【作者】 张豪勇；

【导师】 张卫；

【作者基本信息】 郑州大学 ， 麻醉学， 2011， 硕士

【摘要】 背景与目的芬太尼在我国临床麻醉、术后静脉镇痛以及临床疼痛治疗中已广泛应用,但临床发现应用相同剂量芬太尼后其镇痛效果及不良反应发生如术后呕吐、呼吸抑制等存在明显的个体差异,研究显示静脉注射芬太尼后体内药动学个体差异导致血药浓度的个体差异可能是造成这种现象的重要原因。有研究表明芬太尼主要代谢途径为经肝脏内细胞色素P450(cytochrome P450, CYP)3A4代谢,CYP3A4酶活性在不同个体差异明显,且CYP3A4酶的编码基因存在多态性。研究提示CYP3A4基因多态性可能是造成芬太尼药动学个体差异,进而导致临床药效学差异的原因。CYP3A4*1G是中国人新近发现的突变位点,也是中国人中所有的CYP3A4单核苷酸多态性中突变频率最高的一个位点。既往研究表明该多态性可影响CYP3A4酶活性,并间接造成术后芬太尼静脉镇痛需要量的差异,推测原因是CYP3A4*1G多态性引起芬太尼药动学个体差异所致。本研究拟通过研究女性健康志愿者静脉注射芬太尼后体内药动学、痛阈和CYP3A4*1G多态性的关系,探讨芬太尼个体药效学差异的遗传学因素,为临床芬太尼的个体化用药提供参考。材料与方法1.研究对象与分组女性健康志愿者166人中进行CYP3A4*1G基因型筛选,按照基因型分为野生纯合型组(CYP3A4*1/*1)、突变杂合型组(CYP3A4*1/*1G)、突变纯合型组(CYP3A4*1G/*1G)。依照基因型随机挑选28名志愿者,野生纯合子组10名、突变杂合子组10名、突变纯合子组8名。年龄18-25岁,体重指数在正常范围(1±20%)。所有受试者均无肝肾疾病史及药物过敏史,精神状态良好,全面体格检查均健康(包括心电图、血压、血常规、肝肾功能等),无烟酒嗜好,受试前4周及试验期间未服用过肝脏CYP3A4酶诱导剂或抑制剂。试验方案经郑州大学第一附属医院医学伦理委员会批准,受试者对试验方案和与受试药物有关的可能不良反应表示理解,并签署知情同意书。2.基因型检测受试者抽取外周静脉血2 m1,基因组DNA采用酚-氯仿法提取；CYP3A4*1G多态性位点采用聚合酶链反应-限制性片段长度多态性分析(polymerase chain reaction-restriction fragment length polymorphism, PCR-RFLP)技术进行检测分析。直接测序法验证基因型检测方法的可靠性。3.试验方法28名受试者给药前一天夜12点后开始禁饮食,给药当日早晨入实验室,受试者入室后监测生命体征BP、HR、SPO2等,开放左右上肢静脉通路,置入套管针备用,一侧用于注射药物,一侧用于采集血样。所有受试者予以芬太尼(宜昌人福药业,090903)5μg/kg静脉注射,1分钟注射完毕。注射完毕后予以面罩吸氧并密切观察患者生命体征,若患者出现严重呼吸抑制等不良反应,则予以纳洛酮(北京华素制药,100812)拮抗并剔除出本实验。给药4h、8h后给予统一低脂肪、低蛋白标准餐并予以适量饮水,试验当天及试验前两天严禁饮用茶、酒精、咖啡、可乐以及葡萄柚汁。分别于给药后即刻、2、5、10、15、30、60、90、120、180、300、420、720分钟取静脉血4mL,EDTA抗凝,低温离心10min (4℃,4 OOOr·min- 1),取血浆-80℃保存待测。给药前及给药后45分钟、2.5、4小时时间点采用电刺激法进行痛阈测定。4.芬太尼血药浓度分析芬太尼血药浓度检测采用液相色谱一质谱法进行检测(LC-MS/MS)。5.统计学分析芬太尼的药时曲线用3P97药动学软件作参数估算,采用梯形法计算曲线下面积(AUC)。采用SPSS10.0软件进行数据处理,计量资料以x±s表示,等位基因和基因型分布是否符合Hardy-Weinberg平衡采用χ2检验检测；多组间药动学参数数据比较采用单因素方差分析(ANOVA),各组间两两比较采用LSD法；多组间痛阈资料采用重复测量设计的方差分析；计量资料变量之间的关系采用直线相关分析。检验水准为α=0.05。结果1中国女性CYP3A4*1G等位基因频率166例女性健康志愿者中,97例为野生纯合子组(CYP3A4*1/*1),60例为突变杂合子组(CYP3A4*1/*1G),9例为突变纯合子组(CYP3A4*1G/*1G)CYP3A4*1G等位基因突变率为23.5%。CYP3A4*1G等位基因的野生型纯合子、突变型纯合子、杂合子的分布符合Hardy-Weinberg平衡(P>0.05),表明本研究人群CYP3A4*1G基因型分布已达到遗传平衡,具有群体代表性。2受试者基本情况28例受试者中CYP3A4*1G野生纯合子组(10名)、CYP3A4*1G突变杂合子组(10名)、CYP3A4*1G突变纯合子组(8名)。年龄为19.2±1.4岁,体重指数20.4±2.6 kg/m2。三组受试者间年龄、体重指数及给药前痛阈比较差异无统计学意义(P>0.05)。3 CYP3A4*1G基因多态性对静脉注射芬太尼药动学的影响将三个不同基因型组各时相点芬太尼血药浓度的测定值经3p97药代动力学软件程序处理,得到三组受试者芬太尼药动学相关参数。突变纯合子组的终末消除半衰期t1/2β长于野生纯合子组和突变杂合子组(P<0.05)；AUC 0～∞突变纯合子组高于野生纯合子组和突变杂合子组(P<0.05),但在野生纯合子组和突变杂合子组之间差异无统计学意义。4 CYP3A4*1G基因多态性对静脉注射芬太尼后受试者痛阈的影响三组间给药前痛阈差异无统计学意义,给药后痛阂突变纯合子组高于突变杂合子组和野生纯合子组(P<0.05)；突变杂合子组与野生纯合子组间痛阈差异无统计学意义(P>0.05)5芬太尼血药浓度与受试者电刺激痛阈的相关性三组受试者电刺激痛阈在静脉注射芬太尼后均较给药前增高,且随着芬太尼血药浓度的下降而下降,具有良好的相关性(r1=0.87;r2=0.92；r3=0.91；P均小于0.05)。结论1.CYP3A4*1G突变可导致芬太尼代谢减弱和静脉注射芬太尼后电刺激痛阈增强,可能是引起芬太尼药效学个体差异的原因之一；2.受试者电刺激痛阈与芬太尼血药浓度具有良好的相关性。更多还原

【Abstract】 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.更多还原