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 March 2007; revised 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 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-2,2dimethylpropyl)-7-chloro-5-(2,3-dimethoxy- phenyl)-2-oxo-1,2,3,5-tetrahydro-4,1benzoxazepin-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 biosynthetic 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 Chemical 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 cerivastatininduced myotoxicity, five-week-old male guinea pigs were assigned to three groups to receive the vehicle (0.5% methylcellulose solution, n=16), ceri- vastatin (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 mono- nuclear 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 ml of 90% (w/v) KOH and 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), μm column of 50×4.6 mm using acetonitrile/water (98:2, v/v) as the mobile phase at a flow rate of 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 ceri- vastatin (1 mg/kg, n=4) with lapaquistat (30 mg/kg) Drugs were orally admi- nistered 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 dry- ness under a nitrogen gas stream, and then the residue was dissolved in ace- tonitrile/0.01 mol/l ammonium formate/formic acid (60:40:0.05, v/v/v) The solution was injected into a highperformance liquid chromatography appa- ratus (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, inflammation 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 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 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 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 13 16 16 16 16 0 12 0 0 0 0 0 0 0 Cerivastatin 10 13 11 11 3 0 0 Cerivastatin+lapaquistat 15 16 16 16 16 1 0 13 0 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 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 pre- liminary 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 sig- nificantly induced histopathological changes, necrosis and re- generation 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, coadministration of lapaquistat remarkably prevented the myo- toxicity 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 lapaqui- stat (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 sup- pressed 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 cer- ivastatin (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 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 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 Relationship between cumulative grades of skeletal muscle lesions (histopathology score) and plasma CK levels in guinea pigs 42 T Nishimoto et al / Toxicology and Applied Pharmacology 223 (2007) 39–45 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 cho- lesterol-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 myotoxi- city 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 not cause skeletal myopathy On the other hand, genetic defects of pro- ximal enzymes in cholesterol biosynthesis, such as mevalonate kinase (Fig 1), are associated with a reduction in isoprenoid metabolism and skeletal myopathy This suggests that choles- terol depletion is not the primary cause of myotoxicity, but the depletion of proximal metabolites in the cholesterol biosyn- thetic 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 deple- tion of cholesterol level does not cause myotoxicity in guinea pigs Furthermore, cerivastatin (1 mg/kg) alone significantly Fig 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 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.) 43T Nishimoto et al / Toxicology and Applied Pharmacology 223 (2007) 39 –45 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 myo- toxicity (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 dipho- sphate 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 geranylger- anylation 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 determi- nation 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 References Amano, Y., Nishimoto, T., Tozawa, R., Ishikawa, E., Imura, Y., Sugiyama, Y., 2003 Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia Eur J Pharmacol 466, 155–161 Anderson, K.M., Castelli, W.P., Levy, D., 1987 Cholesterol and mortality 30 years of follow-up from the Framingham Study J Am Med Assoc 257, 2176–2180 Backman, J.T., Kyrklund, C., Neuvonen, M., Neuvonen, P.J., 2002 Gemfibrozil greatly increases plasma concentration of cerivastatin Clin Pharmacol Ther 72, 685–691 Baker, 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TAK-475; Statin; Myotoxicity; Squalene synthase... 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 biosynthetic pathway Since farnesyl... 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