Intrahepatic antiviral quantification in a patient undergoing orthotopic cadaveric liver transplantation

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 −80°C 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 μm 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 −80°C 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 methods.4–6 We successfully isolated 100 000 hepatocytes and quantified all three NAs from the core biopsy samples and paired PBMCs. We then measured the other antiviral agents in the liver tissue and plasma (Table 1). Tenofovir, emtricitabine, atazanavir and ritonavir concentrations were greater in liver tissue compared with plasma, but concentrations of raltegravir were greater in plasma versus the liver. The phosphorylated NA metabolites isolated from hepatocytes were found in higher concentration in PBMCs compared with hepatocytes. Table 1. Antiviral concentrations in peripheral blood, liver tissue and hepatocytes This case illustrates the feasibility of measuring antiviral concentrations in liver tissue and the potential to compare liver concentrations with those in the peripheral blood. There are limited prior reports of measuring antiviral concentrations in the liver in vivo. Using fine needle aspiration (FNA), Talal et al.7 measured telaprevir in 15 patients. Although FNA sampling is less invasive than core needle biopsy (CNB),8 the yield of actual liver tissue is lower. FNA samples contain 43 ± 24% liver tissue versus 95 ± 17% for CNB.8 Vaniprevir has been measured in three subjects using CNB.9 Vaniprevir liver to plasma ratios ranged from 20 to 280. There was wide interpatient variability in intrahepatic concentrations, which investigators speculated may be due to non-uniform drug distribution in the liver, variable numbers of hepatocytes within samples or different degrees of liver fibrosis. Sofosbuvir and ribavirin total metabolite concentrations were 73 and 356 μM, respectively, in liver samples from 25 patients obtained at the time of transplant then flash frozen,10 but concentration results are difficult to interpret without a hepatocyte count or other means of normalizing the result (e.g. per µg of DNA or mg of protein). Our approach differs from previous reports because we isolated fresh hepatocytes and measured the individual NA triphosphate moieties normalized to a cell count. In conclusion, antiviral drugs, including the phosphorylated anabolites of the NAs, can be quantified in liver samples and hepatocytes. Understanding the relationship between plasma and hepatic drug concentrations will inform our treatment of persons with ESLD.