Effects Of CYP3A4*1G Genetic Polymorphism On Fentanyl Pharmacokinetics And Pain-threshold After Intravenous Injection In Female Healthy Volunteers

Background and ObjectiveFentanyl is a opioid widely used for clinical anaesthesia,patient controlled analgesia and therapy for pain. The fentanyl's analgesia effect varies in different patients, which makes it difficult to have the appropriate dose for a patient. Some studies contribute this to the variation of fetanyl pharmacokinetics after intravenous administration. Fentanyl is metabolized predominantly by hepatic cytochrome P4503A4 (CYP3A4) after intravenous administration. CYP3A4 protein expression varies up to 40-folds in liver. The CYP3A4 gene's polymorphism may contribute to interindividual variability in fentanyl metabolism and leading to variability of clinical effect. CYP3A4*1G was found rencently and is a high-frequency allele in Chinese. Previous study show that the CYP3A4*1G allele can decrease the activity of CYP3A4 and then decrease patient-controlled intravenous fentanyl consumption.They speculated variation of fentanyl pharmacokinetics changed by CYP3A4*1G allele maybe the possible reason.Due to the importance of CYP3A4 in the metabolism of fentanyl, we conducted this study to observe the influence of CYP3A4*1G polymorphism on fentanyl pharmacokinetics and pain threshold after injection in female healthy volunteers. The present study provides an important theoretical evidence and foundation for individualization of fentanyl clinical administration.Materials and Methods1. SubjectsTotal one hundred and sixty-six female students of zheng zhou university, aged 18-25 yr, within±20% of ideal body weight were enrolled for gene analysis. Twenty-eight volunteers were assigned to three groups according to their genotypes: wild homozygote group(10), mutation heterozygote group(10) and mutation homozygote group(8). Before entering the study, all volunteers were ascertained to be in good health by medical history, standard hematological and blood-chemistry tests, clinical examination. None of the volunteers suffered hepatic or renal dysfunction, gastrointestinal disease, volunteers who have consumed drugs known to induce or inhibit the expression of CYP3A enzymes in four weeks were also excluded. The study design was approved by Institutional Ethics Committee of the first affiliated hospital of Zheng zhou University and Written informed consent was obtained from all 28 healthy volunteers.2. Genotyping assaysVenous blood samples (2 ml) were collected from all volunteers in this study. The standard phenol/chloroform procedure was used to extracted DNA from leukocytes. Genotype of CYP3A4*1G allele was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results for each genotype were confirmed in randomly selected individuals by direct sequence analysis.3. Study designCentre early in the morning with vital sign monitored after an over night(12h) fast. Subjects had intravenous catheters placed in both forearms, one for drug administration and the other in the opposite arm for blood sampling. All volunteers received 5 u g/kg of intravenous fentanyl(Fentanyl 0.05 mg/ml injection, Yi-chang ren fu,090903.) in 1 min. Naloxone(Narcanti 1 mg/ml, Beijing Hua su,100812) was used if needed to counteract the side effects of fentanyl. The volunteers who used naloxone were also excluded.The volunteers were given standard meals 4 h and 8 h after fentanyl administration. The drinking of alcohol, coffee, tea, grapefruit juice, or cola was forbidden on the test days and for 2 days prior to the study. A baseline blood sample was drawn into a ethylenediaminetetraacetate (EDTA) tube before fentanyl administration, and timed blood samples (4 ml each) were drawn 0,2,5,10,15,30, 60,90,120,180,300,420,720 min after fentanyl administration. Plasma was separated within 30 min by centrifugation (10 min.4 OOOr·min-1) and stored at-80℃until analysis. Pain threshold was measured using electrical stimulation at the time before and 45 minutes^ 2.5 hours^ 4 hours after fentanyl administration.5. Bioanalysis of fentanylPlasma concentrations of fentanyl was quantified by use of an liquid chroma-tography system coupled to tandem mass spectrometry(LC-MS/MS).6. Statistical analysisThe pharmacokinetics parameters of fentanyl were evaluated by 3p97 software.SPSS 10.0 software was used for statistical analyses. Values were reported as x±s. Chi-square test was used to verify Hardy-Weinberg equilibrium; One-way analysis of variance(ANOVA) was used to assess whether significant differences exist in the three genotypes;Linear correlation analysis was used to assess the correlation between fentanyl concentration and pain threshold;The a was set at 0.05.Results1. Frequency of CYP3A4*1G alleleThe frequency of CYP3A4*1G allele in female healthy volunteers was 0.235. The allele frequency of CYP3A4*1G was in Hardy-Weinberg equilibrium (P>0.05).2. General information of volunteersTwenty-eight volunteers were assigned to three groups according to their genotypes:wild homozygote group(10), mutation heterozygote group(10) and mutation homozygote group(8). Aged 18-25 yr, within±20% of ideal body weight. There was no significant difference in age, Body Mass Index and pain threshold before fentanyl injectionCP>0.05).3. Association of CYP3A4*1G gene polymorphism with fentanyl pharmacokineticsFentanyl AUC 0～∞, t 1/2βin mutation homozygote group were significantly increased than those in mutation heterozygote group and Wild homozygote group (P <0.05),but there is no difference between mutation heterozygote group and wild homozygote group(P>0.05).4. Association of CYP3A4*1G gene polymorphism with pain thresholdMutation homozygote group has higher pain threshold than mutation heterozygote group and Wild homozygote group after fentanyl injection(P< 0.05),but there is no difference between mutation heterozygote group and wild homozygote group(P>0.05).5. correlation between fentanyl concentration and pain thresholdThe pain threshold increased in all three groups after fentanyl administration,and then decreased with fentanyl concentration decreasing.There was a good correlation between fentanyl concentration and pain threshold.Conclusions1. CYP3A4*1G polymorphism is related to the pharmacokinetics of fentanyl, and people with CYP3A4*1G variant A allele have a lower metabolic rate of fentanyl and higher pain tolerance. The specific CYP3A4*1G polymorphism may predict the individual requirement of fentanyl.2. There was a good correlation between fentanyl concentration and pain threshold.