TMP-153, a novel ACAT inhibitor, lowers plasma cholesterol through its hepatic action in Golden hamsters

ATHEROSCLEROSIS Atherosclerosis118(1995)145 I53

ELSEVIER

TM&l 53, a novel ACAT inhibitor, lowers plasma cholesterol through its hepatic action in Golden hamsters Yasuo Sugiyama *, Hiroyuki Odaka, Shigekazu Itokawa, Eiichiro Ishikawa, Yoshiaki Tomari, Hitoshi Ikeda

ReceivedI9 January 1995;revision received

4

April 1995:acceptedI9 April 1995

Abstract

The mechanism of the hypocholesterolemic action of N-[4-(2-chlorophenyl)-6,7-dimethyl-3-quinolyl]-~‘-(2, 4-difluorophenyl) urea (TMP-153), a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was studied in Golden hamsters. TMP-153 (0.5-1.5 mg/kg) dose-dependently reduced plasma total- and low density lipoprotein (LDL)-cholesterol without affecting high density lipoprotein (HDL)-cholesterol. TMP-153 markedly reduced the cholesterol influx into the plasma upon intravenous injection of Triton WR-1339. The compound also decreasedcholesterol absorption calculated from dietary intake, biliary secretion and the absorption co-efficient. Hepatic cholesterol secretion was calculated by substracting the cholesterol absorption from the cholesterol influx. In hamsters, the liver accounted for 92% of the cholesterol influx with the remaining 8% coming from the intestine, and both were markedly decreasedby TMP-153. Thus, it is likely that TMP-153 lowers plasma cholesterol through its hepatic action. In the liver, the compound significantly reduced the unesterified cholesterol content in addition to markedly reducing the content of esterified cholesterol. In accordance with this reduction, the half-life time of [12’I]-LDL was significantly shortened by the compound, suggesting an increase in LDL receptors. However, the hepatic cholesterogenesisfrom [14C]acetatewas decreasedby TMP-153 treatment. This effect seemsto be secondary, since the compound did not inhibit cholesterogenesis from [14C]acetatein HepG2 cells. From the data described above, the contribution of hepatic secretion and intestinal absorption of cholesterol to the plasma cholesterol level in Golden hamsters are discussed. Keywords: ACAT inhibitor; Golden hamster; Plasma cholesterol: Cholesterol absorption: Hepatic cholesterol secretion: LDL clearance

1. introduction

Accumulation of esterified cholesterol is a major metabolic change in atherosclerotic lesions [l]. Many clinical studies have shown that decrease in

* Correspondingauthor 002l-9150/95/$09.50

C 1995 Elsevier

SSD/OO21-9150(95)05601-R

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plasma cholesterol lowers the incidence of coronary heart disease through the reduction of esterified cholesterol in atherosclerotic lesions [2-41. Acyl-CoA cholesterol acyltransferase (ACAT, EC 2.3.1.26) catalyzes cholesterol esterification and this enzyme is involved in intestinal cholesterol absorption, hepatic very low density lipoprotein (VLDL)-cholesterol secretion and cholesterol accumulation in the vascular wall [5-61. Therefore, ACAT inhibitors have the potential to lower plasma cholesterol through the suppression of intestinal cholesterol absorption and hepatic VLDL secretion and are under development as hypocholesterolemic and antiatherosclerotic agents [7,8]. N-[4-(2-chlorophenyl)-6,7-dimethyl-3-quinolyl]N’-(2,4-difluorophenyl) urea (TMP-153) is a structurally novel and potent ACAT inhibitor [8]. We reported the ACAT inhibition and plasma cholesterol lowering activities of this compound in rats fed a cholesterol diet [9] and these activities were more potent than those of the other ACAT inhibitors reported so far [7,10- 131. TMP-153 inhibited hepatic ACAT activities in various animals and cholesterol esterification in HepG2 cells, and had a hypocholesterolemic effect in hamsters, which show human-like cholesterol metabolism [9]. In hamsters, the prominent contribution of the hepatic action of TMP-153 in plasma cholesterol lowering was suggested from the observation that the compound was more effective for plasma cholesterol lowering in hamsters fed a stock diet than in those fed the stock diet supplemented with 1% cholesterol [9]. In this article, we describe the effect of TMP-153 on the kinetics and metabolism of cholesterol in hamsters and the contribution of hepatic secretion and intestinal absorption of cholesterol to the plasma cholesterol level are discussed. 2. Materials

and methods

2.1. Animals and diet

Male Golden Syrian hamsters were individually housed in metal cages in a room with controlled temperature (23 f l”C), humidity (55% + 5%) and light (7 am-7 pm). They were weaned at 4 weeks of age, maintained freely on water and a

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stock diet, CE-2 (Clea Japan Inc., Tokyo), and used at 8-10 weeks of age without fasting. The diet contains 35 mg of sterols/lOO g as estimated by the cholesterol oxidase method and the dietary cholesterol intake was calculated to be 3.2 mg/ day/hamster from food intake. 2.2. Drug administration

TMP-153 was synthesized as reported previously [8]. The compound was suspended in 5% gum arabic solution and orally administered via a stomach tube twice a day (9 am and 4 pm), or given as a dietary admixture. 2.3. Hypocholesterolemic

activity

Blood was taken from the orbital sinus of nonfasted hamsters, and plasma cholesterol and triglyceride were measured enzymatically using commercially available assay kits (Iatron Laboratories Inc., Tokyo). 2.4. Separation of lipoproteins

Blood was collected by decapitation and lipoproteins were separated from ethylenediamine tetraacetic acid (EDTA) plasma (4 mmol/l) by ultracentrifugation according to the method of Hatch and Lees [14]. 2.5. Cholesterol influx into plasma at steady state

Cholesterol influx into plasma was measured upon an intravenous injection of Triton WR-1339 (400 mg/kg) [15]. Cholesterol influx into plasma was calculated from the increment in plasma cholesterol levels over the first 2 h following the injection of Triton WR-1339. 2.6. Cholesterol absorption

Cholesterol absorption was measured according to the method of Zilversmit [16]. In brief, hamsters were surgically equipped with a jugular vein cannula under anesthesia (50 mg/kg, Nembutal@, Abbott Laboratories IL) and given free accessto CE-2 diet and water. After 24 h they were orally given 1 ml of lipid emulsion containing 8.3 mg of cholesterol, 4 PCi of [4-‘4C]cholesterol, 48 mg of oleic acid, 48 mg of sodium taurocholate and 8.3 mg of bovine serum albumin (BSA) in saline with or without TMP-I53 (1 mg/kg) and were intra-

Y. Sugiyama

et al. 1 Atherosclerosis

venously given 0.5 ml of saline containing 2.5 mg of BSA and 4 PCi of [1,2-3H]cholesterol. Two days later, the “C/3H ratio in the plasma became constant and was expressed as an absorption coefficient. 2.7. Lipid content in the liver

Hepatic lipids were extracted by the method of Folch [ 171.Total and unesterified cholesterol and phospholipids (Cholesterol C-test, Free cholesterol C-test and Phospholipid-test, Wako Pure Chemical Industries, Ltd., Osaka, Japan) and triglyceride (Cleantech TG-S, Iatron Laboratories Inc., Tokyo) were measured using commercially available assay kits.

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terol esterification and cholesterogenesis were measured by incubating cells in the same medium supplemented with [ 1-‘4C]oleate (2p Ci/ml) and [2-14C]acetate(lpCi/ml), respectively, at 37°C for 2 h. Cellular lipid was extracted and separated by TLC to measure the radioactivity of esterified and unesterified cholesterol fractions [ 181. 2.11. Statistical analysis

The data we‘re expressed as mean F SD. and statistically analyzed by Student’s t-test or Duncan’s multiple range test. 3. Results

2.8. Choksterogenesis in the liver

3.1. Effects on plasma lipid levels and hepatic lipid content

Ten-week-old hamsters were given TMP-153 (0.65 mg/kg/d) or lovastatin (1.2 mg/kg/d) as a dietary admixture for 24 days. Lovastatin (3 mg/ kg) was also given orally to a group of hamsters maintained on a stock diet 1 h before the intraperitoneal injection of [14C]acetate (10 pCci/ hamster). Livers were removed one hour after the injection. Hepatic lipids were extracted by the method of Folch [17] and separated by TLC, and the radioactivity of the unesterified cholesterol fraction was measured [18].

Hamsters were given TMP-153 orally for 21 days, and plasma lipids and hepatic lipid content were measured. Plasma cholesterol was dose-dependently lowered after 8 days of TMP-153 treatment and the reduction in plasma cholesterol reached a constant level after 3 weeks (Fig. 1). The ED,, of TMP-153 for hypocholesterolemic effect was calculated to be 5.6 mg/kg, b.i.d. Lipoproteins were separated by ultracentrifugation and the distribution of cholesterol among the lipoproteins was

2.9. Low density lipoprotein clearance

Eight-week-old hamsters were given TMP-153 (0.68 mg/kg/d) or lovastatin (1.1 mg/kg/d) as a dietary admixture for 12 days. Hamsters were intravenously injected with human [‘*‘I]-low density lipoprotein (LDL) (3pCi/hamster) and blood samples were taken 0.5, 3, 6, 9 and 24 h after the injection. Half-life was calculated from the decay curve of radioactivity in the plasma protein fraction [19]. 2.10. Cholesterol esteriJication and cholesterogenesis in HepG2 cells

HepG2 cells were purchased from the Institute for Fermentation, Osaka (Osaka, Japan). HepG2 cells were cultured in RPMI-1640 containing 10% foetal calf serum (FCS). After confluency, cells were cultured in the presence of TMP-153 (0.5 PM). After 0, 24 and 72 h of the culture, choles-

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Days Fig. I. Effect of TMP-I 53 on plasma cholesterol level in Golden hamsters. Eight-week-old male Golden hamsters were given TMP-153 twice daily for 21 days and plasma cholesterol was measured before and after 3, 8, I4 and 21 days of TMP-153 treatment. Mean + S.D. (n = 5). *P < 0.05 vs. control.

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measured. In the control group, cholesterol levels of high density lipoprotein (HDL), LDL and VLDL plus chyromicron fraction were 70, 92 and 53 mg/dl, respectively. TMP-153 (5 mg/kg, b.i.d.) reduced those levels to 39, 33 and 29 mg/dl, respectively. Thus, the compound most prominently decreased LDL-cholesterol by 64% and reduced the LDL/HDL ratio, an atherogenic index, from 1.32 to 0.85 (data not shown). TMP153 did not affect plasma triglyceride levels in hamsters at the doses tested (data not shown). In the liver, esterified cholesterol content was markedly decreased and unesterified cholesterol content was significantly decreased by TMP-153 treatment (Fig. 2). Since the compound also decreased liver weight dose-dependently by 6%16%, total cholesterol in the liver was decreased by TMP-153 treatment (data not shown). The compound did not affect hepatic triglyceride content (Fig. 2).

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3.2. Cholesterol influx into plasma

TMP-153 was given to hamsters for 14 days and cholesterol influx into plasma was measured upon intravenous injection of Triton WR-1339. TMP-153 (2.3 mg/kg/d) lowered plasma cholesterol to 68% that of the control group and decreased cholesterol influx into the plasma to 35% that of the control without affecting the triglyceride influx into the plasma (Table 1). 3.3. Inhibition

of cholesterol absorption

Effect on cholesterol absorption was measured by the double radioisotope method in hamsters. The absorption coefficient of cholesterol in the control group was 75.3%. TMP-153 treatment (5 mg/kg, b.i.d.) lowered the plasma cholesterol level to 77% of that in the control group in hamsters fed the stock diet. TMP-153 treatment reduced the absorption coefficient to 27.3% (Table 2). 3.4. EfSect on cholesterogenesis in the liver

To test the effect on cholesterogenesis in vivo, hamsters were given TMP-153 or lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitor, for 24 days, and cholesterogenesis from [14C]-acetate was mea-

0

0.5 1.5 5 TMP-153 (mglkg, b.i.d.)

Fig. 2. Effect of TMP-153 on hepatic lipid content in Golden hamsters. Eight-week-old male Golden hamsters were given TMP-153 orally twice daily for 21 days as described in Fig. I, After decapitation, livers were removed and hepatic lipid was measured as described in Materials and methods. Mean i S.D. (n = 5). Different script letters in the figure indicate a significant difference (P < 0.05) between the respective data.

sured. The plasma cholesterol level was clearly reduced to 53% of that in the control group by TMP-153 treatment but was not reduced by lovastatin treatment (Fig. 3). TMP-153 clearly inhibited the incorporation of [14C]-acetate into cholesterol (Fig. 3). On the other hand, lovastatin suppressed cholesterogenesis acutely but not chronically at a dose tested (Fig. 3).

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Table 1 Effect of TMP-153 on lipid influx into plasma in Golden hamsters

Body weight (g) Plasma cholesterol (mg:dl) Cholesterol influx (mg/dl!2 h) Triglyceride influx (mgidli2 h)

Control

TMP-153

l26& 231 f 79.0 f 812 f

125i 6.2 162k 19* 21.5 * 14.4* 1016+ 178

I1 24 22.2 146

Ten-week-old male Golden hamsters were given TMP-I 53 as a dietary admixture for I4 days (2.32 mgjkg;d). They were intravenously injected with Triton WR-1339 and plasma lipid levels were measured before and 2 h after the injection. The difference in lipid levels is expressed as the influx into the plasma. Mean + S.D. (n = 4). *P c 0.05 vs. control.

Control

TMP-153

12000

.!I w

9000

Lovastatin

Control

a -l1

a

3.5. LDL clearance

An increase in plasma LDL clearance may also contribute to the reduction of plasma cholesterol. Therefore, the effect of TMP-153 or lovastatin on LDL clearance was measured using [‘251]-LDL. Both TMP-153 and lovastatin significantly shortened the half-life of [“51]-LDL (Table 3). 3.6. Cholesterol esterljication and cholesteropenesis in HepG2 cells

TMP-153 (0.5 PM) markedly decreasedcholesterol esterification in HepG2 cells without affecting the incorporation of [‘4C]oleate into triglyceride during the 3 day culture period (Fig. 4). The compound did not inhibit cholesterogenesis from [14C]acetatein HepG2 cells at this concentration.

Table 2 Effect of TMP-i53 on cholesterol absorption in Golden hamsters Plasma cholesterol hdd])

Absorption coefficient of cholesterol (xl)

Control 201& 10 75.3 f 14.3 TMP-153 154237* 21.3 + 5.4* _____ Eight-week-old male Golden hamsters were orally given 5 mg/kg of TMP-153 twice daily for 16 days. They were then given [‘%I-cholesterol orally as a lipid emulsion and [‘HIcholesterol intravenously. Mean + SD. (tz= 4 or 5). P -c 0.001 vs. control.

Control

TMP-153

Lovastatin

Lovastatin (acute)

Fig. 3. Etfect of TMP-153 and lovastatin on cholesterogenesis in Golden hamsters. Ten-week-old male Golden hamsters were given TMP-I53 (0.65 mg/kg/d) or lovastatin (1.2 mg/kg/d) as a dietary admixture for 24 days and plasma cholesterol was measured. Lovastatin (3 mg/kg, once) was given orally to another group of hamsters I h before the intraperitoneal injection of [‘Y?]acetate. Livers were removed I h after the injection and the radioactivity in the hepatic cholesterol fraction was measured. Mean f S.D. (n = 5 or 6). Different script letters in the figure indicate a significant difference (P < 0.05) between the respective data.

4. Discussion Since Golden Syrian hamsters have a plasma cholesterol level, a rate of cholesterogensis and lipoprotein metabolism similar to those in humans [20&21], the hamster is thought to be a good model in which to estimate the efficacy of hypocholesterolemic agents in humans. In hamsters, TMP-153 potently inhibits ACAT activity in the liver and the small intestine and shows a hypocholesterolemic effect [9]. Therefore, TMP153 may be efficacious for plasma cholesterol lowering in humans. Interestingly the compound showed more effective hypocholesterolemic activ-

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ity in hamsters fed a stock diet than in those fed the same diet supplemented with 1% cholesterol, suggesting the prominent contribution of the hepatic action of TMP-153 to the plasma cholesterol lowering effects. In this study, we examined the kinetics and metabolism of cholesterol in hamsters fed the stock diet. From the data, the contribution of hepatic secretion and intestinal absorption of cholesterol to the plasma cholesterol level are assessedas follows. Body weight and plasma volume were assumed to be 130 g and 5 ml, respectively. The data were normalized for body weight and the values are expressedas per day and/or per hamster in Fig. 5. The pool size of plasma cholesterol was calculated to be 10.1 mg from the plasma cholesterol concentration and plasma volume in a hamster fed the stock diet (Fig. 5). Absorbed cholesterol (3.8 mg/d) was calculated from the dietary intake (3.2 mg/d), biliary secretion (1.8 mg/d, calculated using the data reported previously [9]) and absorption coefficient of cholesterol (75%). Hepatic cholesterol secretion was calculated to be 43.6 mg/d by subtracting the absorbed cholesterol from the total influx (47.4 mg/d), indicating that more than 92% of cholesterol influx into plasma is derived from the liver. Thus, it was indicated that total cholesterol influx is mainly derived from the liver in hamsters fed the stock diet. The values for TMP-153-treated hamsters are shown in parentheses in Fig. 5. TMP-153 inhibited both the cholesterol absorption by 63% (TMP-153 treated group: 1.4 mg/d vs. control group: 3.8 mg/d) and the hepatic Table 3 Effect of TMP-I 53 on half-life of [‘251]-LDL in Golden hamsters

Control TMP-153 Lovastatin

Plasma cholesterol tmg/dl)

T cd;’

1X&29 134+20* 200 _+33

14.52+ 0.88 13.28+ 0.88* 12.13+ 2.25*

Eight-week-old male Golden hamsters were given TMP-153 (0.68 mg/kg/d) or lovastatin (1.1 mg/kg/d) as a dietary admixture for 12 days. They were given [‘251]-LDL intravenously. Mean + S.D. (n = 5-6). *P < 0.05 vs. control.

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46

72

46

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Fig. 4. Effect of TMP-I 53 on cholesterol esterification, triglyceride formation and cholesterogenesisin HepG2 cells. HepG2 cells were cultured in RPMI-1640 containing 10% FCS with or without TMP-I 53 (0.5 ,uM) for 72 h. Cholesterol esterification and triglyceride formation were measured using [‘4C]oleate, and cholesterogenesis was measured using [14C]acetateas described in Materials and methods.

cholesterol secretion by 77% (TMP-153-treated group: 10.0 mg/d vs. control group: 43.6 mg/d). As a result, 93% of the reduction in cholesterol influx into the plasma is explained by the inhibition of hepatic cholesterol secretion. In addition plasma cholesterol distribution in VLDL plus chyromicron fraction was decreasedfrom 53 to 29 mg/dl by TMP-153 treatment. Since ACAT is reported to regulate apolipoprotein B (apo B)containing lipoproteins in the cultured human liver cells (HepG2) [22] and the perfused primate liver [23] and is required for VLDL formation

[24], TMP-153 is thought to reduce plasma cholesterol level through the suppression of hepatic VLDL-cholesterol secretion. Thus, more prominent inhibition of the hepatic secretion than that of the intestinal absorption by TMP-153 explains at least in part the reason why the compound showed more effective hypocholesterolemic activity in hamsters fed a stock diet than in those fed a cholesterol diet. There has been speculation as to undesirable hepatic efftcts of ACAT inhibitors; ACAT inhibitors increase the hepatic concentration of unesterified cholesterol and cause downregulation of the hepatic LDL receptor. However, TMP-153 slightly but significantly shortened the half-life time of [‘251]-LDL in hamsters and this may be another mechanism for plasma cholesterol lowering. We used human LDL preparation to show LDL receptor-dependent LDL clearance, since human LDL is well known to contain only apo B. However, it is interesting and important to confirm the change of homologous LDL clearance Liver Pool size: 1amg (31.2)

0 Bilii

Plasma Pool size : 10.1 mg (7.7) Chdesterol influx 47.4 rng 43.6 nq (11.4) (10.0) a) 3.6n-q A (1.4) : absorption elfkbnoy 75% (27) 5.0 (5.0)

Intestine

Fig. 5. Effect of TMP-153 on cholesterol dynamics in Golden hamsters. The contribution of hepatic secretion and intestinal absorption of cholesterol to the plasma cholesterol level are assessedas follows. Body weight and plasma volume were assumed to be 130 g and 5 ml. respectively. The data were normalized for body weight and the values are expressed as per day and/or per hamster. The pool size of plasma cholesterol was calculated from the plasma cholesterol concentration and plasma volume. Absorbed cholesterol was calculated from the dietary intake, biliary secretion and absorption coefficient of cholesterol. Hepatic cholesterol secretion was calculated by subtracting the absorbed cholesterol from the total cholesterol influx. The values for TMP- l53-treated hamsters are shown in parentheses.

by TMP-153 treatment in hamsters, becausehamster preparation almost exclusively containing apo B is reported [25]. Since the LDL receptor is regulated in a sterol-mediated manner at the transcription level [26,27] and the compound reduces the unesterified cholesterol content in the liver, the effect of TMP-153 on LDL clearance may be explained by upregulation of LDL receptors. Recently, regulation of the LDL receptor gene expression through non-sterol signals has been suggested[28], and the stimulation of LDL clearance by TMP-153 through a non-sterol mechanism is also possible. Futhermore, the effect of TMP-153 on LDL clearance explains why the reduction of cholesterol distribution to LDL is the most prominent. The upregulation of LDL receptor activity by HMG-CoA reductase inhibitors was reported to contribute to plasma cholesterol lowering although it is ineffective with regard to whole body cholesterogenesisin humans [29]. We showed that lovastatin acutely inhibited hepatic cholesterogenesis by 64°K at a dose of 3 mg/kg. In addition, lovastatin chronically shortened the half-life of [“‘I]-LDL in the piasma at a dose of 1.I mg/kg/ day, but was not effective on plasma cholesterol lowering and hepatic cholesterogenesischronically in hamsters at the dose tested. Ma et al. showed the cholesterol-lowering effect and upregulation of LDL receptor and HMG-CoA reductase by lovastatin (30-70 mgjkglday) in hamsters [30]. Thus stronger upregulation of the LDL receptor and/or other mechanisms, such as a suppression of hepatic cholesterol secretion, may be required to show hypocholesterolemic activity in hamsters. In this context, it is noteworthy that TMP-153 significantly decreasesthe unesterified cholesterol content in addition to markedly reducing the esterified cholesterol in the hamster liver, since ACAT inhibitors would be expected to increase unesterified cholesterol content. These results suggest some changes in hepatic cholesterol metabolisms and we have demonstrated that hepatic cholesterogenesis is clearly decreased by TMP-153 treatment in hamsters. Therefore, the suppression of hepatic cholesterogenesis is also responsible for the reduction in hepatic cholesterol content and subsequent cholesterol secretion

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in addition to the inhibition of hepatic cholesterol esterification in hamsters. Since TMP-153 does not inhibit cholesterogenesis in HepG2 cells directly, the effect seemsto be secondary. Recently, it has been reported that cellular cholesterol is differentially compartmentalized and these cholesterol pools have different functions [31]. Therefore, it is possible that a cholesterol pool responsible for the regulation of HMG-CoA reductase gene expression is increased while total cellular cholesterol content is reduced. Further studies are necessary to elucidate the mechanisms by which TMP-153 decreasesthe hepatic cholesterogenesis in hamsters. Acknowledgements The authors are grateful to Drs. A. Imada and K. Meguro for valuable discussion and encouragement throughout this work. They also thank Miss Y. Sano for her excellent technical assistance. References [I] Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters. J Biol Chem 1980;255:93449352. [2] Ball MJ. Dietary intervention trials - Effect on cardiovascular morbidity and mortality. Curr Opin Lipidol 1993;4:7- 12. [3] Tikkanen MJ. Fibric acid derivatives. Curr Opin Lipidol 1992;3:29-33. [4] Hunninghake DB. HMG GoA reductase inhibitors. Curr Opin Lipidol 1992;3:22-28. (51 Suckling KE, Stange EF. Role of acyl-CoA:cholesterol acyltransferase in cellular cholesterol metabolism. J Lipid Res 1985;26:647-671. [6] Shepherd J, Packard CJ. Pharmacological approaches to the modulation of plasma cholesterol. Trends Pharmacol Sci 1988;9:326-329. [7] Sliskovic DR. White AD. Therapeutic potential of ACAT inhibitors as lipid-lowering and anti-atherosclerotic agents. Trends Pharmacol Sci 1991;12:194- 199. [8] Tawada H, Harcourt M, Kawamura N, Kajino M, Ishikawa E Sugiyama Y, Ikeda H, Meguro K. Novel acyl-CoA:cholesterol acyltransferase inhibitors. Synthesis and biological activity of 3-quinolylurea derivatives. J Med Chem 1994;37:2079-2084. [9] Sugiyama Y, Ishikawa E, Odaka H, Miki N, Kawabata T Kogawa T, Tawada H, Ikeda H. TMP-153, a novel

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lovastatin therapy on the parameters of whole-body cholesterol metabolism. J Clin Invest 1990:85:801-808. [30] Ma PTS. Gil G, Shudhof TC, Bilheimer DW. Goldstein JL Brown MS. Mevinolin. an inhibitor of cholesterol synthesis induces mRNA for low density lipoprotein receptor in livers of hamsters and rabbits. a competitive inhibitor of the reductase. Proc Natl Acad Sci 1986;83:8370 8374. [31] Bilhartz LE, Spady DK, Dietschy JM. Inappropriate hepatic cholesterol synthesis expands the cellular pool of sterol available for recruitment by bile acids in the rat. J Clin invest 1989:84:1181~ 1187.