Effect of ezetimibe on the prevention and dissolution of cholesterol gallstones

Abstract

Background & aims: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Figure 1. Effect of ezetimibe on the prevention of cholesterol gallstones

Ezetimibe significantly reduces, in a dose-dependent fashion, hepatic outputs of (A) biliary cholesterol and (B) phospholipid, but not (C) bile salts. *P<0.05, **P<0.01 and ***P<0.001, compared with mice fed the lithogenic diet and receiving no ezetimibe. (D) There is a clear dose-dependent reduction in intestinal cholesterol absorption efficiency from 50±6% to 4±2% in chow-fed mice, as measured by the fecal dual-isotope ratio method. (E) When doses of ezetimibe are increased from 0 to 4 mg/kg/day, gallstone prevalence rates are reduced from 80% to 10% in mice fed the lithogenic diet for 8 weeks. No gallstones are found in mice treated with ezetimibe at 8 mg/kg/day. (F) The relative lipid compositions of pooled gallbladder biles from mice fed the lithogenic diet and receiving no ezetimibe are located in the central three-phase zone, where biles are composed of solid cholesterol monohydrate crystals, liquid crystals, and saturated micelles at equilibrium. In contrast, administration of the highest dose (8 mg/kg/day) of ezetimibe results in the relative biliary lipid compositions of pooled gallbladder biles plotted in the one-phase micellar zone, even upon the lithogenic diet feeding for 8 weeks. By phase analysis, these biles are composed of unsaturated micelles at equilibrium. A symbol ♦ represents relative lipid compositions of pooled gallbladder biles at 8 weeks on the lithogenic diet supplemented with ezetimibe at 0; ● 0.8; ▲ 4; and ■ 8 mg/kg/day.

Figure 2. Effect of ezetimibe on the dissolution of cholesterol gallstones

(A) For gallstone dissolution experiments, mice with the preexisting gallstones are fed the chow diet alone for 8 weeks, which does not result in a spontaneous dissolution of gallstones. In contrast, treatment with ezetimibe at 0.8 to 8 mg/kg/day induces rapid dissolution of gallstones. Gallstones are completely dissolved by the highest (8 mg/kg/day) dose of ezetimibe. (B) Representative photomicrographs of mucin gel, liquid crystals, cholesterol monohydrate crystals, and gallstones as observed in gallbladder biles at week 8 after ezetimibe treatment. All magnifications are ×800, except for ezetimibe treatment at 0 and 0.8 mg/kg/day, which are ×400, by polarizing light microscopy. (C) The relative lipid compositions of pooled gallbladder biles from mice fed 8 weeks with the chow diet supplemented with varying doses of ezetimibe are plotted on a condensed phase diagram. Because of a 12-week feeding of the lithogenic diet, the relative lipid compositions of pooled gallbladder biles from mice that have formed cholesterol gallstones are located in the central three-phase zone. Although the lithogenic diet is replaced with the chow diet for 8 weeks, the relative biliary lipid compositions of biles are still in Region C, where at equilibrium the biles are composed of solid cholesterol crystals, liquid crystals, and saturated micelles. By feeding varying doses of ezetimibe, the relative lipid compositions of pooled gallbladder biles gradually shift down, and finally, enter the one-phase micellar zone. These alterations explain that gallstones are dissolved through an unsaturated micelle mechanism. A symbol * represents relative lipid compositions of pooled gallbladder biles from mice that have preexisting gallstones and before ezetimibe treatment; ♦ relative lipid compositions of pooled gallbladder biles at end of gallstone dissolution study at week 8 of feeding the chow diet only (control); ● 0.8; ▲ 4; and ■ 8 mg/kg/day of ezetimibe.

Figure 3

Fasting gallbladder volumes (A and B) are significantly increased by the lithogenic diet compared with the chow diet. However, these lithogenic effects on gallbladder dynamics are totally blocked by ezetimibe in doses of higher than or equal to 4 mg/kg/day. *P<0.01 and **P<0.05, compared with the chow group. (C and D) Because of gallbladder emptying and the release of a concentrated gallbladder bile, biliary bile salt output and bile flow are increased sharply and significantly in response to exogenously administered CCK-8 (as shown by arrows) in mice with bile fistulae. Gallbladder contractile function is completely impaired in mice fed the lithogenic diet for 8 weeks and partially in mice fed the lithogenic diet and concomitantly treated with ezetimibe at 0.8 or 4 mg/kg/day. In contrast, there is normal gallbladder contractile function in mice treated with the highest dose of ezetimibe at 8 mg/kg/day, even in challenge to the lithogenic diet. As shown in insets of Figures 3C and 3D, the areas under the curves of bile salt outputs and bile flow rates are significantly increased in a dose-dependent fashion in mice treated with ezetimibe. *P<0.01 and **P<0.05, compared with mice fed the lithogenic diet and receiving no ezetimibe.

Figure 4

Upon the lithogenic diet feeding, compared with mice receiving no ezetimibe, mice treated with ezetimibe at 8 mg/kg/day display significantly reduced expression levels of Abcg5/g8 and Acat2, mostly attributable to secondary reaction in response to decreased amounts of the absorbed cholesterol. Also, ezetimibe treatment significantly reduces expression levels of Npc1l1 in the small intestine. *P<0.01, **P<0.001, and ***P<0.00001, compared with mice fed the lithogenic diet and receiving no ezetimibe.