中国肾移植患者红细胞内嘌呤类药物活性代谢物6-硫鸟嘌呤核苷酸浓度监测及影响因素分析

目的：研究服用硫唑嘌呤（AZA）中国肾移植患者红细胞（RBC）内活性代谢物6-硫鸟嘌呤核苷酸（6-TGNs）分布特征及影响因素，为临床合理应用嘌呤类药物提供依据。方法：以89例中国肾移植患者为研究对象，关联分析年龄、性别、体质量、AZA剂量和TPMT活性对RBC内6-TGNs浓度的影响，并应用SPSS v20.0软件进行多元线性回归分析。结果：89例中国肾移植患者RBC内6-TGNs浓度呈非正态分布（P<0.000 1），6-TGNs浓度中位数为167.60（四分位间距，108.10~300.80） pmol/8×10⁸ RBC，个体间差异约24.3倍。关联分析显示患者年龄、性别、体质量、TPMT活性对6-TGNs浓度均无显著影响（P>0.05）；而AZA剂量与6-TGNs浓度间呈显著正相关性（r_s=0.307 1，P<0.01）。多元线性回归分析显示，RBC内6-TGNs浓度与AZA剂量间呈显著正相关（P<0.001），与TPMT活性呈显著负相关（P<0.05）。结论：AZA剂量和RBC内TPMT活性协同影响嘌呤类药物活性代谢物6-TGNs浓度，进而影响该类药物临床疗效和毒性反应。     

HPLC测定炎症性肠病患者红细胞中6-硫鸟嘌呤核苷酸及6-甲巯基嘌呤的浓度

摘要：目的:建立HPLC法同时测定炎症性肠病（IBD）患者红细胞中硫唑嘌呤代谢物6-硫鸟嘌呤核苷酸（6-TGNs）及6-甲巯基嘌呤（6-MMP）浓度,为硫唑嘌呤（AZA）个体化治疗提供依据。方法:红细胞经高氯酸沉淀,6-TGNs在酸性条件下加热水解生成6-硫鸟嘌呤（6-TG）,6-MMP水解生成4-氨基-5-（甲硫基）羰基咪唑（AMTCI）,以5-溴尿嘧啶（5-BU）为内标,采用HPLC法测定其含量。色谱柱:Kromasil 100-5-C18（150 mm×4.6 mm,5μm）,流动相:乙腈-20 mmol·L1磷酸二氢钾溶液（6∶94）,流速:0.8 ml·min-1,柱温:30℃;6-TG、6-MMP及5-BU检测波长分别为340,303,280 nm。10例IBD患者,口服AZA 50～100 mg·d-11个月以上,检测其6-TGNs及6-MMP浓度,焦磷酸测序检测TPMT基因型。结果:6-TG在8～800pmol﹒（8×108）-1RBC浓度范围内线性关系良好（r=0.994 0）;低、中、高3个浓度的绝对回收率为63.24%～69.09%（n=5）。AMTCI在250～16 000 pmol·（8×108）-1RBC浓度范围内线性关系良好（r=0.997 8）;低、中、高3个浓度的绝对回收率为85.17%～92.28%（n=5）。10例患者TPMT均为野生型。1例患者6-TGNs浓度偏高,白细胞减少;2例患者6-MMP浓度较高,碱性磷酸酶轻度升高。结论:本方法专属性、精密度及稳定性较好,灵敏度高,适用于同时测定炎症性肠病患者红细胞中6-TGNs及6-MMP的浓度。     

硫嘌呤甲基转移酶基因多态性的硫唑嘌呤个体化用药临床研究分析

硫唑嘌呤属于硫嘌呤类药物,是一种免疫抑制药。硫嘌呤甲基转移酶(thiopurine S-methyltransferase, TPMT)是参与硫唑嘌呤代谢的重要酶,TPMT的基因多态性是导致个体间用药差异的重要因素。TPMT基因遗传多态性导致酶活性存在个体差异,从而使接受硫唑嘌呤治疗的人群表现出不同的疗效和药物不良反应。TPMT的遗传多态性能够影响到TPMT酶的活性,而不同TPMT活性的患者在服用硫唑嘌呤后的药效学不同。越来越多的研究证实,在服用硫唑嘌呤之前进行基因型检测,针对不同TPMT活性的患者进行硫唑嘌呤个体化用药指导,可以有效地提高疗效,降低药物不良反应。本研究综述了基于TPMT基因多态性的硫唑嘌呤个体化用药治疗现状,通过讨论TPMT基因型指导下对不同患者服用硫唑嘌呤后的临床结局的影响,以及精准用药的建议,尤其关注了中国人群中TPMT用药的研究现状,以期为不同人群进行硫唑嘌呤治疗提供用药建议。     

多药耐药基因MDR1C1236T多态性与炎症性肠病患者的活性代谢物6-TGNs的相关性

目的考察多药耐药基因MDR1的单核苷酸多态性(SNP)C1236T与炎症性肠病(IBD)患者服用硫嘌呤类药物后的活性代谢产物6-硫鸟苷酸(6-TGNs)的相关性。方法有105名患者纳入研究,收集全血提取DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法,进行MDR1C1236T基因型分析,采用HPLC方法检测红细胞内6-TGNs的浓度,并统计药物不良反应情况。卡方检验分析相关性。结果相同剂量下,6-TGNs浓度在1236CT/TT型携带者比CC型携带者高(P=0.048);同时,1236CT/TT型携带者服药后发生不良反应的风险显著高于1236CC型携带者(P=0.045)。结论 MDR1 1236 C>T突变与高6-TGNs浓度及高不良反应发生率密切相关。     

NUDT15 c.415C>T和TPMT*3C基因多态性与汉族人群服用硫唑嘌呤致白细胞减少关联性研究

目的：探讨NUDT15 c.415C>T和TPMT*3C基因多态性与汉族人群服用硫唑嘌呤致白细胞减少的关联性。方法：选取2017年5月-2018年5月在福建医科大学附属第一医院就诊,接受硫唑嘌呤治疗2周以上的汉族患者,PCR-RFLP法检测患者NUDT15 c.415C>T和TPMT*3C基因型,HPLC法测定患者红细胞内6-硫鸟嘌呤核苷酸谷浓度。根据白细胞值分组,结合临床资料,分析2种基因多态性与硫唑嘌呤致白细胞减少的相关性。结果：共纳入患者129例,白细胞减少组15例（11.6%）,白细胞正常组114例（88.4%）,2组在性别、年龄、日剂量、是否联合使用美沙拉秦或柳氮磺吡啶、沙利度胺、英夫利西及TPMT*3C基因多态性等均无显著性差异（P>0.05）。白细胞减少组53.3%（8/15）携带NUDT15 c.415>T突变基因,与白细胞正常组14.9%（17/114）差异有显著性（P=0.000 372）。携带该等位基因突变型患者出现白细胞减少风险高于携带野生型患者（OR=6.2,95%CI：2.5~15.4,P=0.000 054）,更易发生Ⅲ度以上严重的白细胞减少（P=0.000 022）。同一剂量范围野生型患者与突变型患者的6-硫鸟嘌呤核苷酸谷浓度并没有显著性差异（P>0.05）。NUDT15 c.415>T预测白细胞减少特异度为85.1%,敏感度为53.3%,ROC曲线AUC为0.69。结论：NUDT15c.415C>T基因多态性检测对降低汉族人群服用硫唑嘌呤致白细胞减少的风险具有较好的临床价值。     

HPLC法测定炎症性肠病患者红细胞中硫鸟嘌呤核苷酸浓度

目的:测定硫唑嘌呤与6-巯基嘌呤的活性代谢产物硫鸟嘌呤核苷酸(6-TGNs)在红细胞内的浓度,为提高临床疗效和降低副作用提供参考。方法:红细胞稀释液经高氯酸沉淀,6-TGNs在酸性条件下加热水解生成6-硫鸟嘌呤(6-TG),采用高效液相色谱法测定其含量。色谱柱为Hypersil ODS2,流动相为乙腈-20mmol/L磷酸二氢钾溶液(磷酸调节pH3.3)(5∶95);流速为1.0ml/min;柱温为30℃;检测波长为340nm,以外标法定量。结果:6-TGNs在上述条件下水解生成6-TG,在7.87～787.40pmol(/8×108)红细胞浓度范围内线性关系良好(r=0.9997);日内及日间RSD在0.46%～7.64%之间;平均方法回收率在99.10%～102.07%之间。结论:本方法准确,专属性和稳定性较好,灵敏度高,适用于接受硫唑嘌呤或6-巯基嘌呤治疗的炎症性肠病患者红细胞中6-TGNs浓度监测,可为临床在实施个体化治疗时提供参考。     

速尿、苯氧比酸、硫唑嘌呤对慢性炎症性肠病患者红细胞TPMT活性的影响

目的:研究利尿剂速尿、苯氧比酸及其硫唑嘌呤对慢性炎症性肠病患者红细胞硫嘌呤甲基转移酶(TPMT)活性的影响。方法:应用高效液相法(HPLC)测定慢性炎症性肠病患者红细胞TPMT活性,其中中等活性、正常活性、较高活性的患者各6例,依据浓度效应曲线计算每个药物的IC50。结果:速尿对TPMT活性的抑制作用较强,平均IC50为15～19μmol·L-1,在健康志愿者的正常血浆浓度范围内。苯氧比酸和硫唑嘌呤的平均IC50值分别为300～313μmol·L-1、430～532μmol·L-1,均远高于体内所能达到的血浆浓度。3种药物的IC50值在3个不同TPMT活性组间无明显差异(P>0.05)。结论:速尿可抑制红细胞TPMT活性,当速尿和硫嘌呤类药物合用时,应警惕发生不良相互作用。     

硫嘌呤甲基转移酶遗传多态性检测在6-MP个体化治疗中的意义

目的：研究硫嘌呤甲基转移酶活性和基因型检测对6-巯基嘌呤（6-MP）的个体化治疗的意义。方法-94例口服6-MP维持治疗的白血病患者，19例口服6-Mp后出现血液系统危象的为观察组，同时服用6-MP没有出现血液系统损害的75患者为对照组，采用高效液相色谱法测定硫嘌呤甲基转移酶（TPMT）活性，等位基因特异性PCR和限制性片段长度多态性（RFLP）的方法检测TPMT＊2、TPMT＊3A、TPMT＊3B和TPMT＊3C的等位基因频率。通过临床白细胞记数和骨髓象检查及临床表现判定巯嘌呤的疗效和不良反应。结果：观察组TPMT活性是（6．1±2．1）U·mL^-1 pRBCs显著低于对照组（15．3±2．3）U·mL^-1 pRBCs，而观察组的基因突变率10．7％高于对照组1．2％。结论：TPMT活性低下和／或TPMT基因突变型个体，在服用标准剂量的巯嘌呤后，发生不良反应的危险较高，应及时发现此类患者并积极调整剂量实现安全有效的治疗。     

巯嘌呤甲基转移酶基因检测用于指导6-巯基嘌呤在中国儿童急性淋巴细胞白血病中个体化治疗的循证评价

目的　系统评价6-巯基嘌呤(6-mercaptopurine, 6-MP)治疗亚洲儿童急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)出现骨髓抑制与巯嘌呤甲基转移酶(thiopurine methyltransferase, TPMT)基因多态性的相关性,并分析中国ALL患儿TPMT基因检测的经济性。方法　采用循证医学方法搜集6-MP治疗亚洲儿童ALL相关骨髓抑制与TPMT基因多态性相关的随机对照试验或观察性研究进行Meta分析。借助决策树模型针对中国ALL患儿,对两种6-MP初始给药剂量方案,进行成本效果分析。结果　最终纳入6个观察性研究,共577例亚洲患儿。Meta分析结果显示：TPMT基因多态性与骨髓毒性［OR=5.61,95%CI（2.05,15.34）,P=0.0008］发生有关。在基础数据分析中,针对中国ALL患儿,以上两个方案以严重骨髓抑制发生率为效果指标,增量成本-效果比为10403.83,敏感性分析显示结果稳定。结论　亚洲ALL患儿的TPMT基因多态性与6-MP的骨髓毒性显著相关。决策树模型结果显示,在中国,ALL患儿通过TPMT基因检测调整6-MP初始剂量并不优于标准剂量给药。     

嘌呤类药物代谢酶和代谢物与药物反应关联及影响因素研究进展

临床上嘌呤类药物作为免疫抑制剂广泛应用于治疗实体器官移植、自身免疫性疾病和炎症性肠病、儿童急性淋巴细胞性白血病等疾病,但该类药物体内处置个体间差异大,治疗窗窄,且所致不良反应（如肾毒性和肝毒性）发生率较高。本文综述近年来嘌呤类药物代谢酶（TPMT、NDPK、ITPA、GST、XO/XDH、HGPRT、IMPDH和NUDT15）和代谢物（6-MMPR、6-TGNs、6-TGTP）对该类药物临床疗效和不良反应的影响,以及患者服药剂量和疗程、性别、年龄、体质量、遗传等多因素对代谢酶和代谢物影响的研究进展。研究表明该类药物临床疗效和不良反应与其体内代谢酶活性和代谢物浓度密切相关,但结论并不完全一致,因此有待大规模临床研究进一步验证以上关联性及影响因素。     

Thiopurine treatment in inflammatory bowel disease: clinical pharmacology and implication of pharmacogenetically guided dosing

This review summarises clinical pharmacological aspects of thiopurines in the treatment of chronic inflammatory bowel disease (IBD). Current knowledge of pharmacogenetically guided dosing is discussed for individualisation of thiopurine therapy, particularly to avoid severe adverse effects. Both azathioprine and mercaptopurine are pro-drugs that undergo extensive metabolism. The catabolic enzyme thiopurine S-methyltransferase (TPMT) is polymorphically expressed, and currently 23 genetic variants have been described. On the basis of an excellent phenotype-genotype correlation for TPMT, genotyping has become a safe and reliable tool for determination of a patient's individual phenotype. Thiopurine-related adverse drug reactions are frequent, ranging from 5% up to 40%, in both a dose-dependent and -independent manner. IBD patients with low TPMT activity are at high risk of developing severe haematotoxicity if pharmacogenetically guided dosing is not performed. Based on several cost-benefit analyses, assessment of TPMT activity is recommended prior to thiopurine therapy in patients with IBD. The underlying mechanisms of azathioprine/mercaptopurine-related hepatotoxicity, pancreatitis and azathioprine intolerance are still unknown. Although the therapeutic response appears to be related to 6-thioguanine nucleotide (6-TGN) concentrations above a threshold of 230-260 pmol per 8 x 10(8) red blood cells, at present therapeutic drug monitoring of 6-TGN can be recommended only to estimate patients' compliance.Drug-drug interactions between azathioprine/mercaptopurine and aminosalicylates, diuretics, NSAIDs, warfarin and infliximab are discussed. The concomitant use of allopurinol without dosage adjustment of azathioprine/mercaptopurine leads to clinically relevant severe haematotoxicity due to elevated thiopurine levels. Several studies indicate that thiopurine therapy in IBD during pregnancy is safe. Thus, azathioprine/mercaptopurine should not be withdrawn in strictly indicated cases of pregnant IBD patients. However, breastfeeding is contraindicated during azathioprine/mercaptopurine therapy. Use of azathioprine/mercaptopurine for induction and maintenance of remission in corticosteroid-dependent or corticosteroid-refractory IBD, particularly Crohn's disease, is evidence based. To improve response rates in thiopurine therapy of IBD, comprehensive analyses including metabolic patterns and genome-wide profiling in patients with azathioprine/mercaptopurine treatment are required to identify novel candidate genes.     

Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism

The efficacy of the immunosuppressants azathioprine and 6-mercaptopurine has been well established in the therapy of inflammatory bowel diseases (IBD). However, its use has been complicated by a high incidence of serious adverse drug reactions such as hematotoxicity, hepatotoxicity, pancreatitis and gastrointestinal disturbances. Whereas azathioprine-related pancytopenia has been clearly linked to thiopurine S-methyltransferase (TPMT) polymorphism limited data are available to explain gastrointestinal side effects. In a retrospective analysis of 93 adults with IBD and azathioprine therapy both phenotyping and genotyping was used to explore systematically the relationship between TPMT and azathioprine-related adverse reactions. At time of inclusion, 69 patients were still receiving azathioprine therapy and had never experienced side effects. Azathioprine had been withdrawn in 10 patients for non-medical reasons or lack of response and 14 patients (15%) had stopped medication or were on reduced dose due to severe azathioprine-related side effects. Nine of these 14 patients had developed gastrointestinal side effects (hepatotoxicity, n = 3; pancreatitis, n = 3; others, n = 3), but their normal red blood cell TPMT activities were in accordance to TPMT wild-type. TPMT deficiency in one patient had led to pancytopenia whereas only two of the remaining four patients with hematotoxicity displayed an intermediate phenotype of TPMT. This study demonstrates that azathioprine-related gastrointestinal side effects are independent of the TPMT polymorphism. Nevertheless pharmacogenetic testing for TPMT prior to commencing thiopurine therapy should become routine practice in order to avoid severe hematotoxicity in TPMT deficient patients and lowering the incidence of hematological side effects in individuals heterozygous for TPMT.     

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease

BACKGROUND: Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. AIMS: To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. METHODS: Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. RESULTS: Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. CONCLUSIONS: Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.     

基于连接酶反应检测硫唑嘌呤基因多态性及应用

目的：建立并探讨基于连接酶反应检测硫嘌呤甲基转移酶（thiopurine S—methyltransferase,TPMT）基因分型分布的新方法。方法：建立多重PCR／连接酶检测方法检测TPMT基因多态性,并对50份健康志愿者样本进行检测。结果：本方法可靠、迅速,自动化程度高。研究未发现导致TPMT酶活性降低的基因型^＊2,^＊3A,^＊3B和^＊4,TPMT^＊3C仅发现2例杂合子。结论：本文建立的基因分型技术可为TPMT基因型检测提供可靠的方法。     

中国人CYP3A5和CYP2D6基因多态性与他莫昔芬及其活性代谢物血药浓度的相关性

目的：研究中国汉族人群CYP3A5和CYP2D6基因多态性与乳腺癌患者体内他莫昔芬及其活性代谢物4-羟基他莫昔芬血药浓度的相关性。方法：30例乳腺癌患者,应用PCR方法检测其CYP3A5和CYP2D6基因型,应用LC—MS／MS方法测定患者体内他莫昔芬及其活性代谢物4-羟基他莫昔芬的含量,对试验数据进行统计分析。结果：CYP3A5基因型与乳腺癌患者体内他莫昔芬的浓度差异有统计学意义,比较发现＊1／＊1和＊1／＊3组的他莫昔芬的浓度值明显低于＊3／＊3组（P〈0．01）。CYP2D6基因型与其活性代谢物4-羟基他莫昔芬的浓度差异有统计学意义;＊1／＊1和＊1／＊10两组4-羟基他莫昔芬的浓度值都明显高于＊10／＊10组（P〈0．01）,但＊1／＊1组和＊1／＊10组之间4-羟基他莫昔芬的浓度值差异无统计学意义（P〉0．05）。结论：乳腺癌患者的CYP3A5和CYP2D6基因型影响他莫昔芬的体内代谢,相关基因型的测定可以为他莫昔芬个体化合理用药提供依据。     

Pharmacokinetics of 6-mercaptopurine in patients with inflammatory bowel disease: implications for therapy

Proper prospective pharmacokinetic studies of 6-mercaptopurine (6-MP) in inflammatory bowel disease (IBD) patients are lacking. As a result, conflicting recommendations have been made for metabolite monitoring in routine practice. The authors have evaluated 6-MP pharmacokinetics in IBD patients, including the genetic background for thiopurine methyltransferase (TPMT). Red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine ribonucleotide (6-MMPR) concentrations were measured in 30 IBD patients at 1, 2, 4, and 8 weeks after starting 6-MP, 50 mg once daily. Outcome measures included mean 6-TGN and 6-MMPR concentrations (+/- 95% confidence interval, CI95%) and their associations with TPMT genotype, 6-MP dose, and hematologic, hepatic, pancreatic, and efficacy parameters during the 8-week period. Steady-state concentrations were reached after 4 weeks, indicating a half-life of approximately 5 days for both 6-TGN and 6-MMPR; the concentrations were 368 (CI95% 284-452) and 2837 (CI95% 2101-3573) pmol/8 x 10 RBCs, respectively. Large interpatient variability occurred at all time points. TPMT genotype correlated with 6-TGN concentrations (0.576, P < 0.01), and patients with mutant alleles had a relative risk (RR) of 12.0 (CI95% 1.7-92.3) of developing leukopenia. A 6-MMPR/6-TGN ratio less than 11 was associated with therapeutic efficacy. Based on this pharmacokinetic analysis, therapeutic drug monitoring is essential for rational 6-MP dosing.     

CYP2C9基因多态性对华法林日平均剂量影响的Meta分析

目的:估算CYP2C9基因多态性对华法林日平均剂量(mean daily warfarin dose,MDWD)影响的定量关系,为临床个体化使用华法林提供参考。方法:计算机检索Cochrane Library和Pub Med(²013年12月),根据纳入标准对文献进行筛选和评估,采用Rev Man 5.1软件及Stata 12.0软件对数据进行Meta分析。结果:共纳入包括3 902例患者在内的27个合格英文研究。与CYP2C9*1/*1型相比,*1/*2型、*1/*3型、*2/*2型、*2/*3型和*3/*3型的MDWD分别下降0.20(95%CI:-0.24,-0.16)、0.31(95%CI:-0.35,-0.28)、0.40(95%CI:-0.47,-0.33)、0.52(95%CI:-0.59,-0.46)和0.81个标准单位(95%CI:-0.85,-0.76)。Begg检验未发现明显发表性偏倚。结论:本研究获得了CYP2C9基因多态性与华法林剂量个体间差异相关的定量关系,该定量关系可为临床设计华法林剂量个体化方案提供参考。     

CYP3A4＊1G基因多态性与比索洛尔药动学关系的研究

目的：研究C Y P3A4＊1G基因多态性对健康志愿者体内比索洛尔药动学的影响。方法：采用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）方法分析CYP3A4＊1G基因型。24名健康志愿者分为CC、CT、TT三组;受试者单剂量口服5mg富马酸比索洛尔片后,采用高效液相色谱法测定血药浓度,用3P97程序的一房室模型作药动学参数估算。结果：CC、CT、TT三种基因型受试者之间主要药动学参数t1/2,Cmax及AUC0-32分别为（7.79±1.57）、（7.49±0.77）、（7.88±1.21）h;（37.23±4.00）、（38.33±8.94）、（39.51±6.55）μ g/L;（386.42±91.49）,（375.53±84.51）,（406.24±87.24）μ g·h/L,通过t检验,三种基因型受试者之间的主要药动学参数差异没有显著性（P〉0.05）。结论：比索洛尔药动学在个体间的差异与CYP3A4＊1G基因型没有相关性。