Tác động bảo vệ của chất ức chế squalene

Protective effects of a squalene synthase inhibitor,

lapaquistat acetate (TAK-475), on statin-induced

myotoxicity in guinea pigs

Tomoyuki Nishimoto a,1, Eiichiro Ishikawa a,1, Hisashi

Anayama b, Hitomi Hamajyo b, Hirofumi Nagai b, Masao

Hirakata a, Ryuichi Tozawa a,⁎

a Pharmacology Research Laboratories I, Pharmaceutical

Research Division, Takeda Pharmaceutical Company

Limited, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-

8686, Japan b Development Research Center, Takeda

Pharmaceutical Company Limited, 2-17-85, Jusohonmachi,

Yodogawa-ku, Osaka 532-8686, Japan

Received 6 March 2007; revised 8 May 2007; accepted 13

May 2007 Available online 24 May 2007

Abstract

High-dose statin treatment has been recommended as a

primary strategy for aggressive reduction of LDL

cholesterol levels and protection against coronary

artery disease. The effectiveness of high-dose statins

may be limited by their potential for myotoxic side

effects. There is currently little known about the

molecular mechanisms of statin-induced myotoxicity.

Previously we showed that T-91485, an active metabolite

of the squalene synthase inhibitor lapaquistat acetate

(lapaquistat: a previous name is TAK-475), attenuated

statin-induced cytotoxicity in human skeletal muscle

cells [Nishimoto, T., Tozawa, R., Amano, Y., Wada, T.,

Imura, Y., Sugiyama, Y., 2003a. Comparing myotoxic

effects of squalene synthase inhibitor, T-91485, and 3-

hydroxy-3-methylglutaryl coenzyme A. Biochem.

Pharmacol. 66, 2133–2139]. In the current study, we

investigated the effects of lapaquistat administration

on statin-induced myotoxicity in vivo. Guinea pigs were

treated with either high-dose cerivastatin (1 mg/kg) or

cerivastatin together with lapaquistat (30 mg/kg) for

14 days. Treatment with cerivastatin alone decreased

plasma cholesterol levels by 45% and increased creatine

kinase (CK) levels by more than 10-fold (a marker of

myotoxicity). The plasma CK levels positively

correlated with the severity of skeletal muscle lesions

as assessed by histopathology. Co-administration of

lapaquistat almost completely prevented the

cerivastatin-induced myotoxicity. Administration of

mevalonolactone (100 mg/kg b.i.d.) prevented the

cerivastatin-induced myotoxicity, confirming that this

effect is directly related to HMG-CoA reductase

inhibition. These results strongly suggest that

cerivastatin-induced myotoxicity is due to depletion of

mevalonate derived isoprenoids. In addition, squalene

synthase inhibition could potentially be used

clinically to prevent statin-induced myopathy.

Keywords: Lapaquistat acetate; TAK-475; Statin;

Myotoxicity; Squalene synthase inhibitor; Guinea pigs

Introduction

According to many epidemiological studies, including

the Framingham Heart Study, an elevated plasma level of

low- density lipoprotein (LDL) cholesterol is a

significant risk factor for coronary heart disease

(Gordon et al., 1981; Anderson et al.,

1987).Variouscholesterol-

loweringdrugswithdifferentaction mechanisms have been

developed and used for the treatment of patients with

hypercholesterolemia. Among them, 3-hydroxy-3-

methylglutaryl-coenzyme A (HMG-CoA) reductase

inhibitors, known as statins, are the most common

cholesterol-lowering drugs. Many clinical studies have

revealed that cholesterol- lowering therapy with

statins significantly reduces coronary heart disease

risk (Scandinavian Simvastatin Survival Study Group,

1994; Sacks et al., 1996; Downs et al., 1998).

Recently, clinical studies have suggested that

aggressive cholesterol- lowering therapy produces more

benefits than mild therapy

(HeartProtectionStudyCollaborativeGroup,2002; Cannonet

al., 2004; Nissen et al., 2004). More effective

cholesterol- lowering therapy is required in the

clinical setting; however, high-dose statins, albeit

rarely, increase the risk of toxicity such as

myotoxicity (Illingworth et al., 2001; Brewer, 2003).

This toxicity is thought to result from the reduction

of isoprenylated metabolites such as ubiquinones,

dolichols or isoprenylated

Fig. 1. Cholesterol biosynthetic pathway and its

inhibitors.

proteins in tissues (Thibault et al., 1996; Flint et

al., 1997b; Bliznakov, 2002), but the precise mechanism

of statin-induced myotoxicity remains unclear.

Recently, we discovered a novel squalene synthase

inhibitor, lapaquistat acetate (hereafter abbreviated

to lapaquistat) called as TAK-475(1-[[(3R,5S)-1- (3-

acetoxy-2,2-dimethylpropyl)-7-chloro-5-(2,3-dimethoxy-

phenyl)-2-oxo-1,2,3,5-tetrahydro-4,1-benzoxazepin-3-yl]

acetyl]piperidin-4-yl)acetic acid) previously, which

lowered plasma cholesterol levels in various animals

(Amano et al., 2003; Nishimoto et al., 2003b) and

humans (Perez et al., 2006; Piper et al., 2006).

Squalene synthase catalyzes the conversion of farnesyl

diphosphate to squalene in the cholesterol bio-

synthetic pathway. Since farnesyl diphosphate is a

precursor of isoprenylated metabolites (Fig. 1), they

may be increased by lapaquistat. Thus, the combination

of lapaquistat with statins is expected to prevent the

decrease in isoprenylated metabolites by statins, which

may reduce the frequency of statin-induced myopathy. In

our previous work, T-91485, a pharmacologically active

metabolite of lapaquistat exhibited very little

cytotoxicity in human skeletal myocytes. In addition,

treatment with this compound attenuated statin-induced

cytotoxicity (Nishimoto et al., 2003a). In the present

study, we examined the effects of squalene synthase

inhibition on statin-induced myotoxicity in in vivo

models. Guinea pigs are a particularly useful model for

studying the lipid-lowering drugs because of their

similar- ities to humans in terms of hepatic

cholesterol and lipoprotein metabolism (Fernandez,

2001; West and Fernandez, 2004). We report that

lapaquistat offered near complete protection from

cerivastatin-induced myotoxicity in guinea pigs.

Materials and methods

Materials. Lapaquistat was synthesized by Takeda

Pharmaceutical Company Limited (Osaka, Japan). DL-

Mevalonolactone was purchased from Sigma Che- mical Co.

(St. Louis, MO, U.S.A.). Cerivastatin was purchased

from Sequoia

Research Products (Oxford, U.K.). Other chemicals were

purchased from Wako Pure Chemical Industries (Osaka,

Japan).

Animals. Male guinea pigs (Hartley strain, std grade)

were purchased from Japan SLC Inc. (Shizuoka, Japan).

They were fed a chow diet (Labo G diet; Nippon Nosan,

Kanagawa, Japan) and allowed access to water ad

libitum. All animal experiments were carried out

according to the Takeda Animal Care Guidelines.

Experimental design. To investigate the effect of

lapaquistat on cerivastatin- induced myotoxicity, five-

week-old male guinea pigs were assigned to three groups

to receive the vehicle (0.5% methylcellulose solution,

n=16), cerivastatin (1 mg/kg, n=16) and cerivastatin

(1 mg/kg, n=16) with lapaquistat (30 mg/kg). Drugs were

orally administered once daily for 14 days. On the 15th

day, the guinea pigs were anesthetized using diethyl

ether, and blood samples were collected. Skeletal

muscle samples were quickly exercised from two sites,

musculus biceps femoris and musculus quadriceps

femoris. The left femoral muscles were immediately

frozen on dry ice to measure squalene and cholesterol

levels. The right femoral muscles were fixed with 10%

neutral-buffered formalin and processed for

histopathological examination. Plasma total

cholesterol, creatine kinase (CK), myoglobin, alanine

aminotransferase (ALT) and aspartate aminotransferase

(AST) were measured using a biochemical autoanalyzer

(Hitachi 7070, Hitachi, Tokyo, Japan). To investigate

the effect of mevalonolactone on cerivastatin-induced

myotoxicity, five-week-old male guinea pigs were

assigned to three groups to receive the vehicle (0.5%

methylcellulose solution, n=10), cerivastatin (1 mg/

kg, n=10) and cerivastatin (1 mg/kg, n=10) with

mevalonolactone (100 mg/kg, b.i.d). Drugs were orally

administered once daily (cerivastatin and 0.5%

methylcellulose solution) or twice daily

(mevalonolactone) for 14 days. On the 15th day, the

animals were anesthetized using diethyl ether, and

blood samples were collected and plasma total

cholesterol, CK, myoglobin, ALTand ASTwere measured as

described above.

Histopathological examination. The muscles fixed with

10% neutral- buffered formalin were embedded in

paraffin, sectioned, stained with hema- toxylin and

eosin and examined by a microscope. Histopathological

evaluation was performed by a blinded manner, and the

severity of myofiber necrosis was evaluated according

to the following scoring system: grade 0, within normal

limits; grade 1, isolated single necrotic myofibers;

grade 2, scattered single necrotic myofibers; grade 3,

widespread single necrotic myofibers; grade 4,

widespread small groups of necrotic myofibers; grade 5,

diffuse myofiber necrosis. The severity of inflammation

was evaluated according to the following scoring

system: grade 0, within normal limits; grade 1,

scattered foci of mononuclear cell infiltration in

the endomysium (myophagia); grade 2, widespread

mononuclear cell infiltration and occasional fibroblast

proliferation in and around the endomysium; grade 3,

focal mixed inflammatory cell infiltration in and

around the muscle fascicles; grade 4, diffuse mixed

inflammatory cell infiltration in and around the muscle

fascicles. The rate of myofiber regeneration was

evaluated according to the following scoring system:

grade 0, within normal limits; grade 1, scattered

single regenerative myofibers; grade 2, widespread

single regenerative myofibers; grade 3, widespread

small groups of regenerative myofibers; grade 4,

diffuse myofiber regeneration.

Determination of muscular squalene and cholesterol

levels. We determined squalene and cholesterol levels

in the musculus quadriceps femoris by the method of

Grieveson et al. (1997) with slight modifications.

Muscle (200 mg) was homogenized in nine volumes of

distilled water using a Polytron mixer (PT1300D;

Kinematica AG, Littan/Luzern, Switzerland). One

milliliter of homogenate was saponified with 1 ml of

90% (w/v) KOH and 2 ml of ethanol at 70 °C for 90 min,

and lipids were extracted with petroleum ether. The

solvent was evaporated to dryness under a nitrogen gas

stream, and then the residue was dissolved in

acetonitrile. The solution was injected into a high-

performance liquid chromatography apparatus (LC-10;

Shimadzu Co., Kyoto, Japan), equipped with a Cadenza

CD-C18 (Imtakt, Kyoto, Japan), 3 μm column of 50×4.6 mm

using acetonitrile/water (98:2, v/v) as the mobile

phase at a flow rate of 1 ml/min. The absorbance of the

elution was monitored at 205 nm.

40 T. Nishimoto et al. / Toxicology and Applied

Pharmacology 223 (2007) 39–45

Toxicokinetics analysis of cerivastatin. Five-week-old

male guinea pigs were assigned to two groups to receive

cerivastatin (1 mg/kg, n=4) or cerivastatin (1 mg/kg,

n=4) with lapaquistat (30 mg/kg). Drugs were orally

administered once daily for 14 days. On the 15th day,

blood samples were collected. The plasma concentrations

of cerivastatin in guinea pigs were determined by high-

performance liquid chromatography/tandem mass

spectrometry according to the following methods.

Cerivastatin and the internal standard (atorvastatin)

were extracted from guinea pig plasma using a solid

phase extraction cartridge (Oasis HLB; Waters Co.,

Milford, MA). The solvent was evaporated to dryness

under a nitrogen gas stream, and then the residue was

dissolved in acetonitrile/0.01 mol/l ammonium

formate/formic acid (60:40:0.05, v/v/v). The solution

was injected into a high-performance liquid

chromatography apparatus (LC-10; Shimadzu Co., Kyoto,

Japan), equipped with a Cadenza CD-C18 column (Imtakt,

Kyoto, Japan), using acetonitrile/0.01 mol/l ammonium

formate/formic acid (60:40:0.05, v/v/v) as the mobile

phase. Cerivastatin was detected using a tandem mass

spectrometer (API3000; AB/MDS Sciex Instrument).

Statistical analysis. Statistical analysis was

performed using Student's t- test for biochemical

parameters in the plasma and muscle (vehicle vs. ceri-

vastatin and cerivastatin vs. combination). The grade

of necrosis, inflamma-

tion and regeneration in each muscle was summed in each

animal and then Wilcoxon's test was performed between

groups (vehicle vs. cerivastatin and cerivastatin vs.

combination). Correlation analysis was performed by

Pearson's methods to evaluate whether the plasma CK

level was a predictor of myotoxicity in this model. P-

values of b0.05 were considered statistically

significant.

Results

Protective effects of lapaquistat on cerivastatin-

induced myotoxicity

In our preliminary studies, CEV (0.5 and 2 mg/kg)

lowered plasma cholesterol levels by 40% and 61% and

increased plasma CK levels to 10- and 32-fold,

respectively, while lapaquistat (30 and 100 mg/kg)

lowered plasma cholesterol level by 31% and 53%,

respectively, without increasing plasma CK levels (−29%

and −25% compared to control, respectively). In the

current study, cerivastatin (1 mg/kg) alone decreased

plasma cholesterol

Fig. 2. Effects of co-administration of lapaquistat

with cerivastatin on plasma cholesterol (A), CK (B) and

myoglobin (C) levels. After 14-day administration of

drugs to guinea pigs, plasma cholesterol, CK and

myoglobin levels were determined as described in

Materials and methods. Data represent the mean±SE of 16

animals. *Pb0.01 vs. vehicle-treated group and #Pb0.01

vs. cerivastatin-treated group. Vehicle, 0.5%

methylcellulose; CEV, cerivastatin (1 mg/kg);

CEV+lapaquistat, cerivastatin (1 mg/kg)+lapaquistat (30

mg/kg).

Table 1 Incidence and severity of light microscopic

changes in skeletal muscle Drugsa Gradeb Incidence and

severity of myopathic changesc

Biceps femoris Quadriceps femoris

Necrosis Inflammation Regeneration Necrosis

Inflammation Regeneration

Vehicle 0 13 16 16 4 16 16 1 3 0 0 12 0 0 2 0 0 0 0 0 0

3 0 0 0 0 0 0 Cerivastatin 0 3 10 13 0 4 5 1 6 5 3 4 11

8 2 7 1 0 11 1 3 3 0 0 0 1 0 0 Cerivastatin+lapaquistat

0 15 16 16 3 16 16 1 1 0 0 13 0 0 2 0 0 0 0 0 0 3 0 0 0

0 0 0 a Vehicle, 0.5% methylcellulose; CEV,

cerivastatin (1 mg/kg); CEV+lapaquistat, cerivastatin

(1 mg/kg)+lapaquistat (30 mg/kg). b Diagnostic criteria

of histopathological changes in skeletal muscle are

described in Materials and methods. No animals were

more than grade 4 in all groups. c Number of animals in

each group is 16.

41T. Nishimoto et al. / Toxicology and Applied

Pharmacology 223 (2007) 39 –45

level by 45% and increased plasma CK level, a marker of

myotoxicity, to more than 10-fold (Figs. 2A, B). Since

our preliminary study indicated that using

cerivastatin it was difficult to induce

histopathological change in the musculus soleus predo-

minantly consisting of type I myofibers, we performed a

histopathological examination using the musculus

biceps femoris and quadriceps femoris that consisted of

type I and II fibers. Histopathological examination

revealed that cerivastatin significantly induced

histopathological changes, necrosis and regeneration

of myofibers and inflammation, in both muscles (Pb0.01,

Table 1, Fig. 3). The relationship between the plasma

CK levels and the cumulative grades of skeletal muscle

lesions was plotted (Fig. 4). The plasma CK levels were

well correlated with the grade of myotoxicity. While

co-administration of lapaquistat (30 mg/kg) slightly

enhanced the cholesterol- lowering effects of

cerivastatin (Fig. 2A), co-administration of

lapaquistat significantly suppressed the elevation of

plasma CK (Fig. 2B). According to histopathological

examination, co- administration of lapaquistat

remarkably prevented the myotoxicity induced by

cerivastatin (Pb0.01, Table 1). Plasma myoglobin level

is known as a typical marker of myotoxicity other than

plasma CK level. Cerivastatin (1 mg/kg) alone also

increased the levels of plasma myoglobin to 89-fold,

which was also significantly suppressed by co-

administration of lapaquistat (Fig. 2C). The plasma

myoglobin level was also well correlated with the

histopathological severity of myotoxicity (data not

shown). Cerivastatin also increased plasma ALT and AST

levels in the cerivastatin-treated group to 6.5- and

3.8- fold, respectively, and co-administration of

lapaquistat suppressed the increase in these

parameters, although we could not detect apparent

histopathological changes in the liver of animals

treated by either cerivastatin or lapaquistat. We could

not observe changes in body weight or behavior among

groups through this experiment.

Effects of co-administration of lapaquistat with

cerivastatin on muscular squalene and cholesterol

levels

Cerivastatin (1 mg/kg) alone significantly decreased

the muscular squalene level by 60%. Co-administration

of lapaquistat (30 mg/kg) significantly accentuated its

decrease

by cerivastatin (Fig. 5A). There were no differences in

muscular cholesterol levels among groups (Fig. 5B).

Protective effects of mevalonolactone on cerivastatin-

induced myotoxicity

In order to confirm that cerivastatin-induced

myotoxicity is caused by the inhibition of an HMG-CoA

reductase, we co- administered mevalonolactone (100

mg/kg, b.i.d.) with cerivastatin (1 mg/kg) to guinea

pigs for 14 days. Cerivastatin alone decreased plasma

cholesterol level by 58% and increased plasma CK and

myoglobin levels to 34- and 43-fold, respectively (Fig.

6). Co-administration of mevalonolactone completely

suppressed these elevations (Fig. 6).

Toxicokinetics of cerivastatin

The plasma concentration of cerivastatin was not

affected by co-administration of lapaquistat (30

mg/kg). There were no differences in the Tmax, Cmax and

area under the curve for 0 to 24 h (AUC0–24 h) values

of plasma cerivastatin between groups with and without

lapaquistat. Tmax values for cerivastatin were 0.5 and

0.6 h, Cmax values were 143.2 and 163.7 ng/ml and

Fig. 3. Photomicrographs of skeletal muscle from guinea

pigs. After 14-day administration, HE-stained section

of the musculus quadriceps femoris from cerivastatin-

treated animal (A) showed necrosis of myofibers and

inflammation (arrow head) and the regeneration of

myofibers (arrow). Sections of skeletal muscle from

vehicle-treated (B) and co-administration of

lapaquistat with cerivastatin-treated (C) animals

showed only scattered necrosis of myofibers. Original

magnification ×160.

Fig. 4. Relationship between cumulative grades of

skeletal muscle lesions (histopathology score) and

plasma CK levels in guinea pigs.



AUC0–24 h values were 500.5 and 575.1 ng h/ml with and

without lapaquistat, respectively.

Discussion

Many clinical studies have supported that aggressive

cholesterol-lowering therapy produces more benefits

than mild therapy; however, high-dose statins increase

the risk of toxicity such as myotoxicity (Illingworth

et al., 2001; Brewer, 2003). Indeed, one statin,

cerivastatin, was withdrawn from the world market due

to the high frequency of myotoxicity. In the present

study, we showed that high dose cerivastatin-induced

myotoxicity in guinea pigs. Interestingly,

lapaquistat remarkably prevented cerivastatin-induced

myotoxicity in this model. This is the first evidence

that a squalene synthase inhibitor prevented the

statin-induced myotoxicity in an in vivo animal model.

In addition, the supplementation of mevalonolactone

completely abolished the myotoxicity of cerivastatin,

suggesting that it was caused by the inhibition of an

HMG-CoA reductase. The mechanism by which the

inhibition of an HMG-

CoA reductase induces myotoxicity remains unclear.

Recently, Baker (2005) reviewed the relationship

between genetic defects in cholesterol biosynthetic

enzymes and skeletal myopathy. He suggested that the

genetic defects of terminal enzymes in cholesterol

synthesis, such as 3β-hydroxysterol-delta-24-reduc-

tase or sterol-delta-7-reductase (Fig. 1) cause various

pheno- types including mental retardation, but they do

not cause skeletal myopathy. On the other hand, genetic

defects of proximal enzymes in cholesterol

biosynthesis, such as mevalonate kinase (Fig. 1), are

associated with a reduction in isoprenoid metabolism

and skeletal myopathy. This suggests that cholesterol

depletion is not the primary cause of myotoxicity, but

the depletion of proximal metabolites in the

cholesterol biosynthetic pathway is the critical

cause of myotoxicity. In our study, cerivastatin

significantly induced myotoxicity, and a squalene

synthase inhibitor remarkably prevented cerivastatin-

induced myotoxicity in guinea pigs without any changes

of muscular cholesterol levels (Fig. 5B), strongly

suggesting that the depletion of cholesterol level

does not cause myotoxicity in guinea pigs. Furthermore,

cerivastatin (1 mg/kg) alone significantly

Fig. 5. Effects of co-administration of lapaquistat

with cerivastatin on muscular squalene (A) and

cholesterol (B) levels. After 14-day administration of

drugs to guinea pigs, muscular squalene and cholesterol

levels were determined as described in Materials and

methods. Data represent the mean±SE of 16 animals.

*Pb0.01 vs. vehicle-treated group and #Pb0.01 vs.

cerivastatin-treated group. Vehicle, 0.5%

methylcellulose; CEV, cerivastatin (1 mg/kg);

CEV+lapaquistat, cerivastatin (1 mg/ kg)+lapaquistat

(30 mg/kg).

Fig. 6. Effects of co-administration of mevalonolactone

with cerivastatin on plasma cholesterol (A), CK (B) and

myoglobin (C) levels. After 14-day administration of

drugs to guinea pigs, plasma cholesterol, CK and

myoglobin levels were determined as described in

Materials and methods. Data represent the mean±SE of 10

animals. *Pb0.01 vs. vehicle-treated group and #Pb0.01

vs. cerivastatin-treated group. Vehicle, 0.5%

methylcellulose; CEV, cerivastatin (1 mg/kg); CEV+MVA,

cerivastatin (1 mg/kg)+mevalonolactone (100 mg/kg,

b.i.d.).



decreased muscular squalene levels, and co-

administration of lapaquistat (30 mg/kg) significantly

accentuated the decreases by cerivastatin (Fig. 5A).

This result suggests that the depletion of post-

squalene metabolites is not the primary cause of

statin- induced myotoxicity. In consistent with this

observation, studies using rat skeletal muscle cells

also indicated that both squalene and squalene epoxide

synthesis inhibitors did not cause myotoxicity

(Matzno et al., 1997; Flint et al., 1997a). Based on

the mode of pharmacological action, lapaquistat can

restore the depletion of isoprenylated metabolites by

statins (Fig. 1). In our previous study, the

supplementation of geranylgeranyl diphosphate

improved statin-induced cytotoxicity in human skeletal

muscle cells (Nishimoto et al., 2003a). Johnson et al.

(2004) reported that statins inhibited protein

geranylgeranylation in rat skeletal muscle cells. They

also reported that geranylgeranyl transferase

inhibitors could cause cytotoxicity in rat myocytes.

These results suggest that the depletion of protein

geranylgeranylation is one of the dominant mechanisms

of statin-induced myotoxicity. Now we are investigating

the effects of statins on muscular levels of

isoprenoids and geranylgeranylated protein. Clinical

pharmacokinetics study showed that AUC value of plasma

cerivastatin was 69.9 ng h/ml at a dose of 0.8 mg,

which is the maximum dose in humans (Stein et al.,

1999). The plasma level of cerivastatin observed in

this study was about seven times higher than clinical

levels; thus, the dose level used in this study was

relatively high. Some drugs have been reported to

increase the plasma concentration of cerivastatin by

drug–drug interaction (Plosker et al., 2000; Backman et

al., 2002), one of which, gemfibrozil, was reported to

increase the plasma concentration of cerivastatin to

about 4-fold in a clinical study (Backman et al.,

2002). Taken together, the dose level adopted in this

study is not far from that in the clinical setting. In

conclusion, we confirmed that co-administration of

lapaquistat remarkably prevented the myotoxicity

induced by an HMG-CoA reductase inhibitor,

cerivastatin, in guinea pigs. The present study

suggests that a squalene synthase inhibitor, lapa-

quistat, combined with statins is expected to provide a

novel approach for aggressive cholesterol-lowering

therapy with re- duced risk of statin-induced

myotoxicity.

Acknowledgments

We thank Drs. Yoshimi Imura, Hideaki Nagaya and Takeo

Wada in our laboratories for their continuous advice on

com- pleting this manuscript. We thank Dr. Masanori

Yoshida from Takeda Analytical Research Laboratories,

Ltd. for the determination of the plasma

concentration of cerivastatin. We also thank Dr.

William R. Lagor in the University of Pennsylvania

School of Medicine for his valuable advice on revising

this manuscript.

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