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.

Androgenic-anabolic steroid (AAS) misuse has been associated with depression. It has been proposed that stress has a role in depression and that serotonin is involved in both endocrine responses to stress and depressive physiopathology. Although reports demonstrate that AAS chronic administration modifies components of stress-responsive hypothalamic-pituitary-adrenal axis (HPAA), no study has evaluated AAS effect on the response to stressful stimuli. We studied the effects of the subchronic administration (once a day for 14 days in rats) of a supratherapeutical dose of nandrolone decanoate (ND) on HPAA and cortical serotoninergic system response to acute restraint stress (RS). Acute RS produced the following effects: increase in CORT (in blood) and ACTH (both in blood and in pituitary corticotropes), GR depletion in hippocampus and hypothalamus cytosol and GR translocation in hippocampus nuclear fraction, cortical serotonin re-uptake stimulation and hippocampus cytosolic ERK2 activation. ND by itself, i.e. in non-stressed rats, did not modify these parameters, except for a decrease of plasma CORT and ACTH levels and an increase in hippocampus cytosolic phospho-ERK1/2. On the contrary, in stressed rats ND affected stress-induced plasma ACTH increase and prevented all other above reported stress effects, except the increase in pituitary ACTH positive cell density.Our results show that the prolonged administration of a supratherapeutical dose of ND in rats, albeit did not affect in a notable way HPAA and serotonin transporter activity in the absence of stress, may deregulate the stress-induced hormonal cascade which plays a crucial role in depressive psychopathology. (C) 2011 Elsevier Ltd. All rights reserved.

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.

Fracture consolidation is a crucial goal to achieve as early as possible, but pharmacological stimulation has been neglected so far. Teriparatide has been considered for this purpose for its anabolic properties. We set up a murine model of closed tibial fracture on which different doses of teriparatide were tested. Closed fracture treatment avoids any bias introduced by surgical manipulations. Teriparatide's effect on callus formation was monitored during the first 4 weeks from fracture. Callus evolution was determined by histomorphometric and microhardness assessment. Daily administration of 40 μg/kg of teriparatide accelerated callus mineralization from day 9 onward without significant increase of sizes, and at day 15 the microhardness properties of treated callus were similar to those of bone tissue. Teriparatide considerably improved callus consolidation in the very early phases of bone healing.

Here we describe our 5 years' experience on the use of oral hydroquinidine (HQ) in a newborn with familiar short QT syndrome (SQTS). In July 2005 a female child with a history of SQTS in her family was born in Turin, Italy. She was the first-born daughter of a patient with SQTS and history of syncope during exertion and paroxysmal atrial fibrillation since 20 years of age, belonging to a family with many cases of sudden death in four generations and also in the newborn [1]. The family was genotyped and a mis-sense mutation causing the substitution of asparagine for a positively charged lysine at codon 588 (N588K) in the S5-loop region of the cardiac KV11.1* channel KCNH2 (HERG) was found [2]. The effect of the mutation is to increase the repolarizing currents active during the early phase of the action potential, leading to abbreviation of the action potential and thus to abbreviation of the QT interval. Moreover it reduces the affinity of the channel for drugs with KIR blocking action, such as sotalol, but to a lesser degree for hydroquinidine [3, 4]. Twelve-lead standard electrocardiogram (ECG) recordings were obtained at birth and were consistent with the diagnosis (QTc 310 ms). During the first 2 weeks of life ECG recordings was repeated every 2 days. After discharge from hospital, ECG was repeated at each ambulatory follow-up (Figure 1). Periodically ambulatory ECG monitoring (24 h Holter) was also performed. Representative twelve-lead ECGs at birth (A), at 1 year of age (B) and at 5 years (C). QTc at birth 310 ms (A), at 1 year 390 ms (B), at 5 years 400 ms (C), paper speed 25 mm s−1 She was recognized as being affected by the N588K mutation in KCNH2 (HERG), already identified in the father and in other family members. Anti-arrhythmic prophylaxis with oral HQ was started at 9 days of age. Drug dosage was increased every week, monitoring ECG and plasma concentration of basal HQ (quantified by HPLC-UV). The target was a QTc interval at ECG ≥ 360 ms and HQ basal plasma concentration between 0.6 and 2.0 µg ml−1 (therapeutic range referred from literature). Initial oral drug dosage was 4 mg kg−1 three times daily, while maximum dosage administered to maintain HQ in therapeutic range was 10.9 mg kg−1 three times daily. The mean HQ plasma concentration achieved was 0.66 ± 0.23 µg ml−1 (range 0.27–1.14). HQ dosage kg–1, QTc and HQ plasma concentrations are summarized in Table 1. A significant correlation between HQ plasma concentrations and prolongation of QT interval was observed. No correlation was found between HQ plasma concentrations and drug dosage. No cardiac symptoms or major side effect were observed during a follow-up of 5 years. Only transient abdominal pain was observed for a few days after every increase of drug dosage. During periodical Holter monitoring no arrhythmic events were recorded. SQTS is a rare, recently recognized genetic anomaly [5], characterized by a typical electrocardiogram (ECG) pattern and the risk of major, sometimes lethal, arrhythmias. The ECG shows a short QTc interval (less than 320 ms), with lack of adaptation during increase of heart rate. Arrhythmias associated with SQTS are atrial fibrillation and ventricular tachyarrhythmias. Sudden cardiac death occurs in adult patients and also in infancy, so SQTS is a potential cause of sudden infant death syndrome. To date less then 80 patients with SQTS are described in the literature and most of them are familiar forms. In 2000 a family (a 17-year-old girl with several episodes of paroxysmal atrial fibrillation, her brother and their mother) with QT and QTc intervals <300 ms was described [5]. In 2003 the short QT syndrome was recognized as a new clinical entity related to familial sudden death [1]. Cardiac arrest is the most frequent clinical presentation. SQTS was soon recognized as a genetic disorder with autosomal dominant inheritance. Gain of function mutations in three different genes (KCNH2, KCNQ1, KCNJ2) encoding potassium channels and loss of function mutations in two genes encoding the CaV1.2* calcium channel (CACNA1C and CACNB2b) have been linked with SQTS [6, 7]. Because of the high incidence of sudden death, the first choice therapy in adult patients with SQTS is an implantable cardioverter-defibrillator device (ICD) [8]. In paediatric patients there are technical problems and risk of complications linked to an ICD implant: in small babies an ICD implant is not feasible, so pharmacological prophylaxis is the only alternative. Medical treatment proposed for SQTS is oral HQ, the only anti-arrhythmic drug able to normalize the QT interval at resting heart rates [3, 4, 9]. HQ therapy may induce many different adverse effects both cardiac and not. Thus, monitoring of its plasma concentrations in treated patients may be useful to avoid toxicity. Up to now data about therapeutic HQ monitoring in patients are rarely available in literature, even less about paediatric patients. The case of this infant well explains why therapeutic drug monitoring is considered an important tool in clinical practice in different disciplines. In newborn babies is often difficult to obtain an ECG with a heart rate less than 100 beats min−1 and the correct QT evaluation is not always reliable, so it seems to be strategical to avoid an erroneous HQ dosage by monitoring drug plasma concentration. An increase in drug biotransformation ability reached by liver during the first months of life is probably responsible for the need of greater doses of HQ for maintaining therapeutic plasma concentrations. There are no competing interests to declare.

PURPOSE:Differentiation-inducing factor-1 (DIF-1) is a morphogen originally identified in the amoebozoan Dictyostelium discoideum. In mammalian cells, it has been shown to activate GSK3β, which in turn is expected to reduce levels of β-catenin and cyclin D1, thus mediating DIF-1 antiproliferative properties. Since this could alter the expression and activity of E2F1 transcription factor and consequently those of the prognostic marker/chemotherapy target thymidylate synthase (TS), we evaluated (1) whether DIF-1 could effectively regulate these genes, (2) whether it could interfere with cell viability, and (3) whether DIF-1 activity could enhance the efficacy of the TS inhibitor 5-fluorouracil (5-FU).METHODS:We investigated the effects of DIF-1 in continuous human cell lines derived from two oral tumor histotypes (corresponding to an adenosquamous and a squamous carcinoma) and a gingival epithelium. We evaluated mRNA accumulation by means of quantitative real-time PCR and efficacy of drugs on cell viability by means of MTT assay.RESULTS:DIF-1 inhibited the accumulation of E2F1 mRNA and reduces TS mRNA levels in tumor cell lines, but did not alter mRNA levels in the gingival counterpart. As a result, it inhibited proliferation preferentially of tumor cell in time- and concentration-dependent manner. Moreover, it enhanced cytotoxic effects of 5-FU only in tumor cell, whereas reduced them in the gingival counterpart.CONCLUSIONS:These findings suggest a tumor-specific action of DIF-1 on oral carcinoma cells. Thus, interfering with E2F1 and TS transcription, DIF-1 potentiates TS enzymatic inhibitors.

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.

Silicon carbide quantum dots are highly luminescent biocompatible nanoparticles whose properties might be of particular interest for biomedical applications. In this study we investigated Silicon Carbide Quantum Dots (3C-SiC QDs) cellular localisation and influence on viability and proliferation on oral squamous carcinoma (AT-84 and HSC) and immortalized cell lines (S-G). They clearly localize into the nuclei, but the presence of 3C-SiC QDs in culture medium provoke morphological changes in cultured cells. We demonstrate that 3C-SiC QDs display dose- and time-dependent selective cytotoxicity on cancer versus immortalized cells in vitro. Since one of the limitations of classical antineoplastic drugs is their lack of selectivity, these results open a new way in the search for antiproliferative drugs.

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.

Oral squamous cell carcinoma (OSCC) develops slowly and it is usually preceded by identifiable oral preneoplastic lesions (OPLs): chemoprevention could be a promising approach. Resveratrol (RV) is a plant-based agent characterized by a strong in vitro antineoplastic action, but this effect has not been clinically confirmed owing to its metabolic inactivation. In order to circumvent this limitation and to improve RV efficacy, it was locally applied and complexed with a protective and solubilising vehicle (2-hydroxypropyl-β-cyclodextrin, HPβCD). The experimentation was performed in vitro on 7,12-dimethylbenz[a]anthracene-induced hamster OSCC cell line (HCPC I) and in vivo in the related animal model, by comparison of two RV–HPβCD formulations (cream and mouthwash) and RV alone. Vehicles and RV-formulations were free from toxicity. Antiproliferative action of RV on HCPC I was concentration- and time-dependent, and was improved in HPβCD-formulations. In vivo, RV prevented OPL and OSCC appearance and growth. Here, too, HPβCD-formulations (mainly mouthwash) demonstrated the best chemopreventive effects in terms of lesions prevalence, multiplicity, dimension, and histological signs of malignancy. HPLC detection of RV corroborated that its action is concentration-correlated and is improved by its inclusion in HPβCDs. In summary, our study demonstrates that RV is effective in the chemoprevention of DMBA-induced oral carcinogenesis and when it is complexed with HPβCDs its efficacy is significantly improved.