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.

Agreement between DSM-IV and DSM-5 criteria for alcohol use disorder among outpatients suffering from depressive and anxiety disorders.

BACKGROUND AND OBJECTIVES: Since significant differences have been reported, we estimated agreement between DSM-5 and DSM-IV criteria for alcohol use disorder (AUD). METHODS: We assessed 327 outpatients (mean age: 45.2 ± 13.4) with depressive or anxiety disorders. RESULTS: Absolute differences in prevalence rates between DSM-5 and DSM-IV AUD ranged from -1.1% (subjects with anxiety disorders) to +1.8% (tobacco smokers). The agreement was excellent (k = 0.88), also accounting for specific subgroups (relevant k coefficients >0.80). DISCUSSION AND CONCLUSIONS: DSM-5 criteria did not inflate AUD rates. SCIENTIFIC SIGNIFICANCE: Our results have epidemiological significance since, unlike previous reports, we found diagnostic stability between new and old AUD criteria in this clinical population. (Am J Addict 2017;26:53-56).

Clinical utility of a single-item test for DSM-5 alcohol use disorder among outpatients with anxiety and depressive disorders.

BACKGROUND: There is a lack of studies testing accuracy of fast screening methods for alcohol use disorder in mental health settings. We aimed at estimating clinical utility of a standard single-item test for case finding and screening of DSM-5 alcohol use disorder among individuals suffering from anxiety and mood disorders. METHODS: We recruited adults consecutively referred, in a 12-month period, to an outpatient clinic for anxiety and depressive disorders. We assessed the National Institute on Alcohol Abuse and Alcoholism (NIAAA) single-item test, using the Mini- International Neuropsychiatric Interview (MINI), plus an additional item of Composite International Diagnostic Interview (CIDI) for craving, as reference standard to diagnose a current DSM-5 alcohol use disorder. We estimated sensitivity and specificity of the single-item test, as well as positive and negative Clinical Utility Indexes (CUIs). RESULTS: 242 subjects with anxiety and mood disorders were included. The NIAAA single-item test showed high sensitivity (91.9%) and specificity (91.2%) for DSM-5 alcohol use disorder. The positive CUI was 0.601, whereas the negative one was 0.898, with excellent values also accounting for main individual characteristics (age, gender, diagnosis, psychological distress levels, smoking status). DISCUSSION: Testing for relevant indexes, we found an excellent clinical utility of the NIAAA single-item test for screening true negative cases. Our findings support a routine use of reliable methods for rapid screening in similar mental health settings.

Differentiation-inducing factor-1 enhances 5-fluorouracil action on oral cancer cells inhibiting E2F1 and thymidylate synthase mRNAs accumulation.

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 GSK3ß, which in turn is expected to reduce levels of ß-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.

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.

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.

Preferential killing of cancer cells using silicon carbide quantum dots.

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.

Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch by topical application of resveratrol complexed with 2-hydroxypropyl-beta-cyclodextrin.

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-beta-cyclodextrin, HPbetaCD). 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-HPbetaCD 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 HPbetaCD-formulations. In vivo, RV prevented OPL and OSCC appearance and growth. Here, too, HPbetaCD-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 HPbetaCDs. In summary, our study demonstrates that RV is effective in the chemoprevention of DMBA-induced oral carcinogenesis and when it is complexed with HPbetaCDs its efficacy is significantly improved.

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.

Time-dependent acetylsalicylic acid effects on liver CYP1A and antioxidant enzymes in a rat model of 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis.

7,12-Dimethylbenzanthracene (DMBA) is an abundant environmental contaminant, which undergoes bioactivation, primarily by the CYP1 family, both in liver and extra-hepatic tissues. Dietary acetylsalicylic acid (ASA) has been recently reported to inhibit DMBA-mediated mammary tumour formation in rats. Chemopreventive substances may reduce the risk of developing cancer by decreasing metabolic enzymes responsible for generating reactive species (phase I enzymes) and/or increasing phase II enzymes that can deactivate radicals and electrophiles. To test these hypotheses, Sprague-Dawley female rats were orally administered ASA as lysine acetylsalicylate (50 mg per capita/day for 21 days in water), DMBA (10 mg per capita in olive oil on day 7, 14, and 21), ASA and DMBA in combination, and vehicles only, respectively. Six rats for each group were sacrificed on day 8, 15, and 22. The DMBA-mediated increase in hepatic CYP1A expression and related activities was not significantly affected by ASA, which, conversely, enhanced in a time-dependent manner the liver reduced glutathione content (up to 52%) and the activity of NAD(P)H-quinone oxidoreductase (up to 34%) in DMBA-treated rats. It is proposed that the positive modulation of the hepatic antioxidant systems by ASA may play a role in the chemoprevention of mammary tumourigenesis induced by DMBA in the female rat.

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.

The aspirin metabolite, salicylate, inhibits 7,12-dimethylbenz[a]anthracene-DNA adduct formation in breast cancer cells.

There is evidence that aspirin and other non-steroidal anti-inflammatory drugs may be protective agents against cancer in the gastrointestinal tract. These effects are particularly well documented for the colon and rectum. Some epidemiological and experimental studies have suggested that aspirin could also be a chemopreventive agent against breast cancer. We investigated the effects of the aspirin metabolite, salicylate (SA), on 7,12-dimethylbenz[a]anthracene (DMBA)-DNA adduct formation as well as on the expression of the enzymes involved in the carcinogen bioactivation pathway, in particular cytochrome P450 1A (CYP1A) and cyclooxygenases (COX-1 and COX-2). The effects of the test drug were examined in both the human mammary carcinoma cell line, MCF-7, and mammary cells derived from DMBA-induced rat mammary tumours (RMTCs). In this study, we also reported the effects of SA on cell growth and viability in breast cancer cells (BCCs). The results demonstrated that DMBA-DNA adduct formation in both cancer cell lines was inhibited by SA at concentrations of > or = 2.5 mM. CYP1A was undetectable in RMTCs while CYP1A induction by beta-naphthoflavone in MCF-7 cells was significantly inhibited by SA in a concentration-dependent manner. Aspirin did not affect COX-1 expression in either of the BCCs. COX-2 was not detected in MCF-7 cells, but its expression in RMTCs was inhibited by SA treatment, which also significantly reduced BCC growth, but failed to cause cell death by necrosis or apoptosis. These data suggest that inhibition of DMBA-DNA adduct formation may contribute to aspirin breast cancer chemopreventive action and indicate that this drug can act in the first stage of carcinogenesis.

Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway.

The injury of podocytes is associated with alterations of the glomerular size-selective barrier to proteins. In this study, oxidized LDL (oxLDL) but not native LDL induced apoptosis in human cultured podocytes and reduced Akt activity and P-Akt/Akt ratio. Moreover, oxLDL-induced redistribution and loss of nephrin, an adhesion molecule specific for the glomerular slit diaphragm. Nephrin reduction was preceded by inhibition of nephrin tyrosine phosphorylation and of its association with p85 phosphatidylinositol 3-kinase (PI3K). Moreover, three different statins, mevastatin, pravastatin, and simvastatin, inhibited in a dose-dependent manner apoptosis and loss of nephrin induced by oxLDL by stimulating Akt activity. In addition, simvastatin significantly increased the expression of nephrin protein and mRNA by podocytes. The protective effects of statins were blocked by treatment of podocytes with two unrelated pharmacologic inhibitors of PI3K, LY294002 and wortmannin, suggesting a role for PI3K, and by mevalonate, indicating dependency on HMG-CoA reductase activity. Statins directly stimulated Akt phosphorylation ad activity. Finally, oxLDL induced a retraction of cultured podocytes and an increase in the albumin diffusion across their monolayer that was inhibited by treatment with statins. In conclusion, statins reduced the oxLDL-induced apoptosis and loss of nephrin in glomerular podocytes. The statin-induced Akt activation may protect from the loss of nephrin by an inhibition of its redistribution and shedding and by a stimulation of its synthesis. These data provide a rationale for the anti-proteinuric effect of statins.

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.

Determination of aplidin, a marine-derived anticancer drug, in human plasma, whole blood and urine by liquid chromatography with electrospray ionisation tandem mass spectrometric detection.

A sensitive and highly specific liquid chromatographic method with electrospray ionisation tandem mass spectrometric detection (LC-ESI-MS/MS) is reported for the determination in human plasma, whole blood and urine of Aplidin (APL), a novel depsipeptide derived from the tunicate Aplidium albicans with a potent cytotoxic activity under investigation in clinical studies. Didemnin B was used as internal standard and, after protein precipitation with acetonitrile and liquid-liquid extraction with chloroform, APL was separated by liquid chromatography using a reversed-phase C18 column and a linear gradient of acetonitrile in water (both containing 0.5% formic acid). Detection was performed using a turboionspray source operated in positive ion mode and by multiple reaction monitoring (MRM; m/z 1111 --> 295 for APL and m/z 1113 --> 297 for didemnin B). The method was linear (r > or = 0.9933) over the range 1-250 ng/ml, with intra- and inter-batch precision and accuracy below 12.2% (except at LLOQ < or = 15.4%) for both plasma and urine. Recoveries were moderate, ranging from 54 to 70% in plasma and blood, and from 46 to 60% in urine, for both APL and didemnin B. The LOD was 0.25 ng/ml for both matrices. APL resulted stable in the different matrices at least for 6 h (both at room temperature and 37 degrees C), after freeze and thaw cycles and long term storage at -20 degrees C. The method allowed demonstrating that APL is in a dynamic equilibrium between plasma and blood cells. Moreover, the method was successfully applied to the pharmacokinetic study of Aplidin in cancer patients.

Bifunctional role for VEGF-induced heme oxygenase-1 in vivo: induction of angiogenesis and inhibition of leukocytic infiltration.

Heme-oxygenases (HOs) catalyze the conversion of heme into carbon monoxide and biliverdin. HO-1 is induced during hypoxia, ischemia/reperfusion, and inflammation, providing cytoprotection and inhibiting leukocyte migration to inflammatory sites. Although in vitro studies have suggested an additional role for HO-1 in angiogenesis, the relevance of this in vivo remains unknown. We investigated the involvement of HO-1 in angiogenesis in vitro and in vivo. Vascular endothelial growth factor (VEGF) induced prolonged HO-1 expression and activity in human endothelial cells and HO-1 inhibition abrogated VEGF-driven angiogenesis. Two murine models of angiogenesis were used: (1) angiogenesis initiated by addition of VEGF to Matrigel and (2) a lipopolysaccharide (LPS)-induced model of inflammatory angiogenesis in which angiogenesis is secondary to leukocyte invasion. Pharmacologic inhibition of HO-1 induced marked leukocytic infiltration that enhanced VEGF-induced angiogenesis. However, in the presence of an anti-CD18 monoclonal antibody (mAb) to block leukocyte migration, VEGF-induced angiogenesis was significantly inhibited by HO-1 antagonists. Furthermore, in the LPS-induced model of inflammatory angiogenesis, induction of HO-1 with cobalt protoporphyrin significantly inhibited leukocyte invasion into LPS-conditioned Matrigel and thus prevented the subsequent angiogenesis. We therefore propose that during chronic inflammation HO-1 has 2 roles: first, an anti-inflammatory action inhibiting leukocyte infiltration; and second, promotion of VEGF-driven noninflammatory angiogenesis that facilitates tissue repair.