| ObjectiveEfavirenz (EFV) is a potent nonnucleoside reverse transcriptase inhibitor for the treatment of HIV type Ⅰ (HIV-Ⅰ). The main enzyme that metabolizes efavirenz is P4502B6(CYP2B6). The CYP2B6gene is highly polymorphic and genotyping for functional single nucleotide polymorphisms (SNPs) has proven to be useful in the prediction of efavirenz pharmacokinetics. This study would provide a theoretical evidence for the impact of CYP2B6polymorphisms on efavirenz pharmacokinetics and provide the basis for the drug of efavirenz on the clinical use, realizing individualized medication.Method1. SubjectsWe filtered40healthy Chinese subjects including26males and14females enrolled this sduty. Before enrollment, all subjects underwent clinical screening. Pregnancy tests were negative.All the subjects did not smoke and drink. The blood in the urine routine, liver and kidney function and electrocardiogram examination, the examination indexes were normal. All subjects had no history of drug allergy, and experiments during the two weeks before participate in the test had not taken any other drugs2. Study Design and ProceduresThe subjects were fasted for10hours before drug administration. Each subject received a single dose of600mg efavirenz tablet with240ml warm water at7a.m. Serial blood samples for determination of nifedipine plasma concentration were obtained at time0,0.5,1,2,3,3.5,4,4.5,5,5.5,6,8,12,16,24,48,72,96,120,144,168hours after administration. The samples were centrifuged and the plasma was transferred to polyethylene tubes and stored at-70℃until assayed. All subjects received standardized meals at6hours after drug administration.3.GenotypingThe genome DNA was exacted by hydroxybenzene-chloroform methods from venous blood samples. Genotypes were determined by sequencing of PCR products.4. Determination of efavirenz plasma ConcentrationsSample analysis of efavirenz concentrations in human plasma were decteced by a validated LC-MS/MS. Liquid chromatography conditions:Chromatography was performed on a Phenomenex Synergi4u, Polar-RP (2.0x50mm,4μm) column and the column temperature was25℃. The mobile phase consisted of ammonium acetateC0.8g·L-1, PH=7.5)/acetonitrile(50:50v/v) at a flow rate of1.0ml min-1. Injection volume was20μl. The mass spectrometric condition:The mass spectromter was operated using an electrospray sourse configured to negativeion mode (EIS-) and acquisition was none using multiple reaction-monitoring (MRM).Nitrogen (UHP N2) Served as desolvation gas at389L h-1. The ion transitions secected for MRM detection were:m/z314.0/68.9amu,319.0/68.9amu for efavirenz and IS, respectively.5. Statistical AnalysisThe pharmacokinetic parameters were calculated by DAS2.0software. The data were analyzed by SPSS13.0. Pharmacokinetic data are expressed as x±s. For the normally distributed data, a one-way analysis of variance (ANOVA) was used to test for differences among independent genotype groups. Differences were considered to be statistically significant with P<0.05.Result1. Validation of the LC-MS MethodThe recovery of efavirenz was consistent over the entire calibration range, and was on average97.8%. The R.S.D of inter-day and intra-day were all less than10%. The lowest limit of quantitation could be taken was20ng·ml-1. The standard curve was y=0.000814x+0.000228, with r2=0.9998. The difference after up to three times freezing and thawing was less than5%. All the indexes could fill the bit.2. Effect of CYP2B6polymorphism on pharmacokinetic parameters of efavirenzCYP2B6*1/*1,*1/*6,*6/*6were29,8and3volunteers, respectively. Their t1/2were85.88±23.08,94.25±25.76and162.11±82.55h, respectively, and had a significant difference(P=0.001); their AUC0-t were113674.47±14579.31,134421.24±42135.48and147947.86±34953.87h-ng/ml, respectively, and had a significant difference(P=0.017); The AUC0-INF of them were147630.24±24859.60,188526.79±73715.22and278934.17±120841.84h-ng/ml, respectively, with a significant difference(P=0.000).3. Effect of gender on pharmacokinetic parameters of efavirenzThe t1/2in male and female volunteers were93.33±23.59and121.22±42.20h, respectively, with a significant difference (P=0.001); their AUCo-t were137537.36±28947.29and174646.77±47294.96h-ng/ml, respectively, with a significant difference (P=0.012); The AU0-INF of them were149105.73±34178.49and20761.81±75245.47h·ng/ml, respectively, with a significant difference (P=0.002).ConclusionsCYP2B6*6genetic polymorphism and gender affect the metabolism of efavirenz, and for patients with gene variant and female patients, a reduced drug dose should be prescribed. |
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