Functional and transcriptional induction of aquaporin-1 gene by hypoxia; analysis of promoter and role of Hif-1α.

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O(2) transport. It has been reported that this protein contributes to gas permeation (CO(2), NO and O(2)) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5' proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl(2)) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Effectiveness of the bevacizumab-irinotecan regimen in the treatment of recurrent glioblastoma multiforme: Comparison with other second-line treatments without this regimen.

A retrospective cohort study was conducted to analyse the effectiveness of bevacizumab and irinotecan (BVZ/CPT-11) as a second-line treatment in patients with primary glioblastoma multiforme (GBM) in comparison with a control group that were not administered BVZ/CPT-11 at the first recurrence. The difference in overall survival (OS) between the two groups was used as a predictor of effectiveness. OS was calculated according to prognostic factors and gender. A total of 28 and 32 patients were enrolled in the BVZ/CPT-11 cohort and control group, respectively. The median OS was 17.94 months (95% CI, 14.91-20.96) in the BVZ/CPT-11 treatment cohort and 10.97 months (95% CI, 7.65-14.30) in the control cohort. The results obtained on the effectiveness of BVZ/CPT-11 treatment in patients with primary GBM are consistent with data from previous studies. No significant differences were identified in OS based on prognostic factors; therefore, the latter cannot be used to select patients who would incur the greatest benefits from BVZ/CPT-11 treatment.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

La notificación on line de reacciones adversas a medicamentos, un avance de la Web del Centro Andaluz de Farmacovigilancia

Boletín semanal para profesionales sanitarios de la Secretaría General de Salud Pública y Participación Social de la Consejería de Salud

Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

BACKGROUND In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates. METHODOLOGY/PRINCIPAL FINDINGS KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p=0.0038; HR: 1.40; 95% CI:1.12-1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p=0.0002; HR: 1.55; 95% CI: 1.23-1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p=0.0054; OR: 1.77; 95% CI: 1.18-2.64). CONCLUSIONS/SIGNIFICANCE This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.

Plerixafor and granulocyte colony-stimulating factor for first-line steady-state autologous peripheral blood stem cell mobilization in lymphoma and multiple myeloma: results of the prospective PREDICT trial.

In Europe, the combination of plerixafor + granulocyte colony-stimulating factor is approved for the mobilization of hematopoietic stem cells for autologous transplantation in patients with lymphoma and myeloma whose cells mobilize poorly. The purpose of this study was to further assess the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization in European patients with lymphoma or myeloma. In this multicenter, open label, single-arm study, patients received granulocyte colony-stimulating factor (10 μg/kg/day) subcutaneously for 4 days; on the evening of day 4 they were given plerixafor (0.24 mg/kg) subcutaneously. Patients underwent apheresis on day 5 after a morning dose of granulocyte colony-stimulating factor. The primary study objective was to confirm the safety of mobilization with plerixafor. Secondary objectives included assessment of efficacy (apheresis yield, time to engraftment). The combination of plerixafor + granulocyte colony-stimulating factor was used to mobilize hematopoietic stem cells in 118 patients (90 with myeloma, 25 with non-Hodgkin's lymphoma, 3 with Hodgkin's disease). Treatment-emergent plerixafor-related adverse events were reported in 24 patients. Most adverse events occurred within 1 hour after injection, were grade 1 or 2 in severity and included gastrointestinal disorders or injection-site reactions. The minimum cell yield (≥ 2 × 10(6) CD34(+) cells/kg) was harvested in 98% of patients with myeloma and in 80% of those with non-Hodgkin's lymphoma in a median of one apheresis. The optimum cell dose (≥ 5 × 10(6) CD34(+) cells/kg for non-Hodgkin's lymphoma or ≥ 6 × 10(6) CD34(+) cells/kg for myeloma) was harvested in 89% of myeloma patients and 48% of non-Hodgkin's lymphoma patients. In this prospective, multicenter European study, mobilization with plerixafor + granulocyte colony-stimulating factor allowed the majority of patients with myeloma or non-Hodgkin's lymphoma to undergo transplantation with minimal toxicity, providing further data supporting the safety and efficacy of plerixafor + granulocyte colony-stimulating factor for front-line mobilization of hematopoietic stem cells in patients with non-Hodgkin's lymphoma or myeloma.

Functional Characterization of a Novel Frameshift Mutation in the C-terminus of the Nav1.5 Channel Underlying a Brugada Syndrome with Variable Expression in a Spanish Family.

INTRODUCTION We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS Treatment: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Study of the effectiveness of first-line treatment in renal cell carcinoma.

The emergence of novel drugs corresponds with the determination of the effectiveness of the current treatments used in clinical practice. A retrospective observational study was conducted to evaluate the effectiveness of first-line treatments and to test the influence of the prognostic factors established using the Memorial Sloan-Kettering Cancer Center (MSKCC) and the analysis of Mekhail's study for two or more metastatic sites. The primary endpoints were median progression-free survival (mPFS) and median overall survival (mOS) times. A total of 65 patients were enrolled and the mPFS and mOS of the patients treated with sunitinib (n=51) were 9.0 and 20.1 months, respectively, and for the patients treated with temsirolimus (n=14) these were 3.0 and 6.2 months, respectively. In the poor-prognosis (PP) group, a difference of 1.2 months (P=0.049) was found in mPFS depending on the first-line treatment. A difference of 4.1 months (P=0.023) was also found in mPFS when classified by histology (clear verses non-clear cell) in the sunitinib-treatment group. When stratified by the prognostic group, differences of >7 months (P<0.001) were found between the groups. Therefore, it was concluded that the effectiveness of the treatments was reduced compared to previous studies and differences were found in the PP group when classified by first-line drug and histology. Additionally, the influence of prognostic factors on OS and the value of stratifying patients using these factors have been confirmed.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS: TREATMENT: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Detection of possible variant forms of hemoglobin in HbA1c measurements by chromatography liquid ion exchange high resolution (HPLC)

Background: The glycosylated hemoglobin (HbA1c) is used to help monitor the degree of a diabetic’s hyperglycemia. Security and accuracy of the methods used in its detection are affected by variants forms of Hb or elevations in levels of Fetal Hb (HbF). These interference are the result of a change in the haemoglobin total net charge of the variant due of a substitution of one amino acid in the remaining amino terminal of the beta chain. International Standardization for HbA1c values (NGSP) not include interference assessment as part of the certification program. Therefore, the effect of each variant or the lifting of the HbF on HbA1c result should be examined in each sample depending on the detected variant and the method used for the detection of the same. The objectives were: to describe the possible variants of Hb and their interference in HbA1c measurement by our method, after the implementation of a computer program for their detection. To identify some variants detected by chromatography liquid ion exchange high resolution (HPLC) with DNA molecular sequencing.