Patients with atherogenic dyslipidemia are at high risk forcoronary heart disease (CHD).1) Guidelines from the Na-tional Cholesterol Education Program recommend reductionof low-density lipoprotein (LDL) cholesterol as the primarygoal in cardiovascular risk reduction therapy.2) According tothe guidelines, statins are widely used for LDL cholesterol-lowering therapy. Recently, meta-analyses of prospectivestudies indicated that increased triglyceride (TG) levels werealso an independent risk factor for CHD.3) Furthermore, ab-normal postprandial accumulations of remnants from TG-rich lipoproteins, such as chylomicron and very low-densitylipoprotein (VLDL), have been shown to be linked to CHDregardless of fasting TG levels.4) Fibrates are often used forTG-lowering therapy and have lowering effects on postpran-dial TG levels as well as fasting TG levels.5) However, thereare no potent lipid-lowering agents that reduce both choles-terol and TG levels.

Microsomal TG transfer protein (MTP) plays an importantrole in the assembly of apolipoprotein B (apoB)-containinglipoproteins such as VLDL in the liver and chylomicron inthe intestine.6,7) MTP inhibitors have been shown to reduceboth total cholesterol and TG levels not only in animal exper-iments8,9) but also in clinical studies.8) In addition, MTP in-hibitors reduced the plasma TG rise after oral fat loading inrodents.8,9) Therefore MTP inhibitors should be useful forprevention of atherosclerotic diseases in patients with com-bined hyperlipidemia, such as type IIb and III hyperlipi-demia.

Apolipoprotein E knockout (apoE KO) mice are widelyused as an animal model of atherosclerosis. When fed aWestern-type diet (WD), these mice develop severe hyper-lipidemia and atherosclerosis.10,11) In this mouse model, anMTP inhibitor, (2S)-2-cyclopentyl-2-[4-[(2,4-dimethyl-9H-pyrido[2,3-b]indol-9-yl)methyl]phenyl]-N-[(1S)-2-hydroxy-

1-phenylethyl]ethanamide (implitapide), has been shown toreduce progression of atherosclerosis.12) To elucidate the an-tiatherosclerotic mechanisms of implitapide in the mousemodel of atherosclerosis, we examined the effects on plasmalipid levels, TG elevation after oral fat loading, and develop-ment of atherosclerotic lesions in apoE KO mice fed a WD.

MATERIALS AND METHODS

Materials Implitapide was synthesized at Fujisawa Phar-maceutical Co., Ltd. (Osaka, Japan). Sodium pentobarbital(Nembutal) was purchased from Dainippon PharmaceuticalCo., Ltd. (Osaka, Japan). TG E Test Wako, Cholesterol ETest Wako, and oil red O were purchased from Wako PureChemical (Osaka, Japan). All other reagents were purchasedfrom Nacalai Tesque (Osaka, Japan).

Animals and Diets Homozygous apoE KO mice werebred from breeding pairs obtained from Jackson Laboratory(Bar Harbor, ME, U.S.A.). C57BL/6J mice were purchasedfrom SLC Ltd. (Shizuoka, Japan). Animals were housed in atemperature-controlled facility with a 12-h light/dark cycleand allowed ad libitum access to water. The WD, containing21% (w/w) fat and 0.15% (w/w) cholesterol, and chow diet(CD), containing 5% (w/w) fat, were prepared by OrientalYeast (Osaka, Japan). All animal experimental procedureswere performed in accordance with guidelines of the AnimalExperiment Committee of Fujisawa Pharmaceutical Co., Ltd.

Experimental Protocol Male apoE KO mice aged 6weeks were fed either the CD or the WD. At age 7 weeks,apoE KO mice fed the WD were divided into two groupswith similar mean body weight: apoE KO mice fed the WDand apoE KO mice fed the WD containing implitapide (WI).Age-matched C57BL/6J mice fed the CD were used as anaive control (C57BL).

February 2005 Biol. Pharm. Bull. 28(2) 247—252 (2005) 247

∗ To whom correspondence should be addressed. e-mail: koji_ueshima@po.fujisawa.co.jp © 2005 Pharmaceutical Society of Japan

Implitapide, a Microsomal Triglyceride Transfer Protein Inhibitor,Reduces Progression of Atherosclerosis in Apolipoprotein E KnockoutMice Fed a Western-Type Diet: Involvement of the Inhibition ofPostprandial Triglyceride Elevation

Koji UESHIMA,* Hitomi AKIHISA-UMENO, Akira NAGAYOSHI, Shoji TAKAKURA, Masahiko MATSUO, andSeitaro MUTOH

Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd.; 2–1–6 Kashima, Yodogawa-ku, Osaka532–8514, Japan. Received September 14, 2004; accepted November 8, 2004

Microsomal triglyceride transfer protein (MTP) is essential for the synthesis of both chylomicron in the in-testine and very low-density lipoprotein in the liver. An MTP inhibitor, (2S)-2-cyclopentyl-2-[4-[(2,4-dimethyl-9H-pyrido[2,3-b]indol-9-yl)methyl]phenyl]-N-[(1S)-2-hydroxy-1-phenylethyl]ethanamide (implitapide), has beenshown to suppress atherosclerosis in apolipoprotein E knockout (apoE KO) mice. To elucidate the antiatheroscle-rotic mechanisms of implitapide in the mice, we examined the effects on plasma lipid levels, triglyceride (TG) ele-vation after oral fat loading, and development of atherosclerosis in apoE KO mice fed a Western-type diet. Impli-tapide at a dosage of approximately 3.2 mg/kg/day significantly reduced both total cholesterol and TG levels dur-ing the 8-week treatment period. In addition, implitapide significantly inhibited the increase in plasma TG levelsafter oral olive oil loading tests conducted after 4 weeks of treatment. After the treatment, implitapide signifi-cantly suppressed the atherosclerotic lesion area by 83% compared with a control group. These results providedirect evidence that the antiatherosclerotic effects of implitapide in apoE KO mice are associated with the inhibi-tion of postprandial TG elevation, in addition to the reduction of both plasma total cholesterol and TG levels.

Key words microsomal triglyceride transfer protein; implitapide; atherosclerosis

Implitapide concentrations (14—22 ppm) in the diet wereadjusted once a week to ensure dosage consumption of ap-proximately 3.2 mg/kg/d. This dosage was based on a prelim-inary study, which showed that implitapide at a dosage of ap-proximately 3.2 mg/kg/d, but not at a dosage of approxi-mately 1 mg/kg/d, significantly reduced both total cholesteroland TG levels in apoE KO mice fed a WD. Body weight andaverage food consumption for 3 d were monitored weekly.Before and at 4 and 8 weeks of treatment, blood was col-lected for measurements of plasma lipid levels. At the 4thweek, an oral fat-loading test was performed. At the 5thweek of treatment, feces were collected for determination offecal fat. At the end of 8 weeks of treatment, mice were euth-anized for analysis of atherosclerotic lesions.

Plasma Lipid Measurement and Lipoprotein Fraction-ation Blood was collected from the retroorbital venousplexus. Plasma was separated, and total cholesterol and TGlevels were enzymatically measured with Cholesterol E TestWako and TG E Test Wako, respectively. For lipoproteinfractionation, plasma samples of each group were pooled andelectrophoresed on 1% agarose gel (Titan Gels, Helena Lab-oratories, Saitama, Japan). The cholesterol in lipoproteinfractions was stained with the CHOL/TRIG Combo systemand scanned by a densitometer (EDC, Helena Laboratories).

Oral Fat-Loading Test To assess the postprandial in-crease in TG-rich lipoproteins, oral fat-loading tests wereperformed. Olive oil was chosen for the loading tests becauseof its safety and adequate fluidity. Mice fasted for 16 h wereorally administered 10 ml/kg of olive oil emulsion, contain-ing 30% olive oil, 5% Tween-20, and 65% methylcellulosesolution (0.5% w/v). Blood samples were collected before(0 h), and 2 and 4 h after oral fat loading. Plasma was sepa-rated and TG levels were measured as an index of remnant-like particles, since changes in TG levels after oral fat load-ing were reported to be correlated with changes in remnant-like particles derived from TG-rich lipoproteins.13) The areaunder the curve of plasma TG levels above the initial level(DAUC0—4 h) was calculated.

Determination of Fat in Feces The feces were collectedfor 3 d, dried, and pulverized in a mill. The amount of fat infeces was analyzed using the method of van de Kamer etal.14) Briefly, fatty acids and fat were extracted with petro-leum ether from an acidic, alcoholic solution containing thesample of feces, and the amount of extract was measuredgravimetrically. The amount of fat ingested was determinedby multiplying food intake by the percentage of food as fat.The percentage of fat excreted was calculated by dividing theamount of fat in feces by the amount of fat ingested.

Analysis of Atherosclerotic Lesions After euthanasia

under sodium pentobarbital anesthesia, the thoracic aorta andheart were perfusion-fixed with 4% paraformaldehyde (pH7.4). For the analysis of en face lipid staining, the thoracicaorta was opened longitudinally and pinned out on a blackcorkboard. Fatty streak lesions were stained with oil red O(Wako Pure Chemical). En face images of the aorta weretaken with a digital camera (Nikon, Tokyo, Japan). Quantifi-cation of the images was performed using Mac Scope soft-ware (Mitani, Fukui, Japan). Aortic lesion areas were calcu-lated by dividing oil red O-stained areas by the total area. Forobservation of the aortic root, the heart was embedded inOCT compound (Sakura, Tokyo, Japan) or paraffin. Cryostatsections (6 mm) were stained with hematoxylin and oil red O.Serial 5-mm thick sections of the paraffin-embedded heartwere stained with hematoxylin and eosin or orcein to im-prove the visibility of elastic tissues. Sections were observedunder a light microscope (Nikon, Tokyo, Japan).

Statistical Analysis Data are expressed as mean�S.E.M. if not otherwise stated. Statistical analysis of meandifferences was performed with Student’s t-test and Dun-nett’s multiple comparison test. Values of p�0.05 were re-garded as statistically significant.

RESULTS

Body Weight and Food Consumption Body weightchanges and food consumption during the feeding period areshown in Table 1. The initial body weight of the CD groupwas significantly lower than that of the C57BL group, butthere was no significant difference in body weight gain. Foodconsumption during the feeding period was significantlyhigher in the CD group than that in the C57BL group(p�0.01). The body weight gain of the WD group during thefeeding period was similar to that of the CD group, in spiteof significantly lower food consumption (p�0.01). The WIgroup had significantly lower final body weight as comparedwith that of the WD group (p�0.01), although the WI groupshowed a tendency toward increased food consumption.

Plasma Lipid Levels Both total cholesterol and TG lev-els were higher in the CD group than in the C57BL group(Fig. 1). These lipid elevations in apoE KO mice were aug-mented by the WD feeding. Implitapide significantly reducedthe plasma lipid levels to nearly or below the CD level at 4and 8 weeks of treatment (p�0.01).

In the pooled samples of plasma collected after 4 weeks oftreatment, VLDL and LDL cholesterol levels were obviouslylower in the WI group compared with the WD group (Fig. 2).However, high-density lipoprotein (HDL) cholesterol levelsin the WD group and the WI group were almost the same.

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Table 1. Body Weight and Food Consumption in apoE KO Mice

Group nBody weight (g)

Food consumption

Initial Final Gain(g/mouse/d)

C57BL 10 23.1�0.3 32.2�0.6 9.2�0.5 5.1�0.2CD 8 20.8�0.3* 31.1�0.9 10.3�0.7 6.6�0.2*WD 8 21.0�0.4 30.8�0.8 9.8�0.7 3.6�0.2†

WI 8 20.5�0.3 28.5�0.4†† 8.0�0.5 4.3�0.3

At 7 weeks of age (initial), diets were changed as indicated and were given for 8 weeks. C57BL/6J mice were fed a chow diet (C57BL). ApoE KO mice were fed a chow diet(CD), or a Western-type diet without (WD) or with implitapide approximately 3.2 mg/kg/d (WI). Data are expressed as mean�S.E.M. ∗ p�0.01 compared with C57BL, † p�0.01compared with CD, †† p�0.01 compared with WD.

Plasma TG Levels after Oral Fat Loading An oral fat-loading test was performed after 4 weeks of treatment. After16-h fasting, baseline TG levels of the WI group were signif-icantly lower than those of the WD group (55�10 mg/dl and138�14 mg/dl, respectively, p�0.01) (Fig. 3A). Plasma TGlevels after oral fat loading were also lowered by implitapidetreatment, and the DAUC of TG level in the WI group wassignificantly reduced by 37% compared with that in the WDgroup (p�0.01) (Fig. 3B).

Fecal Fat Excretion At the 5th week of treatment, ex-cretion of fecal fat in the CD group and the WD group was4% and 2% of ingested fat, respectively (Fig. 4). Excretion offecal fat in the WI group was markedly higher (15% of in-gested fat) than that in the WD group.

Atherosclerosis Typical lipid-stained sections of the aor-tic root are shown in Fig. 5. There were no lipid-stained le-

sions in the C57BL group (Fig. 5A). The CD group demon-strated little lipid-stained areas in the entire neointima (Fig.5B), while such lesions containing foam cells were exacer-bated by the WD feeding (Figs. 5C, E). Implitapide markedlysuppressed lipid-stained lesions in the mice fed the WD(Figs. 5D, F). As shown in Figs. 6A and C, the aortic root ob-tained from the WD group demonstrated gross intimal thick-ening and cholesterol crystal accumulation. These alterationsof such lesions were mitigated in the WI group (Figs. 6B, D).Figure 7 shows the lipid-stained lesion areas at the en facesurface thoracic aorta. The lesion area of the WD group wasmarkedly larger than that of the CD group (p�0.01). Impli-tapide significantly decreased lesion area by 83% comparedwith that of the WD group (p�0.01).

DISCUSSION

The present study demonstrated that implitapide signifi-

February 2005 249

Fig. 1. Effects of Implitapide on Plasma Total Cholesterol (A) and TGLevels (B)

C57BL/6J mice fed a chow diet (CD) (C57BL; open triangle; n�10), apoE KO micefed a CD (closed triangle; n�8), apoE KO mice fed a Western-type diet (WD) without(WD; open circle; n�8) or with implitapide at a dosage of approximately 3.2 mg/kg/d(WI; closed circle; n�8). Values are expressed as mean�S.E.M. && p�0.01 comparedwith C57BL, # p�0.05, ## p�0.01 compared with CD, ∗∗ p�0.01 compared with WD.

Fig. 2. VLDL, LDL, and HDL Cholesterol Levels in apoE KO Mice Fed aWD without (WD; Open Column) or with Implitapide (WI) at a Dosage ofApproximately 3.2 mg/kg/d (WI; Closed Column)

Plasma samples were taken after 4 weeks of treatment (n�8), and pooled samplesfrom each group were used for lipoprotein analysis.

Fig. 3. Effects of Implitapide on Plasma TG Levels (A) and the AUC0—4 h

of TG Levels above the Initial Level (B) after Oral Fat Loading in apoE KOMice

An oral fat-loading test was performed after a 4-week treatment period. Mice werefasted for 16 h, and then 10 ml/kg of 30% olive oil emulsion was given orally. Micewere fed a WD without (WD; open circle; n�8) or with implitapide at a dosage of ap-proximately 3.2 mg/kg/d (WI; closed circle; n�7). Values are expressed asmean�S.E.M. ∗ p�0.05, ∗∗ p�0.01 compared with WD.

Fig. 4. Fecal Fat Excretion in apoE KO Mice Fed a CD, a WD without(WD) or with Implitapide at a Dosage of Approximately 3.2 mg/kg/d (WI)

Fecal samples were collected for 3 d after 5 weeks of treatment (n�8), and pooledsamples from each group were used for the determination of fecal fat. Fecal fat excre-tion is expressed as a percentage of dietary ingested fat.

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Fig. 5. Photomicrographs of the Aortic Root in apoE KO Mice after 8 Weeks of Treatment

Cryostat sections from C57BL/6J mice fed a CD (C57BL; A), apoE KO mice fed a CD (B), and apoE KO mice fed a WD without (WD; C and E) or with implitapide at a dosageof approximately 3.2 mg/kg/d (WI; D and F) were stained with hematoxylin and oil red O (original magnifications: �100 in A—D; �400 in E and F). Horizontal bars represent100 mm.

Fig. 6. Photomicrographs of the Aortic Root in apoE KO Mice after 8 Weeks of Treatment

Sections of the paraffin-embedded heart from apoE KO mice fed a WD without (WD; A and C) or with implitapide at a dosage of approximately 3.2 mg/kg/d (WI; B and D)were stained with hematoxylin and eosin (A and B) or orcein (C and D) (original magnifications: �100). Horizontal bars represent 100 mm.

cantly lowered both plasma total cholesterol and TG levelsand reduced the progression of atherosclerotic lesions inapoE KO mice fed a WD. In addition, implitapide also inhib-ited the increase in plasma TG levels after oral fat loading.These results suggest that this MTP inhibitor reduces theprogression of atherosclerosis through the inhibition of post-prandial TG elevation in addition to lowering both plasmatotal cholesterol and TG levels.

ApoE KO mice fed a WD containing implitapide (1, 5, and15 mg/kg/d) for 14 weeks have been shown to reduce signifi-cantly both plaque area (by 66, 78, and 93%, respectively)and lipid moieties within plaque (4.3, 2.6, and 0%, respec-tively, versus 9.5% in controls).12) Our study also showed thatimplitapide at a dosage of approximately 3.2 mg/kg/d signifi-cantly reduced the lipid-stained aortic lesions by 83% inapoE KO mice. Thus it was confirmed that this dose of impli-tapide is sufficient for the suppression of atherosclerosis inapoE KO mice.

MTP inhibitors have been shown to reduce both plasmatotal cholesterol and TG levels in normolipidemic mice,8)

rats, and dogs.9) Similar effects were also observed in Watan-abe-heritable hyperlipidemic rabbits, a model of human ho-mozygous familial hypercholesterolemia.15,16) Our studydemonstrated that implitapide at a dosage of approximately3.2 mg/kg/d significantly lowered both plasma total choles-terol and TG levels in apoE KO mice fed a WD. Thus it wasindicated that this dose of implitapide reduces both lipid lev-els in these mice.

Implitapide was shown to suppress MTP activity using arecombinant human form complexed with protein disulphideisomerase (IC50�10 nM) and inhibit secretion of apoB-con-taining VLDL-like lipoproteins from a human hepatoma cell(HepG2) with an IC50 value of 1.1 nM.12) In our study, impli-tapide treatment lowered the plasma VLDL cholesterol level.Thus implitapide should inhibit hepatic MTP in apoE KOmice. MTP also plays an important role in lipid absorption inthe intestine, since the MTP is essential for the assembly ofchylomicron.6,7) Intestinal absorption of radio-labeled TGwas inhibited by a MTP inhibitor in rats.8) Our resultsshowed that excretion of fecal fat was increased by impli-tapide in apoE KO mice, suggesting that implitapide inhibitslipid absorption via the inhibition of intestinal MTP. There-fore implitapide inhibited both hepatic and intestinal MTP,

and these effects would be involved in the marked reductionof both cholesterol and TG levels in apoE KO mice.

The benefit of cholesterol-lowering therapy in the preven-tion of atherosclerosis is well established, while increasedTG level has been demonstrated to be an independent riskfactor for CHD.3) It has been shown that postprandial accu-mulations of remnants from TG-rich lipoproteins are linkedto CHD.4) Our results demonstrated that implitapide inhibitsplasma TG elevation after oral fat loading. Taken together,these facts suggest that the antiatherogenic effects of impli-tapide in apoE KO mice are attributed to the inhibitory effecton postprandial TG elevation in addition to the reduction ofboth cholesterol and TG levels.

Our findings in pathologic examinations demonstrated thatimplitapide suppressed foam cell lesions in the arterial wallof apoE KO mice fed a WD. In the process of atherogenesis,macrophage uptake of oxidized LDL is thought to play a cen-tral role in foam cell formation.17) In addition, chylomicronremnant, a major plasma lipoprotein produced from dietaryfat during the postprandial state, is known to be an athero-genic lipoprotein stimulating lipid accumulation in suben-dothelial macrophages.4) Our study showed that implitapidenot only reduced LDL cholesterol, but also inhibited post-prandial TG elevation. Thus these effects of implitapide maysynergistically contribute to the suppression of foam cell le-sions, leading to the antiatherogenic effect. It is unknownwhether the effect of implitapide on foam cell lesions is at-tributed to the reduction of foam cell formation and/or the re-gression of foam cells. Further study to elucidate thesepathologic mechanisms are needed.

In conclusion, this study provides direct evidence that theantiatherosclerotic effects of implitapide in apoE KO mice isassociated with the inhibition of postprandial TG elevation,in addition to the reduction of both plasma total cholesteroland TG levels. Inhibition of MTP may be a promising strat-egy for the prevention of atherosclerotic diseases in patientswith combined hyperlipidemia, such as type IIb and III hy-perlipidemia.

Acknowledgments We thank Shinichi Hasegawa fortechnical assistance in the histologic examination. We alsothank Helena Laboratories for technical assistance in themeasurements of lipoproteins.

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February 2005 251

Fig. 7. Effects of Implitapide on en Face Surface Lesions on the ThoracicAorta

Fatty streak lesions in C57BL/6J mice fed a CD (C57BL), apoE KO mice fed a CD(CD), and apoE KO mice fed a WD without (WD) or with implitapide at a dosage ofapproximately 3.2 mg/kg/d (WI) were stained with oil red O after 8 weeks of treatment.Values are expressed as mean�S.E.M. (n�8 per group), && p�0.01 compared withC57BL, ## p�0.01 compared with CD, ∗∗ p�0.01 compared with WD.

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