclerosis. Apo E is an LDL-C receptor ligand that is
`important in the regulation of serum cholesterol and
`lipid levels. Inactivation of the gene coding for apo E is
`associated with high serum cholesterol levels and prema(cid:173)
`ture, spontaneous development of atherosclerosis in
`mice. In one study conducted in this animal model,
`lipoprotein and atherosclerotic changes were determined
`in apo E KO mice fed either a low-fat diet (IO kcal%
`corn oil, 0· 15% cholesterol) or a high-fat (western) diet
`(40 kcal% butter fat, 0· 15% cholesteroJ)C 1 •1. Ezetimibe
`(5 mg . kg- 1 • day- 1) reduced plasma cholesterol levels
`by more than 60% in apo E KO mice fed either diet for
`6 months. These reductions occurred in the very-low(cid:173)
`density lipoprotein and low-density lipoprotein frac(cid:173)
`tions. Ezetimibe increased HDL-C levels more than
`
`Eur Heart J Supplements, Vol. 3 (Suppl E) June 2001
`
`twofold in animals fed either the low-fat or western diet.
`Moreover, ezetimibe inhibited the development and
`progression of aortic atherosclerosis in both the western
`and low-fat groups of apo E KO mice. Ezetimibe also
`reduced carotid artery atherosclerosis in the apo E KO
`mice (Fig. 3). The drug's anti-atherosclerotic effect was
`demonstrated by a 97% reduction in the atherosclerosis
`in the intimal area of the carotid artery as well as a 47-
`87% reduction in surface areas .of aortic lesions.
`
`Co-administration of ezetimibe with a
`statin in dogs
`
`Before evaluating the co-administration of ezetimibe
`and a statin in the dog, investigators wished to verify
`whether czctimibe blocked cholesterol absorption in this
`animal model. Beagle dogs were fed a diet containing 1 %
`cholesterol, 0·2% cholic acid and 5·5% lard alone (con(cid:173)
`trol) or with ezetimibe (0·003, 0·01 or 0·03 mg. kg- 1
`)
`for 7 days. Ezetimibe significantly inhibited the rise in
`plasma total cholesterol levels in
`these cholesterol(cid:173)
`(ED50 =0·007 mg. kg-•. day- 1)
`fed dogs
`(P<0·05
`compared with controls)l1 21.
`A study in normocholesterolaemic dogs was then
`conducted to assess the effects of the co-administration
`of ezetimibe and lovastatinP 2J. Beagle dogs were fed a
`cholesterol-free
`chow diet
`and given
`ezetimibe
`(0·007 mg. kg- 1
`
`), lovastatin (5 mg. kg- 1) or a combi(cid:173)
`nation of ezetimibe and lovastatin for 14 days. Neither
`ezetimibe nor lovastatin alone significantly affected
`plasma choiesterol levels. The combination of ezetimibe
`with lovastatin, however, synergistically reduced plasma
`total cholesterol levels by 50% (P<0·05 compared with
`control and either agent alone) from day 3 of treatment
`to day 14. Thus, combined therapy with ezetimibe and
`lovastatin decreased plasma cholesterol concentrations
`significantly more than either agent alone.
`
`Discussion
`
`Ezetimibe is the first of a new class of lipid-modifying
`drugs, the selective cholesterol absorption inhibitors.
`Ezetimibe inhibited cholesterol absorption and reduced
`plasma total cholesterol concentrations in a number of
`pre-clinical models. Ezetimibe also increased HDL-C
`levels in apo E knockout mice given either a low-fat
`or western diet; furthermore, ezetimibe inhibited the
`progression of atherosclerosis in apo E knockout mice
`under both dietary conditions, suggesting an effect
`independent, at least in part, from the type of diet.
`Ezetimibe effectively reduced plasma cholesterol and
`LDL-C levels in animals fed a high-cholesterol diet.
`Ezetimibe prevented diet-induced hypercholesterolaemia
`and reduced pre-established hypercholesterolaemia in
`rhesus monkeys fed a high-fat, western diet. Because of
`the long duration of action of ezetimibe, the effect of
`
`3 of 5
`
`PENN EX. 2175
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`(a)
`
`(b)
`
`Ezetimibe inhibits cholesterol absorption E9
`
`-- - - - - - - - - - · ----- __ J
`
`Figure 3 Ezetimibe reduces carotid artery atherosclerosis in apo E knockout mice fed a 0· 15% cholesterol diet for
`6 months' 11
`'. (a) Control, (b) 5 mg. kg- 1
`• day- 1 ezetimibe.
`
`treatment persisted for several days after its cessation,
`supporting the contention that once-daily dosing should
`be sufficient for an adequate. therapeutic effect. In
`addition, ezetimibe rapidly reversed pre-existing hyper(cid:173)
`cholesterolaemia, despite a continued high-cholesterol
`diet.
`Cynomolgus monkeys fed a single high-cholesterol
`meal and treated with an ezetimibe analogue also had
`a significant reduction in the chylomicron cholesterol
`content, with no effect on the triglyceride content.
`
`remnants
`Because chylomicrons and chylomicron
`16l, further investigation of this
`may be atherogenic1 13-
`phenomenon might. shed more light on the mech(cid:173)
`anism of the anti-atherogenic effect of ezetimibe. It
`should be noted that while statins may increase the
`clearance of chylomicron remnants they may not
`decrease cholesterol content of chylomicrons. Therefore,
`the combination of a statin and ezetimibe should be
`highly effective in reducing the atherogenic potential of
`chylomicrons.
`
`Eur Heart J Supplements, Vol. 3 (Suppl E) June 2001
`
`4 of 5
`
`PENN EX. 2175
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`EJO A. L. Catapano
`
`Ezetimibe caused only modest reductions in plasma
`cholesterol levels in animals fed cholesterol-free diets,
`which was probably the result of an up-regulation of
`hepatic cholesterol synthesis1121. ·Ezetimibe reduces the
`delivery of dietary and biliary cholesterol to the liver
`from the intestine, resulting in a decrease of hepatic
`cholesterol stores and an up-regulation of hepatic
`HMG-CoA reductase activity. Ezetimibe has a mode of
`action complementary to that of the statins in lower(cid:173)
`ing plasma cholesterol concentrations as it inhibits the
`absorption of cholesterol, while statins decrease the
`synthesis of cholesterol. The synergistic reduction in
`plasma total cholesterol concentration in dogs treated
`with ezetimibe (0·007 mg. kg- 1 . day- 1) and lovastatin
`(5 mg . kg- 1
`
`• day - 1) has also been reported with
`ezetimibe and pravastatin (2·5 mg. kg- 1 • day- 1) or
`fluvastatin (2·5 mg. kg- 1
`• day- 1)1 171.
`The synergistic or additive result of combining
`ezetimibe with a statin is particularly significant because
`of the rule of six1181, which has been demonstrated with
`all of the statins. According to the rule of six, each
`doubling of the statin dose achieves a further reduction
`in LDL-C of only 6%.
`However, at the same time, increasing the statin dose
`increases the potential for toxicity. Ezetimibe has no
`significant effect on
`the activity of major drug(cid:173)
`metabolizing enzymes, and the potential for ezetimibe to
`cause a drug interaction involving the cytochrome P450
`substrates is unlikelyP 91. Clinical benefit could derive
`from co-administration of ezetimibe and a statin in cases
`in which a single agent is not sufficiently effective or in
`which there is concern about higher doses of an effective
`agent.
`In conclusion, studies of hyperlipidaemia and athero(cid:173)
`sclerosis in animal models have shown that ezetimibe,
`given alone or combined with a statin, has significant
`lipid-lowering
`and
`anti-atherosclerotic properties.
`Ezetimibe lowered plasma total cholesterol and LDL-C
`in cholesterol-fed animals. Ezetimibe also
`levels
`inhibited the development of aortic and carotid artery
`atherosclerosis
`in
`apo E knockout mice. Co(cid:173)
`administration of ezetimibe with a statin has a synergis(cid:173)
`tic cholesterol-lowering effect, suggesting that such
`combination therapy for patients with hypercholestero(cid:173)
`laemia may reduce plasma cholesterol more than is
`attainable with either drug alone.
`
`References
`
`[I] van Heek M, France CF, Compton DS et al. In vivo
`metabolism-based discovery of a potent cholesterol absorp(cid:173)
`tion inhibitor, SCH58235, in the rat and rhesus monkey
`
`identification of the active metabolites of
`the
`through
`SCH48461. J Pharmacol Exp Ther 1997; 283: 157-63.
`[2] van Heek M, Farley C, Compton DS et al. Comparison of the
`activity and disposition of the novel cholesterol absorption
`inhibitor, ·scH58235, and its glucuronide, SCH60663. Br J
`Pharmacol 2000; 129: 1748-54.
`[3] van Heek M, Farley C, Compton D, Hoos L, Davis H. The
`novel cholesterol absorption inhibitor, ezetimibe, selectively
`inhibits the intestinal absorption of free cholesterol in the
`presence and absence of exocrine pancreatic function (Abstr).
`Atherosclerosis 2000; 151: 155.
`(4] Guerciolini R. Mode of action of orlistat. Int J Obes Relat
`Metab Disord 1997; 21 (Suppl 3): S12-23.
`[5] Toplak H, Marhardt K. Reduction of obesity and improve(cid:173)
`ment in metabolic parameters by inhibition of intestinal
`lipases: current results with orlistat. Acta Med Austriaca 1998;
`25: 142-5.
`[6] Heck AM, Yanovski JA, Calis KA. Orlistat, a new lipase
`inhibitor for the management of obesity. Pharmacotherapy
`2000; 20: 270-9.
`[7] West RJ, Lloyd JK. The effect of cholestyramine on intestinal
`absorption. Gut 1975; 16: 93-8.
`[8] Matsui MS, Rozovski SJ. Drug-nutrient interaction. Clin
`Ther 1982; 4: 423-40.
`[9] Zbaida S, Alton K, Shannon D et al. In vitro metabolism
`of SCH 58235, l-(4-fluorophenyl)-3R-[3-(4-fluorophenyl)-3S(cid:173)
`hydroxypropyl]-4S-(4-hydroxyphenyl)-2-azetidinone, by liver
`and kidney slices (Abstr). 12th International Symposium on
`Microsomes and Drug Oxidations (ISMDO), Montpellier,
`France, July 20-24, 1998.
`[10] van Heek M, Compton DS, Davis HR. The cholesterol
`absorption inhibitor, ezetimibe, decreases diet-induced hyper(cid:173)
`cholesterolemia in monkeys. Eur J Pharmacol 2001; 415:
`79-84
`(11] Davis HR, Compton DS, Hoos L, TetzloffG. The cholesterol
`inhibitor ezetimibe inhibits the development of atherosclerosis
`in apo E knockout ( - I-) mice fed low fat and western diets
`(Abstr). Atherosclerosis 2000; 151: 133.
`[12] Davis HR, Watkins RW, Compton DS et al. The cholesterol
`absorption inhibitor ezetimibe (SCH 58235) and lovastatin
`synergistically lower plasma cholesterol and inhibit the devel(cid:173)
`opment of atherosclerosis. Metabolism; in press.
`[13] Zilversmit DB. Atherogenic nature of triglycerides, post(cid:173)
`prandial lipemia, and triglyceride-rich remnant lipoproteins.
`Clin Chem 1995; 41: 153-8.
`[14] Cohn JS. Postprandial lipemia: emerging evidence for athero(cid:173)
`genicity of remnant lipoproteins. Can J Cardiol 1998; 14
`(Suppl B): 18B-27B.
`[15] Mamo JC, Proctor SD, Smith D. Retention of chylomicron
`remnants by arterial tissue; importance of an efficient clear(cid:173)
`ance mechanism from plasma. Atherosclerosis 1998; 141
`(Suppl !): S63-9.
`[16] Havel RJ. Remnant lipoproteins as therapeutic targets. Curr
`Opin Lipidol 2000; 11: 615-20.
`[17] Davis HR, van Heek M, Watkins RW et al. The hypo(cid:173)
`cholesterolemic activity of the potent cholesterol absorption
`inhibitor SCH58235 alone and in combination with HMG
`CoA reductase inhibitors (Abstr). Drugs Affecting Lipid
`Metabolism (DALM); Houston, TX; November 1995.
`[18] Knopp RH. Drug treatment of lipid disorders. N Engl J Med
`1999; 341: 498-511.
`[19] Zhu Y, Statkevich P, Kosoglou T et al. Effect of ezetimibe
`(SCH 58235) on the activity of drug metabolizing enzymes in
`vivo (Abstr). Clin Pharmacol Ther 2000; 67: 152.
`
`Eur Heart J Supplements, Vol. 3 (Suppl E) June 2001
`
`5 of 5
`
`PENN EX. 2175
`CFAD V. UPENN
`IPR2015-01835