Chiara Carcieri

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

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

A LC-MS method to quantify tenofovir urinary concentrations in treated patients.

Tenofovir disoproxil fumarate is a prodrug of tenofovir used in the treatment of HIV and HBV infections: it is the most used antiretroviral worldwide. Tenofovir is nucleotidic HIV reverse trascriptase inhibitor that showed excellent long-term efficacy and tolerability. However renal and bone complications (proximal tubulopathy, hypophosphatemia, decreased bone mineral density, and reduced creatinine clearance) limit its use. Tenofovir renal toxicity has been suggested as the consequence of drug entrapment in proximal tubular cells: measuring tenofovir urinary concentrations may be a proxy of this event and it may be used as predictor of tenofovir side effects. No method is currently available for quantifying tenofovir in this matrix: then, the aim of this work was to validate a new LC-MS method for the quantification of urinary tenofovir. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 5 μm T3, 4.6 mm × 150 mm, reversed phase analytical column. Detection of tenofovir and internal standard was achieved by electrospray ionization mass spectrometry in the positive ion mode. Calibration ranged from 391 to 100,000 ng/mL. The limit of quantification was 391 ng/mL and the limit of detection was 195 ng/mL. Mean recovery of tenofovir and internal standard were consistent and stable, while matrix effect resulted low and stable. The method was tested on 35 urine samples from HIV-positive patients treated with tenofovir-based HAARTs and did not show any significant interference with antiretrovirals or other concomitantly administered drugs. All the observed concentrations in real samples fitted the calibration range, confirming the capability of this method for the use in clinical routine. Whether confirmed in ad hoc studies this method may be used for quantifying tenofovir urinary concentrations and help managing HIV-positive patients treated with tenofovir.

UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs, dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat and ritonavir boosters in human plasma

&NA; Rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) are the latest antiretroviral drugs approved for treatment of HIV infection. Currently, poor information is currently available concerning their pharmacokinetic and pharmacodynamic properties, thus making the use of therapeutic drug monitoring for these drugs not useful. This lack of information is partially due to the absence of an high‐throughput method for their simultaneous quantification together with other antiretroviral drugs. In this work, we describe the development and validation of a new UPLC–MS/MS method to quantify these drugs, together with other fourteen antiretroviral agents, in human plasma. One hundred microliters of plasma samples were added with internal standard (6,7‐Dimethyl‐ 2,3‐di(2‐pyridyl) quinoxaline), underwent a simple protein precipitation with methanol:acetonitrile (50:50 v/v) followed by sample dilution with water. Chromatographic separation was performed on a Acquity® UPLC HSS T3 column (150 mm x 2.1 mm I.D) with a particle size of 1.8 &mgr;m and compounds were detected with a tandem mass detector, monitoring two ion transitions for each drugs. The mean recovery of RPV, DTG and EVG were 101%, 87% and 112.3% respectively. Accuracy and precision inter/intra‐day were below 15% for all drugs, in accordance to Food and Drug Administration guidelines requirements. The UPLC–MS/MS method reported here could be used routinely to monitor plasma concentrations of antiviral drugs, including RPV, DTG and EVG. Graphical abstract Typical chromatogram from direct injection of a chemical mix containing all the target analytes at a concentration of 2 &mgr;g/mL. Figure. No caption available. HighlightsUseful UHPLC–MS/MS method to quantify a wide panel of antiretroviral drugs.Validated method following FDA guidelines.Fast and cheap protein precipitation protocol, suitable for clinical routine.The method has been tested on real‐life specimens with good results.

Treatment with daclatasvir and sofosbuvir for 24 weeks without ribavirin in cirrhotic patients who failed first-generation protease inhibitors

BackgroundTreatment of patients with chronic hepatitis C who failed the triple therapy with first generation of protease inhibitors is not still defined. The combined use of sofosbuvir (SOF) and daclatasvir (DCV) seems to be promising due to higher genetic barrier, good tolerance and effectiveness.MethodsWe described the treatment with this drug combination in a real-life cohort of 20 cirrhotic patients with genotype 1 who failed the triple therapy.Results18 of them (90%) with Child–Pugh A, 11 (55%) with genotype 1a, 17 (85%) with more than 1 and 8 (40%) with more than 2 previous failed treatment; all patients had at baseline NS3 resistance-associated variants related to triple therapy failure. RBV was not administered due to anemia in previous treatments. The sustained virological response was 100%.ConclusionTreatment with SOF + DCV without RBV for 24 weeks is safe and effective in cirrhotic patients who failed triple therapy with the first generation of protease inhibitors.

Vitamin D pathway gene polymorphisms as predictors of hepatitis C virus-related mixed cryoglobulinemia.

Mixed cryoglobulinemia (MC) is the most frequent extrahepatic hepatitis C virus (HCV) complication. Vitamin D is a modulator of several biological processes, including immune and skeletal systems and MC presence and systemic vasculitis were associated independently with low levels of vitamin D. Considering the impact of vitamin D, we aimed to evaluate the role of some single nucleotide polymorphisms (SNPs) of vitamin D pathway genes in the prediction of MC in HCV patients treated with pegylated interferon and ribavirin. We investigated SNPs in IL-28B, CYP27B1, CYP27A1, CYP24A1, VDBP, and VDR genes through real-time PCR. VDR gene SNPs were related to baseline viral load: VDR BsmI AA (P=0.018), TaqI CC (P=0.009), and ApaI AA (P=0.004) showed a lower baseline HCV count. Among vitamin D pathway gene polymorphisms, VDR FokI T>C was a factor associated with the presence of MC in the study population (P=0.011): related to C allele carriers (TT vs. TC/CC), we obtained a P-value of 0.003. In the logistic regression analysis to assess which demographic, clinical, or genetic factors could predict the presence of cryoglobulin, the TT/CC IL-28B rs8099917/rs12979860 haplotype [P<0.001; odds ratio (OR) 3.516 (1.951–6.336)], baseline viral load [P<0.001; OR 1.000 (0.999–1.001)], and VDR FokI TC/CC genotypes [0.044; OR 0.463 (0.218–0.981)] remained in the final regression model. These data could help physicians identify patients with a higher probability of developing MC extrahepatic complications.

Cannabinoids concentration variability in cannabis olive oil galenic preparations

Knowledge of the exact concentration of active compounds in galenic preparations is crucial to be able to ensure their quality and to properly administer the prescribed dose. Currently, the need for titration of extracts is still debated. Considering this, together with the absence of a standard preparation method, the aim of this study was to evaluate cannabinoids concentrations variability in galenic olive oil extracts, to evaluate the interlot and interlaboratory variability in the extraction yield and in the preparation composition.

Influence of ABCB11 and HNF4α genes on daclatasvir plasma concentration: preliminary pharmacogenetic data from the Kineti-C study

Background Daclatasvir is an inhibitor of HCV non-structural 5A protein and is a P-glycoprotein substrate. Pharmacogenetics has had a great impact on previous anti-HCV therapies, particularly considering the association of IL-28B polymorphisms with dual therapy outcome. Objectives We investigated the association between daclatasvir plasma concentrations at 2 weeks and 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCB11 and HNF4α). Patients and methods Allelic discrimination was achieved through real-time PCR, whereas plasma concentrations were evaluated through LC-MS/MS. Results Fifty-two patients were analysed, all enrolled in the Kineti-C study. HNF4α 975 C > G polymorphism was found to be associated with the daclatasvir plasma concentrations at 2 weeks (P = 0.009) and 1 month of therapy (P = 0.006). Linear regression analysis suggested that, at 2 weeks of therapy, age, baseline BMI and haematocrit were significant predictors of daclatasvir concentrations, whereas at 1 month of therapy ABCB111131 CC and HNF4α CG/GG genotypes were significant predictors of daclatasvir concentrations. Conclusions These are the first and preliminary results from our clinical study focusing on daclatasvir pharmacogenetics, showing that this approach could have a role in the era of new anti-HCV therapies.

Therapeutic drug monitoring of boosted PIs in HIV-positive patients: undetectable plasma concentrations and risk of virological failure

Background Therapeutic drug monitoring (TDM) of antiretroviral drugs is performed in selected HIV-positive patients. The aim of this study was to estimate the prevalence of undetectable plasma concentrations of ritonavir and boosted PIs and to evaluate the association between those and the 48 week risk of virological failure. Methods A TDM registry study and a retrospective follow-up study were conducted. Plasma concentrations were measured through validated methods. According to PI and ritonavir concentrations, patients were stratified as adherent, partially non-adherent or non-adherent. Virological outcome was evaluated 48 weeks afterwards. Results The TDM registry study included 2468 samples collected from 723 patients (68.1% male, median age 43.5 years). Eighty-seven samples (3.5%, 74 patients) and 68 samples (2.8%, 52 patients) were in the partially non-adherent and non-adherent groups, respectively; more patients on atazanavir/ritonavir (7.9%) versus darunavir/ritonavir (2% twice daily and 1.9% once daily) and lopinavir/ritonavir (1.5%; P  <   0.001) were observed in the partially non-adherent group. Two hundred and ninety patients were included in the follow-up study (64.1% male, median age 40 years). Patients in the adherent group had a higher chance of viral control [81.9% (167/204)] versus the partially non-adherent group and the non-adherent group [71.7% (33/46) and 53.1% (17/32), respectively; P  =   0.001]. Based on multivariate analysis, baseline HIV RNA >50 copies/mL ( P  <   0.001), genotypic susceptibility score ≤2 ( P  =   0.001), lower nadir CD4 cell count ( P  =   0.003) and not being in the adherent group ( P  =   0.029) were independent predictors of HIV RNA >50 copies/mL at 48 weeks. Conclusions The measurement of PI and ritonavir plasma levels can uncover incomplete compliance with treatment; TDM may represent a useful tool for identifying patients in need of adherence-promoting interventions.

Vitamin D pathway genetic variants are able to influence sofosbuvir and its main metabolite pharmacokinetics in HCV mono-infected patients

Vitamin D levels and genetic variants were associated with drug outcome/toxicity and concentrations. The plasma exposure of GS-331007, the main sofosbuvir metabolite, has been related to SVR. We evaluated the impact of polymorphisms in genes (CYP27B1, CYP24A1, VDBP and VDR) related to vitamin D pathway on sofosbuvir and GS-331007 plasma levels in HCV mono-infected patients at one month of treatment. Polymorphisms were investigated through real-time PCR; drug plasma quantification was performed through a UHPLC-MS/MS method. GS-331007 levels were associated with CYP24A1rs2248359 and VDRCdx2 variants in all the analyzed patients and linear regression analysis showed that sex, body mass index, HCV genotype, baseline estimated glomerular filtration rate, VDRCdx2AG/GG and CYP27B1-1260TT genotypes significantly predict concentrations. We performed sub-analyses considering the HCV genotype and the concomitant drug, identifying polymorphisms associated with GS-331007 concentrations. This is the first study focusing on vitamin D pathway gene variants and DAAs concentrations, but further studies are required.

Pharmacokinetic evaluation of oral itraconazole for antifungal prophylaxis in children

Itraconazole is a first‐generation triazole agent with an extended spectrum of activity; it is licensed in adults for superficial and systemic fungal infections; no recommendation has been yet established for use in children patients. Its variable and unpredictable oral bioavailability make it difficult to determine the optimal dosing regimen. Hence, therapeutic drug monitoring, highly available in clinical practice, may improve itraconazole treatment success and safety. The aim of the study was to describe in paediatrics the oral itraconazole pharmacokinetics, used for prophylaxis. Moreover, we evaluated the utility of its therapeutic drug monitoring in this cohort. A fully validated chromatographic method was used to quantify itraconazole concentration in plasma collected from paediatric patients, at the end of dosing interval. Associations between variables were tested using the Pearson test. Mann‐Whitney U test has been used to probe the influence of categorical variables on continuous ones. Any predictive power of the considered variables was finally evaluated through univariate and multivariate linear and logistic regression analyses. A high inter‐individual variability was shown; ethnicity (beta coefficient, β −0.161 and interval of confidence at 95%, IC −395.035; −62.383) and gender (β 0.123 and IC 9.590; 349.395) remained in the final linear regression model with P value of .007 and .038, respectively. This study highlights that therapeutic drug monitoring is required to achieve an adequate target itraconazole serum exposure.