Davide Massano

A new HPLC UV validated method for therapeutic monitoring of deferasirox in thalassaemic patients.

We describe a new high performance liquid chromatography coupled with ultraviolet detection method for the quantification of plasma concentration of oral iron chelating agent deferasirox. A simple protein precipitation extraction procedure was applied on 500 μl of plasma aliquots. Chromatographic separation was achieved on a C18 reverse phase column and eluate was monitored at 295 nm, with 8 min of analytical run. This method has been validated following Food and Drug Administration procedures: mean intra and inter day variability was 4.64 and 10.55%; mean accuracy was 6.27%; mean extraction recovery 91.66%. Calibration curves ranged from 0.078125 to 40 μg/ml. Limit of quantification was set at 0.15625 while limit of detection at 0.078125 μg/ml. We applied methodology developed on plasma samples of thalassaemic patients treated with deferasirox, finding correlation between deferasirox plasma concentrations and serum ferritin levels. This methodology allowed a specific, sensitive and reliable determination of deferasirox, that could be useful to perform its therapeutic monitoring and pharmacokinetic studies in patients plasma.

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.

Role of pharmacogenetics on deferasirox AUC and efficacy

AIM We evaluated deferasirox pharmacokinetic according to SNPs in genes involved in its metabolism and elimination. Moreover, we defined a plasma area under the curve cut-off value predicting therapy response. PATIENTS & METHODS Allelic discrimination was performed by real-time PCR. Drug plasma concentrations were measured by a high performance liquid chromatography system coupled with an ultraviolet method. RESULTS Pharmacokinetic parameters were significantly influenced by UGT1A1 rs887829C>T, UGT1A3 rs1983023C>T and rs3806596A>G SNPs. Area under the curve cut-off values of 360 μg/ml/h for efficacy were here defined and 250 μg/ml/h for nonresponse was reported. UGT1A3 rs3806596GG and ABCG2 rs13120400CC genotypes were factors able to predict efficacy, whereas UGT1A3 rs3806596GG was a nonresponse predictor. CONCLUSION These data show how screening patients genetic profile may help clinicians to optimize iron chelation therapy with deferasirox.

Deferasirox AUC efficacy cutoff and role of pharmacogenetics

Deferasirox (DFX) is a once-daily orally administered iron chelator and its pharmacokinetic significantly correlates with therapy outcome [1]. This drug is metabolized in the liver by UDP-glucuronyltransferase (UGT) 1A1 and 1A3, by cytochrome-P450 (CYP) 1A1, 1A2 and 2D6 enzymes, and it is eliminated via the biliary-enteric circulation through multidrug resistance protein 2 (MRP2) [2]. Our previous pharmacogenetic study, based on DFX trough plasma levels, showed that CYP1A1 rs2606345 AA and CYP1A2 rs2470890 TT genotypes have a negative predictive role of therapy efficacy [3]. Our aim was to retrospectively evaluate DFX pharmacokinetic (AUC, area under the curve) according to single nucleotide polymorphisms (SNPs) in genes involved in its metabolism and elimination in a cohort of adult patients. Moreover, we defined plasma AUC cutoff value predicting an adequate response to therapy. We performed amonocentric cohort study inβ-thalassemic patients treated at San Luigi Gonzaga University Hospital (Orbassano, Turin, Italy) between September 2011 and January 2014. Inclusion criteria were transfusional iron overload, age over 18 years old and treated for at least 6 months with a self-reported adherence of 90 %. DFX administered dose range was 20–40 mg/kg/day. Study protocol (BStudio dei determinanti farmacogenetici nella farmacocinetica e nella risposta clinica del deferasirox^, registration number 79/2012) was approved by the local ethics committee. A written informed consent for the study was obtained from each subject. Plasma DFX concentrations were determined from samples obtained after 5 days of washout or in naïve patients before and after 2, 4, 6 and 24 h drug administration. Drug concentrations were evaluated using a high-performance liquid chromatography system coupled with an ultraviolet determination (HLPC-UV) [4]. AUC values were determined by the mixed log-linear rule (Kinetica software, Waltham, MA, USA). Whole blood was used to isolate genomic DNA (MagnaPure Compact, Roche, Monza, Italy). Genotypes were assessed through a real-time polymerase chain reaction allelic discrimination system (LightCycler96, Roche, Monza, Italy). Analysed polymorphisms were UGT1A1 rs887829 C > T, UGT1A3 rs3806596 A > G and rs1983023 C > T, CYP1A1 rs2606345 C > A and rs4646903 T > C, CYP1A2 rs762551 A > C and rs2470890 C > T, CYP2D6 rs1135840 C > G,MRP2 rs2273697 G > A, BCRP1 rs2231142 G > A and rs13120400 T > C. For descriptive statistics, continuous and non-normal variables were summarized as median values and interquartile range (25th–75th percentiles); categorical variables were described as frequency and percentage. All the variables were tested for normality with the Shapiro-Wilk test. The correspondence of each parameter was evaluated with a normal or non-normal distribution, through the Kolmogorov*UNI EN ISO 9001:2008 Certified Laboratory; Certificate No. IT-64386; ** Certification for: BDESIGN, DEVELOPMENTANDAPPLICATION OFDETERMINATIONMETHODS FOR ANTI-INFECTIVE DRUGS. PHARMACOGENETIC ANALYSES.^

Deferasirox pharmacogenetic influence on pharmacokinetic, efficacy and toxicity in a cohort of pediatric patients

AIM We aimed to evaluate the influence of genetic polymorphisms involved in deferasirox metabolism and transport on its pharmacokinetics and treatment toxicity, in a cohort of β-thalassaemic children. PATIENTS & METHODS Drug plasma concentrations were measured by a HPLC-UV method. Allelic discrimination for UGT1A1, UGT1A3, CYP1A1, CYP1A2, CYP2D6, MRP2 and BCRP1 polymorphisms was performed by real-time PCR. RESULTS CYP1A1 rs2606345AA influenced Ctrough (p = 0.001) and t1/2 (p = 0.042), CYP1A1 rs4646903TC/CC (p = 0.005) and BCRP1 rs2231142GA/AA (p = 0.005) influenced Tmax and CYP2D6 rs1135840CG/GG influenced Cmax (p = 0.044). UGT1A1 rs887829TT (p = 0.002) and CYP1A2 rs762551CC (p = 0.019) resulted as predictive factor of ferritin levels and CYP1A1 rs2606345CA/AA (p = 0.021) and CYP1A2 rs762551AC/CC (p = 0.027) of liver iron concentration. CONCLUSION Our data suggest the usefulness of deferasirox pharmacogenetics in pediatric treatment optimization.

Deferasirox pharmacokinetic evaluation in β-thalassaemia paediatric patients.

Iron chelation in the transfusion‐dependent anaemias management is essential to prevent end‐organ damage and to improve survival. Deferasirox is a once‐daily orally active tridentate selective iron chelator which pharmacokinetic disposition could influence treatment efficacy and toxicity. Therapeutic drug monitoring is an important tool for optimizing drug utilization and doses.

Deferasirox pharmacokinetic and toxicity correlation in β-thalassaemia major treatment

Deferasirox adverse effects include the following: gastrointestinal disturbance, mild elevations in serum creatinine levels and intermittent proteinuria; these events are dose‐dependent and reversible with drug discontinuation, but this solution can lead to an inadequate iron chelation. For these reasons, interindividual variability of drug plasma concentration could help the clinical management of deferasirox dosage. We sought to describe deferasirox plasma exposure in a cohort of 60 adult patients.

Role of CYP24A1 , VDR and GC gene polymorphisms on deferasirox pharmacokinetics and clinical outcomes

β-Thalassemia patients develop deficiency in vitamin D absorption and liver hydroxylation, resulting in extremely low calcitriol levels. We explored the role of single-nucleotide polymorphisms (SNPs) involved in vitamin D metabolism, transport and activity on deferasirox pharmacokinetics and outcomes (effectiveness trough levels (Ctrough) and the area under the curve (AUC) cutoffs of 20 μg ml−1 and 360 μg ml−1 h−1, respectively; nonresponse AUC limit of 250 μg ml−1 h−1). Ninety-nine β-thalassemic patients were enrolled. Drug plasma Ctrough and AUC were measured by the high-performance liquid chromatography system coupled with an ultraviolet determination method. Allelic discrimination for VDR, CYP24A1, CYP27B1 and GC gene SNPs was performed by real-time PCR. CYP24A1 22776 TT significantly influenced Cmin and negatively predicted it in regression analysis. CYP24A1 3999 CC was associated with Ctrough and Cmin and was a negative predictor of Tmax, whereas CYP24A1 8620 GG seemed to have a role in Ctrough, AUC, t1/2 and Cmin, and was an AUC negative predictor factor. Considering treatment outcome, Cdx2 and GC 1296 were retained in regression analysis as AUC efficacy cutoff negative predictors.

Effect of pharmacogenetic markers of vitamin D pathway on deferasirox pharmacokinetics in children

Objectives Patients with &bgr;-thalassemia major have extremely low vitamin D levels, owing to reduced intestinal absorption, subicteric tint, and/or iron-induced higher pigmentation. We investigated whether some polymorphisms within the VDR, CYP24A1, CYP27B1, and GC genes could play a role in deferasirox pharmacokinetics in a cohort of pediatric patients. Patients and methods Eighteen children with &bgr;-thalassemia were enrolled. Drug plasma concentrations at the end of dosing interval (Ctrough) and after 0, 2, 4, 6, and 24 h of drug administration were measured by a HPLC-UV method. Allelic discrimination for VDR (TaqI, FokI, BsmI, Cdx2, and ApaI), CYP24A1 (22776, 3999 and 8620), CYP27B1 (2838 and −1260), and GC (1296) single nucleotide polymorphisms was performed by real-time PCR. Results CYP24A1 8620 AG/GG group negatively predicted Ctrough in regression analysis (P=0.012). ApaI AA genotype resulted as a negative predictor of Ctrough (P=0.025) and area under the concentration curve (P=0.007); FoKI CC genotype remained as area under the concentration curve positive predictor (P=0.008) and TC/CC group as half-life (t1/2) (P=0.003) and volume of distribution (Vd) (P=0.011) negative one; TaqI TC/CC was retained as a negative predictor of drug maximum concentration (Cmax) (P=0.004). Moreover, GC 1296 TG/GG seemed able to predict lower time to reach drug maximum concentration (Tmax) (P=0.033). Conclusion Our preliminary experience suggested the potential usefulness of vitamin D pharmacogenetic to better understand deferasirox interindividual variability, also in pediatric patients.

Role of CYP1A1, ABCG2, CYP24A1 and VDR gene polymorphisms on the evaluation of cardiac iron overload in thalassaemia patients

Objectives Iron-burden-induced arrhythmia and heart failure are among the leading causes of morbidity and mortality in &bgr;-thalassaemia major patients. T2* cardiac magnetic resonance remains the only reliable noninvasive method for the heart iron excess assessment. We explored the role of single nucleotide polymorphisms involved in vitamin D metabolism, transport and activity and in deferasirox (DFX) metabolism on cardiac iron burden. Patients and methods One hundred and five &bgr;-thalassaemia patients, treated with DFX, were enrolled in the present study. Drug plasma Ctrough was measured by a high-performance liquid chromatography-ultraviolet method. Allelic discrimination was carried out using the real-time PCR. Results CYP1A1*1189 CC, ABCG2 421 GA, CYP24A1 8620 GG and VDR TaqI CC single nucleotide polymorphisms influenced T2* values. Age, serum ferritin, ABCG2 421 GA, ABCG2 1194 +928 TC/CC, CYP24A1 22776 TT and VDR TaqI TC/CC were retained in linear regression model. Conclusion Our results suggested, for the first time, the role of DFX and vitamin D pharmacogenetics on cardiac iron overload.