TMP-153, a novel ACAT inhibitor, inhibits cholesterol absorption and lowers plasma cholesterol in rats and hamsters

ATHEROSCLEROSIS Atherosclerosis I 13 (1995) 71-78

TMP-153, a novel ACAT inhibitor, inhibits cholesterol absorption and lowers plasma cholesterol in rats and hamsters Yasuo Sugi.yama* a, Eiichiro Ishikawaa, Hiroyuki Odaka”, Nanami Miki”, Hiroyuki Tawadab, Hitoshi Ikeda” aPharmaceutical

Research

bPharmaceutical

Research

Laboratories II, Pharmaceutical Research Division, Take& 2-l 7-85 Jusohonmachi, Yodogawa-ku. Osaka 532, Japan Laboratories III, Pharmaceutical Research Division, Take& 2-l 7-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan

Chemical

Industries,

Ltd.,

Chemical

Industries,

Ltd.,

Received 3 May 1994; revision received 12 August 1994; accepted 23 August 1994

Abstract Effectsof TMP-153, ~-[4-(2-chlorophenyl)-6,7-dimethyl-3-quinolyI]-N’-(2,4-difluorophenyl)urea,on intestinaland hepaticacyl-CoA:cholesterolacyltransferase(ACAT) activities,cholesterolabsorptionand plasmacholesterollevel in rats and hamsterswere studied.TMP-153 hasIC,values of around S-10 nM for the hepatic and intestinalACAT from variclus animals.The most potent inhibition was observedin the intestinal ACAT from Golden hamsters (KS0= 2.3 nM). The inhibition modeof TMP-153 wasnon-competitivefor rat intestinalACAT. TMP-153 inhibited cholesterolesterificationboth in humancolonic adenocarcinoma cells,LS180,and in humanhepatomacells,HepG2 (ICB = 150nM and 330 nM, respectively).[‘4C]cholesteroland cold cholesterolabsorptionfrom the smallintestine wasmarkedly inhibited by oral administrationof TMP-153 (1 mg/kg)without affecting lymph flow and triglyceride absorption.Whenthe compoundwasgivenasa dietary admixture,plasmacholesterolwasreducedin rats fed a cholesterol diet (IED, = 0.25mg/kg/day),but not in thosefed a stock diet. On the other hand, TMP-153 showedmoreprominenthypocholesterolemic effect in Golden hamstersfed the stock diet (ED, = 0.81 mg/kg/day)than in thosefed the cholesteroldiet (EDSo= 8.01mg/kg/day).In hamstersfed the stock diet, TMP-153 markedlydecreased the hepatic unesterifiedcholesterolin addition to esterifiedcholesterolcontent,but did not affect bileflow andthe biliary secretion of bile acid and lipids. Different mechanisms for plasmacholesterolloweringby TMP-153 betweenrats and hamsters wasdiscussed. Acyl-CoA:cholesterolacyltransferase inhibitor; Cholesterolesterification;Humancell lines;Cholesterolabsorption; Hypocholesterolemic activity; Golden hamsters Keywords:

1. Introrhlction

-* Corresponding author, Tel.: (06) 300 6630; Fax: (06) 300 6306.

Accumulation of ester&d cholesterol is a major metabolic change in the atherosclerotic lesion [ 11. Many clinical studies have shown that reduction in

0021-9150/9Y$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 002 I-9 150(94)05429-M

12

Y. Sugjyama

et al. /Atherosclerosis

plasma cholesterol may lower the incidence of coronary heart disease through the reduction of esterified cholesterol in the atherosclerotic lesion [2-41. Acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) catalyses cholesterol esterification and is involved in cholesterol absorption, hepatic very low density lipoprotein (VLDL)-cholesterol secretion, and cholesterol accumulation in the vascular wall [5]. Therefore, ACAT inhibitors are expected to be cholesterol lowering and antiatherosclerotic agents. TMP-153 is a novel quinolyl derivative showing potent ACAT inhibition [6]. In this article, we describe the effects of TMP-153 on the ACAT activities of various animals, cholesterol absorption and plasma cholesterol levels in rats and hamsters. 2. Materials and methods TMP-153, ZV-[4-(2chlorophenyl)-6,7-dimethyl-3quinolyl]-llr’-(2,4-difluorophenyl)urea was synthesized as reported previously [6]. 2. I. Animals

Male Sprague-Dawley rats, Golden hamsters, Watanabe heritable hyperlipemic (WHHL), rabbits and beagle dogs were individually housed in metal cages in a room with controlled temperature (23 f l”C), humidity (55% f 5%), and light (07:00-19:00 h). They were weaned at 4 weeks of age and maintained freely on water and a stock diet, CE-2 (Clea Japan Inc., Tokyo) for rats and hamsters or RC-4 (Oriental Yeast Co., Ltd., Tokyo) for rabbits. For beagle dogs, 250 g of a laboratory chow diet, CD-5 (Clea Japan Inc., Tokyo) was given once a day (09:OO h). 2.2. ACAT activity

Microsome fractions were prepared from the small intestine and the liver of 7-week-old Sprague Dawley rats, 8-week-old Golden hamsters, 24month-old WHHL rabbits, and lPmonth-old beagle dogs. ACAT activity was measured using [ l14C]oleoyl-CoA as a substrate according to the method of Helgerud et al. [7]. The inhibition mode of TMP-153 was assessed from Lineweaver-Burk plots for various substrate concentrations of oleoyl-CoA.

113 (1995)

71-78

2.3. Cholesterol esterification

in human cells

Both LS180 and HepG2 cells were purchased from the Institute for Fermentation, Osaka (Osaka, Japan). LS 180 cells, derived from human colonic adenocarcinoma, were cultured in MEM containing 10% fetal calf serum (FCS), and HepG2 cells from human hepatoma were cultured in RPMI-1640 containing 10% FCS. After confluency, cells were incubated with [l-‘4C]oleate in the medium at 37°C for 2 h. Cellular lipid was extracted, separated on TLC (Kieselgel 60 FZs4, E. Merck, Frankfurter Strasse 250, FRG) and the radioactivity of esterilied cholesterol fraction was measured [8]. 2.4. Cholesterol absorption in rats

Cholesterol absorption was measured according to the method of Gallo et al. [9]. In brief, sevenweek-old male Sprague-Dawley rats were surgically equipped with a thoracic lymph cannula and a gastric infusion tube under anesthesia (50 mg/kg, Nembutal@‘, Abbott Laboratories, IL). They were placed in restraint cages with free access to saline containing 5% glucose and infused 3 ml/hr of the same solution through the gastric tube. After 24 h, they were orally given lipid emulsion containing 25 mg of cholesterol, 7 PCi of [4-‘4C]cholesterol (Amersham International, England), 145 mg of oleic acid, 144 mg of sodium taurocholate and 25 mg of bovine serum albumin (BSA) with or without TMP153 (1 mg/kg) and lymph fluid was collected for 6 h. Lipids were extracted with chloroform-methanol mixtures [lo], and total cholesterol and triglyceride were measured using commercially available assay kits (cholesterol C-test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Japan, and Cleantech TG-S, Iatron Laboratories Inc., Tokyo). To measure the distribution of radioactive cholesterol, esterified and unesterilied cholesterol was separated on TLC (Kieselgel 60 F,,,, E. Merck, Frankfurter Strasse 250, FRG) using an aliquot of the extracted lipid, and the radioactivity in each fraction was measured. 2.5. Hypocholesterolemic activity in rats and hamsters

Eight-week-old, male Sprague-Dawley rats were given TMP-153 as a dietary admixture of a cholesterol diet containing 1% cholesterol, 0.5% cholic acid and 5% olive oil for 7 days. Ten-week-old, male

Y. Sugjjama et al. /Atherosclerosis 113 (1995) 71-78

Golden hamsters were given TMP-153 as a dietary admixture with or without the cholesterol diet. Plasma cholesterol and triglyceride were measured enzymatically using commercially available kits (Iatron Laboratories Inc., Tokyo). 2.6. Lipid content in the liver Hepatic: lipids were extracted with chloroformmethanol mixtures [lo]. Unesteritied cholesterol and phosphatidyl choline (free cholesterol C-test Wako and phospholipid B-test Wako, Wako Pure Chemical Indust.ries, Ltd., Osaka, Japan), and total cholesterol and triglyceride were measured using commercially available assay kits as described above.

13

Table 1 Inhibition of ACAT activities by TMP-153 Origin of ACAT

IG

Liver Golden hamster Sprague-Dawley rat WHHL rabbit Beagle dog

6.4 9.0 12.0 1.3

Small intestine Golden hamster Sprague-Dawley rat WHHL rabbit Beagle dog

WI)

2.3 6.4

8.0 4.3

2.7. Collection of bile and measurement of biliary composition Hamsters were litted with a cannula on the bile duct and bile was collected for an hour under anesthesia (50 mg/kg, Nembutal@ Abbott Laboratories, IL). Biliary total bile acid (total bile acid test Wako, Wako Pure Chemical Industries, Ltd., Osaka, Ja.pan), and cholesterol and phosphatidylcholine were measured using commercially available kits as described above. 2.8. Statistical analysis The data were expressed as mean f SD. and statistically analysed by Student’s t-test.

o.og0

2

4

6

Oleoyl CoA

8

10

12

(ulUl)

3. Results

3.1. Inhibition of A CAT activity Microsome fractions of the liver and the small intestine were prepared from various species and ACAT activities were measured using [ “C]oleoylCoA as a substrate. TMP-153 potently inhibited activities of ACAT in hamsters, rats, WHHL rabbits and beagle dogs, and 50% inhibitory concentrations (IC& were around 5 to 10 nM (Table 1). The ICsO values for the intestinal enzymes were slightly lower than those for the hepatic ones. The most potent inhibition was observed in the intestinal ACAT from Golden hamsters (ICw, = 2.3 nM). The inhibition mode of TMP-153 for rat intestinal ACAT was shown to be non-competitive (Fig. 1).

-0.75

-0.5

4.25

0.0

0.25 l/S

0.5

Fig. 1. Inhibition of ACAT from rat small intestine by TMP153. A. Effect of TMP-153 on ACAT activity against increasing concentrations of oleoyl CoA. Rat intestinal ACAT activity was measured using oleoyl CoA as substrate in the presence or absenceof TMP-153 (10 nM). B. Lineweaver-Burk plot from data of Fig. 1A.

Y. Sugjyama et al. /Atherosclerosis 113 (1995) 71-78

74

3.2. Inhibition of cholesterolester@ation in human cells

The effect of TMP- 153 on cholesterol esterification was tested using [ “C]oleate as a substrate in LS 180 cells derived from human colonic adenocarcinema and HepG2 cells from human hepatoma. TMP-153 concentration-dependently inhibited oleic acid incorporation into cholesterol ester in both lines of cells (Fig. 2). I& values of TMP- 153 were 150 nM and 330 nM for HepG2 and LS180 cells, respectively.

rats. Rats were surgically equipped with a thoracic lymph cannula and the lymph fluid was collected for 6 h. TMP-153 (1 mg/kg) significantly inhibited cholesterol absorption from the intestine without affecting the lymph flow and triglyceride absorption (Fig. 3). [‘4C]cholesterol absorption was also in-

50*-+-

40

Control TMP-153

T

30-

3.3. Inhibition of cholesterol absorption in rats

The effect of TMP-153 on intestinal cholesterol absorption was examined using [‘4C]cholesterol in

20-

a) HepG2 Cells

--o-

lcTor

0

0.1

,

1

10

TMP-153

6

Hours

8

_i,

4

2

0

F 120w r

Control TMP-153

I

\,

100

1000

(MA)

b) LSl80 Cells

lwr

4

2

0

Hours *-

80 - +

6

T

Control TMP-153

w300 4Q200 .

I&k330

nM

20,

100 0

j/$/l ’ 0

0.1

I 1

I 10

TMP-153

I 100

I 1000

(M)

Fig. 2. Effect of TMP-153 on cholesterol esteritication in HepG2 and LSl80 cells. Cells were incubated in MEM containing [t4Cloleate and bovine serum albumin at 37’C for 2 h. Cellular lipid was extracted and separated by TLC, and the radioactivity of esteritied cholesterol fraction was measured. Mean f S.D. (n = 3).

0

0

I 4

I 2

I 6

Hours

Fig 3. Effect of TMP-153 on lymph flow and lymphatic lipid absorption in rats. Seven-week-old, male Sprague-Dawley rats were surgically equipped with a thoracic lymph cannula and a gastric infusion tube. After 24 h, they were given lipid emulsion containing [ “C]cholesterol with or without TMP-153 (I mgkg) through the gastric tube and lymph fluid was collected for 6 h. Mean f SD. (n = 3-7). *P < 0.05 vs. control.

Y. Sugiyama et al. /Atherosclerosis 113 (1995) 71-78

hibited to 27%-40% of the control during 6 h after the administration (Fig. 4). In the control group, more than 80% of absorbed radioactive cholesterol was distributed to the ester&d cholesterol fraction and less than 20% to the unesterilied cholesterol fraction (Fig. 4). On the contrary, the distribution of absorbed radioactive cholesterol to the esterilied fraction was markedly reduced by TMP- 153 administration, suggesting the inhibition (and gradual restoration) of cholesterol esterification (Fig. 4). 3.4. Hypocholesterolemicactivity in rats andhamsters

ACAT inhibitors are expected to reduce the plasma cholesterol level by two possible mechanisms: through inhibition of cholesterol absorption in the small intestine and suppression of hepatic VLDL secretion.

20 E ‘S P ij P =

-O-+-

15

The hypocholesterolemic effect of TMP-153 was tested in rats and hamsters. TMP-153 effectively lowered plasma cholesterol in cholesterol-fed rats (EDSo = 0.25 mglkglday, Table 2), while the compound was not effective in rats kept on a stock diet even at a dose of 10 mg/kg/day (data not shown). TMP- 153 also dose-dependently reduced plasma cholesterol level in hamsters. Hypocholesterolemic activity of the compound was lo-fold higher in hamsters fed the stock diet (ED,, = 0.81 mglkgiday) than in those fed the cholesterol diet (ED,, = 8.01 mg/kg/day, Table 2). 3.5 Effect of TMP-I53 on hepatic lipid content in Golden hamsters

TMP-153 was given to Golden hamsters as a dietary admixture (mean dose, 0.62 mgikg/day) for

Control TMP-153

2

4

6

Hours Control -r Eateritied cholesterol

G-T-

Unesterified cholesterol

L.

01

1

I

I

2

3

I

I

I

4

5

6

Hours Fig. 4. Effect of TMP-153 on lymphatic (‘4C]cholesterol absorption and its distribution in rats. Lymphatic lipids were extracted with chloroform-methanol mixtures and applied on TLC to separate esterified and unesterified cholesterol, and the radioactivity in each fraction was measured. Other experimental conditions were the same as Fig. 3. Mean f S.D. (n = 3-7). l P < 0.05 vs. control.

76

Y. Sugjyama et al. /Atherosclerosis 113 (1995) 71-78

Table 2 Hypocholesterolemic activity of TMP-153 Cholesterol load Rats Hamsters

+ +

Table 4 Effect of TMP-I53 on liver weight and hepatic lipid content in Golden hamsters

Hypocholesterolemic activity (ED,,, m&g/day) 0.25 0.81 8.01

Seven-week-old, male Sprague-Dawley rats were given TMP153 as a dietary admixture of I% cholesterol diet for 7 days.Tenweek-old, male Golden hamsters were given TMP- 153 as a dietary admixture with or without 1% cholesterol diet for 14 days.

29 days and plasma and hepatic lipids were measured. After 3,14 and 29 days of the treatment, plasma cholesterol level was reduced to 81%, 57% and 43% of that of the control, respectively (Table 3). The cholesterol level was also halved by TMP153 treatment when compared to the initial level. The plasma triglyceride level was not reduced significantly by TMP-153 treatment (Table 3). The hepatic content of both esterified and unesterified cholesterol was increased in control hamsters with advancing age. The esterified cholesterol content was decreased to 72%, 11% and 2.6% of that of the control group after 3, 14 and 29 days of TMP-153 treatment, respectively (Table 4). The hepatic unesteritied cholesterol content was increased in both control and TMP-153-treated hamsters with Table 3 Effect of TMP-153 on body weight, plasma cholesterol and plasma triglyceride in Golden hamsters

Body weight (g)

Plasma cholesterol OwW Plasma triglyceride (Wdl)

Days of treatment

Control

0 3 14 29 0 3 14 29 0 3 I4 29

131 f 6 135 zk 8 134 f 8 147 f 9 158 * 11 171 * 13 199 f I5 198 + 12 213 zt 23 229 f 35 229 f 65 188 + 23

Days of treatment

Control

TMP-153

Liver weight (g)

3 6.82 f 0.73 6.46 f 0.87 14 6.86 f 0.64 6.46 zt 0.61 29 7.94 f 0.61 6.44 f 0.26’ Hepatic lipid content (mg/g liver) Unesteritied 3 1.58 f 0.46 0.99 zt 0.16* cholesterol 14 5.06 zt 1.05 1.57 f 0.05’ 29 6.71 f 0.35 1.43 zk 0.06’ Esteritied 3 6.26 f 0.95 4.48 ziz 1.17* cholesterol I4 11.6 zt 1.15 1.34 zt 0.31’ 29 15.5 f 3.52 0.40 f 0.18* Triglyceride 3 6.28 f I.16 5.25 f 0.62 14 5.66 ztz0.73 3.18 zt 0.37* 29 6.81 f 0.28 2.94 f 0.42’ Phosphatidylchohne 3 15.0 zt 1.40 14.8 zt 2.56 14 15.7 f 0.97 16.6 + 2.03 29 15.5 + 2.15 17.4 f 1.32 Ten-week-old, male Golden hamsters were given TMP-153 as a dietary admixture for 29 days (0.62 mg/kg/day). Mean f SD. (n = 5 or 6). *P < 0.05 vs. control.

advancing age, but was decreased to 21%-63% of the control by TMP-153 treatment (Table 4). Hepatic content of triglyceride and phosphatidylcholine was not altered with advancing age. TMP-153 gradually reduced the hepatic triglyceride content to 43% of control value after 29 days of the treatment, but the compound did not affect the hepatic phosphatidylcholine content (Table 4).

TMP-153 130 * 10 130 Et 10 137 f 8 147 f I 161 f 13 131 * 14* 113 l 14’ 88 f 10; 214 f 33 187 f 26 210 f 43 150 zt 32

Ten-week-old, male Golden hamsters were given TMP-153 as a dietary admixture for 29 days (0.62 mg/kg/day). Mean * SD. (n = 5 or 6). *P < 0.05 vs. control.

Table 5 Effect of TMP-153 on bile flow and biliary composition in Golden hamsters Control Bile flow (&h/100 g) Biliary concentration (mg/dl) Total cholesterol Total bile acid Phosphatidylcholine Total cholesterohphosphatidylcholine ratio

TMP- 153

226 f 109 24.1 f 1029 f 203 zt 0.12 f

3.3 248 9 0.01

223 f 91 26.4 i 1183 l 261 A 0.10 f

5.6 297 71 0.01

Nine-week-old, male Golden hamsters were given TMP- 153 as a dietary admixture for 5 days (0.75 mgIkg/day). Bile was collected from the bile duct for an hour under anesthesia. Mean f S.D. (a = 5 or 6). *P < 0.05 vs. control.

Y. Sugjyama et al. / AIherosclerosis 113 (1995) 71-78

3.6. Bile ji!ow and biliary cholesterol secretion

TMP-153 was given to Golden hamsters as a dietary admixture (mean dose, 0.75 mg/kg/day) for 5 days and the bile was collected. Plasma cholesterol level was significantly reduced to 68% of the control by the compound. However, bile flow and the biliary concentrations of cholesterol, bile acid, and phosphatidylcholine were not affected by TMP153 (Table 4). 4. Mscussion ACAT i,sinvolved in cholesterol absorption in the intestine, VLDL secretion from the liver, and esterificatlon and accumulation of cholesterol in macrophages and smooth muscle cells of the arterial intima [5,11]. Therefore, with ACAT inhibitors, it is possible to lower plasma cholesterol through the suppression of cholesterol absorption in the intestine and VLDL secretion from the liver. They are under development as antiatherosclerotic agents [ 121. TMP-153 is a structurally novel quinolyl derivative with potent ACAT inhibitory action [6]. TMP-153 had an IC,, value of 6.4 nM for rat intestinal ACAT and has 0.25 mgikgday of EDso value for plasma cholesterol lowering in rats fed a cholesterol diet. Such activities suggest one of the most potent ACAT inhibitors reported [12-161. Regarding the inhibitory activity of TMP- 153 on cholesterol absorption, we have demonstrated that the compound inhibited intestinal ACAT activities in various animals and cholesterol esterilication in human colonic adenocarcinoma cells, LS 180. Therefore, the compound is expected to lower plasma cholesterol through intestinal cholesterol absorption. In spite of such an expectation, it was reported that an ACAT inhibitor, CL 277082, failed to inhibit cholesterol absorption in healthy subjects [ 171. Two possibilities have been suggested for such a failure: (1) insufficient inhibitory potency of the drug, and (2) irrelevance of ACAT as the ratelimiting enzyme for cholesterol absorption in humans. The effectiveness of ACAT inhibitors in inhibition oF cholesterol absorption in humans should he clarified using other ACAT inhibitors, including TMP-153, which is 80 times more potent than CL 277082, at least for the inhibition of the rat intestinal ACAT. In the liver, ACAT is reported to regulate apo B-containing lipoproteins in cultured human liver cells (HepG2) [ 181and perfused primate

II

liver [19], and is required for VLDL formation [201. TMP-153 inhibited hepatic ACAT activities from various animals and cholesterol esterihcation in HepG2 cells from human origin. The compound is expected to reduce plasma cholesterol through the suppression of hepatic VLDL secretion. In fact, TMP- 153 effectively lowered plasma cholesterol in hamsters fed a stock diet. In addition, a IO-fold higher dose of the compound was required for plasma cholesterol lowering in hamsters fed the diet containing 1% cholesterol than in hamsters fed the stock diet, indicating different mechanisms from the inhibition of intestinal cholesterol absorption, which is thought to be a main mechanism for plasma cholesterol lowering in rats, for hypocholesterolemic action of TMP-153 in hamsters. In this context, it is interesting that TMP-153 markedly reduced hepatic esterified cholesterol content in hamsters fed the stock diet. Because esteritied cholesterol is necessary for the editing and the secretion of VLDL [18-201, a decrease in hepatic esterified cholesterol may reduce VLDL-cholesterol secretion from the liver. This is a possible mechanism for hypocholesterolemic action of TMP-153 in hamsters. It is noteworthy that TMP-153 also reduced unesterified cholesterol content signiI?cantly in hamster liver, although it may be a secondary/chronic effect because ACAT inhibitors inhibit cholesterol esterification and increase unesterified cholesterol at least in an ACAT-accessible cholesterol pool. Reduction in unesterified cholesterol concentration may upregulate the low density lipoprotein (LDL) receptor in the liver and the resultant increase in LDL clearance is also a possible mechanism to lower plasma cholesterol level in hamsters. The destination of the decreased cholesterol mass in the liver is also interesting. Loss of hepatic cholesterol by TMP-153 treatment is calculated from hepatic cholesterol content and liver weight and the values are 18,95 and 164 mg/hamster at 3, 14 and 29 days of TMP-153 treatment, respectively. We measured bile flow and biliary secretion of bile acid and cholesterol; they were not alfected by TMP- 153, suggesting that the bile did not become lithogenic by TMP-153 treatment in hamsters. Thus, it is not likely that the increase in catabolism or biliary secretion of cholesterol is the mechanism which lowers plasma cholesterol and explains the destination of decreased hepatic cholesterol content. The differ-

18

Y. Sugjyama et al. /Atherosclerosis 113 (1995) 71-78

ence in hepatic cholesterol content is calculated on the assumption of similar hepatic cholesterogenesis between TMP-153~treated and non-treated hamsters. Therefore, it is important to know whether hepatic cholesterogenesis may be decreased by TMP-153 treatment. If that is the case, the data may resolve the question on the destination of cholesterol and also explain why unesterified cholesterol in addition to esterified cholesterol content is reduced by TMP-153 treatment in the hamster liver. In conclusion, TMP-153 is a novel and potent inhibitor of ACAT and may be useful as a hypocholesterolemic agent. Further studies are necessary to elucidate the contribution of the hepatic action of the compound and its mechanism for plasma cholesterol lowering. Acknowledgements Authors gratefully acknowledge the valuable discussions and encouragement throughout this work of Drs. A. Imada and K. Meguro. They also thank Mr. T. Kawabata and Miss T. Kogawa for their excellent technical assistance. References 111Brown, MS., Ho, Y.K. and Goldstein, J.L., The cholesteryl ester cycle in macrophage foam cells. Continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters, J. Biol. Chem., 255 (1980) 9344. 121 Kannel, W.B., Casteri, W.P., Gordon, T. and McNamara, P.M., Serum cholesterol, lipoproteins, and the risk of coronary heart disease: the Framingham study, Ann. Intern. Med., 74 (1971) 1. 131 Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial Results. I, Reduction in the incidence of coronary heart disease,J. Am. Med. Assoc., 251 (1984) 351. 141 Frick, M.H., Elso, O., Haapa, K. et al., Helsinki Heart study: Primary-prevention trial with gemfibrozil in middleaged men with dyslipidemia: safety of treatment, changes in risk factors, and incidence of coronary heart disease, N. Engl. J. Med., 317 (1987) 1237. PI Suckling, K.E. and Stange, E.F., Role of acyl-CoA:cholesterol acyltransferase in cellular cholesterol metabolism, J. Lipid Res., 26 (1985) 647. WI Tawada, H., Harcourt, M., Kawamura, N. et al., Novel acyl-CoA:cholesterol acyltransferase inhibitors. Synthesis and biological activity of 3-quinolylurea derivatives, J. Med. Chem., 37 (1994) 2079.

Helgerud, P., Saarem, K. and Norum, K.R., AcylCoAcholesterol acyltransferase in human small intestine: its activity and some properties of the enzyme reaction, J. Lipid Res., 22 (1981) 271. PI Oram, J.F., Receptor madiated transport of cholesterol between cultured cells and high-density lipoproteins, Methods Enzymol., 129B (1986) 645. [91 Gallo, L.L., Wadsworth, J.A. and Vahouny, G.V., Normal cholesterol absorption in rats deficient in intestinal acyl co-enzyme A: cholesterol acyltransferase activity, J. Lipid Res., 28 (1987) 381. 1101Folch, J., Lees, M. and Sloane-Stanley, G.H., Simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. 1111Shepherd, J. and Packard, C.J., Pharmacological approaches to the modulation of plasma cholesterol, Trends Pharmacol. Sci., 9 (1988) 326. WI Sliskovic, D.R. and White, A.D., Therapeutic potential of ACAT inhibitors as lipid lowering and anti-atherosclerotic agents, Trends Pharmacol. Sci., 12 (1991) 194. iI31 Heider, J.G., Pickens, C.E. and Kelly, L.A., Role of acyl CoA: cholesterol acyltransferase in cholesterol absorption and its inhibition by 57-l 18 in rabbits, J. Lipid Res., 24 (1983) 1127. 1141 Krause, B.R., Bousley, R.F., Kieft, K.A. and Stanfield, R.L., Effect of the ACAT inhibitor (X-976 on plasma cholesterol concentrations and distribution in hamsters fed zero- and low-cholesterol diets, Clin. Biochem., 25 (1992) 371. 1151 Bell, F.P., Gammill, R.B. and St. John, L.C., U-73482: A novel ACAT inhibitor that elevates HDL-cholesterol, lowers plasma triglyceride and facilitates hepatic cholesterol mobilization in the rat, Atherosclerosis, 92 (1992) 115. 1161 Balasubramaniam, S., Simons, L.A., Chang, S., Roach, P.D. and Nestel, P.J., On the mechanism by which an ACAT inhibitor (CL 277082) influences plasma lipoproteins in the rat, Atherosclerosis, 82 (1990) 1. 1171 Harris, W.S., Djovne, C.A., Von Bergmann, K., Neal, J., Akester, J., Windsor, S.L., Greene, D. and Look, Z., Effectsof an ACAT inhibitor CL 277082 on cholesterol metabolism in humans, Clin. Pharamacol. Therap., 48 (1990) 189. WI Cianflone, K.M., Yasruel, Z., Rodriguez, M.A. and Sniderman, A.D., Regulation of apo B secretion from HepG2 cells: evidence for a critical role for cholesteryl ester synthesis in the response to a fatty acid challenge, J. Lipid Res., 31 (1990) 2045. 1191 Carr, T.P. and Rudell, L.L., Partial inhibition of ACAT decreases apo B secretion by the liver of African Green Monkeys, Arteriosclerosis, 10 (1990) 823a (Abstr. 4540). WI Khan, B.V., Fungwe T.V., Wilcox, H.G. and Heimberg, M., Cholesterol is required for the secretion of the very low density lipoprotein: in vivo studies, B&him. Biophys. Acta, 1044 (1990) 297. [71