REGULATION OF HEPATIC P450S BY LOVASTATIN AND OXYSTEROLS

3-羟基-3-甲基戊二酰辅酶A抑制剂洛伐他汀 还原酶被广泛用于治疗高胆固醇血症。 冠状动脉疾病发展的主要危险因素。近期 该实验室的证据表明，洛伐他汀也是一种 细胞色素P450在培养大鼠肝细胞中的新诱导剂 单独给药时，P450 2B2和4A1的mRNAs，并增强 苯巴比妥对培养细胞2B1/2和3A1基因的诱导作用 或者克霉唑。洛伐他汀显著诱导培养的P450 3A6。 兔肝细胞，与现有的人类数据一致。感应式或 洛伐他汀增强人类P45O的诱导可能 降低其治疗效果或增加其毒性 同时给药。这项提议的假设是 洛伐他汀通过完全抑制胆固醇的生物合成，防止 抑制P450的特异性氧化甾醇的合成和积累 表情。推而广之，其他改变P450表达的化学物质， 如咪唑类抗真菌药物，可抑制甾醇中的酶 代谢途径，导致调节氧固醇水平的变化。 这项建议的具体目的是： (1)确定HMG-CoA还原酶抑制剂(即，洛伐他汀， 普伐他汀、辛伐他汀和Bmy-22089对大鼠肺泡灌洗液中2B、3A类分子表达的影响 和4A P45Os或兔P450 3A6的mRNA(Northern杂交分析)， 蛋白质(通过蛋白质印迹分析)和酶活性水平 原代培养的肝细胞， (2)明确洛伐他汀对大鼠肺泡巨噬细胞B、3A、B类分子表达的影响。 4A P45O和兔P450 3A6在mRNA、蛋白和酶活性上的差异 体内的水平， (3)确定洛伐他汀对P450影响的一般机制 表达(如目标1和2中所定义)，酌情审查， 处理对转录速率的影响(通过核运行 分析)，信使核糖核酸稳定性(通过测量抑制后的信使核糖核酸水平 转录)、蛋白质稳定性(通过脉冲追逐实验)或酶 活动, (4)确定氧合甾醇治疗的效果(即甲氧戊酸，25- 羟基胆固醇、低密度脂蛋白和角鲨素1) 大鼠2B、3A和4A类P45O或兔P450 3A6在 培养肝细胞的mRNA、蛋白质和酶活性水平 (5)观察洛伐他汀和咪唑类抗真菌药物对血管紧张素转换酶的影响。 [~(14)C]标记的乙酸酯或甲氧丙戊酸掺入氧甾醇 高效液体培养大鼠或兔肝细胞 层析，并将特定氧化甾醇的变化与 P450的表达。 这项工作的意义在于它将提供以下信息 可能会影响洛伐他汀在人体内的安全性和有效性 允许对肝脏P450细胞基础的新假说进行检验 归纳，并可能确定更多发展的新目标 有效和/或毒性较低的降胆固醇药物。

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.