Mode of Action of Probucol in Reducing Serum Cholesterol in Mice

Katsuhiko TAWARA, Munehiro TOMIKAWA and Yasushi ABIKO

Laboratory of Biochemistry, Research Institute, Daiichi Seiyaku Co., Ltd., Edogawa-ku, Tokyo 134, Japan

Accepted September 20, 1985

Abstract-The mode of action of probucol in reducing serum cholesterol was studied in normal and cholesterol-fed mice. Probucol did not affect intestinal absorption of radioactive cholesterol in normal and cholesterol-fed mice. In normal mice, probucol treatment resulted in inhibition of incorporation of [14C]-acetate into cholesterol in the liver, while it stimulated the incorporation in the small intestines. Incorporation of [14C] -mevalonate into cholesterol was not affected by the treatment. These results were consistent with the finding that the HMG CoA reductase activity was decreased in the liver, but increased in the intestinal tissues of the treated mice. In cholesterol-fed mice, probucol treatment had no effect on cholesterol synthesis in the liver, while it increased the intestinal cholesterol synthesis. The over-all effect of this drug on cholesterol synthesis was not sig nificant, although it tended to be inhibitory in normal mice and stimulatory in cholesterol-fed mice. On the other hand, probucol treatment resulted in acceleration of the clearance of [14C]-cholesterol-derived radioactivity from the circulation and resulted also in a significant increase in fecal excretion of the radioactivity, cholesterol and bile acids without changes in lipid composition of the bile. Cholesterol content in and radioactivity distribution among the tissues were not affected by probucol. Hepatic cholesterol 7a-hydroxylase activity was increased by probucol. These findings indicate that probucol lowers serum cholesterol mainly by increasing catabolic excretion of cholesterol into bile.

Probucol is a potent hypocholesterolemic cholesterol may contribute, to some extent, agent in many animal species including man. to reduction of plasma cholesterol by this Although there are a number of papers agent (9, 10). describing the cholesterol lowering action of More studies are required for clarifying this agent in pharmacological and clinical these conflicting findings and for better studies, the mechanism of its action has not understanding the mode of action of probucol. been well defined (1-8). The results of some The present paper describes the effect of studies with animal and human subjects probucol on intestinal absorption of suggested a possible involvement of stimu cholesterol, hepatic and intestinal cholesterol lation of catabolic excretion of cholesterol synthesis and catabolic excretion of (9-11) and inhibition of hepatic cholesterol cholesterol in normal and cholesterol-fed synthesis at early stages (9, 10, 12), although mice, under the conditions where their serum with some variation (4, 10, 12), in the cholesterol was significantly reduced by cholesterol-lowering action of probucol. probucol treatment. However, other studies indicated a significant reduction of fecal excretion of neutral and Materials and Methods acid sterols together with decrease in hepatic Animals: Animals used were age-matched cholesterol 7a-hydroxylase activity by treat male STD:ddY mice (4 weeks old at the start ment (12). Impaired intestinal absorption of of experiment, Shizuoka Agricultural Co operative Association for Laboratory Animals, separated using a solvent of n-hexane-diethyl Shizuoka). These mice were fed on a standard ether-acetic acid (73:25:2, v/v/v). The lipid laboratory mouse chow (Funabashi Farm , spots were visualized by spraying with 0.03% Ltd., Funabashi). Hypercholesterolemia was rhodamine 6G, and those corresponding to induced by feeding a high cholesterol diet free cholesterol and cholesterol ester were composed of 1 % cholesterol, 0.5% cholic acid, separately scrapped into scintillation vials. 5% olive oil and 93% standard mouse chow One ml of methanol and 15 ml of liquid (Funabashi Farm, Ltd.). Probucol (Lot no. scintillation mixture (dioxane scintillator) 634N, Dow-Lepetit-Japan Co., Ltd., Tokyo) were added, and the radioactivity was was dissolved in olive oil at 40 mg/ml and measured by a liquid scintillation spectro administered by a stomach tube at a dose of meter. Total cholesterol was also determined 400 or 800 mg/kg-day. Control animals by the method of Allain et al. (15). received olive oil alone (10 or 20 ml/kg). Fecal excretion and tissue distribution of Chemicals: Sodium [1-14C]-acetate (1.9 cholesterol: Mice were intravenously injected mCi/mmol), DL-[2-14C]-mevalonate (DBEB with [14C] -cholesterol (100 ,uCi/kg) which salt, 40.8 mCi/mmol), [4-14C] -cholesterol was dissolved in 20% ethanol in saline at (54.0 and 52.5 mCi/mmol) and DL-[glutaryl 100 fCi/10 ml. Blood samples were taken 3-14C] -3-hydroxy-3-methylglutaryl CoA from the tail vein of each mouse before and (57.6 mCi/mmol) were obtained from New consecutively after the injection of [14C] England Nuclear, Boston. Other chemicals cholesterol for determination of radioactivity used were of reagent grade. and cholesterol concentration in serum. All Intestinal absorption of cholesterol: feces were collected separately from each Intestinal absorption of exogenous cholesterol animal for 6 days (normal mice) or 5 days was studied according to Iritani and Nogi (cholesterol-fed mice) after the cholesterol (13) by measuring radioactivity of serum injection for determination of fecal excretion samples taken consecutively after oral admin of radioactivity, cholesterol and bile acids. istration of [14C] -cholesterol. The labeled Then the mice were sacrificed for determi cholesterol was suspended in 0.2% Tween nation of radioactivity and cholesterol 80 at 1 aCi (25 mg)/ml and was given to content in the tissues including the liver, mice at a dose of 0.4 aCi/mouse after kidneys, whole small intestines, lungs, overnight fasting. In the experiments with spleen, heart and aortas. Fecal bile acids and cholesterol-fed mice, the animals were not neutral sterols were extracted according to fasted before the test. Blood samples were Grundy (16) and determined enzymatically taken consecutively from the tail vein for (17) and colorimetrically (18), respectively. determination of radioactivity and cholesterol Fecal radioactivity was measured by the content in serum. combustion method. Total lipids in tissues Biosynthesis of cholesterol: This was were extracted according to Schoenheimer studied by measuring incorporation of et al. (14). Cholesterol and radioactivity radioactive precursors into cholesterol in the were determined by colorimetry (18) and liver and small intestines. [14C] -Acetate (100 liquid scintillation spectrometry, respectively. ,aCi/kg) or [14C] -mevalonate (50 ,aCi/kg) Assay of HMG-CoA reductase and was intravenously injected to mice as a saline cholesterol 7a-hydroxylase: The liver and the solution (10 ml/kg). The animals were small intestine were homogenized with 10 sacrificed 1 hr after the injection of the labeled volumes of 0.25 M sucrose-0.01 M Tris precursors to obtain serum, the liver and the buffer, pH 7.4. The homogenates were whole small intestines. Lipids were extracted subjected to ultracentrifugation to obtain from serum and the tissues by mixing or microsomes according to Shefer et al. (19). homogenization with 20 volumes of acetone The microsomal fraction was resuspended in ethanol (1 :1, v/v) according to Schoenheimer the buffer solution. HMG-CoA reductase (14). Aliquots of the extracts were spotted on activity of hepatic and intestinal microsomes TLC plates (Kiesel gel 60F254; Merck, was assayed by the method of Shapiro et al. Darmstadt), and major lipid classes were (20) using the radioactive substrate, R,S [3-14C]-HMG-CoA. Cholesterol 7a-hydrox Probucol was orally administered to ylase activity in the liver microsomes was cholesterol-fed mice at a daily dose of 800 assayed according to Lakshmanan and Veech mg/kg for 9 days. [14C] -Cholesterol was (21) with the radioactive substrate [4-14C] orally given 1 hr after the 7th dose of probucol. cholesterol (specific activity 52.5 mCi/ Blood samples were taken 3, 6, 24 and 72 hr mmole). The radioactive products were after the labeled cholesterol ingestion. As separated on TLC plates (Silica gel G, Merck, shown in Table 2, serum cholesterol levels Darmstadt). The activity was expressed as were significantly lower in the treated group pmoles of a product formed per min by 1 mg than in the control group. However, there microsomal protein. Protein was determined was no significant difference in serum by the method of Lowry et al. (22). radioactivity between the 2 groups at each Analysis of data: Statistical significance of point of measurement except for the values data was analyzed by Student's t-test. Data at 72 hr. are presented as the mean±S.E. As a complementary experiment, the effect of probucol was studied in partially hepatec Results tomized mice with reduced hepatic cholesterol Effect of probucol on intestinal absorption synthesis and catabolism. Probucol (400 of cholesterol: Probucol was orally adminis mg/kg; probucol-treated mice) or vehicle tered to normal mice at a daily dose of 400 (olive oil, 1 ml/kg; control mice) was orally mg/kg for 7 days. [14C]-Cholesterol was given to approximately 60%-hepatectomized then given orally 1 hr after the last dose of mice and sham-operated mice at 4 and 24 hr probucol. Blood samples were taken 3, 6 after the operation. Serum cholesterol was and 24 hr after [14C]-cholesterol ingestion for found to be not affected in the hepatectomized determination of serum cholesterol and radio mice at 18 hr after the last dose (106.8±1 5.4 activity derived from absorbed [14C] and 122.6±11.7 mg/dl in the probucol cholesterol. As shown in Table 1, serum treated and the control mice, respectively, cholesterol levels were significantly lower in n=12, P>0.1), while it was significantly the probucol-treated mice than in the controls. lower in the sham-operated and probucol The radioactivity in serum was progressively treated mice (79.1 ±3.2 mg/dl, n=1 2, increased without significant difference be P<0.01) than in the sham-operated control tween the 2 groups during at least 6 hr. mice (117.8±3.4 mg/dl, n=12). However, the treated mice showed a sig Effect of probucol on biosynthesis of nificant lower radioactivity at 24 hr than the cholesterol: Probucol was given to mice at a controls. daily oral dose of 400 mg/kg for 7 days. Almost identical results were obtained with [14C] -Acetate or [14C] -mevalonate was then mice which were fed the high cholesterol diet. intravenously injected 5 hr after the last dose

Table 1. Effect of probucol on intestinal absorption of [14C]-cholesterol in normal mice Table 2. Effect of probucol on intestinal absorption of [14C] -cholesterol in cholesterol-fed mice

Table 3. Effect of probucol on incorporation of [14C]-acetate or [14C] -mevalonate into cholesterol in normal mice

of probucol, and their incorporation into as shown in Table 4, probucol treatment at cholesterol was measured in the liver and the a daily dose of 800 mg/kg for 7 days no small intestines. As shown in Table 3, longer affected the incorporation of [14C] probucol treatment resulted in a significant acetate into cholesterol in the liver, but it reduction of incorporation of [14C] -acetate still stimulated cholesterol synthesis in the into cholesterol in the liver, whereas it small intestines. In this batch of experiments, contrastively stimulated the incorporation of probucol (800 mg/kg) was found again to the labeled acetate into cholesterol in the inhibit cholesterol synthesis in the liver and intestines. Probucol did not affect the to stimulate it in the intestines in normal incorporation of [14C] -mevalonate into mice (Table 4). cholesterol in both tissues. In these experi In all these experiments, tissue cholesterol ments, there was no difference in specific concentration was not affected by probucol, radioactivity of cholesterol in serum between except for serum cholesterol levels which the treated and the control groups. were significantly lower in the treated mice In the cholesterol-fed mice, endogenous than in the control mice. cholesterogenesis was suppressed by about Since probucol exerted dural effects on 90% in the liver and by about 50% in the small cholesterol synthesis in mice, inhibition in intestines (Table 4). Under these conditions, the liver and stimulation in the small Cholesterol Lowering Effect of Probucol intestines, the over-all effect of this drug was calculated in each mouse. As shown in Table 5, there was no significant difference in the sum of the incorporation of [14C]-acetate into cholesterol in the whole liver and intestines between the probucol-treated (400 mg/kg in normal and 800 mg/kg in cholesterol -fed) and the control groups , although the over-all effect tended to be inhibitory in normal mice and stimulatory in cholesterol fed mice. At a high dose of probucol (800 mg/kg), the over-all effect was found to be significantly inhibitory in normal mice. Effect of probucol on catabolic excretion of cholesterol: Probucol was administered to normal mice at a daily oral dose of 400 mg/kg for 9 days. [14C]-Cholesterol was intra venously injected on the third day. As shown in Fig. la, serum cholesterol levels were significantly decreased to reach a bottom level (87.4±5.0 mg/dl) on day 5, which remained low thereafter. Serum radioactivity derived from the injected [14C]-cholesterol was significantly lower at every point of measure ment (1, 3, 5, 20, 28, 48, 72 and 120 hr after [14C]-cholesterol injection) in the treated mice than in the controls (Fig. 1 b). There was no significant difference in serum radioactivity within 50 min (3, 10, 20, 30, 40 and 50 min) Fig. 1. Changes in serum cholesterol levels and after [14C] -cholesterol injection between the radioactivity in probucol-treated and control mice 2 groups (data not shown). Half life times of fed a basal diet. a: Changes in serum cholesterol radioactivity decay in serum were calculated levels in normal mice during daily treatment with to be 31.9±1.3 hr for the treated group and probucol at 400 mg/kg (0) or vehicle (10 ml/kg) (A) for 9 days. Each point represents the mean 37.8±1.2 hr for the control group (P<0.01). value from 7 (probucol) or 8 mice (control). Bars Table 6 shows fecal excretion of radio indicate±S.E. Double star symbols indicate sig activity, cholesterol and bile acids for 6 days nificant difference of P<0.01 from control values. after [14C]-cholesterol injection. Fecal cho b: Changes in serum radioactivity in normal mice lesterol was significantly increased in the after [14C]-cholesterol injection during daily treat treated group, but there was no significant ment with probucol (0) or vehicle (A). [14C]_ difference in fecal bile acids and radioactivity cholesterol (100 pCi/kg) was intravenously injected between the 2 groups, although a slight after the 3rd dose of probucol or vehicle (arrow). increase in fecal radioactivity was observed Each point represents the mean value from 7 in the treated group. At sacrifice of animals, (probucol) or 8 mice (control). there was no significant difference in cho lesterol content in and radioactivity dis They were then divided into 2 groups of 8 tribution among various tissues including the mice each and fed on normal laboratory chow liver, kidneys, small intestines, lungs, spleen, for 5 days thereafter. One group of mice was heart and aortas between the 2 groups (data orally given probucol at a daily dose of 800 not shown). mg/kg, and the other was given vehicle Another experiment was carried out with (olive oil, 20 ml/kg). At the time of diet cholesterol-fed mice. Mice were maintained exchange, [14C] -cholesterol was intra on the high cholesterol diet for 3 weeks. venously injected. As shown in Fig. 2, serum Table 6. Effect of probucol on fecal excretion of the radioactivity, cholesterol and bile acids in normal mice

Fig. 2. Changes in serum cholesterol levels in probucol-treated and control mice fed a high cholesterol diet. Changes in serum cholesterol levels in cholesterol-fed mice before (40) and during daily treatment with probucol at 800 mg/kg (0) or vehicle (20 ml/kg) (A) for 5 days. Each point represents the mean value from 8 mice. Bars indicate±S.E. Single and double star symbols indicate significant difference of P<0.05 and P<0.01, respectively, from control values. o indicates the mean value from 6 normal mice. cholesterol levels were maintained high mice, respectively. during feeding the high cholesterol diet and Table 7 shows fecal excretion of radio were gradually decreased after the diet was activity, total cholesterol and total bile acids exchanged to normal chow. The decrease in which were all significantly increased in the serum cholesterol levels was significantly treated mice. At sacrifice of the animals, there greater in the probucol-treated group than in was no significant difference in cholesterol the control. Serum radioactivity derived from content in and radioactivity distribution the injected [14C]-cholesterol was decreased among various tissues including the liver, significantly faster in the treated mice with a kidneys, small intestines, lungs, spleen, heart half life time of 26.9±1.2 hr than in the and aortas between the 2 groups. control mice with a half life time of 38.0±1.7 hr Effect of probucol on HMG-CoA reductase (n=8, P<0.01) (Fig. 3). Fractional removal and cholesterol 7a-hydroxylase activities: rate of serum cholesterol was calculated to Probucol was orally administered to normal be 0.015±0.002 hr-' and 0.012±0.002 hr-1 mice at a daily dose of 400 mg/kg for 7 days. (P<0.05) for the treated and the control About 20 hr after the last dose, blood samples were taken for determination of serum cho cholesterol 7a-hydroxylase activities. As lesterol, and the animals were sacrificed to shown in Table 8, HMG-CoA reductase obtain the liver and small intestines for assay activity was decreased by 30% in the liver, of microsomal HMG-CoA reductase and aut increased by 53% in the intestines from

Fig. 3. Changes in serum radioactivity in cholesterol-fed mice after [14C]-cholesterol injection during daily treatment with probucol at 800 mg/kg (C) or vehicle (20 ml/kg) (A) for 5 days. ['4C]-Cholesterol (100 pCi/kg) was intravenously injected immediately after the 1 st dose of probucol or vehicle. Each point represents the mean value from 8 mice.

Table 7. Effect of probucol on fecal excretion of radioactivity, cholesterol and bile acids in cholesterol fed mice

Table 8. Effect of probucol on hepatic and intestinal HMG-CoA reductase and cholesterol 7a hydroxylase activities in normal mice the treated mice, although the effects were experiment may be too simple to lead to this not statistically significant. Cholesterol 7a conclusion. Effect of a drug on cholesterol hydroxylase activity was significantly in absorption is usually studied by the combined creased by 86% in the treated mice. Serum use of 2 differently radiolabeled cholesterols cholesterol levels were also significantly as reported by Barnhart et al. with probucol decreased by the treatment. (10). However, the above conclusion was supported by the finding that probucol had Discussion no effect on serum cholesterol in hepatec Cholesterol concentration in the circulating tomized mice with reduced liver functions but blood is influenced by several factors with intact gastrointestinal functions. including intestinal absorption of exogenous The effect of probucol on cholesterol cholesterol, biosynthesis of cholesterol in synthesis was rather complicated. In normal tissues, mainly in the liver and the small mice, probucol was an inhibitor of cholesterol intestines, and catabolic excretion of cho synthesis in the liver but was a stimulator in lesterol into bile as well as reabsorption of the small intestines when [14C]-acetate was excreted cholesterol through the enter used as a precursor of cholesterol synthesis ohepatic circulation. Hypocholesterolemic (Tables 3 and 4). These effects were, however, agents are thought to reduce serum choles not observed with [14C]-mevalonate as a terol levels in animals by affecting some of precursor, indicating the site of action of these factors. probucol was on HMG-CoA reductase Clofibrate, a widely used hypocholes (Table 3). This was supported by the finding terolemic agent, did not lower serum cho that the probucol -treated mice showed a lesterol in mice with a 2 week administration lower HMG-CoA reductase activity in the as reported before (3). On the other hand, liver and a higher activity in the intestines probucol reduced serum cholesterol levels (Table 8). Also, in the cholesterol-fed mice effectively in clofibrate-resistant mice. These whose cholesterogenesis was suppressed by findings suggest that the mode of the the feed-back inhibition of cholesterol, hypocholesterolemic effect of probucol may probucol still stimulated cholesterol synthesis be different from clofibrate. So we have from acetate in the intestines (Table 4). As studied the mode of action of probucol in the liver and the small intestines are the main reducing serum cholesterol in mice. In this organs that produce cholesterol in the body, sort of a study, it seems important to examine these results indicate that the over-all effect some metabolic and biochemical changes in of probucol on cholesterol synthesis is not test animals whose serum cholesterol is involved in lowering serum cholesterol, actually reduced by the drug tested. In the although the effect of a very high dose (800 present study, therefore, probucol was orally mg/kg) could be partially responsible for the administered at a daily dose of 400 mg/kg to reduction of serum cholesterol in normal mice normal and at 800 mg/kg to cholesterol-fed (Table 5). These findings give rise to some mice on the basis of the finding reported questions about the previous conclusion that previously (3). inhibition of cholesterol synthesis is involved The present results (Tables 1 and 2) in the hypocholesterolemic action of this demonstrated no difference in serum radio drug (9, 10, 12). The previous conclusion activity after oral administration of radioactive was drawn only from the study on hepatic cholesterol between the probucol-treated and cholesterol synthesis. The precise mechanism the control mice. This indicates no effect of of this dural action of probucol is not clear probucol on intestinal absorption of yet. exogenous cholesterol. Some difference in The effect of probucol on catabolic serum radioactivity which was observed 24 excretion of cholesterol was the most reliable 72 hr after [14C] -cholesterol ingestion was one in the present study to explain the mode thought to be due to some effect of the drug of the hypocholesterolemic action of this to stimulate catabolism of cholesterol drug. Probucol treatment significantly absorbed (see later). The procedure of this accelerated the decay of the injected radio

in Atherosclerosis, Edited by Noseda, G., Lewis, B. and Paoletti, R., p. 199, Raven Press, New York (1980)

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Nutr. 23, 1229-1233 (1970) Acceleration of catabolic excretion of 5 Drake, J.W., Brandford, R.H., McDearmon, M. cholesterol was also reported as a main mode and Furman, R.H.: The effect of 4,4-(iso of action of probucol by several workers with propylidenedithio) -bis-(2,6-di-t-butyl phenol) cholesterol-fed monkeys (10) and hyper (DH-581) on serum lipids and lipoproteins in human subjects. Metabolism 18, 916-925 cholesterolemic patients (9, 11). However, Li et al. (12) reported the decrease in fecal (1969) 6 Lelorier, J., Dubreuil-Quidoz, S., Lussier-Cacan, sterols and hepatic cholesterol 7a-hy S., Haung, Y.S. and Davignon, J.: Diet and droxylase activity -in the probucol-treated Probucol in lowering cholesterol concentrations. rats, the opposite findings to other workers Arch. Intern. Med. 137, 1429-1434 (1977) (9-11 ) and our present finding. 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