【作者】 寇惠娟；

【导师】 李太生；

【作者基本信息】 北京协和医学院 ， 内科学， 2012， 博士

【摘要】 研究背景高效抗逆转录病毒疗法(Highly active antiretroviral therapy, HAART)被认为是现今治疗HIV/AIDS行之有效的方法。奈韦拉平(nevirapine, NVP)是非核苷类逆转录酶抑制剂的一种,目前我国使用HAART方案治疗的患者中大部分仍以NVP作为初始治疗的首选药物。因此,优化NVP临床用药方案,观察NVP血药浓度与病毒学应答的关系,探讨NVP药物相关肝毒性和皮疹发生的影响因素及发生机制等具有重要的临床意义和价值。洛匹那韦(lopinavir, LPV)和利托那韦(ritonavir,RTV)均是蛋白酶抑制剂,替诺福韦(tenofovir, TNF)是核苷酸类逆转录酶抑制剂的一种,目前在我国主要应用于初治治疗失败的HIV/AIDS患者,研究这两种药物在中国HIV/AIDS患者体内的药代动力学特征对促进其临床安全有效应用具有重要价值。研究目的探讨NVP在中国HIV/AIDS人群中的临床药效学和药动学特点；建立高效液相色谱法测定人血浆中TNF浓度,观察TNF在中国HIV/AIDS人群中临床药代动力学特征；建立高效液相色谱法同时测定人血浆中LPV和RTV浓度,观察LPV在中国HIV/AIDS人群中的临床药动学特征。研究对象与方法筛选2005～2011年期间接受包含NVP治疗方案、规律随诊、自愿签署知情同意书的828例HIV/AIDS患者,于服药前或服药后2-4小时采集静脉血2mL并分离血浆,应用高效液相色谱-紫外检测法(HPLC-UV)测定人血浆中药物浓度,并详细记录治疗前及每次随诊时患者的一般情况、血尿常规、肝肾功能、淋巴细胞亚群计数及病毒载量等结果,进行NVP相关临床药动学和药效学分析。招募接受包含TNF和/或LPV抗病毒治疗方案的16位患者住院治疗,于服药前以及服药后0.5,1,1.5,2,2.5,3,4,6,8,10,12,16,24小时,采集静脉血并分离冻存血浆,应用本研究建立的HPLC-UV法分别测定人血浆中TNF和LPV药物浓度,采用WINONLIN软件计算TNF和LPV临床药动学参数。研究结果导入期末NVP血浆谷浓度(Ctrough)为4.26μg/mL(IQR3.05-5.61),峰浓度(Cmax)为5.07μg/mL (IQR3.92-6.44),均已达到国际公认有效治疗浓度下限(3.0μg/mL),但是显著低于稳态NVP-Ctrough (6.15μg/mL, IQR4.63-8.20, P=0.000)和NVP-Cmax (6.51μg/mL, IQR5.22-8.41, P=0.000)。分层分析发现,基线ALT≥1.5×ULN患者的导入期末NVP-Ctrough显著高于ALT<1.5×ULN患者(4.86v.s.4.25μg/mL,P=0.045)：随着用药时间的延长,HIV合并HCV感染患者NVP-Ctrough逐渐升高,至随访第48周Ctrough显著高于未合并HCV感染患者(8.16v.s.6.15μg/mL,P=0.004)。发生病毒学失败(viralogocal failure, VF)的患者NVP-Ctrough均值显著低于完全病毒学抑制和部分病毒学抑制患者组(3.23μg/mL v.s.6.73和6.60μg/mL,P=0.000)。当NVP-Ctrough<3.9μg/mL时,发生VF的风险是NVP-Ctrough≥3.9μg/mL患者的6.15倍,预测VF发生的灵敏度和特异度分别为87.9%和90%。单因素分析提示合并HCV感染、ALT和AST≥1.25ULN、CD4计数>250/mm3与肝损发生相关,女性、体重<55Kg与CD4计数≥250/mm3与重度肝损发生相关,进一步分层分析发现男性合并HCV感染会增加肝损发生风险,女性合并HCV感染则未观察到这一现象；多因素logistic回归分析发现ALT和AST水平升高以及高CD4计数与肝损发生密切相关,低体重和高CD4计数与重度肝损发生密切相关。男性患者肝损当次NVP-Ctrough≥10μg/mL时发生重度肝损的比例是NVP-Ctrough<10μg/mL患者的3.77倍,女性患者则未观察到这一现象。当肝损前次NVP-Ctrough≥5μg/mL时,发生肝损的风险是NVP-Ctrough<5μg/mL患者的2.36倍,预测肝损发生的灵敏度和特异度分别为61.9%和59.3%。当导入期NVP-Ctrough≥6μg/mL时,发生肝损的风险是NVP-Ctrough<6μg/mL患者的2.21倍,预测肝损发生的灵敏度和特异度分别为60.0%和59.5%。女性NVP相关皮疹发生风险是男性患者的1.73倍,合并HBV/HCV感染或高CD4计数不会增加服药患者皮疹发生风险；皮疹患者皮疹发生前次、当次和后次MIG、IP-10和IFN-y因子水平均显著高于正常健康人群,皮疹患者其当次MIG和IP-10因子水平均显著高于皮疹发生前次和后次。建立同时测定人血浆中LPV和RTV浓度的高效液相色谱方法,LPV在0.5～20gg/mL范围内,RTV在0.05～5μg/mL范围内线性良好(r=0.9995和0.9997)；建立测定人血浆中TNF浓度的高效液相色谱方法,TNF在20～2000ng/mL范围内线性良好(r=0.9997)。两种高效液相色谱检测方法灵敏度高,操作简便,精密度、准确度及稳定性结果考察良好。WinNonlin软件计算显示,替诺福韦符合二室模型,计算的药动参数分别为Tmax (1.20±0.47h)、Cmax (361.51±219.04ng/mL)、AUC(4077.50±1554.88ng*h/mL)、t1/2(29.53±14.12h)和CL/F(80.89±21.49L/h),统计学分析发现未合并LPV的患者其TNF血浆t1/2显著大于合并LPV的患者(38.04v.s.22.09h,P=0.023)。TNF的非房室模型药动参数分别为T1/2(21.84±7.64h)、Tmax (1.33±0.45h)、Cmax (447.09±217.39ng/mL)、Ctrough (98.66±36.66ng/m)、AUC(4074.7±1551.86ng*h/mL)和CL/F(45.77±13.05L/h)。LPV在中国服药人群具有达峰时间晚、个体差异大等特点结论NVP200mg一天一次的给药方式在中国HIV/AIDS人群中可能具有可行性；基线肝脏功能异常和合并HCV感染会对NVP药物代谢产生影响,临床应予以重视；NVP-Ctrough降低与病毒学失败的发生相关,与国际通用的NVP-Ctrough3.0μg/mL阈值相比,在中国人群中以3.9μg/mL作为预测病毒学失败发生的治疗浓度阈值具有较高灵敏度；NVP-Ctrough升高与肝毒性发生相关特别是在男性患者中,肝损前次NVP-Ctrough≥5μg/mL及导入期NVP-Ctrough≥6μg/mL预测肝损发生有一定的临床意义；女性患者易出现NVP相关皮疹,IP-10和MIG等炎症性趋化因子可能通过介导Thl型免疫应答反应促进组织损伤从而参与了NVP相关皮疹的发生发展；中国HIV/AIDS患者服用TNF的稳态血浆T1/2、Cmax、trough及AUC均显著高于国外研究水平,Tmax与国外研究报道类似,合并LPV用药可能会对TNF的药物消除产生影响,临床应予以重视；LPV在中国服药人群具有达峰时间晚、个体差异大等特点。更多还原

【Abstract】 BackgroundHighly active antiretroviral therapy (HAART) has been considered as the most effective way of treatment of HIV infection and/or AIDS diseases. Nevirapine is one of non-nucleoside reverse transcriptase inhibitors and was recommended as the first-line antiretroviral regimen by the guidelines of international AIDS society and USA panel. It is important and necessary to observe the relationship between nevirapine concentration and viralogical response and to evaluate the influence of patient characteristics on nevirapine concentrations and drug-related liver toxicity and skin rash. Due to the higher incidence of drug-related liver toxicity and skin rash in Chinese HIV-infected patients with the conventional dosage regimen, it is of interest to investigate the potential of nevirapine200mg once daily regimen. Lopinavir is one protease inhibitor and tenofovir is one of nucleotide reverse transcriptase inhibitors. As the major antiretrovials in salvage regimen, tenofovir and lopinavir have been proven valid in Chinese HIV-infected adults with first-line treatment failure. Thus it is important to investigate their pharmacokinetic features in Chinese HIV-infected patients for their effective and safe use.ObjectivesTo investigate the potential of nevirapine200mg once daily regimen in clinical practice and evaluate the relationship between nevirapine concentration and viralogical response and drug-related adverse events; to develop two high performance liquid chromatography methods coupled with UV detection for quantitative determination of lopinavir and ritonavir and tenofovir in human plasma and to investigate the pharmacokinetic features of lopinavir and tenofovir in the Chinese patients with HIV infection.MethodsThis was a prospective, multicentre cohort study with828HIV-infected patients receiving nevirapine as a part of their initial antiretroviral therapy between Jan2005and Dec2011. The trial was approved by institutional review boards and carried out in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice. Written informed consent was obtained from each patient and the study protocol was approved by independent ethics committee of participating hospitals. During the treatment period, patients were monitored at baseline, week2,4,12,24,36, and48for clinical features (particularly severe adverse events), plasma nevirapine concentrations and laboratory values including blood routine examination, hepatic and renal function, hepatitis B (HBV) or hepatitis C (HCV) serological state, CD4cell counts and plasma viral load. Nevirapine plasma concentrations during the lead-in (200mg once daily) and steady-state (200mg twice daily) periods were compared.A total of16patients receiving tenofovir as a part of antiretroviral regimen were enrolled into the pharmacokinetic study. Written informed consent was obtained from each patient and the study protocol was approved by the ethics committee of Peking Union Medical College Hospital. Blood samples were collected before and0.5,1,1.5,2,2.5,3,4,6,8,10,12,16and24h after administration. Plasma samples were analyzed for lopinavir and tenofovir concentrations by using the developed HPLC assays in present study. WINONLIN software was used to compute the pharmacokinetic parameters.ResultsThe median nevirapine trough and peak concentration during the lead-in period were4.26μg/mL (IQR3.05-5.61) and5.07μg/mL (IQR3.92-6.44) respectively, which both exceeded the recommended thresholds of nevirapine plasma concentrations, e.g.3.0μg/mL and3.9μg/mL, even though significantly lower than the steady-state levels (6.15and6.51μg/mL, P=0.000). Baseline hepatic function had a moderate effect on median nevirapine trough concentrations at week2(4.25μg/mL vs.4.86μg/mL, for ALT<1.5×ULN and≥1.5×ULN, respectively, P=0.045). No significant difference was observed in median nevirapine trough concentration between lead-in and steady-state periods in patients with baseline ALT and AST level≥1.5×ULN, which was different from the patients with ALT/AST level<1.5ULN. The median trough concentrations were significantly higher in HIV/HCV co-infected patients than those without HCV at week48(8.16μg/mL vs.6.15μg/mL, P=0.004).The median nevirapine trough concentrations were3.23,6.60and6.73μg/mL in patients with viralogical failure, partial viralogical suppression and complete viralogical suppression respectively, however, the differences of nevirapine peak concentrations were not observed among the three groups. Compared to the patients with nevirapine trough concentration greater than3.0or3.9μg/mL, the patients with nevirapine trough concentration lower than3.0or3.9μg/mL had a higher risk of occurrence of viralogical failure. Compared with3.0ug/mL,3.9μg/mL was a better threshold of nevirapine trough concentration in Chinese population to predict viralogical failure with sensitivity of87.9%and specificity of90.0%.The univariate stratification analysis showed that HIV/HCV coinfection particularly in males, ALT and AST>1.25ULN, CD4>250/mm3were associated with liver toxicity, female, Wt<55Kg and CD4>250/mm were associated with severe liver toxicity. The multivariate logistic regression indicated that increased levels of ALT and AST and CD4cell counts were significantly associated with liver toxicity, increased CD4cell counts and low body weight were significantly associated with severe liver toxicity. The risk of liver toxicity was increased3.23times when NVP-Ctrough>H Hg/mL, particularly, the risk was increased3.77times when NVP-CtrOugh>10ug/mL in males. The risk of liver toxicity was increased2.36times when the last-time NVP-Ctrough was>5u.g/mL. ROC analysis showed that NVP-Ctrough threshold of>5ug/mL had high sensitivity and specificity to predict the occurrence of liver toxicity. The risk of liver toxicity was increased2.21times when the lead-in NVP-Ctrough was>6ug/mL. ROC analysis showed that NVP-Ctrough threshold of>6ug/mL had high sensitivity and specificity of predicting the occurrence of liver toxicity.The risk of skin rash was increased1.73times in females, however, HIV/HBV or HIV/HCV coinfection and elevated CD4cell counts were not associated with the drug related skin rash. The levels of MIG, IP-10and IFN-y in patients with rash were significantly higher than the healthy volunteers. Additionaly, the levels of MIG and IP-10at the time rash occurrence were significantly higher than the levels before and after the appearance of rash.The calibration curves were linear in the range of0.5-20ug/mL for LPV (r=0.9995) and0.05-5ug/mL for RTV (r=0.9999). The range of the calibration curve of tenofovir was20-2000ng/mL (r=0.9997). The precision, accuracy and stability of the assays were satisfactory. The pharmacokinetics of tenofovir in15Chinese HIV-infected patients complied with two-compartment model, and the steady pharmacokinetic parameters were as follows: Tm29.53±14.12h, Tmax1.20±0.47h, Cmax361.51±219.04ng/mL, AUC4077.50±1554.88ng*h/mL, CL/F80.89±21.49L/h respectively. Additionaly, the patients who were not coadministered with LPV showed longer T1/2compared to the patients with LPV coadministration. The non-compartment model PK parameters were as follows: T1/221.84±7.64h, Tmax1.33±0.45h, Cmax447.09±217.39ng/mL, Ctrough98.66±36.66ng/mL and AUC4074.7±1551.86ng*h/mL, CL/F45.77±13.05L/h respectively.ConclusionsThe200mg once daily regimen of nevirapine might be comparable to twice daily in plasma pharmacokinetics in Chinese patients with HIV infection. Hepatic function prior to nevirapine treatment and HIV/HCV coinfection were significantly associated with nevirapine concentrations.The incidence of viralogical failure was increased with the low nevirapine trough concentration;3.9μg/mL was a better threshold of nevirapine trough concentration in Chinese population to predict viralogical failure with sensitivity of87.9%and specificity of90.0%higher than the3.0μg/mLPatients with high NVP-Ctrough were at greater risk of liver toxicity, especially in males. The last-time NVP-Ctrough of≥5μg/mL or lead-in NVP-Ctrough of≥6μg/mL was a good target to predict the incidence of hepatotoxicity.The female patients were vulnerable to nevirapine related skin rash; the inflammatory cytokines of MIG and IP-10may play a main role in the immune pathogenesis of nevirapine related skin rash through the abnormality of immune response.Two simple, validated, sensitive and reproducible methods for quantifying the concentration of tenofovir, lopinavir and ritonavir in human plasma by high performance liquid chromatography coupled with UV detection have been reported. The pharmacokinetics of tenofovir in Chinese patients demonstrated longer half-life, increased Cmax, Ctrough and AUC compared with those in Caucasians. Coadministration with LPV would potentially affect the pharmacokinetics of TNF. The pharmacokinetic profiles of lopinavir in Chinese HIV-infected patients demonstrated delayed Tmax and significant individual differences.更多还原