Potent Cholesterol Absorption Inhibitor Research Articles

Transporters as Drug Targets: Discovery and Development of NPC1L1 Inhibitors

The potent cholesterol absorption inhibitor ezetimibe was developed as a first-in-class drug for treating hypercholesterolemia even before its molecular target, Niemann-Pick C1-like 1 (NPC1L1), had been identified. The NPC1L1 protein mediates sterol transport across the enterocyte brush border membrane and is essential for intestinal cholesterol absorption, a major pathway controlling whole-body cholesterol homeostasis. An elucidation of the mechanism underlying NPC1L1-dependent cholesterol absorption would greatly facilitate the discovery and development of new cholesterol-lowering agents for treating hypercholesterolemia and other cholesterol-related metabolic disorders.

Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

The Niemann-Pick C1 Like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1,332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homo-logy to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1), and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of human NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH2-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. human NPC1L1 contains seven small cytoplasmic loops--four small and three large luminal loops--one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, NPC1, and SCAP. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of the different domains of NPC1L1.

Effects of ezetimibe add-on therapy for high-risk patients with dyslipidemia

BackgroundEzetimibe (Zetia®) is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia. Statin, an inhibitor of cholesterol synthesis, is the first-choice drug to reduce low-density lipoprotein-cholesterol (LDL-C) for patients with hypercholesterolemia, due to its strong effect to lower the circulating LDL-C levels. Because a high dose of statins cause concern about rhabdomyolysis, it is sometimes difficult to achieve the guideline-recommended levels of LDL-C in high-risk patients with hypercholesterolemia treated with statin monotherapy. Ezetimibe has been reported to reduce LDL-C safely with both monotherapy and combination therapy with statins.ResultsTo investigate the effect of ezetimibe as "add-on" therapy to statin on hypercholesterolemia, we examined biomarkers and vascular endothelial function in 14 patients with hypercholesterolemia before and after the 22-week ezetimibe add-on therapy. Ezetimibe add-on therapy reduced LDL-C by 24% compared with baseline (p < 0.005), with 13 patients (93%) reaching their LDL cholesterol goals. Of the Ezetimibe add-on therapy significantly improved not only LDL-C, high-density lipoprotein-cholesterol (HDL-C), and apolipoprotein (apo)B levels, but also reduced levels of triglyceride (TG), the ratio of LDL/HDL-C, the ratio of apoB/apoA-I, and a biomarker for oxidative stress (d-ROMs). Furthermore, ezetimibe add-on therapy improved vascular endothelial function in high-risk patients with hypercholesterolemia.ConclusionIn conclusion, ezetimibe as add-on therapy to statin might be a therapeutic good option for high-risk patients with atherosclerosis.

Vascular protective effects of ezetimibe in ApoE-deficient mice

One of the major risk factors for ischemic disease is hyperlipidemia, which is mainly regulated by endogenous cholesterol synthesis in the liver and dietary absorption in the small intestine. In this study, we evaluated the vascular protective effects of a potent cholesterol absorption inhibitor, ezetimibe. ApoE-deficient mice were fed a chow or high-fat diet with or without ezetimibe (5mg/(kgday)) for 3 months. Co-treatment with ezetimibe significantly reduced plasma cholesterol (by 76%; from 1592 to 381mg/dL) and LDL cholesterol (by 78%; from 1515 to 319mg/dL), and increased HDL cholesterol (by 187%; from 16 to 46mg/dL) in high-fat diet mice. Consistently, a marked inhibitory effect of ezetimibe on the development of lipid-rich plaque was observed, as assessed by oil red O staining. Of importance, treatment with ezetimibe significantly improved endothelial dysfunction as assessed by the vasodilator response to acetylcholine, accompanied by inhibition of interleukin-6 mRNA and an increase in endothelial nitric oxide synthase (eNOS) mRNA in the aorta. Ezetimibe also suppressed oxidative stress and the ubiquitination–proteasome system in the aorta. Although changes in body weight and several tissue weights were similar in the groups with and without ezetimibe administration, only liver weight was significantly decreased in the ezetimibe-treated group. Interestingly, ezetimibe markedly inhibited lipid accumulation in the liver. Furthermore, ezetimibe increased the mRNA expression of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase as a counteraction in the liver, but not in the aorta. Overall, ezetimibe significantly prevented atherosclerosis through not only lipid-lowering effects, but also other direct and/or indirect vascular protective actions in ApoE-deficient mice.

Effect of Ezetimibe on the Prevention and Dissolution of Cholesterol Gallstones

Cholesterol cholelithiasis is one of the most prevalent and most costly digestive diseases in developed countries and its incidence has increased markedly in Asian countries owing to the adoption of Western-type dietary habits. Because animal experiments showed that high efficiency of intestinal cholesterol absorption contributes to gallstone formation, we explored whether the potent cholesterol absorption inhibitor ezetimibe could prevent gallstones and promote gallstone dissolution in mice and reduce biliary cholesterol content in human beings. Male gallstone-susceptible C57L mice were fed a lithogenic diet and concomitantly administered with ezetimibe at 0, 0.8, 4, or 8 mg/kg/day for 8 or 12 weeks. Gallbladder biles and gallstones were examined by microscopy. Gallbladder emptying in response to cholecystokinin octapeptide was measured gravimetrically. Biliary lipid outputs were analyzed by physical-chemical methods. Cholesterol absorption efficiency was determined by fecal dual-isotope ratio and mass balance methods. Lipid changes in gallbladder biles of gallstone patients vs overweight subjects without gallstones were examined before (day 0) and at 30 days after ezetimibe treatment (20 mg/day). Ezetimibe prevented gallstones by effectively reducing intestinal cholesterol absorption and biliary cholesterol secretion, and protected gallbladder motility function by desaturating bile in mice. Treatment with ezetimibe promoted the dissolution of gallstones by forming an abundance of unsaturated micelles. Furthermore, ezetimibe significantly reduced biliary cholesterol saturation and retarded cholesterol crystallization in biles of patients with gallstones. Ezetimibe is a novel approach to reduce biliary cholesterol content and a promising strategy for preventing or treating cholesterol gallstones by inhibiting intestinal cholesterol absorption.

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.

New azetidinone cholesterol absorption inhibitors

Since their initial discovery, azetidinones have been extensively studied as potent cholesterol absorption inhibitors. Ezetimibe is the first-in-class and only member in the clinics either as a monotherapy or in combination with statins for treatment of hypercholesterolemia. The recent identification of Niemann–Pick C1-like 1 protein in the brush border membrane of the small intestine as a possible molecular target has opened the door for the design and synthesis of new anticholesterol drugs. This article reviews the important literature and patents on cholesterol absorption inhibitors.

Cholesterol absorption inhibitors as a therapeutic option for hypercholesterolaemia

The development of cholesterol-lowering drugs (including a variety of statins, bile acid-binding resins and recently discovered inhibitors of cholesterol absorption) has expanded the options for cardiovascular prevention. Recent treatment guidelines emphasise that individuals at substantial risk for atherosclerotic coronary heart disease should meet defined targets for LDL cholesterol concentrations. Combination therapy with drugs that have different or complementary mechanisms of action is often needed to achieve lipid goals. Existing approaches to the treatment of hypercholesterolaemia are still ineffective in halting the progression of coronary artery disease in some patients despite combination therapies. Other patients are resistant to conventional drug treatment and remain at high risk for the development and progression of atherosclerotic cardiovascular disease and alternative approaches are needed. The discovery and development of ezetimibe (a novel, selective and potent cholesterol absorption inhibitor) has advanced the treatment of hypercholesterolaemia. New agents including the phytostanol preparation FM-VP4 and inhibitors of acyl coenzyme A:cholesterol acyltransferase, the apical Na+-dependent bile acid transporter and microsomal triglyceride transfer protein may also play a future role in combination therapy. This review focuses on the recent progress in the molecular mechanisms of intestinal cholesterol absorption and transport, and novel therapeutic approaches to inhibit the cholesterol absorption process.

Selective Compensatory Induction of Hepatic HMG-CoA Reductase in Response to Inhibition of Cholesterol Absorption

The effect of the potent inhibitor of cholesterol absorption, ezetimibe, on serum cholesterol levels was tested in diabetic and thyroidectomized male Sprague-Dawley rats. Feeding diets supplemented with 1% cholesterol to the diabetic rats raised serum cholesterol levels from 132 to 514 mg/dl while decreasing hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein and mRNA levels. Addition of 10 mg/kg/day of ezetimibe to the diets of these animals lowered the serum cholesterol level to 90 mg/dl and produced a large compensatory increase in hepatic HMG-CoA reductase protein without significantly increasing mRNA levels, indicating a post-transcriptional effect. Hepatic LDL receptor protein levels in these diabetic rats were unaffected by ezetimibe treatment. In contrast, ezetimibe treatment of these young normal Sprague-Dawley rats, known to express high levels of hepatic HMG-CoA reductase, did not lower serum cholesterol levels. In thyroidectomized rats, dietary cholesterol increased serum cholesterol levels from 116 to 135 mg/dl and ezetimibe treatment lowered these elevated cholesterol levels to 85 mg/dl. Cholesterol feeding of thyroidectomized rats severely reduced hepatic HMG-CoA reductase protein, while ezetimibe treatment restored reductase protein to normal levels. Again, hepatic LDL receptor protein levels were unaffected by ezetimibe treatment of cholesterol-fed thyroidectomized rats. The data demonstrate that the cholesterol absorption inhibitor ezetimibe profoundly lowers serum cholesterol levels in animals expressing very low rates of hepatic cholesterol synthesis and produces large compensatory increases in hepatic HMG-CoA reductase expression without significantly affecting expression of hepatic LDL receptors. This indicates that ezetimibe should be most effective in lowering serum cholesterol levels in people with low rates of cholesterol synthesis/high rates of cholesterol absorption.

New Insights Into the Genetic Regulation of Intestinal Cholesterol Absorption

The small intestine is a unique organ providing dietary and reabsorbed biliary cholesterol to the body. However, the molecular mechanisms whereby cholesterol is absorbed have not yet been fully understood. Recent research suggests that the newly identified Niemann-Pick C1-like 1 protein (NPC1L1) is expressed at the apical surface of enterocytes and plays a critical role in the absorption of intestinal cholesterol. Furthermore, adenosine triphosphate (ATP)-binding cassette (ABC) transporters ABCG5 and ABCG8 represent apical sterol export pumps that promote active efflux of cholesterol and plant sterols from enterocytes back into the intestinal lumen for excretion. This provides an explanation why cholesterol absorption is a selective process, with plant sterols and other noncholesterol sterols being absorbed poorly or not at all. These findings strongly support the concept that cholesterol absorption is a multistep process, which is regulated by multiple genes at the enterocyte level. The absorption efficiency of cholesterol is most likely determined by the net effect between influx and efflux of intraluminal cholesterol molecules across the brush border of the enterocyte. Combination therapy using a novel, specific, and potent cholesterol absorption (NPC1L1) inhibitor (ezetimibe) and HMG-CoA reductase inhibitors (statins) offers an efficacious new approach to the prevention and treatment of hypercholesterolemia.

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.

An in vitro assay for evaluation of small-molecule inhibitors of cholesterol absorption.

Giving cholesterol the brush off: An intestinal brush border membrane vesicle assay has been devised for the convenient in vitro testing of small molecules for inhibition of cholesterol absorption. The assay was used to identify new nonhydrolyzable glycosides as potent cholesterol-absorption inhibitors and an oxazolidinone as an effective replacement of the β-lactam scaffold of ezetimibe (1).

Sterol absorption by the small intestine.

Cholesterol absorption is a selective process in that plant sterols and other non-cholesterol sterols are absorbed poorly or not at all. Recent research on the sterol efflux pumps adenosine triphosphate-binding cassette transporter G5 and adenosine triphosphate-binding cassette transporter G8 has not only provided an explanation for this selectivity, but also, together with the discovery of a new class of cholesterol absorption inhibitor, has yielded new insights into the mechanisms that potentially regulate the flux of cholesterol across the enterocyte. This review discusses these recent developments and their importance to the regulation of whole body cholesterol homeostasis. Adenosine triphosphate-binding cassette transporters G5/8 regulate plant sterol absorption and also the secretion into bile of cholesterol and non-cholesterol sterols. Loss of adenosine triphosphate-binding cassette transporter G5/8 function results in sitosterolemia. Ezetimibe, a novel, potent and selective inhibitor of cholesterol absorption which is effective in milligram doses, lowers plasma plant sterol concentrations in sitosterolemic subjects, thus suggesting that this drug might be inhibiting the activity of a putative sterol permease in the brush border membrane of the enterocyte that actively facilitates the uptake of cholesterol as well as other non-cholesterol sterols. Intestinal cholesterol absorption represents a major route for the entry of cholesterol into the body's miscible pools and therefore can potentially impact the plasma LDL-cholesterol concentration. The combined use of agents that inhibit the absorption and synthesis of cholesterol provides a powerful new approach to the prevention and treatment of atherosclerosis.

Synthesis of fluorescent biochemical tools related to the 2-azetidinone class of cholesterol absorption inhibitors

Fluorescent analogues of the cholesterol absorption inhibitor (CAI), Sch 58235, have been designed and synthesized as single enantiomers. Biological testing reveals that they are potent CAIs and are suitable tools for the investigation of the azetidinone CAI mechanism of action (MOA).

Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function.

1. Ezetimibe potently inhibits the transport of cholesterol across the intestinal wall, thereby reducing plasma cholesterol in preclinical animal models of hypercholesterolemia. The effect of ezetimibe on known absorptive processes was determined in the present studies. 2. Experiments were conducted in the hamster and/or rat to determine whether ezetimibe would affect the absorption of molecules other than free cholesterol, namely cholesteryl ester, triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid. In addition, to determine whether exocrine pancreatic function is involved in the mechanism of action of ezetimibe, a biliary anastomosis model, which eliminates exocrine pancreatic function from the intestine while maintaining bile flow, was established in the rat. 3. Ezetimibe reduced plasma cholesterol and hepatic cholesterol accumulation in cholesterol-fed hamsters with an ED(50) of 0.04 mg kg(-1). Utilizing cholesteryl esters labelled on either the cholesterol or the fatty acid moiety, we demonstrated that ezetimibe did not affect cholesteryl ester hydrolysis and the absorption of fatty acid thus generated in both hamsters and rats. The free cholesterol from this hydrolysis, however, was not absorbed (92 - 96% inhibition) in the presence of ezetimibe. Eliminating pancreatic function in rats abolished hydrolysis of cholesteryl esters, but did not affect the ability of ezetimibe to block absorption of free cholesterol (-94%). Ezetimibe did not affect the absorption of triglyceride, ethinylestradiol, progesterone, vitamins A and D, and taurocholic acid in rats. 4. Ezetimibe is a potent inhibitor of intestinal free cholesterol absorption that does not require exocrine pancreatic function for activity. Ezetimibe does not affect the absorption of triglyceride as a pancreatic lipase inhibitor (Orlistat) would, nor does it affect the absorption of vitamin A, D or taurocholate, as a bile acid sequestrant (cholestyramine) would.

Ezetimibe, a potent cholesterol absorption inhibitor, normalizes combined dyslipidemia in obese hyperinsulinemic hamsters.

Ezetimibe potently and selectively inhibits cholesterol absorption in the intestine, thereby reducing plasma cholesterol in preclinical models of hypercholesterolemia. Clinical trials have demonstrated that ezetimibe lowers LDL cholesterol and raises HDL cholesterol in humans. The effect of ezetimibe on other dyslipidemias, particularly hypertriglyceridemia, is not yet known. In the present studies, we assessed the effect of ezetimibe on combined hypercholesterolemia and hypertriglyceridemia in obese hyperinsulinemic hamsters. Hamsters were fed chow, chow with cholesterol (0.12%), or the same cholesterol diet containing different dietary triglycerides (15%) in the absence or presence of 1 mg/kg ezetimibe (in diet) for up to 84 days. Body weight, serum insulin, leptin, glucose, cholesterol, and triglyceride levels were analyzed. Cholesterol and triglyceride levels were also determined in VLDL+IDL, LDL, and HDL. Hamsters maintained on high-fat diets became obese, hyperinsulinemic, hyperleptinemic, hypercholesterolemic, and hypertriglyceridemic. Ezetimibe did not affect body weight, insulin, or leptin, but ablated the combined hypercholesterolemia and hypertriglyceridemia induced by high-fat diets. Ezetimibe normalized VLDL+IDL cholesterol and triglyceride and significantly decreased LDL cholesterol to below chow-fed levels. The ratio of HDL to LDL cholesterol increased significantly with the addition of ezetimibe. Ezetimibe completely eliminated the accumulation of cholesteryl ester and free cholesterol in liver that was induced under the various dietary conditions in the absence of drug. In conclusion, ezetimibe is very effective in correcting the combined dyslipidemia in diet-induced obese hyperinsulinemic hamsters and may be an effective therapy for ameliorating combined dyslipidemia in obese insulin-resistant and/or type 2 diabetic humans.

The potent cholesterol absorption inhibitor, ezetimibe, is glucuronidated in the intestine, localizes to the intestine, and circulates enterohepatically

The potent cholesterol absorption inhibitor, ezetimibe, is glucuronidated in the intestine, localizes to the intestine, and circulates enterohepatically

The potent cholesterol absorption inhibitor, ezetimibe, ablates hypercholesterolemia and hypertriglyceridemia in a model of combined hyperlipidemia

The potent cholesterol absorption inhibitor, ezetimibe, ablates hypercholesterolemia and hypertriglyceridemia in a model of combined hyperlipidemia

Rapid normalization of pre-established hypercholesterolemia in rhesus monkeys by the potent cholesterol absorption inhibitor ezetimibe (SCH58235)

Rapid normalization of pre-established hypercholesterolemia in rhesus monkeys by the potent cholesterol absorption inhibitor ezetimibe (SCH58235)