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Striatal astrocytes produce neuroblasts in an excitotoxic model of Huntington's disease

In the adult brain, subsets of astrocytic cells residing in well-defined neurogenic niches constitutively generate neurons throughout life. Brain lesions can stimulate neurogenesis in otherwise non-neurogenic regions, but whether local astrocytic cells generate neurons in these conditions is unresolved. Here, through genetic and viral lineage tracing in mice, we demonstrate that striatal astrocytes become neurogenic following an acute excitotoxic lesion. Similar to astrocytes of adult germinal niches, these activated parenchymal progenitors express nestin and generate neurons through the formation of transit amplifying progenitors. These results shed new light on the neurogenic potential of the adult brain parenchyma. Highlighted article: Brain lesion in mice reveals a latent neurogenic potential in striatal astrocytes, which can proliferate and generate new neuroblasts.

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.

Cannabinoids concentration variability in cannabis olive oil galenic preparations

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.

A simple high performance liquid chromatography–mass spectrometry method for Therapeutic Drug Monitoring of isavuconazole and four other antifungal drugs in human plasma samples

Graphical abstract Overlaid chromatograms showing the retention times of Fluconazole (FLU), Voriconazole (VRC), Posaconazole (PSC), Isavuconazole (ISC), Itraconazole (ITC) and their corresponding internal standards in a calibration standard sample. Figure. No Caption available. HighlightsSimple HPLC–MS method to quantify 5 antifungal triazoles in human plasma.Fast protein precipitation protocol, ideal for clinical routine use.Full validation following FDA and EMA guidelines.High expected applicability, due to wide use of TDM in antifungal treatments. Abstract Triazoles chanced the prevention and treatment of invasive fungal infections, but their pharmacokinetic properties are still unclear. In particular, isavuconazole (ISC) is a new broad‐spectrum antifungal triazole approved in 2015 as first‐line treatment for intravenous and oral use against invasive aspergillosis and for mucormycosis. Nowadays, the optimal management of the treatments with triazoles requires the use of Therapeutic Drug Monitoring (TDM), in order to prevent sub‐therapeutic or toxic concentrations. In turn, the routine use of TDM requires reliable quantification methods The aim of this work was the development and full validation of a HPLC‐mass spectrometry assay for the simultaneous quantification of fluconazole, itraconazole, isavuconazole, posaconazole and voriconazole in human samples. Both standards and quality controls were prepared in human plasma. After the addition of internal standard (6,7‐dimethyl‐2,3‐di(2‐pyridyl)quinaxoline for voriconazole, posaconazole and itraconazole; stable isotope labeled compounds for fluconazole and isavuconazole), protein precipitation with acetonitrile and dilution with water were performed. Chromatographic separation was performed on Atlantis® T3 5 &mgr;m 4.6 × 150 mm column, with a gradient of water and acetonitrile, both added with 0.05% formic acid. Accuracy, intra‐day and inter‐day imprecision fitted FDA and EMA guidelines, while matrix effects and recoveries resulted stable between samples for each analyte. Stability results were in accordance with previously published data. Finally, we tested this method by monitoring plasma concentrations in real patients and using external quality controls with good results. This method resulted very simple, fast, cheap and very useful for TDM application, to improve clinical management of antifungal therapy in critically ill patients.

UHPLC-MS/MS method with sample dilution to test therapeutic adherence through quantification of ten antihypertensive drugs in urine samples

Graphical abstract Figure. No caption available. HighlightsRobust UHPLC–MS/MS method to quantify a wide panel of antihypertensive drugs.Urine sampling allows immediate and non‐invasive testing.Fast sample preparation, suitable for rapid adherence‐screening purpose.The method has been tested on real‐life specimens with good results. Abstract Nowadays, hypertension represents an important health problem, particularly in developed countries. In some cases the standard therapeutic approaches are not able to reestablish the normal blood pressure values: this condition is called “resistant hypertension”. However, a fraction of cases of resistant hypertension are actually due to poor adherence to the prescribed therapy. Therapeutic Drug Monitoring could represent a direct and useful tool to correctly identify non‐compliant patients. Nevertheless, high throughput methods for the simultaneous monitoring of a wide panel of drugs in the same analysis are lacking and, furthermore, there is not a generally acknowledged “standard” matrix for this test (plasma or urine). In this work, we validated a UHPLC–MS/MS assay to quantify ten among the most used antihypertensive agents in urine samples, covering all the current classes: amlodipine, atenolol, clonidine, chlortalidone, doxazosin, hydrochlorothiazide, nifedipine, olmesartan, ramipril and telmisartan. Both standards and quality controls were prepared in urine matrix. Only 100 &mgr;L of each sample were added with 40 &mgr;L of internal standard and 860 &mgr;L of water:acetonitrile 90:10, acidified with 0.05% formic acid. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 &mgr;m 2.1 × 150 mm 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 resulted reproducible among different urine lots. Method performances were tested on urine samples from hypertensive patients with good results. This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Influence of ABCB11 and HNF4α genes on daclatasvir plasma concentration: preliminary pharmacogenetic data from the Kineti-C study

Background Daclatasvir is an inhibitor of HCV non-structural 5A protein and is a P-glycoprotein substrate. Pharmacogenetics has had a great impact on previous anti-HCV therapies, particularly considering the association of IL-28B polymorphisms with dual therapy outcome. Objectives We investigated the association between daclatasvir plasma concentrations at 2 weeks and 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCB11 and HNF4α). Patients and methods Allelic discrimination was achieved through real-time PCR, whereas plasma concentrations were evaluated through LC-MS/MS. Results Fifty-two patients were analysed, all enrolled in the Kineti-C study. HNF4α 975 C > G polymorphism was found to be associated with the daclatasvir plasma concentrations at 2 weeks (P = 0.009) and 1 month of therapy (P = 0.006). Linear regression analysis suggested that, at 2 weeks of therapy, age, baseline BMI and haematocrit were significant predictors of daclatasvir concentrations, whereas at 1 month of therapy ABCB111131 CC and HNF4α CG/GG genotypes were significant predictors of daclatasvir concentrations. Conclusions These are the first and preliminary results from our clinical study focusing on daclatasvir pharmacogenetics, showing that this approach could have a role in the era of new anti-HCV therapies.

Vitamin D pathway genetic variants are able to influence sofosbuvir and its main metabolite pharmacokinetics in HCV mono-infected patients

Vitamin D levels and genetic variants were associated with drug outcome/toxicity and concentrations. The plasma exposure of GS-331007, the main sofosbuvir metabolite, has been related to SVR. We evaluated the impact of polymorphisms in genes (CYP27B1, CYP24A1, VDBP and VDR) related to vitamin D pathway on sofosbuvir and GS-331007 plasma levels in HCV mono-infected patients at one month of treatment. Polymorphisms were investigated through real-time PCR; drug plasma quantification was performed through a UHPLC-MS/MS method. GS-331007 levels were associated with CYP24A1rs2248359 and VDRCdx2 variants in all the analyzed patients and linear regression analysis showed that sex, body mass index, HCV genotype, baseline estimated glomerular filtration rate, VDRCdx2AG/GG and CYP27B1-1260TT genotypes significantly predict concentrations. We performed sub-analyses considering the HCV genotype and the concomitant drug, identifying polymorphisms associated with GS-331007 concentrations. This is the first study focusing on vitamin D pathway gene variants and DAAs concentrations, but further studies are required.

Pharmacogenetics of the anti-HCV drug sofosbuvir: a preliminary study

Background Sofosbuvir is a potent nucleotide HCV NS5B polymerase inhibitor that is also a P-glycoprotein (encoded by the ABCB1 gene) and breast cancer resistance protein (encoded by the ABCG2 gene) substrate. Concerning previous anti-HCV therapies, pharmacogenetics had a significant impact, particularly considering the association of interleukin28B polymorphisms with dual-therapy (ribavirin + pegylated IFN) outcomes. Objectives In this work, we investigated the association between sofosbuvir and its prevalent metabolite (GS-331007) plasma concentrations at 1 month of therapy and genetic variants (SNPs) in genes encoding transporters and nuclear factors (ABCB1, ABCG2 and HNF4α) related to sofosbuvir transport. Patients and methods Allelic discrimination was performed through real-time PCR, whereas plasma concentrations were evaluated through liquid chromatography. One hundred and thirteen patients were enrolled. Results Sofosbuvir concentrations were below the limit of quantification since the drug was converted into its GS-331007 metabolite. ABCB1 2677 G>T (P = 0.044) and HNF4α 975 C>G (P = 0.049) SNPs were associated with GS-331007 metabolite plasma concentrations. In linear multivariate analysis, liver stiffness, insulin resistance, baseline haemoglobin and haematocrit and SNPs in the ABCB1 gene (3435 CT/TT and 1236 TT genotypes) were significant predictors of GS-331007 concentrations. Furthermore, we performed sub-analyses considering the anti-HCV concomitant drug and HCV genotype, identifying specific polymorphisms associated with GS-331007 plasma concentrations: ABCB1 3435 C>T and HNF4α975 C>G in patients treated with daclatasvir, ABCB1 2677 G>T with ledipasvir and ABCB1 3435 C>T, ABCB1 2677 G>T, ABCG2 421 C>A and ABCG2 1194 + 928 C>A with ribavirin. Conclusions In this study we suggested sofosbuvir GS-331007 metabolite plasma levels were affected by variants in the ABCB1 and HNFα genes.

Effect of ABCC2 and ABCG2 Gene Polymorphisms and CSF-to-Serum Albumin Ratio on Ceftriaxone Plasma and Cerebrospinal Fluid Concentrations

We measured ceftriaxone pharmacokinetics in patients’ plasma and cerebrospinal fluid (CSF) and assessed the influence of biometric, demographic, genetic (ABCB1, ABCC2, ABCB11, ABCG2, and SLCO1A2 polymorphisms) and pathological features. Adult patients with signs and symptoms of central nervous system infections, receiving intravenous ceftriaxone, were enrolled. Ceftriaxone plasma and CSF concentrations were measured by high‐precision liquid chromatographic methods; allelic discrimination was performed by real‐time polymerase chain reaction. Forty‐three patients were included: median ceftriaxone maximal concentration was 15,713 ng/mL in plasma and 3512 ng/mL in CSF with a CSF‐to‐plasma ratio of 0.3. ABCC2 1249 rs2273697 (P = .027) and ABCG2 1194+928 rs13120400 (P = .015) variants were significantly associated with CSF concentrations and CSF‐to‐plasma ratios. At linear regression analysis, CSF‐to‐serum albumin ratio was an independent predictor of ceftriaxone CSF concentrations (P = .001; also in those with intact blood‐brain barrier: P = .031) and CSF‐to‐plasma ratio (P = .001; also in those with blood‐brain barrier impairment: P = .040). We here report the role of transporters’ genetic variants as well as of blood‐brain barrier permeability in predicting ceftriaxone exposure in the central nervous system.

Role of CYP1A1, ABCG2, CYP24A1 and VDR gene polymorphisms on the evaluation of cardiac iron overload in thalassaemia patients

Objectives Iron-burden-induced arrhythmia and heart failure are among the leading causes of morbidity and mortality in &bgr;-thalassaemia major patients. T2* cardiac magnetic resonance remains the only reliable noninvasive method for the heart iron excess assessment. We explored the role of single nucleotide polymorphisms involved in vitamin D metabolism, transport and activity and in deferasirox (DFX) metabolism on cardiac iron burden. Patients and methods One hundred and five &bgr;-thalassaemia patients, treated with DFX, were enrolled in the present study. Drug plasma Ctrough was measured by a high-performance liquid chromatography-ultraviolet method. Allelic discrimination was carried out using the real-time PCR. Results CYP1A1*1189 CC, ABCG2 421 GA, CYP24A1 8620 GG and VDR TaqI CC single nucleotide polymorphisms influenced T2* values. Age, serum ferritin, ABCG2 421 GA, ABCG2 1194 +928 TC/CC, CYP24A1 22776 TT and VDR TaqI TC/CC were retained in linear regression model. Conclusion Our results suggested, for the first time, the role of DFX and vitamin D pharmacogenetics on cardiac iron overload.