Inhibition of interferon-gamma-mediated microvascular endothelial cell major histocompatibility complex class II gene activation by HMG-CoA reductase inhibitors.

Bender JR.

Section of Cardiovascular Medicine, Yale University, New Haven, Connecticut 06520, USA.

BACKGROUND: Graft vascular disease, a major cause of late graft failure in cardiac transplant patients, is associated with the presence of class II major histocompatibility complex molecules on the endothelium. 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, e.g., simvastatin, have been shown to reduce the incidence of graft vascular disease. We studied the effect of simvastatin on interferon (IFN)-gamma-induced human leukocyte antigen (HLA)-DR expression in human microvascular endothelial cells (MVECs). METHODS AND RESULTS: Simvastatin pretreatment inhibited MVEC HILA-DR induction by IFN-gamma, as detected by flow cytometry. Simvastatin's inhibitory effect was reversed by the cholesterol synthesis pathway intermediates mevalonate and geranylgeranyl pyrophosphate but not squalene, indicating the involvement of protein prenylation in this process. Reverse transcription-polymerase chain reaction analysis demonstrated that induction of class II transactivator (CIITA), and consequently, HLA-DRalpha mRNA, is abrogated by simvastatin. Although signal transducer and activator of transcription (STAT)-1 is a critical CIITA gene transactivator, immunofluorescence studies, Western blotting, and electrophoretic mobility shift assays demonstrated that IFN-gamma-induced STAT-1 phosphorylation, nuclear translocation, and DNA binding are not affected by simvastatin. However, simvastatin inhibited IFN-gamma-induced transactivation of a CIITA promoter IV reporter construct, indicating the involvement of this promoter in the inhibitory effect of simvastatin. CONCLUSIONS: Simvastatin pretreatment inhibits CIITA and consequent HLA-DR induction by IFN-gamma in MVECs through interference with protein prenylation. This inhibitory effect occurs at the level of transcription and is directed, at least in part, at the CIITA promoter IV. These results explain some of the beneficial effects of HMG-CoA reductase inhibitors in cardiac transplantation.

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Mutual amplification of apoptosis by statin-induced mitochondrial stress and doxorubicin toxicity in human rhabdomyosarcoma cells.

Hohenegger M.

Institute of Pharmacology, Medical University of Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria.

Besides their cholesterol-lowering effect, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) show antiproliferative behaviour, which has been suggested as a promising anticancer strategy. However, the signalling cascades leading to statin-induced death of cancer cells are poorly characterized. Here we show that statins activate the mitochondrial pathway of apoptosis in rhabdomyosarcoma RD cells via translocation of Bax from the cytosol to mitochondria. The prototypical representative of statins, simvastatin, induced consecutive activation of caspase 9 and 3 in a concentration-dependent manner. The permeability transition pore inhibitor bongkrekic acid was capable of completely preventing simvastatin-induced caspase 9 and 3 activity, corroborating the mitochondrial pathway of apoptosis as the sole mechanism of statin action. Alternative pathways via death receptors, that is, caspase 8 or calpain activation, were not triggered by simvastatin. Simvastatin-treated RD cells could be completely rescued from apoptosis by the co-application of mevalonic acid, indicating that deprivation of cholesterol precursors is essential for statin-induced apoptosis. However, pretreatment with subthreshold concentrations of simvastatin was sufficient to augment doxorubicin toxicity via the mitochondrial apoptotic machinery. Moreover, the presence of doxorubicin increased the potency of simvastatin to trigger caspase activation. Taken together, these data highlight the therapeutic anticancer potential of statins and their additivity and mutual sensitization, in combination with doxorubicin in human rhabdomyosarcoma cells. British Journal of Pharmacology (2004).

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HMG-CoA reductase inhibitor stabilizes rabbit atheroma by increasing basal NO and decreasing superoxide.

Iguchi A.

Department of Geriatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

Male rabbits fed a 0.5% cholesterol diet for 8 wk were divided into three groups. Group 1 was hypercholesterolemic; group 2 was fed a regular diet for an additional 12 wk; and group 3 was fed a regular diet with simvastatin (5 mg x kg(-1) x day(-1)). Simvastatin treatment reduced the atherosclerotic area and total and esterified cholesterol concentrations in the thoracic aorta. Tone-related basal nitric oxide (NO) release was highest in group 3. Acetylcholine-induced, NO-dependent relaxation was improved in group 3 compared with group 2. Amount of endothelial nitric oxide synthase (eNOS) mRNA in vessels increased in group 1, compared with normal aorta, and decreased in group 2; however, it did not decrease in group 3. The amount of O released from vessels increased in group 1 and group 2 compared with normal rabbits; however, it decreased in group 3, especially in the endothelial cells. Peroxynitrite determined by nitrotyrosine staining decreased in group 3. Additionally, the arteries of rabbits fed a regular diet with or without simvastatin were investigated. The aorta from simvastatin-treated group showed increase of tone-related basal NO release and eNOS mRNA and decrease of O release. Taken together, upregulation of eNOS and decrease of O treatment were observed in vivo in the process of the sufficient stabilization of atheroma following simvastatin.

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Drug interaction between simvastatin and itraconazole in male and female rats.

Sugiyama Y.

Drug Metabolism and Pharmacokinetics Research Laboratories, New Drug Development Division and Product Strategy Department, Sankyo Co., Ltd., Shinagawa-ku, Tokyo, Japan.

Taking into account the species and sex differences in drug interactions based on the inhibition of cytochrome P450 (P450)-mediated drug metabolism, we examined whether the interaction between simvastatin and itraconazole observed in humans could also occur in rats, the most commonly used animal species for pharmacokinetic studies. Itraconazole inhibited the in vitro metabolism of simvastatin in female rat liver microsomes, but not in male rat liver microsomes. Using anti-P450 antisera, the main P450 isozyme responsible for the metabolism of simvastatin was identified as CYP3A in female rats and CYP2C11 in male rats. Therefore, the sex difference in the inhibition of simvastatin metabolism by itraconazole seems to be caused by a difference in the P450 isozymes responsible for the metabolism of simvastatin in male and female rats and the different ability of itraconazole to inhibit CYP3A and CYP2C11. In addition, the effect of itraconazole on the pharmacokinetics of simvastatin in rats was also investigated. The area under the curve value of simvastatin was increased approximately 1.6-fold by the concomitant use of itraconazole (50 mg/kg) in female rats, whereas in male rats, itraconazole had no effect. In conclusion, it was found that the results obtained in male rats did not reflect the results in humans as far as the inhibition of simvastatin metabolism by itraconazole was concerned. The P450 isozymes involved in the metabolism of drugs should be taken into consideration when rats are used as a model animal for humans in the investigation of drug interactions.

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Inhibitory effects of verapamil and diltiazem on simvastatin metabolism in human liver microsomes.

Yeo WW.

Section of Clinical Pharmacology & Therapeutics, University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield. k.r.yeo sheffield.ac.uk

AIMS: To determine the effects of verapamil and diltiazem on simvastatin metabolism in human liver microsomes and to compare their inhibitory potencies and CYP3A4 inactivation parameters with those reported previously for mibefradil. METHODS: Simvastatin metabolism was investigated in human liver microsomes in the presence and absence of verapamil or diltiazem (0.1-250 microM). Kinetics of CYP3A4 inactivation by verapamil and diltiazem were determined using testosterone as the substrate. RESULTS: When verapamil was coincubated with simvastatin, IC50 values ranged from 23 to 26 microM for all major metabolites. The IC50 values ranged from 4.8 to 5.6 microM on preincubation of verapamil for 30 min in the presence of an NADPH-generating system. Corresponding IC50 values for diltiazem ranged from 110-127 microM and from 21-27 microM, respectively. Verapamil and diltiazem inhibited testosterone 6beta-hydroxylation in a time- and concentration-dependent manner, key features of mechanism-based inactivation. Values for the inactivation parameters kinact and KI were 0.15 +/- 0.04 min-1 (mean +/- s.d.) and 2.9 +/- 0.6 microM, respectively, for verapamil and 0.07 +/- 0.01 min-1 and 3.3 +/- 1.5 microM, respectively, for diltiazem. CONCLUSIONS: The IC50 values for coincubation of verapamil and diltiazem were 46- and 220-fold higher, respectively, than those reported previously for mibefradil, and 16- and 71-fold higher, respectively, for preincubation. Thus, the results of this study suggest that verapamil and diltiazem are less likely than mibefradil to cause acute drug interactions with simvastatin in vivo. However, verapamil and diltiazem are moderate mechanism-based inhibitors of CYP3A4 and therefore may still cause significant inhibition of simvastatin metabolism in vivo during chronic therapy.

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The combined effect of inhibiting both ACAT and HMG-CoA reductase may directly induce atherosclerotic lesion regression.

Sliskovic DR.

Department of Cardiovascular Therapeutics, Pfizer Global Research and Development, Ann Arbor Laboratories, Pfizer Inc., 2800 Plymouth Road, Ann Arbor, MI 48105, USA. thomas.bocan pfizer.com

We hypothesized that coadministration of avasimibe and simvastatin would limit size, composition and extent of atherosclerotic lesions and potentially promote lesion regression, since bioavailable ACAT inhibitors decrease monocyte-macrophage enrichment and HMG-CoA reductase inhibitors limit smooth muscle cell migration and proliferation. Male New Zealand white rabbits were sequentially fed a 0.5% cholesterol, 3% peanut oil, 3% coconut oil diet for 9 weeks and a chow-fat diet for 6 weeks prior to drug administration. A time zero control group was necropsied prior to drug administration and the progression control was fed various diets but untreated. Avasimibe (10 mg/kg), simvastatin (2.5 mg/kg) or combination of avasimibe (10 mg/kg) with simvastatin (2.5 mg/kg) were administered in the chow-fat diet for 8 weeks. Plasma total cholesterol exposure was unchanged by avasimibe but was reduced 21% by both simvastatin alone and in combination with avasimibe. Combination of avasimibe and simvastatin decreased VLDL-cholesterol exposure by 56%. VLDL+IDL lipid composition was similar in the progression control and simvastatin-treated animals. Administration of avasimibe alone or in combination with simvastatin reduced the cholesteryl ester fraction and increased the triglyceride fraction to comparable extents. Relative to the progression control, avasimibe plus simvastatin markedly decreased thoracic aortic cholesteryl ester content and lesion coverage by 50% and aortic arch lesion size and macrophage area by 75 and 73%, respectively. With respect to lesion regression, avasimibe+simvastatin decreased aortic arch lesion size by 64% and monocyte-macrophage area by 73% when compared to time zero. Based on these data, we conclude that despite changes in plasma total and lipoprotein cholesterol exposure and lipoprotein composition comparable to monotherapy, inhibition of both ACAT and HMG-CoA reductase may not only directly blunt lesion progression but also promote regression of pre-established atherosclerotic lesions.

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