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GUI Mingzhu, LI Jing, LI Zhiling. Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202402020
Citation: GUI Mingzhu, LI Jing, LI Zhiling. Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202402020

Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children

doi: 10.12206/j.issn.2097-2024.202402020
  • Received Date: 2024-02-19
  • Rev Recd Date: 2024-04-12
  •   Objective  To explore the effects of CYP2C19, CYP2C9 and CYP3A5 genotypes on the plasma concentration of voriconazole in children.   Methods  Collected blood samples from 50 hospitalized children with invasive fungal infections who received intravenous voriconazole from January 2020 to December 2020. High performance liquid chromatography was used to detect the blood trough concentration of voriconazole, and the time-of-flight mass spectrometry detection system was used to detect the genotypes of CYP2C19, CYP2C9 and CYP3A5, and the effects of children’s genotyping on the plasma concentration, efficacy and adverse reactions of voriconazole were analyzed.   Results  The total effective rate of 50 children with IFI was 84% (42/50) after voriconazole treatment. The incidence of adverse reactions was 20% (10/50 cases). The measured plasma concentration of voriconazole ranged from 0.56~7.62 μg/ml. Combined with the different mutation types of CYP2C19 gene loci, three metabolic activities were produced: fast, medium and slow, and the test results showed that there were 16 cases of fast metabolism, 27 cases of intermediate metabolism and 7 cases of slow metabolism. There was a significant difference in plasma concentrations between the three groups (F=15.359, P< 0.001), and the drug concentrations in the fast metabolic group were significantly lower than those in the intermediate metabolic and slow metabolic groups. The mutations of CYP2C9 and CYP3A5 had no significant effect on the plasma concentrations of the drugs, which were (F=2.213, P=0.086)and (F=0.757, P=0.475).   Conclusion  Voriconazole had significant efficacy in the treatment of invasive fungal infections in children, and the adverse reactions were mild. CYP2C19 genotype was significantly related to the rate of drug metabolism and was an important factor affecting blood drug concentration, the detection of drug concentration and genotype of voriconazole was helpful to adjust the effective drug dose clinically and achieve more scientific and individualized treatment.
  • [1] FERRERAS-ANTOLÍN L, SHARLAND M, WARRIS A. Management of invasive fungal disease in neonates and children[J]. Pediatr Infect Dis J, 2019, 38(6S Suppl 1): S2-S6.
    [2] WARRIS A, LEHRNBECHER T, ROILIDES E, et al. ESCMID-ECMM guideline: diagnosis and management of invasive aspergillosis in neonates and children[J]. Clin Microbiol Infect, 2019, 25(9):1096-1113.
    [3] MCCREARY EK, DAVIS MR, NARAYANAN N, et al. Utility of triazole antifungal therapeutic drug monitoring: Insights from the Society of Infectious Diseases Pharmacists: Endorsed by the Mycoses Study Group Education and Research Consortium[J]. Pharmacotherapy. 2023, 43(10): 1043-1050.
    [4] 中华医学会儿科学分会, 中华儿科杂志编辑委员会. 儿童侵袭性肺部真菌感染临床实践专家共识(2022版)[J]. 中华儿科志, 2022, 60(4):274-282.
    [5] LEE J, NG P, HAMANDI B, et al. Effect of therapeutic drug monitoring and cytochrome P450 2C19 genotyping on clinical outcomes of voriconazole: a systematic review[J]. Ann Pharmacother, 2021, 55(4):509-529. doi:  10.1177/1060028020948174
    [6] 陈恳, 张相林, 克晓燕, 等. 《伏立康唑个体化用药指南》解读[J]. 临床药物治疗杂志, 2019, 17(3):47-52,78. doi:  10.3969/j.issn.1672-3384.2019.03.012
    [7] 中国医药生物技术协会药物性肝损伤防治技术专业委员会, 中华医学会肝病学分会药物性肝病学组. 中国药物性肝损伤诊治指南(2023年版)[J]. 中华肝脏病杂志, 2023, 31(4):355-384. doi:  10.3760/cma.j.cn501113-20230419-00176-1
    [8] CHEN K, ZHANG X L, KE X Y, et al. Individualized medication of voriconazole: a practice guideline of the division of therapeutic drug monitoring, Chinese Pharmacological Society[J]. Ther Drug Monit, 2018, 40(6):663-674. doi:  10.1097/FTD.0000000000000561
    [9] PATTERSON T F, THOMPSON G R, DENNING D W, et al. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America[J]. Clin Infect Dis, 2016, 63(4):e1-e60.
    [10] MORIYAMA B, OBENG A O, BARBARINO J, et al. Clinical pharmacogenetics implementation consortium(CPIC)guidelines for CYP2C19 and voriconazole therapy[J]. Clin Pharmacol Ther, 2017, 102(1):45-51.
    [11] TAHER KW, ALMOFADA R, ALOMAIR S, et al. Therapeutic drug monitoring of voriconazole in critically Ill pediatric patients: a single-center retrospective study. Paediatr Drugs, 2024, 26(2): 197-203.
    [12] PASCUAL A, CSAJKA C, BUCLIN T, et al. Challenging recommended oral and intravenous voriconazole doses for improved efficacy and safety: population pharmacokinetics-based analysis of adult patients with invasive fungal infections[J]. Clin Infect Dis, 2012, 55(3):381-390. doi:  10.1093/cid/cis437
    [13] 郭一萌, 安琳娜, 陈恳, 等. 伏立康唑在不同年龄段使用中安全性和有效性以及药代动力学差异的系统评价[J]. 中国临床药理学杂志, 2016, 32(3):261-263.
    [14] BAHAR MA, SETIAWAN D, HAK E, et al. Pharmacogenetics of drug-drug interaction and drug-drug-gene interaction: a systematic review on CYP2C9, CYP2C19 and CYP2D6[J]. Pharmacogenomics, 2017, 18(7):701-739. doi:  10.2217/pgs-2017-0194
    [15] SHI C C, XIAO Y B, MAO Y, et al. Voriconazole: a review of population pharmacokinetic analyses[J]. Clin Pharmacokinet, 2019, 58(6):687-703.
    [16] 邵贝贝, 赵宁民, 段虹飞, 等. 基于基因多态性的伏立康唑药代动力学研究状况[J]. 中国临床药理学杂志, 2016, 32(7):663-666.
    [17] FAN X, ZHANG H, WEN Z, et al. Effects of CYP2C19, CYP2C9 and CYP3A4 gene polymorphisms on plasma voriconazole levels in Chinese pediatric patients. Pharmacogenet Genomics[J]. 2022, 32(4): 152-158.
    [18] AIUCHI N, NAKAGAWA J, SAKURABA H, et al. Impact of polymorphisms of pharmacokinetics-related genes and the inflammatory response on the metabolism of voriconazole[J]. Pharmacol Res Perspect, 2022, 10(2):e00935. doi:  10.1002/prp2.935
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Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children

doi: 10.12206/j.issn.2097-2024.202402020

Abstract:   Objective  To explore the effects of CYP2C19, CYP2C9 and CYP3A5 genotypes on the plasma concentration of voriconazole in children.   Methods  Collected blood samples from 50 hospitalized children with invasive fungal infections who received intravenous voriconazole from January 2020 to December 2020. High performance liquid chromatography was used to detect the blood trough concentration of voriconazole, and the time-of-flight mass spectrometry detection system was used to detect the genotypes of CYP2C19, CYP2C9 and CYP3A5, and the effects of children’s genotyping on the plasma concentration, efficacy and adverse reactions of voriconazole were analyzed.   Results  The total effective rate of 50 children with IFI was 84% (42/50) after voriconazole treatment. The incidence of adverse reactions was 20% (10/50 cases). The measured plasma concentration of voriconazole ranged from 0.56~7.62 μg/ml. Combined with the different mutation types of CYP2C19 gene loci, three metabolic activities were produced: fast, medium and slow, and the test results showed that there were 16 cases of fast metabolism, 27 cases of intermediate metabolism and 7 cases of slow metabolism. There was a significant difference in plasma concentrations between the three groups (F=15.359, P< 0.001), and the drug concentrations in the fast metabolic group were significantly lower than those in the intermediate metabolic and slow metabolic groups. The mutations of CYP2C9 and CYP3A5 had no significant effect on the plasma concentrations of the drugs, which were (F=2.213, P=0.086)and (F=0.757, P=0.475).   Conclusion  Voriconazole had significant efficacy in the treatment of invasive fungal infections in children, and the adverse reactions were mild. CYP2C19 genotype was significantly related to the rate of drug metabolism and was an important factor affecting blood drug concentration, the detection of drug concentration and genotype of voriconazole was helpful to adjust the effective drug dose clinically and achieve more scientific and individualized treatment.

GUI Mingzhu, LI Jing, LI Zhiling. Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202402020
Citation: GUI Mingzhu, LI Jing, LI Zhiling. Correlation between plasma concentration of voriconazole and polymorphisms in CYP2C19, CYP2C9 and CYP3A5 genes in children[J]. Journal of Pharmaceutical Practice and Service. doi: 10.12206/j.issn.2097-2024.202402020
  • 儿童侵袭性真菌感染的诊断越来越多,其中以念珠菌和曲霉菌感染最为常见[1,2]。伏立康唑因其抗菌谱广、耐药率低的特点,被国内外推荐作为应对侵袭性曲霉菌、念珠菌血症等严重威胁生命的真菌感染的一线治疗药物[3,4],适用于2岁以上的儿童。伏立康唑血液稳态谷浓度与临床疗效及不良反应有着密不可分的关系。肝脏细胞色素P450同工酶[5](如CYP2C19、CYP2C9、CYP3A5)影响伏立康唑的代谢,等位基因的突变使代谢酶的活性增强或降低。儿童因器官发育不成熟,生理状态等不同于成人,为达到有效的临床治疗,降低不良反应,急需探索基因多态性在伏立康唑儿童个体化用药中的相关性影响,对指导临床安全有效用药,实现儿童个体化的给药方案有重大的意义。

    本研究通过收集2020年1月至2020年12月期间在我院与上海市儿童医院接受伏立康唑静脉治疗的50例侵袭性真菌感染住院儿童的血液标本,检测其血药浓度与基因型,分析相关基因分型对伏立康唑血药浓度、疗效以及不良反应的影响。

    • 2020年1月至2020年12月住院治疗的侵袭性真菌感染患儿。本研究共收集病例50例,男29例(56%),女21例(44%),年龄2岁至12岁,平均年龄(5.7±3.5)岁。纳入标准:①诊断为侵袭性真菌感染的儿童;②接受伏立康唑治疗持续用药至少3 d;③接受伏立康唑血药浓度监测及CYP2C19、CYP3A4、CYP3A5基因检测;④患儿家属在入院时签署预留血样本知情同意书。排除标准:①接受伏立康唑治疗后持续用药不足3 d;②未进行伏立康唑血药浓度监测与基因检测;③肝肾功能不全;④缺乏完整的试验室评价指标,不能进行疗效和安全性评价。

      50例患儿均给予静脉注射用伏立康唑,由美国辉瑞公司生产,规格:200 mg/瓶,生产批号:Z572301。单次剂量 8 mg/kg,在1~2 h内滴注完成,q12h。安全性评价:参照《中国伏立康唑个体化用指南》[6],使用伏立康唑期间患者是否有视觉障碍、畏光、失眠兴奋、肝损害、及与伏立康唑有关的不良反应。肝损伤定义为患者使用伏立康唑后ALT(alanine transaminase)、ALP(alkaline phosphatase)检测值升高并≥3倍正常上限值,或TBil(total bilirubin)≥ 3倍正常上限值[7]。本研究经医院伦理委员会审批,伦理批准号:2019-LDYY-036,所有患儿家属均签署知情同意书。

    • 研究对象接受伏立康唑治疗至少 3 d后检测血清谷浓度。使用全自动二维液相色谱耦合仪LC-100HP(湖南德米特仪器有限公司)进行HPLC法测定药物浓度:血样本采集后静置至自然凝固,4 500 r/min 离心 5 min,获取血清样本;取 300 μl血清加入900 μl去蛋白溶液,涡旋震荡1 min,15 000 r/min离心8 min,待分层完全后,取 1 000 μl上清液加入100 μl APC溶液进样,在色谱条件下检测,记录峰面积并计算伏立康唑谷浓度和血清伏立康唑浓度 。

    • 本实验使用DP-TOF飞行时间质谱检测系统(浙江迪谱诊断技术有限公司)进行质谱检测,准备多重PCR引物和延伸引物,以人基因组DNA为模板,在一个反应体系内对多重PCR引物所在区域同时进行扩增,延伸,形成分子量差异。使用飞行时间质谱系统对不同延伸产物的分子量差异进行检测,通过数据分析,就可得到各突变位点的具体基因型。

    • 采用SPSS22.0统计学软件对数据进行统计学分析,对样本数据呈正态分布的计量资料以均数±标准差表示;计数资料采用χ检验,两样本比较采用独立样本 t 检验,多组间比较采用单因素方差分析,P<0.05有统计学意义。

    • 50例患儿均为静脉给药伏立康唑,q12h,使用疗程为10~56 d,中位时间13.50(10.50 ~ 20.30)d,所测药物浓度值在0.56~7.62 μg/ml之间,平均(3.56±1.87) μg/ml。本研究总体有效率为84%(42/50),总体不良反应发生率20%(10/50),给药后50例患儿中共有10例出现不良反应,其中1例总胆红素(TBIL>正常值上限10倍)伴谷草转氨酶(AST)升高,3例谷丙转氨酶(ALT>正常值上限3倍以上)升高,5例ALT与AST均升高,以及1例发生皮疹。此10例患儿均未终止治疗,其中6例继续观察,3例予以保肝药物治疗,1例予以抗过敏治疗。

      参考中国与美国指南[8,9]推荐的血浆稳态谷浓度值范围(1.0~5.5/ml),将本研究病例按照实测结果分为3组。组A<1.0 μg/ml(13例)、组B 1.0~5.5 μg/ml(30例)、组C > 5.5 g/ml(7例)。结果A、B和C 3组有效率分别为61.5%、93.3%和85.7%,统计对比显示3组有效率存在显著差异(χ=6.840,P=0.033)。3组的不良反应发生率分别为3例(3/13例, 23.1%),3例(3/30例, 10.0%)和4例(4/7例, 57.1%),统计对比结果显示,3组不良反应发生率存在显著差异(χ=7.988, P=0.018),具体数据见表1

      组别(μg/ml) 临床疗效 不良反应
      例数(n 有效(%) 无效(%) 例数(n/%)
      <1.0 13 8(61.5) 5(38.5) 3(23.1)
      1.0~5.5 30 28(93.3) 2(6.7) 3(10.0)
      >5.5 7 6(85.7) 1(14.3) 4(57.1)
      χ2 6.84 7.988
      P 0.033 0.018
    • 根据CYP2C19*2与CYP2C19*3基因位点突变形成的不同酶代谢活性[10]类型,分别为快代谢型(EM)16例,占32%;中间代谢型(IM)27例,占54%;慢代谢型(PM)7例,占14%。三组患儿的伏立康唑谷浓度出现显著的统计学差异(F=15.359, P<0.001),EM患儿的药物浓度明显低于IM与PM(EM vs IM, P=0.019, EM vs PM, P<0.001, IM vs PM, P<0.001),IM药物浓度低于PM,差异均具有统计学意义,见表2

      代谢速度 CYP2C19
      基因型
      酶活性 例数
      n
      药物浓度
      ρB/μg·ml−1
      *1/*1 16 1.30±0.24
      *1/*2 25
      3.45±0.20
      *1/*3 2
      *2/*2 1 5.92±0.41
      *2/*3 5
      *3/*3 1
      F 15.359
      P <0.001
    • 检测可能影响CYP2C9代谢活性的SNPs:CYP2C9*3(rs1057910),在纳入研究的患儿中,CYP2C9*3(rs1057910)野生纯合子AA型占92%(46/50),突变杂合子CA型占8%(4/50),未发现突变纯合子CC型。研究结果显示,CYP2C9*3的突变对伏立康唑血浆谷浓度未产生显著的影响,差异无统计学意义(F=2.213, P=0.086),结果见表3

      (CYP2C9*3基因型 例数(n 药物浓度(ρB/μg·ml−1
      野生纯合子(AA) 46 2.78±0.34
      突变杂合子(CA) 4 4.02±0.13
      突变纯合子(CC) 0
      F 2.213
      P   0.086
      注:“−” 表示未检测。
    • 检测可能影响CYP3A5代谢活性的SNPs:CYP3A5*3(rs776746)。在纳入研究的患儿中,CYP3A5*3野生纯合子CC型占54%(27/50),突变杂合子TC型占44%(22/50),突变纯合子TT型占2%(1/50)。CYP3A5*3位点的突变对伏立康唑血浆谷浓度未产生显著的影响,差异无统计学意义(F=0.757, P=0.475),结果见表4

      (CYP3A5*3基因型例数(n药物浓度(ρB/μg·ml−1
      野生纯合子(CC)273.43±0.36
      突变杂合子(TC)223.86±0.29
      突变纯合子(TT)12.85±0
      F0.757
      P 0.475
    • 伏立康唑药动学个体差异大,治疗窗较窄,其血药浓度与临床疗效和不良反应有显著相关性。从婴幼儿到青少年,随着身体功能的不断发生发展,肝、肾器官代谢功能的不断成熟,导致了伏立康唑药动学的个体差异性。为避免血药浓度过高产生药物毒性或血药浓度过低导致治疗失败,中国药理学会治疗药物监测研究专业委员会在制定《中国伏立康唑个体化用药指南推荐意见》[6]时,建议进行伏立康唑药物浓度监测和基因型检测。

      参考国内外成人的伏立康唑有效目标浓度范围(1.0~5.5 μg/ml)[5,6],本研究发现,当药物浓度为1.0~5.5 mg/L时,临床有效率达93.3%,不良反应发生率为10%;浓度<1.0 mg/L时,临床有效率61.5%,不良反应发生率为23.1%;而浓度>5.5 mg/L时临床有效率为85.7%,发生的不良反应达50%以上(χ2=7.988, P=0.018)。可见低血药浓度并未降低不良反应的发生,而高药物浓度并未提高临床有效率,反而增加不良反应的发生。同样在监测了治疗药物浓度的情况下,TACHER等[11]报道伏立康唑临床有效率在80%,PASCUAL等[12]报道的有效率达85%。本文临床总有效率达84%(42/50),与PASCUAL等的研究结果较接近。

      已有不少研究关注血浆伏立康唑浓度与肝毒性的关系,在一篇Meta分析[13]中发现,儿童组(2~12岁)发生肝毒性的比例较青少年和成人组更高。本文研究结果显示,参与研究的患者总体不良反应发生率为20%(10/50),以肝损害为主,其中9例为肝毒性,1例为过敏性皮疹(红色斑丘疹)。需要警惕的是,在本研究低浓度组(<1.0 mg/L)中,不良反应发生率为23.1%,这是由于儿童的肝脏组织结构发育不成熟,代谢功能弱,即使较低药物浓度亦可能导致肝损害,值得引起重视。本组资料中肝毒性均为可逆性,在临床可控范围内,给予保肝药物后恢复正常,无须停药,不影响临床治疗。结合本研究结果,推荐1.0~5.5 mg/L为儿童目标谷浓度,可在确保显著疗效的基础上,提高临床安全性。

      CYP2C19[14]是CYP450酶第二亚家族中的重要成员,在肝脏中有表达较多,是人体重要的代谢酶。目前研究[15]较多的等位基因有*17(酶活性增强),*1(正常酶活性),*2、*3~*8(酶无功能活性)。在东亚人群中,CYP2C19*2与CYP2C19*3两个突变位点是导致相关弱代谢遗传缺陷的主要突变点,其突变率达到95%左右。有研究显示[10],亚洲人属于弱代谢者;强代谢者中纯合子代谢最快,弱代谢者中杂合子的代谢最慢。本研究中儿童慢代谢与中间代谢占本研究对象所有代谢型的68%(34/50),接近我国汉族群体中的变异比例[8,16],3种代谢型患者体内伏立康唑药物浓度有显著性差异。快代谢儿童药物浓度明显低于中间代谢和慢代谢儿童。这是因为药物在进入体内后,快代谢者体内药物很快被清除,只剩余较少的药物来抗真菌感染,这不仅很难清除体内的真菌,还可能引起耐药;而对慢代谢者,药物进入体内后很难被清除,大量的药物在组织蓄积,不仅提高了血药浓度,还会产生组织器官损害。可见,快代谢者适当增加给药剂量,可获得有效的治疗浓度。慢代谢者根据药物暴露量,建议相应的降低给药剂量,以防药物在体内蓄积,对各脏器功能产生不良影响。

      CYP2C9基因[17]上是P450-IIC酶家族的主要组成部分,占肝微粒体中CYP总量的20%,参与16%的临床药物的代谢。CYP2C9*3基因的突变使酶与底物的亲和力降低,影响药物的代谢,可提高药物浓度。本研究中测得的突变基因仅有4例,其血药浓度虽高于未突变者,但并未提示有统计学意义,可能与本研究中的样本量较少有关。

      CYP3A5基因[18]主要在肠道和肝脏表达,其编码区表达剪接缺陷,突变后形成无功能的蛋白,酶活性缺失,可能降低药物的代谢,提高药物浓度。本研究中CYP3A5*3(rs776746)部分突变和全突变占40%,但对伏立康唑的药物浓度无明显影响。提示可能多数在肠道表达的CYP3A5基因对于主要通过肝脏代谢的伏立康唑,并不是其关键性的影响因素。

      本研究显示,CYP2C19的基因多态性是导致儿童体内伏立康唑代谢个体间差异的主要影响因素,进行基因分型有助于为患儿选择适当的伏立康唑给药剂量,并且推荐1.0~5.5 mg/L为儿童目标谷浓度,临床可根据CYP2C19基因型与药物浓度监测结果相应调整药物剂量。

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