Sarah Allegra

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.

Vitamin D pathway gene variants and HCV-2/3 therapy outcomes.

BACKGROUND The combination of ribavirin and pegylated interferon-α is considered the standard of care for HCV-2/3 genotypes. The immune system plays a key role in the achievement of a sustained virological response (SVR). Vitamin D seems to influence antiviral response in chronic hepatitis C and its pathway is controlled by polymorphic genes such as CYP27B1, CYP24A1 and VDR. In this study, we have investigated the correlation among the treatment outcomes and single nucleotide polymorphisms (SNPs) in the above-mentioned genes and IL28B genes. METHODS A total of 112 HCV-2/3 patients treated with interferon plus ribavirin were retrospectively studied; allelic discrimination was performed by real-time PCR. RESULTS CYP24A1rs2585428, IL28Brs12979860 and rs8099917 SNPs affected treatment failure and body mass index (BMI), Metavir score, IL28Brs8099917TT and CYP24A1rs2585428GG were the only factors able to predict it. SVR was predicted by Metavir score, HCV RNA at baseline and early virological response (EVR). IL28Brs12979860 SNP and HCV RNA were also related to rapid virological response. EVR was predicted by BMI, Metavir score and CYP24A1rs2585428 SNP. IL28Brs8099917TT and FokITT were relapse prediction factors. CONCLUSIONS In addition to non-genetic factors, SNPs in the vitamin D pathway seem to have a role in HCV-2/3 therapy outcomes. This study reveals the likely usefulness of pharmacogenetic-based ribavirin and interferon therapy to help identify patients for whom therapy could be successful or not, also considering new future expensive therapy options. To date, no similar data were published on these viral genotypes, but further studies in different and bigger cohorts are needed.

Role of IL28-B polymorphisms in the treatment of chronic hepatitis B HBeAg-negative patients with peginterferon

Interleukin (IL)28-B polymorphism has been related to interferon response in the treatment of hepatitis C, but its role in chronic hepatitis B (CHB) therapy is still poorly understood. We aimed to investigate the effect of IL28-B polymorphisms in the treatment with pegylated-interferon (PEG-IFN) of patients with CHB. We retrospectively analyzed 190 patients with chronic hepatitis B e antigen (HBeAg) negative, genotype A (22%), B (12%), C (10%), D (33%), E (20%), treated with PEG-IFN alfa-2a for 48weeks; genotype analysis was performed for IL28-B polymorphisms rs12979860, rs8099917 and rs12980275 according to virological, serological and biochemical response. During 2years of follow-up 12 patients (6.3%) cleared hepatitis B surface antigen (HBsAg) with seroconversion, 40 (21%) obtained a negative viral load and 104 (54.7%) gained a biochemical response. We found a difference of distribution of rs12979860 CC genotype among different ethnicity (p=0.013). Rs12979860 CC genotype was significantly associated with serological and virological response (p<0.001); rs8099917 TT and rs12980275 AA genotypes were mostly related with virological response (p<0.001). In multivariate logistic analysis rs12979860 CC was predictive of virological response (OR=4.290; CI=1.589-11.580, p=0.004) and serological response (OR=10.129; CI=2.440-42.044; p<0.001). Rs8099917 TT was predictive only of virological response (OR=3.746, CI=1.235-11.355; p=0.020). The E genotype was a negative predictive factor of virological response (OR=0.057; CI=0.014-0.238; p<0.001). IL28-B polymorphisms are related to different response in the treatment of CHB HBeAg-negative with PEG-IFN, and the E genotype is a novel negative predictive factor.

Efavirenz pharmacogenetics in a cohort of Italian patients

The pharmacogenetics and pharmacokinetics of efavirenz (EFV) have been widely studied, although data in the Italian population are limited. Single nucleotide polymorphisms (SNPs) in the CYP2B6 gene have been associated with increased EFV plasma concentrations and central nervous system toxicity. The aim of this work was to evaluate EFV plasma exposure according to SNPs in genes involved in drug metabolism and elimination in a cohort of Italian HIV-1-positive patients treated with EFV. Plasma samples were used to measure EFV concentrations at 12h after intake (C12) by a validated HPLC/PDA system. Whole blood was used to identify SNPs in ABCB1, MRP2, CYP2B6, CYP2A6, UGT2B7, NR1I2 (PXR), NR1I3 (CAR) and HNF4α by real-time PCR. The association between SNPs and EFV plasma levels was evaluated through non-parametric tests. Among 201 patients, the median EFV C12 was 2618.5ng/mL. No significant associations were found for MRP2, CYP2A6, UGT2B7, PXR and CAR SNPs; conversely, an association of CYP2B6 516G>T, ABCB1 3435C>T and 2677G>T, and HNF4α 975C>G polymorphisms with EFV C12 was observed. In multivariate analysis, only CYP2B6 516 TT and ABCB1 3435 TT genotypes were independently associated with an EFV C12 of >4000ng/mL (toxicity cut-off). This study confirmed the role of CYP2B6 and ABCB1 polymorphisms, showed a relationship with HNF4α, and the lack of association of CYP2A6, UGT2B7, NR1I2 and NR1I3 SNPs on EFV plasma exposure. Data regarding some of the studied SNPs are the first obtained in an Italian cohort of HIV patients and lead to a global vision about EFV pharmacogenetics.

Influence of single-nucleotide polymorphisms on deferasirox C trough levels and effectiveness

Deferasirox (DFX) is the only once-daily oral chelator for iron overload and its pharmacokinetic has been related with response to therapy. Our aim was to evaluate DFX plasma concentrations according to single-nucleotide polymorphisms in genes involved in its metabolism (UGT1A1, UGT1A3, CYP1A1, CYP1A2 and CYP2D6) and elimination (MRP2 and BCRP1). Further aim was to define a plasma concentration cutoff value predicting an adequate response to therapy. Plasma concentrations were determined at the end of dosing interval (Ctrough) using an high-performance liquid chromatography–ultraviolet method. Allelic discrimination was performed by real-time PCR. Ctrough levels were influenced by UGT1A1C>T rs887829, CYP1A1C>A rs2606345, CYP1A2A>C rs762551, CYP1A2C>T rs2470890 and MRP2G>A rs2273697 polymorphisms. A DFX plasma efficacy cutoff value of 20 000 ng ml−1 was identified; CYP1A1C>A rs2606345 AA and CYP1A2C>T rs2470890 TT genotypes may predict this value, suggesting a negative predictive role in therapy efficacy. Our data suggest the feasibility of a pharmacogenetic-based DFX dose personalization.

Pharmacogenetics of ribavirin-induced anemia in HCV patients.

Dual therapy (pegylated interferon plus ribavirin) was considered the standard of care for hepatitis C virus (HCV) treatment until 2011, when the first-wave direct-acting antivirals were added to this regimen for HCV genotype-1 patients to increase the sustained virological response rate. The second-wave direct-acting antivirals entered the clinical use also in some ribavirin (RBV)- and/or interferon-free combinations. Nevertheless, since some of the new therapeutic regimens also include RBV and its use results still associated with hemolytic anemia, this requires countermeasures to be prevented. These include the identification of several host predictive factors involved in RBV absorption, distribution, metabolism, elimination and many others that might influence this toxic effect. For this reason, we provided an overview of the potential role of pharmacogenomics in predisposing RBV-treated HCV patients to anemia.

Intracellular accumulation of boceprevir according to plasma concentrations and pharmacogenetics.

Boceprevir (BOC) is a directly-acting antiviral agent for the treatment of hepatitis C virus genotype 1 (HCV-1) infection. It is a mixture of two stereoisomers, the inactive R and the active S isomers. No data have previously been published on BOC intracellular accumulation. In this study, BOC isomer concentrations in peripheral blood mononuclear cells (PBMCs) and plasma were determined. The influence of various single nucleotide polymorphisms (SNPs) on plasma and intracellular drug exposure at Week 4 of triple therapy were also evaluated. Plasma and intracellular BOC concentrations were determined at the end of the dosing interval (C(trough)) using a UPLC-MS/MS validated method. Allelic discrimination was performed through real-time PCR. Median plasma concentrations were 65.97 ng/mL for the S isomer and 36.31 ng/mL for the R isomer; the median S/R plasma concentration ratio was 1.66. The median PBMC concentration was 2285.88 ng/mL for the S isomer; the R isomer was undetectable within PBMCs. The median S isomer PBMC/plasma concentration ratio was 28.59. A significant positive correlation was found between plasma and PBMC S isomer concentrations. ABCB1 1236, SLC28A2 124 and IL28B rs12979860 SNPs were associated with the S isomer PBMC/plasma concentration ratio. In regression models, S isomer plasma levels and FokI polymorphism were able to predict S isomer intracellular exposure, whereas SNPs in AKR1, BCRP1 and SLC28A2 predicted the S isomer PBMC/plasma concentration ratio. No similar data regarding BOC pharmacogenetics and pharmacokinetics have been published previously. This study adds a novel and useful overview of the pharmacological properties of this drug.

ABCB11 and ABCB1 gene polymorphisms impact on telaprevir pharmacokinetic at one month of therapy

In 2011 direct-acting antivirals, including telaprevir, have been developed to achieve a better antiviral effect. It was reported that telaprevir is a substrate of P-glycoprotein (ABCB1) and cytochrome P450 3A4. The aim of this retrospective study was the evaluation of the influence of some single nucleotide polymorphisms (SNPs) of genes (ABCB1, SLC28A2/3, SLC29A1) involved in TLV and RBV transport and their correlation with plasma TLV drug exposure at 1 month of therapy. We also investigated the association of a SNP in ABCB11 gene, whose role in TLV transport was not yet shown. Twenty-nine HCV-1 patients treated with telaprevir, ribavirin and pegylated-interferon-α were retrospectively analyzed; allelic discrimination was performed by real-time PCR. Telaprevir Ctrough levels were influenced by Metavir score (P=0.023), ABCB1 2677 G>T (P=0.006), ABCB1 1236 C>T (P=0.015) and ABCB11 1131 T>C (P=0.033) SNPs. Regarding ABCB1 3435 C>T, a not statistically significant trend in telaprevir plasma concentration was observed. Metavir score (P=0.002, OR -336; 95% CI -535;-138), ABCB1 2677 (P=0.020, OR 497; 95% CI 86; 910), ABCB11 1131 (P=0.002, OR 641; 95% CI 259;1023) and CNT2 -146 (P=0.006, OR -426; 95% CI -721;-132) were able to predict telaprevir plasma levels in the regression analysis. Other SNPs showed no association. This study reveals BSEP implication in telaprevir transport and confirms the involvement and influence of P-glycoprotein on telaprevir plasma levels. To date, no similar data concerning pharmacogenetics and pharmacokinetics were published, but further studies in different and bigger cohorts are needed.

Identification of naïve HVC-4 patients who may be treated with pegylated-interferon and ribavirin according to IL28B polymorphisms

BACKGROUND The current treatment of HCV-4 patients is dual therapy with PEG-IFN and ribavirin; however, new drugs against this genotype will be available within few months. Despite the evidenced good virological response in IFN-free regimens, the high cost of these new therapies will require patient selection. In our paper we propose the use of both rs8099917 and rs12979860 IL28-B polymorphisms, in order to identify potentially categories of SVR, null-responder and relapse and consequently to choose the dual therapy or novel approach. METHODS One hundred and sixty-nine patients with chronic hepatitis C and genotype 4 treated with pegylated interferon and ribavirin for 48weeks were retrospectively studied. All patients were genotyped for rs8099917 and rs12979860 interleukin-28B polymorphisms. RESULTS 80 patients with SVR (88.8%) had the TT/CC or TT/TC (rs8099917/rs12979860) (p<0.001) genotypes; the null-responders (n=13), 9 (69.2%) showed the GG/TT allelic distribution (p<0.001); relapsers showed a prevalent distribution of the TG/TC genotype (83.3%) (p<0.001). The 6 (100%) breakthrough patients showed TT/TC genotype, while the partial responders patients did not show any particular IL-28B genetic profile. Genetic profiles different from TT/CC showed 94.9% negative predictive value for SVR, with 92.6% of sensitivity and 65.2% of specificity. Insulin-resistance, diabetes and liver fibrosis were not relevant in our multivariate analysis. CONCLUSIONS The combination of both rs8099917/rs12979860 polymorphisms is useful for early identification of SVR, null-responders and relapsers. This could be used to chose between standard dual therapy or novel approach with IFN-free regimens.

Ethambutol plasma and intracellular pharmacokinetics: A pharmacogenetic study

We evaluated ethambutol plasma and intracellular pharmacokinetic according to single nucleotide polymorphisms in ABCB1, OATP1B1, PXR, VDR, CYP24A1 and CYP27B1 genes. Mycobacterium tubercolosis infected patients were enrolled. Standard weight-adjusted antitubercular treatment was administered intravenously for 2 weeks and then orally. Allelic discrimination was performed by real-time PCR. Ethambutol plasma and intracellular concentrations were measured by UPLC-MS/MS methods. Twenty-four patients were included. Considering weeks 2 and 4, median plasma Ctrough were 73 ng/mL and 247 ng/mL, intracellular Ctrough were 16,863 ng/mL and 13,535 ng/mL, plasma Cmax were 5627 ng/mL and 2229 ng/mL, intracellular Cmax were 133,830 ng/mL and 78,544 ng/mL. At week 2, ABCB1 3435 CT/TT (p=0.023) and CYP24A1 8620 AG/GG (p=0.030) genotypes for plasma Ctrough, BsmI AA (p=0.036) for intracellular Ctrough and BsmI AA (p<0.001) and ApaI AA (p=0.048) for intracellular Cmax, remained in linear regression analysis as predictive factors. Concerning week 4 only ABCB1 3435 CT/TT (p=0.035) and Cdx2 AG/GG (p=0.004) genotypes for plasma Ctrough and BsmI AA (p=0.028) for plasma Cmax were retained in final regression model. We reveal, for the first time, the possible role of single nucleotide polymorphisms on ethambutol plasma and intracellular concentrations; this may further the potential use of pharmacogenetic for tailoring antitubercular treatment.