Silvia De Francia

New HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma.

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple protein precipitation extraction procedure was applied on 250 microl of plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase analytical column with 20min of analytical run, at flow rate of 1 ml/min. Mean intra-day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was 1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from 10,000 to 62.5 ng/ml. The limit of quantification was set at 78.1 ng/ml for imatinib and at 62.5 ng/ml for dasatinib and nilotinib. This novel developed methodology allows a specific, sensitive and reliable simultaneous determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in plasma of patients affected by chronic myeloid leukemia.

Prospective evaluation of mitotane toxicity in adrenocortical cancer patients treated adjuvantly

Toxicity of adjuvant mitotane treatment is poorly known; thus, our aim was to assess prospectively the unwanted effects of adjuvant mitotane treatment and correlate the findings with mitotane concentrations. Seventeen consecutive patients who were treated with mitotane after radical resection of adrenocortical cancer (ACC) from 1999 to 2005 underwent physical examination, routine laboratory evaluation, monitoring of mitotane concentrations, and a hormonal work-up at baseline and every 3 months till ACC relapse or study end (December 2007). Mitotane toxicity was graded using NCI CTCAE criteria. All biochemical measurements were performed at our center and plasma mitotane was measured by an in-house HPLC assay. All the patients reached mitotane concentrations >14 mg/l and none of them discontinued definitively mitotane for toxicity; 14 patients maintained consistently elevated mitotane concentrations despite tapering of the drug. Side effects occurred in all patients but were manageable with palliative treatment and adjustment of hormone replacement therapy. Mitotane affected adrenal steroidogenesis with a more remarkable inhibition of cortisol and DHEAS than aldosterone. Mitotane induced either perturbation of thyroid function mimicking central hypothyroidism or, in male patients, inhibition of testosterone secretion. The discrepancy between salivary and serum cortisol, as well as between total and free testosterone, is due to the mitotane-induced increase in hormone-binding proteins which complicates interpretation of hormone measurements. A low-dose monitored regimen of mitotane is tolerable and able to maintain elevated drug concentrations in the long term. Mitotane exerts a complex effect on the endocrine system that may require multiple hormone replacement therapy.

Phase II study of weekly paclitaxel and sorafenib as second/third-line therapy in patients with adrenocortical carcinoma

BACKGROUND There is a strong rationale in the use of antiangiogenic therapy in the management of adrenocortical carcinoma (ACC). Metronomic administration of chemotherapy and antiangiogenic drugs can be synergistic in targeting endothelial cells. OBJECTIVE We assessed the activity of sorafenib plus metronomic paclitaxel as second/third-line therapy in advanced ACC patients. We also tested the activity of sorafenib and paclitaxel against NCI-H295R in vitro. DESIGN Multicenter, prospective phase II trial. Setting Referral centers for ACC. METHODS Twenty-five consecutive metastatic ACC patients who progressed after mitotane plus one or two chemotherapy lines were planned to be enrolled. The patients received a combination of i.v. paclitaxel (60 mg/m(2) every week) and oral sorafenib (400 mg twice a day) till progression. The primary aim was to measure the progression-free survival rate after 4 months and the secondary aims were to assess the objective response rate and toxicity. RESULTS Tumor progression was observed in nine evaluable patients at the first assessment. These results led to the premature interruption of the trial. The treatment was well tolerated. The most relevant toxicities were fatigue, being grade 2 or 3 in four patients, and hypophosphatemia, being grade 3 in three patients. In the in vitro study, sorafenib impaired the viability of H295R cells with dose-response and time-response relationships. The in vitro sorafenib activity was not increased in combination with paclitaxel. CONCLUSIONS Despite the in vitro activity, sorafenib plus weekly paclitaxel is an inactive salvage treatment in patients with advanced ACC and should not be recommended.

HPLC–MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib and nilotinib in human peripheral blood mononuclear cell (PBMC)

A new method using high performance liquid chromatography coupled with electrospray mass spectrometry is described for the quantification of PBMC concentration of tyrosine kinase inhibitors imatinib, dasatinib and nilotinib. A simple PBMC isolation and extraction procedure were applied on 10-14 mL of blood aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was achieved with a gradient (acetonitrile and water+formic acid 0.05%) on a C18 reverse phase analytical column with 25 min of analytical run, at flow rate of 0.25 mL/min. Mean intra- and inter-day precision for all compounds were 8.76 and 12.20%; mean accuracy was -3.86%; extraction recovery ranged within 79 and 91%. Calibration curves ranged from 50.0 to 0.25 ng. The limit of quantification was set at 0.25 ng for all the analyzed drugs. This novel developed methodology allows a specific, sensitive and reliable simultaneous intracellular determination of the three tyrosine kinase inhibitors imatinib, dasatinib and nilotinib in a single chromatographic run, useful for drugs estimation in PBMC of patients affected by chronic myeloid leukemia.

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 &mgr;g/ml; P=0.008) and 6 months (17.70 vs. 9.75 &mgr;g/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.

Practical treatment using mitotane for adrenocortical carcinoma.

Purpose of reviewDescription of novel findings about the mechanism of action of mitotane and its activity as an adjunctive postoperative measure, or for treatment of advanced adrenocortical carcinoma. Recent findingsSeveral in-vitro studies have shown that mitotane suppresses gene transcription of different enzymatic steps of the steroidogenetic pathway. Moreover, mitotane induces CYP3A4 expression, thus accelerating the metabolic clearance of a variety of drugs including steroids. Retrospective studies provided evidence that adjunctive mitotane can prolong recurrence-free survival of treated patients. The concept of a therapeutic window of mitotane plasma concentrations was confirmed also for adjunctive treatment, but the relationship between mitotane concentration and given dose is loose. Genetic variability of the P450-dependent enzymes metabolizing mitotane may explain individual differences. SummaryMitotane concentration of 14–20 mg/l should be reached and maintained during treatment also in an adjunctive setting. In advanced adrenocortical carcinoma, a high-dose starting regimen should be employed when mitotane is used as monotherapy. The combination of mitotane with other drugs should consider the possibility of pharmacologic interactions due to mitotane-induced activation of drug metabolism. This concept applies also to steroid replacement in mitotane-treated patients, who need higher doses to adjust for increased steroid metabolism.

Differential expression of determinants of glucocorticoid sensitivity in androgen-dependent and androgen-independent human prostate cancer cell lines.

Glucocorticoids (GCs) are widely used for the treatment of hormone refractory prostate cancer. However, few data are available on the expression and regulation of glucocorticoid and mineralocorticoid receptors (GR and MR) and 11beta-hydroxysteroid dehydrogenase (11beta-HSD) 1 and -2 activities in prostate cancer cells. Here we show that GR is expressed in both the androgen-independent PC-3 cell line and, at very low levels, in the androgen-dependent LNCaP cells, and MR is expressed in both cell lines. IL-1beta increased GR expression in both cell lines. In LNCaP cells IL-1beta also increased MR expression. Significant 11beta-HSD oxidase activity and 11beta-HSD2 protein were found in LNCaP cells, but not in PC3 cells, and no ketoreductase activity was detected in either cell lines. GR function was assessed by measuring the inhibitory effect of dexamethasone on constitutive and IL-1beta-inducible IL-6 and osteoprotegerin (OPG) production. In PC-3 cells, IL-1beta stimulated IL-6 and OPG release, and dexamethasone dose-dependently inhibited IL-1beta-inducible IL-6 release, and constitutive and IL-1beta-inducible OPG release. In LNCaP cells, IL-1beta stimulated only OPG release. While dexamethasone was ineffective, cortisol dose-dependently inhibited IL-1beta-inducible OPG release. Eplerenone (Epl), a selective mineralocorticoid antagonist, reverted this effect. We conclude that different patterns of expression of receptors and 11beta-HSD activity were associated with different responsiveness to GCs in terms of regulated gene expression. GR and MR expression may vary as a function not only of the malignant phenotype, but also of local conditions such as the degree of inflammation. Inhibition of IL-6 and OPG release by GCs may contribute to the antitumor efficacy in prostate cancer.

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.

The combined low-dose dexamethasone suppression corticotropin releasing-hormone test as a tool to rule out Cushing's syndrome

OBJECTIVE It remains to be evaluated whether the combined low-dose dexamethasone suppression corticotropin-releasing hormone test (LDDST-CRH test) may add to the diagnostic approach of patients suspected to have Cushings syndrome (CS). The aim of the present study was to evaluate whether the LDDST-CRH test may have a place in the diagnostic strategy of CS. DESIGN Prospective evaluation of a consecutive series of patients with suspected CS from 2004 to 2006. METHODS All the subjects underwent the same screening protocol including 1 mg dexamethasone suppression test, 24-h urinary free cortisol (UFC), and midnight serum cortisol, followed by the LDDST-CRH test whose results were not used to establish a definitive diagnosis. Plasma dexamethasone concentration was measured 2 h after the last dose of dexamethasone. Patients qualified for CS when at least two screening tests were positive. RESULTS Sixteen patients had CS while in the remaining 15 subjects CS was excluded. Even if not statistically significant, the sensitivity and the negative predictive value of the cortisol 15 min after CRH were better than the other tests; on the other hand, the test specificity was lower. All of the patients classified as indeterminate were correctly diagnosed by the LDDST-CRH test. Nevertheless, the repeated assessment of the screening tests and the active follow-up gave the same correct results. In all of the patients misclassified by the LDDST-CRH test, the plasma dexamethasone concentrations were in the normal range. CONCLUSIONS Based on our findings, we suggest that the LDDST-CRH test may still find a place as a rule-out procedure in patients who present with indeterminate results after screening and may be unavailable to repeat testing during follow-up.

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.