The effect of vitamin D pathway genes and deferasirox pharmacogenetics on liver iron in thalassaemia major patients.

Monitoring and treating iron overload is crucial in transfusion-dependent thalassaemia patients. Liver stiffness measurement by transient elastography and T2* magnetic resonance imaging represent non-invasive ways to evaluate the adequacy of the iron chelation treatment. We explored the role of single nucleotide polymorphisms involved in vitamin D metabolism, transport and activity, and in deferasirox metabolism on liver iron burden parameters. One-hundred and five beta-thalassaemia patients, treated with deferasirox, have been enrolled. Drug plasma Ctrough and AUC were measured by a HPLC-UV method. Allelic discrimination was performed by real-time PCR. Age, UGT1A1-364 CT/TT and CYP27B1 -1260 GT/TT positively predicted liver stiffness values. Deferasirox dose and serum ferritin negatively predicted T2* data, whereas age and CYP2D6 1457 GG genotype positively influenced these values. The discoveries of this research may be useful for personalized medicine and the proposed method could be applied in patients with hereditary hemochromatosis and myelodysplastic syndromes.

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

OBJECTIVES: Patients with ß-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 ß-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 ß-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 ß-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.

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 (P < 0.001). Close voriconazole pharmacokinetics monitoring should help individualize antifungal therapy for children.

Clinical relevance of deferasirox trough levels in ß-thalassemia patients.

We evaluated the role of deferasirox therapeutic drug monitoring in order to avoid toxicity or treatment failure. Plasma concentrations, measured between two consecutive liver iron determinations, were determined at the end of dosing interval. Fifty-four ß-thalassemic adult patients were enrolled: 50% were males; median age was 32.3 years (IQR 19.1-41.7 years) and median body mass index was 22.25 kg/m2 (IQR 20.24-23.75 kg/m2 ). The mean deferasirox dose was 28.6 ± 6.3 mg/kg/d and mean plasma concentration was 17.3 ± 16.8 µg/mL. Drug levels showed lower results in males. Deferasirox concentration was significantly correlated with serum creatinine levels (P = .01) and serum ferritin (P < .0001). The assessment of deferasirox therapeutic drug monitoring could help clinicians to predict patient responses and to optimize the therapy.

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, ß -0.161 and interval of confidence at 95%, IC -395.035; -62.383) and gender (ß 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.

Plasma and intracellular imatinib concentrations in patients with chronic myeloid leukemia.

BACKGROUND: Imatinib (Gleevec, STI-571), a 2-phenylaminopyrimidine-type competitive inhibitor of Bcr-Abl kinase, is the current frontline therapy for patients with chronic myeloid leukemia, and it induces durable responses and prolonging event-free and progression-free survival. Monitoring imatinib trough plasma concentration is a simple and rapid way to determine if the drug exposure exceeds the clinical efficacy threshold (1 mcg/mL). Because the target enzyme is located within cells, adequate drug intracellular concentrations are needed to inhibit its function. METHODS: Chromatographic methods were used to quantify imatinib concentrations in both plasma and peripheral blood mononuclear cells collected from adult patients with chronic myeloid leukemia at the Department of Hematology. Samples were collected at steady state, and trough concentrations (24 ± 2 hours after last drug intake) were evaluated. Associations between variables were tested using the Pearson test; results are presented as mean (±SD). RESULTS: Thirty-five samples from 24 patients were collected; patients were mainly men (16, 66.7%), aged 60 years old (±13.1) and with a body mass index of 24.8 (±4.4). A positive and significant correlation (r = 0.203; P = 0.027) was found between imatinib plasma and intracellular concentrations. CONCLUSIONS: The observed correlation between plasma and intracellular imatinib concentrations suggests that they may be used to monitor drug exposure and treatment efficacy.

Influence of the CYP2B6 polymorphism on the pharmacokinetics of mitotane.

OBJECTIVE: The aim of this study was to assess the potential impact of the pharmacogenetic variability of CYP2B6 and ABCB1 genes on the pharmacokinetics of mitotane. METHODS: A retrospective analysis was carried out on 27 patients with adrenocortical carcinoma on postoperative adjunctive mitotane. CYP2B6 and ABCB1 polymorphisms were genotyped and tested for an association with plasma trough concentration after 3, 6, 9, and 12 months of therapy. RESULTS: Patients with the GT/TT genotype had higher mitotane plasma concentrations compared with patients with GG at 3 months (14.80 vs. 8.01 µg/ml; P=0.008) and 6 months (17.70 vs. 9.75 µg/ml; P=0.015). Multivariate logistic regression analysis showed that only the CYP2B6 rs3745274GT/TT genotype (odds ratio=10.7; P=0.017) was a predictor of mitotane plasma concentrations of at least 14 µg/ml after 3 months of treatment. Mitotane concentrations were not influenced by the polymorphisms of the ABCB1 gene. CONCLUSION: Evaluation of the CYP2B6 polymorphism enabled prediction of the individual response to adjuvant mitotane treatment.

Pharmacogenetic of voriconazole antifungal agent in pediatric patients.

AIM: We explored the role of SNPs within the SLCO1B3, SLCO1B1, SLC22A6, ABCB1, ABCG2, SLCO3A1, CYP2C19, ABCC2, SLC22A1, ABCB11 and NR1I2 genes on voriconazole pharmacokinetics. PATIENTS & METHODS: 233 pediatric patients were enrolled. Drug plasma Ctrough was measured by a HPLC-MS method. Allelic discrimination was performed by qualitative real-time PCR. RESULTS: SLCO1B3 rs4149117 c.334 GT/TT (p = 0.046), ABCG2 rs13120400 c.1194 + 928 CC (p = 0.029) and ABCC2 rs717620 c.-24 GA/AA (p = 0.025) genotype groups significantly influenced Ctrough. ethnicity (p = 0.042), sex (p = 0.033), SLCO1B3 rs4149117 c.334 GT/TT (p = 0.041) and ABCB1 rs1045642 c.3435 TT (p = 0.016) have been retained in linear regression model as voriconazole predictor factors. CONCLUSION: Understanding how some gene polymorphisms affect the voriconazole pharmacokinetic is essential to optimally dose this agent.

Deferasirox pharmacokinetic evaluation in ß-thalassaemia paediatric patients.

OBJECTIVES: 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. METHODS: A fully validated chromatographic method was used to quantify deferasirox concentration in plasma collected from paediatric patients with ß-thalassaemia. Samples obtained after 5 days of washout or in naïve patients before and after 2, 4, 6 and 24 h drug administration were evaluated. KEY FINDINGS: Associations between variables were tested using the Pearson test. Twenty paediatric patients were enrolled; they were mainly men (13.65%), with median age of 6.35 years and body mass index of 15.45 kg/m2 . Concerning pharmacokinetic parameters, a higher interindividual variability was shown. A positive, but not significant, correlation (r = 0.363; P = 0.115) was found between deferasirox area under the concentration curve over 24 h (AUC) and drug dose. CONCLUSIONS: Monitoring plasma deferasirox concentrations appears beneficial for guiding appropriate patient treatment, enhancing effectiveness and minimizing toxicity.

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.

Circannual variation of mitotane and its metabolites plasma levels in patients with adrenocortical carcinoma.

OBJECTIVES: Mitotane is the reference drug for the adrenocortical carcinoma treatment; its pharmacological activity seems to depend on drug transformation in two active metabolites: o,p'-DDE (dichlorodiphenylethene) and o,p'-DDA (dichlorodiphenylacetate). Mitotane and metabolites are lipophilic agents; thus, they tend to accumulate into adipose tissues (white and brown), which change their prevalence seasonally. Aim of the work was to evaluate mitotane and metabolites plasma levels variation over the year, in adrenocortical cancer patients treated with Lysodren® for at least 6 months. METHODS: We enrolled a group of 86 adrenocortical carcinoma diagnosed patients, who underwent radical surgery and started mitotane as adjuvant treatment. For drug and metabolites plasma level (from samples collected ~12 h after the dose administration of mitotane, just before the subsequent administration) determination, a validated chromatographic method was used. KEY FINDINGS: Results showed an evidence of a seasonal trend for the three substance (o,p'-DDD, o,p'-DDE and o,p'-DDA) plasma levels, in terms of acrophases and lower values. Furthermore, it came out that male patients need a higher significant mitotane drug dose than female patients to reach mitotane therapeutic window. CONCLUSIONS: In conclusion, this is the first study assessing a mitotane plasma level variation over the year, but further studies in larger cohorts are required.

A new simple HPLC method for measuring mitotane and its two principal metabolites Tests in animals and mitotane-treated patients.

A new C18 reversed-phase column and UV HPLC method for the detection of mitotane, its principal metabolites, dichlorodiphenylacetate and dichlrodiphenylethene, and its precursor DDT is described. In this article mitotane, dichlorodiphenylacetate, and dichlrodiphenylethene concentrations in organs of rats fed on a mitotane diet, and the effects of erythromycin and grapefruit juice as cytochrome P450 common inhibitors are presented. Tissue accumulation of mitotane and dichlrodiphenylethene, the acquired ability to eliminate dichlorodiphenylacetate, and inhibition of beta-hydroxylation by both inhibitors are illustrated here. Blood samples from mitotane-treated patients revealed two correlations: plasma mitotane/dichlrodiphenylethene and plasma mitotane/red cell mitotane.

Deferasirox pharmacokinetic and toxicity correlation in ß-thalassaemia major treatment.

OBJECTIVES: 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. METHODS: A fully validated chromatographic method was used to quantify deferasirox concentration in plasma collected from ß-thalassaemia adult patients. Samples obtained before and after 2, 4, 6 and 24 h drug administration were evaluated. Associations between variables were tested using the Pearson test. KEY FINDINGS: Concerning pharmacokinetic parameters, a higher interindividual variability was shown. A positive correlation was found between deferasirox area under the concentration curve over 24 h and serum creatinine (r = 0.314; P = 0.018) and between area and drug dose (r = 0.311; P = 0.016). Moreover, a negative correlation resulted among area under the concentration curve over 24 h and serum ferritin (r = -0.291; P = 0.026) and among drug half-life and its dose (r = -0.319; P = 0.013). CONCLUSIONS: Treatment decision based on the individual characteristics could strongly contribute to minimize toxicity and increase efficacy of deferasirox therapy.

Deferasirox pharmacokinetics evaluation in a woman with hereditary haemochromatosis and heterozygous ß-thalassaemia.

We present the deferasirox pharmacokinetics evaluation of a female patient on iron chelation, for the interesting findings from her genetic background (hereditary haemochromatosis and heterozygous ß-thalassaemia) and clinical history (ileostomy; iron overload from transfusions). Drug plasma concentrations were measured by an HPLC-UV validated method, before and after ileum resection. Area under deferasirox concentration curve over 24h (AUC) values were determined by the mixed log-linear rule, using Kinetica software. AUC was low also with high deferasirox dose as well as tolerability. Non invasive tissue iron quantification by magnetic resonance imaging or superconducting quantum interference device were prevented by a metal hip replacement. Good efficacy and normalisation of iron markers was obtained on long term. Therapeutic drug monitoring in patient in critical conditions may help to understand reasons for non response and set individualised treatment.

Bioequivalence of Branded and Generic Oxaliplatin: From Preclinical Assessment to Clinical Incidence of Hypersensitivity Reactions.

BACKGROUND: Generic anticancer drugs represent an opportunity in terms of cost savings but there are some concerns about their tolerability. The safety profiles of generic versus branded oxaliplatin formulations have never been studied in detail. PATIENTS AND METHODS: We tested in vitro concentrations, stability and efficacy of branded versus generic oxaliplatin formulations, then we retrospectively collected data about hypersensitivity reactions (HSR) of 427 colorectal cancer patients treated with oxaliplatin-based regimens. RESULTS: No significant difference in oxaliplatin concentration or time-dependent antiproliferative activity between branded and generic oxaliplatin was detected. The incidence of HSR was 12.1% (33/273 patients) in those treated with branded and 9.8% (15/154 patients) in those treated with generic oxaliplatin (p=0.46). The occurrence of grade III-IV HSRs and severe HSRs leading to oxaliplatin discontinuation were comparable. CONCLUSION: No difference between generic and branded formulations of oxaliplatin were demonstrated in preclinical nor in clinical settings. Generic oxaliplatin can be considered a safe alternative to branded formulation.

Salicylate inhibition of rat mammary carcinogenesis and angiogenesis in female rat compatible with misoprostol administration.

Epidemiological data suggest that non-steroidal anti-inflammatory drugs prevent colon cancer. The evidence for other types of tumour is less conclusive, though animal and in vitro studies indicate that they may be effective against mammary cancer cells. We assessed the effect of dietary acetylsalicylic and salicylic acid against dimethylbenzanthracene-induced rat tumours. Tumour angiogenesis was also investigated to explore the mechanism responsible for salicylate effect. Mammary tumours were induced in female Sprague-Dawley rats fed with different amounts of acetylsalicylic and salicylic acid. Serum vascular endothelial growth factor concentrations were measured and vascularization of basement membrane proteins injected in vivo (Matrigel) was determined by evaluation of haemoglobin content to assess the extent to which angiogenesis was inhibited. Dimethylbenzanthracene-induced carcinogenesis was inhibited by both acids and there was a log-dose/response correlation between the tumour diameter and salicylate concentration. Salicylic acid seems more effective than acetylsalicylic acid. Vascular endothelial growth factor was less concentrated in treated animals than in the controls and so was Matrigel haemoglobin. The mechanism involved, however, is still uncertain, though concomitant inhibition of tumour angiogenesis may be an important component. The documented salicylate serum VEGF modulation is interesting also for presence of the flk-1 receptor in mammary tumour cells of our model. Although misoprostol is a prostaglandin analogous its concomitant administration did not compromise the salicylate anti-tumour effect.

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.

A new HPLC-UV validated method for therapeutic drug monitoring of tyrosine kinase inhibitors in leukemic patients.

Development and validation of simple, rapid, and reliable high-performance liquid chromatography (HPLC)-UV method for quantification of major tyrosine kinase inhibitors, imatinib, dasatinib, and nilotinib, in human plasma is presented. Chromatographic separation of the drugs is achieved on an RP-C(18) column at flow rate of 0.9 mL/min at 35°C; eluate is monitored at 267 nm. Mean intra-day and inter-day precision for all compounds are 2.5 and 13.3%; mean accuracy is 13.9%; extraction recovery ranges within 40.24 and 81.81%. Calibration curves range from 10 to 0.005 µg/mL. Limits of detection are 10 ng/mL for imatinib and nilotinib, 50 ng/mL for dasatinib; limits of quantitation are 50 ng/mL for imatinib and nilotinib, 100 ng/mL for dasatinib. Although this method allows the detection of dasatinib, levels found in patients plasma are close to the limit of detection, then below the limit of quantitation. Quantification with HPLC-mass spectrometry, then, is required for dasatinib to give a correct evaluation. In conclusion, the sensitivity of this new method is sufficient to perform therapeutic monitoring and pharmacokinetic studies of imatinib and nilotinib but not dasatinib in CML patients.