European Journal of Pharmacology 431 2001 127–131
`Ž.
`
`www.elsevier.comrlocaterejphar
`
`MTP inhibitor decreases plasma cholesterol levels in LDL
`receptor-deficient WHHL rabbits by lowering the VLDL secretion
`Masashi Shiomi), Takashi Ito
`Institute for Experimental Animals, Kobe UniÕersity School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
`Received 28 June 2001; received in revised form 19 September 2001; accepted 25 September 2001
`
`Abstract
`To examine whether a microsomal triglyceride transfer protein MTP -inhibitor is effective in patients with homozygous familial
`Ž.
`hypercholesterolemia, we administered 2S -2-cyclopentyl-2- 4- 2,4-dimethyl-9H-pyrido 2,3-b indol-9-yl methyl phenyl -N- 1S -2-hy-
`Ž.
`
`.
`4
`wŽ
`wŽ.
`w
`x
`x
`droxy-1-phenylethyl ethanamide Implitapide , a new MTP inhibitor, to low-density lipoprotein LDL -receptor-deficient Watanabe
`.
`Ž.
`Ž
`x
`heritable hyperlipidemic WHHL rabbits at doses of 3, 6, and 12 mgrkg for 4 weeks. In the 12 mgrkg group, the plasma cholesterol and
`Ž.
`triglyceride levels were decreased by 70% and 45%, respectively, and the very low-density lipoprotein VLDL secretion rate was
`Ž.
`decreased by 80%. The composition of newly secreted VLDL was similar in each group. This suggests that Implitapide diminished the
`number of VLDL particles secreted from the liver. Although the ratio of vitamin ErLDL was not altered by Implitapide, triglyceride
`accumulation and a decrease in vitamin E were observed in the liver. In conclusion, an inhibition of VLDL secretion led to a decrease of
`plasma LDL in WHHL rabbits, and MTP inhibitors should have hypolipidemic effects against homozygous familial hypercholesterolemia.
`q2001 Elsevier Science B.V. All rights reserved.
`Keywords: MTP inhibitor; Implitapide; VLDL very low-density lipoprotein secretion; Hypolipidemic effect; WHHL, rabbit
`Ž.
`
`1. Introduction
`In patients with homozygous familial hypercholes-
`terolemia, especially low-density lipoprotein LDL -recep-
`Ž.
`tor null
`type,
`therapy using inhibitors of 3-hydroxy-3-
`methylglutaryl coenzyme A HMG-CoA reductase statin ,
`Ž.
`Ž
`.
`which competitively inhibits cholesterol biosynthesis and
`increase the LDL-receptor function, has almost no effect.
`To reduce the plasma cholesterol levels of homozygous
`familial hypercholesterolemia, reduction in the secretion of
`very low-density lipoprotein VLDL -cholesterol from the
`Ž.
`liver may be one of the important approaches.
`Microsomal triglyceride transfer protein MTP plays an
`Ž.
`important role in the assembly of VLDL particles in the
`liver and of chylomicron particles in the intestine Wet-
`Ž
`terau et al., 1992; Sharp et al., 1993; Sorbera et al., 2000 .
`.
`In vitro studies have demonstrated that if the assembly of
`VLDL is suppressed, secretion of the lipoproteins is re-
`
`duced Jamil et al., 1996; Gruetzmann et al., 2000 . The
`Ž.
`main mechanisms of hypolipidemic effects of MTP in-
`hibitors are largely different from inhibitors for HMG-CoA
`reductase. Therefore, MTP inhibitors have the possibility
`of lowering the plasma cholesterol levels of patients with
`homozygous familial hypercholesterolemia.
`Recently, Wetterau et al. 1998 reported the effect of
`Ž.
`an MTP inhibitor on lipoprotein lipid levels and the
`triglyceride secretion rate using rats and hamsters. They
`also showed an MTP inhibitor that normalized the plasma
`lipid levels of homozygous Watanabe heritable hyperlipi-
`demic WHHL rabbits, which is an LDL receptor-defi-
`Ž.
`cient animal model Tanzawa et al., 1980; Kita et al.,
`Ž
`1981 . There are no original studies reporting whether
`.
`MTP inhibitors suppress secretion of VLDL particles from
`the liver in vivo. Therefore, we attempted to examine the
`effects of an MTP inhibitor on the VLDL secretion rate,
`lipoprotein levels, and plasma vitamin E levels in homozy-
`gous WHHL rabbits, using another MTP inhibitor 2S -2-
`Ž.
`cyclopentyl-2- 4- 2,4-dimethyl-9H-pyrido 2,3-b indol-
`
`w Ž
`x
`w
`9-yl methyl phenyl -N-
`1S -2-hydroxy-1-phenylethyl
`4
`w Ž.
`.
`x
`x
`ethanamide Implitapide Sorbera et al., 2000 .
`Ž.
`Ž
`.
`0014-2999r01r$ - see front matter q2001 Elsevier Science B.V. All rights reserved.
`PII: S0014-2999 01 01419-4
`Ž.
`
`) Corresponding author. Tel.: q81-78-382-5660; fax: q81-78-382-
`5679.
`E-mail address: ieakusm@med.kobe-u.ac.jp M. Shiomi .
`Ž.
`
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`128
`2. Materials and methods
`
`2.1. Materials
`
`)
`(
`M. Shiomi, T. ItorEuropean Journal of Pharmacology 431 2001 127–131
`concentration was measured enzymatically and the protein
`concentration was determined by the method of Lowry et
`al. 1951 . The increased rate of VLDL was calculated by
`Ž.
`dividing the difference between the VLDLs obtained be-
`fore and at 6 h after the Triton injection by 6 h.
`
`Implitapide was provided by Bayer Yakuhin Osaka,
`Ž
`Japan . Triton WR-1339 4- 1,1,3,3-tetramethylbutyl phe-
`.Ž
`Ž
`.
`nol polymer with formaldehyde and oxirane was pur-
`.
`chased from Nakarai Tesque Tokyo, Japan .
`Ž.
`
`2.2. Animals
`
`Twenty male homozygous WHHL rabbits Shiomi et
`Ž
`al., 1992 aged 6 months were divided into four groups,
`.
`i.e., a placebo group and Implitapide-treated groups admin-
`istered daily doses of 3, 6, and 12 mgrkg, respectively.
`Implitapide was suspended in 0.5% methylcellulose every
`day and was administered to WHHL rabbits orally for 4
`weeks. All animal experimentation and care were con-
`ducted according to the Guidelines of Animal Experimen-
`tation of Kobe University.
`
`2.3. Fractionation of plasma lipoprotein and measurement
`of lipid leÕels
`
`After overnight fasting, blood samples were taken every
`week from the marginal ear vein. The plasma lipid levels
`were measured every week by enzymatic methods. Before
`and at the end of the treatment, lipoprotein was fraction-
`ated by ultracentrifugation to yield the following fractions:
`VLDL d-1.006 grml , intermediate-density lipoprotein
`Ž.
`IDL, 1.006-d-1.019 grml , LDL 1.019-d-1.063
`Ž.
`Ž
`grml , and high-density lipoprotein HDL, d)1.063
`.Ž
`grml .
`.
`
`2.4. Determination of VLDL secretion rate
`
`At the end of Implitapide treatment, we determined the
`VLDL secretion rate Shiomi et al., 1994; Shiomi and Ito,
`Ž
`1994 . The VLDL secretion rate was determined by intra-
`.
`venous injection of Triton WR-1339 after overnight fasting
`to eliminate the influence of chylomicrons. It was reported
`that Triton WR-1339 blocks degradation of VLDL Shotz
`Ž
`et al., 1957; Borenztajn et al., 1976 and that the VLDL
`.
`secretion rate determined by a Triton injection reflects well
`the secretion rate of VLDL in vivo Guettet et al., 1989;
`Ž
`Maeda et al., 1993 . In addition, the apolipoprotein B of
`.
`liver perfusates was contained almost exclusively in VLDL
`in WHHL rabbits Yamada et al., 1987 . Prior to Triton
`Ž.
`injection, Implitapide was administered to rabbits in the
`treated group. Triton WR-1339 at 200 mgrml in 0.15 M
`NaCl solution was injected into an ear vein at a dose of
`400 mgrkg body weight. Before and at 6 h after Triton
`injection, blood samples were obtained and the VLDL
`fraction was prepared by ultracentrifugation. The lipid
`
`2.5. Measurement of plasma Õitamin E leÕels
`
`Before and at the end of the Implitapide administration,
`the vitamin E levels were measured in the plasma samples
`by analysis in a commercial
`laboratory SRL, Tokyo,
`Ž
`Japan . Vitamin E was extracted with hexane, and a-, b-,
`.
`and g-tocopherol were separated and measured by high-
`performance liquid chromatography employing a column
`Ž.Unisil Q NH , GL Science, Tokyo, Japan and a spectro-
`2
`fluorometer FP-821, JASCO, Tokyo, Japan .
`Ž.
`
`2.6. Measurement of lipid contents in the liÕer
`
`After the Triton experiment, rabbits were anesthetized
`by an intravenous injection of sodium pentobarbital 25
`Ž
`mgrkg and perfused with saline solution. After the perfu-
`.
`sion, the liver was excised. Using 5 g of the tissue, the
`lipid accumulated in the liver was extracted according to
`the method of Folch et al. 1957 . The concentration of
`Ž.
`extracted lipid and vitamin E were determined as described
`above.
`
`2.7. Statistical analysis
`
`Values are presented as the means"SEM. Statistical
`analysis was carried out by Williams–Shirley multiple
`comparison test. Shirley, 1977 .
`Ž.
`
`3. Results
`
`3.1. Effect of Implitapide on the plasma and lipoprotein
`lipid leÕels
`
`All plasma lipid levels were decreased dose-depen-
`dently by Implitapide treatment. Comparing the highest
`dose group to the placebo group, the decrease in plasma
`lipid levels was 70% P-0.01 for cholesterol 20.4 "1.0
`Ž.
`Ž
`vs. 6.2 "0.8 mM and 45% P-0.01 for triglyceride
`.Ž
`.
`Ž.2.0 "0.4 vs. 1.1 "0.1 mM . In Fig. 1, each lipoprotein
`containing apolipoprotein-B100 was decreased dose-de-
`pendently by Implitapide treatment. In the highest dose
`group, the VLDL fraction was markedly decreased: the
`decrease was 79% P-0.01 for cholesterol and 65%
`Ž.
`Ž.P-0.05 for triglyceride, and the decrease in the LDL
`fraction was similar to that of the plasma lipid levels.
`However, the HDL-lipid levels did not show significant
`changes.
`
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`M. Shiomi, T. ItorEuropean Journal of Pharmacology 431 2001 127–131
`
`129
`
`Fig. 1. Effects of Implitapide on the lipoprotein lipid levels of homozy-
`, 3 mgrkg of Implitapide
`gous WHHL rabbits. I, placebo ns5 ;
`Ž.
`ns5 ;
`, 6 mgrkg of Implitapide ns5 ; and X, 12 mgrkg of
`Ž.
`Ž.
`Implitapide ns5 . Values are presented as means"SEM. Statistical
`Ž.
`analyses were carried out with the Williams–Shirley multiple comparison
`test
`P -0.05; and †P -0.01 vs. placebo group . The plasma choles-
`Ž)
`.
`terol levels were 20"1 mM in the placebo group, 16"1 mM in the 3
`mgrkg group, 13"1 mM in the 6 mgrkg group, and 6"1 mM in the 12
`mgrkg group.
`
`3.2. Effect of Implitapide on VLDL secretion
`The VLDL secretion rate was decreased dose-depen-
`dently Fig. 2 . Using all parameters, the VLDL secretion
`Ž.
`rate of the highest dose group was decreased significantly
`by about 80% P-0.05 or P-0.01 compared with the
`Ž.
`placebo group. Fig. 3 shows the composition of newly
`secreted VLDL at 6 h after Triton injection. Guettet et al.
`1989 and Maeda et al. 1993 reported that VLDL ob-
`Ž.
`Ž.
`tained after injection of Triton WR-1339 more closely
`resembled nascent VLDL rather than circulating VLDL.
`Therefore, VLDL obtained after Triton injection is consid-
`ered newly secreted VLDL. The VLDL composition of
`
`Fig. 3. Effects of Implitapide on the lipid composition of the newly
`secreted VLDL of homozygous WHHL rabbits. We analyzed the VLDL
`fraction obtained at 6 h after Triton injection as newly secreted VLDL.
`Values are presented as means"SEM. There were no significant differ-
`ences among the groups ns5 by the Williams–Shirley multiple com-
`Ž.
`parison test.
`
`newly secreted VLDL showed almost no changes in all the
`groups.
`
`3.3. Plasma Õitamin E leÕels
`
`Before the experiment, the plasma a-tocopherol level
`was 64"4 mM ns20 and the b- or g-tocopherol levels
`Ž.
`were below 1 mM. At the end of the treatment Fig. 4 , the
`Ž.
`plasma a-tocopherol level in the highest dosage group was
`decreased by 76% P-0.01 compared with the placebo
`Ž.
`group. The decrease was dose-dependent. However, the
`plasma a-tocopherol level was closely correlated with the
`LDL-cholesterol level
`rs0.8842, P-0.001 . In addi-
`Ž.
`tion,
`the ratio of plasma a-tocopherolrplasma LDL-
`cholesterol was 2.9 "0.3 ns5 in the highest dose group
`Ž.
`at the end of the treatment, and this was similar to the ratio
`
`Fig. 2. Effects of Implitapide on the VLDL secretion rate of homozygous
`, 3 mgrkg of Implitapide ns5 ;
`WHHL rabbits. I, placebo ns5 ;
`Ž.
`Ž.
`, 6 mgrkg of Implitapide ns5 ; and X, 12 mgrkg of Implitapide
`Ž.
`Ž.ns5 . Values are presented as means"SEM. Statistical analyses were
`carried out with the Williams–Shirley multiple comparison test
`P -
`Ž)
`0.05 and †P -0.01 versus placebo group .
`.
`
`Fig. 4. Relation between the plasma a-tocopherol levels and the LDL
`cholesterol levels of homozygous WHHL rabbits treated with Implitapide,
`an MTP inhibitor. e, placebo
`ns5 ;
`, 3 mgrkg of Implitapide
`Ž.
`ns5 ;
`, 6 mgrkg of Implitapide ns5 ; and v, 12 mgrkg of
`Ž.
`Ž.
`Implitapide ns5 . The plasma a-tocopherol levels were 62"4 mM in
`Ž.
`the placebo group, 44"3 mM in the 3 mgrkg group, 31"2 mM in the 6
`mgrkg group, and 15"2 mM in the 12 mgrkg group.
`
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`130
`
`)
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`M. Shiomi, T. ItorEuropean Journal of Pharmacology 431 2001 127–131
`
`Fig. 5. Accumulation of lipids and a-tocopherol in the livers of homozygous WHHL rabbits treated with Implitapide, an MTP inhibitor. I, placebo
`ns5 ;
`, 3 mgrkg of Implitapide ns5 ;
`, 6 mgrkg of Implitapide ns5 ; and X, 12 mgrkg of Implitapide ns5 . Values are presented as
`Ž.
`Ž.
`Ž.
`Ž.
`means"SEM. Statistical analyses were carried out with the Williams–Shirley multiple comparison test
`P-0.05 and †P-0.01 vs. placebo group .
`Ž)
`.
`
`in each group. This suggests that reduction in the plasma
`a-tocopherol levels by Implitapide was due to the reduc-
`tion in the plasma LDL levels.
`3.4. Lipid accumulation in the liÕer
`Fig. 5 shows accumulation of lipids and a-tocopherol in
`the liver after 4 weeks administration of Implitapide. Al-
`though the cholesterol content was similar in each group,
`the triglyceride accumulation was increased dose-depen-
`dently. In the highest dose group, the triglyceride content
`was about three-fold greater than that of the placebo group.
`In addition, the a-tocopherol content was decreased dose-
`dependently.
`
`4. Discussion
`In this study, we examined whether inhibition of VLDL
`secretion by MTP inhibitor administration could reduce the
`plasma cholesterol levels even in the LDL receptor-defi-
`cient state. We found that 12 mgrkg of Implitapide
`markedly decreased the level of atherogenic apolipo-
`protein-B100-containing lipoproteins and the VLDL secre-
`tion rate in homozygous WHHL rabbits. This suggests that
`a potent inhibition of VLDL secretion led to a marked
`decrease in LDL in the plasma even in the LDL-receptor
`deficient state.
`A previous study by Wetterau et al. 1998 reported the
`Ž.
`effect of another MTP inhibitor on the triglyceride secre-
`tion rate but not on the VLDL secretion rate. Implitapide
`reduced the VLDL secretion rate by about 80%, using not
`only cholesterol as an index but also triglyceride or protein
`as an index, and did not affect the VLDL composition.
`These results suggest that Implitapide decreased the num-
`ber of VLDL particles secreted from the liver. This is a
`novel observation in vivo, although in vitro studies demon-
`strated that MTP inhibitors reduced apolipoprotein-B se-
`cretion from HepG2 cells by an inhibition in the assembly
`of VLDL particles
`Jamil et al., 1996; Wetterau et al.,
`Ž
`1998 . The present findings suggest that the potent hy-
`.
`polipidemic effect of Implitapide was mainly due to sup-
`pression of VLDL particle secretion from the liver.
`
`the hypolipidemic effects of
`In the statin treatment,
`statins are mainly the induction of the LDL-receptor func-
`tion in patients with normal LDL-receptor function. In
`WHHL rabbit studies using statins,
`the hypolipidemic
`effects were due to an induction of LDL-receptor mRNA
`Ž.Kuroda et al., 1992 and a reduction of cholesterol content
`in the VLDL secreted from the liver Shiomi et al., 1994;
`Ž
`Shiomi and Ito, 1994 . In this latter case, the secretion of
`.
`VLDL-triglyceride was not affected despite the reduction
`in the secretion of VLDL-cholesterol. Although the reduc-
`tion in VLDL-cholesterol secretion was about 20% in the
`statin-treated WHHL rabbits Shiomi et al., 1994; Shiomi
`Ž
`and Ito, 1994 , Implitapide 12 mgrkg deceased the
`.Ž
`.
`VLDL secretion by about 80% in the present study. Impli-
`tapide was very efficient
`in the suppression of VLDL
`secretion compared with statin treatments. Therefore, this
`compound has the possibility of being an effective agent
`for patients with homozygous
`familial hypercholes-
`terolemia, although statins have almost no effects.
`Wetterau et al. 1998 and Sorbera et al. 2000 reported
`Ž.
`Ž.
`MTP inhibitors decreased HDL levels. In the present study,
`the HDL cholesterol level did not show any significant
`decrease. Although we have no findings to explain this
`disagreement, this difference may be due to the very low
`HDL levels in WHHL rabbits compared with normal
`rabbits.
`The plasma vitamin E levels were decreased by the
`MTP inhibitor treatment. This decrease was due to a
`reduction in LDL. It is well known that vitamin E binds to
`lipoproteins Cohn et al., 1992 . The ratio of vitamin
`Ž.
`ErLDL-cholesterol did not decrease in the present study.
`In addition, the plasma vitamin E level of age-matched
`normal rabbits was about one-third of the highest dose
`group of the present study data not shown . This suggests
`Ž.
`that MTP inhibitors do not
`interfere with antioxidative
`effects on LDL particle.
`In the present study, we did not examine any side
`effects of Implitapide because the purpose of this study
`was to examine the effects on the VLDL secretion and
`lipoprotein levels in the LDL-receptor deficient state. In
`the experimental period, no rabbits show steatorrhoea or
`other clinical findings, and there were no significant differ-
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`M. Shiomi, T. ItorEuropean Journal of Pharmacology 431 2001 127–131
`ences in food consumption or body weight changes in each
`group. However, in examination of lipid accumulation in
`the liver, Implitapide showed a dose-dependent increase in
`the triglyceride levels and a dose-dependent decrease in
`the a-tocopherol levels despite the normal external appear-
`ance. Since the liver triglyceride content of hamsters treated
`with another MTP inhibitor was increased temporally
`Ž.Wetterau et al., 1998 , it may be inappropriate to conclude
`that the present 4 weeks treatment demonstrates that Impli-
`tapide treatment causes fatty liver. At
`the dose of 3
`mgrkg, Implitapide decreased plasma cholesterol levels by
`23% 20.4 "1.0 vs. 15.7 "0.5 mM, Ps0.003 by Stu-
`Ž
`dent’s t-test . At
`this dose,
`the liver triglyceride and
`.
`a-tocopherol levels did not show any significant changes
`compared with the placebo group. Although Implitapide
`showed potent hypolipidemic effects, it is important to
`select a safe dose and to measure activities of plasma
`enzymes, which reflect liver damage.
`It is well known that there are no effective compounds
`to reduce the plasma cholesterol levels of patients with
`homozygous
`familial hypercholesterolemia,
`especially
`LDL-receptor null type. However, the present results sug-
`gest that potent inhibition of VLDL secretion can reduce
`the plasma cholesterol levels of homozygous WHHL rab-
`bits, and MTP inhibitors are thus considered candidates as
`compounds effective for treatment of homozygous familial
`hypercholesterolemia patients.
`
`Acknowledgements
`We acknowledge Bayer Yakuhin for providing Impli-
`tapide. This work was supported in part by a research grant
`from Bayer Yakuhin.
`
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`CFAD Ex. 1033 (5 of 5)