Effects of sofosbuvir-based hepatitis C treatment on the pharmacokinetics of tenofovir in HIV/HCV-coinfected individuals receiving tenofovir disoproxil fumarate

Effects of sofosbuvir-based hepatitis C treatment on the pharmacokinetics of tenofovir in... Abstract Background The nucleotide analogues tenofovir and sofosbuvir are considered to have low potential for drug interactions. Objectives To determine the effect of sofosbuvir-based HCV treatment on plasma concentrations of tenofovir and cellular concentrations of tenofovir diphosphate. Methods HIV-infected participants with acute HCV were treated for 12 weeks with sofosbuvir + ribavirin in Cohort 1 or 8 weeks with ledipasvir/sofosbuvir in Cohort 2 of AIDS Clinical Trials Group study 5327. Only participants taking tenofovir disoproxil fumarate were included in this analysis. Tenofovir in plasma, tenofovir diphosphate in dried blood spots and tenofovir diphosphate in PBMCs were measured pre-HCV therapy and longitudinally during the study using validated LC/MS-MS. Results Fifteen and 22 men completed Cohorts 1 and 2, respectively. In Cohort 1, tenofovir diphosphate was 4.3-fold higher (95% CI geometric mean ratio 2.46–7.67; P = 0.0001) in dried blood spots and 2.3-fold higher (95% CI 1.09–4.92; P = 0.03) in PBMCs following 12 weeks of sofosbuvir + ribavirin versus study entry. Tenofovir in the plasma was unchanged. In Cohort 2, tenofovir diphosphate was 17.8-fold higher (95% CI 12.77–24.86; P < 0.0001) in dried blood spots after 8 weeks of ledipasvir/sofosbuvir versus study entry. Tenofovir plasma concentrations were 2.1-fold higher (95% CI 1.44–2.91; P = 0.0005). Despite the increase in cellular tenofovir diphosphate concentrations, only a small decline in CLCR (6%–7%) was observed in both cohorts between study entry and end of treatment. Conclusions These data indicate an unexpected drug interaction with tenofovir disoproxil fumarate and sofosbuvir at the cellular level. Additional studies are needed to determine the mechanism and clinical significance. Introduction Globally, an estimated 2.3 million people are coinfected with HIV and HCV owing to shared routes of transmission.1 Prison inmates, MSM and persons who inject drugs are most affected.1 HIV accelerates the progression of liver disease in individuals coinfected with HCV.2,3 Thus, treatment is imperative to reduce the complications of chronic liver disease in this population. In the pegylated interferon and ribavirin era, treatment of HCV in the acute phase resulted in higher rates of sustained virological response with a shortened course of therapy compared with treating individuals with chronic infection.4–7 However, there are limited data on the optimal use of contemporary all-oral, direct-acting antiviral agents (DAAs) in individuals with acute HCV. AIDS Clinical Trials Group study 5327 (‘SWIFT-C’, NCT02128217) was an open-label, two-cohort clinical trial of sofosbuvir-based treatment of acute HCV in individuals with chronic HIV-1 infection.8,9 Cohort 1 of SWIFT-C assessed the safety and efficacy of 12 weeks of 400 mg sofosbuvir in combination with weight-based ribavirin. Cohort 2 assessed the safety and efficacy of 8 weeks of 90 mg ledipasvir in combination with 400 mg sofosbuvir. The sustained virological response rate was 100% in Cohort 2 of SWIFT-C, but only 59% in Cohort 1.9 The high rate of relapse in Cohort 1 was unexpected and we suspected suboptimal ribavirin adherence contributed to this outcome. Although pill counts and participant recall suggested good ribavirin adherence, as previously reported, we found that individuals that relapsed after treatment with sofosbuvir + ribavirin had lower ribavirin plasma concentrations during treatment.9,10 Ribavirin has a long half-life in plasma (∼9 days)11 and, thus, ribavirin concentrations in plasma reflect, in part, cumulative drug dosing and long-term adherence to this drug. Thus, the lower ribavirin plasma concentrations in patients that relapsed in Cohort 1 of SWIFT-C likely reflected suboptimal adherence to ribavirin, given that the patients who achieved sustained virological response had levels comparable with historical data.9,10 Though there are no historical data establishing the ribavirin exposures observed with perfect adherence, mean ribavirin plasma concentrations in SWIFT-C participants that relapsed were lower than those observed in prior studies of sofosbuvir + ribavirin.12,13 Suboptimal adherence to medications leads to inadequate response to treatment and increased healthcare costs.14 The WHO estimates that 50%–60% of individuals are non-adherent to the medications they have been prescribed to treat chronic illnesses.15 Suboptimal adherence to ART leads to drug resistance, an increase in AIDS-related illnesses and death, and higher rates of HIV transmission.16–18 Adherence is also an issue for illnesses with a finite treatment duration. Consider tuberculosis in which 16%–49% of individuals do not complete treatment.19 There is evidence of suboptimal adherence in clinical trials of DAAs, despite the fact that HCV treatment is only 8–24 weeks in duration.20–27 Traditional tools to assess adherence (e.g. refill records, pill counts, self-reporting) have significant shortcomings and typically overestimate adherence.28 Pharmacokinetic-based adherence measures provide objective evidence of drug ingestion.29 For compounds with short half-lives, the information gained is qualitative (i.e. whether a dose was taken recently) but compounds with longer half-lives provide information on cumulative drug dosing and a more quantitative measure of drug-taking behaviour.30 Tenofovir disoproxil fumarate is an acyclic nucleoside phosphonate diester analogue of adenosine monophosphate and is initially converted to tenofovir by diester hydrolysis in the gut, liver and plasma.31,32 Tenofovir is taken up by cells,31 then phosphorylated by cellular kinases to the active form of the drug, tenofovir diphosphate. Tenofovir diphosphate competes with 2′-deoxyadenosine 5′-triphosphate (dATP) for incorporation into replicating viral DNA. If tenofovir diphosphate is incorporated into the viral DNA, rather than dATP, chain termination occurs.33 The tenofovir diphosphate half-life in PBMCs is 3–7 days34–36 and in red blood cells it is 17 days.37 Thus, like ribavirin concentrations in plasma, tenofovir diphosphate concentrations in cells can be used to assess cumulative drug exposure and long-term average adherence.37 Concentrations of tenofovir diphosphate in dried blood spots, which contain ∼12 million red blood cells, are correlated with the risk of acquiring HIV in studies of tenofovir disoproxil fumarate and emtricitabine for HIV prevention.38,39 Plasma concentrations of tenofovir have also been used for assessing adherence, but the plasma half-life of tenofovir is approximately 10–15 h33 and, thus, tenofovir concentrations in plasma reflect only recent dosing. Given that we found lower ribavirin levels in participants that failed treatment with sofosbuvir + ribavirin in Cohort 1 of SWIFT-C,9,10 which suggested suboptimal adherence to ribavirin, the primary aim of this analysis was to evaluate whether similar trends were evident for antiretroviral adherence (using established methods37) among SWIFT-C participants by comparing tenofovir diphosphate concentrations in dried blood spots before, during and after sofosbuvir + ribavirin treatment. In the course of this analysis, we identified an unexpected drug interaction between tenofovir disoproxil fumarate and sofosbuvir. Thus, we also compared tenofovir diphosphate in PBMCs and tenofovir in plasma before, during and after sofosbuvir + ribavirin treatment and evaluated the interaction in Cohort 2 participants receiving ledipasvir/sofosbuvir. Methods Participants ≥18 years old with chronic HIV-1 infection and documented confirmation of acute HCV infection within 6 months prior to entry were recruited from eight different AIDS Clinical Trials Group sites in the USA. We obtained ethics committee approval at all participating centers in accordance with the principles of the 2008 Declaration of Helsinki. All participants provided written informed consent before undergoing any protocol-specified procedures. Acute HCV was defined as9 (i) new HCV RNA in patients with documented negative serology in the past 6 months or (ii) in those with no documented serology in the past 6 months, positive HCV RNA plus a new ALT elevation [>5 × upper limit of normal (ULN)] with normal ALT in the past 12 months or >10 × ULN with no measured ALT in the past 12 months, as well as exclusion of other causes of acute hepatitis. Participants were not required to be taking ART, but those receiving ART were required to be stable on a regimen for at least 8 weeks, with an HIV-1 RNA <50 copies/mL and CD4 + T cell count >200 cells/mm3. Participants’ adherence was assessed using a 4 day recall and by pill count. For Cohort 1 participants, plasma, dried blood spots and PBMCs were analysed at entry, week 12 of sofosbuvir + ribavirin treatment and 12 weeks following the end of sofosbuvir + ribavirin treatment (EOT + 12). In Cohort 2 participants, plasma samples were analysed for each participant at entry, week 8 of ledipasvir/sofosbuvir treatment and EOT + 12 and dried blood spot samples were analysed from each participant at entry, week 1, week 2, week 4 and week 8 of ledipasvir/sofosbuvir treatment and EOT + 2, EOT + 4, EOT + 8 and EOT + 12. PBMCs were not collected in Cohort 2. All pharmacology samples were collected at convenience times post-dose. Tenofovir diphosphate concentrations in a 3 mm dried blood spot punch and in PBMCs were analysed using a validated LC/MS-MS method.40 The dynamic range of the assay was 25–6000 fmol/sample for tenofovir diphosphate in dried blood spots and 2.50–2000 fmol/sample for PBMCs.40 Accuracy was ≤ ± 15% from nominal and precision was ≤15% coefficient of variation (CV). Lack of assay interference between analytes was confirmed. Plasma tenofovir concentrations were also analysed using a validated LC/MS-MS method; the dynamic range of the assay was 10–1500 ng/mL.41 Tenofovir and tenofovir diphosphate concentrations were log transformed and compared between study visits using paired t-tests. Results Study patients Seventeen HIV-infected male participants entered Cohort 1 of SWIFT-C and all 17 completed 12 weeks of sofosbuvir + ribavirin treatment for acute HCV. All 17 were on ART and 15 were taking tenofovir disoproxil fumarate as part of their antiretroviral regimen. Among these 15, 11 were Hispanic and 4 were non-Hispanic white with a mean (±SD) age of 44.3 (±9.5) years and a mean (±SD) weight of 76.5 (±10.3) kg. Complete demographic information can be found in Table 1. Table 1. Demographic information Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Table 1. Demographic information Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Twenty-seven HIV-infected male participants entered Cohort 2 of SWIFT-C and all 27 completed 8 weeks of ledipasvir/sofosbuvir treatment for acute HCV. All 27 were on ART and 22 were taking tenofovir disoproxil fumarate as part of their antiretroviral regimen. Among these 22, 6 were Hispanic and 16 were non-Hispanic white with a mean (±SD) age of 44.4 (±8.8) years and a mean (±SD) weight of 76.9 (±9.6) kg. Complete demographic information can be found in Table 1. Tenofovir diphosphate and tenofovir concentrations In Cohort 1, at study entry, the geometric mean (CV%) tenofovir diphosphate concentration in dried blood spots was 1687 (31) fmol/punch; consistent with historical data in HIV-infected and -uninfected individuals with high adherence.29,30,37,42 However, at week 12 of sofosbuvir + ribavirin treatment, the geometric mean (%CV) tenofovir diphosphate concentration in dried blood spots was 6607 (74) fmol/punch. In the 12 participants with both entry and week 12 tenofovir diphosphate concentrations in dried blood spots, the geometric mean ratio for tenofovir diphosphate was 4.3 versus entry (95% CI 2.46–7.67; P = 0.0001). By EOT + 12, tenofovir diphosphate in dried blood spots had declined and the geometric mean (%CV) was 2101 (36) fmol/punch (P = 0.0072) (Figure 1a). Tenofovir diphosphate concentrations were also determined in PBMCs. At entry, week 12 and EOT + 12, geometric mean (%CV) tenofovir diphosphate concentrations in PBMCs were 79 (48), 149 (91) and 81 (52) fmol/million cells, respectively. The tenofovir diphosphate concentrations pre-sofosbuvir + ribavirin in PBMCs were similar to historical data in HIV-infected individuals with high adherence,29,37 but for the 10 participants with both entry and week 12 concentrations, the tenofovir diphosphate concentrations in PBMCs were 2.3-fold higher at week 12 (95% CI 1.09–4.92; P = 0.03) (Figure 1b). Despite the increased levels of tenofovir diphosphate in PBMCs and red blood cells, tenofovir concentrations in plasma were similar to historical data and unchanged across visits (P = 0.83). At entry, week 12 and EOT + 12, geometric mean (%CV) tenofovir concentrations were 98 (63), 96 (57) and 94 (76) ng/mL, respectively (Figure 1c). Figure 1. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/million cells) in PBMC samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (c) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). The tenofovir concentrations were similar to historical data and unchanged across visits (P = 0.83). GMR, geometric mean ratio. Figure 1. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/million cells) in PBMC samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (c) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). The tenofovir concentrations were similar to historical data and unchanged across visits (P = 0.83). GMR, geometric mean ratio. In Cohort 2, at study entry, the geometric mean (%CV) tenofovir diphosphate concentrations in dried blood spots were 1516 (36) fmol/punch, which was similar to that for Cohort 1 and consistent with historical data in HIV-infected and -uninfected individuals with high adherence.29,37,42 By week 1 of ledipasvir/sofosbuvir treatment, tenofovir diphosphate concentrations were 6.7-fold higher versus entry (Figure 2a). At week 8 of ledipasvir/sofosbuvir treatment, for the 20 participants with both entry and week 8 tenofovir diphosphate concentrations in dried blood spots, the geometric mean ratio for tenofovir diphosphate was 17.8 (95% CI 12.77–24.86; P < 0.0001) compared with study entry. The geometric mean (%CV) at week 8 of tenofovir diphosphate was 26 846 (49) fmol/punch. Tenofovir concentrations in plasma were increased 2.1-fold (95% CI 1.44–2.91; P = 0.0005), at week 8 of ledipasvir/sofosbuvir treatment with a geometric mean (%CV) of 155 (112) ng/mL (Figure 2b). Three participants in Cohort 2 switched from tenofovir disoproxil fumarate to tenofovir alafenamide at week 1, week 7 and week 15 of study, respectively; thus, tenofovir diphosphate in dried blood spots and tenofovir in plasma samples for these individuals were excluded from analyses following the switch. Figure 2. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting ledipasvir/sofosbuvir), weeks 1, 2, 4 and 8 of ledipasvir/sofosbuvir treatment and 2, 4, 8 and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 2, EOT + 4, EOT + 8 and EOT + 12). Concentrations of tenofovir diphosphate during ledipasvir/sofosbuvir treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting ledipasvir/sofosbuvir), after 8 weeks of ledipasvir/sofosbuvir treatment and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 12). The tenofovir concentrations were 2.1-fold higher at week 8 versus study entry (P = 0.0005). GMR, geometric mean ratio. Figure 2. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting ledipasvir/sofosbuvir), weeks 1, 2, 4 and 8 of ledipasvir/sofosbuvir treatment and 2, 4, 8 and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 2, EOT + 4, EOT + 8 and EOT + 12). Concentrations of tenofovir diphosphate during ledipasvir/sofosbuvir treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting ledipasvir/sofosbuvir), after 8 weeks of ledipasvir/sofosbuvir treatment and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 12). The tenofovir concentrations were 2.1-fold higher at week 8 versus study entry (P = 0.0005). GMR, geometric mean ratio. Prior data in healthy volunteers suggested administering ledipasvir/sofosbuvir concomitantly with a boosted antiretroviral regimen and tenofovir disoproxil fumarate could result in tenofovir exposures that exceed the range for established renal safety data.43 In this study, Cohort 1 participants who were taking either ritonavir or cobicistat for pharmacokinetic boosting did not have higher tenofovir diphosphate or tenofovir concentrations compared with those not taking a boosting agent at any study visit. In Cohort 2, participants taking a boosted antiretroviral regimen had higher tenofovir diphosphate concentrations at baseline (before ledipasvir/sofosbuvir). The geometric mean (%CV) tenofovir diphosphate concentrations prior to the addition of ledipasvir/sofosbuvir were 1912 (29) fmol/punch and 1279 (32) fmol/punch for boosted versus unboosted regimens, respectively (P = 0.005). However, this was not observed at subsequent visits and, rather, participants not taking a boosting agent actually had numerically (though not statistically) higher tenofovir diphosphate concentrations. Renal function The mean (SD) CLCR in Cohort 1 participants was 125.4 mL/min (24) at study entry and 117.7 mL/min (20) at week 12 of sofosbuvir + ribavirin treatment (P = 0.18). The mean (SD) CLCR in Cohort 2 participants was 107.5 mL/min (16) at study entry and 99.8 mL/min (18) at week 8 of ledipasvir/sofosbuvir treatment (P =  0.01). Two participants, both on tenofovir disoproxil fumarate plus a boosting agent in Cohort 2, had an increase in serum creatinine >0.4 mg/dL (confirmed within 2 weeks), which was a protocol-defined renal toxicity. However, neither participant required a dose reduction of tenofovir disoproxil fumarate during ledipasvir/sofosbuvir treatment. There were no premature study discontinuations. Discussion In our investigations using drug concentrations to assess antiretroviral adherence in HIV-infected men treated for acute HCV, we identified an unexpected increase in cellular tenofovir diphosphate concentrations with both sofosbuvir + ribavirin and to a much larger extent with ledipasvir/sofosbuvir. The interaction was not observed in plasma for sofosbuvir + ribavirin and was observed with ledipasvir/sofosbuvir, but at a much smaller magnitude compared with the effect on tenofovir diphosphate in dried blood spots. We believe this finding suggests the presence of a previously undescribed drug–drug interaction between tenofovir disoproxil fumarate and sofosbuvir. The mechanism for this interaction is unclear, but given that the increase in tenofovir diphosphate concentrations occurred with both sofosbuvir + ribavirin and ledipasvir/sofosbuvir it suggests sofosbuvir is perpetrating this interaction. However, the interaction occurred to a much larger extent in those who received ledipasvir in addition to sofosbuvir, so ledipasvir is also a significant contributor to this interaction. Whether the contribution is via a direct effect of ledipasvir on tenofovir or tenofovir disoproxil fumarate, perhaps through inhibition of P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP), or the result of ledipasvir causing an increase in sofosbuvir AUC, which has been observed in prior studies,44–46 is unknown. In healthy volunteers, sofosbuvir increases the maximum concentration of tenofovir in plasma by 25% with no change in AUC.47 Ledipasvir/sofosbuvir increases tenofovir plasma concentrations by 30%–80% depending on the concomitant antiretrovirals.43,48 There are several potential mechanisms for the sofosbuvir-mediated increase in tenofovir diphosphate. A study in Caco-2 monolayers found that tenofovir disoproxil fumarate recovery (from the apical to basolateral membrane) was increased in the presence of sofosbuvir.46 Recent work suggests sofosbuvir inhibits carboxylesterase-2 (CES2).49 Tenofovir disoproxil fumarate is converted to tenofovir by two separate ester hydrolysis steps.32 Carboxylesterases are involved in this conversion of tenofovir disoproxil to tenofovir.50,51 Carboxylesterase enzymes are expressed in liver, small intestine, kidneys, lungs, plasma and other cell types.52 Thus, sofosbuvir inhibition of CES2 could result in greater prodrug delivery and increased cellular uptake. Other potential mechanisms include alterations in drug phosphorylation or dephosphorylation or changes in cellular uptake or efflux. In terms of potential alterations in phosphorylation, it is possible that sofosbuvir (or its intracellular metabolites) enhance phosphorylation of tenofovir or inhibit tenofovir diphosphate dephosphorylation through up-regulation or down-regulation of the involved kinases or phosphatases. The effect of sofosbuvir on enzymes responsible for tenofovir phosphorylation (e.g. adenylate kinases 1 and 2, pyruvate kinase, creatine kinase) or tenofovir diphosphate dephosphorylation (e.g. 5′-nucleotidases)53–57 has not been evaluated to our knowledge. In terms of the potential for sofosbuvir to increase cellular uptake of tenofovir disoproxil fumarate or tenofovir, tenofovir is a substrate for the uptake transporter, organic anion transporter (OAT1), and this transporter mediates the nephrotoxicity observed with tenofovir disoproxil fumarate.58 Tenofovir may also be a substrate for equilibrative nucleoside transporters (ENTs). One study found tenofovir diphosphate concentrations also may be increased in individuals with variant polymorphisms in the genes encoding ENTs.59 The potential for sofosbuvir (or its metabolites) to induce OAT1, ENT1 or other uptake transporters has not been evaluated to our knowledge. In terms of the potential for sofosbuvir to block cellular efflux, sofosbuvir does not inhibit P-gp, BCRP, MDR-associated protein 2 (MRP2) or bile salt export pump. Ledipasvir does not inhibit MRP2 or MRP4, but does inhibit intestinal P-gp and BCRP. As evidence of its effect on P-gp and BCRP, ledipasvir increases sofosbuvir (a P-gp and BCRP substrate) AUC and Cmax by 2.3- and 2.2-fold, respectively,44–46 and this may explain why the effect of ledipasvir/sofosbuvir on tenofovir diphosphate is greater than that observed with sofosbuvir + ribavirin, as more tenofovir disoproxil fumarate may cross the gut barrier and reach the portal blood if intestinal P-gp and/or BCRP are inhibited. In addition to uncovering the potential mechanism for this interaction, it is important to determine its clinical relevance. Chronic tenofovir disoproxil fumarate use can result in proximal tubular damage and acute and chronic kidney injury.60,61 Some studies suggest these toxicities are concentration dependent (i.e. higher plasma tenofovir or intracellular tenofovir diphosphate concentrations are associated with a greater risk of toxicity).62–64 Tenofovir diphosphate concentrations would be difficult to measure in renal proximal tubule cells, but if tenofovir diphosphate concentrations were also increased by sofosbuvir in this cell type, this could theoretically lead to a higher risk of renal toxicity. We did not find a large decline in estimated CLCR overall in our cohorts or in any individual participant, but sofosbuvir dosing was for a relatively short period of time, 8–12 weeks. Longer treatment may require closer monitoring. SWIFT-C participants also had normal renal function at baseline (Table 1), so we cannot determine whether the interaction would result in renal toxicity in individuals with impaired renal function. An important factor to consider for this drug–drug interaction is that HCV therapy is finite. Thus, this interaction may not have serious clinical implications for HIV-coinfected individuals, but there may be consequences for other nucleotide analogues that may require longer durations of treatment. In addition, the implications of this interaction in the context of the new tenofovir prodrug, tenofovir alafenamide, which has 5- to 7-fold higher tenofovir diphosphate concentrations in PBMCs,65 is also unknown. These data also raise the question of which form of the nucleos(t)ide analogue (the prodrug, parent drug or triphosphate) should be evaluated in drug–drug interaction studies. In summary, this study uncovered an unexpected drug–drug interaction between tenofovir disoproxil fumarate and sofosbuvir and ledipasvir at the cellular level. The much higher levels of tenofovir diphosphate were not associated with large declines in CLCR in these participants with good baseline renal function; however, this interaction precludes using tenofovir diphosphate in dried blood spots to assess adherence during sofosbuvir-based HCV treatments pending further study. Additional research is needed to determine the mechanism for this interaction and the potential clinical implications. Acknowledgements The study team would like to thank all participants for participating in this study, the AIDS Clinical Trials Group, Statistical Data Management Center, participating Clinical Research Sites and Specialty Laboratories. We also thank Gilead Sciences for providing sofosbuvir and ribavirin and ledipasvir/sofosbuvir as well as funding for the HCV RNA testing.  A5327 study team members: Beverly Alston-Smith, Laura Weichmann, Thucuma Sise, Emily Cosimano, Cheryl Jennings, Sikhulile Moyo and Oswald Dadson.  A5327 site investigators: Annie Luetkemeyer and Jay Dwyer—UCSF AIDS CRS (Site 801) Grant 5UM1AI069496; Valery Hughes and Joanne Grenade—Weill Cornell Uptown CRS (Site 7803) Grant 5UM1 AI069419, UL1 TR000457; Todd Stroberg and Tiina Ilmet—Weill Cornell Chelsea CRS (Site 7804) Grant 5UM1 AI069419, UL1 TR000457; Sarah Henn and Kristi Kiger—Whitman-Walker Health (Site 31791) Grant UM1AI069465; Teri Flynn and Amy Sbrolla—Massachusetts General Hospital (Site 101) Grant 2UM1AI069412–09; Kathleen Nuffer and David Wyles—UCSD AVRC (Site 701) Grant AI069432; Donna McGregor and Claudia Hawkins—Northwestern University CRS (Site 2701) Grant AI 069471; Brett Williams and Tondria Green—Rush University Medical Center CRS (Site 2702) Grant U01 AI069471; Pablo Tebas and Deborah Kim—Penn Therapeutics CRS (Site 6201) Grants ACTG- UM-AI069534–09, CFAR –P30- AI045008–17; Roger Bedimo and Holly Wise—Trinity Health and Wellness Center CRS (Site 31443) Grant U01 AI069471; and Roberto C. Arduino and Aristoteles Villamil-Houston AIDS Research Team (HART) (Site 31473) Grant 2 UM1 AI069503. Funding Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers UM1 AI068634, UM1 AI068636 and UM1 AI106701. The research was also supported in part by a grant from Gilead Sciences. J. J. K. was supported by R01 DA040499 from the National Institute on Drug Abuse. Transparency declarations K. M. M. receives research funding (paid to her institution) from Gilead Sciences, Bristol-Myers Squibb and Merck&Co. D. S. F. owns common stock in Gilead Sciences. S. N. has received research funding (paid to her institution) from Vertex Pharmaceuticals, Merck, Gilead Sciences, Janssen Pharmaceuticals, AbbVie, Bristol-Meyers Squibb and Tacere, and has served as a consultant and scientific advisor for Vertex Pharmaceuticals, Merck, Gilead Sciences, Janssen Pharmaceuticals, AbbVie and Bristol-Meyers Squibb. R. T. C. receives research support (paid to his institution) from AbbVie, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, Janssen Therapeutics, MassBiologics and Merck&Co. A. Y. K. receives research support (paid to his institution) from AbbVie and Gilead Sciences. M. G. P. serves on advisory boards for AbbVie, Genentech, Gilead Sciences, Janssen Therapeutics and Merck&Co. D. M. B. is an employee of Gilead Sciences and holds stock options. P. L. A. receives research funding (paid to his institution) and donated study medication from Gilead Sciences. J. J. K. receives research funding (paid to her institution) from ViiV Healthcare, Janssen and Gilead Sciences, and donated study medication from Gilead Sciences for an NIH-sponsored study. C. E. M., M. D. H., S. M. S., J. R. C.-M. and L. R. B.: none to declare. Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1 Platt L , Easterbrook P , Gower E et al. Prevalence and burden of HCV co-infection in people living with HIV: a global systematic review and meta-analysis . Lancet Infect Dis 2016 ; 16 : 797 – 808 . Google Scholar CrossRef Search ADS PubMed 2 Martinez-Sierra C , Arizcorreta A , Diaz F et al. Progression of chronic hepatitis C to liver fibrosis and cirrhosis in patients coinfected with hepatitis C virus and human immunodeficiency virus . Clin Infect Dis 2003 ; 36 : 491 – 8 . Google Scholar CrossRef Search ADS PubMed 3 Mastroianni CM , Lichtner M , Mascia C et al. 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Microbiology Review—Division of Antiviral Drug Products—HFD- 530. 2001 . https://www.accessdata.fda.gov/drugsatfda_docs/nda/2001/21-356_Viread_microbr.pdf. 32 Kearney BP , Flaherty JF , Shah J. Tenofovir disoproxil fumarate: clinical pharmacology and pharmacokinetics . Clin Pharmacokinet 2004 ; 43 : 595 – 612 . Google Scholar CrossRef Search ADS PubMed 33 Product Information: Viread®, Tenofovir Disoproxil Fumarate Tablets . Foster City, CA, USA : Gilead Sciences, Inc ., 2003 . https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/021356s055,022577s011lbl.pdf. 34 Louissaint NA , Cao YJ , Skipper PL et al. Single dose pharmacokinetics of oral tenofovir in plasma, peripheral blood mononuclear cells, colonic tissue, and vaginal tissue . AIDS Res Hum Retroviruses 2013 ; 29 : 1443 – 50 . Google Scholar CrossRef Search ADS PubMed 35 Jackson A , Moyle G , Watson V et al. Tenofovir, emtricitabine intracellular and plasma, and efavirenz plasma concentration decay following drug intake cessation: implications for HIV treatment and prevention . J Acquir Immune Defic Syndr 2013 ; 62 : 275 – 81 . Google Scholar CrossRef Search ADS PubMed 36 Baheti G , Kiser JJ , Havens PL et al. Plasma and intracellular population pharmacokinetic analysis of tenofovir in HIV-1-infected patients . Antimicrob Agents Chemother 2011 ; 55 : 5294 – 9 . Google Scholar CrossRef Search ADS PubMed 37 Castillo-Mancilla JR , Zheng JH , Rower JE et al. Tenofovir, emtricitabine, and tenofovir diphosphate in dried blood spots for determining recent and cumulative drug exposure . AIDS Res Hum Retroviruses 2013 ; 29 : 384 – 90 . Google Scholar CrossRef Search ADS PubMed 38 Grant RM , Anderson PL , McMahan V et al. Uptake of pre-exposure prophylaxis, sexual practices, and HIV incidence in men and transgender women who have sex with men: a cohort study . Lancet Infect Dis 2014 ; 14 : 820 – 9 . Google Scholar CrossRef Search ADS PubMed 39 Gandhi M , Glidden DV , Liu A et al. Strong correlation between concentrations of tenofovir (TFV) emtricitabine (FTC) in hair and TFV diphosphate and FTC triphosphate in dried blood spots in the iPrEx open label extension: implications for pre-exposure prophylaxis adherence monitoring . J Infect Dis 2015 ; 212 : 1402 – 6 . Google Scholar CrossRef Search ADS PubMed 40 Zheng JH , Rower C , McAllister K et al. Application of an intracellular assay for determination of tenofovir-diphosphate and emtricitabine-triphosphate from erythrocytes using dried blood spots . J Pharm Biomed Anal 2016 ; 122 : 16 – 20 . Google Scholar CrossRef Search ADS PubMed 41 Delahunty T , Bushman L , Fletcher CV. Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS . J Chromatogr B Analyt Technol Biomed Life Sci 2006 ; 830 : 6 – 12 . Google Scholar CrossRef Search ADS PubMed 42 Seifert SM , Castillo-Mancilla J , Erlandson K et al. Cumulative tenofovir exposure is associated with decreased BMD in young and old HIV-infected adults on tenofovir based regimens. In: Abstracts of the Seventeenth International Workshop on Clinical Pharmacology of HIV and Hepatitis Therapy, Washington, DC, USA, 2016. Abstract 9. 43 German P , Garrison K , Pang PS et al. Drug interactions between the anti-HCV regimen ledipasvir/sofosbuvir and ritonavir-boosted protease inhibitors plus emtricitabine/tenofovir DF. In: Abstracts of the Twenty-second Conference on Retroviruses and Opportunistic Infections, Seattle, WA, USA, 2015. Foundation for Retrovirology and Human Health, Alexandria, VA, USA. Abstract 82. 44 Talavera Pons S , Boyer A , Lamblin G et al. Managing drug-drug interactions with new direct-acting antiviral agents in chronic hepatitis C . Br J Clin Pharmacol 2017 ; 83 : 269 – 93 . 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The 5'-nucleotidases as regulators of nucleotide and drug metabolism . Pharmacol Ther 2005 ; 107 : 1 – 30 . Google Scholar CrossRef Search ADS PubMed 58 Kohler JJ , Hosseini SH , Green E et al. Tenofovir renal proximal tubular toxicity is regulated by OAT1 and MRP4 transporters . Lab Invest 2011 ; 91 : 852 – 8 . Google Scholar CrossRef Search ADS PubMed 59 Seifert SM , Chen X , Clayton CW et al. Transporter genetics and TFV-DP/FTC-TP cellular pharmacology in vivo. In: Abstracts of the Twenty-third Conference on Retroviruses and Opportunistic Infections, Boston, MA, USA, 2016. Foundation for Retrovirology and Human Health, Alexandria, VA, USA. Abstract 445. 60 Hall AM , Hendry BM , Nitsch D et al. Tenofovir-associated kidney toxicity in HIV-infected patients: a review of the evidence . Am J Kidney Dis 2011 ; 57 : 773 – 80 . Google Scholar CrossRef Search ADS PubMed 61 Monteiro N , Branco M , Peres S et al. 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Tenofovir alafenamide vs. tenofovir disoproxil fumarate in single tablet regimens for initial HIV-1 therapy: a randomized phase 2 study . J Acquir Immune Defic Syndr 2014 ; 67 : 52 – 8 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Effects of sofosbuvir-based hepatitis C treatment on the pharmacokinetics of tenofovir in HIV/HCV-coinfected individuals receiving tenofovir disoproxil fumarate

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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10.1093/jac/dky146
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Abstract

Abstract Background The nucleotide analogues tenofovir and sofosbuvir are considered to have low potential for drug interactions. Objectives To determine the effect of sofosbuvir-based HCV treatment on plasma concentrations of tenofovir and cellular concentrations of tenofovir diphosphate. Methods HIV-infected participants with acute HCV were treated for 12 weeks with sofosbuvir + ribavirin in Cohort 1 or 8 weeks with ledipasvir/sofosbuvir in Cohort 2 of AIDS Clinical Trials Group study 5327. Only participants taking tenofovir disoproxil fumarate were included in this analysis. Tenofovir in plasma, tenofovir diphosphate in dried blood spots and tenofovir diphosphate in PBMCs were measured pre-HCV therapy and longitudinally during the study using validated LC/MS-MS. Results Fifteen and 22 men completed Cohorts 1 and 2, respectively. In Cohort 1, tenofovir diphosphate was 4.3-fold higher (95% CI geometric mean ratio 2.46–7.67; P = 0.0001) in dried blood spots and 2.3-fold higher (95% CI 1.09–4.92; P = 0.03) in PBMCs following 12 weeks of sofosbuvir + ribavirin versus study entry. Tenofovir in the plasma was unchanged. In Cohort 2, tenofovir diphosphate was 17.8-fold higher (95% CI 12.77–24.86; P < 0.0001) in dried blood spots after 8 weeks of ledipasvir/sofosbuvir versus study entry. Tenofovir plasma concentrations were 2.1-fold higher (95% CI 1.44–2.91; P = 0.0005). Despite the increase in cellular tenofovir diphosphate concentrations, only a small decline in CLCR (6%–7%) was observed in both cohorts between study entry and end of treatment. Conclusions These data indicate an unexpected drug interaction with tenofovir disoproxil fumarate and sofosbuvir at the cellular level. Additional studies are needed to determine the mechanism and clinical significance. Introduction Globally, an estimated 2.3 million people are coinfected with HIV and HCV owing to shared routes of transmission.1 Prison inmates, MSM and persons who inject drugs are most affected.1 HIV accelerates the progression of liver disease in individuals coinfected with HCV.2,3 Thus, treatment is imperative to reduce the complications of chronic liver disease in this population. In the pegylated interferon and ribavirin era, treatment of HCV in the acute phase resulted in higher rates of sustained virological response with a shortened course of therapy compared with treating individuals with chronic infection.4–7 However, there are limited data on the optimal use of contemporary all-oral, direct-acting antiviral agents (DAAs) in individuals with acute HCV. AIDS Clinical Trials Group study 5327 (‘SWIFT-C’, NCT02128217) was an open-label, two-cohort clinical trial of sofosbuvir-based treatment of acute HCV in individuals with chronic HIV-1 infection.8,9 Cohort 1 of SWIFT-C assessed the safety and efficacy of 12 weeks of 400 mg sofosbuvir in combination with weight-based ribavirin. Cohort 2 assessed the safety and efficacy of 8 weeks of 90 mg ledipasvir in combination with 400 mg sofosbuvir. The sustained virological response rate was 100% in Cohort 2 of SWIFT-C, but only 59% in Cohort 1.9 The high rate of relapse in Cohort 1 was unexpected and we suspected suboptimal ribavirin adherence contributed to this outcome. Although pill counts and participant recall suggested good ribavirin adherence, as previously reported, we found that individuals that relapsed after treatment with sofosbuvir + ribavirin had lower ribavirin plasma concentrations during treatment.9,10 Ribavirin has a long half-life in plasma (∼9 days)11 and, thus, ribavirin concentrations in plasma reflect, in part, cumulative drug dosing and long-term adherence to this drug. Thus, the lower ribavirin plasma concentrations in patients that relapsed in Cohort 1 of SWIFT-C likely reflected suboptimal adherence to ribavirin, given that the patients who achieved sustained virological response had levels comparable with historical data.9,10 Though there are no historical data establishing the ribavirin exposures observed with perfect adherence, mean ribavirin plasma concentrations in SWIFT-C participants that relapsed were lower than those observed in prior studies of sofosbuvir + ribavirin.12,13 Suboptimal adherence to medications leads to inadequate response to treatment and increased healthcare costs.14 The WHO estimates that 50%–60% of individuals are non-adherent to the medications they have been prescribed to treat chronic illnesses.15 Suboptimal adherence to ART leads to drug resistance, an increase in AIDS-related illnesses and death, and higher rates of HIV transmission.16–18 Adherence is also an issue for illnesses with a finite treatment duration. Consider tuberculosis in which 16%–49% of individuals do not complete treatment.19 There is evidence of suboptimal adherence in clinical trials of DAAs, despite the fact that HCV treatment is only 8–24 weeks in duration.20–27 Traditional tools to assess adherence (e.g. refill records, pill counts, self-reporting) have significant shortcomings and typically overestimate adherence.28 Pharmacokinetic-based adherence measures provide objective evidence of drug ingestion.29 For compounds with short half-lives, the information gained is qualitative (i.e. whether a dose was taken recently) but compounds with longer half-lives provide information on cumulative drug dosing and a more quantitative measure of drug-taking behaviour.30 Tenofovir disoproxil fumarate is an acyclic nucleoside phosphonate diester analogue of adenosine monophosphate and is initially converted to tenofovir by diester hydrolysis in the gut, liver and plasma.31,32 Tenofovir is taken up by cells,31 then phosphorylated by cellular kinases to the active form of the drug, tenofovir diphosphate. Tenofovir diphosphate competes with 2′-deoxyadenosine 5′-triphosphate (dATP) for incorporation into replicating viral DNA. If tenofovir diphosphate is incorporated into the viral DNA, rather than dATP, chain termination occurs.33 The tenofovir diphosphate half-life in PBMCs is 3–7 days34–36 and in red blood cells it is 17 days.37 Thus, like ribavirin concentrations in plasma, tenofovir diphosphate concentrations in cells can be used to assess cumulative drug exposure and long-term average adherence.37 Concentrations of tenofovir diphosphate in dried blood spots, which contain ∼12 million red blood cells, are correlated with the risk of acquiring HIV in studies of tenofovir disoproxil fumarate and emtricitabine for HIV prevention.38,39 Plasma concentrations of tenofovir have also been used for assessing adherence, but the plasma half-life of tenofovir is approximately 10–15 h33 and, thus, tenofovir concentrations in plasma reflect only recent dosing. Given that we found lower ribavirin levels in participants that failed treatment with sofosbuvir + ribavirin in Cohort 1 of SWIFT-C,9,10 which suggested suboptimal adherence to ribavirin, the primary aim of this analysis was to evaluate whether similar trends were evident for antiretroviral adherence (using established methods37) among SWIFT-C participants by comparing tenofovir diphosphate concentrations in dried blood spots before, during and after sofosbuvir + ribavirin treatment. In the course of this analysis, we identified an unexpected drug interaction between tenofovir disoproxil fumarate and sofosbuvir. Thus, we also compared tenofovir diphosphate in PBMCs and tenofovir in plasma before, during and after sofosbuvir + ribavirin treatment and evaluated the interaction in Cohort 2 participants receiving ledipasvir/sofosbuvir. Methods Participants ≥18 years old with chronic HIV-1 infection and documented confirmation of acute HCV infection within 6 months prior to entry were recruited from eight different AIDS Clinical Trials Group sites in the USA. We obtained ethics committee approval at all participating centers in accordance with the principles of the 2008 Declaration of Helsinki. All participants provided written informed consent before undergoing any protocol-specified procedures. Acute HCV was defined as9 (i) new HCV RNA in patients with documented negative serology in the past 6 months or (ii) in those with no documented serology in the past 6 months, positive HCV RNA plus a new ALT elevation [>5 × upper limit of normal (ULN)] with normal ALT in the past 12 months or >10 × ULN with no measured ALT in the past 12 months, as well as exclusion of other causes of acute hepatitis. Participants were not required to be taking ART, but those receiving ART were required to be stable on a regimen for at least 8 weeks, with an HIV-1 RNA <50 copies/mL and CD4 + T cell count >200 cells/mm3. Participants’ adherence was assessed using a 4 day recall and by pill count. For Cohort 1 participants, plasma, dried blood spots and PBMCs were analysed at entry, week 12 of sofosbuvir + ribavirin treatment and 12 weeks following the end of sofosbuvir + ribavirin treatment (EOT + 12). In Cohort 2 participants, plasma samples were analysed for each participant at entry, week 8 of ledipasvir/sofosbuvir treatment and EOT + 12 and dried blood spot samples were analysed from each participant at entry, week 1, week 2, week 4 and week 8 of ledipasvir/sofosbuvir treatment and EOT + 2, EOT + 4, EOT + 8 and EOT + 12. PBMCs were not collected in Cohort 2. All pharmacology samples were collected at convenience times post-dose. Tenofovir diphosphate concentrations in a 3 mm dried blood spot punch and in PBMCs were analysed using a validated LC/MS-MS method.40 The dynamic range of the assay was 25–6000 fmol/sample for tenofovir diphosphate in dried blood spots and 2.50–2000 fmol/sample for PBMCs.40 Accuracy was ≤ ± 15% from nominal and precision was ≤15% coefficient of variation (CV). Lack of assay interference between analytes was confirmed. Plasma tenofovir concentrations were also analysed using a validated LC/MS-MS method; the dynamic range of the assay was 10–1500 ng/mL.41 Tenofovir and tenofovir diphosphate concentrations were log transformed and compared between study visits using paired t-tests. Results Study patients Seventeen HIV-infected male participants entered Cohort 1 of SWIFT-C and all 17 completed 12 weeks of sofosbuvir + ribavirin treatment for acute HCV. All 17 were on ART and 15 were taking tenofovir disoproxil fumarate as part of their antiretroviral regimen. Among these 15, 11 were Hispanic and 4 were non-Hispanic white with a mean (±SD) age of 44.3 (±9.5) years and a mean (±SD) weight of 76.5 (±10.3) kg. Complete demographic information can be found in Table 1. Table 1. Demographic information Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Table 1. Demographic information Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Cohort 1 (N = 15) Cohort 2 (N = 22) Male, n (%) 15 (100) 22 (100) Race, n (%)  Hispanic 11 (73) 6 (27)  non-Hispanic white 4 (27) 16 (73) Baseline CLCR (mL/min), mean ± SD 125.4 ± 23.4 107.5 ± 15.7 Age (years), mean ± SD 44.3 ± 9.5 44.4 ± 8.8 Weight (kg), mean ± SD 76.5 ± 10.3 76.9 ± 9.6 Antiretroviral regimen, n (%)  NNRTI 7 (47) 8 (36)  integrase 5 (33) 11 (50)  boosted 6 (40) 9 (41)   cobicistat 3 (50) 4 (44)   ritonavir 3 (50) 5 (56) Twenty-seven HIV-infected male participants entered Cohort 2 of SWIFT-C and all 27 completed 8 weeks of ledipasvir/sofosbuvir treatment for acute HCV. All 27 were on ART and 22 were taking tenofovir disoproxil fumarate as part of their antiretroviral regimen. Among these 22, 6 were Hispanic and 16 were non-Hispanic white with a mean (±SD) age of 44.4 (±8.8) years and a mean (±SD) weight of 76.9 (±9.6) kg. Complete demographic information can be found in Table 1. Tenofovir diphosphate and tenofovir concentrations In Cohort 1, at study entry, the geometric mean (CV%) tenofovir diphosphate concentration in dried blood spots was 1687 (31) fmol/punch; consistent with historical data in HIV-infected and -uninfected individuals with high adherence.29,30,37,42 However, at week 12 of sofosbuvir + ribavirin treatment, the geometric mean (%CV) tenofovir diphosphate concentration in dried blood spots was 6607 (74) fmol/punch. In the 12 participants with both entry and week 12 tenofovir diphosphate concentrations in dried blood spots, the geometric mean ratio for tenofovir diphosphate was 4.3 versus entry (95% CI 2.46–7.67; P = 0.0001). By EOT + 12, tenofovir diphosphate in dried blood spots had declined and the geometric mean (%CV) was 2101 (36) fmol/punch (P = 0.0072) (Figure 1a). Tenofovir diphosphate concentrations were also determined in PBMCs. At entry, week 12 and EOT + 12, geometric mean (%CV) tenofovir diphosphate concentrations in PBMCs were 79 (48), 149 (91) and 81 (52) fmol/million cells, respectively. The tenofovir diphosphate concentrations pre-sofosbuvir + ribavirin in PBMCs were similar to historical data in HIV-infected individuals with high adherence,29,37 but for the 10 participants with both entry and week 12 concentrations, the tenofovir diphosphate concentrations in PBMCs were 2.3-fold higher at week 12 (95% CI 1.09–4.92; P = 0.03) (Figure 1b). Despite the increased levels of tenofovir diphosphate in PBMCs and red blood cells, tenofovir concentrations in plasma were similar to historical data and unchanged across visits (P = 0.83). At entry, week 12 and EOT + 12, geometric mean (%CV) tenofovir concentrations were 98 (63), 96 (57) and 94 (76) ng/mL, respectively (Figure 1c). Figure 1. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/million cells) in PBMC samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (c) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). The tenofovir concentrations were similar to historical data and unchanged across visits (P = 0.83). GMR, geometric mean ratio. Figure 1. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/million cells) in PBMC samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). Concentrations at week 12 of sofosbuvir + ribavirin treatment were compared with entry using a paired t-test. (c) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting sofosbuvir + ribavirin), after 12 weeks of sofosbuvir + ribavirin treatment and 12 weeks after completing sofosbuvir + ribavirin treatment (EOT + 12). The tenofovir concentrations were similar to historical data and unchanged across visits (P = 0.83). GMR, geometric mean ratio. In Cohort 2, at study entry, the geometric mean (%CV) tenofovir diphosphate concentrations in dried blood spots were 1516 (36) fmol/punch, which was similar to that for Cohort 1 and consistent with historical data in HIV-infected and -uninfected individuals with high adherence.29,37,42 By week 1 of ledipasvir/sofosbuvir treatment, tenofovir diphosphate concentrations were 6.7-fold higher versus entry (Figure 2a). At week 8 of ledipasvir/sofosbuvir treatment, for the 20 participants with both entry and week 8 tenofovir diphosphate concentrations in dried blood spots, the geometric mean ratio for tenofovir diphosphate was 17.8 (95% CI 12.77–24.86; P < 0.0001) compared with study entry. The geometric mean (%CV) at week 8 of tenofovir diphosphate was 26 846 (49) fmol/punch. Tenofovir concentrations in plasma were increased 2.1-fold (95% CI 1.44–2.91; P = 0.0005), at week 8 of ledipasvir/sofosbuvir treatment with a geometric mean (%CV) of 155 (112) ng/mL (Figure 2b). Three participants in Cohort 2 switched from tenofovir disoproxil fumarate to tenofovir alafenamide at week 1, week 7 and week 15 of study, respectively; thus, tenofovir diphosphate in dried blood spots and tenofovir in plasma samples for these individuals were excluded from analyses following the switch. Figure 2. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting ledipasvir/sofosbuvir), weeks 1, 2, 4 and 8 of ledipasvir/sofosbuvir treatment and 2, 4, 8 and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 2, EOT + 4, EOT + 8 and EOT + 12). Concentrations of tenofovir diphosphate during ledipasvir/sofosbuvir treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting ledipasvir/sofosbuvir), after 8 weeks of ledipasvir/sofosbuvir treatment and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 12). The tenofovir concentrations were 2.1-fold higher at week 8 versus study entry (P = 0.0005). GMR, geometric mean ratio. Figure 2. View largeDownload slide (a) Geometric mean (%CV) tenofovir diphosphate concentrations (TFV-DP in fmol/punch) in dried blood spot samples at study entry (before starting ledipasvir/sofosbuvir), weeks 1, 2, 4 and 8 of ledipasvir/sofosbuvir treatment and 2, 4, 8 and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 2, EOT + 4, EOT + 8 and EOT + 12). Concentrations of tenofovir diphosphate during ledipasvir/sofosbuvir treatment were compared with entry using a paired t-test. (b) Geometric mean (%CV) tenofovir concentrations (TFV in ng/mL) in plasma samples at study entry (before starting ledipasvir/sofosbuvir), after 8 weeks of ledipasvir/sofosbuvir treatment and 12 weeks after completing ledipasvir/sofosbuvir treatment (EOT + 12). The tenofovir concentrations were 2.1-fold higher at week 8 versus study entry (P = 0.0005). GMR, geometric mean ratio. Prior data in healthy volunteers suggested administering ledipasvir/sofosbuvir concomitantly with a boosted antiretroviral regimen and tenofovir disoproxil fumarate could result in tenofovir exposures that exceed the range for established renal safety data.43 In this study, Cohort 1 participants who were taking either ritonavir or cobicistat for pharmacokinetic boosting did not have higher tenofovir diphosphate or tenofovir concentrations compared with those not taking a boosting agent at any study visit. In Cohort 2, participants taking a boosted antiretroviral regimen had higher tenofovir diphosphate concentrations at baseline (before ledipasvir/sofosbuvir). The geometric mean (%CV) tenofovir diphosphate concentrations prior to the addition of ledipasvir/sofosbuvir were 1912 (29) fmol/punch and 1279 (32) fmol/punch for boosted versus unboosted regimens, respectively (P = 0.005). However, this was not observed at subsequent visits and, rather, participants not taking a boosting agent actually had numerically (though not statistically) higher tenofovir diphosphate concentrations. Renal function The mean (SD) CLCR in Cohort 1 participants was 125.4 mL/min (24) at study entry and 117.7 mL/min (20) at week 12 of sofosbuvir + ribavirin treatment (P = 0.18). The mean (SD) CLCR in Cohort 2 participants was 107.5 mL/min (16) at study entry and 99.8 mL/min (18) at week 8 of ledipasvir/sofosbuvir treatment (P =  0.01). Two participants, both on tenofovir disoproxil fumarate plus a boosting agent in Cohort 2, had an increase in serum creatinine >0.4 mg/dL (confirmed within 2 weeks), which was a protocol-defined renal toxicity. However, neither participant required a dose reduction of tenofovir disoproxil fumarate during ledipasvir/sofosbuvir treatment. There were no premature study discontinuations. Discussion In our investigations using drug concentrations to assess antiretroviral adherence in HIV-infected men treated for acute HCV, we identified an unexpected increase in cellular tenofovir diphosphate concentrations with both sofosbuvir + ribavirin and to a much larger extent with ledipasvir/sofosbuvir. The interaction was not observed in plasma for sofosbuvir + ribavirin and was observed with ledipasvir/sofosbuvir, but at a much smaller magnitude compared with the effect on tenofovir diphosphate in dried blood spots. We believe this finding suggests the presence of a previously undescribed drug–drug interaction between tenofovir disoproxil fumarate and sofosbuvir. The mechanism for this interaction is unclear, but given that the increase in tenofovir diphosphate concentrations occurred with both sofosbuvir + ribavirin and ledipasvir/sofosbuvir it suggests sofosbuvir is perpetrating this interaction. However, the interaction occurred to a much larger extent in those who received ledipasvir in addition to sofosbuvir, so ledipasvir is also a significant contributor to this interaction. Whether the contribution is via a direct effect of ledipasvir on tenofovir or tenofovir disoproxil fumarate, perhaps through inhibition of P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP), or the result of ledipasvir causing an increase in sofosbuvir AUC, which has been observed in prior studies,44–46 is unknown. In healthy volunteers, sofosbuvir increases the maximum concentration of tenofovir in plasma by 25% with no change in AUC.47 Ledipasvir/sofosbuvir increases tenofovir plasma concentrations by 30%–80% depending on the concomitant antiretrovirals.43,48 There are several potential mechanisms for the sofosbuvir-mediated increase in tenofovir diphosphate. A study in Caco-2 monolayers found that tenofovir disoproxil fumarate recovery (from the apical to basolateral membrane) was increased in the presence of sofosbuvir.46 Recent work suggests sofosbuvir inhibits carboxylesterase-2 (CES2).49 Tenofovir disoproxil fumarate is converted to tenofovir by two separate ester hydrolysis steps.32 Carboxylesterases are involved in this conversion of tenofovir disoproxil to tenofovir.50,51 Carboxylesterase enzymes are expressed in liver, small intestine, kidneys, lungs, plasma and other cell types.52 Thus, sofosbuvir inhibition of CES2 could result in greater prodrug delivery and increased cellular uptake. Other potential mechanisms include alterations in drug phosphorylation or dephosphorylation or changes in cellular uptake or efflux. In terms of potential alterations in phosphorylation, it is possible that sofosbuvir (or its intracellular metabolites) enhance phosphorylation of tenofovir or inhibit tenofovir diphosphate dephosphorylation through up-regulation or down-regulation of the involved kinases or phosphatases. The effect of sofosbuvir on enzymes responsible for tenofovir phosphorylation (e.g. adenylate kinases 1 and 2, pyruvate kinase, creatine kinase) or tenofovir diphosphate dephosphorylation (e.g. 5′-nucleotidases)53–57 has not been evaluated to our knowledge. In terms of the potential for sofosbuvir to increase cellular uptake of tenofovir disoproxil fumarate or tenofovir, tenofovir is a substrate for the uptake transporter, organic anion transporter (OAT1), and this transporter mediates the nephrotoxicity observed with tenofovir disoproxil fumarate.58 Tenofovir may also be a substrate for equilibrative nucleoside transporters (ENTs). One study found tenofovir diphosphate concentrations also may be increased in individuals with variant polymorphisms in the genes encoding ENTs.59 The potential for sofosbuvir (or its metabolites) to induce OAT1, ENT1 or other uptake transporters has not been evaluated to our knowledge. In terms of the potential for sofosbuvir to block cellular efflux, sofosbuvir does not inhibit P-gp, BCRP, MDR-associated protein 2 (MRP2) or bile salt export pump. Ledipasvir does not inhibit MRP2 or MRP4, but does inhibit intestinal P-gp and BCRP. As evidence of its effect on P-gp and BCRP, ledipasvir increases sofosbuvir (a P-gp and BCRP substrate) AUC and Cmax by 2.3- and 2.2-fold, respectively,44–46 and this may explain why the effect of ledipasvir/sofosbuvir on tenofovir diphosphate is greater than that observed with sofosbuvir + ribavirin, as more tenofovir disoproxil fumarate may cross the gut barrier and reach the portal blood if intestinal P-gp and/or BCRP are inhibited. In addition to uncovering the potential mechanism for this interaction, it is important to determine its clinical relevance. Chronic tenofovir disoproxil fumarate use can result in proximal tubular damage and acute and chronic kidney injury.60,61 Some studies suggest these toxicities are concentration dependent (i.e. higher plasma tenofovir or intracellular tenofovir diphosphate concentrations are associated with a greater risk of toxicity).62–64 Tenofovir diphosphate concentrations would be difficult to measure in renal proximal tubule cells, but if tenofovir diphosphate concentrations were also increased by sofosbuvir in this cell type, this could theoretically lead to a higher risk of renal toxicity. We did not find a large decline in estimated CLCR overall in our cohorts or in any individual participant, but sofosbuvir dosing was for a relatively short period of time, 8–12 weeks. Longer treatment may require closer monitoring. SWIFT-C participants also had normal renal function at baseline (Table 1), so we cannot determine whether the interaction would result in renal toxicity in individuals with impaired renal function. An important factor to consider for this drug–drug interaction is that HCV therapy is finite. Thus, this interaction may not have serious clinical implications for HIV-coinfected individuals, but there may be consequences for other nucleotide analogues that may require longer durations of treatment. In addition, the implications of this interaction in the context of the new tenofovir prodrug, tenofovir alafenamide, which has 5- to 7-fold higher tenofovir diphosphate concentrations in PBMCs,65 is also unknown. These data also raise the question of which form of the nucleos(t)ide analogue (the prodrug, parent drug or triphosphate) should be evaluated in drug–drug interaction studies. In summary, this study uncovered an unexpected drug–drug interaction between tenofovir disoproxil fumarate and sofosbuvir and ledipasvir at the cellular level. The much higher levels of tenofovir diphosphate were not associated with large declines in CLCR in these participants with good baseline renal function; however, this interaction precludes using tenofovir diphosphate in dried blood spots to assess adherence during sofosbuvir-based HCV treatments pending further study. Additional research is needed to determine the mechanism for this interaction and the potential clinical implications. Acknowledgements The study team would like to thank all participants for participating in this study, the AIDS Clinical Trials Group, Statistical Data Management Center, participating Clinical Research Sites and Specialty Laboratories. We also thank Gilead Sciences for providing sofosbuvir and ribavirin and ledipasvir/sofosbuvir as well as funding for the HCV RNA testing.  A5327 study team members: Beverly Alston-Smith, Laura Weichmann, Thucuma Sise, Emily Cosimano, Cheryl Jennings, Sikhulile Moyo and Oswald Dadson.  A5327 site investigators: Annie Luetkemeyer and Jay Dwyer—UCSF AIDS CRS (Site 801) Grant 5UM1AI069496; Valery Hughes and Joanne Grenade—Weill Cornell Uptown CRS (Site 7803) Grant 5UM1 AI069419, UL1 TR000457; Todd Stroberg and Tiina Ilmet—Weill Cornell Chelsea CRS (Site 7804) Grant 5UM1 AI069419, UL1 TR000457; Sarah Henn and Kristi Kiger—Whitman-Walker Health (Site 31791) Grant UM1AI069465; Teri Flynn and Amy Sbrolla—Massachusetts General Hospital (Site 101) Grant 2UM1AI069412–09; Kathleen Nuffer and David Wyles—UCSD AVRC (Site 701) Grant AI069432; Donna McGregor and Claudia Hawkins—Northwestern University CRS (Site 2701) Grant AI 069471; Brett Williams and Tondria Green—Rush University Medical Center CRS (Site 2702) Grant U01 AI069471; Pablo Tebas and Deborah Kim—Penn Therapeutics CRS (Site 6201) Grants ACTG- UM-AI069534–09, CFAR –P30- AI045008–17; Roger Bedimo and Holly Wise—Trinity Health and Wellness Center CRS (Site 31443) Grant U01 AI069471; and Roberto C. Arduino and Aristoteles Villamil-Houston AIDS Research Team (HART) (Site 31473) Grant 2 UM1 AI069503. Funding Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers UM1 AI068634, UM1 AI068636 and UM1 AI106701. The research was also supported in part by a grant from Gilead Sciences. J. J. K. was supported by R01 DA040499 from the National Institute on Drug Abuse. Transparency declarations K. M. M. receives research funding (paid to her institution) from Gilead Sciences, Bristol-Myers Squibb and Merck&Co. D. S. F. owns common stock in Gilead Sciences. S. N. has received research funding (paid to her institution) from Vertex Pharmaceuticals, Merck, Gilead Sciences, Janssen Pharmaceuticals, AbbVie, Bristol-Meyers Squibb and Tacere, and has served as a consultant and scientific advisor for Vertex Pharmaceuticals, Merck, Gilead Sciences, Janssen Pharmaceuticals, AbbVie and Bristol-Meyers Squibb. R. T. C. receives research support (paid to his institution) from AbbVie, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, Janssen Therapeutics, MassBiologics and Merck&Co. A. Y. K. receives research support (paid to his institution) from AbbVie and Gilead Sciences. M. G. P. serves on advisory boards for AbbVie, Genentech, Gilead Sciences, Janssen Therapeutics and Merck&Co. D. M. B. is an employee of Gilead Sciences and holds stock options. P. L. A. receives research funding (paid to his institution) and donated study medication from Gilead Sciences. J. J. K. receives research funding (paid to her institution) from ViiV Healthcare, Janssen and Gilead Sciences, and donated study medication from Gilead Sciences for an NIH-sponsored study. C. E. M., M. D. H., S. M. S., J. R. C.-M. and L. R. B.: none to declare. Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. References 1 Platt L , Easterbrook P , Gower E et al. Prevalence and burden of HCV co-infection in people living with HIV: a global systematic review and meta-analysis . Lancet Infect Dis 2016 ; 16 : 797 – 808 . Google Scholar CrossRef Search ADS PubMed 2 Martinez-Sierra C , Arizcorreta A , Diaz F et al. Progression of chronic hepatitis C to liver fibrosis and cirrhosis in patients coinfected with hepatitis C virus and human immunodeficiency virus . Clin Infect Dis 2003 ; 36 : 491 – 8 . Google Scholar CrossRef Search ADS PubMed 3 Mastroianni CM , Lichtner M , Mascia C et al. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 9, 2018

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