Ritonavir Concentrations Research Articles

Effect of quercetin on the transport of ritonavir to the central nervous system in vitro and in vivo.

The aim of this study was to identify an effective flavonoid that could improve the intracellular accumulation of ritonavir in human brain-microvascular endothelial cells (HBMECs). An in vivo experiment on Sprague-Dawley rats was then designed to further determine the flavonoid's impact on the pharmacokinetics and tissue distribution of ritonavir. In the accumulation assay, the intracellular level of ritonavir was increased in the presence of 25 mmol L(-1) of flavonoids in HBMECs. Quercetin showed the strongest effect by improving the intracellular accumulation of ritonavir by 76.9 %. In the pharmacokinetic study, the presence of quercetin in the co-administration group and in the pretreatment group significantly decreased the area under the plasma concentration-time curve (AUC0-t) of ritonavir by 42.2 % (p < 0.05) and 53.5 % (p < 0.01), and decreased the peak plasma concentration (cmax) of ritonavir by 23.1 % (p < 0.05) and 45.8 % (p < 0.01), respectively, compared to the control group (ritonavir alone). In the tissue distribution study, the ritonavir concentration in the brain was significantly increased 2-fold (p < 0.01), during the absorption phase (1 h) and was still significantly higher (p < 0.05) during the distribution phase (6 h) in the presence of quercetin.

Simultaneous Quantification of Novel Antiretroviral Drug Combination by Stability-indicating High Performance Liquid Chromatography Method

Research in the many areas of human immunodeficiency virus treatment, eradication and prevention has necessitated measurement of antiretroviral concentrations in nontraditional specimen types. For HIV infection, drug combinations are typically used as highly active antiretroviral therapy, intended to maximize viral suppression. A novel four drugs combination was used, which contains two nucleoside analog reverse transcriptase inhibitors (lamivudine and tenofovir) and two protease inhibitors (darunavir and ritonavir). A new simple, efficient, and sensitive reverse phase high performance liquid chromatographic method has been developed for simultaneously extraction and determination of the concentrations of lamivudine, tenofovir, darunavir and ritonavir in bulk and their tablets. Four compounds were separated on a reversed-phase C18 column at 30±2.0° using a gradient mobile phase combination containing potassium dihydrogen phosphate, acetonitrile and methanol. The pH was adjusted to 3.5±0.05 by the addition of ortho phosphoric acid. The samples were detected using a UV detector, 260 nm for lamivudine, tenofovir and darunavir and 240 nm for ritonavir. The procedure separated analytes and its potential degradation products, in an overall analysis time of about 15 min with lamivudine, tenofovir, darunavir and ritonavir eluting at about 2.385, 4.055, 11.353 and 14.010 min, respectively. The linear range of lamivudine, tenofovir, darunavir and ritonavir was 58.32-174.96 μg/ml, 58.32-174.96 μg/ml, 72.00-216.00 μg/ml and 112.50-337.5 μg/ml, respectively. The relative standard deviation for precision was less than 2.0%. The drug was subjected to acid, alkaline peroxide and photolytic stress conditions and the performance of the method was validated according to the present as per the International Conference on Harmonization guidelines for speci?city, linearity, accuracy, precision and robustness.

Simulation of the impact of rifampicin on once-daily darunavir/ritonavir pharmacokinetics and dose adjustment strategies: a population pharmacokinetic approach.

Treatment of HIV/TB coinfection is challenging due to potent drug-drug interactions between antiretrovirals and rifampicin. The effect of rifampicin on darunavir/ritonavir has not been studied. Population pharmacokinetic modelling was applied to investigate the interaction and generate alternative doses to inform clinical trial design. Darunavir/ritonavir concentrations were modelled simultaneously, including data from three studies in HIV patients (n = 51, 7 female). The darunavir/ritonavir-rifampicin interaction was assumed to mimic that previously observed with lopinavir/ritonavir. Daily darunavir/ritonavir 800/100 mg was simulated as a reference (n = 1000; -rifampicin). Simulations with apparent oral clearance increased by 71% and 36% and relative bioavailability decreased by 20% and 45% for darunavir and ritonavir, respectively, were performed for +rifampicin, 600 mg once daily (n = 1000). Darunavir/ritonavir 1200/200 mg once daily, 1600/200 mg once daily, 800/100 mg twice daily and 1200/150 mg twice daily +rifampicin were simulated. Darunavir parameters for each dose +rifampicin were compared with -rifampicin by geometric mean ratio (90% CI). A maximum effect model, with ritonavir inhibiting darunavir clearance, best described the relationship between the drugs. Compared with -rifampicin, simulated darunavir AUC0-24 was 57%, 26%, 1% and 16% lower for 800/100 mg once daily, 1200/200 mg once daily, 1600/200 mg once daily and 800/100 mg twice daily +rifampicin, respectively; but 39% higher with 1200/150 mg twice daily +rifampicin. Darunavir/ritonavir 1600/200 mg once daily, 800/100 mg twice daily and 1200/150 mg twice daily could potentially overcome reduced darunavir concentrations with rifampicin. In the absence of clinical data, modelling and simulation may be useful to predict drug-drug interactions and aid optimal dose selection.

Altered Oligodendrocyte Maturation and Myelin Maintenance: The Role of Antiretrovirals in HIV-Associated Neurocognitive Disorders.

Despite effective viral suppression through combined antiretroviral therapy (cART), approximately half of HIV-positive individuals have HIV-associated neurocognitive disorders (HAND). Studies of antiretroviral-treated patients have revealed persistent white matter abnormalities including diffuse myelin pallor, diminished white matter tracts, and decreased myelin protein mRNAs. Loss of myelin can contribute to neurocognitive dysfunction because the myelin membrane generated by oligodendrocytes is essential for rapid signal transduction and axonal maintenance. We hypothesized that myelin changes in HAND are partly due to effects of antiretroviral drugs on oligodendrocyte survival and/or maturation. We showed that primary mouse oligodendrocyte precursor cell cultures treated with therapeutic concentrations of HIV protease inhibitors ritonavir or lopinavir displayed dose-dependent decreases in oligodendrocyte maturation; however, this effect was rapidly reversed after drug removal. Conversely, nucleoside reverse transcriptase inhibitor zidovudine had no effect. Furthermore, in vivo ritonavir administration to adult mice reduced frontal cortex myelin protein levels. Finally, prefrontal cortex tissue from HIV-positive individuals with HAND on cART showed a significant decrease in myelin basic protein compared with untreated HIV-positive individuals with HAND or HIV-negative controls. These findings demonstrate that antiretrovirals can impact myelin integrity and have implications for myelination in juvenile HIV patients and myelin maintenance in adults on lifelong therapy.

Darunavir and ritonavir total and unbound plasmatic concentrations in HIV-HCV-coinfected patients with hepatic cirrhosis compared to those in HIV-monoinfected patients.

Our objective was to describe the pharmacokinetic (PK) parameters of total and unbound darunavir and ritonavir concentrations in HIV-hepatitis C virus (HCV)-coinfected patients with cirrhosis, as ritonavir-boosted darunavir is mainly metabolized in the liver, and hepatic cirrhosis might modify darunavir-ritonavir concentrations. This was a prospective, case-control, and unicenter study. HIV-HCV-coinfected patients with compensated cirrhosis (cases) and HIV-monoinfected patients with normal liver function (controls) were included. Darunavir-ritonavir was given at 800/100 mg once daily. Patients were followed for 24 weeks to assess safety and efficacy. A steady-state 12-h PK study was performed. Total and unbound concentrations were determined by liquid chromatography-tandem mass spectrometry. The unbound fraction was obtained by ultrafiltration. The plasma area under the concentration-time curve (AUC) and oral clearance (CL/F) were assessed by noncompartmental models. Thirty patients (20 cases and 10 controls) were included. Among cirrhotic patients, the Child-Pugh score was C in 4 cases, B in 1 case, and A in 15 cases; the median (interquartile range) transient elastography values were 20 kPa (14 to 26 kPa), and 5 patients had prior clinical decompensations. There were no significant differences in the darunavir PK parameters between cases and controls except for longer time to maximum plasma concentrations (Tmax) and half-lives in the cirrhotic patients. There were no significant differences in ritonavir total concentrations, but the unbound concentrations were higher in cirrhotic patients. There were significant correlations between the darunavir total and unbound concentrations in both cirrhotic patients and controls. There were no differences in PK parameters based on Child-Pugh score, liver elasticity, gender, or use of concomitant medications. In conclusion, in HIV-HCV-coinfected patients with clinically compensated cirrhosis receiving darunavir-ritonavir at 800/100 mg once daily, the darunavir total and unbound concentrations are similar to those observed in noncirrhotic patients, and dose adjustments are not necessary.

Unbound Ritonavir Concentrations in Rat and Human Hepatocytes

Knowledge regarding intracellular drug exposure is crucial to gain mechanistic understanding of hepatic disposition. This study aims to develop an approach to determine unbound intracellular concentrations (Cu,cell ) of ritonavir. Ritonavir was selected as a model drug as incubations with high ritonavir concentrations inhibited all saturable processes involved in ritonavir disposition including metabolism and transporter-mediated membrane passage. Following this incubation, hepatocytes were re-equilibrated in fresh protein-containing medium before determination of extracellular unbound ritonavir concentrations. In the absence of metabolism and transport, unbound intracellular and unbound extracellular concentrations were identical. In parallel, total intracellular ritonavir concentrations (Ccell ) were determined, enabling the calculation of intracellular free fractions (fu,cell ). Additionally, Ccell was determined after exposing hepatocytes to a therapeutically relevant concentration (0.5 μM). Multiplication of this concentration with fu,cell resulted in Cu,cell . Finally, Kpu,u (intracellular unbound drug accumulation ratio) was calculated. Exposure of rat and human hepatocytes to 0.5 μM ritonavir resulted in Cu,cell of 12 ± 1 and 8 ± 1 nM. Corresponding Kpu,u values were 2.7 ± 0.5 and 1.4 ± 0.2. We present an in vitro method to determine Cu,cell of ritonavir in intact hepatocytes. Cu,cell obtained at clinically relevant extracellular concentrations are in accordance with concentrations known to inhibit cytochrome P450 and are achieved because of ritonavir accumulation in hepatocytes.

A rapid spin through oil results in higher cell-associated concentrations of antiretrovirals compared with conventional cell washing.

Determination of cell-associated antiretroviral drug concentrations is necessary for research into reservoirs of HIV. Variation exists in cell-associated drug concentrations among research groups. One cause for this may be washing cells during processing. We explored spinning cells through oil to minimize this variability. Raltegravir, atazanavir, darunavir, efavirenz, lopinavir and ritonavir concentrations were assessed in CEM.ss T cells washed with HBSS and oil-spun cells. Oil-spun cells had significantly higher concentrations for all drugs compared with samples washed with HBSS. The decline in cell-associated drug concentrations with saline washes compared with a single spin through oil shows the utility of a spin through oil. Oil centrifugation results in high cell-associated drug concentrations, and can be done in a fast, efficient manner.

Determination of Intrahepatic Exposure to the HIV Protease Inhibitor Ritonavir

Accurate knowledge regarding intracellular drug concentrations is crucial for mechanistic understanding of drug efficacy, toxicity and exposure. The aim of this study was to develop an in vitro method to determine the unbound intrahepatic concentration of the model compound ritonavir. First, metabolism and transporter-mediated membrane passage were inhibited by incubating rat hepatocytes with high ritonavir concentrations. Hepatocytes were subsequently allowed to re-equilibrate in fresh albumin-containing medium before determination of extracellular unbound ritonavir concentrations by equilibrium dialysis. The absence of metabolism and carrier-mediated processes allowed deriving free intracellular from free extracellular concentrations. In parallel, total intracellular ritonavir concentrations were determined, enabling calculation of intracellular free fraction (fu,cell). Multiplying the total intracellular ritonavir concentration measured after incubation at a clinically relevant concentration (0.5 µM) with fu,cell resulted in unbound intracellular concentrations. Finally, Kpu,u (intra- to extracellular unbound concentration) was calculated. Fu,cell for ritonavir in rat hepatocytes was 0.0085±0.0023. The Kpu,u in hepatocytes exposed to 0.5 µM ritonavir was 2.83±0.90. This finding is consistent with substantial intrahepatic accumulation of ritonavir, an important property of this widely used pharmacokinetic booster (via CYP3A inhibition). In conclusion we have developed a new in vitro approach to determine the free intracellular concentrations of ritonavir in rat hepatocytes. Experiments are ongoing to determine the free intracellular concentrations of ritonavir in human hepatocytes and of other HIV protease inhibitors.

Anti-HIV Drug-Combination Nanoparticles Enhance Plasma Drug Exposure Duration as Well as Triple-Drug Combination Levels in Cells Within Lymph Nodes and Blood in Primates

HIV patients on combination oral drug therapy experience insufficient drug levels in lymph nodes, which is linked to viral persistence. Following success in enhancing lymph node drug levels and extending plasma residence time of indinavir formulated in lipid nanoparticles, we developed multidrug anti-HIV lipid nanoparticles (anti-HIV LNPs) containing lopinavir (LPV), ritonavir (RTV), and tenofovir (PMPA). These anti-HIV LNPs were prepared, characterized, scaled up, and evaluated in primates with a focus on plasma time course and intracellular drug exposure in blood and lymph nodes. Four macaques were subcutaneously administered anti-HIV LNPs and free drug suspension in a crossover study. The time course of the plasma drug concentration as well as intracellular drug concentrations in blood and inguinal lymph nodes were analyzed to compare the effects of LNP formulation. Anti-HIV LNPs incorporated LPV and RTV with high efficiency and entrapped a reproducible fraction of hydrophilic PMPA. In primates, anti-HIV LNPs produced over 50-fold higher intracellular concentrations of LPV and RTV in lymph nodes compared to free drug. Plasma and intracellular drug levels in blood were enhanced and sustained up to 7 days, beyond that achievable by their free drug counterpart. Thus, multiple antiretroviral agents can be simultaneously incorporated into anti-HIV lipid nanoparticles to enhance intracellular drug concentrations in blood and lymph nodes, where viral replication persists. As these anti-HIV lipid nanoparticles also prolonged plasma drug exposure, they hold promise as a long-acting dosage form for HIV patients in addressing residual virus in cells and tissue.

Long-acting three-drug combination anti-HIV nanoparticles enhance drug exposure in primate plasma and cells within lymph nodes and blood.

Insufficient HIV drug levels in lymph nodes have been linked to viral persistence. To overcome lymphatic drug insufficiency, we developed and evaluated in primates a lipid-drug nanoparticle containing lopinavir, ritonavir, and tenofovir. These nanoparticles produced over 50-fold higher intracellular lopinavir, ritonavir and tenofovir concentrations in lymph nodes compared to free drug. Plasma and intracellular drug levels in blood were enhanced and sustained for 7 days after a single subcutaneous dose, exceeding that achievable with current oral therapy.

Pharmacokinetics, biodistribution, and toxicity of folic acid-coated antiretroviral nanoformulations.

The drug delivery platform for folic acid (FA)-coated nanoformulated ritonavir (RTV)-boosted atazanavir (FA-nanoATV/r) using poloxamer 407 was developed to enhance cell and tissue targeting for a range of antiretroviral drugs. Such formulations would serve to extend the drug half-life while improving the pharmacokinetic profile and biodistribution to reservoirs of human immunodeficiency virus (HIV) infection. To this end, we now report enhanced pharmacokinetics and drug biodistribution with limited local and systemic toxicities of this novel nanoformulation. The use of FA as a targeting ligand for nanoATV/r resulted in plasma and tissue drug concentrations up to 200-fold higher compared to equimolar doses of native drug. In addition, ATV and RTV concentrations in plasma from mice on a folate-deficient diet were up to 23-fold higher for mice administered FA-nanoATV/r than for mice on a normal diet. Compared to earlier nanoATV/r formulations, FA-nanoATV/r resulted in enhanced and sustained plasma and tissue ATV concentrations. In a drug interaction study, ATV plasma and tissue concentrations were up to 5-fold higher in mice treated with FA-nanoATV/r than in mice treated with FA-nanoATV alone. As observed in mice, enhanced and sustained plasma concentrations of ATV were observed in monkeys. NanoATV/r was associated with transient local inflammation at the site of injection. There were no systemic adverse reactions associated with up to 10 weeks of chronic exposure of mice or monkeys to FA-nanoATV/r.

Population Pharmacokinetic/Pharmacogenetic Model of Lopinavir/Ritonavir in HIV-Infected Patients

This study aims to develop a population pharmacokinetic/pharmacogenetic model for lopinavir/ritonavir (LPV/r) in European HIV-infected patients. A total of 693 LPV/r plasma concentrations were assessed and 15 single-nucleotide polymorphisms were genotyped. The population pharmacokinetic/pharmacogenetic model was created using a nonlinear mixed-effect approach (NONMEM® v.7.2.0., ICON Development Solutions, Dublin, Ireland). Covariates significantly related to LPV/r apparent clearance (CL/F) were ritonavir trough concentration (RTC), BMI, high-density lipoprotein cholesterol (HDL-C) and certain single-nucleotide polymorphisms in genes encoding for metabolizing enzymes, which are representable as follows: CL/F = (0.216BMI + 0.0125HDL-C) × 0.713RTC × 1.26rs28371764[C/T] × 0.528rs6945984[C/C] × 0.302 CYP3A4[1461insA/del] Conclusion: The LPV/r standard dose appears to be appropriate for the rs28371764[C/T] genotype. However, lower doses should be recommended for the rs6945984[C/C] and CYP3A4[1461insA/del] genotypes and even for those patients without any of these variants, as the standard dose seems to be higher than that which is required in order to achieve therapeutic levels.

Intracellular accumulation of atazanavir/ritonavir according to plasma concentrations and OATP1B1, ABCB1 and PXR genetic polymorphisms.

The rate of accumulation of atazanavir and ritonavir within cells is still debated due to methodological limitations. Our aim was to measure peripheral blood mononuclear cell (PBMC) concentrations of atazanavir and ritonavir and investigate whether single-nucleotide polymorphisms of OATP, ABCB1, CYP3A4 and PXR genes are involved in intracellular drug penetration. HIV-positive patients administered 300 mg of atazanavir/100 mg of ritonavir were enrolled. Blood sampling was performed at the end of the dosing interval (Ctrough). PBMC-associated and plasma atazanavir and ritonavir concentrations were measured by validated HPLC coupled with a single mass detector (HPLC-MS) and HPLC-photodiode array (PDA) methods, respectively. Cell count and mean cellular volume were determined using a Coulter counter. Genotyping was conducted using real-time PCR. Thirty-five patients were enrolled. Median atazanavir and ritonavir intracellular concentrations were 1844 and 716 ng/mL, respectively. Median plasma concentrations were 645 ng/mL for atazanavir and 75 ng/mL for ritonavir, while median intracellular/plasma concentration ratios were 2.4 and 9.2, respectively. Median ritonavir intracellular concentrations were higher for OATP1B1 521 T→C TC or CC carriers and for PXR 44477 A→G AG or GG carriers. Atazanavir intracellular/plasma concentration ratios were higher in patients GG for the ABCB1 2677 G→T single-nucleotide polymorphism (SNP) compared with GT and TT groups. Our study showed a higher intracellular ritonavir accumulation than previously reported. Ritonavir intracellular concentrations were associated with OATP1B1 521 and PXR 44477 SNPs while intracellular atazanavir exposure was associated with the ABCB1 2677 SNP. Further clinical studies are necessary in order to confirm these data.

Intracellular antiviral activity of low-dose ritonavir in boosted protease inhibitor regimens.

Protease inhibitors are largely used for the treatment of HIV infection in combination with other antiretroviral drugs. Their improved pharmacokinetic profiles can be achieved through the concomitant administration of low doses of ritonavir (RTV), a protease inhibitor currently used as a booster, increasing the exposure of companion drugs. Since ritonavir-boosted regimens are associated with long-term adverse events, cobicistat, a CYP3A4 inhibitor without antiviral activity, has been developed. Recently, high intracellular concentrations of ritonavir in lymphocytes and monocytes were reported even when ritonavir was administered at low doses, so we aimed to compare its theoretical antiviral activity with those of the associated protease inhibitors. Intracellular concentrations of ritonavir and different protease inhibitors were determined through the same method. Inhibitory constants were obtained from the literature. The study enrolled 103 patients receiving different boosted protease inhibitors, darunavir-ritonavir 600 and 100 mg twice daily and 800 and 100 mg once daily (n = 22 and 4, respectively), atazanavir-ritonavir 300 and 100 mg once daily (n = 40), lopinavir-ritonavir 400 and 100 mg twice daily (n = 21), or tipranavir-ritonavir 500 and 200 mg twice daily (n = 16). According to the observed concentrations, we calculated the ratios between the intracellular concentrations of ritonavir and those of the companion protease inhibitor and between the theoretical viral protease reaction speeds with each drug, with and without ritonavir. The median ratios were 4.04 and 0.63 for darunavir-ritonavir twice daily, 2.49 and 0.74 for darunavir-ritonavir once daily, 0.42 and 0.74 for atazanavir-ritonavir, 0.57 and 0.95 for lopinavir-ritonavir, and 0.19 and 0.84 for tipranavir-ritonavir, respectively. Therefore, the antiviral effect of ritonavir was less than that of the concomitant protease inhibitors but, importantly, mostly with darunavir. Thus, further in vitro and in vivo studies of the RTV antiviral effect are warranted.

Potential association between rosuvastatin use and high atazanavir trough concentrations in ritonavir-treated HIV-infected patients.

Emerging evidence shows that standard atazanavir (ATV) dosages are associated with considerable interindividual variability in plasma drug concentrations. Given the potential impact of ATV exposure on clinical outcome, in this retrospective study we sought to assess demographic and clinical factors influencing ATV plasma concentrations in a large cohort of HIV-infected patients. HIV-infected patients treated with ATV for at least 3 months and with at least one assessment of ATV trough concentrations were enrolled. Blood trough samples were collected from all patients immediately before the next drug intake. Plasma ATV and ritonavir concentrations were quantified by a validated chromatographic method coupled with tandem mass spectrometric detection. Univariate and multivariate regression analyses were performed using ATV plasma concentrations as the dependent variable, with demographic and clinical characteristics as the independent ones. A total of 273 HIV-infected adult patients were included in the present study. Factors significantly associated with increased ATV concentrations by univariate analysis were ATV dosage, number of concomitant drugs, ritonavir or statin use. However, with multivariate regression analysis, the only factors independently and significantly associated with ATV concentrations were ritonavir use (r=0.291, P<0.0001) and concomitant rosuvastatin therapy (r=0.315, P<0.0001). We found that patients on ATV/ritonavir given rosuvastatin concomitantly have ATV concentrations that exceed the upper therapeutic threshold of 800 ng/ml known to be associated with a high risk of experiencing ATV-related side effects. In agreement with previous findings we confirmed the lack of association between other demographic and clinical characteristics such as age, gender, body weight and ATV exposure.

A novel ritonavir paediatric powder formulation is bioequivalent to ritonavir oral solution with a similar food effect.

A novel ritonavir oral powder formulation has been developed to eliminate the alcohol and propylene glycol contents in the current ritonavir oral solution for paediatric use. Two clinical studies were conducted to assess the bioequivalence of the powder formulation to the marketed oral solution and to evaluate the effect of food and vehicles on bioavailability. Study 1 was a randomized, partial-crossover, 4-period study in 48 subjects. Regimens included: oral solution under moderate-fat conditions, powder formulation in water under fasting, moderate-fat or high-fat conditions, and powder formulation in chocolate milk or pudding under moderate-fat conditions. Study 2 was a randomized, crossover, 4-period study in 24 subjects. Subjects were randomized to a sequence of the oral solution and powder formulation in water, infant formula and apple sauce, all under moderate-fat conditions. Bioavailability comparisons were assessed by the 90% CIs for the geometric least-squares mean ratios. Ritonavir powder formulation in water was found to be bioequivalent to the marketed oral solution. Ritonavir powder formulation administered in chocolate milk, pudding, infant formula or apple sauce was bioequivalent to the powder formulation administered in water. Compared with fasting conditions, moderate-fat and high-fat meals were associated with approximately 25-40% and 35-50% reduction in ritonavir concentrations, respectively. The novel ritonavir powder formulation is bioequivalent to marketed ritonavir oral solution under moderate-fat conditions with a similar effect of meals. None of the vehicles tested negatively affected the bioavailability, which suggests the potential for use of a broad range of vehicles for dose preparation.

Low-density lipoprotein and ritonavir: an interaction between antiretrovirals and lipids mediated by P-glycoprotein

Antiretroviral therapy has considerably reduced HIV disease progression, but complete eradication of HIV cannot actually be achieved. Moreover, prolonged use of protease inhibitors (PIs) causes profound changes in lipid metabolism with an increased risk of cardiovascular diseases. P-glycoprotein (P-gp) is expressed on many cell types, playing an important role in the efflux of drugs including PIs, limiting their intracellular concentration. Furthermore, several studies showed that P-gp is involved in lipid homeostasis and its activity is regulated by cholesterol. THP-1 monocytes were used to study: (i) the influence of low-density lipoprotein (LDL) on P-gp expression and function, assessed by flow cytometry and quantitative real-time PCR analysis and measuring ritonavir and rhodamine-123 dye efflux, respectively; and (ii) the influence of ritonavir on cholesterol mobilization. The intracellular levels of ritonavir or cholesterol were measured by HPLC-UV and filipin staining, respectively. In THP-1 cells, LDL was able to yield an increase in both P-gp expression and activity. THP-1 cells treated with LDL showed a decrease in the intracellular ritonavir concentration in a dose-dependent manner. Notably, ritonavir induced reduced cholesterol mobilization in THP-1 cells, probably due to its inhibitory action on P-gp activity. Our data indicate a potential interplay between LDL and ritonavir mediated by P-gp. This interaction could influence both therapy effectiveness and cellular lipid metabolism, with important implications in the management of HIV patients treated with boosted PIs.

Novel Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Detection of Anti-HIV Drugs Lopinavir, Ritonavir, and Tenofovir in Plasma

For HIV infection, anti-HIV drug combinations are typically used as highly active antiretroviral therapy (HAART), intended to maximize viral suppression. Three drugs used frequently in combination are the protease inhibitors lopinavir and ritonavir and the nucleotide reverse transcriptase inhibitor tenofovir. We have successfully developed a simple, efficient, and sensitive method to simultaneously extract and determine the concentrations of lopinavir, ritonavir, and tenofovir in plasma samples. The plasma extractions were performed using a liquid-liquid extraction followed by protein precipitation of the remaining aqueous layer. The collected fractions were combined, dried, and reconstituted in the mobile phase. The drugs were quantified using liquid chromatography coupled with tandem mass spectrometry. The assay was applied to a study of plasma drug levels in two primates (Macaca nemestrina). The bioanalytical assay was optimized and validated to exhibit a high extraction efficiency and good sensitivity and reproducibility. When the assay was applied in a primate study, all three drugs could be detected in plasma within minutes of subcutaneous dosing. This validated assay will be useful for evaluation of drug concentrations in an efficient, selective, and sensitive manner.

Sex differences in responses to antiretroviral treatment in South African HIV-infected children on ritonavir-boosted lopinavir- and nevirapine-based treatment

BackgroundWhile studies of HIV-infected adults on antiretroviral treatment (ART) report no sex differences in immune recovery and virologic response but more ART-associated complications in women, sex differences in disease progression and response to ART among children have not been well assessed. The objective of this study was to evaluate for sex differences in response to ART in South African HIV-infected children who were randomized to continue ritonavir-boosted lopinavir (LPV/r)-based ART or switch to nevirapine-based ART.MethodsART outcomes in HIV-infected boys and girls in Johannesburg, South Africa from 2005–2010 were compared. Children initiated ritonavir-boosted lopinavir (LPV/r)-based ART before 24 months of age and were randomized to remain on LPV/r or switch to nevirapine-based ART after achieving viral suppression. Children were followed for 76 weeks post-randomization and then long-term follow up continued for a minimum of 99 weeks and maximum of 245 weeks after randomization. Viral load, CD4 count, lipids, anthropometrics, drug concentrations, and adherence were measured at regular intervals. Outcomes were compared between sexes within treatment strata.ResultsA total of 323 children (median age 8.8 months, IQR 5.1-13.5), including 168 boys and 155 girls, initiated LPV/r-based ART and 195 children were randomized. No sex differences in risk of virological failure (confirmed viral load >1000 copies/mL) by 156 weeks post-randomization were observed within either treatment group. Girls switched to nevirapine had more robust CD4 count improvement relative to boys in this group through 112 weeks post-randomization. In addition, girls remaining on LPV/r had higher plasma concentrations of ritonavir than boys during post-randomization visits. After a mean of 3.4 years post-randomization, girls remaining on LPV/r also had a higher total cholesterol:HDL ratio and lower mean HDL than boys on LPV/r.ConclusionsSex differences are noted in treated HIV-infected children even at a young age, and appear to depend on treatment regimen. Future studies are warranted to determine biological mechanisms and clinical significance of these differences.Trial registrationClinicalTrials.gov Identifier: NCT00117728

Two-way interaction study between ritonavirboosted danoprevir, a potent HCV protease inhibitor, and ketoconazole in healthy subjects

Danoprevir is a potent, highly selective, macrocyclic, orally bioavailable inhibitor of the hepatitis C virus protease, and a substrate of cytochrome P450 (CYP) 3A. It is co-administered with low-dose ritonavir, a potent CYP3A inhibitor, to enhance danoprevir pharmacokinetics. Ketoconazole is a substrate for and potent selective inhibitor of CYP3A. In this open-label, 3-period study, the 2-way interaction potential between ritonavir-boosted danoprevir (danoprevir/r) and ketoconazole was investigated in 18 healthy subjects. Subjects initially received ketoconazole 200 mg q24h for 4 days (Period 1) followed by a 7-day washout period. Danoprevir/r 100/100 mg q12h was then given for 10 days (Period 2) followed by a further 4 days of danoprevir/r 100/100 mg q12h plus ketoconazole 200 mg q24h (Period 3). Serial blood samples were collected for the determination of danoprevir, ritonavir and/or ketoconazole plasma concentrations, and calculation of pharmacokinetic parameters. Safety and tolerability were monitored throughout the study. Co-administration of ketoconazole with danoprevir/r modestly increased the danoprevir AUCτ by 1.44-fold, with no effect on danoprevir Cmax. Co-administration of danoprevir/r with ketoconazole substantially increased ketoconazole AUCτ and Cmax by 3.71-fold and 1.73-fold, respectively. Danoprevir/r was well tolerated when administered alone or with ketoconazole. These results indicate that the effect of potent CYP3A inhibitors, such as ketoconazole, on danoprevir/r pharmacokinetics is not likely to be clinically relevant.