REGULATION OF HEPATIC P450S BY LOVASTATIN AND OXYSTEROLS

洛伐他汀，3-羟基-3-甲基戊二酰（HMG）-CoA抑制剂 还原酶，广泛用于治疗高胆固醇血症， 冠心病的主要危险因素。最近 该实验室的证据表明，洛伐他汀也是一种 在培养的大鼠肝细胞中细胞色素P450的新诱导剂，诱导 P450 2B 2和4A 1 mRNA单独给药时， 在与苯巴比妥共处理的培养物中诱导2B 1/2和3A 1 mRNA 或克霉唑。洛伐他汀对P450 3A 6有明显的诱导作用。 兔肝细胞，与现有人类数据一致。感应或 用洛伐他汀处理的人中P450的增强诱导可以 降低治疗效果或增加毒性， 同时服用药物。这一提议的假设是， 洛伐他汀通过完全抑制胆固醇的生物合成， 抑制P450特定氧化固醇的合成和积累 表情推而广之，其他修饰P450表达的化学物质， 例如咪唑类抗霉菌药物，可以抑制甾醇中的酶， 代谢途径，导致调节氧化固醇水平改变。 这项建议的具体目标是： (1)定义了HMG-CoA还原酶抑制剂的作用（即，洛伐他汀， 普伐他汀、辛伐他汀和BMY-22089）对大鼠2B类、3A类 和4A P450或兔P450 3A 6的mRNA（通过北方印迹分析）， 蛋白质（通过Western印迹分析）和酶活性水平， 原代培养的肝细胞， (2)确定洛伐他汀对大鼠2B、3A和 4A P450或兔P450 3A 6的mRNA、蛋白和酶活性 体内水平， (3)确定洛伐他汀对P450影响的一般机制 表达（如目标1和2所定义），酌情审查， 处理对转录速率的影响（通过核运行） 分析）、mRNA稳定性（通过测量抑制后的mRNA水平）、 转录）、蛋白质稳定性（通过脉冲追踪实验）或酶 活动， (4)定义氧甾醇治疗的效果（即，甲羟戊酸，25- 羟基胆固醇、低密度脂蛋白和角鲨素1） 大鼠2B、3A和4A类P450或兔P450 3A 6在 培养肝细胞中的mRNA、蛋白质和酶活性水平， (5)检查洛伐他汀和咪唑类抗霉菌药物对 将[14 C]标记的乙酸盐或甲羟戊酸盐掺入氧甾醇中， 高效液相色谱法培养大鼠或兔肝细胞 色谱法，并将特定氧固醇的变化与 P450表达。 这项工作的意义在于它将提供信息， 可能会影响洛伐他汀在人体中的安全性和有效性， 允许测试肝P450的细胞基础的新假设 诱导，并可能确定新的目标，为发展更多的 有效和/或毒性较小的降胆固醇药物。

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, is widely used in the treatment of hypercholesterolemia, a major risk factor for the development of coronary artery disease. Recent evidence from this laboratory has demonstrated that lovastatin is also a novel inducer of cytochromes P450 in cultured rat hepatocytes, inducing P450 2B2 and 4A1 mRNAs when administered alone, and potentiating the inductions of 2B1/2 and 3A1 mRNAs in cultures cotreated with phenobarbital or clotrimazole. Also, lovastatin markedly induced P450 3A6 in cultured rabbit hepatocytes, consistent with existing human data. Induction or potentiated induction of P45Os in humans treated with lovastatin could decrease the therapeutic efficacies, or increase the toxicities, of simultaneously administered drugs. The hypothesis of this proposal is that lovastatin, by completely inhibiting cholesterol biosynthesis, prevents the synthesis and accumulation of specific oxysterols that suppress P450 expression. By extension, other chemicals that modify P450 expression, such as the imidazole antimycotic drugs, may inhibit enzymes in the sterol metabolic pathway, resulting in altered levels of regulatory oxysterols. The specific aims of this proposal are to: (1) define the effects of HMG-CoA reductase inhibitors (i.e., lovastatin, pravastatin, simvastatin and BMY-22089) on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA (by Northern blot analysis), protein (by Western blot analysis) and enzymatic activity levels in primary cultured hepatocytes, (2) define the effects of lovastatin on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA, protein and enzymatic activity levels in vivo, (3) determine the general mechanisms for the effects of lovastatin on P450 expression (as defined in aims 1 and 2) by examining, as appropriate, effects of treatments on rates of transcription (by nuclear run on analysis), mRNA stability (by measuring mRNA levels after inhibition of transcription), protein stability (by pulse-chase experiments) or enzyme activity, (4) define the effects of oxysterol treatments (i.e., mevalonate, 25- hydroxycholesterol, low density lipoproteins and squalistatin 1) on expression of rat class 2B, 3A and 4A P45Os or rabbit P450 3A6 at the mRNA, protein and enzymatic activity levels in cultured hepatocytes and (5) examine the effects of lovastatin and imidazole antimycotic drugs on incorporation of [14C)labeled acetate or mevalonate into oxysterols in cultured rat or rabbit hepatocytes by high performance liquid chromatography, and correlate changes in specific oxysterols to changes in P450 expression. The significance of this work is that it will provide information that could impact on the safety and efficacy of lovastatin in humans, will allow testing of a new hypothesis for the cellular basis of hepatic P450 induction, and may identify new targets for the development of more effective and/or less toxic hypocholesterolemic drugs.