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Effects of CS-514 on plasma lipids and lipoprotein composition in hypercholesterolemic subjects
Atherosclerosis, 71 (1988) 95-101 Elsevier Scientific Publishers Ireland,
95 Ltd.
ATH 04124
Effects of CS-514 on plasma lipids and lipoprotein composition in hypercholesterolemic subjects Gen Yoshino I, Tsutomu Kazumi 2, Masahide Iwai I, Ippei Iwatani I, Kohji Matsuba I, Toshio Kasama 2, Masayuki Matsushita ‘, Makoto Otsuki ’ and Shigeaki Baba ’ ’Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ky Kobe 650 (Japan), and 2 Faculty of Medicine, Hyogo Medical Center for Adults, Kitaohji-cho, Akashi 673 (Japan) (Received 16 September, 1987) (Revised, received 23 November, 1987) (Accepted 25 November, 1987)
The effect of CS-514 (eptastatin, Sankyo Co., Tokyo), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was investigated in 47 patients with hypercholesterolemia (WHO type IIa: 27, IIb: 20). Ten or 20 mg of CS-514 was administered daily for 3 months. In both types of patient, total cholesterol and phospholipid levels were significantly reduced by CS-514. The triglyceride, cholesterol and phospholipid content of low density lipoprotein (LDL) and the plasma levels of apolipoprotein B were also decreased in both groups. In contrast, total triglyceride, very low density lipoprotein (VLDL)-triglyceride and apolipoprotein C-II were decreased only in type IIb subjects. Also the levels of high density lipoprotein (HDL)-cholesterol and apolipoproteins A-I and A-II were increased by CS-514 in IIb but not in IIa patients. In both groups, no change occurred in either the cholesterol/ triglyceride or phospholipid ratio in any lipoprotein fraction, nor in the ratio of HDL-cholesterol to apolipoprotein A-I or A-II, respectively. Therefore, CS-514 suppresses plasma levels of cholesterol in hypercholesterolemic patients without modifying lipoprotein composition. Moreover, this drug has different effects on the levels of plasma triglyceride and HDL-cholesterol of type IIa and IIb patients.
Key words: CS-514; Cholesterol; Triglyceride; HDL-cholesterol; Hypercholesterolemia
Introduction
An increased plasma level of cholesterol is a primary risk factor for atherosclerotic heart disCorrespondence to: Dr. G. Yoshino, 2nd Department of Internal Medicine, Kobe University School of Medicine, Chuoku, Kobe 650, Japan. 0021-9150/88/$03.50
0 1988 Elsevier Scientific
Publishers
Ireland,
Lipoprotein
composition;
ease [1,2]. Patients with familial hypercholesterolemia are especially at risk of premature atherosclerosis [3,4]. Because of an inborn error in the function of cellular LDL receptors [5,6] these heterozygous subjects have plasma levels of cholesterol 2-3 times higher than normal. Various kinds of treatment have been used to lower plasma levels of cholesterol in these patients. However, Ltd.
96
since cholesterol-lowering diets have only a limited effect, the development of an effective cholesterollowering drug is necessary. A promising drug for this condition is CS-514, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. CS-514 is a derivative of ML-236B which reduces plasma LDLcholesterol levels in experimental animals [7,8]. A preliminary study suggested that CS-514 effectively lowers plasma levels of cholesterol in healthy normocholesterolemic volunteers [9]. Recently, we reported the clinical efficacy and safety of this drug [lO,ll]. According to other recent reports [12,13], CS-514 exerts a dose-dependent hypocholesterolemic effect on non-familial and heterozygous familial hypercholesterolemia. However, for further clinical use it is important to know whether this inhibitor of endogenous cholesterol biosynthesis can suppress plasma lipid levels without modifying lipoprotein composition. Therefore, we have examined the lipid-to-lipid ratio in each lipoprotein fraction and the lipoprotein-lipid-to-apolipoprotein ratio during CS-514 treatment. Subjects and methods Forty-seven subjects (20 men and 27 women) with hypercholesterolemia (WHO type IIa 27, IIb 20) aged 28-68 years, gave informed consent and were included in this study (Table 1). Five type IIa patients were diagnosed as heterozygous familial hypercholesterolemia based on family history and Achilles tendon xanthoma. Eleven patients were diabetics (5 type IIa, 6 type IIb and 5
on sulfonylurea, 6 on diet) under excellent control for more than 6 months before this study. None of the patients had clinical or laboratory evidence of renal, hepatic or thyroid disorders. After stabilizing plasma lipid levels by at least 1 month of lipid-lowering diet, 10 or 20 mg per day of CS-514 was administered. Blood samples were obtained in the morning after a 14-h overnight fast. Determination of complete blood count, blood urea nitrogen (BUN), serum transaminases (SGOT and SGPT), serum y-glutaryltranspeptidase, serum bilirubin (direct and indirect), blood glucose (FBG), serum uric acid (UA), serum creatinine (Cr) and creatinine phosphokinase were performed using standard autoanalyser methods. Separation of plasma lipoproteins was carried out by ultracentrifugation [14]. Cholesterol, phospholipid and triglyceride in plasma and in each lipoprotein fraction were measured enzymatically. Apolipoproteins A-I, A-II, B, C-II, C-III and E in plasma were measured by a single radial immunodiffusion method [15] using kits commercially available in Japan (Daiichi Kagaku Co., Tokyo). The assay procedure was the same as that of Ishibashi et al. [16] and Mabuchi et al. [13]. Compliance with diet and medications was assessed by oral questioning. Statistical analysis was performed using a paired t-test. Results Seven type IIa subjects received 20 mg daily of CS-514 while the other subjects received 10 mg. We found a similar dose-dependent change in plasma cholesterol to that of previous reports
TABLE 1 CLINICAL
DATA IN SUBJECTS
WITH HYPERCHOLESTEROLEMIA
Data are expressed as mean f SE. FH: familial hypercholesterolemia. n = number of subjects. WHO
diabetes
FH
n
type
Age (yrs)
mg/dl Plasma cholesterol
Plasma triglyceride
HDLcholesterol
IIa
+
+ -
16 5 6
52zt 5 59* 3 47* 9
214 f 10 353f 6 279fll
91+42 133 f 28 106k21
61k 4 51+ 6 58kll
IIb
+
-
15 5
51+10 48*13
308 k 18 216 f 25
279 f 38 349 f 95
45* 45*
3 4
97 Phospholipld
Triglyceride
Cholesterol
(w/dQ)l
(nrS/dQ)l
(w/dQ)l
300-
300-
I
%_ -.\_
II b
(Month) Fig. 1. Effects of G-514 on plasma cholesterol (panel A), triglyceride (panel B) and phospholipid (panel C) levels of WHO type IIa (solid line) and IIb (broken line) hyperlipidemic subjects. Vertical bars represent mean + SE. * Significantly different from 0 month value (P < 0.05 or less).
[12,13]. Since the focus of this study was on the effect of G-514 on lipoprotein composition, the results for the 10 or 20 mg groups were combined. Total cholesterol and phospholipid levels in the plasma of both type IIa and IIb patients were decreased significantly after 3 months of CS-514 (Fig. 1). A significant reduction in total plasma triglyceride was found in type IIb patients but not in type IIa (Fig. 1, panel B). These results did not differ even if the patients were subdivided as shown in Table 1. Within the VLDL fraction of Cholesterol
type IIb patients, the levels of triglyceride and phospholipid were decreased significantly (Fig. 2, panels B and C). The 3 lipids in the VLDL of type IIa patients showed no change (Fig. 2). In the LDL fraction of both types of patients, CS-514 significantly reduced the levels of cholesterol, triglyceride and phospholipid (Fig. 3). In the HDL fraction, phospholipid and cholesterol levels were significantly increased in type IIb patients by CS514 (Fig. 4). In these subjects there was a concomitant significant increase in apolipoproteins A-I Phospholtpld
Triglyceride
I
(Month)
Fig. 2. Effects of CS-514 on the 3 lipid levels in VLDL
fraction of type IIa and type IIb hyperlipidemic legends of symbols.
subjects. See Fig. 1 for
Cholesterol
Triglyceride
Phospholipid
100
A)
Cl
01 0
3
1
6
0
3
(Month)
Fig. 3. Effects of CS-514 on the 3 lipid levels in LDL fraction
of type IIa and type IIb hyperlipidemic
subjects.
See Fig. 1 for legends
of symbols.
Phospholipid
loo-
l_---
.%
r
50 -
loi8) 1
I
0
3
tL C) I 0
I 3
(Month)
Fig. 4. Effects of CS-514 on the 3 lipid levels in HDL fraction
A- I
of type IIa and type IIb hyperlipidemic of symbols.
subjects.
See Fig. 1 for legends
B
A-II
(ma/d4
%.
,..4 .x
150-
-* . ..***. i
150-
f-*--.*-
I
\
lOO-
loo-
t A?
,
0
3
L 0
3
(Month)
Fig. 5. Effects
of CS-514
on plasma
apolipoproteins A-I (panel A), A-II (panel B) and B (panel hyperlipidemic subjects. See Fig. 1 for legends of symbols.
C) of type
IIa and type
IIb
99
c-m
E
-. L -.._ %_ %_
-3 .I.
B)
Cl
I
1
I
,
0
3
0
3
(Month) Fig. 6. Effects of CS-514 on plasma apolipoproteins C-II (panel A), C-III (panel B) and E (panel C) of type IIa and type IIb hyperlipidemic subjects. See Fig. 1 for legends of symbols.
and A-II (Fig. 5). In contrast, apolipoprotein B significantly decreased in both groups (Fig. 5, panel C). Apolipoprotein C-II in type IIb and apolipoprotein E in type IIa were also significantly decreased by CS-514 (Fig. 6). There was no significant change in the lipid-tolipid ratio (cholesterol-to-triglyceride or phospholipid) in any lipoprotein fraction. Interestingly, the cholesterol/ triglyceride ratio in the HDL fraction of type IIb subjects did increase but not significantly. The ratio of HDL-cholesterol/ apolipoprotein A-I or apolipoprotein A-II showed no change in either group. The drug was well tolerated by the patients. Subjective side-effects were not mentioned. No serious adverse effects were noted on hematopoietic, hepatic or renal function. A slight elevation of SGOT and SGBT was observed in one case but enzyme levels returned to normal immediately after discontinuation of the drug. Discussion The HMG-CoA reductase inhibitors exert their effects on plasma cholesterol levels by competitive inhibition of this rate-limiting enzyme in cholesterol biosynthesis [17]. The similarity of their structure to HMG-CoA allows them to bind to the enzyme’s active site and inhibit enzyme activity [18]. The subsequent reduction in intracellular
cholesterol leads to a compensatory induction of LDL receptors [19] and an increase in the fractional catabolic rate of LDL [20]. The significant fall in plasma triglyceride in type IIb subjects after CS-514 treatment is important. The drug may enhance removal of precursors of LDL [21] that can be bound by the LDL receptors. Alternatively, the drug may reduce VLDL production by the liver. Grundy and Vega [22] have reported a decrease in LDL synthesis after the administration of mevinolin, which might be secondary to a decrease in VLDL secretion. In support of this, other preliminary experiments have revealed a significant decrease in the triglyceride secretion rate of CS-514 treated normal rats (unpublished data). The reduction of triglyceride and phospholipid in VLDL and of plasma apolipoprotein C-II after treatment support either possibility. The absence of a reduction in plasma triglyceride levels in type IIa after CS-514 agrees with previous reports on heterozygous familial hypercholesterolemia [13]. The production and removal of VLDL are at basal levels in these subjects. Therefore, CS-514 may fail to exert an effect on VLDL metabolism in type IIa individuals. The effects of HMG-CoA reductase inhibitors on HDL-cholesterol levels are controversial. Our study clearly demonstrates that the effect of this drug differs when administered to type IIa or to
100 IIb patients. Not only HDL-cholesterol but also apolipoprotein A-I and A-II were elevated in type IIb subjects after CS-514. These changes were absent in type IIa subjects. The mechanism for the elevation of the HDL fraction in type IIb subjects is not known. However, it is possible that the production of new HDL particles was stimulated by CS-514 enhancing the catabolism of VLDL [21,23]. In support of this suggestion, a significant decrease in VLDL-triglyceride was also seen in the type IIb but not type IIa subjects. The changes induced in the HDL fraction of the type IIb patients by CS-514 indicate a clinical efficacy for this drug. Recent reports [24,25] demonstrate that a long-term increase in HDL-cholesterol is independently associated with a reduced coronary heart disease morbidity and mortality and an inhibited progress of coronary artery atherosclerosis. Furthermore, the Leiden [26] and Helsinki [27] Heart Studies uniformly suggest that an elevation of HDL-cholesterol may reduce progression of coronary atherosclerosis. Our finding of an almost complete absence of changes in the lipid/lipid ratio in each lipoprotein fraction, despite a specific inhibition of cholesterol biosynthesis by this drug [28] is of great interest. Since CS-514 suppresses only hepatic and intesti nal cholesterol biosynthesis [28], it might be expected that the patient’s lipoproteins would become cholesterol-depleted. However, only the cholesterol/ triglyceride ratio in the LDL fraction of type IIa subjects tended to decrease and this was not significant. The mechanism by which an inhibitor of cholesterol biosynthesis suppresses plasma lipid levels without changing the lipid composition of lipoprotein particles is not known. One possibility is that cellular cholesterol biosynthesis has a key role in lipoprotein production and secretion. Except for discoidal HDL, the structure of plasma lipoproteins is spherical. One model for lipoprotein structure [29] indicates that they have triglyceride and esterified cholesterol as core components with apolipoprotein C-II on the surface. Therefore, Ishibashi et al. [13] reported that the cubic root of plasma triglyceride ( 3fi) er square root of plasma apolipoprotein C-II ( ?--. apoC-II ) is constant even in severe hypertriglyceridemia. Our previous observation [30] of a similarity in the
regression lines between ‘fi and ,/m of 40 hyperlipidemic subjects before and after treatment with CS-514 suggests that lipoprotein structure was not affected by this drug. Together with our previous observation [30] we conclude here that CS-514 suppresses plasma cholesterol levels in hypercholesterolemic subjects without modifying lipoprotein composition or lipoprotein structure. In type IIb subjects, the drug also significantly suppresses plasma triglyceride level and elevates apolipoprotein A-I and A-II as well as HDL-cholesterol. It appears therefore that this drug is clinically useful for both type IIa and type IIb hyperlipidemic patients. References Kannel, W.B., Castelli, W.D. and McNamara, P.M., Serum cholesterol lipoproteins, and risk of coronary heart disease, The Framingham Study, Ann. Intern. Med., 74 (1971) 1. Gotto, Jr., A.M., Status report - plasma lipids, lipoproteins and coronary heart disease, Atheroscler. Rev., 4 (1979) 17. Slack, J., Risks of ischaemic heart disease in familial hyperlipoproteinaemia states, Lancet, ii (1969) 1380. Goldstein, J.L. and Brown, MS., Familial hypercholesterolemia. In: Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., (Eds.), The Metabolic Basis of Inherited Disease, 5th edn., McGraw-Hill, New York, 1983, p. 672. Goldstein, J.L. and Brown, M.S., Familial hypercholesterolemia: identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol, Proc. Natl. Acad. Sci. USA, 70 (1973) 2804. Goldstein, J.L., Brown, M.S. and Stone, N.J., Genetics of the LDL receptor. Evidence that the mutations affecting binding and internalization are allelic, Cell, 12 (1977) 629. Endo, A., Kuroda, M. and Tsujita, Y., ML-236A, ML-236B and ML-236C, new inhibitors of cholesterogenesis produced by penicillium citrinum, J. Antibiot., 29 (1976) 1346. Endo, A., Tsujita, Y., Kuroda, M. and Tanzawa, K., Inhibition of cholesterol synthesis in vitro and in vivo by ML-236A and ML-236B, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Eur. J. Biochem., 37 (1978) 313. 9 Nakaya, N., Homma, Y., Tamachi, H. and Goto, Y., The effect of CS514, an inhibitor of HMG-CoA reductase, on serum lipids in healthy volunteers, Atherosclerosis, 61(1986) 125. 10 Yoshino, G., Kazumi, T., Uenoyama, R., Inui, A., Kasama, T., Iwatani, I., Iwai, M., Yokono, K., Otsuki, M. and Baba, S., Probucol versus eptastatin in hypercholesterolemic diabetics, Lancet, i (1986) 740. 11 Kazumi, T., Yoshino, G., Kasama, T., Iwatani, I., Iwai, M., Morita, S. and Baba, S., Effect of CS-514, a new inhibitor
101 of HMG-CoA reductase, on plasma lipids, lipoproteins and apoproteins in patients with primary hypercholesterolemia, Horm. Metab. Res., 18 (1986) 654. 12 Nakaya, N., Homma, Y., Tamachi, H., Shigematsu, H., Hata, Y. and Goto, Y., The effect of CS-514 on serum lipids and apolipoproteins in hypercholesterolemic subjects, 13
14
15
16
17
18
19
20
21
J. Am. Med. Assoc. 257 (1987) 3088. Mabuchi, H., Kamon, N., Fujita, H., Michishita, I., Takeda, M., Kajinami, K., Itoh, H., Wakasugi, T. and Takeda, R., Effects of CS-514 on serum lipoprotein lipid and apolipoprotein levels in patients with familial hypercholesterolemia, Metabolism, 36 (1987) 475. Havel, R.J., Eder, H.A. and Bragdon, J.H., Distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. Sakai, Y., Itakura, K., Kanada, T., Ebata, N., Suga, K., Aikawa, H., Nakamura, K. and Sata, T., Quantitation of apolipoprotein A-I in pooled human serum by single radial immunodiffusion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Anal. Biochem., 137 (1984) 1. Ishibashi, S., Murase, T., Takahashi, K., Mori, N., Kawakami, M., and Takaku, F., Plasma apolipoprotein CII levels in hypertriglyceridemia, Metabolism, 35 (1986) 781. Brown, M.S. and Goldstein, J.L., Multivalent feedback regulation of HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth, J. Lipid Res., 21 (1980) 505. Endo, A., Biological and pharmacological activity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Trends B&hem. Sci., 6 (1981) 10. Kovanen, P.T., Bilheimer, D.W., Goldstein, J.L., Jaramillo, J.J. and Brown, M.S., Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog, Proc. Natl. Acad. Sci. USA, 78 (1987) 1194. Bilheimer, D.W., Grundy, S.M., Brown, MS. and Goldstein, J.L., Mevinolin and colestipol stimulate receptormedicated clearance of low density lipoprotein from plasma in familial hypercholesterolemia heterozygotes, Proc. Natl. Acad. Sci. USA, 80 (1983) 4124. Goldstein, J.L., Kita, T. and Brown, MS., Defective lipoprotein receptors and atherosclerosis. Lessons from an animal counterpart of familial hypercholesterolemia, N. Engl. J. Med., 309 (1983) 288.
22 Grundy,
23 24
25
26
27
28
29
30
M.S. and Vega, L.L., Influence
of mevinolin
on
metabolism of low density lipoproteins in primary moderate hypercholesterolemia, J. Lipid Res., 26 (1985) 1464. Eisenberg, S., High density lipoprotein metabolism, J. Lipid Res., 25 (1984) 1017. Lipid Research Clinics Program, The Lipid Clinics Coronary Primary Prevention Trial results. Part 1. Reduction in incidence of coronary heart disease. J. Am. Med. Assoc., 251 (1984) 351. Levy, RI., Brensike, J.F., Epstein, S.E., Kelsey, S.F., Passamani, E.R., Richardson, J.M., Loh, I.K., Stone, N.J., Aldrick, R.F., Battaglini, J.W., Moriarty, D.J., Fisher, M.L., Friedman, L., Friedewald, W. and Detre, K.M., The influence of changes in lipid values induced by cholestyramine and diet on progression of coronary artery disease Results of the NHLBI Type II Coronary Intervention Study, Circulation, 69 (1984) 325. Amtzenius, A.C., Kromhout, D., Barth, J.D., Reiber, J.H.C., Bruschke, A.V.G., Buis B., Van Gent, C.M., KempenVoogd, N., Strikwerda, S. and Van der Velde, E.A., Diet, lipoproteins, and the progression of coronary atherosclerosis, N. Engl. J. Med., 312 (1985) 805. N&kill, E.A., Viikinkoski, P., Valle, M. and Frick, M.H., Prevention of progression of coronary atherosclerosis by treatment of hyperlipidemia - A seven year prospective angiographic study, Br. Med. J., 289 (1984) 220. Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai. M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y. and Fujii, S., CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaql coenzyme A reductase: tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species, Biochim. Biophys. Acta., 877 (1986) 50. Morrisett, J.D., Jackson, R.J. and Gotto, Jr., A.M., Lipid protein interactions in the plasma lipoproteins, Biochim. Biophys. Acta, 472 (1977) 93. Yoshino, G., Kazumi, T., Iwai, M., Kasama, T., Iwatani, I., Matsuba, K., Inui, A., Uenoyama, R., Yokono, K., Otsuki, M. and Baba, S., CS-514 suppresses plasma triglyceride in hypertriglyceridemic subjects without modifying a lipoprotein structure model, Horm. Metab. Res., 19 (1987) 513.
95 Ltd.
ATH 04124
Effects of CS-514 on plasma lipids and lipoprotein composition in hypercholesterolemic subjects Gen Yoshino I, Tsutomu Kazumi 2, Masahide Iwai I, Ippei Iwatani I, Kohji Matsuba I, Toshio Kasama 2, Masayuki Matsushita ‘, Makoto Otsuki ’ and Shigeaki Baba ’ ’Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ky Kobe 650 (Japan), and 2 Faculty of Medicine, Hyogo Medical Center for Adults, Kitaohji-cho, Akashi 673 (Japan) (Received 16 September, 1987) (Revised, received 23 November, 1987) (Accepted 25 November, 1987)
The effect of CS-514 (eptastatin, Sankyo Co., Tokyo), a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was investigated in 47 patients with hypercholesterolemia (WHO type IIa: 27, IIb: 20). Ten or 20 mg of CS-514 was administered daily for 3 months. In both types of patient, total cholesterol and phospholipid levels were significantly reduced by CS-514. The triglyceride, cholesterol and phospholipid content of low density lipoprotein (LDL) and the plasma levels of apolipoprotein B were also decreased in both groups. In contrast, total triglyceride, very low density lipoprotein (VLDL)-triglyceride and apolipoprotein C-II were decreased only in type IIb subjects. Also the levels of high density lipoprotein (HDL)-cholesterol and apolipoproteins A-I and A-II were increased by CS-514 in IIb but not in IIa patients. In both groups, no change occurred in either the cholesterol/ triglyceride or phospholipid ratio in any lipoprotein fraction, nor in the ratio of HDL-cholesterol to apolipoprotein A-I or A-II, respectively. Therefore, CS-514 suppresses plasma levels of cholesterol in hypercholesterolemic patients without modifying lipoprotein composition. Moreover, this drug has different effects on the levels of plasma triglyceride and HDL-cholesterol of type IIa and IIb patients.
Key words: CS-514; Cholesterol; Triglyceride; HDL-cholesterol; Hypercholesterolemia
Introduction
An increased plasma level of cholesterol is a primary risk factor for atherosclerotic heart disCorrespondence to: Dr. G. Yoshino, 2nd Department of Internal Medicine, Kobe University School of Medicine, Chuoku, Kobe 650, Japan. 0021-9150/88/$03.50
0 1988 Elsevier Scientific
Publishers
Ireland,
Lipoprotein
composition;
ease [1,2]. Patients with familial hypercholesterolemia are especially at risk of premature atherosclerosis [3,4]. Because of an inborn error in the function of cellular LDL receptors [5,6] these heterozygous subjects have plasma levels of cholesterol 2-3 times higher than normal. Various kinds of treatment have been used to lower plasma levels of cholesterol in these patients. However, Ltd.
96
since cholesterol-lowering diets have only a limited effect, the development of an effective cholesterollowering drug is necessary. A promising drug for this condition is CS-514, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. CS-514 is a derivative of ML-236B which reduces plasma LDLcholesterol levels in experimental animals [7,8]. A preliminary study suggested that CS-514 effectively lowers plasma levels of cholesterol in healthy normocholesterolemic volunteers [9]. Recently, we reported the clinical efficacy and safety of this drug [lO,ll]. According to other recent reports [12,13], CS-514 exerts a dose-dependent hypocholesterolemic effect on non-familial and heterozygous familial hypercholesterolemia. However, for further clinical use it is important to know whether this inhibitor of endogenous cholesterol biosynthesis can suppress plasma lipid levels without modifying lipoprotein composition. Therefore, we have examined the lipid-to-lipid ratio in each lipoprotein fraction and the lipoprotein-lipid-to-apolipoprotein ratio during CS-514 treatment. Subjects and methods Forty-seven subjects (20 men and 27 women) with hypercholesterolemia (WHO type IIa 27, IIb 20) aged 28-68 years, gave informed consent and were included in this study (Table 1). Five type IIa patients were diagnosed as heterozygous familial hypercholesterolemia based on family history and Achilles tendon xanthoma. Eleven patients were diabetics (5 type IIa, 6 type IIb and 5
on sulfonylurea, 6 on diet) under excellent control for more than 6 months before this study. None of the patients had clinical or laboratory evidence of renal, hepatic or thyroid disorders. After stabilizing plasma lipid levels by at least 1 month of lipid-lowering diet, 10 or 20 mg per day of CS-514 was administered. Blood samples were obtained in the morning after a 14-h overnight fast. Determination of complete blood count, blood urea nitrogen (BUN), serum transaminases (SGOT and SGPT), serum y-glutaryltranspeptidase, serum bilirubin (direct and indirect), blood glucose (FBG), serum uric acid (UA), serum creatinine (Cr) and creatinine phosphokinase were performed using standard autoanalyser methods. Separation of plasma lipoproteins was carried out by ultracentrifugation [14]. Cholesterol, phospholipid and triglyceride in plasma and in each lipoprotein fraction were measured enzymatically. Apolipoproteins A-I, A-II, B, C-II, C-III and E in plasma were measured by a single radial immunodiffusion method [15] using kits commercially available in Japan (Daiichi Kagaku Co., Tokyo). The assay procedure was the same as that of Ishibashi et al. [16] and Mabuchi et al. [13]. Compliance with diet and medications was assessed by oral questioning. Statistical analysis was performed using a paired t-test. Results Seven type IIa subjects received 20 mg daily of CS-514 while the other subjects received 10 mg. We found a similar dose-dependent change in plasma cholesterol to that of previous reports
TABLE 1 CLINICAL
DATA IN SUBJECTS
WITH HYPERCHOLESTEROLEMIA
Data are expressed as mean f SE. FH: familial hypercholesterolemia. n = number of subjects. WHO
diabetes
FH
n
type
Age (yrs)
mg/dl Plasma cholesterol
Plasma triglyceride
HDLcholesterol
IIa
+
+ -
16 5 6
52zt 5 59* 3 47* 9
214 f 10 353f 6 279fll
91+42 133 f 28 106k21
61k 4 51+ 6 58kll
IIb
+
-
15 5
51+10 48*13
308 k 18 216 f 25
279 f 38 349 f 95
45* 45*
3 4
97 Phospholipld
Triglyceride
Cholesterol
(w/dQ)l
(nrS/dQ)l
(w/dQ)l
300-
300-
I
%_ -.\_
II b
(Month) Fig. 1. Effects of G-514 on plasma cholesterol (panel A), triglyceride (panel B) and phospholipid (panel C) levels of WHO type IIa (solid line) and IIb (broken line) hyperlipidemic subjects. Vertical bars represent mean + SE. * Significantly different from 0 month value (P < 0.05 or less).
[12,13]. Since the focus of this study was on the effect of G-514 on lipoprotein composition, the results for the 10 or 20 mg groups were combined. Total cholesterol and phospholipid levels in the plasma of both type IIa and IIb patients were decreased significantly after 3 months of CS-514 (Fig. 1). A significant reduction in total plasma triglyceride was found in type IIb patients but not in type IIa (Fig. 1, panel B). These results did not differ even if the patients were subdivided as shown in Table 1. Within the VLDL fraction of Cholesterol
type IIb patients, the levels of triglyceride and phospholipid were decreased significantly (Fig. 2, panels B and C). The 3 lipids in the VLDL of type IIa patients showed no change (Fig. 2). In the LDL fraction of both types of patients, CS-514 significantly reduced the levels of cholesterol, triglyceride and phospholipid (Fig. 3). In the HDL fraction, phospholipid and cholesterol levels were significantly increased in type IIb patients by CS514 (Fig. 4). In these subjects there was a concomitant significant increase in apolipoproteins A-I Phospholtpld
Triglyceride
I
(Month)
Fig. 2. Effects of CS-514 on the 3 lipid levels in VLDL
fraction of type IIa and type IIb hyperlipidemic legends of symbols.
subjects. See Fig. 1 for
Cholesterol
Triglyceride
Phospholipid
100
A)
Cl
01 0
3
1
6
0
3
(Month)
Fig. 3. Effects of CS-514 on the 3 lipid levels in LDL fraction
of type IIa and type IIb hyperlipidemic
subjects.
See Fig. 1 for legends
of symbols.
Phospholipid
loo-
l_---
.%
r
50 -
loi8) 1
I
0
3
tL C) I 0
I 3
(Month)
Fig. 4. Effects of CS-514 on the 3 lipid levels in HDL fraction
A- I
of type IIa and type IIb hyperlipidemic of symbols.
subjects.
See Fig. 1 for legends
B
A-II
(ma/d4
%.
,..4 .x
150-
-* . ..***. i
150-
f-*--.*-
I
\
lOO-
loo-
t A?
,
0
3
L 0
3
(Month)
Fig. 5. Effects
of CS-514
on plasma
apolipoproteins A-I (panel A), A-II (panel B) and B (panel hyperlipidemic subjects. See Fig. 1 for legends of symbols.
C) of type
IIa and type
IIb
99
c-m
E
-. L -.._ %_ %_
-3 .I.
B)
Cl
I
1
I
,
0
3
0
3
(Month) Fig. 6. Effects of CS-514 on plasma apolipoproteins C-II (panel A), C-III (panel B) and E (panel C) of type IIa and type IIb hyperlipidemic subjects. See Fig. 1 for legends of symbols.
and A-II (Fig. 5). In contrast, apolipoprotein B significantly decreased in both groups (Fig. 5, panel C). Apolipoprotein C-II in type IIb and apolipoprotein E in type IIa were also significantly decreased by CS-514 (Fig. 6). There was no significant change in the lipid-tolipid ratio (cholesterol-to-triglyceride or phospholipid) in any lipoprotein fraction. Interestingly, the cholesterol/ triglyceride ratio in the HDL fraction of type IIb subjects did increase but not significantly. The ratio of HDL-cholesterol/ apolipoprotein A-I or apolipoprotein A-II showed no change in either group. The drug was well tolerated by the patients. Subjective side-effects were not mentioned. No serious adverse effects were noted on hematopoietic, hepatic or renal function. A slight elevation of SGOT and SGBT was observed in one case but enzyme levels returned to normal immediately after discontinuation of the drug. Discussion The HMG-CoA reductase inhibitors exert their effects on plasma cholesterol levels by competitive inhibition of this rate-limiting enzyme in cholesterol biosynthesis [17]. The similarity of their structure to HMG-CoA allows them to bind to the enzyme’s active site and inhibit enzyme activity [18]. The subsequent reduction in intracellular
cholesterol leads to a compensatory induction of LDL receptors [19] and an increase in the fractional catabolic rate of LDL [20]. The significant fall in plasma triglyceride in type IIb subjects after CS-514 treatment is important. The drug may enhance removal of precursors of LDL [21] that can be bound by the LDL receptors. Alternatively, the drug may reduce VLDL production by the liver. Grundy and Vega [22] have reported a decrease in LDL synthesis after the administration of mevinolin, which might be secondary to a decrease in VLDL secretion. In support of this, other preliminary experiments have revealed a significant decrease in the triglyceride secretion rate of CS-514 treated normal rats (unpublished data). The reduction of triglyceride and phospholipid in VLDL and of plasma apolipoprotein C-II after treatment support either possibility. The absence of a reduction in plasma triglyceride levels in type IIa after CS-514 agrees with previous reports on heterozygous familial hypercholesterolemia [13]. The production and removal of VLDL are at basal levels in these subjects. Therefore, CS-514 may fail to exert an effect on VLDL metabolism in type IIa individuals. The effects of HMG-CoA reductase inhibitors on HDL-cholesterol levels are controversial. Our study clearly demonstrates that the effect of this drug differs when administered to type IIa or to
100 IIb patients. Not only HDL-cholesterol but also apolipoprotein A-I and A-II were elevated in type IIb subjects after CS-514. These changes were absent in type IIa subjects. The mechanism for the elevation of the HDL fraction in type IIb subjects is not known. However, it is possible that the production of new HDL particles was stimulated by CS-514 enhancing the catabolism of VLDL [21,23]. In support of this suggestion, a significant decrease in VLDL-triglyceride was also seen in the type IIb but not type IIa subjects. The changes induced in the HDL fraction of the type IIb patients by CS-514 indicate a clinical efficacy for this drug. Recent reports [24,25] demonstrate that a long-term increase in HDL-cholesterol is independently associated with a reduced coronary heart disease morbidity and mortality and an inhibited progress of coronary artery atherosclerosis. Furthermore, the Leiden [26] and Helsinki [27] Heart Studies uniformly suggest that an elevation of HDL-cholesterol may reduce progression of coronary atherosclerosis. Our finding of an almost complete absence of changes in the lipid/lipid ratio in each lipoprotein fraction, despite a specific inhibition of cholesterol biosynthesis by this drug [28] is of great interest. Since CS-514 suppresses only hepatic and intesti nal cholesterol biosynthesis [28], it might be expected that the patient’s lipoproteins would become cholesterol-depleted. However, only the cholesterol/ triglyceride ratio in the LDL fraction of type IIa subjects tended to decrease and this was not significant. The mechanism by which an inhibitor of cholesterol biosynthesis suppresses plasma lipid levels without changing the lipid composition of lipoprotein particles is not known. One possibility is that cellular cholesterol biosynthesis has a key role in lipoprotein production and secretion. Except for discoidal HDL, the structure of plasma lipoproteins is spherical. One model for lipoprotein structure [29] indicates that they have triglyceride and esterified cholesterol as core components with apolipoprotein C-II on the surface. Therefore, Ishibashi et al. [13] reported that the cubic root of plasma triglyceride ( 3fi) er square root of plasma apolipoprotein C-II ( ?--. apoC-II ) is constant even in severe hypertriglyceridemia. Our previous observation [30] of a similarity in the
regression lines between ‘fi and ,/m of 40 hyperlipidemic subjects before and after treatment with CS-514 suggests that lipoprotein structure was not affected by this drug. Together with our previous observation [30] we conclude here that CS-514 suppresses plasma cholesterol levels in hypercholesterolemic subjects without modifying lipoprotein composition or lipoprotein structure. In type IIb subjects, the drug also significantly suppresses plasma triglyceride level and elevates apolipoprotein A-I and A-II as well as HDL-cholesterol. It appears therefore that this drug is clinically useful for both type IIa and type IIb hyperlipidemic patients. References Kannel, W.B., Castelli, W.D. and McNamara, P.M., Serum cholesterol lipoproteins, and risk of coronary heart disease, The Framingham Study, Ann. Intern. Med., 74 (1971) 1. Gotto, Jr., A.M., Status report - plasma lipids, lipoproteins and coronary heart disease, Atheroscler. Rev., 4 (1979) 17. Slack, J., Risks of ischaemic heart disease in familial hyperlipoproteinaemia states, Lancet, ii (1969) 1380. Goldstein, J.L. and Brown, MS., Familial hypercholesterolemia. In: Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Goldstein, J.L. and Brown, M.S., (Eds.), The Metabolic Basis of Inherited Disease, 5th edn., McGraw-Hill, New York, 1983, p. 672. Goldstein, J.L. and Brown, M.S., Familial hypercholesterolemia: identification of a defect in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity associated with overproduction of cholesterol, Proc. Natl. Acad. Sci. USA, 70 (1973) 2804. Goldstein, J.L., Brown, M.S. and Stone, N.J., Genetics of the LDL receptor. Evidence that the mutations affecting binding and internalization are allelic, Cell, 12 (1977) 629. Endo, A., Kuroda, M. and Tsujita, Y., ML-236A, ML-236B and ML-236C, new inhibitors of cholesterogenesis produced by penicillium citrinum, J. Antibiot., 29 (1976) 1346. Endo, A., Tsujita, Y., Kuroda, M. and Tanzawa, K., Inhibition of cholesterol synthesis in vitro and in vivo by ML-236A and ML-236B, competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Eur. J. Biochem., 37 (1978) 313. 9 Nakaya, N., Homma, Y., Tamachi, H. and Goto, Y., The effect of CS514, an inhibitor of HMG-CoA reductase, on serum lipids in healthy volunteers, Atherosclerosis, 61(1986) 125. 10 Yoshino, G., Kazumi, T., Uenoyama, R., Inui, A., Kasama, T., Iwatani, I., Iwai, M., Yokono, K., Otsuki, M. and Baba, S., Probucol versus eptastatin in hypercholesterolemic diabetics, Lancet, i (1986) 740. 11 Kazumi, T., Yoshino, G., Kasama, T., Iwatani, I., Iwai, M., Morita, S. and Baba, S., Effect of CS-514, a new inhibitor
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