Intrahepatic antiviral quantification in a patient undergoing orthotopic cadaveric liver transplantation.
Intrahepatic antiviral quantification in a patient undergoing orthotopic cadaveric liver transplantation.
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接受原位尸体肝移植的患者的肝内抗病毒定量。
DOI：
10.1093/jac/dku334
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Kiser,JenniferJ
中科院分区：
文献类型：
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作者：
Moorehead,KaitlynJ;BurtonJr,JamesR;Everson,GregoryT;Zheng,Jia-Hua;Kerr,BeckyJo;Bushman,LaneR;Wang,Meng;Ju,Cynthia;Nydam,Trevor;Kiser,JenniferJ
先生，与没有明显肝功能损害的患者相比，终末期肝病（ESLD）患者对丙型肝炎病毒（HCV）治疗的持续病毒学应答率较低。 1 可能存在与 ESLD 相关的病理生理学改变，这可能会改变肝脏中的药物渗透或药物激活。 2 比较肝脏和血液中抗病毒药物的浓度可能有助于 ESLD 的药物选择。这项工作的目的是量化肝组织中的抗病毒药物以及从新鲜肝外植体获得的肝细胞中活性磷酸化形式的核（t）ide类似物（NA），并将这些值与配对血浆和PBMC中的药物浓度进行比较。一名 50 岁男性，肝移植名单上 HIV/HCV 合并感染，因急性肾损伤（血清肌酐 4.11 mg/dL）转入我院，ESLD 评分为 40 分。入院前一年，患者的抗逆转录病毒治疗方案包括富马酸替诺福韦二吡呋酯 300 mg 每日一次加恩曲他滨 200 mg 每日一次，达芦那韦 800 毫克，每日一次；利托那韦 100 毫克，每日一次；拉替拉韦 400 毫克，每日两次。该患者的 HIV-1 RNA 为 20 个拷贝/mL，CD4 计数为 420 个细胞/mm3。第 1 天，停用富马酸替诺福韦二吡呋酯/恩曲他滨。第2天开始使用拉米夫定并根据肾功能调整剂量。第 3 天停用达芦那韦并用阿扎那韦替代。拉替拉韦和利托那韦剂量保持不变。患者提供了书面知情同意书，同意对来自其外植肝脏的血液、PBMC 和肝组织中的抗病毒药物进行定量并发表研究结果。转院六天后，患者接受了原位尸体肝移植。在手术室中，获取全血以定量血浆和 PBMC 中的抗病毒药物。四十五分钟后，肝硬化肝脏被切除。使用 18 号针活检从肝脏外植体中提取组织核心，用于分离肝细胞和定量磷酸化核苷酸。还从外植体的手术刀切割中收集小肝组织样品，用于母体药物的定量。将肝组织样品称重并用电匀浆器均质化，并在2808C下冷冻直至母体药物定量。从核心活检中分离肝细胞以定量磷酸化代谢物。简而言之，将核心活检样本浸泡在温热的灌注介质中。添加消化培养基并摇动样品。通过 100 mm 细胞滤网过滤解离的细胞聚集体并用洗涤介质冲洗。将样品离心，弃去上清液，将沉淀重新悬浮在洗涤介质中并计数。分离的肝细胞用70:30的甲醇/水裂解，然后储存在2808°C直至进行细胞内药物水平分析。使用经过验证的液相色谱串联质谱 (LC-MS/MS) 方法对 PBMC 和分离的肝细胞中的替诺福韦二磷酸盐、恩曲他滨三磷酸盐和拉米夫定三磷酸盐进行定量。 3 使用经过验证的 LC/MS 或 HPLC/UV 测量血浆和肝组织匀浆中的替诺福韦、恩曲他滨、拉替拉韦、阿扎那韦和利托那韦
Sir, Patients with end-stage liver disease (ESLD) have lower rates of sustained virological response to hepatitis C virus (HCV) treatment compared with those without significant hepatic impairment. 1 There may be pathophysiological alterations associated with ESLD, which may alter drug penetration or drug activation in the liver. 2 Comparing the concentrations of antiviral drugs in liver versus blood may inform drug selection in ESLD. The objective of this work was to quantify antiviral drugs in liver tissue and the active, phosphorylated forms of nucleos (t) ide analogues (NAs) in hepatocytes obtained from a fresh liver explant and to compare these values with drug concentrations in paired plasma and PBMCs. A 50-year-old male with HIV/HCV coinfection on the liver transplant list was transferred to our hospital with acute kidney injury (serum creatinine 4.11 mg/dL) with a Model for ESLD score of 40. In the year prior to hospitalization, the patient’s antiretroviral regimen included 300 mg of tenofovir disoproxil fumarate once daily plus 200 mg of emtricitabine once daily, 800 mg of darunavir once daily, 100 mg of ritonavir once daily and 400 mg of raltegravir twice daily. The patient had an HIV-1 RNA of, 20 copies/mL and a CD4 count of 420 cells/mm3. On day 1, tenofovir disoproxil fumarate/emtricitabine was discontinued. On day 2, lamivudine was initiated and dose adjusted based on renal function. Darunavir was discontinued and replaced with atazanavir on day 3. Raltegravir and ritonavir doses remained unchanged. The patient provided written informed consent for quantification of antiviral drugs in blood, PBMCs and hepatic tissue from his explant liver and publication of study findings. Six days following transfer to our hospital, the patient underwent orthotopic cadaveric liver transplantation. In the operating room, whole blood was obtained for quantification of antiviral drugs in plasma and PBMCs. Forty-five minutes later, the cirrhotic liver was removed. An 18-gauge needle biopsy was used to extract tissue cores from the liver explant for isolation of hepatocytes and quantification of phosphorylated nucleotides. Small liver tissue samples were also collected from scalpel cuts of the explant for quantification of parent drugs.Liver tissue samples were weighed and homogenized with an electro-homogenizer and frozen at 2808C until quantification of parent drugs. Hepatocytes were isolated from core biopsies for quantification of phosphorylated metabolites. Briefly, the core biopsy samples were soaked in warm perfusion medium. Digestion medium was added and samples were shaken. Dissociated cell aggregates were filtered through a 100 mm cell strainer and rinsed with wash medium. Samples were centrifuged, the supernatant discarded and the pellet re-suspended in wash medium and counted. The isolated hepatocytes were lysed with 70: 30 methanol/water then stored at 2808C until intracellular drug level analysis. Tenofovir diphosphate, emtricitabine triphosphate and lamivudine triphosphate were quantified in PBMCs and isolated hepatocytes using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. 3 Tenofovir, emtricitabine, raltegravir, atazanavir and ritonavir were measured in plasma and liver tissue homogenate using validated LC/MS or HPLC/UV