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Carmen Fucile,1 Francesca Mattioli,1 Valeria Marini,1 Massimo Gregori,2 Aurelio Sonzogni,3 Antonietta Martelli,1 Natalia Maximova4 1Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy; 2Department of Pediatric Radiology, Institute for Maternal and Child Health – IRCCS Burlo Garofalo, Trieste, Italy; 3Department of Pathology, Ospedale Beato Papa Giovanni XIII, Bergamo, Italy; 4Bone Marrow Transplant Unit, Institute for Maternal and Child Health – IRCCS Burlo Garofalo, Trieste, Italy Abstract: To date, in pediatric field, various hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Iron overload and systemic siderosis often occur in this particular cohort of patients and are associated with poor prognosis. We describe herein the case of two allo-HSCT patients, on treatment with deferasirox; they showed histopathological elements compatible with venoocclusive disease or vanishing bile duct syndrome in ductopenic evolution before deferasirox started. The first patient developed drug-induced liver damage with metabolic acidosis and the second one a liver impairment with Fanconi syndrome. After withdrawing deferasirox treatment, both patients showed improvement. Measurements of drug plasma concentrations were performed by HPLC assay. The reduction and consequent disappearance of symptoms after the suspension of deferasirox substantiate its role in inducing hepatic damage, probably enabling the diagnosis of drug-induced liver damage. But the difficulties in diagnosing drug-related toxicity must be underlined, especially in compromised subjects. For these reasons, in patients requiring iron-chelating therapy, close and careful drug therapeutic monitoring is strongly recommended. Keywords: deferasirox, allogeneic hematopoietic stem cell transplantation, allo-HSCT, pediatric, ductopenia, therapeutic drug monitoring, adverse events

Background The thiopurine S-methyltransferase (TPMT)/azathioprine (AZA) gene-drug pair is one of the most well-known pharmacogenetic markers. Despite this, few studies investigated the implementation of TPMT testing and the combined evaluation of genotype and phenotype in multidisciplinary clinical settings where patients are undergoing chronic therapy with AZA. Methods A total of 356 AZA-treated patients for chronic autoimmune diseases were enrolled. DNA was isolated from whole blood and the samples were analyzed for the c.460G>A and c.719A>G variants by the restriction fragment length polymorphism (RFLP) technique and sequenced for the c.238G>C variant. The TPMT enzyme activity was determined in erythrocytes by a high-performance liquid chromatography (HPLC) assay. Results All the patients enrolled were genotyped while the TPMT enzyme activity was assessed in 41 patients. Clinical information was available on 181 patients. We found no significant difference in the odds of having adverse drug reactions (ADRs) in wild-type patients and variant allele carriers, but the latter had an extra risk of experiencing hematologically adverse events. The enzyme activity was significantly associated to genotype. Conclusions TPMT variant allele carriers have an extra risk of experiencing hematologically adverse events compared to wild-type patients. Interestingly, only two out of 30 (6.6%) patients had discordant results between genotype, phenotype and onset of ADRs.

Metformin is an antidiabetic drug which possesses antiproliferative activity in cancer cells when administered at high doses, due to its unfavorable pharmacokinetics. The aim of this work was to develop a pharmacological tool for the release of metformin in proximity of the tumor, allowing high local concentrations, and to demonstrate the in vivo antitumor efficacy after a prolonged metformin exposition. A 1.2% w/w metformin thermoresponsive parenteral formulation based on poloxamers P407 and P124, injectable at room temperature and undergoing a sol-gel transition at body temperature, has been developed and optimized for rheological, thermal and release control properties; the formulation is easily scalable, and proved to be stable during a 1-month storage at 5 °C. Using NOD/SCID mice pseudo-orthotopically grafted with MDA-MB-231/luc+ human breast cancer cells, we report that multiple administrations of 100 mg of the optimized metformin formulation close to the tumor site cause tissue accumulation of the drug at levels significantly higher than those observed in plasma, and enough to exert antiproliferative and pro-apoptotic activities. Our results demonstrate that this formulation is endowed with good stability, tolerability, thermal and rheological properties, representing a novel tool to be pursued in further investigations for adjuvant cancer treatment.

Aim: Monitoring of blood levels of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) is necessary for optimization of administration of medical cannabis. We describe the validation of a ultra-HPLC–MS/MS method for quantifying THC and CBD from plasma and decoctions and its application for therapeutic drug monitoring.Materials & methods: Analyses were performed by using a TSQ Quantiva™ Triple Quadrupole coupled to a Ultimate 3000 UHPLC system with atmospheric pressure chemical ionization after sample preparation with a straightforward method with deuterated internal standards. Results: The method has been validated following EMA guidelines and is linear in plasma from 0.16 to 10 ng/ml for both THC and CBD and in decoctions from 4.7 to 600 ng/ml. Conclusion: Given the unpredictable pharmacokinetic behavior of THC and CBD in patients, monitoring of plasma concentrations is strongly recommended for patients under treatment with medical cannabis.

The objective of this study was to assess the achievement of pharmacokinetic/pharmacodynamic (PK/PD) targets of meropenem (MEM) in critically-ill patients with bloodstream infections (BSI) due to Klebsiella pneumoniae-carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) with MEM minimum inhibitory concentrations (MICs) ≥16 mg/L. Nineteen critically-ill patients with KPC-Kp BSI were given combination therapy including MEM, tigecycline, plus colistin or gentamicin (according to susceptibility testing). MEM was administered as an extended 3-hour infusion of 2 g every 8 hours, or adjusted according to renal function. MEM plasma concentrations were determined by high-performance liquid chromatography. PK/PD targets for MEM were defined as T > 40% 1×MIC and T > 40% 4×MIC. Possible synergisms between MEM and coadministered agents were assessed by time-kill assays based on plasma levels for MEM and on fixed plasma concentrations for the other agents. In none of 19 patients MEM reached any PK/PD target. The actual MEM MICs were 256, 512, and 1024 mg/L in 1, 3, and 15 isolates, respectively. However, theoretically, the PK/PD target of T > 40% 1×MIC could have been achieved in 95%, 68%, 32% and 0% of the isolates for MIC equal to 8, 16, 32, and 64 mg/L, respectively. No synergisms were observed between MEM and coadministered agents. In conclusion, high-dose MEM failed to reach PK/PD targets in 19 patients with BSI due to KPC-Kp with very high MEM MICs. On a theoretical basis, our results suggest a possible usefulness of MEM against resistant blood isolates with MICs up to 32 mg/L.

The purpose of this study was to evaluate the aqueous humor concentrations of bromfenac ophthalmic solution 0.09 % in patients undergoing phacoemulsification. Patients requiring cataract extraction received one drop (50 µL) of bromfenac 0.09 % solution in the eye to be operated, before bedtime the day before surgery or the morning of the surgery. The last administration was recorded. At the time of paracentesis, an aqueous humor sample was collected with a 30-gauge needle attached to a TB syringe and was later analyzed by high-performance liquid chromatography for drug concentration. 188 treated volunteers and 48 control, untreated, subjects were included in the study. The mean aqueous concentration of bromfenac in the treated group was 37.60 ± 68.86 and 0 nM (nmol/L) in the control group (p

// Natalia Maximova 1 , Massimo Gregori 2 , Roberto Simeone 3 , Aurelio Sonzogni 4 , Giulia Boz 5 , Carmen Fucile 6 , Valeria Marini 6 , Antonietta Martelli 6 and Francesca Mattioli 6 1 Bone Marrow Transplant Unit, Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy 2 Department of Pediatric Radiology, Institute for Maternal and Child Health - IRCCS Burlo Garofolo, Trieste, Italy 3 University of Trieste, Trieste, Italy 4 Department of Pathology, Ospedale Beato Papa Giovanni XIII, Bergamo, Italy 5 University of Cagliari, Faculty of Medicine, Cagliari, Italy 6 Pharmacology and Toxicology Unit, University of Genoa, Genoa, Italy Correspondence to: Maximova Natalia, email: natalia.maximova@burlo.trieste.it Keywords: deferasirox, allogeneic hematopoietic stem cell transplantation (allo-HSCT), pediatric, ductopenia, therapeutic drug monitoring Received: April 05, 2017 Accepted: May 23, 2017 Published: June 28, 2017 ABSTRACT 42 pediatric patients with iron overload, who underwent liver biopsy and DFX treatment after hematopoietic stem cell transplantation were included in the study group. The patients were divided into two groups diversified according to deferasirox trough plasma concentrations (DFX C trough ) with cut-off equal to10 mcg/mL. The average dose of DFX was 25.9 mg/kg in the DFX C trough 10 mcg/mL group ( p =0,0003). The mean duration of DFX treatment was 135.7 days in the DFX C trough 10 mcg/mL group ( p 10 mcg/mL group ( p 10 mcg/mL group had ductopenia which was complete in 47.6% of them and severe in 52.4%. All patients with particularly high C trough (> 25 mcg/mL) were found to have total ductopenia. 90.5% of all deferasirox-related adverse events and 100% of major adverse events occurred in the DFX C trough > 10 mcg/mL group. In the DFX C trough 10 mcg/mL group. We would recommend a close monitoring in pediatric hematopoietic transplant recipients subjected to deferasirox-based therapy because we have observed a high incidence of adverse events and discontinuation of chelation treatment.

Background The aim of the study was to recreate in-vitro artificial aqueous humor with the same physico-chemical properties of human aqueous humor to be used as a standard matrix in chromatography to assess drug concentration in the anterior and posterior chamber of the human eye. Methods The artificial aqueous humor was prepared according to the human aqueous humor chemical compositions reported in the literature. The artificial matrix was then analysed via the HPLC-UV method and compared with aqueous humor from 15 patients who underwent cataract surgery. Known concentrations of widely-used ophthalmological drugs were added to the artificial aqueous humor in order to assess whether it can be used to explore ocular disposition towards topically or systemically administered drugs. Results No significant differences were found between the two examined aqueous humor types. There were no significant qualitative differences between examined fluids in terms of presence of ophthalmological drugs. Conclusions The composition of artificial, in-vitro recreated aqueous humor was similar to that of the human kind. The absence of significant differences in the analysis of tested drugs both in the artificial and in human aqueous humor indicates that artificial aqueous humor may be used to generate a matrix-based standard curve for analytical method validation.

Introduction The A118G (rs 1799971) polymorphism in the mu-opioid receptor gene (OPRM1) has been reported to be associated with alcohol addiction. Methods In this study 109 patients diagnosed with alcohol dependence in accordance with DMS-IV criteria and 95 healthy subjects were enrolled and everyone has been genotyped. Results The percentage of alcoholic patients with higher than normal gamma-glutamyl transferase (GGT) levels significantly decreased after six months of standard detoxification treatment, both in patients with A/A genotype and in the other ones with A/G genotype. However, the percentage of alcohol dependent patients with the A/A genotype recorded a slight decrease of the GGT and the mean corpuscolar volume of erythrocytes (MCV) combination marker after six months of therapy (30% vs 12%), while subjects with the A/G genotype showed no variation. Conclusion This finding suggests that alcohol dependent patients with the A/A genotype could have a faster restoration of their liver function than those ones with the A/G genotype: it might be possible that the presence of G allele confers on these patients a reduced ability in abstaining from drinking alcohol.

Background Cancer stem cells (CSCs) are considered the cell subpopulation responsible for breast cancer (BC) initiation, growth, and relapse. CSCs are identified as self-renewing and tumor-initiating cells, conferring resistance to chemo- and radio-therapy to several neoplasias. Nowadays, th (about 10mM)e pharmacological targeting of CSCs is considered an ineludible therapeutic goal. The antidiabetic drug metformin was reported to suppress in vitro and in vivo CSC survival in different tumors and, in particular, in BC preclinical models. However, few studies are available on primary CSC cultures derived from human postsurgical BC samples, likely because of the limited amount of tissue available after surgery. In this context, comparative oncology is acquiring a relevant role in cancer research, allowing the analysis of larger samples from spontaneous pet tumors that represent optimal models for human cancer. Methods Isolation of primary canine mammary carcinoma (CMC) cells and enrichment in stem-like cell was carried out from fresh tumor specimens by culturing cells in stem-permissive conditions. Phenotypic and functional characterization of CMC-derived stem cells was performed in vitro, by assessment of self-renewal, long-lasting proliferation, marker expression, and drug sensitivity, and in vivo, by tumorigenicity experiments. Corresponding cultures of differentiated CMC cells were used as internal reference. Metformin efficacy on CMC stem cell viability was analyzed both in vitro and in vivo. Results We identified a subpopulation of CMC cells showing human breast CSC features, including expression of specific markers (i.e. CD44, CXCR4), growth as mammospheres, and tumor-initiation in mice. These cells show resistance to doxorubicin but were highly sensitive to metformin in vitro. Finally, in vivo metformin administration significantly impaired CMC growth in NOD-SCID mice, associated with a significant depletion of CSCs. Conclusions Similarly to the human counterpart, CMCs contain stem-like subpopulations representing, in a comparative oncology context, a valuable translational model for human BC, and, in particular, to predict the efficacy of antitumor drugs. Moreover, metformin represents a potential CSC-selective drug for BC, as effective (neo-)adjuvant therapy to eradicate CSC in mammary carcinomas of humans and animals. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1235-8) contains supplementary material, which is available to authorized users.