Neither ritonavir nor darunavir affect cell surface expression of tetherin or Vpu-mediated tetherin down-modulation
3
Citation
15
Reference
10
Related Paper
Abstract:
Protease inhibitors act late in the HIV-1 life cycle, following viral assembly at the cellular membrane, to inhibit protease-mediated viral maturation. Virological outcome associated with the use of protease inhibitors is correlated with levels of pharmacokinetic exposure, which can be affected by drug metabolism and active removal of drugs from target cells [1]. Protease inhibitors serve as a substrate for several membrane-spanning drug transporters and efflux pumps [2,3]. Ritonavir (RTV) is a protease inhibitor that can increase both intracellular and extracellular concentrations of other protease inhibitors, partly by direct inhibition of drug transporters or efflux pumps, and is used as a boosting agent for other drugs in antiretroviral therapy [4,5]. Controversy exists as to whether RTV also interferes with calpain-mediated proteasomal degradation of drug transporters and other cell-membrane proteins. Whereas calpain was shown to be inhibited by RTV in cell culture models [6], these results were not confirmed in biochemical studies [7]. Tetherin (BST-2/CD317/HM1.24) is an interferon-inducible integral membrane protein that contributes to innate cellular defense against infection by HIV-1 and other enveloped viruses by tethering nascent viral particles to the cell surface and inhibiting viral release [8,9]. In HIV-1 infection, the viral protein Vpu counters this effect by promoting tetherin down-modulation from the cell surface as well as its subsequent endosomal/lysosomal or proteasomal degradation, leading to increased viral release [10,11]. Through its transmembrane domain, which also possesses an ion channel function, Vpu interacts with the transmembrane domain of tetherin [12,13]. The fact that RTV can block cell surface membrane exporters makes it important to understand whether this drug and other protease inhibitors might also impact on levels of tetherin expression and Vpu-mediated tetherin down-modulation in HIV-1-infected cells. To investigate tetherin expression, we used Sup-T1 cells that contain the human tetherin gene [12]. Tetherin expression was induced by adding either 0.1 or 1 μg/ml doxycycline (dox) (Sigma, St Louis, Missouri, USA). Doxycycline-induced cells were infected with either VSV-G-pseudotyped wt HIV-1 or with a Δvpu clonal derivative termed BR4-3-IRES-eGFP, which expresses enhanced green fluorescent protein (eGFP) from an internal ribosomal entry site downstream of nef[14]. Induced Sup-T1 cells were infected to a percentage of 20–30%, as assessed by eGFP detection at 48 h postinfection (p.i.) by flow cytometry. Drugs were added at concentrations similar to those of plasma levels attained in patients receiving protease inhibitor drugs [darunavir (DRV), 10 μM; RTV, 5 μM; r/DRV, 5 μM RTV/10 μM DRV] [15,16]. Tetherin was stained using a rabbit antitetherin antibody with a secondary PerCP-labeled antirabbit antibody. Levels of cell surface tetherin were assessed by flow cytometry for PerCP at 48 h p.i. Uninfected and infected cells were distinguished by virus-derived eGFP expression. Data from at least three independent experiments were analyzed for statistical significance by one-way analysis of variance and Dunnett's post-test. We found that cell surface tetherin levels, induced by 0.1 and 1 μg/ml dox, were significantly down-regulated in wt-infected cells compared to uninfected and Δvpu-infected cells under two different induction conditions (0.1 μg/ml dox, P = 0.03; 1 μg/ml dox, P = 0.02). This confirms that, in our system, tetherin down-modulation in HIV-1 infection is Vpu-dependent. Moreover, we did not detect any statistically significant effect of the presence of either RTV, DRV, or both together on the cell surface presence of tetherin (0.1 and 1 μg/ml dox) in uninfected cells (0.1 μg/ml dox, P = 0.19; 1 μg/ml dox, P = 0.65) or cells infected with wt virus (0.1 μg/ml dox, P = 0.12; 1 μg/ml dox, P = 0.56) or Δvpu virus (0.1 μg/ml dox, P = 0.68; 1 μg/ml dox, P = 0.75) compared to controls not exposed to protease inhibitors (Fig. 1).Fig. 1: Tetherin cell surface expression levels are not significantly affected by protease inhibitors. Infected and uninfected cells were assessed for tetherin cell surface expression in the absence or presence of 5 μmol/l ritonavir (RTV), 10 μmol/l darunavir (DRV), or the combination of 5 μmol/l RTV/10 μmol/l DRV. Tetherin expression was induced by 0.1 μg/ml (a, c) or 1 μg/ml doxycyclin (dox) (b, d). Cell surface expression of tetherin was assessed by PerCP detection within Sup-T1 cell populations containing wt-infected (a, b) or Δvpu-infected (c, d) cells. Data are presented as geometric means; error bars represent the standard error of the mean.In summary neither RTV nor DRV appear to modulate tetherin levels at the surface of uninfected cells or of cells infected with wt or vpu-deficient virus. These results also imply that these protease inhibitors do not affect Vpu-mediated down-modulation of tetherin and that tetherin degradation is probably calpain-independent. Therefore, exposure to RTV is unlikely to lead to elevated levels of cell surface tetherin and it is unlikely that the antiviral activity of boosted protease inhibitor regimens can be attributed to any tetherin-related effects. Acknowledgements We are grateful to Professor Frank Kirchhoff for providing the viral clone pBR-NL43-IRES-eGFP and Dr Klaus Strebel for providing the hBST-2 antibody; both were provided through the NIH AIDS Research and Reference Reagent program. This research was supported by grants from the Canadian Institutes of Health Research.Keywords:
Tetherin
Ritonavir
Darunavir
Cell membrane
Darunavir is a new protease inhibitor (PI) active against wild-type and drug-resistant HIV-1 strains and has recently been approved for use in treatment-experienced adult patients. The main clinical trials, POWER and TITAN, have been conducted in treatment-experienced, HIV-1-infected patients. The Phase IIb POWER 1 and 2 trials compared the efficacy and safety of darunavir plus low-dose ritonavir (darunavir/ritonavir) with currently available PIs in highly treatment-experienced patients receiving an optimized background regimen. At 24 and 48 weeks in POWER 1 and 2, patients receiving darunavir/ritonavir had significantly higher efficacy responses than those receiving currently available PIs (45 vs 10% of patients reached a viral load <50 copies/ml at week 48). The highest responses were observed with the darunavir/ritonavir 600/100 mg twice daily dose (also evaluated in the POWER 3 analysis), which was selected for the subsequent Phase III TITAN study. TITAN evaluated darunavir/ritonavir 600/100 mg twice daily in comparison with lopinavir/ritonavir in early treatment-experienced, lopinavir-naive patients. For the primary end point of a viral load of less than 400 copies/ml, darunavir/ritonavir was noninferior to lopinavir/ritonavir (77 vs 68%; 95% confidence interval [CI]: 2–16); for a viral load of less than 50 copies/ml, darunavir/ritonavir was again noninferior to the lopinavir/ritonavir group (71 vs 60%; 95% CI: 3–19). Both regimens were well tolerated, although discontinuations due to diarrhea and liver and lipid abnormalities were less frequent in the darunavir/ritonavir group. Darunavir/ritonavir has been shown to be effective in patients with a broad range of treatment experience and has the potential to become used across the HIV treatment spectrum, including in antiretroviral-naive patients.
Darunavir
Ritonavir
Regimen
Enfuvirtide
Cite
Citations (3)
S/GSK1349572 is an unboosted, once-daily integrase inhibitor with a novel resistance profile. As standard of care for patients infected with HIV is combination therapy, the potential interaction between S/GSK1349572 and ritonavir-boosted protease inhibitors was evaluated. In an open-label, repeat-dose, 2-period, 2-sequence crossover study in healthy participants, S/GSK1349572 was administered at 30 mg once daily for 5 days, followed by randomization to lopinavir/ritonavir 400/100 mg twice daily or darunavir/ritonavir 600/100 mg twice daily coadministered with S/GSK1349572 30 mg once daily for 14 days. There was no washout between periods. Serial pharmacokinetic (PK) samples and safety assessments were obtained throughout the study. Thirty of 31 participants completed the study (15 participants per group). Treatment comparisons of steady-state S/GSK1349572 PK parameters demonstrated that coadministration of lopinavir/ritonavir had no significant effect on steady-state PK of S/GSK1349572. Coadministration of darunavir/ritonavir resulted in a nonclinically significant reduction in steady-state plasma S/GSK1349572 exposures. Plasma S/GSK1349572 AUC((0-τ)), C(max), and C(τ) decreased by 22%, 11%, and 38%, respectively, on average. S/GSK1349572 was well tolerated with no serious adverse events (AEs) or withdrawals due to drug-related AEs. The most frequent drug-related AEs were diarrhea, dizziness, and headache. No dosage adjustment for S/GSK1349572 is required when used with lopinavir/ritonavir or darunavir/ritonavir.
Darunavir
Ritonavir
Lopinavir
Lopinavir/ritonavir
Crossover study
Cite
Citations (57)
Darunavir (DRV) is an HIV-1 protease inhibitor that is used together with a low boosting dose of ritonavir as part of an antiretroviral therapy (ART) regimen in treatment-experienced and naïve HIVpositive patients. In naïve and experienced patients with no DRV-mutations, DRV is licensed at the dose of 800 mg plus 100 mg of ritonavir once daily. We report our results in seven ART-experienced HIV-infected patients, in whom a reduced dose of darunavir/ritonavir (600/100 mg once daily) successfully controlled viral replication
Darunavir
Ritonavir
Regimen
Protease inhibitor (pharmacology)
Cite
Citations (3)
This was an open-label, crossover study to investigate the pharmacokinetic interaction between darunavir (TMC114), coadministered with low-dose ritonavir (darunavir/ritonavir), and the protease inhibitor saquinavir in HIV-negative healthy volunteers. Thirty-two volunteers were randomized into two cohorts (panel 1 and panel 2). In two separate sessions, panel 1 received 400/100 mg darunavir/ritonavir twice a day and 400/1000/100 mg darunavir/saquinavir/ritonavir twice a day; panel 2 received 1000/100 mg saquinavir/ritonavir twice a day and 400/1000/100 mg darunavir/saquinavir/ritonavir twice a day. All treatments were administered orally under fed conditions for 13 days with an additional single morning dose on day 14. Treatment sessions were separated by a washout period of at least 14 days. Twenty-six volunteers completed the study (n = 14, panel 1; n = 12, panel 2), whereas six discontinued as a result of adverse events. Coadministration of saquinavir with darunavir/ritonavir resulted in decreases of darunavir area under the curve and maximum and minimum plasma concentrations of 26%, 17%, and 42%, respectively, compared with administration of darunavir/ritonavir alone. Relative to treatment with saquinavir/ritonavir alone, saquinavir exposure was not significantly different with the addition of darunavir. Ritonavir area under the curve12h increased by 34% when saquinavir was added to treatment with darunavir/ritonavir. The coadministration of darunavir/saquinavir/ritonavir was generally well tolerated. Similar findings are expected with the approved 600/100 mg darunavir/ritonavir twice-a-day dose. The combination of saquinavir and darunavir/ritonavir is currently not recommended.
Saquinavir
Darunavir
Cite
Citations (24)
Darunavir
Ritonavir
Lopinavir
Emtricitabine
Lopinavir/ritonavir
Cite
Citations (8)
Background Darunavir, a second‐generation protease inihibitor, is used with a low boosting dose of ritonavir to improve its clinical efficacy. The boosting dose of ritonavir acts as an inhibitor of CYP3A4, thereby increasing darunavir bioavailability. Recently, ritonavir tablet has been on sale in place of soft capsule. However, pharmacokinetic study of darunavir by changing ritonavir form is still not clear. In this study, we aimed to compare with plasma darunavir concentrations by switching ritinavir soft capsule to tablet in Japanese HIV‐1 infected patients. Methods We analyzed 34 Japanese HIV‐1 infected patients (32 males: 2 females) recruited at the National Hospital Organization Nagoya Medical Center. All patients had been administered with 800/100 mg darunavir/ritonavir once daily in combination with other antiretrovirals. Plasma darunavir concentrations were determined by an HPLC method. A pared t‐test was used to compare with their concentrations by switching from ritonavir soft capsule to tablet. Results The mean of age, body weight, and duration of antiretroviral therapy for 34 patients were 41.9 (range: 24–62) years, 66.3 (range: 51.4–90.0) kg, and 436 (range: 182–739) days, respectively. The mean±SD darunavir concentration was 3.44±1.78 µg/ml when ritonavir soft capsule was co‐administered. After switching to ritonavir tablet, the mean±SD darunavir concentration was 3.30±2.02 µg/ml. Statistical difference was not found in plasma trough darunavir concentration between ritonavir soft capsule and tablet (P=0.826). On the other hand, the mean of viral load was 78 copies/ml when ritonavir soft capsule was administered. After switching to ritonavir tablet, the mean viral load was 33 copies/ml. Conclusion Recruited all patients have been sustained an ndetectable viral load (less than 40 copies/ml) after switching to ritonavir tablet. In this study, switching to ritonavir tablet had no significant difference on plasma darunavir concentrations in Japanese HIV‐1 infected patients.
Darunavir
Ritonavir
Capsule
Cite
Citations (0)
Darunavir (DRV) is an HIV-1 protease inhibitor that is used together with a low boosting dose of ritonavir as part of an antiretroviral therapy (ART) regimen in treatment-experienced and naïve HIVpositive patients. In naïve and experienced patients with no DRV-mutations, DRV is licensed at the dose of 800 mg plus 100 mg of ritonavir once daily. We report our results in seven ART-experienced HIV-infected patients, in whom a reduced dose of darunavir/ritonavir (600/100 mg once daily) successfully controlled viral replication
Darunavir
Ritonavir
Regimen
Protease inhibitor (pharmacology)
Cite
Citations (0)
Atazanavir/ritonavir and darunavir/ritonavir are common protease inhibitor-based regimens for treating patients with HIV. Studies comparing these drugs in clinical practice are lacking. We conducted a retrospective cohort study of antiretroviral naïve participants in the Canadian Observational Cohort (CANOC) collaboration initiating atazanavir/ritonavir- or darunavir/ritonavir-based treatment. We used separate Fine and Gray competing risk regression models to compare times to regimen failure (composite of virologic failure or discontinuation for any reason). Additional endpoints included virologic failure, discontinuation due to virologic failure, discontinuation for other reasons, and virologic suppression. We studied 222 patients treated with darunavir/ritonavir and 1791 patients treated with atazanavir/ritonavir. Following multivariable adjustment, there was no difference between darunavir/ritonavir and atazanavir-ritonavir in the risk of regimen failure (adjusted hazard ratio 0.76, 95% CI 0.56 to 1.03) Darunavir/ritonavir-treated patients were at lower risk of virologic failure relative to atazanavir/ritonavir treated patients (aHR 0.50, 95% CI 0.28 to 0.91), findings driven largely by high rates of virologic failure among atazanavir/ritonavir-treated patients in the province of British Columbia. Of 108 discontinuations due to virologic failure, all occurred in patients starting atazanavir/ritonavir. There was no difference between regimens in time to discontinuation for reasons other than virologic failure (aHR 0.93; 95% CI 0.65 to 1.33) or virologic suppression (aHR 0.99, 95% CI 0.82 to 1.21). The risk of regimen failure was similar between patients treated with darunavir/ritonavir and atazanavir/ritonavir. Although darunavir/ritonavir was associated with a lower risk of virologic failure relative to atazanavir/ritonavir, this difference varied substantially by Canadian province and likely reflects regional variation in prescribing practices and patient characteristics.
Darunavir
Ritonavir
Atazanavir
Medical microbiology
Cite
Citations (8)
The potential for once-daily use of darunavir in combination with low-dose ritonavir (darunavir/ritonavir) in antiretroviral-naive patients has been evaluated in the Phase III ARTEMIS trial. This followed the demonstration of sustained efficacy and a favorable safety profile of darunavir/ritonavir 600/100 mg twice daily in patients with a broad range of treatment experience in the Phase IIb POWER and Phase III TITAN studies. The 48-week primary analysis of ARTEMIS demonstrated that once-daily darunavir/ritonavir 800/100 mg was noninferior to lopinavir/ritonavir, given either 400/100 mg twice daily or 800/200 mg every day, in treatment-naive, HIV-1-infected patients (84 vs 78% of patients, respectively, had confirmed plasma HIV-1 RNA less than 50 copies/ml by per-protocol time-to-loss of virologic response at week 48). In patients with baseline HIV RNA more than 100,000 copies/ml, darunavir/ritonavir achieved higher response rates than lopinavir/ritonavir (79 vs 67%; p <0.05). In addition to being at least as effective as lopinavir/ritonavir, once-daily darunavir/ritonavir had tolerability advantages compared with lopinavir/ritonavir, including lower incidences of grade 2–4 gastrointestinal adverse events (mainly diarrhea), and triglyceride and total cholesterol elevations. These benefits, along with the convenience of once-daily darunavir/ritonavir 800/100 mg and its low propensity to the development of resistance, suggest darunavir/ritonavir is a valuable treatment option for treatment-naive patients.
Darunavir
Ritonavir
Lopinavir
Tolerability
Cobicistat
Cite
Citations (1)
This study investigated the steady-state pharmacokinetic interaction between the HIV protease inhibitor, darunavir (TMC114), administered with low-dose ritonavir (darunavir/ritonavir), and clarithromycin in HIV-negative healthy volunteers. In a 3-way crossover study, 18 individuals received darunavir/ritonavir 400/100 mg bid, clarithromycin 500 mg bid, and darunavir/ritonavir 400/100 mg bid plus clarithromycin 500 mg bid in 3 separate sessions for 7 days, with a washout period of at least 7 days between treatments. Pharmacokinetic assessment was performed on day 7. Safety and tolerability of the study medication were monitored throughout. Coadministration of darunavir/ritonavir with clarithromycin resulted in a reduction in darunavir maximum plasma concentration (Cmax) and area under the curve from administration until 12 hours postdose (AUC12 h) of 17% and 13%, respectively. Ritonavir Cmax and AUC12 h were unchanged. During coadministration with darunavir/ritonavir, clarithromycin Cmax and AUC12 h increased by 26% and 57%, respectively; 14-hydroxy-clarithromycin plasma concentrations were reduced to below the lower limit of quantification (<50 ng/mL). The study medication was generally well tolerated. Based on these pharmacokinetic findings, neither clarithromycin nor darunavir/ritonavir dose adjustments are necessary when clarithromycin is coadministered with darunavir/ritonavir.
Darunavir
Ritonavir
Crossover study
Tolerability
Cite
Citations (22)