Association Study Of CYP3A4*18B, CYP3A5*3 And MDR1C3435T Genetic Polymorphism With Lipid-lowering Efficacy Of Atorvastatin

Background and Objective3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme of cholesterol formation in the liver and other tissues, which plays an important role in cholesterol synthesis. Atorvastatin is a kind of HMG-CoA reductase inhibitor. It has been widely used for the treatment of hypercholesterolaemia and hypertriglyceridaemia. In vitro studies showed that atorvastatin has affinity for CYP3A4, the metabolism of atorvastatin in human liver microsomes is catalyzed primarily (≥80%) by CYP3A4/5. Atorvastatin is also a substrate for P-glycoprotein (P-gp). P-gp is encoded by multidrug-resistance 1 (MDR1). The interindividual heterogeneity in enzymatic activity of CYP3A4 as well as CYP3A5 and P-gp in the small intestine and liver has contributed to the variability on lipid-lowering efficacy of atorvastatin in Chinese hyperlipidemic patients. Several studies showed that the lipid-lowering efficacy of atorvastatin were influenced by CYP3A5*3 and MDR1C3435T polymorphisms. However, there are no report on the association of CYP3A4*18B, CYP3A5*3 and MDR1C3435T genotype with the lipid-lowering efficacy of atorvastatin in Chinese hyperlipidemic patients. Our research aims to determine the frequencies of CYP3A4 alleles (CYP3A4*18B), CYP3A5 alleles (CYP3A5*3),MDR1 alleles (MDR1C3435T) in Chinese hyperlipidemic patients and to observe the impact of CYP3A4*18B, CYP3A5*3 and MDR1C3435T genetic polymorphism on lipid-lowering efficacy of atorvastatin. The present study provides an important foundation and theoretical evidence for the gene-directed rationalization and individualization of medication in the hyperlipidemic patients.Materials and Methods Subjects A group of 221 unrelated Han Chinese hyperlipidemic patients(92 men, 119 women) (triglycerides(TG)>1.78 mmol/l or total cholesterol(TC)>6mmol/l or low-density lipoprotein cholesterol (LDL-C) >3.36mmol/l or high-density lipoprotein cholesterol(HDL-C)<0.82mmol/l) with a mean age 53±7.9 years were recruited from hospitalized patients and non-hospitalized patients in the first affiliated hospital of Zhengzhou University and Pingdingshan first municipal people's hospital. These participants were healthy, as assessed by medical history, clinical laboratory test results of liver and kidney function, blood test for human hepatitis B or C. They didn't use any lipid-lowering drug at least 2 weeks before intake of atorvastatin. The study design was approved by the Committee on Human Research of Zhengzhou University.Genotyping Venous blood (2-3ml) was obtained from the unrelated hyperlipidemic patients. DNA was isolated from peripheral leucocytes by classic hydroxybenzene-chloroform methods. Genotyping of CYP3A4*18B, CYP3A5*3 and MDR1C3435T alleles were conducted by the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Several samples each different genotype were sequenced to confirm the expected squences of each genotype.Evaluation of lipid-lowering efficacy of atorvastatin The hyperlipidemic patients were treated with 20 mg atorvastatin daily for 4 weeks. Serum triglyceride(TG), total cholesterol(TC), low density lipoprotein cholesterol(LDL-C) and high density lipoprotein(HDL-C) levels were determined using an automated analyzer(Roche 800, Japan). The primary effects measured at week 4 were the mean percentage change in TC, TG, LDL-C and HDL-C from baseline. We observed the effect of CYP3A4*18B, CYP3A5*3 and MDR1C3435T genetic polymorphism on the lipid-lowering efficacy of atorvastatin.Statistical analysis The hyperlipidemic patients were divided into three groups according to the genotypes, and allele frequencies were estimated from the observed numbers of each specific allele. SPSS 10.0 software was used for statistical analyses. Chi-square test was used to verify Hardy-Weinberg equilibrium. Interethnic differences in allelic frequencies were analyzed by Chi-square test or Fisher's exact test of probabilities. ANOVA was used to compare the differences on the lipid-lowering effects of atorvastatin, LSD was used to compare the two groups. P-values <0.05 were considered statistically significant.ResultsFrequencies of CYP3A4*18B, CYP3A5*3 and MDR1C3435T The frequencies of CYP3A4*18B, CYP3A5*3 and MDR1C3435T in Chinese hyperlipidemic patients were 27.9%, 73.7% and 39.9%, respectively. The allelic frequency was consistent with Hardy-Weinberg equilibrium. The allelic frequency of CYP3A4*18B in Chinese hyperlipidemic patients was similar to that reported in Japanese(24.9%)(P>0.05). The allelic frequency of CYP3A5*3 in Chinese hyperlipidemic patients was in accordance with that reported in Japanese(76.8%)(P>0.05), but lower than Caucasian(91.7%)(P<0.05), higher than African(47.5%)(P < 0.05). The allelic frequency of MDR1C3435T in Chinese hyperlipidemic patients was lower than British Caucasian(48%)(P<0.05) and southeast Asian (66%)(P<0.05), higher than normal African(16%)(P < 0.05).Effect of CYP3A4*18B, CYP3AS*3, MDR1C3435T genetic polymorphism on lipid-lowering efficacy of atorvastatin After oral intake of atorvastatin 20 mg daily for 4 weeks, the change of serum lipid in CYP3A4*18B/*18B, CYP3A4*1/*18B or CYP3A4*1/*1 groups showed a significant difference, with a mean decrease in TC of 20.9%±5.0% versus 17.8%±3.8%(P<0.01) or 16.8%±3.3% (P< 0.01). There was also a statistically significant difference in the reduction of TC between subjects carrying the CYP3A4*1/*18B and CYP3A4*1/*1 genotype (17.8%±3.8% versus 16.8%±3.3%, P>0.05). Furthermore, there was a statistically significant difference in the increase of HDL-C between subjects carrying the CYP3A4*1/*18B and CYP3A4*18B/*18B genotype (-2.4%±7.8% versus 3.6%±10.4%, P>0.05). However, we found no statistically significant differences in the increase of HDL-C between subjects carrying the CYP3A4*18B/*18B and CYP3A4*1/*1 genotype (3.6%±10.4% versus 1.2%±10.1%, P>0.05). In contrast, there was no significant relation between lipid-lowering efficacy of atorvastatin with CYP3A5*3 or MDR1C3435T polymorphisms (P>0.05).Effect of haplotype on lipid-lowering efficacy of atorvastatin For CYP3A4/CYP3A5 haplotype, the change of serum lipid in hyperlipidemic patients possessing CYP3A4*18B/CYP3A5*3 haplotype(CYP3A4*1/*18B or CYP3A4*18B/*18B, CYP3A5*1/*3 or CYP3A5*3/*3), possessing CYP3A4*1/CYP3A5*3 haplotype(CYP3A4*1/*1, CYP3A5*1/*3 or CYP3A5*3/*3) and possessing CYP3A4*18B/CYP3A5*1 haplotype(CYP3A4*1/*18B or CYP3A4*18B/*18B, CYP3A5*1/*1) showed a significant difference, with a mean decrease in TC of 18.5%±4.1% versus 16.9%±3.2%(P<0.05) or 16.3%±2.8% (P<0.05). For CYP3A4/MDR1 haplotype, the change of TC level in the hyperlipidemic patients possessing CYP3A4*18B/MDR13435T haplotype (CYP3A4*1/*18B or CYP3A4*18B/*18B, CT or TT) and hyperlipidemic patients possessing CYP3A4*1/MDR13435T haplotype(CYP3A4*1/*1, CT or TT) showed a significant difference(18.4%±4.2% versus 16.6%±3.0%, P < 0.05). For CYP3A5/MDR1C3435T haplotype, we found no statistically significant differences in the change of lipid level (TG, TC, LDL-C, HDL-C) in hyperlipidemic patients possessing CYP3A5*3/MDR13435T haplotype(CYP3A5*1/*3 or CYP3A5*3/*3, CT or TT), CYP3A5*3/MDR13435CC haplotype(CYP3A5*1/*3 or CYP3A5*3/*3, CC) or CYP3A5*1/MDR13435T haplotype(CYP3A*1/*1, CC).Conclusions1. The frequencies of CYP3A4 alleles (CYP3A4*18B), CYP3A5 alleles (CYP3A5*3) and MDR1 alleles (MDR1C3435T) in Chinese hyperlipidemic patients are 27.9%, 73.7%, 39.9%, respectively.2. The carrying CYP3A4*18B increase the lipid-lowering efficacy of atorvastatin. CYP3A5*3 and MDR1C3435T genetic polymorphism may have no significant effect on atorvastatin treatment.3. For CYP3A4/CYP3A5 haplotype, CYP3A4*18B/CYP3A5*3 increase the lipid-lowering efficacy of atorvastatin. For CYP3A4/MDR1 haplotype, CYP3A4*18B/MDR13435T has a significant effect on the lipid-lowering efficacy of atorvastatin. For CYP3A5/MDR1 haplotype, CYP3A5*3/MDR1C3435T has no significant effect on the lipid-lowering effects of atorvastatin.