Antiviral Therapy

Entecavir is an efficient inhibitor of HBV reverse transcriptase (RT) and widely used for therapy of chronic hepatitis B. Entecavir treatment of HBV patients with lamivudine-resistant viral strains, however, often fails, but the mechanism of cross-resistance development is not fully understood.

Using non-linear regression models, dose– response curves of cloned HBV strains from patients pre-treated with RT inhibitors were established in human hepatoma cell lines after transfection with HBV genomes containing HBV polymerase genes from patient isolates. 50% and 90% inhibitory concentrations (IC₅₀ and IC₉₀) and corresponding antiviral resistance factors (RF₅₀ and RF₉₀) were calculated.

The entecavir dose–response curve of lamivudine-resistant HBV RT mutants rtM204 for the replication of HBV decreased less than expected with increasing drug dose. Remarkably, due to the fat dose–response curves, RF₉₀ values against entecavir of samples with rtM204 substitutions were up to 30x higher than their RF₅₀ values.

The unexpectedly high IC₉₀ indicates a strong residual replication capacity of lamivudine-resistant HBV rtM204 variants under entecavir therapy, although IC₅₀ values are initially within the therapeutic range of ente-cavir. This characteristic favours rapid selection of additional mutants with overt resistance against entecavir. Thus, the current phenotypic resistance assays should include determination of IC₉₀.

The introduction of highly sensitive HIV-1 viral load assays with a lower quantification limit of 20 copies/ml uncovered that in a number of patients on ART, the viral load systematically fluctuates around or slightly above the detection limit of the assays. This study aimed to analyse the presence or occurrence of drug resistance mutations in HIV-1-infected patients during long-term persistent low-level viraemia (PLLV) under ART.

A retrospective study was carried out in which baseline and on-therapy presence of drug resistance mutations in the HIV-1 protease and reverse transcriptase genes were analysed in patients with PLLV between 20 and 250 copies/ml. For all available plasma samples collected during PLLV, resistance analysis was attempted with an ultrasensitive amplification and sequencing protocol.

Resistance analysis was successful for 154 samples collected longitudinally from 23 patients over a median period of 4.7 years (IQR 3.3–5.7). Twenty of these patients were on a boosted protease inhibitor (PI)-based regimen (87%). Single drug resistance mutations were detected in isolated samples of 4 patients, 2 of the 3 patients who initiated a non-nucleoside reverse transcriptase inhibitor-based regimen and 2 of the 20 on a PI-based regimen. Only one of the detected mutations decreased susceptibility to the therapy regimen taken at the time of sample collection. Drug resistance mutations were not found in the three patients who developed virological failure (viral load >250 copies/ml) during the study.

Long episodes of PLLV in patients on boosted PI-based regimens rarely result in the selection of new drug-resistant variants.

This study describes first dose and steady state pharmacokinetics of raltegravir (RAL) in cervicovaginal fluid (CVF) and blood plasma (BP).

Three cohorts of women were enrolled sequentially in a single-site, open-label pharmacokinetic study of oral raltegravir 400 mg twice daily: HIV-negative premenopausal, HIV-infected premenopausal and HIV-infected post-menopausal women. BP and CVF were collected over 12 h after a single observed dose and at steady state. RAL concentrations were measured by HPLC-MS methods. Data are expressed as median (IQR). The ANOVA rank-sum test was used to evaluate between-group differences in steady state raltegravir exposure (area under the concentration-time curve over the 12-h dosing interval [AUC_{0–12 h}]).

First dose pharmacokinetics were obtained in HIV-negative premenopausal women and HIV-infected post-menopausal women only. The median (IQR) BP AUC_{0–12 h} was 3,099 (985–5,959) and 4,239 (2,781–13,695) ng•h/ml and the median (IQR) CVF AUC_{0–12 h} was 1,720 (305–5,288) and 13,797 (11,066–19,563) ng•h/ml for HIV-negative pre-menopausal and HIV-infected post-menopausal women, respectively. All cohorts contributed to steady-state pharmacokinetic profiles. Median (IQR) BP AUC_{0–12 h} did not differ between the groups: 8,436 (3,080–10,111), 5,761 (1,801–10,095) and 6,180 (5,295–8,282) ng•h/ml in HIV-negative premenopausal, HIV-infected premenopausal and HIV-infected post-menopausal women, respectively. There was a trend for lower CVF AUC_{0–12 h} among HIV-negative women 3,164 (1,156–9,540) compared to 11,465 (9,725– 17,138) and 9,568 (4,271–24,306) ng•h/ml HIV-infected premenopausal and HIV-infected post-menopausal women, respectively, but this was not statistically significant (P=0.08). HIV-negative premenopausal women had a median (IQR) CVF:BP AUC_{0–12 h} ratio of 0.46 (0.2–1.1), whereas HIV-infected premenopausal and post-menopausal women had median (IQR) CVF:BP AUC_{0–12 h} ratio of 3.9 (1.2–6.7) and 1.4 (0.7–4.3), respectively.

This is the first study to investigate RAL exposure in BP and CVF in premenopausal HIV-negative and pre- and post-menopausal HIV-infected women. These data indicate HIV and menopausal status may influence antiretroviral distribution into the female genital tract.

Lambda interferons (IFNLs) have potent antiviral activity against HCV, and polymorphisms within the IFNL gene cluster near the IFNL3 gene strongly predict spontaneous- and treatment-related HCV infection outcomes. The mechanism(s) linking IFNL polymorphisms and HCV control is currently elusive.

IFNL induction was studied in primary human hepatocytes (PHH) from 18 human donors, peripheral blood mononuclear cells (PBMCs) from 18 human donors, multiple cell lines and induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-hepatocytes) from 7 human donors. After stimulation with intracellular RNA and infectious HCV, quantitative PCR (qPCR) primers and probes were designed to distinguish and quantify closely related IFNL messenger (m)RNAs from IFNL1, IFNL2 and IFNL3.

PHH demonstrated the most potent induction of IFNLs, although had lower pre-stimulation levels compared to PBMCs, monocytes and cell lines. PHH stimulation with cytoplasmic poly I:C induced >1,000-fold expression of IFNL1, IFNL2 and IFNL3. PHH from donors who were homozygous for the favourable IFNL3 allele (IFNL3-CC) had higher IFNL3 induction compared to PHH from IFNL3-TT donors (P=0.03). Baseline IFNL mRNA expression and induction was also tested in iPSC-hepatocytes: iPSC-hepatocytes had significantly higher baseline expression of IFNLs compared to PHH (P<0.0001), and IFNL3 induction was marginally different in iPSC-hepatocytes by IFNL genotype (P=0.07).

Hepatocytes express IFNLs when stimulated by a synthetic viral RNA that signals the cell through the cytoplasm. IFNL induction may be greater in persons with the favourable IFNL3 allele. These data provide insight into the strong linkage between IFNL3 genetics and control of HCV infection.

The influenza antiviral oseltamivir is not licensed for infants aged <1 year in most countries outside the United States. More information is needed on oseltamivir safety at different dosing levels in this vulnerable age group.

In this prospective, observational, non-randomized study, infants aged <1 year with laboratory-confirmed influenza were treated with oral oseltamivir for 5 days. Cohorts 1, 2 and 3 (aged 91–364, 31–90 and 0–30 days, respectively), received twice-daily dosages of 3, 2.5 and 2 mg/kg, respectively. Assessments included pharmacokinetics, on-treatment adverse events, resistance testing and viral shedding.

A total of 65 patients were enrolled: 40, 20 and 5 in cohorts 1, 2 and 3, respectively. Systemic exposure to oseltamivir carboxylate (active metabolite) reached therapeutic levels in all patients, with an adequate safety margin. On-treatment adverse events (n=48) were reported by 32 patients (49%). At least one adverse event was reported by 43%, 65% and 40% of infants in cohorts 1, 2 and 3, respectively; most frequently vomiting and diarrhoea. Eight serious adverse events were reported, all of which were considered unrelated to treatment by the investigator. No deaths occurred and no patient had treatment withdrawn. Oseltamivir resistance mutations were detected in eight patients.

Oseltamivir dosages of 2–3 mg/kg were well tolerated in infants aged <1 year and achieved therapeutic exposure levels. The current study supports the adoption of a universal dosing recommendation for infants. Clinicaltrials.gov unique identifier NCT00988325.

Hepatitis C, caused by a single-stranded RNA virus, has become a global health problem. Infecting millions of individuals in the United States alone, chronic HCV infection can lead to devastating medical problems including cirrhosis and hepatocellular carcinoma. These problems create millions of dollars in health-care costs for treatment and management. This study determines the cost-effectiveness of hepatitis C treatment with the new generation of oral protease inhibitors.

A Markov model was constructed to simulate the progression of genotype-1 chronic hepatitis C disease in a cohort of 50-year-old patients. A decision tree, along with the Markov model, was then used to determine duration of disease, treatment success, progression of disease and mortality. At the end of each stage in the model, the cost and quality-adjusted life years (QALY) were summed for each individual. These were then used to calculate the overall cost-effectiveness ratio (CER) using QALY as the unit of effectiveness. Four treatment options were modelled: sofosbuvir with pegylated interferon-α and ribavirin (SOF/PEG-IFN+RBV), sofosbuvir with ribavirin (SOF/RBV), simeprevir with pegylated interferon-α and ribavirin (SMV/ PEG-IFN+RBV) and simeprevir with sofosbuvir (SMV/SOF).

SOF/PEG-IFN+RBV yielded a CER ratio of $6,796.22/QALY, SMV/PEG-IFN+RBV of $7,642.60/QALY and SMV/SOF of $8,959.11/QALY. SOF/RBV had a higher CER of $16,295.30/QALY. It is important to note however that SMV/SOF had the highest QALY at 19.08.

After consideration of quality of life, treatment regimens and treatment side effects, the SMV/SOF regimen yields acceptable cost-effectiveness ratios with high QALY.

Little is known about the early steps of the HBV life cycle due to the lack of susceptible cells permissive for viral infection. Hence, viral entry has not been exploited for antiviral targets, but the recent seminal discovery of sodium taurocholate co-transporting polypeptide (NTCP) as the cellular receptor for HBV entry opened up many avenues of investigation, making HBV entry amenable to therapeutic intervention.

In order to exploit HBV entry, we established a HepG2-NTCP cell line that supports HBV infection. Over 70% of cells were infected at a dose of 10⁴ genome equivalents (GEq) per cell. Several FDA-approved drugs with NTCP-inhibiting activity were tested for their ability to inhibit HBV infection of the cell line.

Consistent with their NTCP inhibitory activities, our results showed that several of them inhibit HBV infection. In particular, irbesartan, a drug used for the treatment of hypertension, inhibits HBV infection at the 50% effective concentration value of 35 μM.

The observation that the pharmacological inhibitors of the NTCP transporter could block HBV entry suggests that NTCP represents an attractive molecular target for therapeutic intervention in HBV infection.

Some drugs that are actively taken up into the liver exhibit greater than dose proportional increases in plasma exposure, although human liver-to-plasma concentration ratios have rarely been evaluated. Understanding these relationships has implications for drug concentrations at the target site for certain classes of compounds, such as direct-acting antivirals, targeted towards HCV.

Treatment-experienced, chronic HCV non-cirrhotic patients (n=3) received vaniprevir (600 mg or 300 mg twice daily) on days 1–3 and (600 mg or 300 mg single dose) on day 4. Core needle biopsy was performed at 6 or 12 h post-dose on day 4. Blood samples were collected pre-dose on days 1 and 4, and for 24 h post-dose on day 4. The primary study objective was the hepatic concentration of vaniprevir at 6 and 12 h post-dose.

Vaniprevir plasma pharmacokinetic parameters increased in a greater than dose-proportional manner between the 300 mg and 600 mg doses, with approximately fivefold increases in AUC_0–12 and C_max associated with a twofold increase in dose (AUC_0–12, 10.6 μM/h to 59.5 μM/h; C_max, 2.60 μM to 13.5 μM). In the 300 mg and 600 mg dose groups, mean liver concentrations of vaniprevir were 84.6 μM and 169 μM at 6 h post-dose, and 29.4 μM and 53.7 μM at 12 h post-dose. Liver concentrations were higher than plasma with liver-to-plasma concentration ratios of approximately 20–280.

These data confirm higher vaniprevir concentrations in human liver compared with plasma and demonstrate that measurement of human liver drug concentration using needle biopsy is feasible.

DRIVESHAFT is a randomized, open-label, 48-week clinical trial that examined virological outcomes and safety of antiretroviral simplification among virologically suppressed, treatment-experienced HIV-infected patients switching from darunavir/ritonavir (DRV/r) twice-daily-based regimens to a once-daily DRV/r component.

HIV-infected adults with a stable antiretroviral regimen including DRV/r 600/100 mg twice daily plus a minimum of two other antiretrovirals, <2 historical DRV-associated mutations and HIV RNA<40 copies/ml for at least 12 weeks prior to entry were eligible. Participants were randomized 1:1 to switch DRV/r to 800/100 mg once daily or maintain their current regimen. The primary end point was HIV-1 RNA<40 copies/ml at week 48 using the Snapshot algorithm.

Demographics and baseline characteristics were similar between arms. Virological suppression was greater in the DRV/r once-daily (n=30) versus twice-daily (n=30) arm at week 48 (90.0% versus 83.3%; 95% CI: -11.5, 24.8). Three subjects discontinued the once-daily arm, with four discontinuations and one virological failure in the twice-daily arm. No discontinuations were related to adverse events. Reduction in LDL was significantly greater in the once-daily arm at week 24 (-8.0 mg/dl versus 3.3 mg/dl; P=0.04). There was a trend towards suboptimal adherence <90% to antiretrovirals among subjects taking twice-daily versus once-daily DRV/r by week 48 (12.0% versus 0.0%; P=0.06).

Switching from twice-daily to once-daily DRV/r in virologically suppressed patients maintains viro-logical control, with greater reduction in LDL cholesterol by 24 weeks. This study provides pilot data that could be used to design a non-inferiority study to definitively answer the question of whether switching from twice-daily to once-daily DRV/r maintains viral suppression. ClinicalTrials.gov number: NCT01423812.

Post-treatment control of viraemia after discontinuation of antiretroviral therapy begun during primary HIV-1 infection is considered a potential path toward a sustained remission of infection.

Subjects enrolled in an observational primary infection cohort who received at least 11 months of highly active antiretroviral therapy beginning within the first 12 weeks of HIV-1 infection and who subsequently discontinued therapy were evaluated for post-treatment control.

Within a cohort of 389 subjects with primary HIV-1 infection enrolled over 22 years, only 22 met criteria for evaluation of post-treatment control. Among these subjects, 21 (95%) had loss of viral control (HIV-1 RNA>500 copies/ml) within 18 months after treatment discontinuation, and only 1 (4.5%, 95% CI 0.32, 18.9) controlled viral load to levels <500 copies/ml for at least 24 months. The median time to virological failure was 2.17 (IQR 1.18–3.39) months.

Our data suggest a low likelihood of post-treatment control even when highly active antiretroviral therapy is started within 12 weeks of HIV-1 infection.

The HCV NS3/4A serine protease inhibitors (PIs) boceprevir (BOC), telaprevir (TVR) and simeprevir (SMV) are approved for treatment of chronic hepatitis C infection in combination with pegylated interferon and ribavirin. The present study investigated the prevalence of HCV NS3 drug resistance mutations (DRMs) associated with HCV genotype-1-infected individuals at baseline and in viral breakthrough following BOC and TVR treatment.

HCV genotype-1-infected individuals were enrolled in a multicentre, prospective outcomes study. The HCV NS3 viral protease was analysed for DRMs at baseline (n=164) and at viral breakthrough (n=18) following BOC/ TVR treatment.

Viral NS3 protease subtype analysis showed 65.2% (107/164) were HCV subtype-1a and 34.8% (57/164) were HCV subtype-1b infections. Naturally occurring PI DRMs in NS3 (V36L, T54S, V55A, Q80K/R and I132V) were identified in 57.3% (94/164) cases at baseline. The NS3 Q80K polymorphism was found in 43/107 (40.2%) of HCV subtype-1a and exclusively in clade 1 (43/82; 52.4%) versus clade 2 viruses (0/25; 0%, P<10^-6). The pretreatment I132V variant was found in 78.9% (45/57) of subtype-1b. Of 18 patients who had viral breakthrough, the majority was subtype-1a (77.8%, 14/18). BOC/TVR-associated DRMs were detected in 94.4% (17/18), of which 64.7% (11/17) emerged on-treatment.

To ensure the most appropriate direct-acting antiviral-based treatment regimen is employed, baseline reporting of clade and resistance mutations for HCV sub-type-1a using nucleotide sequence-based analysis is warranted prior to commencement of therapy.