Giovanna Fatiguso

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.

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.

Intracellular and Plasma Trough Concentration and Pharmacogenetics of Telaprevir

PURPOSEnTriple therapy for HCV-1 infection consists in boceprevir or telaprevir, ribavirin and PEG-interferon. Telaprevir is a P-glycoprotein substrate and it is metabolized by CYP3A4/5. No data have been published on intracellular penetration of telaprevir. We determined peripheral blood mononuclear cells (PBMCs) and trough plasma S and R telaprevir isomers concentrations; moreover, we evaluated the influence of some single nucleotide polymorphisms (SNPs) on these pharmacokinetic data after 1 month of triple therapy in humans.nnnMETHODSnPlasma and intracellular telaprevir concentrations were determined at the end of dosing interval (Ctrough) using ULPC-MS/MS validated methods; allelic discrimination was performed through real-time PCR.nnnRESULTSnMedian telaprevir Ctrough plasma concentrations were 2579 ng/mL and 2233 ng/mL for the pharmacologically more active S, and R, enantiomers, respectively, with median S/R plasma ratio of 1.11. In PBMC, the medians were 6863 ng/mL and 1096 ng/mL for S and R, respectively, with median S/R being 5.73. The PBMC:plasma ratio for S was 2.59 for R. Plasma ribavirin concentrations were directly correlated with plasma S-telaprevir concentrations. In linear regression analysis, only CYP24A1_rs2585428 SNP (p=0.003) and body mass index (p=0.038) were able to predict S-telaprevir PBMC concentrations.nnnCONCLUSIONSnOur preliminary data could increase the understanding of mechanisms underlying telaprevir intracellular and plasma exposure, suggesting the implementation of pharmacogenetics in these drug kinetic studies.

A simple high performance liquid chromatography–mass spectrometry method for Therapeutic Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples

Graphical abstract Overlaid chromatograms showing the retention times of Fluconazole (FLU), Voriconazole (VRC), Posaconazole (PSC), Isavuconazole (ISC), Itraconazole (ITC) and their corresponding internal standards in a calibration standard sample. Figure. No Caption available. HighlightsSimple HPLC–MS method to quantify 5 antifungal triazoles in human plasma.Fast protein precipitation protocol, ideal for clinical routine use.Full validation following FDA and EMA guidelines.High expected applicability, due to wide use of TDM in antifungal treatments. Abstract Triazoles chanced the prevention and treatment of invasive fungal infections, but their pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad‐spectrum antifungal triazole approved in 2015 as first‐line treatment for intravenous and oral use against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent sub‐therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification methods The aim of this work was the development and full validation of a HPLC‐mass spectrometry assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole and voriconazole in human samples. Both standards and quality controls were prepared in human plasma. After the addition of internal standard (6,7‐dimethyl‐2,3‐di(2‐pyridyl)quinaxoline for voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and isavuconazole), protein precipitation with acetonitrile and dilution with water were performed. Chromatographic separation was performed on Atlantis® T3 5 &mgr;m 4.6 × 150 mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra‐day and inter‐day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable between samples for each analyte. Stability results were in accordance with previously published data. Finally, we tested this method by monitoring plasma concentrations in real patients and using external quality controls with good results. This method resulted very simple, fast, cheap and very useful for TDM application, to improve clinical management of antifungal therapy in critically ill patients.

Therapeutic drug monitoring of voriconazole for treatment and prophylaxis of invasive fungal infection in children

Voriconazole therapeutic drug monitoring is not consistently recommended due to its high interpatient and intrapatient variability. Here, we aimed to describe our experience with voriconazole for treatment and prophylaxis of invasive fungal infections in paediatric patients. A fully validated high-performance liquid chromatography-mass spectrometry method was used to quantify voriconazole concentration in plasma, at the end of dosing interval. A high interindividual variability was shown. We enrolled 237 children, 83 receiving intravenous and 154 oral voriconazole. A positive correlation between drug dose and drug plasma exposure was observed. Considering intravenous route, patients with higher serum creatinine had higher voriconazole concentrations; a positive correlation was found among drug exposure and age. Sex significantly influenced drug levels: males had higher median drug concentrations than females (Pxa0<xa00.001). Close voriconazole pharmacokinetics monitoring should help individualize antifungal therapy for children.

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.

Evaluation of Posaconazole Pharmacokinetics in Adult Patients with Invasive Fungal Infection

Mortality and morbidity due to invasive fungal infections have increased over the years. Posaconazole is a second-generation triazole agent with an extended spectrum of activity, which shows a high interindividual variability in its plasma levels, rendering dosing in many patients inconsistent or inadequate. Hence, posaconazole therapeutic drug monitoring, which is easily available in clinical practice, may improve treatment success and safety. The aim of the study was to describe posaconazole pharmacokinetics, and to evaluate the utility of therapeutic drug monitoring for therapy and prophylaxis in a cohort of adult patients. A fully validated chromatographic method was used to quantify posaconazole concentration in plasma collected from adult patients at the end of the dosing interval. Associations between variables were tested using the Pearson test. The Mann-Whitney test was used to probe the influence of categorical variables on continuous ones. A high inter-individual variability was shown. Of the 172 enrolled patients, among those receiving the drug by the oral route (N = 170), gender significantly influenced drug exposure: males showed greater posaconazole concentration than females (p = 0.028). This study highlights the importance of therapeutic drug monitoring in those with invasive fungal infections and its significant clinical implications; moreover we propose, for the first time, the possible influence of gender on posaconazole exposure.

Effect of ABCC2 and ABCG2 Gene Polymorphisms and CSF-to-Serum Albumin Ratio on Ceftriaxone Plasma and Cerebrospinal Fluid Concentrations

We measured ceftriaxone pharmacokinetics in patients’ plasma and cerebrospinal fluid (CSF) and assessed the influence of biometric, demographic, genetic (ABCB1, ABCC2, ABCB11, ABCG2, and SLCO1A2 polymorphisms) and pathological features. Adult patients with signs and symptoms of central nervous system infections, receiving intravenous ceftriaxone, were enrolled. Ceftriaxone plasma and CSF concentrations were measured by high‐precision liquid chromatographic methods; allelic discrimination was performed by real‐time polymerase chain reaction. Forty‐three patients were included: median ceftriaxone maximal concentration was 15,713 ng/mL in plasma and 3512 ng/mL in CSF with a CSF‐to‐plasma ratio of 0.3. ABCC2 1249 rs2273697 (P = .027) and ABCG2 1194+928 rs13120400 (P = .015) variants were significantly associated with CSF concentrations and CSF‐to‐plasma ratios. At linear regression analysis, CSF‐to‐serum albumin ratio was an independent predictor of ceftriaxone CSF concentrations (P = .001; also in those with intact blood‐brain barrier: P = .031) and CSF‐to‐plasma ratio (P = .001; also in those with blood‐brain barrier impairment: P = .040). We here report the role of transporters’ genetic variants as well as of blood‐brain barrier permeability in predicting ceftriaxone exposure in the central nervous system.

Pharmacogenetic of voriconazole antifungal agent in pediatric patients

AIMnWe explored the role of SNPs within the SLCO1B3, SLCO1B1, SLC22A6, ABCB1, ABCG2, SLCO3A1, CYP2C19, ABCC2, SLC22A1, ABCB11 and NR1I2 genes on voriconazole pharmacokinetics.nnnPATIENTS & METHODSn233 pediatric patients were enrolled. Drug plasma Ctrough was measured by a HPLC-MS method. Allelic discrimination was performed by qualitative real-time PCR.nnnRESULTSnSLCO1B3 rs4149117 c.334 GT/TT (pxa0=xa00.046), ABCG2 rs13120400 c.1194xa0+xa0928 CC (pxa0=xa00.029) and ABCC2 rs717620 c.-24 GA/AA (pxa0=xa00.025) genotype groups significantly influenced Ctrough. ethnicity (pxa0=xa00.042), sex (pxa0=xa00.033), SLCO1B3 rs4149117 c.334 GT/TT (pxa0=xa00.041) and ABCB1 rs1045642 c.3435 TT (pxa0=xa00.016) have been retained in linear regression model as voriconazole predictor factors.nnnCONCLUSIONnUnderstanding how some gene polymorphisms affect the voriconazole pharmacokinetic is essential to optimally dose this agent.