Vitamin D-Related Genetics as Predictive Biomarker of Clinical Remission in Adalimumab-Treated Patients Affected by Crohn's Disease: A Pilot Study.

Adalimumab (ADA) is a human anti-tumor necrosis factor (TNF-α) monoclonal antibody used in inflammatory bowel diseases, such as Crohn's disease (CD). Vitamin-D (VD) is important for biological functions, such as the modulation of expression of genes encoding enzymes and transporters involved in drug metabolism and transport. ADA trough levels were associated with VD concentrations in patients with IBD, but no data are present in the literature concerning VD pathway-related gene single-nucleotide polymorphisms (SNPs) in affecting clinical outcomes. For this reason, the aim of this study was to evaluate the ability of VD-related genetics to predict clinical remission at 3 and 12 months in patients affected by CD treated with ADA. Patients affected by CD were included in this study. SNPs in CYP27B1, CYP24A1, GC, and VDR genes were analyzed through real-time PCR. A total of 63 patients were enrolled. Calprotectin, hemoglobin, and C-reactive protein levels were influenced by SNPs in VDR, CYP27B1, and GC genes. After 3 months of therapy, clinical remission was predicted by smoke, systemic steroids, and VDR BsmI, whereas at 12 months by GC 1296AA/AC and VD supplementation. This study reports the association between VD pathway-related genetics and ADA treatment. Further studies are needed to confirm these promising data.

Influence of Vitamin D in Advanced Non-Small Cell Lung Cancer Patients Treated with Nivolumab.

Nivolumab is one of the most commonly used monoclonal antibodies for advanced non-small cell lung cancer treatment, to the extent that the presence of its anti-antibody is considered a negative prognostic factor. Vitamin D (VD) modulates expression of the genes involved in drug metabolism and elimination. Immune system regulation and immunodeficiency is frequent in non-small cell lung cancer patients. To date, no data have been reported about the relationship between nivolumab and VD. The aim of this study was to quantify plasma 25-hydroxyVD (25-VD) and 1,25-VD, nivolumab, and its anti-antibody before starting treatment (baseline) and at 15, 45 and 60 days of therapy. VD-pathway-associated gene single nucleotide polymorphisms (SNPs) were also evaluated. Molecules were quantified through enzyme-linked immunosorbent assay, and SNPs through real-time PCR. Forty-five patients were enrolled. Median nivolumab concentrations were 12.5 ug/mL, 22.3 ug/mL and 27.1 ug/mL at 15, 45 and 60 days respectively. No anti-nivolumab antibodies were found. Correlations were observed between nivolumab concentrations and 25-VD levels. Nivolumab concentrations were affected by VD-pathway-related gene SNPs. VDBP AC/CC genotype and baseline 25-VD < 10 ng/mL predicted a nivolumab concentration cut-off value of <18.7ug/mL at 15 days, which was associated with tumor progression. This is the first study showing VD marker predictors of nivolumab concentrations in a real-life context of non-small cell lung cancer treatment.

Cannabinoids concentration variability in cannabis olive oil galenic preparations.

OBJECTIVES: Knowledge of the exact concentration of active compounds in galenic preparations is crucial to be able to ensure their quality and to properly administer the prescribed dose. Currently, the need for titration of extracts is still debated. Considering this, together with the absence of a standard preparation method, the aim of this study was to evaluate cannabinoids concentrations variability in galenic olive oil extracts, to evaluate the interlot and interlaboratory variability in the extraction yield and in the preparation composition. METHODS: Two hundred and one extracts (123 (61.2%) from Bedrocan® , 54 (26.9%) from Bediol® , 11 (5.5%) from Bedrolite® , and 13 (6.5%) from mixed preparations) were analysed by liquid chromatography coupled with tandem mass spectrometry, quantifying cannabinoids (THC, CBD, THCA, CBDA and CBN) concentrations. KEY FINDINGS: The RSD% of THC and CBD concentrations resulted higher than 50%. Specifically for Bedrocan® , Bediol® , Bedrolite® (5 g/50 ml), these were THC 82%, THC 53% and CBD 91%, THC 58% and CBD 59%, respectively. The median extraction yields were greater than 75% for all preparations. CONCLUSIONS: Our results highlighted a wide variability in THC and CBD concentrations that justify the need for titration and opens further questions about other pharmaceutical preparations without regulatory indication for this procedure.

UHPLC-MS/MS method with protein precipitation extraction for the simultaneous quantification of ten antihypertensive drugs in human plasma from resistant hypertensive patients.

Today the management of resistant hypertension is a critical health problem: the main difficulty on this field is the discrimination of cases of poor therapeutic adherence from cases of real resistance. This gives rise to the need of high throughput and reliable quantification methods for the Therapeutic Drug Monitoring (TDM) of antihypertensive drugs. The aim of this work was the development and validation of a UHPLC-Tandem mass spectrometry assay for this application and its use in plasma from patients with resistant hypertension. The novelty of this method resides in the ability to simultaneously quantify a wide panel of antihypertensive drugs: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Moreover, this method stands out for its simplicity and cheapness, resulting feasible for clinical routine. Both standards and quality controls were prepared in human plasma. After the addition of internal standard, each sample underwent protein precipitation with acetonitrile and was then dried. Extracts were resuspended in water:acetonitrile 90:10 (0.05% formic acid) and then injected into the chromatographic system. Chromatographic separation was performed on an Acquity(®) UPLC HSS T3 1.8µm 2.1×150mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra-day and inter-day precision fitted FDA guidelines for all analytes, while matrix effects and recoveries resulted stable between samples for each analyte. Finally, we tested this method by monitoring plasma concentrations in 22 hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Efavirenz pharmacogenetics in a cohort of Italian patients.

The pharmacogenetics and pharmacokinetics of efavirenz (EFV) have been widely studied, although data in the Italian population are limited. Single nucleotide polymorphisms (SNPs) in the CYP2B6 gene have been associated with increased EFV plasma concentrations and central nervous system toxicity. The aim of this work was to evaluate EFV plasma exposure according to SNPs in genes involved in drug metabolism and elimination in a cohort of Italian HIV-1-positive patients treated with EFV. Plasma samples were used to measure EFV concentrations at 12h after intake (C12) by a validated HPLC/PDA system. Whole blood was used to identify SNPs in ABCB1, MRP2, CYP2B6, CYP2A6, UGT2B7, NR1I2 (PXR), NR1I3 (CAR) and HNF4α by real-time PCR. The association between SNPs and EFV plasma levels was evaluated through non-parametric tests. Among 201 patients, the median EFV C12 was 2618.5ng/mL. No significant associations were found for MRP2, CYP2A6, UGT2B7, PXR and CAR SNPs; conversely, an association of CYP2B6 516G>T, ABCB1 3435C>T and 2677G>T, and HNF4α 975C>G polymorphisms with EFV C12 was observed. In multivariate analysis, only CYP2B6 516 TT and ABCB1 3435 TT genotypes were independently associated with an EFV C12 of >4000ng/mL (toxicity cut-off). This study confirmed the role of CYP2B6 and ABCB1 polymorphisms, showed a relationship with HNF4α, and the lack of association of CYP2A6, UGT2B7, NR1I2 and NR1I3 SNPs on EFV plasma exposure. Data regarding some of the studied SNPs are the first obtained in an Italian cohort of HIV patients and lead to a global vision about EFV pharmacogenetics.

UHPLC-MS/MS method with automated on-line solid phase extraction for the quantification of entecavir in peripheral blood mononuclear cells of HBV+ patients.

To date five nucleoside analogs are used in the treatment of chronic hepatitis B: among these, entecavir is the most used. Nevertheless a few information about its distribution in tissues is currently known. Since the determination of entecavir disposition in the hepatocytes is impracticable because of its invasiveness, the quantification in an "easier-to-obtain" cellular model could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry assay based on an automated on-line SPE, to quantify entecavir concentrations in peripheral blood mononucleated cells (PBMCs), in both its phosphorylated and un-phosphorylated forms. To achieve this, each PBMC isolate was divided in two aliquots, one was treated with acid phosphatase to convert entecavir phosphorylated metabolites into free form, the other one was not-treated. Standards and quality controls were prepared in PBMCs, isolated from healthy donors, and underwent the same process. 20 µL of the resulting solutions were injected in the on-line SPE system. Thymidine was used as internal standard. Calibration curves fitted a linear model for entecavir levels in a range from 0.039 ng to 5 ng (mean r(2)=0.998). Accuracy, intra-day and inter-day precision of the method fitted FDA guidelines recommendations. Moreover, recovery was consistent and matrix effect resulted low and reproducible. We tested this method by monitoring entecavir concentrations in PBMCs from 28HBV mono-infected patients, confirming its reliability and suitability for the evaluation of intracellular entecavir penetration.

Physical and Chemical Stability of Urapidil in 0.9% Sodium Chloride in Elastomeric Infusion Pump.

Urapidil is an antihypertensive agent, usually administered through intravenous bolus injection, slow-intravenous infusion, or continuous-drug infusion by perfusor. Since to date no evidences are available on drug stability in elastomeric pumps, patients have to be hospitalized. The purpose of this study was to validate an ultra-performance liquid chromatographic method to evaluate urapidil stability in an elastomeric infusion pump, in order to allow continuous infusion as home-care treatment. Analyses were conducted by diluting urapidil in an elastomeric pump. Two concentrations were evaluated: 1.6 mg/mL and 3.3 mg/mL. For the analyses, a reverse-phase ultra-performance liquid chromatographic- photodiode array detection instrument was used. Stressed degradation, pH changes, and visual clarity were used as stability indicators up to 10 days after urapidil solution preparation. The drug showed no more than 5% degradation during the test period at room temperature. No pH changes and no evidences of incompatibility were observed. Stress tests resulted in appreciable observation of degradation products. Considering the observed mean values, urapidil hydrochloride in sodium chloride 0.9% in elastomeric infusion pumps is stable for at least 10 days. These results indicate that this treatment could be administered at home for a prolonged duration (at least 7 days) with a satisfactory response.

In Vitro Stabilifighty Evaluation of Different Pharmaceutical Products Containing Meropenem.

BACKGROUND: Meropenem is a beta-lactam antibiotic for treating multidrug-resistant gram-negative bacilli infections. The expiry of the drug's patent (Merrem) allowed the production of generics to be commercialized by a few companies, including Hospira and Hikma. The stability of these medicines after reconstitution as reported on a data sheet report is 6 hours for Merrem and 1 hour for generics. OBJECTIVES: The aim of this work was to evaluate the stability profile of 3 products in 0.9% sodium chloride until 6 hours. METHODS: Six polyolefin bags (2 for each drug, stored in the light and in the dark) were prepared for every test run (n =10) at concentrations of 4 and 10 mg/mL. All solutions were stored at controlled room temperature (25°C ± 3°C) and sampled immediately after preparation and at every hour until 6 hours had passed. The concentrations, pH changes, and the visual clarity were used as stability and compatibility indicators. RESULTS: All 3 drugs retained over 95% of the initial concentration at 3 to 4 hours. At the sixth hour, all the concentrations decayed 8% to 10%. No statistical differences were observed in the percentage deviation values of the stability profile between generics and the branded drug. CONCLUSION: The stability profile of the products in polyolefin bags, at 4 and 10 mg/mL, was superimposable during the period of analysis and seems to show small values of deviation (1%-2%). These data do not affect the pharmacokinetics because these variations could be attributed to the intra- and interindividual variability between patients. The products showed the same stability, and consequently they could be used interchangeably in hospital pharmacy.