Effect of the ACAT inhibitor CI-976 on plasma cholesterol concentrations and distribution in hamsters fed zero- and low-cholesterol diets

Clin Biochem, Vol. 25, pp. 371-377, 1992 Printed in the USA. All rights reserved.

0009-9120/92 $5.00 + .00 Copyright v 1992 The Canadian Society of Clinical Chemists.

Effect of the ACAT Inhibitor CI-976 on Plasma Cholesterol Concentrations and Distribution in Hamsters Fed Zero- and Low-Cholesterol Diets BRIAN R. KRAUSE, RICHARD F. BOUSLEY, KAREN A. KIEFT, and RICHARD L. STANFIELD

Atherosclerosis Section, Department of Pharmacology, Parke-Davis Pharmaceutical Division, Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, MI 48105, USA The overall objective of the present study was to determine if the ACAT inhibitor CI-976 can lower plasma cholesterol in hamsters fed zero or low, "human-like" levels of cholesterol. With a purified diet containing zero dietary cholesterol, CI-976 significantly lowered VLDL cholesterol (VLDL-C), but not total plasma cholesterol (TPC). When 0.06% cholesterol was added to this diet, reductions in both VLDL and LDL cholesterol (LDL-C) lowered TPC. Efficacy was still greater with 0.2% dietary cholesterol, but not potency. Mixing Cl-976 into the purified diet resulted in greater decreases in VLDL-C compared to gavage administration, but LDL-C reductions with 0.2% cholesterol were optimal with gavage. With nonpurified, chow-based diets efficacy was markedly greater with diet-admix administration, regardless of the amount of dietary cholesterol. CI-976 inhibited cholesterol absorption with chow-based diets more potently compared to nonabsorbable agents (e.g., beta-sitosterol, tigogenin cellobioside), and the lowering of LDL-C was greatest when inhibition of cholesterol absorption was maximal. We conclude that the ACAT inhibitor Cl-976 is efficacious in hamster models which utilize human-like levels of dietary cholesterol. Moreover, the data suggest that the pharmacologic responses to lipophilic ACAT inhibitors in the hamster, or even other lipidregulating drugs, are likely to depend not only on the type of basal diet but also on the mode of drug administration.

KEY WORDS: cholesterol; lipoproteins; cholesteryl esters; cholesterol acyltransferase.

Introduction AT (acyl-CoA:cholesterol acyltransferase, EC A~ .3.1.26) is a microsomal enzyme responsible for the esterification of cholesterol in a variety of tissues from different species (1). Considerable evidence has accumulated supporting a role for ACAT in the esterification and hence the absorption of most exogenous (dietary) and endogenous (biliary) cholesterol. In addition, the liver enzyme is thought to determine the amount of cholesteryl esters, and possibly apoB, secreted in the form of very low density lipoproteins (VLDL). Finally, it is generally appreciated that ACAT-derived cholesteryl esters in arterial macrophages are the hallmark of the ath-

Correspondence: Brian R. Krause, Ph.D. Manuscript received April 2, 1992; revised July 6, 1992; accepted July 13, 1992. CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

erosclerotic process, especially in the formation of the initial fatty streaks. Much effort has gone into the discovery and development of specific inhibitors of this enzyme. The diverse chemical structures and pharmacologic profiles of many ACAT inhibitors have recently been described (2). Essentially all of the pharmacologic data on ACAT inhibitors is derived from only two animal species as experimental models, namely, rats and rabbits. However, an emerging concept in our laboratory and elsewhere is that the cholesterol-fed rat represents an extreme case with respect to ACAT. The ACAT activities and capacity to store and/or secrete cholesteryl esters in lipoprotein-producing organs in this animal model are very high, and thus it is relatively easy to demonstrate efficacy in the form of plasma cholesterol lowering with ACAT inhibitors. Likewise, plasma cholesterol can be reduced in rabbits with very weak ACAT inhibitors such as octimibate (3) and melinamide (4). Therefore, we have initiated studies in another animal species to further characterize the ACAT inhibitor, CI-976. This compound has been shown to lower plasma cholesterol in rats (5) and rabbits (6) fed relatively high levels of dietary cholesterol. The purpose of the present experiments was to determine the conditions in which CI-976 can optimally lower plasma cholesterol in hamster models that utilize lower, more human-like levels of dietary cholesterol.

Methods All animals were male Golden Syrian hamsters from Sasco (Omaha, NE, USA) weighing 100-110 g. They were randomly assigned to treatment groups. Both purified and nonpurified (chow) diets were used in these experiments. The composition of the purified diet is listed in Table 1. It is basically similar to the purified, cholesterol-containing hamster diet described by Turley, Daggy, and Dietschy (8). The nonpurified chow diet was Certified Rodent Chow #5002 (Ralston-Purina, St. Louis, MO, USA) supplemented with hydrogenated coconut oil (10%) and cholesterol at the indicated amounts. Plasma 371

KRAUSE, BOUSLEY,

TABLE 1 Composition of the Purified Diet Ingredient

Amount (% by Weight)

Sucrose Casein Corn starch Corn oil Hydrogenated coconut oil Cellulosea DL-Methionine Choline bitartrate Salt mixb

36.5 20.0 20.0 1.0 10.0 8.0 0.3 0.2 3.5

Vitamin mix b Cholesterol

1.0 0, 0.06, 0.2

This diet was formulated by Dr. E. Ulman, Research Diets, Inc., New Brunswick, NJ, USA a Avicel, PH101, FMC Corporation, Philadelphia, PA, USA. b AIN-76A mixes (7). cholesterol (9) and triglyceride (10) concentrations were determined enzymatically using an Abbott VP Series II Bichromatic Analyzer (Abbott Laboratories, Chicago, IL, USA) and reagents from Boehringer-Mannheim (Indianapolis, IN, USA). Liver samples were homogenized and extracted into 20 volumes of isopropanol. Aliquots of these extracts were assayed for cholesterol by a modification of the method of Cooper (11). The mass of cholesterol per g of wet tissue was multiplied by the liver weight to obtain total liver cholesterol content. The distribution of cholesterol among plasma lipoproteins was determined by high-performance gel chromatography (HPGC) using a Superose 6HR column (PharmacialLKB, Piscataway, NJ, USA). The method was essentially the same as we described earlier (12) except that the computer-controlled Beckman System Gold was used (Beckman Instruments, San Ramon, CA, USA). This technique allows the direct, automated determination of VLDL-cholesterol (VLDLC), LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) using microliter q u a n t i t i e s of whole plasma. In one experiment cholesterol absorption was e s t i m a t e d by o r a l l y a d m i n i s t e r i n g 3Hcholesterol (10 uCi/animal) dispersed into a stabilized triglyceride/phospholipid emulsion (Intralipid, KabiVitrum, Inc., Alameda, CA, USA). Twenty-four hours later the radioactivity in plasma and liver was expressed as a percentage of the administered dose and compared to untreated controls. This represents a crude indirect measure of cholesterol absorption; and we are currently attempting to validate an isotopic technique in hamsters. CI-976 was administered either by daily oral gavage (0800-0900 h) using a carboxymethylcellulose/Tween20 (1.5%/0.2%, v/v) suspension in water or by mixing the drug in the diet (diet-admix), as indicated. In gavage experiments the animals were dosed 6 h before sacrifice. All experiments were of 2 weeks duration unless otherwise stated and drug and diets were started at the same time (i.e., no prefeeding). Regardless of the 372

KIEFT,

A N D STANFIELD

mode of drug administration, animals were sacrificed in the nonfasted state. The animals were euthanized by exsanguination using ether anesthesia. In all experiments there were six to seven animals per treatment group and body weight gain was not different between groups. Statistical differences between control and t r e a t m e n t values were determined using unpaired, one-tailed t-tests. CI-976 and CL 277,082 were synthesized in the Department of Chemistry at the Parke-Davis Pharmaceutical Division, Warner-Lambert Company. Beta-sitosterol (No. 102886) was from ICN Biochemicals (Cleveland, OH, USA), and tigogenin cellobioside (CP88818) was the kind gift of Dr. John Stam, Pfizer Central Research Division (Groton, CT, USA). The chemical structures of these compounds are shown in Figure 1. Results

Table 2 lists the data for nonfasted control animals using the diets described previously. First, the cholesterol-free purified diet results in plasma total cholesterol values that are about two-fold higher than normal chow diets. Second, with both basal diets the percentage of HDL-C decreases from about H3CO~ [ , "

OC~3

~'~

HN,'~(CH2)eCH3

OCH3

CI-976 O

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CL-277,082 .

CH2CH3

2V --q2 HO-

v

-,~

~-sitosterol

HO'~-~

HO,---~ ,O~ ~

H~ ~"~ ~OH HO'~W bH

_~~ H

CP-88,818 Figure 1 -- Chemical structures of cholesterol absorption inhibitors.

CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

A C A T INHIBITION IN H A M S T E R S TABLE 2

Control Lipid and Lipoprotein Data for Hamsters Fed Purified and Nonpurified, Chow-Based Diets

Basal Diet P P P NP NP NP NP NP

n

Dietary Cholesterol (%)

Supplemental Fat

VLDL-C (%)

LDL-C (%)

HDL-C (%)

TPC (retool/L)

TG (retool/L)

13 13 23 23 7 14 7 12

0 0.06 0.20 0 0.06 0.06 0.20 0.20

NA NA NA No No Yes No Yes

11-+ 2 16-+ 5 33-+ 9 8 -+ 2 13 -+ 4 28 -+ 10 16 -+ 2 37 -+ 14

18-+ 4 23-+ 4 30+- 5 26 + - 7 32 -+ 5 43 -+ 14 47 -+ 8 46 -+ 9

72+-4 60-+4 37-+9 65-+5 56 -+ 5 26 -+ 5 37 -+ 6 17 -+ 4

6.1-+ 0.4 7.3-+ 0.6 1 0 . 4 - + 2.4 3.5-+ 0.1 6.5 -+ 0.4 9.4 -+ 2.9 6.6 -+ 1.5 23.7 -+ 10.3

5.6-+1.1 6.8-+1.7 12.8-+5.9 2.5-+0.2 2.9 -+ 0.6 13.2 -+ 6.3 8.7 -+ 0.6 12.6 -+ 7.1

Values are the mean -+ SD for the percentages of cholesterol found in VLDL, LDL, and HDL for control animals used in all experiments with CI-976. For total plasma cholesterol (TPC) and triglycerides (TG) the mean absolute value -+ SD is shown. The supplemental fat was 10% hydrogenated coconut oil. P, Purified; NP, nonpurified; NA, not applicable; n, number of animals. 60-70% in animals fed no dietary cholesterol to less t h a n 40% when 0.2% cholesterol is fed. A level of 0.06% dietary cholesterol is similar to t h a t of Western m a n (150 mg/1,000 kcal of diet) (13). Third, with purified diets LDL-C represents only 20-30% of the total cholesterol, regardless of the a m o u n t of dietary cholesterol. But with chow-based diets almost 50% of the cholesterol is LDL-C with 0.06% cholesterol (with supplementary fat) or with 0.2% cholesterol. Finally, hamsters are hyper-responders (relative to rats) with respect to changes in plasma lipids when chow is supplemented with both cholesterol and saturated fat. Under these conditions it is not uncommon to find values in the range of 1 3 - 2 6 mmol/L for cholesterol and >11 mmol/L for triglycerides (Table 2). If the animals are fasted 24 h, however, these lipid values are 50-70% lower compared to nonfasted values (data not shown). Generally, efficacy of drugs affecting cholesterol absorption is greater (i.e., percentage changes in plasma lipids) if t r e a t m e n t groups are compared in the nonfasted state, as in the following experiments. In the first series we administered CI-976 by gavage while the animals consumed the cholesterolfree, purified diet containing different amounts of cholesterol (0, 0.06%, or 0.2%). The reductions in plasma total cholesterol in hamsters fed the cholesterol-free, purified diet were nonsignificant at the doses indicated [Figure 2(A)]. However, when animals were fed 0.06% dietary cholesterol, plasma cholesterol was reduced significantly at doses of 10 mg/ kg and higher. At a dietary cholesterol level of 0.2%, reductions in cholesterol approaching 50% were observed at 30 and 100 mg/kg. Thus, efficacy in terms of plasma cholesterol reduction was clearly dependent upon the level of d i e t a r y cholesterol under these conditions. Reductions in liver cholesterol content also occurred with increasing dietary cholesterol level [Figure 2(B)], and with 0.06% dietary cholesterol changes in the liver exceeded, on a percentage b a s i s , t h o s e in t h e p l a s m a . T h u s , l i v e r cholesterol is a more sensitive efficacy parameter at CLINICALBIOCHEMISTRY,VOLUME 25, OCTOBER 1992

low levels of d i e t a r y cholesterol. A l t h o u g h t h e amount of free cholesterol tended to decrease at every dose, the decreases in liver cholesterol were primarily due to changes in cholesteryl ester concentrations (data not shown), consistent with the assumption t h a t these stored liver esters are ACATderived.

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Figure 2 -- Changes in (A) plasma total cholesterol and (B) liver cholesterol content due to CI-976 in h~msters fed a purified basal diet containing zero (O), 0.06% (O), or 0.2% (I) dietary cholesterol. The animals were dosed by daily gavage (AM) for 2 weeks while fed the diets ad libiturn. Values are the m e a n - S E M for seven animals/ treatment group. Asterisks indicate significant changes

from control (p

<

0.05). 373

KRAUSE, BOUSLEY, KIEFT, AND STANFIELD

To determine which lipoprotein(s) accounted for the changes in plasma total cholesterol, the plasma samples from these experiments were subjected to HPGC analysis (Figure 3). Even though the changes in total cholesterol in animals fed the cholesterolfree diet were less than 10% and nonsignificant, the changes in VLDL-C were significant at doses of 10 mg/kg and higher [Figure 3(A)]. VLDL-C represents only about 10% of the total cholesterol under these conditions (Table 2), so the absolute change is minimal and does not result in a change in total cholesterol. In contrast, changes in LDL-C were significant only when cholesterol was fed. With both levels of dietary cholesterol CI-976 lowered VLDL-C and LDL-C, the lowest significant dose for both being 10 mg/kg [Figure 3(B,C)]. CI-976 did not elevate HDL-C with these diets, and in fact lowered it at two doses with 0.2% dietary cholesterol. With the puri-

fled diets efficacy was the same regardless of whether the animals were dosed in the morning or in the late afternoon (data not shown). For comparison, we dosed the ACAT inhibitor CL 277,082 (14) at 100 mg/kg in the hamsters fed 0.2% cholesterol [Figure 2(C), open symbols]. This compound failed to lower LDL-C, but it had the same effect as CI-976 on VLDL-C (-50%). Unlike CI-976, it also lowered HDL-C ( - 2 6 % ) . Since many drugs that act during the lumenal phase of lipid absorption (e.g., beta-sitosterol, resins, surformer) are most efficacious when mixed into the diet of animals, we decided to evaluate CI-976 in this fashion using these purified diets. Specifically, we compared the ability of CI-976 to lower VLDL-C and LDL-C using the two cholesterol-containing diets (0.06% and 0.2% dietary cholesterol) and the two intermediate drug doses (10 and 30 mg/kg). VLDL-C was reduced more by diet-admix compared to gavage administration with both levels of dietary cholesterol (Figure 4). In contrast, under conditions where CI-976 lowered LDL-C significantly (0.2% dietary cholesterol) efficacy was greater with gavage com-

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Figure 3 - - Changes in VLDL-C (O), LDL-C (B), and HDL-C (A) due to CI-976 in hamsters fed a purified basal

diet containing (A) zero, (B) 0.06%, and (C) 0.2% dietary cholesterol. The animals were dosed by daily gavage (AM) for 2 weeks while fed the diets ad libitum. Values are the mean -+ S E M for seven animals/treatment group. Open

symbols (C) are the data for CL 277,082 dosed at 100 mg/ kg. Asterisks indicate significant changes from control (p < 0.05). 374

VLD

-90 10

LDL-C 30

10

B.

30

Dose (mg/kg)

Figure 4 - - Comparison of gavage (open bars) or dietadmix (closed bars) administration of CI-976 on VLDL-C and LDL-C in hamsters fed a purified diet containing (A) 0.06% and (B) 0.2% dietary cholesterol. Values are the mean -+ S E M for six to seven animals/treatment group. Asterisks indicate a significant difference between the two modes of drug administration (p < 0.05). CLINICAL BIOCHEMISTRY, VOLUME 25, OCTOBER 1992

ACAT INHIBITION IN HAMSTERS

pared to diet-admix [Figure 4(B)]. Liver cholesterol was not evaluated in these diet-admix administration experiments. In preliminary experiments, CI-976 by gavage or diet-admix did not lower plasma total cholesterol significantly in hamsters fed chow alone or chow plus 10% hydrogenated coconut oil. Therefore in the next series of experiments we evaluated efficacy using a chow-based diet supplemented with both cholesterol and this saturated fat. Saturated fat accentuates the effect of dietary cholesterol on plasma LDL clearance in this animal model (13). We tested CI-976 in hamsters fed both 0.06% and 0.2% dietary cholesterol using either gavage or diet-admix administration (Figure 5). Using this nonpurified basal diet, we observed marked differences in efficacy between these two modes of drug administration. With both levels of dietary cholesterol efficacy was far greater with diet-admix compared to daily gavage. In a separate experiment efficacy was improved with PM versus AM gavage using 0.2% dietary cholesterol, but not to the extent found with diet-admix drug administration (data not shown). Cholesterol d i s t r i b u t i o n was d e t e r m i n e d by HPGC in animals fed the human-like amounts of dietary cholesterol (0.06% cholesterol) and administered CI-976 by diet-admix using the chow-based diet. VLDL-C was lowered by 36%, 69%, and 66% at dose levels of 3, 10, and 30 mg/kg, respectively, but LDL-C was not changed significantly at these doses. Although we have not dosed longer than 2 weeks with 0.06% dietary cholesterol, in a separate experiment designed to evaluate aortic lesions in animals fed 0.2% cholesterol it was observed that increasing the duration to 8 weeks did result in greater druginduced decreases in LDL-C, especially at lower doses (data not shown). Last, we addressed the question concerning the

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.....

--A

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~

i

i

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10

30

40

50

60

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(mg/kg)

Figure 5 -- Comparison of gavage (---) and diet-admix ( ) administration of CI-976 on plasma total cholesterol in hamsters fed a nonpurified, chow-based diet supplemented with 0.06% (b, A) and 0.2% (O, C)) dietary cholesterol and 10% hydrogenated coconut oil. Values are the means of seven animals/group. All values for dietadmix are significantly different from gavage at each drug dose. CLINICALBIOCHEMISTRY,VOLUME25, OCTOBER1992

possible mechanism of action of CI-976 using the chow-based diets. Cholesterol absorption was estimated after the oral administration of radiolabeled cholesterol. Even though LDL-C was not significantly lowered using 0.06% dietary cholesterol, the absorption of the labeled cholesterol decreased by 37% compared to control (Table 3). Thus, modest inhibition of cholesterol absorption can cause decreases in VLDL-C but not LDL-C. As a positive control large amounts of beta-sitosterol inhibited cholesterol absorption by 74%, r e s u l t i n g in a marked fall in both VLDL-C and LDL-C. We compared these data to an experiment in which 0.2% dietary cholesterol was used without supplemental dietary fat. With this diet plasma cholesterol and triglycerides are lower but the percentage of cholesterol in LDL is still about 50% (Table 2). Under these conditions CI-976 was more potent with respect to lowering LDL-C and inhibiting cholesterol absorption (e.g., 45% lowering of LDL-C at 20 mg/ kg, 50% reduction in cholesterol absorption at 5 mg/ kg). As expected (15), B-tigogenin cellobioside (CP88818) was efficacious, but not as potent as CI-976. CL 277,082 has not been evaluated in this animal model for inhibition of cholesterol absorption. Discussion

The hamster has recently become an important animal model for the study of nutritional factors affecting lipoprotein metabolism. For example, hamsters respond much like humans to various dietary fats (16), fibers (8), and proteins (17) with respect to changes in plasma lipids and lipoproteins. However, less is known concerning the effects of plasma lipidregulating drugs in hamsters. Certain HMG-CoA reductase inhibitors have been shown to lower plasma cholesterol in chow-fed hamsters at doses greater than 50-60 mg/kg (18), and cholestyramine dose-dependently lowers plasma cholesterol in chow-fed hamsters (19). But fenofibrate (20) has no effect on plasma cholesterol in cholesterol-fed hamsters. We have found only two reports of hamsters being treated with ACAT inhibitors. Octimibate (100 mg/kg) lowered plasma cholesterol only 6% in chow-fed hamsters after 1 week when mixed into the diet (21). In another study (22) in which hamsters were fed high amounts of cholesterol (0.5%), Sail 58-035 (100 mg/kg) was without effect, but CL 277,082 lowered plasma cholesterol 21% (50 mg/kg, gavage with corn oil, i week). It was with this background that we selected this species to further profile the ACAT inhibitor, CI-976. The feeding of cholesterol-free, purified diets in hamsters results in plasma cholesterol and triglyceride values that are about two-fold higher than those found in normal, chow-fed hamsters, and these in turn are higher than in chow-fed rats. However, with both basal diets most of the cholesterol in hamster plasma resides in HDL particles. Thus, although hamsters are an excellent model for studying LDL metabolism (13,16), it should be recognized 375

KRAUSE, BOUSLEY, KIEFT, AND STANFIELD TABLE 3

Effect of CI-976 on Plasma Cholesterol Concentration, Distribution and Cholesterol Absorption in Cholesterol-Fed Hamsters Treatment Group Controls CI-976 B-sitosterol Controls CI-976 CI-976 CI-976 CP-88818

Diet

Dose (mg/kg)

TPC (mmol]L)

VLDL-C (retool/L)

LDL-C (retool/L)

Cholesterol Absorption (%-inhibition)

A A A B B B B B

-30 3000 -5 20 50 200

10.27 +- 0.65a 8.49 -+ 0.335 4.08 - 0.23c 7.17 -+ 0.44a 6.25 -+ 0.215 4.44 -+ 0.2c 4.05 - 0.31¢ 4.15 - 0.3¢

3.02 -+ 0.59 a 0.88 -+ 0.185 0.26 -+ 0.03c 1.14 -+ 0.08 ~ 0.77 -+ 0.055 0.28 -+ 0.03 ~'a 0.26 -+ 0.03d 0.46 +- 0.13d

4.72 -+ 0.13a 4.80 -+ 0.23a 1.03 -+ 0.085 3.41 -+ 0.39~ 2.90 +- 0.18 a 1.88 -+ 0.155 1.52 -+ 0.155 1.50 +- 0.235

--37 + 5 - 7 4 -+ 4 -- 5 0 -+ 6 - 7 5 +- 3 - 7 9 -+ 4 -71 +-- 12

Values are the mean -+ SEM (mrnol]L) or +- SD (%-inhibition), n -- 6/group. CI-976 was mixed into the diets at the doses indicated. Diet A: Chow + 0.06% cholesterol + 10% hydrogenated coconut oil. Diet B: Chow + 0.2% cholesterol (no supplemental fat). TPC = Total plasma cholesterol concentration. For each efficacy parameter (column) values with different superscripts are significantly different (p < 0.05) within each experiment (diet) group. t h a t hamsters fed purified diets have relatively low levels of LDL-C, and therefore any absolute differences in LDL-C values between control and drugtreated groups would be necessarily small. However, with chow-based diets, LDL-C concentrations can reach 2.5 mmol/L or higher, even when only 0.06% dietary cholesterol is supplied (with supplemental saturated fat). But methods used to determine cholesterol distribution must be insensitive to the high levels of plasma triglyceride, so precipitation techniques are not ideal under these conditions. That is one additional advantage of the HPGC system used here and described previously (12). The overall objective of our study was achieved in the sense t h a t we identified conditions in which CI976 can lower plasma cholesterol in hamsters fed h u m a n - l i k e levels of d i e t a r y cholesterol (0.06%) (13). In attempting to demonstrate this activity we further found t h a t the changes in cholesterol distribution, especially LDL-C, were dependent upon the type of diet and the mode of drug administration. That these findings cannot be extrapolated to other lipophilic ACAT inhibitors is demonstrated by the finding t h a t CL 277,082 failed to lower LDL-C under conditions where CI-976 was effective. Apparently, CL 277,082 does not lower LDL-C in hamsters even at higher levels of dietary cholesterol (22). It is tempting to speculate t h a t such animal data are predictive of the response in h u m a n s since CL 277,082 failed to lower LDL-C in one hypercholesterolemic h u m a n subject (23). With the purified diets, we consistently observed a marked decrease (%-basis) in VLDL-C with CI-976 treatment, even when plasma total cholesterol was not changed significantly. This appears to be the most sensitive plasma efficacy parameter with most diets, and we have observed VLDL-C reductions in other species fed cholesterol, such as rats, rabbits, and minipigs (Krause and Bocan, unpublished data) and in rabbits fed a cholesterol-free, casein diet (24). This effect was accentuated with diet-admix admin376

istration in the present study with hamsters. Since the animals were nonfasted, VLDL also contains chylomicrons and chylomicron remnants. This contribution m a y be quite significant in the h a m s t e r since, unlike man, there is a marked difference between "fed" and "fasted" plasma cholesterol concentrations. When the drug is mixed into the diet it is obviously present whenever dietary cholesterol is being absorbed, and this m a y be optimal for inhibition of cholesterol absorption. Therefore, the VLDL fraction, which contains the above intestinal particles, is decreased to the greatest extent. Alternatively, drug mixed into the diet m a y be better absorbed and therefore have greater direct effects on hepatic VLDL secretion. Unfortunately, data on the absorption and tissue distribution of CI-976 in this species to t e s t t h e s e possibilities are p r e s e n t l y lacking. LDL-C was lowered only about 15% or less with 0.06% cholesterol, regardless of the mode of drug administration or type of basal diet. With more dietary cholesterol, however, optimal LDL-lowering was observed using gavage administration with the purified diet and with diet-admix (but not gavage) using the nonpurified diet. Reasons for these interactions are presently unknown but are likely to be the result of diet-dependent differences in drug absorption, and m a y not be predictive for other ACAT inhibitors. CI-976 inhibits cholesterol absorption in hamsters fed both types of basal diets. In this respect it is more potent t h a n beta-sitosterol or tigogenin cellobioside, agents t h a t act during the lumenal phase of cholesterol absorption (15). Interestingly, CI-976 was more effective at inhibiting cholesterol absorption when no fat was included in the diet. This could be due to the fact t h a t cholesterol absorption in the h a m s t e r m a y actually be higher in the absence of supplemental saturated fat compared to its presence (16), and therefore an absorption inhibitor might be expected to be more efficacious without supplemental coconut CLINICALBIOCHEMISTRY,VOLUME 25, OCTOBER 1992

ACAT INHIBITIONIN HAMSTERS oil. But since there is a hepatic component to the estimate of cholesterol absorption in the present study, greater inhibition of cholesterol absorption m i g h t also be a reflection of more drug directly reaching the liver via the portal route t h a t m a y predominate in the relative absence of significant chylomicron production (i.e., no dietary fat). This would result in greater inhibition of hepatic ACAT and potentially the secretion of VLDL and/or of apoB (1). Finally, LDL-lowering in these two experiments (Table 3) was greatest under conditions where inhibition of cholesterol absorption was maximal. Thus, cholesterol absorption m a y be a major determinant for the a m o u n t of circulating LDL-C in this animal model.

12.

13.

14.

15.

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16.

17.

18.

19. 20.

21.

22.

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