Analysis of virological response to therapy and resistance profile in treatment-experienced and naive HIV-1 infected Romanian patients receiving regimens containing darunavir boosted with ritonavir or cobicistat.

77% of Romanians infected with HIV receive antiretroviral therapy, with the challenge of maintaining long-term therapeutic success (the viral load becoming/remaining undetectable). The main purpose of this study was to provide comparative analysis of the long-term virological response to therapeutic regimens containing pharmacokinetically enhanced darunavir (DRV) with ritonavir (RTV) or cobicistat (COBI). The second aim was to evaluate the viral resistance profile to therapy, by number/type/frequency of viral mutations. This retrospective study was conducted on 462 patients infected with subtype F HIV-1, registered at the "Matei Bals" National Institute of Infectious Diseases, between 2018 and 2021: 384 patients received (among other ARV) DRV 600 mg, enhanced with RTV 100 mg (twice daily) and 78 patients received DRV 800 mg boosted with COBI 150 mg (once daily). The virological response was measured by determining the viral load (HIV-1 RNA copies/mL), while the incidence of viral resistance to therapy was assessed by genotyping tests. Comparing the patients with undetectable viremia, from the 1st visit to the 3rd one, the outcomes showed that at the last visit, 84.6% subjects in the DRV/c group achieved virological efficiency over those from DRV/r group (76.8%). The differences observed between this time points are statistically significant p < 0.05. DRV/c administered in combination with other ARV, in subtype F HIV-1 infected patients, proved to be more virologically effective, maintaining a favorable long-time result. When comparing the outcomes of the two groups, a statistically significant difference of p < 0.05 was obtained. 32 patients (27 from DRV/r group and 5 from DRV/c group) were evaluated with persistent HIV-1 ARN plasma load > 1000 copies/mL, during all 3 clinical visits. They formed a research sub-group evaluated in terms of resistance to therapy and were reported as virological failures. 28.12% of the sub-group with persistent HIV-1 RNA > 1000 copies/mL were from the DRV/r group and only 3.12% from the DRV/c group. Drug mutations (DRM) involved in antiretroviral resistance/sensitivity occurred both in the protease gene, and in the reverse transcriptase gene, with the involved ARV classes being protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors. 16 different types of mutations were evaluated in the PR gene and 20 mutations were evaluated in RT gene.

The effect of ivermectin alone and in combination with cobicistat or elacridar in experimental Schistosoma mansoni infection in mice.

Schistosoma mansoni is less susceptible to the antiparasitic drug ivermectin than other helminths. By inhibiting the P-glycoprotein or cytochrome P450 3A in mice host or parasites in a murine model, we aimed at increasing the sensitivity of S. mansoni to the drug and thus preventing infection. We assigned 124 BALB/c mice to no treatment, treatment with ivermectin only or a combination of ivermectin with either cobicistat or elacridar once daily for three days before infecting them with 150 S. mansoni cercariae each. The assignment was done by batches without an explicit randomization code. Toxicity was monitored. At eight weeks post-infection, mice were euthanized. We determined number of eggs in intestine and liver, adult worms in portal and mesenteric veins. Disease was assessed by counting granulomas/cm2 of liver and studying organ weight indices and total weight. IgG levels in serum were also considered. No difference between groups treated with ivermectin only or in combination with cobicistat or elacridar compared with untreated, infected controls. Most mice treated with ivermectin and elacridar suffered severe neurological toxicity. In conclusion, systemic treatment with ivermectin, even in the presence of pharmacological inhibition of P-glycoprotein or cytochrome P450 3A, did not result in effective prophylaxis for S. mansoni infection in an experimental murine model.

Pharmacokinetic Enhancement of HIV Antiretroviral Therapy During Pregnancy.

Pharmacokinetic boosting of antiretroviral (ARV) therapies with either ritonavir or cobicistat is used to achieve target drug exposure, lower pill burden, and provide simplified dosing schedules. Several ARVs require boosting, including the integrase inhibitor elvitegravir as well as protease inhibitors such as darunavir, atazanavir, and lopinavir. The use of boosted regimens in pregnant women living with HIV has been studied for a variety of ARVs; however, a recent recommendation by the US Food and Drug Administration advised against cobicistat-boosted regimens in pregnancy due to substantially lower drug exposures observed in clinical pharmacokinetic studies. The objectives of this article are to review pharmacokinetic enhancement of ARVs with ritonavir and cobicistat during pregnancy and postpartum, describe clinical implications, and provide recommendations for future research.

Interactions of cobicistat and ritonavir in patients with HIV and its clinical consequences. / Interacciones de cobicistat y ritonavir en pacientes con VIH y sus consecuencias clínicas.

INTRODUCTION: The prescription of antiretroviral treatment (ART) that contains pharmacokinetic enhancers such as ritonavir and cobicistat is frequent. The objective of this stdy was to analyze the potential interactions of ART that include these molecules in their formulation with the patient's home medication, as well as the clinical management of those potentially serious. METHODS: Prospective study conducted in the pharmacy care clinic of a third level hospital between January and December of 2018. Those HIV+patients with an ART containing cobicistat or ritonavir were included in the study. Potential interactions between ART and concomitant medication were analysed in three databases (Micromedex®, Drugs.com and Liverpool), the interventions carried out were detailed, and adverse drug reactions analysed. RESULTS: 968 patients were included with a total of 2,148 prescriptions (274 different medications). A total of 86 interventions were performed regarding potential interactions in patients. The most frequent were substitutions of corticoid treatments, treatment suspensions and closer monitoring of treatments. A total of possible adverse drug reactions were analysed. The degree of agreement in the severity classification of the interactions for cobicistat and ritonavir was good among the three databases. It was remarkable Micromedex® as the most complete because it has more registered medications. CONCLUSION: The interactions between ART with pharmacokinetic enhancers in its composition and concomitant medication is frequent and requires a significant variety of interventions. The check of interactions in different databases is recommended since they can cause adverse drug reactions.

Iatrogenic Cushing syndrome due to drug interaction between inhaled fluticasone and cobicistat.

In this paper we report a case of iatrogenic Cushing syndrome due to a pharmacological interaction between fluticasone and cobicistat. Inhaled corticosteroids were previously thought to be safe, but increasing numbers of cases of iatrogenic Cushing syndrome are being reported, especially in patients taking cytochrome P450 inhibitors, including cobicistat. Although the drug interaction between cobicistat and fluticasone has been described elsewhere, to our knowledge we present one of the first descriptions of iatrogenic Cushing syndrome due to this pharmacological interaction.

No Inhibition of MATE1/2K-Mediated Renal Creatinine Secretion Predicted With Ritonavir or Cobicistat.

Cobicistat has been reported to increase serum creatinine clinically without affecting glomerular filtration. This was ascribed to transient inhibition of MATE1-mediated renal creatinine secretion. Interestingly, a structurally similar drug, ritonavir, has not been associated with serum creatinine increases at the pharmacoenhancer dose. The present study was aimed to investigate the translation of in vitro MATE1/2K inhibition to clinical creatinine increase (cobicistat) and lack of it (ritonavir) considering their intracellular concentrations in renal proximal tubules. Uptake studies showed ritonavir and cobicistat are unlikely substrates for OCT2. The steady-state unbound concentration in the cytosol of human renal proximal tubule epithelial cells was comparable with the extracellular unbound concentration, suggesting that the entry of these compounds is predominantly mediated by passive diffusion. Ritonavir and cobicistat are MATE1 and MATE2K inhibitors with IC50 values of 3.1 and 90 µM (ritonavir), and 4.4 and 3.2 µM (cobicistat), respectively. However, the unbound cytosolic concentrations (Cu,cytosol) of ritonavir and cobicistat in human renal proximal tubule epithelial cells, 0.065 and 0.10 µM, respectively, after incubation with the clinical maximum total plasma concentrations at pharmacoenhancer doses does not support inhibition in vivo; Cu,cytosol >30 fold lower than IC50s. These results demonstrate that MATE1/2K inhibition is unlikely the mechanism of the clinical creatinine elevations with cobicistat.

Exogenous steroid-induced hypoadrenalism in a person living with HIV caused by a drug-drug interaction between cobicistat and intrabursal triamcinolone.

We report a diagnosis of exogenous steroid-induced hypoadrenalism in a person living with HIV caused by a drug-drug interaction (DDI) between intrabursal triamcinolone and the pharmacokinetic booster cobicistat. A 53-year-old woman living with HIV, managed with dolutegravir and cobicistat-boosted darunavir, presented to the orthopaedic clinic with worsening hip pain. She was diagnosed with greater trochanteric pain syndrome (GTPS) of the hip and was treated with intrabursal injection of bupivacaine and triamcinolone. Seven days following this injection, she presented with Cushingoid features, an undetectable cortisol and was diagnosed with exogenous steroid-induced hypoadrenalism. Cobicistat is a cytochrome P450 3A inhibitor and in this case inhibited clearance of intrabursal triamcinolone, leading to exogenous glucocorticoid excess and adrenal suppression. This is the first report to describe this predictable DDI with cobicistat following intrabursal glucocorticoid injection. This case highlights the complexities in managing non-HIV-related chronic morbidities in people living with HIV.

Darunavir-cobicistat-emtricitabine-tenofovir alafenamide: safety and efficacy of a protease inhibitor in the modern era.

A fixed-dose combination consisting of darunavir (Drv), cobicistat (Cobi), emtricitabine (2',3'-dideoxy-5-fluoro-3'-thiacytidine [FTC]), and tenofovir alafenamide (Taf) has been recently approved by the European Medicines Agency for the treatment of HIV infection, and is the first ever protease-inhibitor-based single-tablet regimen. This article provides a detailed description of its pharmacokinetic, efficacy, and safety profile. The pharmacokinetics of single compounds were analyzed, with a special focus on contrasts between Drv/Cobi and Drv/ritonavir (Rtv). When comparing Cobi and Rtv, multiple interactions must be taken into account: in comparison to Rtv, Cobi is a more selective CYP3A4 inhibitor and has no clinical effect on other isoenzymes inhibited by Rtv (eg, 2C8 and 2C9). Moreover, unlike Cobi, Rtv shows in vivo induction activity on some CYP isoenzymes (eg, 1A2, 2C19, 2C8, 2C9, and 2B6), glucuronyltransferases (eg, UGT1A4), and Pgp. Drv-Cobi-FTC-Taf has recently been demonstrated to be of equal efficacy to Drv-Rtv and other protease inhibitors in both experienced (EMERALD study) and naïve (AMBER study) patients. Moreover, kidney and bone safety profiles have been shown to be good, as has central nervous system tolerance. Total cholesterol:low-density-lipoprotein cholesterol and total cholesterol:high-density-lipoprotein cholesterol ratios are generally high in Drv-Cobi-FTC-Taf vs Rtv-Drv-FTC + tenofovir disoproxil fumarate. An unlikely role of Drv in influencing cardiovascular risk in HIV infection has also been reported. Kidney safety profile is influenced by Cobi, with an increase in creatinine plasma concentration of 0.05-0.1 mg/dL and a parallel glomerular filtration-rate reduction of 10 mL/min within the first 4 weeks after Cobi introduction, which remains stable during treatment. Bone and central nervous system safety profiles were found to be good in randomized clinical trials of both experienced and naïve patients. The efficacy and safety of Drv/Cobi/FTC/Taf are comparable to other drug regimens recommended for HIV treatment.

Brief Report: Efficacy and Safety of Switching to Coformulated Elvitegravir, Cobicistat, Emtricitabine, and Tenofovir Alafenamide (E/C/F/TAF) in Virologically Suppressed Women.

BACKGROUND: The integrase inhibitor regimen [elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (TDF)] demonstrated superior efficacy when compared with a protease inhibitor regimen [ritonavir-boosted atazanavir (ATV + RTV) and FTC/TDF] in 575 treatment-naive women at week 48. We investigated the efficacy, safety, and tolerability of switching to a TAF-based, single-tablet regimen containing elvitegravir, cobicistat, FTC, and tenofovir alafenamide (E/C/F/TAF) versus remaining on ATV + RTV plus FTC/TDF. METHODS: After completing the initial randomized, blinded phase, virologically suppressed (HIV-1 RNA <50 copies/mL) women on ATV + RTV plus FTC/TDF were rerandomized (3:1) to receive open-label E/C/F/TAF versus remaining on their current regimen. The primary end point was proportion of participants with plasma HIV-1 RNA <50 copies per milliliter at week 48 (U.S. FDA snapshot algorithm), with a prespecified noninferiority margin of 12%. Safety [adverse events (AEs)] and tolerability were also assessed. RESULTS: Of 575 women originally randomized and treated in the blinded phase, 159 were rerandomized to switch to E/C/F/TAF and 53 to remain on ATV + RTV plus FTC/TDF. At week 48, virologic suppression was maintained in 150 (94%) of women on E/C/F/TAF and 46 (87%) on ATV + RTV plus FTC/TDF [difference 7.5% (95% confidence interval -1.2% to 19.4%)], demonstrating noninferiority of E/C/F/TAF to ATV + RTV and FTC/TDF. Incidence of AEs was similar between groups; study drug-related AEs were more common with E/C/F/TAF (11% versus 4%). CONCLUSIONS: Switching to E/C/F/TAF was noninferior to continuing ATV + RTV plus FTC/TDF in maintaining virologic suppression and was well tolerated at 48 weeks.

In vitro inhibition of human UGT isoforms by ritonavir and cobicistat.

1. Ritonavir and cobicistat are pharmacokinetic boosting agents used to increase systemic exposure to other antiretroviral therapies. The manufacturer's data suggests that cobicistat is a more selective CYP3A4 inhibitor than ritonavir. However, the inhibitory effect of ritonavir and cobicistat on human UDP glucuronosyltransferase (UGT) enzymes in Phase II metabolism is not established. This study evaluated the inhibition of human UGT isoforms by ritonavir versus cobicistat. 2. Acetaminophen and ibuprofen were used as substrates to evaluate the metabolic activity of the principal human UGTs. Metabolite formation rates were determined by HPLC analysis of incubates following in vitro incubation of index substrates with human liver microsomes (HLMs) at different concentrations of ritonavir or cobicistat. Probenecid and estradiol served as positive control inhibitors. 3. The 50% inhibitory concentrations (IC50) of cobicistat and ritonavir were at least 50 µM, which substantially exceeds usual clinical plasma concentrations. Probenecid inhibited the glucuronidation of acetaminophen (IC50 0.7 mM), but not glucuronidation of ibuprofen. At relatively high concentrations, estradiol inhibited ibuprofen glucuronidation (IC50 17 µM). 4. Ritonavir and cobicistat are unlikely to produce clinically important drug interactions involving drugs metabolized to glucuronide conjugates by UGT1A1, 1A3, 1A6, 1A9, 2B4 and 2B7.

A Review of the Efficacy and Safety of Genvoya® (Elvitegravir, Cobicistat, Emtricitabine, and Tenofovir Alafenamide) in the Management of HIV-1 Infection.

INTRODUCTION: This review evaluates the efficacy and safety of Genvoya® (elvitegravir, cobicistat, emtricitabine, and tenofovir alafenamide [EVG/c/TAF/FTC]), a single-tablet regimen used for the management of HIV-1 infection. Phase II and III randomized clinical trials evaluate the efficacy and safety of EVG/c/TAF/FTC and tenofovir disoproxil fumerate (TDF)-containing arms; renal impairment, bone mineral density, metabolic effects, and other adverse events are topics explored within this review. METHODS: A MEDLINE with full text and PubMed literature search was conducted for the past 5 years, up to April 2016. RESULTS: Virologic suppression was similar between the EVG/c/TAF/FTC and TDF-containing groups (<50 copies/mL) at week 48. The bone mineral density in the hip and spine showed a significant reduction in the TDF-containing groups. The glomerular filtration rate increased in patients in the EVG/c/TAF/FTC arm and there were significant differences in total proteinuria, albuminuria, and tubular proteinuria in patients switching to EVG/c/TAF/FTC. The most common adverse events were diarrhea, nausea, and headache. DISCUSSION: The coformulated Genvoya regimen is well tolerated and effective in treatment-naive and virologically suppressed patients. Data seem to suggest it may also be effective and safe in patients with mild to moderate renal impairment. The lower-dosed single-tablet regimen has significantly reduced bone and renal side effects.

Inhibition of human cytochromes P450 in vitro by ritonavir and cobicistat.

OBJECTIVES: Ritonavir and cobicistat are strong inhibitors of human cytochrome P450-3A (CYP3A) isoforms, and are used clinically as pharmacokinetic boosting agents for other antiretroviral drugs. Data reported by the manufacturer suggest that cobicistat is a more selective inhibitor of CYP3A than ritonavir. However, this claim has not been validated in clinical studies. This study evaluated the in-vitro inhibitory potency of ritonavir and cobicistat vs a series of human CYP isoforms. METHOD: The model system utilized human liver microsomes and isoform-selective index substrates. KEY FINDINGS: Ritonavir and cobicistat both were strong inhibitors of CYP3A4, with IC50 values of 0.014 and 0.032 µm, respectively. A component of inhibition was time-dependent (mechanism-based). Neither drug meaningfully inhibited CYP1A2 (IC50  > 150 µm). CYP2B6, CYP2C9, CYP2C19 and CYP2D6 were inhibited by both drugs, but with IC50 values exceeding 6 µm. CONCLUSIONS: Consistent with previous reports, both ritonavir and cobicistat were highly potent inhibitors of CYP3A. Both drugs were weaker inhibitors of other human CYPs, with IC50 values at least two orders of magnitude higher. There was no evidence of a meaningful difference in selectivity between the two drugs.

Tenofovir alafenamide, emtricitabine, elvitegravir, and cobicistat combination therapy for the treatment of HIV.

INTRODUCTION: Tenofovir alafenamide (TAF) is a novel prodrug of the nucleotide analogue reverse-transcriptase inhibitor, tenofovir. TAF has been co-formulated with emtricitabine (FTC), elvitegravir (EVG) and the EVG metabolic enhancer, cobicistat (COBI) as a single-tablet regimen being the first TAF-containing antiretroviral combination available. Areas covered: This article summarizes the available information on the pharmacology of the novel compound TAF and overviews TAF/FTC/EVG/COBI use for HIV-1 infected patients, with specific focus on clinical efficacy and safety data. Information sources include peer-reviewed scientific literature, conference proceedings and publically available regulatory reports. Compared to tenofovir disoproxil fumarate, TAF results in higher concentrations of the active metabolite tenofovir diphosphate within lymphoid cells, whereas plasma tenofovir exposure is about 90% lower. The efficacy and safety of TAF/FTC/EVG/COBI in treatment-naïve HIV-infected patients has been assessed in phase-III randomized trials, showing non-inferior virological suppression in comparison with TDF/FTC/EVG/COBI, and significantly lower renal and bone toxicity. In addition, TAF/FTC/EVG/COBI has demonstrated efficacy and safety as a switching strategy in suppressed HIV-1 infected individuals, including those with mild or moderate renal impairment. Expert commentary: Approval of the single-tablet TAF/FTC/EVG/COBI regimen is an important advance in HIV therapy, as it is associated with very high efficacy and a better kidney and bone safety profile compared to TDF-containing regimens due to the incorporation of TAF.

Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Alafenamide: A Review in HIV-1 Infection.

Tenofovir alafenamide (tenofovir AF) is a novel oral prodrug of the nucleos(t)ide reverse transcriptase inhibitor (NRTI) tenofovir that has several pharmacological advantages over tenofovir disoproxil fumarate (tenofovir DF), including increased plasma stability and reduced tenofovir systemic exposure. Tenofovir AF has been coformulated with elvitegravir, cobicistat and emtricitabine as a once-daily, single-tablet regimen (elvitegravir/cobicistat/emtricitabine/tenofovir AF; Genvoya(®)) for the treatment of adults and adolescents with HIV-1 infection. With regard to establishing and/or maintaining virological suppression over 48 weeks in randomized, phase III trials, elvitegravir/cobicistat/emtricitabine/tenofovir AF was noninferior to elvitegravir/cobicistat/emtricitabine/tenofovir DF in antiretroviral therapy (ART)-naive adults, and statistically superior (subsequent to established noninferiority) to ongoing treatment with tenofovir DF-containing regimens in ART-experienced adults with virological suppression. In single-arm, phase III trials, elvitegravir/cobicistat/emtricitabine/tenofovir AF also provided high rates of virological suppression among ART-naive adolescents and ART-experienced adults with stable renal impairment. In general, elvitegravir/cobicistat/emtricitabine/tenofovir AF was well tolerated and associated with more favourable renal and bone parameters, but a less favourable lipid profile, than tenofovir DF-containing regimens. Thus, elvitegravir/cobicistat/emtricitabine/tenofovir AF is an alternative single-tablet regimen for adults and adolescents with HIV-1 infection, particularly those with an estimated creatinine clearance of ≥30 to <50 mL/min or an increased risk of tenofovir DF-related bone toxicity.

Aspectos farmacológicos de darunavir/cobicistat / Pharmacological aspects of darunavir/cobicistat

Rezolsta® (darunavir/cobicistat) es el primer inhibidor de la proteasa potenciado en combinación fija con un nuevo potenciador farmacológico. Darunavir es actualmente el inhibidor de la proteasa de uso preferente con un perfil de eficacia y seguridad bien conocido. Cobicistat es un inhibidor del citocromo P450 3A sin actividad antirretroviral que se usa como potenciador farmacológico y que puede ser una alternativa al ritonavir. Los parámetros farmacológicos de darunavir con cobicistat son similares a los que se producen cuando el potenciador es ritonavir. Los ensayos clínicos de darunavir/cobicistat han demostrado gran eficacia y buena tolerabilidad. El cobicistat inhibe de forma más específica el citocromo P450 3A sin efecto inductor, por lo que presenta un menor perfil de interacciones farmacológicas que el ritonavir. El cobicistat está también coformulado a dosis fijas con otros fármacos: elvitegravir, tenofovir diproxil fumarato y emtricitabina (Stribild®), elvitegravir, tenofovir alafenamina y emtricitabina (Genvoya®) y con atazanavir (Evotaz®). En este capítulo se revisan los aspectos farmacológicos de la combinación darunavir/cobicistat (Rezolsta®)

Rezolsta® (darunavir/cobicistat) is the first fixed-dose combination tablet with a new boosting agent. Darunavir is currently the preferred protease inhibitor and has a well-known safety and efficacy profile. Cobicistat is a P450 3A cytochrome inhibitor without antiretroviral activity that is used as a boosting agent and can be an alternative to ritonavir. The pharmacological values of darunavir with cobicistat are similar to those produced when the booster is ritonavir. Clinical trials of darunavir/cobicistat have shown high efficacy and good tolerability of the combination. Cobicistat produces more specific inhibition of cytochrome P450 3A induction effect, and consequently has a lower drug interaction profile than ritonavir. Cobicistat is also coformulated in fixed-dose combinations with other drugs: elvitegravir, tenofovir diproxil fumarate and emtricitabine (Stribild®), elvitegravir, tenofovir alafenamine and emtricitabine (Genvoya®) and with atazanavir (Evotaz®). The present article reviews the pharmacological features of the combination darunavir/cobicistat (Rezolsta®)

Cobicistat versus ritonavir boosting and differences in the drug-drug interaction profiles with co-medications.

Nearly all HIV PIs and the integrase inhibitor elvitegravir require a pharmacokinetic enhancer in order to achieve therapeutic plasma concentrations at the desired dose and frequency. Whereas ritonavir has been the only available pharmacokinetic enhancer for more than a decade, cobicistat has recently emerged as an alternative boosting agent. Cobicistat and ritonavir are equally strong inhibitors of cytochrome P450 (CYP) 3A4 and consequently were shown to be equivalent pharmacokinetic enhancers for elvitegravir and for the PIs atazanavir and darunavir. Since cobicistat is a more selective CYP inhibitor than ritonavir and is devoid of enzyme-inducing properties, differences are expected in their interaction profiles with some co-medications. Drugs whose exposure might be altered by ritonavir but unaltered by cobicistat are drugs primarily metabolized by CYP1A2, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 or drugs undergoing mainly glucuronidation. Thus, co-medications should be systematically reviewed when switching the pharmacokinetic enhancer to anticipate potential dosage adjustments.

Drug Susceptibility and Viral Fitness of HIV-1 with Integrase Strand Transfer Inhibitor Resistance Substitution Q148R or N155H in Combination with Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Resistance Substitutions.

In clinical trials of coformulated elvitegravir (EVG), cobicistat (COBI), emtricitabine (FTC), and tenofovir disoproxil fumarate (TDF), emergent drug resistance predominantly involved the FTC resistance substitution M184V/I in reverse transcriptase (RT), with or without the tenofovir (TFV) resistance substitution K65R, accompanied by a primary EVG resistance substitution (E92Q, N155H, or Q148R) in integrase (IN). We previously reported that the RT-K65R, RT-M184V, and IN-E92Q substitutions lacked cross-class phenotypic resistance and replicative fitness compensation. As a follow-up, the in vitro characteristics of mutant HIV-1 containing RT-K65R and/or RT-M184V with IN-Q148R or IN-N155H were also evaluated, alone and in combination, for potential interactions. Single mutants displayed reduced susceptibility to their corresponding inhibitor classes, with no cross-class resistance. Viruses with IN-Q148R or IN-N155H exhibited reduced susceptibility to EVG (137- and 40-fold, respectively) that was not affected by the addition of RT-M184V or RT-K65R/M184V. All viruses containing RT-M184V were resistant to FTC (>1,000-fold). Mutants with RT-K65R had reduced susceptibility to TFV (3.3- to 3.6-fold). Without drugs present, the viral fitness of RT and/or IN mutants was diminished relative to that of the wild type in the following genotypic order: wild type > RT-M184V ≥ IN-N155H ≈ IN-Q148R ≥ RT-M184V + IN-N155H ≥ RT-M184V + IN-Q148R ≥ RT-K65R/M184V + IN-Q148R ≈ RT-K65R/M184V + IN-N155H. In the presence of drug concentrations approaching physiologic levels, drug resistance counteracted replication defects, allowing single mutants to outcompete the wild type with one drug present and double mutants to outcompete single mutants with two drugs present. These results suggest that during antiretroviral treatment with multiple drugs, the development of viruses with combinations of resistance substitutions may be favored despite diminished viral fitness.

Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors.

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 µM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.