肾移植受者中ABCC2基因多态性与环孢素导致肝损伤的相关性研究

摘 要 目的：探讨肾移植受者中ABCC2基因多态性与环孢素致肝损伤易感性的相关性。方法: 将入组的肾移植受者分为肝损伤组和对照组,利用高通量技术对ABCC2基因的五个单核苷酸多态性位点（rs4919395、rs2804398、rs4148394、rs4148397和rs3740065）进行基因分型,分析这些位点在两组间分布差异。结果:在35例肝损伤组和182例对照组间,ABCC2基因的5个SNP等位基因和基因型差异均无统计学意义（P>0.05）,且所选取的SNP单倍型组间也无显著差异（P>0.05）。结论:ABCC2的rs4919395,rs2804398,rs4148394,rs4148397和rs3740065位点与环孢素致肝损伤并无关联。     

ABCC2-24C/T基因多态性与肾移植患者术后吗替麦考酚酯所致不良反应的相关性研究

摘 要 目的： 探究肾移植受者ABCC2-24C/T基因多态性是否与吗替麦考酚酯（MMF）所致相关不良反应有关。方法: 将236例肾移植患者按不良反应类型分为骨髓抑制组、胃肠道反应组、感染组和未发生不良反应的对照组,采用高效液相 荧光检测器法测定患者的霉酚酸(MPA)血浆药物浓度,采用限制性片段长度多态性（polymerase chain reaction restriction fragment length polymorphism,PCR-RFLP)法检测患者ABCC2-24C/T位点多态性,并将PCR产物送测序公司直接测序以验证结果的准确性,将患者基因型与患者的年龄、性别、体质量、体质指数（BMI）、透析时间、是否尸肾以及移植术后3,6,12,24,36个月时的MMF剂量、浓度进行统计学分析。结果: 本研究中共有36例骨髓抑制,15例胃肠道反应,26例感染,124例为未发生不良反应,ABCC2-24C/T基因突变频率为21.34%,野生纯合子为58.05%,杂合子为37.71%,突变纯合子为4.24%,骨髓毒性组CC基因型分布显著高于对照组（P<0.05）,CT基因型组与CC基因型组在年龄上存在显著差异（P<0.05）,各不良反应组与对照组在BMI值和用药时间上差异均有统计学意义（P<0.05）,骨髓毒性组在透析时间上与对照组差异均有统计学意义（P<0.05）。不同时间点各基因型MMF谷浓度的差异无统计学意义（P>0.05）。Logistic回归模型显示,ABCC2-24CC基因型、用药时间、术后3月的MMF谷浓度是肾移植术后发生MMF所致骨髓抑制毒性的危险因素。结论:ABCC2-24C/T位点的多态性与肾移植术后MMF所致相关不良反应有关,携带ABCC2-24CC基因型的患者更容易发生MMF所致骨髓抑制毒性。     

ABCC2 1249G>A基因多态性与肾移植患者术后吗替麦考酚酯所致不良反应的相关性研究

摘 要 目的:探究肾移植受者ABCC2 1249G>A基因多态性是否与吗替麦考酚酯（MMF）所致相关不良反应有关。方法：对236例患者按不良反应类型分为骨髓抑制组、胃肠道反应组、感染组和对照组,并对患者的肾源、年龄、性别、身高、质量、BMI值、透析时间、移植年月等临床资料以及患者移植术后3,6,12,24,36个月时的MMF剂量进行统计学分析;采用高效液相 荧光检测器法测定肾移植受者的霉酚酸(MPA)血药浓度;采用限制性片段长度多态性（PCR-RFLP）法检测236例患者的ABCC2 1249G>A位点多态性。结果：36例骨髓抑制,15例胃肠道反应,26例感染。ABCC2 1249G>A基因突变频率为12.08%,术后12月时GG基因型组的谷浓度显著高于GA基因型组（P<0.05）,GG基因型组与GA基因型组在BMI值上的差异有统计学意义（P＜0.01）。Logistic回归模型显示,用药时间、术后3月的MMF谷浓度是肾移植术后发生MMF所致骨髓抑制毒性的危险因素。结论：ABCC2 1249G>A位点的多态性与肾移植术后MMF所致相关不良反应无关。     

基因芯片法检测中国人乙醛脱氢酶2的基因多态性

摘 要 目的： 对中国人乙醛脱氢酶2（ALDH2）的基因多态性进行检测,指导临床硝酸甘油的合理用药。方法: 采用显色基因芯片方法测定ALDH2基因（Glu504Lys）多态性。结果:对1 026例中国人群临床样本的检测,获得3种基因型,分布频率为：ALDH2（504Glu）野生纯合子61.7%(623例),ALDH2（Glu 504Lys）杂合子31.6%(319例),ALDH2（504Lys）突变纯合子6.6%(67例)。结论: 中国人群对硝酸甘油无效概率高的患者大约占1/3。本文建立的检测方法适合医院开展常规临床指导用药的基因检测。     

ABCB1（1199G/A）基因多态性对甲磺酸伊马替尼转运的分子机制

摘 要 目的：探讨P 糖蛋白（P-gp）活性和所介导的甲磺酸伊马替尼胞内累积量及药物跨膜渗透性的影响。 方法: 将构建的ABCB1 1199G/wt和1199A/mut重组质粒分别转染HEK293细胞,利用RT-PCR法考察细胞中P-gp的mRNA表达水平。CCK-8法检测药物对细胞的毒性,高效液相色谱法（HPLC）检测细胞中药物浓度和胞内累积量,跨膜电阻实验考察药物跨膜渗透率,评价P-gp活性对药物转运的作用。结果: 细胞毒性实验表明,转染细胞内的药物浓度均低于对照组,证明P-gp具有介导药物转出胞外的作用。HPLC和跨膜电阻实验表明,与野生型ABCB1(1199G)细胞相比,突变型ABCB1(1199A)细胞抗甲磺酸伊马替尼的作用更强,P-gp对介导甲磺酸伊马替尼外排转运的作用较强且药物的跨膜渗透性也相应较强。结论:实验表明ABCB1（1199G/A）位点突变导致其编码蛋白P-gp活性改变,该位点多态性会导致甲磺酸伊马替尼清除率增加,抑制了药物在靶细胞中有效药物浓度,因此临床上应对ABCB1基因进行分型,指导甲磺酸伊马替尼的个体化用药。     

Caco-2细胞中药物转运蛋白的mRNA表达及活力评价

目的 系统研究Caco-2细胞中各药物转运蛋白的mRNA表达水平及转运活力,对比其与人正常肠道中药物转运蛋白表达的差异。方法 实时荧光定量PCR（qRT-PCR）方法测定Caco-2细胞中人肠道相关转运蛋白MDR1、BCRP、MRP2、OATP1A2、OATP2B1和PEPT1的表达水平;将Caco-2细胞接种于Transwell板内培养21 d并给予不同药物转运蛋白的底物及抑制剂,评价Caco-2细胞中相关转运蛋白的转运活性。结果 qRT-PCR结果表明,药物转运蛋白MDR1、MRP2、BCRP和OATP2B1在Caco-2细胞中均有相对高的表达,表达量的顺序为：MDR1 > MRP2 > OATP2B1 > BCRP,在正常人肠道表达量顺序为BCRP > MDR1 > MRP2 > OATP2B1;转运蛋白活力评价表明,各药物转运蛋白的活力测试结果均为阳性,验证了基因的表达水平结果。结论 Caco-2细胞中表达正常人体肠道表达的部分药物转运蛋白（MDR1、MRP2、BCRP和OATP2B1）,表达水平与正常人体肠道中大致相当,但也存在一定差异。     

药物基因组学；单核苷酸多态性；药物代谢酶；药物转运体；药物靶标

摘 要药物基因组学是研究个体患者的遗传差异对药物作用的影响,继而优化药效并减少药物的不良反应的一门学科。利用基因组信息预测个体对药物的反应是实现个体化药物治疗最有效的途径之一,也是实现精准医疗的手段之一。随着基因检测技术的飞速发展,使得高通量、可衡量、低成本的体外个体基因组检测项目越来越多的应用到医疗实践中,指导制订个体化给药方案。本文介绍单核苷酸多态性以及相关临床应用进展,阐述药物基因组学的个体化治疗评价和其根据人体的基因多样性指导医疗的战略性作用。     

NUDT15c.415C>T和TPMT*3C基因多态性检测方法的比较与临床应用

目的 建立准确、快速、经济的方法,检测NUDT15 c.415C>T和TPMT*3C基因多态性,探讨临床应用价值。方法 收集2017年5月-2018年5月期间福建汉族患者服用硫唑嘌呤2周以上的血清样本,提取DNA或白细胞后分别采用PCR-RFLP法、PCR-Sanger测序法和荧光定量PCR法对NUDT15 c.415C>T和TPMT*3C进行基因多态性分型,比较这3种方法的准确性、简便性及经济性。根据白细胞值分组,结合临床资料,探讨基因多态性等因素与硫唑嘌呤致白细胞减少的相关性。结果 共纳入129例患者,其中硫唑嘌呤致白细胞减少15例（11.6%）。3种方法的基因多态性检测结果一致,TPMT*3C未发现突变纯合子。携带NUDT15c.415C>T突变等位基因者服用硫唑嘌呤致白细胞减少的风险高于携带野生等位基因者（OR=6.2,95%CI：2.5~15.4,P=0.000 054）,而携带TPMT*3C突变等位基因者与野生等位基因者出现白细胞减少比例并无显著性差异（P=0.393）。NUDT15c.415C>T基因多态性预测白细胞减少敏感度为53.3%,特异度为85.1%,ROC曲线AUC为0.69。结论 3种方法都可用于临床检测NUDT15 c.415C>T和TPMT*3C基因多态性。PCR-RFLP法不需要专用试剂盒,也不需要昂贵的仪器设备,成本较低,过程简单,易于操作,特别适合条件有限的单位开展工作。福建汉族患者在服用硫唑嘌呤前进行NUDT15c.415C>T基因多态性检测比TPMT*3C更具临床价值。     

颗粒状角膜营养不良Ⅱ型家系的TGFBI基因筛查

目的 对颗粒状角膜营养不良II型（GCD2）的家系进行TGFBI基因筛查，寻找患病原因。方法 对GCD2家系患者TGFBI基因的所有外显子进行突变检测，以GCD2家系中的健康成员和100例无亲缘关系的正常志愿者DNA样本作为对照。结果 GCD2家系所有患者TGFBI基因4号外显子的第370位碱基发生纯合或杂合的G>A突变，导致TGFBI基因编码的蛋白质的第124位氨基酸R变为H。结论 错义纯合和杂合R124H突变是导致GCD2家系角膜营养不良不同临床表型的主要病因。     

6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响

目的 研究6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响。方法 体外培养稳定高表达OATP1B1和OATP1B1基因多态性A388G、T521C的人胚肾细胞（HEK293）株,高表达空白载体（Mock）的HEK293细胞为空白对照,实时荧光定量PCR（qRT-PCR）法检测各转运体细胞中mRNA表达;放射性标记化合物³Hestrone sulfate作为转运底物、利福平作为阳性抑制剂验证各高表达细胞的转运活性;测定30 μmol·L^-1的达比加群、辛伐他汀、替格瑞洛、卡培他滨、多西他赛、依那普利对各细胞³H-estrone sulfate摄入活性的抑制作用,并依据抑制试验结果,进一步测定辛伐他汀、替格瑞洛、多西他赛对转运体细胞的半数抑制浓度（IC₅₀）。结果 HEK293细胞内导入的各种转运体基因都呈现良好的复制表达;OATP1B1、OATP1B1/A388G、OATP1B1/T521C对底物³H-estrone sulfate（5 μmol·L^-1）的转运活性分别为Mock细胞的39、49和48倍,30 μmol·L^-1利福平添加后,可将细胞的转运活性抑制到50%以下;辛伐他汀、替格瑞洛、多西他赛对OATP1B1的抑制作用较强,30 μmol·L^-1给药的转运活性分别为对照组的（40.09±1.95）%、（33.82±0.61）%、（45.08±0.22）%;辛伐他汀对OATP1B1、OATP1B1/A388G、OATP1B1/T521C的IC₅₀分别为14.2、＞100、＞100 μmol·L^-1,替格瑞洛的IC₅₀分别为19.1、68.4、＞100 μmol·L^-1,多西他赛的IC₅₀分别为17.6、22.9、19.3 μmol·L^-1。结论 OATP1B1基因多态性在一定程度上改变了抑制剂对转运体活性的影响程度。     

Genetic polymorphisms of ATP-binding cassette transporters ABCB1 and ABCC2 and their impact on drug disposition

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes, and act as efflux proteins. ABC transporters are characterised by the presence of genetic polymorphisms mainly represented by single nucleotide polymorphisms (SNPs), some of which having an impact on their activity. Besides physiological substances, drugs are also substrates of some ABC transporters, mainly ABCB1, ABCC1, ABCC2, ABCC3 and ABCG2. Identifying the impact of these polymorphisms on the pharmacokinetics (PK) of these drugs may have important clinical implications, certainly for those characterised by a narrow therapeutic index and significant inter- and intra-patient PK variability. This review focuses specifically on ABCB1 and ABCC2 and critically analyses important publications dealing with the influence of ABCB1 and/or ABCC2 polymorphisms on drug disposition in humans. For different reasons discussed in this paper, the effect of ABCB1 and/or ABCC2 polymorphisms on drug concentrations in blood is not always easy to interpret and to correlate with pharmacological effects. In contrast, intracellular or target tissue drug concentrations appear more directly influenced by these polymorphisms, as illustrated with intralymphocyte concentrations for immunosupressants and antiretrovirals or with cerebrospinal fluid (CSF) concentrations for antiepileptics and antidepressants. Further research on intracellular and/or target tissue drug concentrations are still needed to better characterise the PK-PG (pharmacogenetics) relationship involving ABC transporters.     

Pharmacogenetics of aldo-keto reductase 1C (AKR1C) enzymes

Introduction: Genetic variation in metabolizing enzymes contributes to variable drug response and disease risk. Aldo-keto reductase type 1C (AKR1C) comprises a sub-family of reductase enzymes that play critical roles in the biotransformation of various drug substrates and endogenous compounds such as steroids. Several single nucleotide polymorphisms have been reported among AKR1C encoding genes, which may affect the functional expression of the enzymes.

Areas covered: This review highlights and comprehensively discusses previous pharmacogenetic reports that have examined genetic variations in AKR1C and their association with disease development, drug disposition, and therapeutic outcomes. The article also provides information about the effect of AKR1C genetic variants on enzyme function in vitro.

Expert opinion: The current evidence that links the effect of AKR1C gene polymorphisms to disease progression and development is inconsistent and needs further validation, despite of the tremendous knowledge available. Information about association of AKR1C genetic variants and drug efficacy, safety, and pharmacokinetics is limited, thus, future studies that advance our understanding about these relationships and their clinical relevance are needed. It is imperative to achieve consistent findings before the potential translation and adoption of AKR1C genetic variants in clinical practice.     

Pharmacogenetics of telatinib,a VEGFR-2 and VEGFR-3 tyrosine kinase inhibitor,used in patients with solid tumors

Purpose Telatinib is an orally active small-molecule tyrosine kinase inhibitor of kinase insert domain receptor (KDR; VEGFR-2) and fms-related tyrosine kinase 4 (FLT4; VEGFR-3). This study aims at the identification of relationships between single nucleotide polymorphisms (SNPs) in genes encoding for transporter proteins and pharmacokinetic parameters in order to clarify the significant interpatient variability in drug exposure. In addition, the potential relationship between target receptor polymorphisms and toxicity of telatinib is explored. Methods Blood samples from 33 patients enrolled in a phase I dose-escalation study of telatinib were analyzed. For correlation with dose normalized AUC_(0–12), ATP-binding cassette (ABC) B1 (ABCB1), ABCC1, and ABCG2 were the genes selected. For correlation with telatinib toxicity, selected genes were the drug target genes KDR and FLT4. Results No association between dose normalized AUC_(0–12) and drug transporter protein polymorphisms was observed. In addition, no association between toxicity and KDR or FLT4 genotype or haplotype was seen. Conclusions Our pharmacogenetic analysis could not reveal a correlation between relevant gene polymorphisms and clinical and pharmacokinetic observations of telatinib.     

Pharmacogenomics of human ABC transporters: detection of clinically important SNPs by SmartAmp2 method

Genetic polymorphisms and mutations in drug metabolizing enzymes, transporters, receptors, and other drug targets (e.g., toxicity targets) are linked to inter-individual differences in the efficacy and toxicity of many medications as well as risk of genetic diseases. Validation of clinically important genetic polymorphisms and the development of new technologies to rapidly detect clinically important variants are critical issues for advancing personalized medicine. A key requirement for the advancing personalized medicine resides in the ability of rapidly and conveniently testing patients' genetic polymorphisms and/or mutations. We have recently developed a rapid and cost-effective method, named Smart Amplification Process 2 (SmartAmp2), which enables us to detect genetic polymorphisms or mutations in target genes within 30 to 45 min under isothermal conditions without DNA isolation and PCR amplification. Detection of mutations or single nucleotide polymorphisms (SNPs) in human ABC transporter genes is becoming more important, since their functional impairments are reportedly associated with inherited diseases. Thus, certain genetic polymorphisms of ABC transporters are considered important biomarkers for diagnosis of inherited diseases and/or risk of drug-induced adverse reactions. In this review article, we will present the new technology of the SmartAmp2 method and its clinical applications for detection of SNPs in human ABC transporter genes, i.e., ABCC4 and ABCC11.     

Polymorphisms of MRP1 (ABCC1) and related ATP-dependent drug transporters

Genetic variations in drug metabolizing enzymes and targets are established determinants of adverse drug reactions and interactions, but less is known about the role of genetic polymorphisms in membrane transport proteins. MRP1 (ABCC1) is one of 13 polytopic membrane proteins that comprise the 'C' subfamily of the ATP-binding cassette (ABC) superfamily of transport proteins. MRP1 and related ABCC family members, including MRP2, 3, 4 and 5 (ABCC2, 3, 4 and 5), each have a distinctive pattern of tissue expression and substrate specificity. Together, these five transporters play important roles in the disposition and elimination of drugs and other organic anions, and in maintenance of blood-tissue barriers, as confirmed by enhanced chemosensitivity of respective knockout mice. Moreover, Mrp2 (Abcc2) deficient animals display mild conjugated hyperbilirubinemia, corresponding to a human condition known as Dubin-Johnson syndrome (DJS). Naturally occurring mutations in MRP/ABCC-related drug transporters have been reported, some of which are non-synonymous single nucleotide polymorphisms. The consequences of the resulting amino acid changes can sometimes be predicted from in vitro site-directed mutagenesis studies or from knowledge of mutations of analogous (conserved) residues in ABCC proteins that cause DJS, Pseudoxanthoma elasticum (ABCC6), cystic fibrosis (CFTR/ABCC7) or persistent hyperinsulinemic hypoglycemia of infancy (SUR1/ABCC8). Continual updating of databases of sequence variants and haplotype analysis, together with in vitro biochemical validation assays and pharmacological studies in knockout animals, should make it possible to determine how genetic variation in the MRP-related transporters contributes to the range of responses to drugs and chemicals observed in different human populations.     

Pharmacogenomics-based RNA interference nanodelivery: focus on solid malignant tumors

Introduction: RNA interference represents one of the most promising strategies in fighting disease. However, small RNA interference faces substantial challenges for in vivo application due to the inherent instability of the RNA interference molecule. Among the nonviral gene delivery carriers, nanoparticles have attracted interest due to their success in various model systems. Nanomaterials have unique properties compared to conventional bulk materials that may be applicable in this setting. The nanoparticle complex carrying small interference RNA can undergo surface modification to achieve targeted modification for tissue-specific delivery. However, toxicity issues of the delivery systems need to be addressed and they require a pharmacogenomic profile of their own.

Areas covered: The authors review pharmacogenomics, toxicogenomics, nanoparticle-based drug delivery, and small interference RNA, with a focus on how logically engineered nanoparticle delivery systems can be used for personalized medicine in malignant tumors.

Expert opinion: Pharmacogenomics may be helpful in addressing possible individualized drug response for both the gene silencing capability of the delivered siRNA and the nanoparticle drug delivery system as both complete and distinct units. This may be done by assessing variations in gene expressions and single nucleotide polymorphisms. Patient profiling may be key as patient noncompliance due to toxicity plays a major role in treatment failure.     

ABC multidrug transporters: structure, function and role in chemoresistance

Three ATP-binding cassette (ABC)-superfamily multidrug efflux pumps are known to be responsible for chemoresistance; P-glycoprotein (ABCB1), MRP1 (ABCC1) and ABCG2 (BCRP). These transporters play an important role in normal physiology by protecting tissues from toxic xenobiotics and endogenous metabolites. Hydrophobic amphipathic compounds, including many clinically used drugs, interact with the substrate-binding pocket of these proteins via flexible hydrophobic and H-bonding interactions. These efflux pumps are expressed in many human tumors, where they likely contribute to resistance to chemotherapy treatment. However, the use of efflux-pump modulators in clinical cancer treatment has proved disappointing. Single nucleotide polymorphisms in ABC drug-efflux pumps may play a role in responses to drug therapy and disease susceptibility. The effect of various genotypes and haplotypes on the expression and function of these proteins is not yet clear, and their true impact remains controversial.     

Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture

Abstract

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2).

2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)?>?BCRP (9)?>?OCT1 (8)?>?OATP1B1 (5)?>?MRP3 (2).

3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2.

4. Structure–activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.     

The SmartAmp Method: Rapid Detection of SNPs in Thiopurine S-Methyltransferase and ABC Transporters ABCC4 and ABCG2

Genetic polymorphisms of drug transporters as well as drug metabolizing enzymes have been documented to play a significant role in patients' responses to medication. A key requirement for advancing personalized medicine is the ability to rapidly and conveniently test for patients' genetic polymorphisms. We have recently developed a rapid and cost-effective method for single nucleotide polymorphism (SNP) detection, named Smart Amplification Process (SmartAmp), which enables us to detect genetic polymorphisms or mutations in 30 to 45 min under isothermal conditions without the need for DNA isolation and PCR amplification. This article presents the SmartAmp-based detection of SNPs in the thiopurine S-methyltransferase gene as well as in the ATP-binding cassette (ABC) transporter ABCC4 and ABCG2 genes that are critically involved in drug-induced adverse reactions. The SmartAmp method is expected to provide a practical and cost-effective tool for pharmacogenomics-based personalized medicine.