- Email: [email protected]
Mechanisms of action in the liver of crilvastatin, a new hydroxymethylglutaryl-coenzyme A reductase inhibitor
European Journal of Pharmacology, 235 (1993) 59-68
59
Elsevier Science Publishers B.V.
EJP 53022
Mechanisms of action in the liver of crilvastatin, a new hydroxymethylglutaryl-coenzyme A reductase inhibitor T h i e r r y Clerc, M i c h e l J o m i e r , M a g a l i C h a u t a n , H e n r y P o r t u g a l ~, M i c h b l e Senft, A n n e - M a r i e Pauli ~, C l a u d e L a r u e l l e b, Olivier M o r e l b, H u g u e t t e L a f o n t a n d F r a n q o i s e C h a n u s s o t INSERM, Unit~ 130, 18 At,enue Mozart, 13009 Marseille, France, '~Laboratoire Central, H3pital Ste Marguerite, 13009 Marseille, France and b PAN MEDICA, BP 27, 06511 Carros, France
Received 16 July 1992, revised MS received 19 January 1993, accepted 26 January 1993
Crilvastatin is a drug from the pyrrolidone family that had been shown to induce non-competitive inhibition of rat hydroxymethylglutaryl-coenzyme A reductase activity in vitro. The aim of this study was to evaluate the activity of crilvastatin on the hepatic metabolism of cholesterol in rats. Crilvastatin increased low density lipoprotein (LDL)-cholesterol uptake by the liver more than high density lipoprotein (HDL) uptake, thus increasing by up 30% the clearance of excess plasma cholesterol. In normolipidemic rats, crilvastatin significantly enhanced acyl coenzyme A : cholesterol acyl transferase and cholesterol 7a-hydroxylase activity. In rats with a previous high cholesterolemia, crilvastatin also enhanced cholesterol 7a-hydroxylase activity and did not increase liver acyl coenzyme A:cholesterol acyl transferase activity. These findings suggest that a drug such as crilvastatin could have a hypocholesterolemic effect by a mechanism other than the sole inhibition of cholesterol synthesis, possibly by stimulating cholesterol and bile salt secretion via the biliary tract in previously hypercholesterolemic rats. Hypocholesterolemic drugs; Liver; Cholesterol; Bile; Lipoproteins
1. Introduction Hypocholesterolemic agents (Alberts, 1988a, b ) s u c h as compactin (Endo et al., 1976) and mevinolin (A1berts et al., 1980) belong to a new generation of choleso terol-lowering drugs that appear highly effective (Aubert et al., 1988). In the cell, their primary action is to nhibit competitively hepatic hydroxymethylglutarylcoenzyme A reductase activity, the rate limiting enzyme in cholesterol synthesis (Endo et al., 1976; A1berts et al., 1980). The hypocholesterolemic effect of these drugs is related to the stimulation of liver uptake of low density lipoproteins (LDL), which are known to be atherogenic. Thus mevinolin increases L D L receptors which could account for the lowering of the L D L level (Alberts et al., 1980). We previously tested another new and non-competitive hydroxymethylglutaryl-coenzyme A reductase inhibitor from the pyrrolidone group, crilvastatin (Pan Medica, Carros, France). The purpose of the present work was to investigate the mechanisms other than
Correspondence to: F. Chanussot, INSERM Unit6 130, 18 Avenue Mozart, 13009 Marseille, France. Tel. 33-91.75.47.05, fax 3391.75.15.62.
those already described (Esnault et al., 1988), by which crilvastatin acts on the liver. We focused on the balance between circulating lipoprotein cholesterol, and the hepatic cholesterol metabolising enzymes wich are implicated in the bile secretion of cholesterol and bile salts. Bile salts and cholesterol in the bile originate mostly from free cholesterol supplied by high density lipoproteins ( H D L ) and L D L (Halloran et al., 1978). Thus, we first investigated a possible hypocholesterolemic effect of crilvastatin on hypercholesterolemic rats treated chronically with the drug during a period (17 weeks), sufficient for evaluation of this hypocholesterolemic effect. We then investigated the effect of crilvastatin on uptake and metabolism of free cholesterol-HDL and - L D L by the liver and on levels of cholesterol and bile salts secreted in bile. Acyl coenzyme A : cholesterol acyltransferase and cholesterol 7a-hydroxylase activities in the liver were studied in order to evaluate the effect of crilvastatin on hepatic cholesterol storage and cholesterol catabolism to bile salts. We tested the effect of crilvastatin administered over a short period (8 days) that was adequate for the study of possible changes in H D L and L D L metabolism in normal rats. Changes in liver enzyme activity were evaluated in rats with a high cholesterolemia after a period (10 weeks)
6O of administration sufficient long for such modifications to appear,
2. Materials and methods
2.1. Chemicals Chemicals of the highest purity were obtained from Sigma, Sanofi, Carlo Erba and Bio-Rad. Crilvastatin (Pan Medica Lab., Carros, F r a n c e ) i s a synthetic derivative of pyrrolidone carboxylic acid (cycloalkyl ester of pyroglutamic acid) (fig. 1). Previous study had shown that this chemical inhibits hydroxymethylglutaryl-coenzyme A reductase activity in vitro by a non-competitive manner through a mechanism that involves the substrate concentration and alters the Vma× of the incubation reaction (Esnault et al., 1988). The K m of the enzyme remains unchanged. The inhibitory effect of this drug seemed to result from changes in the substrates and reaction products. The kinetic data obtained with crilvastatin showed that this drug was a more potent inhibitor than fenofibrate; mevinolin acted faster than crilvastatin, however the maximum inhibition at the same concentration of these two drugs was identical (Esnault et al., 1988).
2.2. Experimental procedures Three different experiments were carried out. In the first experiment, we tested the possible hypocholesterolemic effect of crilvastatin, administered chronically during a sufficiently long time (17 weeks) in control or hypercholesterolemic rats, by assaying plasma cholesterol. In the second experiment, the fate of intravenously injected [14C]free cholesterol-HDL and -LDL was evaluated after 8-day crilvastatin treatment of normolipemic rats. In the third experiment the effect of a 10-week crilvastatin treatment on enzyme activities iraplicated in the homeostasis of hepatic cholesterol was investigated in hypercholesterolemic rats. At the end of all treatments, rats were anesthetized with sodium pentobarbital (5 m g / 1 0 0 g body weight).
o o ~
,, ~
"
~_~%,¢H3C_ ,,I ell3
II
H,,,~,,/
\
"c ~ 0~'~ ~'~CH cr~v~,s~n
H
Fig. ~. C h e m i c a l s t r u c t u r e of crilvastatin,
3
Plasma was collected from the 10-week and 17-week treatment groups. These rats were fasted overnight prior to plasma collection to allow proper evaluation of cholesterol in the plasma and lipoproteins. Bile was collected from the 10-week treatment group from the common bile duct cannulated with polyethylene tubing (Clay Adams No. 10).
2.2.1. 17-week crih~astatin treatment 2.2.1.1. Animals and drug treatment. Thirty-two 3week-old male Wistar rats raised at Iffa-Credo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). For 6 months, the rats received either a low fat diet (control, 5% lipids as 3% lard, 2% corn oil) or a high fat diet (29.2% lipids as 26% lard, 2% corn oil, 1.2% cholesterol). Both control and high fat diets contained the same amount of protein (24% casein). Thus, the requirement for essential fatty acids and protein was supplied by both diets. In the course of 17 weeks, four experimental groups (8 rats per group) were constituted: control, c o n t r o l / crilvastatin; high fat; high fat/crilvastatin. For the control/crilvastatin and high fat/crilvastatin groups, crilvastatin was previously solubilized in corn oil and mixed with the food. The quantity of drug incorporated was calculated to be such that the oral intake of each rat was 200 mg crilvastatin/kg of body weight per day. The body weight of the animals at the end of the experiment was not modified by the drug (respectively 402 _+ 51 g in the control group, 435 + 122 g in the control/crilvastatin group, 500_+ 70 g in the high fat group and 465 _+ 26 g in the high fat/crilvastatin group).
2.2.2. 8-day crih, astatin treatment 2.2.2.1. Animals and drug treatment. Twenty 15week-old male Wistar rats raised at Iffa-Credo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). They were divided into four groups of five animals. Two control groups as well as two other (control/crilvastatin) groups previously treated with crilvastatin received an [14C]HDL or [14C]LDL injection. All rats received the control diet. Crilvastatin was emulsified by sonication in water (2/1 v / v crilvast a t i n / w a t e r ) and administered by intraperitoneal injection at a dose of 150 mg crilvastatin/kg body weight per day. 2.2.2.2. Preparation of lipoprotein ,fractions. Free cholesterol in LDL and H D L was labelled with [14C] according to a method described by Esnault-Dupuy et al. (1987). Serums 20 ml from non-fasted donor Wistar rats was incubated for 24 h at 10°C with 2 ml of phosphatidylcholine/cholesterol liposomes prepared as follows. Phosphatidylcholine (111.5 mg, Sigma, Paris,
61 France) and [14C]free cholesterol (4.8 ~Ci; specific activity 50 m C i / m m o l ) (CEA, France) were mixed in benzene then evaporated under nitrogen. The dry residue was suspended in 3 ml of 0.9% NaC1 solution then was cooled to 5°C, and sonicated for 1 hour under nitrogen using 30-s pulses. The liposome mixture obtained was centrifuged at 100000 × g for 30 min to eliminate large particles. The homogeneity of the liposomes was verified by A4 agarose (IBF, France) gel filtration using a Tris 0.5 M, pH 7.5 buffer as eluant, Under these conditions, the [~4C]cholesterol liposomes eluted in a single peak. Serum lipoproteins (HDL, LDL) were isolated by sequential ultracentrifugation after adjustement to the required density with sucrose according to the technique of Hatch and Less (1968). LDL were obtained at a flotation density of 1.063 after centrifugation at 120000 × g for 22 h at 12°C. H D L were obtained at a flotation density of 1.21 after centrifugation at 120000 × g for 22 h at 12°C. The H D L preparation was labelled by direct [14C]free cholesterol exchange, since cholesterol is easily exchangeable between H D L and others lipoproteins or membrane surfaces. The H D L and L D L fractions were then ultra-centrifuged at 1 2 0 0 0 0 × g for 1 h at 12°C to separate [~4C]lipoproteins and unbound [~4C]free cholesterol. The final label of the H D L represented 60-65% of the [~4C]free cholesterol incubated with the HDL. Radioactive LDL represented about 50% of the label contained on [~4C]liposomes. Thin-layer chromatography was performed to check that the [~aC]cholesterol in the injected lipoproteins was exclusively on the non-esterifled cholesterol fraction. The mass of radioactive serum lipoproteins, especially H D L and LDL. 2.2.2.3. Injection of radiolabelled materials. In the 8-day treatment group, 20 min after the beginning of the experiment, either [~4C]LDL or [~4C]HDL were injected for 7 and 10 min respectively via the jugular vein. The dose of [ ~ C ] L D L was 0.682 p~Ci [~4C]free cholesterol which is equivalent to 0.07-0.14 /xmol of free cholesterol-LDL (0.36-0.60 ~mol of total cholesterol). The dose of [~4C]HDL was 0.363 /zCi [~aC]free cholesterol which is equivalent to 0.03-0.15 p~mol of free cholesterol-HDL (0.16-0.52 tzmol of total cholesterol). The amount of cholesterol injected was negligible in comparison to the 20 mmol or more of circulating native total cholesterol,
2.2.3. lO-week crilr,astatin treatment 2.2.3.1. Animals and drug treatment. Thirty-four 9week-old male Wistar rats raised at IffaoCredo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). For 3 weeks the rats received either the control diet or the high fat diet. The rats were then divided into four groups for the various
10-week treatments: control (number of rats: n = 10); control/crilvastatin (n = 9); high fat (n = 5); high fat/crilvastatin (n = 10); the mean body weight were respectively 465 _+ 6 g; 472 _+ 27 g; 469 +_ 17 g; 428 _+ 18 g, the mean liver weights were respectively 11.45 _+ 0.38 g; 10.05 _+ 0.43 g; 20.64 _+ 1.41 g; 20.35 _+ 1.13 g. Crilvastatin also was incorporated in the diets.The oral intake of each rat was 200 mg crilvastatin/kg body weight per day. At the end of the 10-week crilvastatin treatment, the body weight of the animals (as well as the liver weight) was not modified by the administration of the drug. 2.2.3.2. Preparation of litter microsomes. Microsomes from livers of rats treated for 10 weeks with crilvastatin were used for the acyl coenzyme A : cholesterol acyltransferase and cholesterol 7a-hydroxylase activity determinations. Microsomes were isolated from fresh liver as follows: 2 g of liver were homogenized in 10 ml of potassium phosphate pH 7.4 buffer (50 mM KH2PO4, 0.1 M sucrose, 30 mM EDTA, 70 mM KCI, 1 mM dithiothreitol) with a polytron PT 10-35 homogenizer. The homogenate was then centrifuged at 10000 × g at 4°C for 15 min. The supernatant was centrifuged again at 1 0 0 0 0 × g for 15 rain and the resulting supernatant was centrifuged at 100000 × g at 4°C for 60 rain. The microsomal pellet obtained was resuspended in 1 ml of the working buffer and was stored frozen at -80°C. The purity of the microsome preparation was checked by assessing the activity of rotenone insensitive N A D P H cytochrome C reductase (EC 1.6.2.4) (Sottocosa et al., 1967). 2.2.3.3. Microsomal enzymatic assays. Acyl coenzyme A:cholesterol acyltransferase and cholesterol 7a-hydroxylase activity were determined on microsomal preparations. Acyl coenzyme A cholesterol acyltransferase activity was measured as described by Chautan et al. (1988) using [14C]l-oleoyl-CoA (NEN, Paris, France, specific activity 57.4 m C i / m m o l ) as substrate. Free cholesterol and esterified cholesterol were separated by chromatography on silica gel microcolumns and elution of esterified cholesterol with petroleum ether/diethyl ether ( 9 8 / 2 v/v). Cholesterol 7a-hydroxylase activity was measured as described elsewhere (Junker and Story, 1985). However, the best results were obtained when liposomes were incubated with dioleoyl-phosphatidylcholine instead of various phospholipids. For 0.5 mg microsomal protein the composition of the incubated liposomes was: 0.5 mg dioleoyl phosphatidycholine and 0.018 /zCi of [~4C]free cholesterol ( C E A , F r a n c e specific activity 50 mCi/mmol). [~4C]Cholesterol and [~4C]7a-hydroxycholesterol were separated as previously described by thin layer chromatography on silica-gel plates using an ethyl a c e t a t e / h e p t a n e 80/20 v / v mixture. The components were visualized under iodine vapor.
62
2.3. Sampling From the 10-week crilvastatin treatment group the bile samples were weighed then immediatly frozen. At the end of the experiment the animals were killed by aortal puncture. Rats treated 17 weeks were killed similarly and blood was collected either for freezer storage of plasma or on ethylenediaminetetraacetic acid (0.1 m g / m 1) for ultra-centrifugation of the lipoproteins. The liver was immediately removed and weighed. Erythrocytes, spleen, kidneys, heart and lungs were also frozen.
2.4. Radioactit,ity measurements Bile radioactivity was counted in 50 /~1 aliquots dissolved in 10 ml of scintillation liquid (Beckman Ready Solv MP). Radioactivity was also measured in bile salts and cholesterol isolated from the bile by thin-layer silica gel F 1500 chromatography (Schleicher-Schiill, Dassel, Germany) with a medium composed of 40% isoamyl acetate, 30% propionic acid, 20% isopropyl alcohol and 10% water (v/v). After identification at 350 rim, the spots were scraped and counted in 10 ml of scintillation liquid (Beckman Ready solv MP). L D L and H D L radioactivity was determined after extraction in a small volume of petroleum ether, Free cholesterol, esterified cholesterol or 7a-hydroxycholesterol were extracted with c h l o r o f o r m / m e t h a n o l 2 / 1 v / v and were separated on thin-layer silica gel F 1500 (Schleicher-Schiill, Dassel, Germany) using a mixture (v/v) containing 90 heptane, 30 sulfuric ether and 1 acetic acid (for free cholesterol and esterified cholesterol), and 80 ethyl acetate and 20 heptane (for free cholesterol and 7a-hydroxycholesterol). The fractions were visualised with iodine vapor. The spots were scraped and counted in 10 ml of scintillation liquid (Beckman Insta-fluor for organic solution),
2.5. Gra~,imetric assays 2.5.1. In the bile Phospholipids (Amic et al., 1972), bile salts (Domingo et al., 1972) and cholesterol (Lie and al., 1976) were assayed by a semi-automatic colorimetric method.
2.5.2. In the plasma Triacylglycerols (Bucolo and David, 1973), total and free cholesterol (Leon and Stasiw, 1976) in the plasma, LDL and HDL, total protein (Skeggs and Hochstrasser, 1964) and protein in L D L and H D L (Bio-Rad assay, 1985) were measured along with alkaline phosphatase (Morgenstern and al., 1965), alanine aminotranferase (Kessler and al., 1975) and aspartate aminotransferase (Kessler and al., 1975).
2.5.3. In the litter When no microsomal preparations were required for analyses, the frozen livers were thawed and homogenized in 1 mM Tris buffer pH 7.4 containing 250 mM sucrose, in a polytron PT 10-35 homogenizer (Beaufay et al., 1974). Total protein (Skeggs and Hochstrasser, 1964), triacylglycerols (Bucolo and David, 1973), total cholesterol (Leon and Stasiw, 1976), alkaline phosphatase (Morgenstern and al., 1965), aspartate aminotransferase and alanine aminotransferase (Kessler and al., 1975) were assayed in these homogenates.
2.6. Statistics The significance of differences was analyzed with Student's t-test.
3. Results
3.1. Effect of lO-week crih~astatin treatment The numbers of plasma and bile samples are not the same for all groups, specially the high fat and high fat/crilvastatin groups, as the samples were not always used for the same purposes.
TABLE1 Cholesterolin the plasma after 10-week crilvastatin treatment. VLDL: very low density lipoprotein; LDL: low density lipoprotein; HDL: high density lipoprotein; CR: crilvastatin. The results are expressedasmeans_+S.E.M. Total EsterifiedFree cholesterol cholesterol cholesterol (retool/l) (mmol/l) (mmol/l) Total plasma (n - 10) 2.33_+0.05 1.82+0.03 0.54_+0.01 VLDL(n=8) 0.15_+0.10 0.11+0.09 0.04_+0.01 Control LDL(n-8) 0.52_+0.12 0.42+0.12 0.07_+0.04 HDL(n = 8) 1.79_+0.80 1.37+0.83 0.40_+0.25 Total plasma (n = 9) VLDL(n=8) Control/CR LDL(n=8) HDL(n=8) Total plasma (n = 5) VLDL (n = 5) HF(n = 5) LDL(n = 5) HDL(n=5)
2.26_+0.01 1.72+0.01 0.53_+0.01 0.13_+0.08 0.10_+0.10 0.03_+0.01 0.48_+0.17 0.40_+0.14 0.11_+0.02 1.76+0.42 1.32_+0.35 0.40+0.07
3 . 4 7 + 0 . 2 8 2.84_+0.21 0.63+0.16 0.48+0.27 0.34_+0.22 0.06+0.02 1.12~-0.14 0.79_+0.15 0.20+0.09 1.84+0.60 1.52_+0.51 0.43+0.15 1.a, Total plasma(n = 5). 2.64_+0.20 2.14+0.27 0.51_+0.18 VLDL (n = 5) 0.26+0.05 0.17_+0.04 0.03+0.02 HF/CR L D L ( n = 5) 0.50_+0.13 0.46_+0.12 0.06+0.3 HDL(n=5) 1.84+0.73 1.55_+0.59 1/.36_+0.21 Differenceswere analyzed with Student's t-test and are significant at ~P < 0.05; a high fat/crilvastatin (bIF/CR) vs. high fat (HF).
63
3.1.1. In the plasma (table 1) T h e total cholesterol levels were 27% lower in the plasma of d r u g - t r e a t e d rats with previously high c h o l e s t e r o l e m i a t h a n in p l a s m a of u n t r e a t e d hyperc h o l e s t e r o l e m i c rats, L D L were 45% lower a n d V L D L were 54% lower. Both free a n d esterified cholesterol decreased, b u t the cholesterol level in H D L did not decrease (high f a t / c r i l v a s t a t i n versus high fat). Crilvastatin also led to a 35% decrease in triacylglycerol level in the control a n d high fat groups. T h e level of p l a s m a a n d l i p o p r o t e i n phospholipids as well as p r o t e i n s in the l i p o p r o t e i n fractions r e m a i n e d unc h a n g e d (respectively 1.7 m m o l / 1 in the plasma phospholipids a n d 22 m g / 1 in the L D L a n d 2500 m g / l in the H D L proteins),
3.1.2. In the bile (table 2) Crilvastatin s t i m u l a t e d bile flow in both control a n d h y p e r c h o l e s t e r o l e m i c rats. T h e drug significantly stimulated the secretion of bile salts a n d bile cholesterol only in rats with a high cholesterolemia. All these differences are seen clearly w h e n the results are expressed in terms of flow ( m i n / g liver) or of s e c r e t i o n / m i n , o n c o m p a r i s o n of the control a n d c o n t r o l / c r i l v a s t a t i n groups, or also of the high fat a n d high f a t / c r i l v a s t a t i n groups. T h e fact that liver weight is identical in all four groups allows this comparison.
3.1.3. In the litter T h e drug had n o effect on the cholesterol level in the liver ( t r e a t e d versus n o n - t r e a t e d groups), particularly in the h y p e r c h o l e s t e r o l e m i c rats (100 m g / g liver), T h e s e findings could be explained by the fact that the d r u g - i n d u c e d e n h a n c e m e n t of the decrease in hepatic cholesterol, secreted t h r o u g h the bile pathway, was
c o m p e n s a t e d for the s t i m u l a t i o n of hepatic uptake of cholesterol from l i p o p r o t e i n s such as LDL.
3.2. Effect of 17-week crih,astatin treatment C r i l v a s t a t i n s i g n i f i c a n t l y r e d u c e d the c h o l e s t e r o l e m i a of previously hypercholesterolemic rats. Thus, in the plasma, the total cholesterol level, like the esterified cholesterol (3.82-3.22 m m o l / l ) and free cholesterol (1.31-0.84 m m o l / l ) , d e c r e a s e d significantly from 5.13 to 4.06 m m o l / l after crilvastatin t r e a t m e n t , however, this effect was not observed in the control rats, since their cholesterol status is always above norreal a n d is very difficult to decrease, as observed elsewhere ( Y a m a u c h i et al., 1991). T h e changes in cholest e r o l e m i a were not due to weight loss. Crilvastatin did not decrease significantly the body weight of the animals; the diet intake as well as p r o t e i n absorption were n o r m a l as we f o u n d no n i t r o g e n loss in any of the groups.
3.3. Fate of [14C]free cholesterol-LDL and [~4C]free cholesterol-HDL in the litter (8 days treatment with crilt,astatin) 3.3.1. Experiment with [14C]HDL A f t e r a d m i n i s t r a t i o n of [14C]HDL, most of the radioactivity was f o u n d in the liver, bile, plasma and erythrocytes (table 3). T h e a m o u n t of [14C] t a k e n up by the heart, kidneys, lungs a n d spleen was very low (less t h a n 1% of the radioactivity injected) a n d these values are not shown. A certain a m o u n t of [~4C] free cholest e r o l - H D L must have b e e n t a k e n up by p e r i p h e r a l tissues since 1 7 - 3 3 % of the total radioactivity injected was not f o u n d back in the organs studied.
TABLE 2 Bile secretion after 10-week crilvastatin treatment. The results are expressed as means_+S.E.M. (the means were calculated for a bile collection period of 120 rain). Control
Control/CR
HF
HF/CR
Bile flow ~l/min per g liver Bile flow /zl/min
1.27+ 6.03 (n = 10) 14.4 _+ 0.4 (n = 10)
2.23_+ 0.11 3'~ (n = 9) 24.0 + 1.3 3.a (n = 9)
0.72+ 0.01 (n = 5) 14.8 _+ 0.20 (n = 5)
1.04+ 0.01 3.~ (n = 10) 21.16_+ 0.20 3.,, (n = 10)
Bile salts nmol/min per g liver Bile salts nmol/min
27.44_+ 3.76 (n = 9) 315.0 _+43.1 (n = 9)
28.91+ 2.20 (n = 9) 292.5 +22.2 (n = 9)
27.27_+ 1.18 (n = 5) 559.8 _+22.2 (n = 5)
32.25_+ 2.25 ~~ (n = 9) 658.3 _+43 (n = 9)
Bile cholesterol nmol/min per g liver Bile cholesterol nmol/min
0.37_+ 0.05 (n = 9) 4.13+ 0.56 (n = 9)
0.28_+ 0.03 (n = 9) 2.84_+ 0.30 (n = 9)
0.28+ 0.01 (n = 5) 5.72_+ 0.20 (n = 5)
0.35_+ 0.1)13.~ (n = 7) 7.15_+ 0.18 3.,~ (n = 7)
Differences were analyzed with Student's t-test and are significant at 1 p < 0.05; 2 p < 0.01; 3 p < 0.001; a control vs. control/crilvastatin (CR) or high fat (HF) vs. high fat/crilvastatin (HF/CR).
64 TABLE 3
Radioactivity distribution in various tissues after [14C]cholesterol lipoprotein injection (% [14C]cholesterol injected), EC: esterified cholesterol; FC: free cholesterol; BS: bile salts. VLDL: very low density lipoprotein; LDL: low density lipoprotein; HDL: high density lipoprotein. The results are expressed as means_+ S.E.M. (n = 5).
Liver %EC %FC Bile %BS % FC Plasma VLDL LDL HDL Erythrocytes
[14C]HDL injection
[14C]LDLinjection
Control (n = 5) 20.2_+ 1.0 0 20.2 21.2_+0.9 19.1 2.1 9.5 +0.8 2.1_+0.4 2.4_+0.7 5.0+ 1.7 9.2_+1.0
Control Crilvastatin (n = 5) (n = 5) 27.6_+1.1 31.0+0.8 ~'" 1.1 1.1 26.5 29.9
Crilvastatin (n = 5) 35.2+ 1.6 3~, 2.8 32.4 15.4-+1.1 2"a
14.2 1.2 12.0-+1.3 2.3+1.1 3.7_+0.4 6.0_+0.3 10.9_+1.1
6.5+0.6
9.2-+0.3 2'a
6.0 8.5 0.5 0.7 30.6_+3.2 193 _+1.9 ~,a 1.1_+0.1 1.7+0.4 25.7+5.4 135 _+1.0 1.a 3.8-+0.8 4.1 +0.4 15.8_+1.8 4.6-+0.83.~
Differences were analyzed by Student's t-test and are significant at: t P < 0.05; 2 p < 0.01; 3 p < 0.001. After [t4C]cholesterol HDL or [tac]cholesterol LDL injection: '~ Control vs. crilvastatin. In c r i l v a s t a t i n - t r e a t e d rats, t h e level o f [14C] was i n c r e a s e d significantly as c o m p a r e d to t h e c o n t r o l s in t h e liver and, to a lesser d e g r e e in p l a s m a a n d was significantly d e c r e a s e d in the bile. E r y t h r o c y t e l a b e l l i n g p r o b a b l y r e s u l t e d m a i n l y from e x c h a n g e s b e t w e e n [14C] c h o l e s t e r o l l i p o p r o t e i n s a n d t h e n o n - l a b e l l e d cholesterol in e r y t h r o c y t e m e m b r a n e s . In the liver, all the r a d i o a c t i v i t y was f o u n d in mainly free c h o l e s t e r o l . In bile (fig. 2), [14C] r a d i o a c t i v i t y which is s e c r e t e d m a i n l y in the f o r m o f bile salts a p p e a r e d 15 min a f t e r crilvastatin t r e a t m e n t . T h e d r u g did not affect the distribution of the r a d i o a c t i v i t y b e t w e e n the bile salts a n d bile free c h o l e s t e r o l . Crilvastatin t r e a m e n t d i d not have a m a r k e d effect on the level of [14C] in p l a s m a (table 3). 3.3.2. E x p e r i m e n t with [ ~ 4 C ] L D L A f t e r a d m i n i s t r a t i o n o f [14C]LDL, [ 14C] radioactivity
ity b e t w e e n bile salts a n d cholesterol. Thus, t h e % bile s a l t s / % free c h o l e s t e r o l r a t i o for [t4C] radioactivity in the bile was a b o u t 10 w h e n [ t 4 C ] H D L w e r e i n j e c t e d ( b o t h c o n t r o l a n d crilvastatin groups) a n d 12 w h e n [~4C]LDL w e r e injected ( b o t h c o n t r o l a n d crilvastatin groups). In plasma, a significant d e c r e a s e in the level o f [14C] c o r r e s p o n d e d to a d e c r e a s e in the level o f L D L (table 3). 3.4. E f f e c t o f crilvastatin on acyl c o e n z y m e A : cholesterol acyltransferase a n d on cholesterol 7a-hydroxylase T h e activity o f t h e s e two e n z y m e s was m e a s u r e d in m i c r o s o m e s i s o l a t e d f r o m t h e livers of control a n d h y p e r c h o l e s t e r o l e m i c rats t r e a t e d chronically with crilvastatin. T h e activities of t h e s e two e n z y m e s a r e exp r e s s e d in c o n v e n t i o n a l units ( p e r mg m i c r o s o m a l p r o tein). T h e s e units allow c o m p a r i s o n s b e t w e e n the control a n d t h e c o n t r o l / c r i l v a s t a t i n groups, a n d b e t w e e n the high fat a n d high f a t / c r i l v a s t a t i n groups, since t h e liver weights in t h e c o n t r o l a n d c o n t r o l / c r i l v a s t a t i n g r o u p s w e r e identical a n d the b o d y weights w e r e unc h a n g e d , b o t h in the high fat a n d high f a t / c r i l v a s t a t i n groups.
% [14 C] cholesterol HDL injected/rain
0.2
1
~
,
0.~ ~k__~z. ~. . ......~ ...... ~ ..... ~...... ~. "~ ~ ~ ~"~ ~
/ ~
30
~'o
~o--~o ~o time (rain)
~o ~o
~o
% [14 c] holesterol LDL injected.10-2/min 7
was f o u n d m a i n l y in the liver, p l a s m a , bile a n d e r y t h r o cytes (table 3). A s in the e x p e r i m e n t with [~4C]HDL, r e s i d u a l a m o u n t s o f [14C] were also d e t e c t e d in the o t h e r o r g a n s s t u d i e d i.e. heart, lungs, s p l e e n , a n d kidneys, a n d s o m e was t a k e n up by p e r i p h e r a l tissues, A f t e r crilvastatin t r e a t m e n t t h e a m o u n t o f [~4C] in t h e liver a n d bile was significantly i n c r e a s e d in c o m p a r ison to controls. Also, the a m o u n t of [ ~ C ] available in the p l a s m a was g r e a t l y d e c r e a s e d a f t e r d r u g t r e a t m e n t . Crilvastatin led to an e n h a n c e m e n t of biliary s e c r e t i o n (fig. 2), as c o m p a r e d to t h e controls. T h e q u a n t i t i e s of [14C] s e c r e t e d w e r e significantly g r e a t e r in the c o n t r o l / crilvastatin g r o u p t h a n in t h e c o n t r o l group, in the bile, t h e d r u g h a d no effect on t h e d i s t r i b u t i o n of r a d i o a c t i v -
~ ~. 4
a 2~ ~ 0 14
~°~:~.~, ..'.i TM /i ..... ~..~..~i...~,..fi,..i ~ ~w-~-~-~-~"_-~ ~
/
3'0
6'0
9'0
'~
1~0 1~0 1~0 2i0 2~0 time (min)
14
Fig. 2. [ C]Cholesterol bile lipid secretion. After [ CJcholesterol 14 HDL injection; after [ C]cholesterol LDL injection. The results are expressed as means_+S.E.M. (n = 5). • Control • crilvastatin. Differences were analyzed with Student's t-test and are significant at: 1, P < 0.05; 2, P < 0.01; 3, P < 0.001. a Control vs. crilvastatin.
65
%pmol/min/mgprotein 200] 1 ~ _ _ is0
1
3.4.2. Cholesterol 7a-hydroxylase actiuity (fig. 4) ~ increase in cholesterol 7a-hydroxylase activity was observed in both groups and the increase was significant in the high fat group.
~
~
too
3.5. Other biochemical data from the li~'er
50 .
o
Control
Control/Crilvastatin
%pmol/min/mgprotein a007
is0~ ~ ............ [............
10o-~ ~
sot
::::::::::::::::::::::::::::::::::::::::::::: Hf ~
HF/Crilvastatin
Fig. 3. Microsomal hepatic acyl coenzyme A : c h o l e s t e r o l actyltransferase activity without cholesterol addition in the incubation m e d i u m (n = 4). The results are expressed as m e a n s _+S.E.M. Differences were analyzed with Student's t-test and are significant at: 1, P < 0.05.
a Control(c)vs. control/crilvastatin(C/CR).
3.4.1. Acyl coenzyme A cholesterol acyltransferase activity (fig. 3) Enzyme activity was stimulated in the control rats. in high fat rats the drug led to a non-significant decrease in acyl coenzyme A:cholesterol acyltransferase activity,
%pmol/min/rngprotein
400 ~00 J a00lo0 0
~
Control
Control/Crilvastatin
n=5
n=5
°/opmol/min/mgprotein 400-q, 3oo-~
la
" 200-
_
10o-
0
Alanine aminotransferase activity was enhanced in animals fed the high fat diet (3689 IU/liver in the control group versus 5736 IU/liver in the high fat group). Drug treatment had no effect. Alkaline phosphatase activity varied from 25 IU/liver in the control group to 45 IU/liver in the high fat group. Crilvastatin had no effect.
HF n=6
HF/Crilvestetin n=6
Fig. 4. Microsomal activity of the cholesterol 7-a-hydroxylase. The results are expressed as m e a n s _+S.E.M. Differences were analyzed by Student's t-test and are significant at: 1, P < 0.05; a High fat ( H F )
vs. highfat/crilvastatin(HF/CR).
4. Discussion
Crilvastatin is a synthetic derivative of pyrrolidone carboxylic acid, it was shown that, under experimental conditions long-term crilvastatin treatment had no effect on the level of plasma cholesterol in normolipemic control rats. However, like mevinolin and related como pounds (compactin, synvinolin, eptastalin) (Alberts, 1988a, b; Alberts et al., 1980) as well as tazasubstrate (Schulze et al., 1986; Diekmann et al., 1986), crilvastatin significantly enhanced the hepatic uptake of LDL-cholesterol. One could speculated that, like other statins (Brown and Goldstein, 1983), crilvastatin stimulates LDL receptors. In vitro studies showed that the primary action of crilvastatin is non-competitive inhibition of hydroxymethylglutaryl-coenzyme A reductase activity in the liver (Esnault et al., 1988). Thus, the hypocholesterolemic effect of statins must be due to other mechanisms of action, since it was shown that, in the rat, long-term lovastatin treatment increased hepatic cholesterol synthesis (Yamauchi et al., 1991). In our experiments, the LDL-cholesterol level did not vary in normal rats treated with crilvastatin. In contrast, in a population of rats with high cholesterolemia crilvastatin decreased plasma cholesterol by 20-30% and in LDL cholesterol even further. These results were obtained in two experiments with two different values for induced hypercholesterolemia. It appears that the efficacy of crilvastatin was comparable in the two situations, even when the hypercholesterolemia reached an aggravated level (experiment: '17-week crilvastatin treatment'). These results were clearly due to the drug, since the body weight of the rats, whether treated or not with crilvastatin, did not change, the diet intake thus being unchanged. These results are comparable to those obtained with cornpactin in animal models such as the dog (Endo, 1988) and the human with primary hypercholesterolemia (Biiheimer et al., 1983; Duane et al., 1988).
66 However the clearance of HDL-[14C]cholesterol from the plasma was not significantly changed in the treated and the untreated group. The HDL-cholesterol level in the plasma remains stable when crilvastatin is administered, in spite of very active and rapid exchanges of cholesterol between H D L and tissue membranes, such as liver membranes. Thus, we observed an increase of radiolabelled material in the liver after injection of [~4C]HDL in 8-day crilvastatin-treated rats; this effect is independent of the bile secretory process, this process not being stimulated by H D L cholesterol, Modulations in cholesterol levels, particulary as induced by hypocholesterolemic drugs, cannot yet be fully explained on the basis of molecular biology. On the one hand, it has been shown that simvastatin-induced inhibition of synthesis and esterification of cholesterol results in a post-transcriptional decrease of apoE and apo A-I, the major component of rat H D L (Ribeiro et al., 1991). Thus, these results can explain the biochemical lipid data. On the other hand, synthesis of apoB, a major component of L D L seems not to be modulated by hepatic cholesterol or triacylglycerol synthesis (Ribeiro et al., 1991) and cannot explain the biochemical data for LDL. Thus, it would be interesting to determine the in vivo apoprotein particulary apoE, levels on treatment with crilvastatin, it can be expected that the most important hypocholesterolemic effect of crilvastatin concerns the LDL more than the HDL. It follows that the effects of long-term crilvastatin treatment (17 w e e k s ) o n acyl coenzyme A:cholesterol acyltransferase and cholesterol 7a-hydroxylase activity in normal and hypercholesterolemic rats could be understood on the basis of the intrahepatic cholesterol flux: with crilvastatin treatment, the excess of hepatic cholesterol provided by L D L in the control group is not diverted to bile secretion. However, the increased amount of [J4C]-labelled sterol in the bile of control rats treated for 8 days with crilvastatin, after [~4C]LDL injection, is not the sole reflection of the drug effect on bile secretion, since [~4C]HDL do not show any stimulation of the secretion of [J4C] material into the bile (and expressed in terms of total volume of bile secreted per min). Such differences in biliary cholesterol transfer from H D L or from LDL, can be explained by a selective mode of transport of crilvastatin or its biliary metabolite(s). The physiological carrier of crilvastatin was shown to be the L D L rather than the H D L (unpublished data). Thus, there results stimulation of the biliary secretion of [~4C] material from [~4C]LDL, [~4C]HDL not involving such effects, The excess of hepatic cholesterol provided by stimulation of hepatic L D L uptake after the 17-week drug treatment, may be converted to the esterified form in the liver, destined to be secreted as plasma lipoproteins. Thus, while acyl coenzyme A : cholesterol acyl-
transferase activity of normolipidemic rats increases significantly, cholesterol 7a-hydroxylase activity does not change significantly. Our results show clearly that in the control rats, crilvastatin acts at the biliary pole through a bile salt-independent and probable ionic choleresis process, related to a hydrophilic metabolite form(s) of crilvastatin, as described for another statin, compactin (Kempen et al., 1984). Our results are different from those obtained in normolipemic subjects chronically treated with lovastatin, showing that synthesis of bile acids as well as of bile cholesterol is reduced (Mitchell et al., 1991). In our 10-week experiment, the bile acid level in bile of normolipemic rats was unchanged by the drug treatment, and bile cholesterol decreased non-significantly. This decrease could be related partly to the inhibitory effect of the drug on hepatic hydroxymethylglutaryl coenzyme A reductase, and partly to a decrease in the contribution of the main lipoprotein implicated in bile sterol secretion in normolipemic rats: the HDL. Our results also differ from those obtained with fibrates, which reduce both acyl coenzyme A:cholesterol acyltransferase and cholesterol 7~-hydroxylase activity (Stahlberg et al., 1989). However, the most promising effect of the statins must be that detected in hypercholesterolemic models. Thus, with crilvastatin, in the high fat group, the excess of hepatic cholesterol provided by LDL is diverted to bile sterol secretion by a bile salt-dependent choleresis process. This excess of cholesterol is not converted into the esterified form, the acyl coenzyme A : cholesterol acyltransferase activity remains stable and the cholesterol 7a-hydroxylase activity increases significantly. Our results, showing stimulation of bile salt secretion in hypercholesterolemic rats treated with crilvastatin, are not comparable to those obtained with fibrates such as clofibrate (the Coronary Drug Project, 1975) and gemfibrozil (Leiss et al., 1985). With these drugs, the saturation index of gallbladder bile increases. However, no such effect is observed during treatment of human IIa or IIb hypercholesterolemia with simvastatin, which decreases the cholesterol saturation index of gallbladder bile (Duane et al., 1988) in spite of the inhibitory effect on cholesterol 7a-hydroxylase (Bj6rkhem, 1986). This preclinical study suggested that Crilvastatin can be considered as a potent and promising drug for the treatment of hypercholesterolemia in our particular model. Our results showed for the first time that crilvastatin has a particular action in the liver since it stimulates cholesterol 7a-hydroxylase activity in hypercholesterolemic rats, by stimulating bile clearance of excess cholesterol from circulating LDL in the form of cholesterol and bile salts. This effect is enhanced as there is no increase in esterification of cholesterol by acyl coenzyme A : cholesterol acyltransferase in the liver
67
of these rats. These effects are especially interesting since the level of H D L remains stable. The effect of crilvastatin as well as of the other statins, on the interrelations between apoprotein synthesis and hepatic cholesterol metabolism need to be investigated further,
Acknowledgements This work was supported by INSERM and PAN MEDICA Laboratories (Grant No. 89031). We wish to thank Mrs Michelle Bonneil for secretarial support and Mr Patrick Garzino for animal care.
References Alberts~ A.W., 1988a, Discovery, biochemistry and biology of lovastatin, Am. J. Cardiol. 62, 10j. Alberts, A.W., 1988b, Lovastatine et simvastatine, Cah. Nutr. DiSt. 23, 231. Alberts, A.W., J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonber, O. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, 1980, Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent, Proc. Natl. Acad. Sci. U.S.A. 77, 3957. Amic, J., D. Lairon and J. Hauton, 1972, Technique de dosage automatique de l'orthophosphate de grande fiabilit6, Clin. Chim. Acta 40, 107. Aubert, I., J. Emmerich, Y. Charpak, B. Chanu, C. Dachet, D. Herlich, M. Besseau, J. Rouffy and B. Jacotot, 1988, Effets de la simvastatine sur les lipides et apoprot~ines (A1 et B) plasmatiques. 24 cas d'hypercholest6rol~mie primaire majeure, La Presse M~dicale 17, 901. Beaufay, H., A. Amar-Costesec, E. Feytmans, D. Thines-Sempoux, M. Wibo, M. Robi and J. Berthet, 1974, Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods, J. Cell. Biol. 61, 188. Bilheimer, D.W., S.M. Grundy, M.S. Brown and J.L. Goldstein, 1983, Mevinolin and colestipol stimulate receptor-mediated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes, Proc. Natl. Acad. Sci. U.S.A. 80, 4124. Bio-Rad assay, Chromatography electrophoresis. Immunochemistry HPLC, and Catalog K., 1985, p. 233. Bj6rkhem, I., 1986, Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutarylcoenzyme A (HMG CoA reductase) reductase and cholesterol 7 alpha-hydroxylase activity, Biochim. Biophys. Acta 877, 43. Brown, M.S. and J.L. Goldstein, 1983, Lipoprotein receptors in the liver, control signals for plasma cholesterol traffic, J. Clin. Invest. 72, 743. Bucolo, G. and H. David, 1973, Quantitative determination of serum triglycerides by use of enzymes, Clin. Chem. 19, 475. Chautan, M., E. Termine, G. Nalbone and H. Lafont, 1988, Acylcoenzyme A cholesterol acyltransferase assay: silica gel column separation of reaction products, Anal. Biochem. 173, 436. Diekmann, H.W., H. Sailer, A. Garbe, H. Nowak, M.R. Lovati, L. Allievi, M. Galbussera and C.R. Sirtori, 1986, Mechanism of the plasma cholesterol lowering effect of tazasubrate in rats: accelerated plasma cholesterol transport with increased liver lipoprotein receptor activity, Pharmacol. Res. Commun. 18, 203.
Domingo, N., J. Amic and J. Hauton, 1972, Dosage automatique des sels biliaires conjugu~s de la bile par la 3 alpha-hydroxystero'ide d6shydrog6nase, Clin. Chim. Acta 37, 399. Duane, W.C., D.B. Hunninghake, M.L. Freeman, P.A. Pooler, L.A. Schlasner and R.L. Gebhard, 1988, Simvastatin, a competitive inhibitor of Hydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase, lowers cholesterol saturation index of gallbladder bile, Hepatology 8, 1147. Endo, A., 1988, Chemistry, biochemistry, and pharmacology of [lydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase inhibitors, Klin. Wochenschr. 66, 42l. Endo, A., M. Kuroda and K. Tanzawak, 1976, Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML 236 A and ML 236 B, fungal metabolites hypocholesterolemic activity, FEBS Lett. 72, 323. Esnault, C., H. Lafont, F. Chanussot, M. Chautan, J. ttauton and C. Laruelle, 1988, Inhibition of hepatic HMG-CoA reductase activity by two new hypocholesterolemic drugs, in: Liver Cells and Drugs. ed. A. Guillouzo, (Colloque INSERM/John Libbey Eurotext Ltd.) 164, p. 99. Esnault-Dupuy, C., F. Chanussot, H. Lafont, C. Chabert and J. Hauton, 1987, The relationship between HDL-LDL-, liposomesfree cholesterol, biliary cholesterol and bile salts in the rat, Biochimie 69, 45. Halloran, L.G., C.C. Schwartz, Z.R. Vlahcevic, R.M. Nisman and L. Swell, and 1978, Evidence for high-density lipoprotein free cholesterol as the primary precursor for bile-acid synthesis in man, Surgery 84, 1. Hatch, F.T. and R.S. Less, 1968, Practical methods for plasma lipoprotein K analysis, Adv. Lipid Res. 6, 1. Junker, L.H. and J.A. Story, 1985, An improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate~ Lipids 20, 712. Kempen, H.J.M., J. De Lange, M.P.M. Vos-Van Holstein, P. Van Wachem, R., Havinga and R.J. Vonk, 1984, Effect of ML-236B (compactin) on biliary excretion of bile salts and lipids, and on bile flow, in the rat, Biochim. Biophys. Acta 794, 435. Kessler, G., S. Morgenstern, L. Snyder and R. Varady, 1975, lmproved point assays for ALT and AST in serum using the Technicon SMAC high speed computer controlled, biochemical analyser to eliminate the common errors found in enzyme analysis, 9th international Congress for Clinical Chemistry, Toronto. Leiss, O., K. Von Bergmann, A. Gnasso and J. Augustin, 1985, Effect of gemfibrozil on biliary lipid metabolism in normolipemic subjects, Metabolism 34, 74. Leon, P. and R.O. Stasiw, 1976, Performance of automated enzymatic cholesterol on SMA 12/60 and auto analyser II instruments, Clin. Chem. 22, 1220. Lie, R.F., J.M. Smitz, K.J. Pierre and N. Gochman, 1976, Cholesterol oxidase-based determination oby continuous flow analysis of total and free cholesterol in serum, Clin. Chem. 22, 1627. Mitchell, J.C., B.G. Stone, G.M. Logan and C. Duane, 1991, Role of cholesterol synthesis in regulation of bile acid synthesis and biliary cholesterol secretion in humans, J. Lipid Res. 32, 1143. Morgenstern, S., G. Kessler, J. Auerbach, R.V. Flor and B. Klein, 1965, An automated p-nitrophenylphosphate serum alkaline phosphatase procedure for the autoanalyser, Clin. Chem. 11,876. Ribeiro, A., M. Mangeney, C. Loriette, G. Thomas, D. Pepin, B. Janvier, J. Chambaz and G. Bereziat, 1991, Effect of simvastatin on the synthesis and secretion of lipoproteins in relation to the metabolism of cholesterol in cultured hepatocytes, Biochim. Biophys. Acta 1086, 279. Schulze, E., J. Obermaier, C.A. Seyfried, H.W. Diekmann and H. Nowak, 1986, Pharmacological profile of tazasubrate. A new cholesterol-lowering agents, Atherosclerosis 59, 137. Skeggs, L.T. and H. Hochstrasser, 1964, Multiple automatic sequential analysis, Clin. Chem. 10, 918.
68 Sottocosa, G.L., B. Kuylenstierna, L. Ernster and A. Bergstrand, 1967, An electron transport system associated with the outer membrane of liver mitochondria, J. Cell. Biol. 32, 415. Stahlberg, D., B. Angelin and K. Einarsson, 1989, Effects of treatmerit with clofibrate, bezafibrate and ciprofibrate on the metabolism of cholesterol in rat liver microsomes, J. Lipid Res. 30, 953.
The Coronary Drug project Research Group, Coronary Drug Project, 1975, Clofibrate and niacin in coronary heart disease, J. Am. Med. Assoc. 231,360. Yamauchi, S., W.G. Linscheer and D.H. Beach, 1991, Increase in serum and K bile cholesterol and Hydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase by Iovastatin in rats, Am. J. Physiol. 23, G. 625.
59
Elsevier Science Publishers B.V.
EJP 53022
Mechanisms of action in the liver of crilvastatin, a new hydroxymethylglutaryl-coenzyme A reductase inhibitor T h i e r r y Clerc, M i c h e l J o m i e r , M a g a l i C h a u t a n , H e n r y P o r t u g a l ~, M i c h b l e Senft, A n n e - M a r i e Pauli ~, C l a u d e L a r u e l l e b, Olivier M o r e l b, H u g u e t t e L a f o n t a n d F r a n q o i s e C h a n u s s o t INSERM, Unit~ 130, 18 At,enue Mozart, 13009 Marseille, France, '~Laboratoire Central, H3pital Ste Marguerite, 13009 Marseille, France and b PAN MEDICA, BP 27, 06511 Carros, France
Received 16 July 1992, revised MS received 19 January 1993, accepted 26 January 1993
Crilvastatin is a drug from the pyrrolidone family that had been shown to induce non-competitive inhibition of rat hydroxymethylglutaryl-coenzyme A reductase activity in vitro. The aim of this study was to evaluate the activity of crilvastatin on the hepatic metabolism of cholesterol in rats. Crilvastatin increased low density lipoprotein (LDL)-cholesterol uptake by the liver more than high density lipoprotein (HDL) uptake, thus increasing by up 30% the clearance of excess plasma cholesterol. In normolipidemic rats, crilvastatin significantly enhanced acyl coenzyme A : cholesterol acyl transferase and cholesterol 7a-hydroxylase activity. In rats with a previous high cholesterolemia, crilvastatin also enhanced cholesterol 7a-hydroxylase activity and did not increase liver acyl coenzyme A:cholesterol acyl transferase activity. These findings suggest that a drug such as crilvastatin could have a hypocholesterolemic effect by a mechanism other than the sole inhibition of cholesterol synthesis, possibly by stimulating cholesterol and bile salt secretion via the biliary tract in previously hypercholesterolemic rats. Hypocholesterolemic drugs; Liver; Cholesterol; Bile; Lipoproteins
1. Introduction Hypocholesterolemic agents (Alberts, 1988a, b ) s u c h as compactin (Endo et al., 1976) and mevinolin (A1berts et al., 1980) belong to a new generation of choleso terol-lowering drugs that appear highly effective (Aubert et al., 1988). In the cell, their primary action is to nhibit competitively hepatic hydroxymethylglutarylcoenzyme A reductase activity, the rate limiting enzyme in cholesterol synthesis (Endo et al., 1976; A1berts et al., 1980). The hypocholesterolemic effect of these drugs is related to the stimulation of liver uptake of low density lipoproteins (LDL), which are known to be atherogenic. Thus mevinolin increases L D L receptors which could account for the lowering of the L D L level (Alberts et al., 1980). We previously tested another new and non-competitive hydroxymethylglutaryl-coenzyme A reductase inhibitor from the pyrrolidone group, crilvastatin (Pan Medica, Carros, France). The purpose of the present work was to investigate the mechanisms other than
Correspondence to: F. Chanussot, INSERM Unit6 130, 18 Avenue Mozart, 13009 Marseille, France. Tel. 33-91.75.47.05, fax 3391.75.15.62.
those already described (Esnault et al., 1988), by which crilvastatin acts on the liver. We focused on the balance between circulating lipoprotein cholesterol, and the hepatic cholesterol metabolising enzymes wich are implicated in the bile secretion of cholesterol and bile salts. Bile salts and cholesterol in the bile originate mostly from free cholesterol supplied by high density lipoproteins ( H D L ) and L D L (Halloran et al., 1978). Thus, we first investigated a possible hypocholesterolemic effect of crilvastatin on hypercholesterolemic rats treated chronically with the drug during a period (17 weeks), sufficient for evaluation of this hypocholesterolemic effect. We then investigated the effect of crilvastatin on uptake and metabolism of free cholesterol-HDL and - L D L by the liver and on levels of cholesterol and bile salts secreted in bile. Acyl coenzyme A : cholesterol acyltransferase and cholesterol 7a-hydroxylase activities in the liver were studied in order to evaluate the effect of crilvastatin on hepatic cholesterol storage and cholesterol catabolism to bile salts. We tested the effect of crilvastatin administered over a short period (8 days) that was adequate for the study of possible changes in H D L and L D L metabolism in normal rats. Changes in liver enzyme activity were evaluated in rats with a high cholesterolemia after a period (10 weeks)
6O of administration sufficient long for such modifications to appear,
2. Materials and methods
2.1. Chemicals Chemicals of the highest purity were obtained from Sigma, Sanofi, Carlo Erba and Bio-Rad. Crilvastatin (Pan Medica Lab., Carros, F r a n c e ) i s a synthetic derivative of pyrrolidone carboxylic acid (cycloalkyl ester of pyroglutamic acid) (fig. 1). Previous study had shown that this chemical inhibits hydroxymethylglutaryl-coenzyme A reductase activity in vitro by a non-competitive manner through a mechanism that involves the substrate concentration and alters the Vma× of the incubation reaction (Esnault et al., 1988). The K m of the enzyme remains unchanged. The inhibitory effect of this drug seemed to result from changes in the substrates and reaction products. The kinetic data obtained with crilvastatin showed that this drug was a more potent inhibitor than fenofibrate; mevinolin acted faster than crilvastatin, however the maximum inhibition at the same concentration of these two drugs was identical (Esnault et al., 1988).
2.2. Experimental procedures Three different experiments were carried out. In the first experiment, we tested the possible hypocholesterolemic effect of crilvastatin, administered chronically during a sufficiently long time (17 weeks) in control or hypercholesterolemic rats, by assaying plasma cholesterol. In the second experiment, the fate of intravenously injected [14C]free cholesterol-HDL and -LDL was evaluated after 8-day crilvastatin treatment of normolipemic rats. In the third experiment the effect of a 10-week crilvastatin treatment on enzyme activities iraplicated in the homeostasis of hepatic cholesterol was investigated in hypercholesterolemic rats. At the end of all treatments, rats were anesthetized with sodium pentobarbital (5 m g / 1 0 0 g body weight).
o o ~
,, ~
"
~_~%,¢H3C_ ,,I ell3
II
H,,,~,,/
\
"c ~ 0~'~ ~'~CH cr~v~,s~n
H
Fig. ~. C h e m i c a l s t r u c t u r e of crilvastatin,
3
Plasma was collected from the 10-week and 17-week treatment groups. These rats were fasted overnight prior to plasma collection to allow proper evaluation of cholesterol in the plasma and lipoproteins. Bile was collected from the 10-week treatment group from the common bile duct cannulated with polyethylene tubing (Clay Adams No. 10).
2.2.1. 17-week crih~astatin treatment 2.2.1.1. Animals and drug treatment. Thirty-two 3week-old male Wistar rats raised at Iffa-Credo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). For 6 months, the rats received either a low fat diet (control, 5% lipids as 3% lard, 2% corn oil) or a high fat diet (29.2% lipids as 26% lard, 2% corn oil, 1.2% cholesterol). Both control and high fat diets contained the same amount of protein (24% casein). Thus, the requirement for essential fatty acids and protein was supplied by both diets. In the course of 17 weeks, four experimental groups (8 rats per group) were constituted: control, c o n t r o l / crilvastatin; high fat; high fat/crilvastatin. For the control/crilvastatin and high fat/crilvastatin groups, crilvastatin was previously solubilized in corn oil and mixed with the food. The quantity of drug incorporated was calculated to be such that the oral intake of each rat was 200 mg crilvastatin/kg of body weight per day. The body weight of the animals at the end of the experiment was not modified by the drug (respectively 402 _+ 51 g in the control group, 435 + 122 g in the control/crilvastatin group, 500_+ 70 g in the high fat group and 465 _+ 26 g in the high fat/crilvastatin group).
2.2.2. 8-day crih, astatin treatment 2.2.2.1. Animals and drug treatment. Twenty 15week-old male Wistar rats raised at Iffa-Credo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). They were divided into four groups of five animals. Two control groups as well as two other (control/crilvastatin) groups previously treated with crilvastatin received an [14C]HDL or [14C]LDL injection. All rats received the control diet. Crilvastatin was emulsified by sonication in water (2/1 v / v crilvast a t i n / w a t e r ) and administered by intraperitoneal injection at a dose of 150 mg crilvastatin/kg body weight per day. 2.2.2.2. Preparation of lipoprotein ,fractions. Free cholesterol in LDL and H D L was labelled with [14C] according to a method described by Esnault-Dupuy et al. (1987). Serums 20 ml from non-fasted donor Wistar rats was incubated for 24 h at 10°C with 2 ml of phosphatidylcholine/cholesterol liposomes prepared as follows. Phosphatidylcholine (111.5 mg, Sigma, Paris,
61 France) and [14C]free cholesterol (4.8 ~Ci; specific activity 50 m C i / m m o l ) (CEA, France) were mixed in benzene then evaporated under nitrogen. The dry residue was suspended in 3 ml of 0.9% NaC1 solution then was cooled to 5°C, and sonicated for 1 hour under nitrogen using 30-s pulses. The liposome mixture obtained was centrifuged at 100000 × g for 30 min to eliminate large particles. The homogeneity of the liposomes was verified by A4 agarose (IBF, France) gel filtration using a Tris 0.5 M, pH 7.5 buffer as eluant, Under these conditions, the [~4C]cholesterol liposomes eluted in a single peak. Serum lipoproteins (HDL, LDL) were isolated by sequential ultracentrifugation after adjustement to the required density with sucrose according to the technique of Hatch and Less (1968). LDL were obtained at a flotation density of 1.063 after centrifugation at 120000 × g for 22 h at 12°C. H D L were obtained at a flotation density of 1.21 after centrifugation at 120000 × g for 22 h at 12°C. The H D L preparation was labelled by direct [14C]free cholesterol exchange, since cholesterol is easily exchangeable between H D L and others lipoproteins or membrane surfaces. The H D L and L D L fractions were then ultra-centrifuged at 1 2 0 0 0 0 × g for 1 h at 12°C to separate [~4C]lipoproteins and unbound [~4C]free cholesterol. The final label of the H D L represented 60-65% of the [~4C]free cholesterol incubated with the HDL. Radioactive LDL represented about 50% of the label contained on [~4C]liposomes. Thin-layer chromatography was performed to check that the [~aC]cholesterol in the injected lipoproteins was exclusively on the non-esterifled cholesterol fraction. The mass of radioactive serum lipoproteins, especially H D L and LDL. 2.2.2.3. Injection of radiolabelled materials. In the 8-day treatment group, 20 min after the beginning of the experiment, either [~4C]LDL or [~4C]HDL were injected for 7 and 10 min respectively via the jugular vein. The dose of [ ~ C ] L D L was 0.682 p~Ci [~4C]free cholesterol which is equivalent to 0.07-0.14 /xmol of free cholesterol-LDL (0.36-0.60 ~mol of total cholesterol). The dose of [~4C]HDL was 0.363 /zCi [~aC]free cholesterol which is equivalent to 0.03-0.15 p~mol of free cholesterol-HDL (0.16-0.52 tzmol of total cholesterol). The amount of cholesterol injected was negligible in comparison to the 20 mmol or more of circulating native total cholesterol,
2.2.3. lO-week crilr,astatin treatment 2.2.3.1. Animals and drug treatment. Thirty-four 9week-old male Wistar rats raised at IffaoCredo (L'Arbresle, France) were placed at random in stainless steel cages (1/cage). For 3 weeks the rats received either the control diet or the high fat diet. The rats were then divided into four groups for the various
10-week treatments: control (number of rats: n = 10); control/crilvastatin (n = 9); high fat (n = 5); high fat/crilvastatin (n = 10); the mean body weight were respectively 465 _+ 6 g; 472 _+ 27 g; 469 +_ 17 g; 428 _+ 18 g, the mean liver weights were respectively 11.45 _+ 0.38 g; 10.05 _+ 0.43 g; 20.64 _+ 1.41 g; 20.35 _+ 1.13 g. Crilvastatin also was incorporated in the diets.The oral intake of each rat was 200 mg crilvastatin/kg body weight per day. At the end of the 10-week crilvastatin treatment, the body weight of the animals (as well as the liver weight) was not modified by the administration of the drug. 2.2.3.2. Preparation of litter microsomes. Microsomes from livers of rats treated for 10 weeks with crilvastatin were used for the acyl coenzyme A : cholesterol acyltransferase and cholesterol 7a-hydroxylase activity determinations. Microsomes were isolated from fresh liver as follows: 2 g of liver were homogenized in 10 ml of potassium phosphate pH 7.4 buffer (50 mM KH2PO4, 0.1 M sucrose, 30 mM EDTA, 70 mM KCI, 1 mM dithiothreitol) with a polytron PT 10-35 homogenizer. The homogenate was then centrifuged at 10000 × g at 4°C for 15 min. The supernatant was centrifuged again at 1 0 0 0 0 × g for 15 rain and the resulting supernatant was centrifuged at 100000 × g at 4°C for 60 rain. The microsomal pellet obtained was resuspended in 1 ml of the working buffer and was stored frozen at -80°C. The purity of the microsome preparation was checked by assessing the activity of rotenone insensitive N A D P H cytochrome C reductase (EC 1.6.2.4) (Sottocosa et al., 1967). 2.2.3.3. Microsomal enzymatic assays. Acyl coenzyme A:cholesterol acyltransferase and cholesterol 7a-hydroxylase activity were determined on microsomal preparations. Acyl coenzyme A cholesterol acyltransferase activity was measured as described by Chautan et al. (1988) using [14C]l-oleoyl-CoA (NEN, Paris, France, specific activity 57.4 m C i / m m o l ) as substrate. Free cholesterol and esterified cholesterol were separated by chromatography on silica gel microcolumns and elution of esterified cholesterol with petroleum ether/diethyl ether ( 9 8 / 2 v/v). Cholesterol 7a-hydroxylase activity was measured as described elsewhere (Junker and Story, 1985). However, the best results were obtained when liposomes were incubated with dioleoyl-phosphatidylcholine instead of various phospholipids. For 0.5 mg microsomal protein the composition of the incubated liposomes was: 0.5 mg dioleoyl phosphatidycholine and 0.018 /zCi of [~4C]free cholesterol ( C E A , F r a n c e specific activity 50 mCi/mmol). [~4C]Cholesterol and [~4C]7a-hydroxycholesterol were separated as previously described by thin layer chromatography on silica-gel plates using an ethyl a c e t a t e / h e p t a n e 80/20 v / v mixture. The components were visualized under iodine vapor.
62
2.3. Sampling From the 10-week crilvastatin treatment group the bile samples were weighed then immediatly frozen. At the end of the experiment the animals were killed by aortal puncture. Rats treated 17 weeks were killed similarly and blood was collected either for freezer storage of plasma or on ethylenediaminetetraacetic acid (0.1 m g / m 1) for ultra-centrifugation of the lipoproteins. The liver was immediately removed and weighed. Erythrocytes, spleen, kidneys, heart and lungs were also frozen.
2.4. Radioactit,ity measurements Bile radioactivity was counted in 50 /~1 aliquots dissolved in 10 ml of scintillation liquid (Beckman Ready Solv MP). Radioactivity was also measured in bile salts and cholesterol isolated from the bile by thin-layer silica gel F 1500 chromatography (Schleicher-Schiill, Dassel, Germany) with a medium composed of 40% isoamyl acetate, 30% propionic acid, 20% isopropyl alcohol and 10% water (v/v). After identification at 350 rim, the spots were scraped and counted in 10 ml of scintillation liquid (Beckman Ready solv MP). L D L and H D L radioactivity was determined after extraction in a small volume of petroleum ether, Free cholesterol, esterified cholesterol or 7a-hydroxycholesterol were extracted with c h l o r o f o r m / m e t h a n o l 2 / 1 v / v and were separated on thin-layer silica gel F 1500 (Schleicher-Schiill, Dassel, Germany) using a mixture (v/v) containing 90 heptane, 30 sulfuric ether and 1 acetic acid (for free cholesterol and esterified cholesterol), and 80 ethyl acetate and 20 heptane (for free cholesterol and 7a-hydroxycholesterol). The fractions were visualised with iodine vapor. The spots were scraped and counted in 10 ml of scintillation liquid (Beckman Insta-fluor for organic solution),
2.5. Gra~,imetric assays 2.5.1. In the bile Phospholipids (Amic et al., 1972), bile salts (Domingo et al., 1972) and cholesterol (Lie and al., 1976) were assayed by a semi-automatic colorimetric method.
2.5.2. In the plasma Triacylglycerols (Bucolo and David, 1973), total and free cholesterol (Leon and Stasiw, 1976) in the plasma, LDL and HDL, total protein (Skeggs and Hochstrasser, 1964) and protein in L D L and H D L (Bio-Rad assay, 1985) were measured along with alkaline phosphatase (Morgenstern and al., 1965), alanine aminotranferase (Kessler and al., 1975) and aspartate aminotransferase (Kessler and al., 1975).
2.5.3. In the litter When no microsomal preparations were required for analyses, the frozen livers were thawed and homogenized in 1 mM Tris buffer pH 7.4 containing 250 mM sucrose, in a polytron PT 10-35 homogenizer (Beaufay et al., 1974). Total protein (Skeggs and Hochstrasser, 1964), triacylglycerols (Bucolo and David, 1973), total cholesterol (Leon and Stasiw, 1976), alkaline phosphatase (Morgenstern and al., 1965), aspartate aminotransferase and alanine aminotransferase (Kessler and al., 1975) were assayed in these homogenates.
2.6. Statistics The significance of differences was analyzed with Student's t-test.
3. Results
3.1. Effect of lO-week crih~astatin treatment The numbers of plasma and bile samples are not the same for all groups, specially the high fat and high fat/crilvastatin groups, as the samples were not always used for the same purposes.
TABLE1 Cholesterolin the plasma after 10-week crilvastatin treatment. VLDL: very low density lipoprotein; LDL: low density lipoprotein; HDL: high density lipoprotein; CR: crilvastatin. The results are expressedasmeans_+S.E.M. Total EsterifiedFree cholesterol cholesterol cholesterol (retool/l) (mmol/l) (mmol/l) Total plasma (n - 10) 2.33_+0.05 1.82+0.03 0.54_+0.01 VLDL(n=8) 0.15_+0.10 0.11+0.09 0.04_+0.01 Control LDL(n-8) 0.52_+0.12 0.42+0.12 0.07_+0.04 HDL(n = 8) 1.79_+0.80 1.37+0.83 0.40_+0.25 Total plasma (n = 9) VLDL(n=8) Control/CR LDL(n=8) HDL(n=8) Total plasma (n = 5) VLDL (n = 5) HF(n = 5) LDL(n = 5) HDL(n=5)
2.26_+0.01 1.72+0.01 0.53_+0.01 0.13_+0.08 0.10_+0.10 0.03_+0.01 0.48_+0.17 0.40_+0.14 0.11_+0.02 1.76+0.42 1.32_+0.35 0.40+0.07
3 . 4 7 + 0 . 2 8 2.84_+0.21 0.63+0.16 0.48+0.27 0.34_+0.22 0.06+0.02 1.12~-0.14 0.79_+0.15 0.20+0.09 1.84+0.60 1.52_+0.51 0.43+0.15 1.a, Total plasma(n = 5). 2.64_+0.20 2.14+0.27 0.51_+0.18 VLDL (n = 5) 0.26+0.05 0.17_+0.04 0.03+0.02 HF/CR L D L ( n = 5) 0.50_+0.13 0.46_+0.12 0.06+0.3 HDL(n=5) 1.84+0.73 1.55_+0.59 1/.36_+0.21 Differenceswere analyzed with Student's t-test and are significant at ~P < 0.05; a high fat/crilvastatin (bIF/CR) vs. high fat (HF).
63
3.1.1. In the plasma (table 1) T h e total cholesterol levels were 27% lower in the plasma of d r u g - t r e a t e d rats with previously high c h o l e s t e r o l e m i a t h a n in p l a s m a of u n t r e a t e d hyperc h o l e s t e r o l e m i c rats, L D L were 45% lower a n d V L D L were 54% lower. Both free a n d esterified cholesterol decreased, b u t the cholesterol level in H D L did not decrease (high f a t / c r i l v a s t a t i n versus high fat). Crilvastatin also led to a 35% decrease in triacylglycerol level in the control a n d high fat groups. T h e level of p l a s m a a n d l i p o p r o t e i n phospholipids as well as p r o t e i n s in the l i p o p r o t e i n fractions r e m a i n e d unc h a n g e d (respectively 1.7 m m o l / 1 in the plasma phospholipids a n d 22 m g / 1 in the L D L a n d 2500 m g / l in the H D L proteins),
3.1.2. In the bile (table 2) Crilvastatin s t i m u l a t e d bile flow in both control a n d h y p e r c h o l e s t e r o l e m i c rats. T h e drug significantly stimulated the secretion of bile salts a n d bile cholesterol only in rats with a high cholesterolemia. All these differences are seen clearly w h e n the results are expressed in terms of flow ( m i n / g liver) or of s e c r e t i o n / m i n , o n c o m p a r i s o n of the control a n d c o n t r o l / c r i l v a s t a t i n groups, or also of the high fat a n d high f a t / c r i l v a s t a t i n groups. T h e fact that liver weight is identical in all four groups allows this comparison.
3.1.3. In the litter T h e drug had n o effect on the cholesterol level in the liver ( t r e a t e d versus n o n - t r e a t e d groups), particularly in the h y p e r c h o l e s t e r o l e m i c rats (100 m g / g liver), T h e s e findings could be explained by the fact that the d r u g - i n d u c e d e n h a n c e m e n t of the decrease in hepatic cholesterol, secreted t h r o u g h the bile pathway, was
c o m p e n s a t e d for the s t i m u l a t i o n of hepatic uptake of cholesterol from l i p o p r o t e i n s such as LDL.
3.2. Effect of 17-week crih,astatin treatment C r i l v a s t a t i n s i g n i f i c a n t l y r e d u c e d the c h o l e s t e r o l e m i a of previously hypercholesterolemic rats. Thus, in the plasma, the total cholesterol level, like the esterified cholesterol (3.82-3.22 m m o l / l ) and free cholesterol (1.31-0.84 m m o l / l ) , d e c r e a s e d significantly from 5.13 to 4.06 m m o l / l after crilvastatin t r e a t m e n t , however, this effect was not observed in the control rats, since their cholesterol status is always above norreal a n d is very difficult to decrease, as observed elsewhere ( Y a m a u c h i et al., 1991). T h e changes in cholest e r o l e m i a were not due to weight loss. Crilvastatin did not decrease significantly the body weight of the animals; the diet intake as well as p r o t e i n absorption were n o r m a l as we f o u n d no n i t r o g e n loss in any of the groups.
3.3. Fate of [14C]free cholesterol-LDL and [~4C]free cholesterol-HDL in the litter (8 days treatment with crilt,astatin) 3.3.1. Experiment with [14C]HDL A f t e r a d m i n i s t r a t i o n of [14C]HDL, most of the radioactivity was f o u n d in the liver, bile, plasma and erythrocytes (table 3). T h e a m o u n t of [14C] t a k e n up by the heart, kidneys, lungs a n d spleen was very low (less t h a n 1% of the radioactivity injected) a n d these values are not shown. A certain a m o u n t of [~4C] free cholest e r o l - H D L must have b e e n t a k e n up by p e r i p h e r a l tissues since 1 7 - 3 3 % of the total radioactivity injected was not f o u n d back in the organs studied.
TABLE 2 Bile secretion after 10-week crilvastatin treatment. The results are expressed as means_+S.E.M. (the means were calculated for a bile collection period of 120 rain). Control
Control/CR
HF
HF/CR
Bile flow ~l/min per g liver Bile flow /zl/min
1.27+ 6.03 (n = 10) 14.4 _+ 0.4 (n = 10)
2.23_+ 0.11 3'~ (n = 9) 24.0 + 1.3 3.a (n = 9)
0.72+ 0.01 (n = 5) 14.8 _+ 0.20 (n = 5)
1.04+ 0.01 3.~ (n = 10) 21.16_+ 0.20 3.,, (n = 10)
Bile salts nmol/min per g liver Bile salts nmol/min
27.44_+ 3.76 (n = 9) 315.0 _+43.1 (n = 9)
28.91+ 2.20 (n = 9) 292.5 +22.2 (n = 9)
27.27_+ 1.18 (n = 5) 559.8 _+22.2 (n = 5)
32.25_+ 2.25 ~~ (n = 9) 658.3 _+43 (n = 9)
Bile cholesterol nmol/min per g liver Bile cholesterol nmol/min
0.37_+ 0.05 (n = 9) 4.13+ 0.56 (n = 9)
0.28_+ 0.03 (n = 9) 2.84_+ 0.30 (n = 9)
0.28+ 0.01 (n = 5) 5.72_+ 0.20 (n = 5)
0.35_+ 0.1)13.~ (n = 7) 7.15_+ 0.18 3.,~ (n = 7)
Differences were analyzed with Student's t-test and are significant at 1 p < 0.05; 2 p < 0.01; 3 p < 0.001; a control vs. control/crilvastatin (CR) or high fat (HF) vs. high fat/crilvastatin (HF/CR).
64 TABLE 3
Radioactivity distribution in various tissues after [14C]cholesterol lipoprotein injection (% [14C]cholesterol injected), EC: esterified cholesterol; FC: free cholesterol; BS: bile salts. VLDL: very low density lipoprotein; LDL: low density lipoprotein; HDL: high density lipoprotein. The results are expressed as means_+ S.E.M. (n = 5).
Liver %EC %FC Bile %BS % FC Plasma VLDL LDL HDL Erythrocytes
[14C]HDL injection
[14C]LDLinjection
Control (n = 5) 20.2_+ 1.0 0 20.2 21.2_+0.9 19.1 2.1 9.5 +0.8 2.1_+0.4 2.4_+0.7 5.0+ 1.7 9.2_+1.0
Control Crilvastatin (n = 5) (n = 5) 27.6_+1.1 31.0+0.8 ~'" 1.1 1.1 26.5 29.9
Crilvastatin (n = 5) 35.2+ 1.6 3~, 2.8 32.4 15.4-+1.1 2"a
14.2 1.2 12.0-+1.3 2.3+1.1 3.7_+0.4 6.0_+0.3 10.9_+1.1
6.5+0.6
9.2-+0.3 2'a
6.0 8.5 0.5 0.7 30.6_+3.2 193 _+1.9 ~,a 1.1_+0.1 1.7+0.4 25.7+5.4 135 _+1.0 1.a 3.8-+0.8 4.1 +0.4 15.8_+1.8 4.6-+0.83.~
Differences were analyzed by Student's t-test and are significant at: t P < 0.05; 2 p < 0.01; 3 p < 0.001. After [t4C]cholesterol HDL or [tac]cholesterol LDL injection: '~ Control vs. crilvastatin. In c r i l v a s t a t i n - t r e a t e d rats, t h e level o f [14C] was i n c r e a s e d significantly as c o m p a r e d to t h e c o n t r o l s in t h e liver and, to a lesser d e g r e e in p l a s m a a n d was significantly d e c r e a s e d in the bile. E r y t h r o c y t e l a b e l l i n g p r o b a b l y r e s u l t e d m a i n l y from e x c h a n g e s b e t w e e n [14C] c h o l e s t e r o l l i p o p r o t e i n s a n d t h e n o n - l a b e l l e d cholesterol in e r y t h r o c y t e m e m b r a n e s . In the liver, all the r a d i o a c t i v i t y was f o u n d in mainly free c h o l e s t e r o l . In bile (fig. 2), [14C] r a d i o a c t i v i t y which is s e c r e t e d m a i n l y in the f o r m o f bile salts a p p e a r e d 15 min a f t e r crilvastatin t r e a t m e n t . T h e d r u g did not affect the distribution of the r a d i o a c t i v i t y b e t w e e n the bile salts a n d bile free c h o l e s t e r o l . Crilvastatin t r e a m e n t d i d not have a m a r k e d effect on the level of [14C] in p l a s m a (table 3). 3.3.2. E x p e r i m e n t with [ ~ 4 C ] L D L A f t e r a d m i n i s t r a t i o n o f [14C]LDL, [ 14C] radioactivity
ity b e t w e e n bile salts a n d cholesterol. Thus, t h e % bile s a l t s / % free c h o l e s t e r o l r a t i o for [t4C] radioactivity in the bile was a b o u t 10 w h e n [ t 4 C ] H D L w e r e i n j e c t e d ( b o t h c o n t r o l a n d crilvastatin groups) a n d 12 w h e n [~4C]LDL w e r e injected ( b o t h c o n t r o l a n d crilvastatin groups). In plasma, a significant d e c r e a s e in the level o f [14C] c o r r e s p o n d e d to a d e c r e a s e in the level o f L D L (table 3). 3.4. E f f e c t o f crilvastatin on acyl c o e n z y m e A : cholesterol acyltransferase a n d on cholesterol 7a-hydroxylase T h e activity o f t h e s e two e n z y m e s was m e a s u r e d in m i c r o s o m e s i s o l a t e d f r o m t h e livers of control a n d h y p e r c h o l e s t e r o l e m i c rats t r e a t e d chronically with crilvastatin. T h e activities of t h e s e two e n z y m e s a r e exp r e s s e d in c o n v e n t i o n a l units ( p e r mg m i c r o s o m a l p r o tein). T h e s e units allow c o m p a r i s o n s b e t w e e n the control a n d t h e c o n t r o l / c r i l v a s t a t i n groups, a n d b e t w e e n the high fat a n d high f a t / c r i l v a s t a t i n groups, since t h e liver weights in t h e c o n t r o l a n d c o n t r o l / c r i l v a s t a t i n g r o u p s w e r e identical a n d the b o d y weights w e r e unc h a n g e d , b o t h in the high fat a n d high f a t / c r i l v a s t a t i n groups.
% [14 C] cholesterol HDL injected/rain
0.2
1
~
,
0.~ ~k__~z. ~. . ......~ ...... ~ ..... ~...... ~. "~ ~ ~ ~"~ ~
/ ~
30
~'o
~o--~o ~o time (rain)
~o ~o
~o
% [14 c] holesterol LDL injected.10-2/min 7
was f o u n d m a i n l y in the liver, p l a s m a , bile a n d e r y t h r o cytes (table 3). A s in the e x p e r i m e n t with [~4C]HDL, r e s i d u a l a m o u n t s o f [14C] were also d e t e c t e d in the o t h e r o r g a n s s t u d i e d i.e. heart, lungs, s p l e e n , a n d kidneys, a n d s o m e was t a k e n up by p e r i p h e r a l tissues, A f t e r crilvastatin t r e a t m e n t t h e a m o u n t o f [~4C] in t h e liver a n d bile was significantly i n c r e a s e d in c o m p a r ison to controls. Also, the a m o u n t of [ ~ C ] available in the p l a s m a was g r e a t l y d e c r e a s e d a f t e r d r u g t r e a t m e n t . Crilvastatin led to an e n h a n c e m e n t of biliary s e c r e t i o n (fig. 2), as c o m p a r e d to t h e controls. T h e q u a n t i t i e s of [14C] s e c r e t e d w e r e significantly g r e a t e r in the c o n t r o l / crilvastatin g r o u p t h a n in t h e c o n t r o l group, in the bile, t h e d r u g h a d no effect on t h e d i s t r i b u t i o n of r a d i o a c t i v -
~ ~. 4
a 2~ ~ 0 14
~°~:~.~, ..'.i TM /i ..... ~..~..~i...~,..fi,..i ~ ~w-~-~-~-~"_-~ ~
/
3'0
6'0
9'0
'~
1~0 1~0 1~0 2i0 2~0 time (min)
14
Fig. 2. [ C]Cholesterol bile lipid secretion. After [ CJcholesterol 14 HDL injection; after [ C]cholesterol LDL injection. The results are expressed as means_+S.E.M. (n = 5). • Control • crilvastatin. Differences were analyzed with Student's t-test and are significant at: 1, P < 0.05; 2, P < 0.01; 3, P < 0.001. a Control vs. crilvastatin.
65
%pmol/min/mgprotein 200] 1 ~ _ _ is0
1
3.4.2. Cholesterol 7a-hydroxylase actiuity (fig. 4) ~ increase in cholesterol 7a-hydroxylase activity was observed in both groups and the increase was significant in the high fat group.
~
~
too
3.5. Other biochemical data from the li~'er
50 .
o
Control
Control/Crilvastatin
%pmol/min/mgprotein a007
is0~ ~ ............ [............
10o-~ ~
sot
::::::::::::::::::::::::::::::::::::::::::::: Hf ~
HF/Crilvastatin
Fig. 3. Microsomal hepatic acyl coenzyme A : c h o l e s t e r o l actyltransferase activity without cholesterol addition in the incubation m e d i u m (n = 4). The results are expressed as m e a n s _+S.E.M. Differences were analyzed with Student's t-test and are significant at: 1, P < 0.05.
a Control(c)vs. control/crilvastatin(C/CR).
3.4.1. Acyl coenzyme A cholesterol acyltransferase activity (fig. 3) Enzyme activity was stimulated in the control rats. in high fat rats the drug led to a non-significant decrease in acyl coenzyme A:cholesterol acyltransferase activity,
%pmol/min/rngprotein
400 ~00 J a00lo0 0
~
Control
Control/Crilvastatin
n=5
n=5
°/opmol/min/mgprotein 400-q, 3oo-~
la
" 200-
_
10o-
0
Alanine aminotransferase activity was enhanced in animals fed the high fat diet (3689 IU/liver in the control group versus 5736 IU/liver in the high fat group). Drug treatment had no effect. Alkaline phosphatase activity varied from 25 IU/liver in the control group to 45 IU/liver in the high fat group. Crilvastatin had no effect.
HF n=6
HF/Crilvestetin n=6
Fig. 4. Microsomal activity of the cholesterol 7-a-hydroxylase. The results are expressed as m e a n s _+S.E.M. Differences were analyzed by Student's t-test and are significant at: 1, P < 0.05; a High fat ( H F )
vs. highfat/crilvastatin(HF/CR).
4. Discussion
Crilvastatin is a synthetic derivative of pyrrolidone carboxylic acid, it was shown that, under experimental conditions long-term crilvastatin treatment had no effect on the level of plasma cholesterol in normolipemic control rats. However, like mevinolin and related como pounds (compactin, synvinolin, eptastalin) (Alberts, 1988a, b; Alberts et al., 1980) as well as tazasubstrate (Schulze et al., 1986; Diekmann et al., 1986), crilvastatin significantly enhanced the hepatic uptake of LDL-cholesterol. One could speculated that, like other statins (Brown and Goldstein, 1983), crilvastatin stimulates LDL receptors. In vitro studies showed that the primary action of crilvastatin is non-competitive inhibition of hydroxymethylglutaryl-coenzyme A reductase activity in the liver (Esnault et al., 1988). Thus, the hypocholesterolemic effect of statins must be due to other mechanisms of action, since it was shown that, in the rat, long-term lovastatin treatment increased hepatic cholesterol synthesis (Yamauchi et al., 1991). In our experiments, the LDL-cholesterol level did not vary in normal rats treated with crilvastatin. In contrast, in a population of rats with high cholesterolemia crilvastatin decreased plasma cholesterol by 20-30% and in LDL cholesterol even further. These results were obtained in two experiments with two different values for induced hypercholesterolemia. It appears that the efficacy of crilvastatin was comparable in the two situations, even when the hypercholesterolemia reached an aggravated level (experiment: '17-week crilvastatin treatment'). These results were clearly due to the drug, since the body weight of the rats, whether treated or not with crilvastatin, did not change, the diet intake thus being unchanged. These results are comparable to those obtained with cornpactin in animal models such as the dog (Endo, 1988) and the human with primary hypercholesterolemia (Biiheimer et al., 1983; Duane et al., 1988).
66 However the clearance of HDL-[14C]cholesterol from the plasma was not significantly changed in the treated and the untreated group. The HDL-cholesterol level in the plasma remains stable when crilvastatin is administered, in spite of very active and rapid exchanges of cholesterol between H D L and tissue membranes, such as liver membranes. Thus, we observed an increase of radiolabelled material in the liver after injection of [~4C]HDL in 8-day crilvastatin-treated rats; this effect is independent of the bile secretory process, this process not being stimulated by H D L cholesterol, Modulations in cholesterol levels, particulary as induced by hypocholesterolemic drugs, cannot yet be fully explained on the basis of molecular biology. On the one hand, it has been shown that simvastatin-induced inhibition of synthesis and esterification of cholesterol results in a post-transcriptional decrease of apoE and apo A-I, the major component of rat H D L (Ribeiro et al., 1991). Thus, these results can explain the biochemical lipid data. On the other hand, synthesis of apoB, a major component of L D L seems not to be modulated by hepatic cholesterol or triacylglycerol synthesis (Ribeiro et al., 1991) and cannot explain the biochemical data for LDL. Thus, it would be interesting to determine the in vivo apoprotein particulary apoE, levels on treatment with crilvastatin, it can be expected that the most important hypocholesterolemic effect of crilvastatin concerns the LDL more than the HDL. It follows that the effects of long-term crilvastatin treatment (17 w e e k s ) o n acyl coenzyme A:cholesterol acyltransferase and cholesterol 7a-hydroxylase activity in normal and hypercholesterolemic rats could be understood on the basis of the intrahepatic cholesterol flux: with crilvastatin treatment, the excess of hepatic cholesterol provided by L D L in the control group is not diverted to bile secretion. However, the increased amount of [J4C]-labelled sterol in the bile of control rats treated for 8 days with crilvastatin, after [~4C]LDL injection, is not the sole reflection of the drug effect on bile secretion, since [~4C]HDL do not show any stimulation of the secretion of [J4C] material into the bile (and expressed in terms of total volume of bile secreted per min). Such differences in biliary cholesterol transfer from H D L or from LDL, can be explained by a selective mode of transport of crilvastatin or its biliary metabolite(s). The physiological carrier of crilvastatin was shown to be the L D L rather than the H D L (unpublished data). Thus, there results stimulation of the biliary secretion of [~4C] material from [~4C]LDL, [~4C]HDL not involving such effects, The excess of hepatic cholesterol provided by stimulation of hepatic L D L uptake after the 17-week drug treatment, may be converted to the esterified form in the liver, destined to be secreted as plasma lipoproteins. Thus, while acyl coenzyme A : cholesterol acyl-
transferase activity of normolipidemic rats increases significantly, cholesterol 7a-hydroxylase activity does not change significantly. Our results show clearly that in the control rats, crilvastatin acts at the biliary pole through a bile salt-independent and probable ionic choleresis process, related to a hydrophilic metabolite form(s) of crilvastatin, as described for another statin, compactin (Kempen et al., 1984). Our results are different from those obtained in normolipemic subjects chronically treated with lovastatin, showing that synthesis of bile acids as well as of bile cholesterol is reduced (Mitchell et al., 1991). In our 10-week experiment, the bile acid level in bile of normolipemic rats was unchanged by the drug treatment, and bile cholesterol decreased non-significantly. This decrease could be related partly to the inhibitory effect of the drug on hepatic hydroxymethylglutaryl coenzyme A reductase, and partly to a decrease in the contribution of the main lipoprotein implicated in bile sterol secretion in normolipemic rats: the HDL. Our results also differ from those obtained with fibrates, which reduce both acyl coenzyme A:cholesterol acyltransferase and cholesterol 7~-hydroxylase activity (Stahlberg et al., 1989). However, the most promising effect of the statins must be that detected in hypercholesterolemic models. Thus, with crilvastatin, in the high fat group, the excess of hepatic cholesterol provided by LDL is diverted to bile sterol secretion by a bile salt-dependent choleresis process. This excess of cholesterol is not converted into the esterified form, the acyl coenzyme A : cholesterol acyltransferase activity remains stable and the cholesterol 7a-hydroxylase activity increases significantly. Our results, showing stimulation of bile salt secretion in hypercholesterolemic rats treated with crilvastatin, are not comparable to those obtained with fibrates such as clofibrate (the Coronary Drug Project, 1975) and gemfibrozil (Leiss et al., 1985). With these drugs, the saturation index of gallbladder bile increases. However, no such effect is observed during treatment of human IIa or IIb hypercholesterolemia with simvastatin, which decreases the cholesterol saturation index of gallbladder bile (Duane et al., 1988) in spite of the inhibitory effect on cholesterol 7a-hydroxylase (Bj6rkhem, 1986). This preclinical study suggested that Crilvastatin can be considered as a potent and promising drug for the treatment of hypercholesterolemia in our particular model. Our results showed for the first time that crilvastatin has a particular action in the liver since it stimulates cholesterol 7a-hydroxylase activity in hypercholesterolemic rats, by stimulating bile clearance of excess cholesterol from circulating LDL in the form of cholesterol and bile salts. This effect is enhanced as there is no increase in esterification of cholesterol by acyl coenzyme A : cholesterol acyltransferase in the liver
67
of these rats. These effects are especially interesting since the level of H D L remains stable. The effect of crilvastatin as well as of the other statins, on the interrelations between apoprotein synthesis and hepatic cholesterol metabolism need to be investigated further,
Acknowledgements This work was supported by INSERM and PAN MEDICA Laboratories (Grant No. 89031). We wish to thank Mrs Michelle Bonneil for secretarial support and Mr Patrick Garzino for animal care.
References Alberts~ A.W., 1988a, Discovery, biochemistry and biology of lovastatin, Am. J. Cardiol. 62, 10j. Alberts, A.W., 1988b, Lovastatine et simvastatine, Cah. Nutr. DiSt. 23, 231. Alberts, A.W., J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonber, O. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, 1980, Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent, Proc. Natl. Acad. Sci. U.S.A. 77, 3957. Amic, J., D. Lairon and J. Hauton, 1972, Technique de dosage automatique de l'orthophosphate de grande fiabilit6, Clin. Chim. Acta 40, 107. Aubert, I., J. Emmerich, Y. Charpak, B. Chanu, C. Dachet, D. Herlich, M. Besseau, J. Rouffy and B. Jacotot, 1988, Effets de la simvastatine sur les lipides et apoprot~ines (A1 et B) plasmatiques. 24 cas d'hypercholest6rol~mie primaire majeure, La Presse M~dicale 17, 901. Beaufay, H., A. Amar-Costesec, E. Feytmans, D. Thines-Sempoux, M. Wibo, M. Robi and J. Berthet, 1974, Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods, J. Cell. Biol. 61, 188. Bilheimer, D.W., S.M. Grundy, M.S. Brown and J.L. Goldstein, 1983, Mevinolin and colestipol stimulate receptor-mediated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes, Proc. Natl. Acad. Sci. U.S.A. 80, 4124. Bio-Rad assay, Chromatography electrophoresis. Immunochemistry HPLC, and Catalog K., 1985, p. 233. Bj6rkhem, I., 1986, Effects of mevinolin in rat liver: evidence for a lack of coupling between synthesis of hydroxymethylglutarylcoenzyme A (HMG CoA reductase) reductase and cholesterol 7 alpha-hydroxylase activity, Biochim. Biophys. Acta 877, 43. Brown, M.S. and J.L. Goldstein, 1983, Lipoprotein receptors in the liver, control signals for plasma cholesterol traffic, J. Clin. Invest. 72, 743. Bucolo, G. and H. David, 1973, Quantitative determination of serum triglycerides by use of enzymes, Clin. Chem. 19, 475. Chautan, M., E. Termine, G. Nalbone and H. Lafont, 1988, Acylcoenzyme A cholesterol acyltransferase assay: silica gel column separation of reaction products, Anal. Biochem. 173, 436. Diekmann, H.W., H. Sailer, A. Garbe, H. Nowak, M.R. Lovati, L. Allievi, M. Galbussera and C.R. Sirtori, 1986, Mechanism of the plasma cholesterol lowering effect of tazasubrate in rats: accelerated plasma cholesterol transport with increased liver lipoprotein receptor activity, Pharmacol. Res. Commun. 18, 203.
Domingo, N., J. Amic and J. Hauton, 1972, Dosage automatique des sels biliaires conjugu~s de la bile par la 3 alpha-hydroxystero'ide d6shydrog6nase, Clin. Chim. Acta 37, 399. Duane, W.C., D.B. Hunninghake, M.L. Freeman, P.A. Pooler, L.A. Schlasner and R.L. Gebhard, 1988, Simvastatin, a competitive inhibitor of Hydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase, lowers cholesterol saturation index of gallbladder bile, Hepatology 8, 1147. Endo, A., 1988, Chemistry, biochemistry, and pharmacology of [lydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase inhibitors, Klin. Wochenschr. 66, 42l. Endo, A., M. Kuroda and K. Tanzawak, 1976, Competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by ML 236 A and ML 236 B, fungal metabolites hypocholesterolemic activity, FEBS Lett. 72, 323. Esnault, C., H. Lafont, F. Chanussot, M. Chautan, J. ttauton and C. Laruelle, 1988, Inhibition of hepatic HMG-CoA reductase activity by two new hypocholesterolemic drugs, in: Liver Cells and Drugs. ed. A. Guillouzo, (Colloque INSERM/John Libbey Eurotext Ltd.) 164, p. 99. Esnault-Dupuy, C., F. Chanussot, H. Lafont, C. Chabert and J. Hauton, 1987, The relationship between HDL-LDL-, liposomesfree cholesterol, biliary cholesterol and bile salts in the rat, Biochimie 69, 45. Halloran, L.G., C.C. Schwartz, Z.R. Vlahcevic, R.M. Nisman and L. Swell, and 1978, Evidence for high-density lipoprotein free cholesterol as the primary precursor for bile-acid synthesis in man, Surgery 84, 1. Hatch, F.T. and R.S. Less, 1968, Practical methods for plasma lipoprotein K analysis, Adv. Lipid Res. 6, 1. Junker, L.H. and J.A. Story, 1985, An improved assay for cholesterol 7 alpha-hydroxylase activity using phospholipid liposome solubilized substrate~ Lipids 20, 712. Kempen, H.J.M., J. De Lange, M.P.M. Vos-Van Holstein, P. Van Wachem, R., Havinga and R.J. Vonk, 1984, Effect of ML-236B (compactin) on biliary excretion of bile salts and lipids, and on bile flow, in the rat, Biochim. Biophys. Acta 794, 435. Kessler, G., S. Morgenstern, L. Snyder and R. Varady, 1975, lmproved point assays for ALT and AST in serum using the Technicon SMAC high speed computer controlled, biochemical analyser to eliminate the common errors found in enzyme analysis, 9th international Congress for Clinical Chemistry, Toronto. Leiss, O., K. Von Bergmann, A. Gnasso and J. Augustin, 1985, Effect of gemfibrozil on biliary lipid metabolism in normolipemic subjects, Metabolism 34, 74. Leon, P. and R.O. Stasiw, 1976, Performance of automated enzymatic cholesterol on SMA 12/60 and auto analyser II instruments, Clin. Chem. 22, 1220. Lie, R.F., J.M. Smitz, K.J. Pierre and N. Gochman, 1976, Cholesterol oxidase-based determination oby continuous flow analysis of total and free cholesterol in serum, Clin. Chem. 22, 1627. Mitchell, J.C., B.G. Stone, G.M. Logan and C. Duane, 1991, Role of cholesterol synthesis in regulation of bile acid synthesis and biliary cholesterol secretion in humans, J. Lipid Res. 32, 1143. Morgenstern, S., G. Kessler, J. Auerbach, R.V. Flor and B. Klein, 1965, An automated p-nitrophenylphosphate serum alkaline phosphatase procedure for the autoanalyser, Clin. Chem. 11,876. Ribeiro, A., M. Mangeney, C. Loriette, G. Thomas, D. Pepin, B. Janvier, J. Chambaz and G. Bereziat, 1991, Effect of simvastatin on the synthesis and secretion of lipoproteins in relation to the metabolism of cholesterol in cultured hepatocytes, Biochim. Biophys. Acta 1086, 279. Schulze, E., J. Obermaier, C.A. Seyfried, H.W. Diekmann and H. Nowak, 1986, Pharmacological profile of tazasubrate. A new cholesterol-lowering agents, Atherosclerosis 59, 137. Skeggs, L.T. and H. Hochstrasser, 1964, Multiple automatic sequential analysis, Clin. Chem. 10, 918.
68 Sottocosa, G.L., B. Kuylenstierna, L. Ernster and A. Bergstrand, 1967, An electron transport system associated with the outer membrane of liver mitochondria, J. Cell. Biol. 32, 415. Stahlberg, D., B. Angelin and K. Einarsson, 1989, Effects of treatmerit with clofibrate, bezafibrate and ciprofibrate on the metabolism of cholesterol in rat liver microsomes, J. Lipid Res. 30, 953.
The Coronary Drug project Research Group, Coronary Drug Project, 1975, Clofibrate and niacin in coronary heart disease, J. Am. Med. Assoc. 231,360. Yamauchi, S., W.G. Linscheer and D.H. Beach, 1991, Increase in serum and K bile cholesterol and Hydroxymethylglutaryl-coenzyme A (HMG CoA reductase) reductase by Iovastatin in rats, Am. J. Physiol. 23, G. 625.