Measurement of autophagy flux in the nervous system in vivo.

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.

Pharmacological properties of S1RA, a new sigma-1 receptor antagonist that inhibits neuropathic pain and activity-induced spinal sensitization.

BACKGROUND AND PURPOSE: The sigma-1 (σ(1) ) receptor is a ligand-regulated molecular chaperone that has been involved in pain, but there is limited understanding of the actions associated with its pharmacological modulation. Indeed, the selectivity and pharmacological properties of σ(1) receptor ligands used as pharmacological tools are unclear and the demonstration that σ(1) receptor antagonists have efficacy in reversing central sensitization-related pain sensitivity is still missing. EXPERIMENTAL APPROACH: The pharmacological properties of a novel σ(1) receptor antagonist (S1RA) were first characterized. S1RA was then used to investigate the effect of pharmacological antagonism of σ(1) receptors on in vivo nociception in sensitizing conditions and on in vitro spinal cord sensitization in mice. Drug levels and autoradiographic, ex vivo binding for σ(1) receptor occupancy were measured to substantiate behavioural data. KEY RESULTS: Formalin-induced nociception (both phases), capsaicin-induced mechanical hypersensitivity and sciatic nerve injury-induced mechanical and thermal hypersensitivity were dose-dependently inhibited by systemic administration of S1RA. Occupancy of σ(1) receptors in the CNS was significantly correlated with the antinociceptive effects. No pharmacodynamic tolerance to the antiallodynic and antihyperalgesic effect developed following repeated administration of S1RA to nerve-injured mice. As a mechanistic correlate, electrophysiological recordings demonstrated that pharmacological antagonism of σ(1) receptors attenuated the wind-up responses in spinal cords sensitized by repetitive nociceptive stimulation. CONCLUSIONS AND IMPLICATIONS: These findings contribute to evidence identifying the σ(1) receptor as a modulator of activity-induced spinal sensitization and pain hypersensitivity, and suggest σ(1) receptor antagonists as potential novel treatments for neuropathic pain.

Comparative bioavailability between two Tramadol once-daily oral formulations.

The aim of this study was to compare the pharmacokinetic profile and oral bioavailability of Tramadol Contramid once-daily (o.d.) 200 mg tablets (Labopharm, Canada) with that of Zytram 200 mg tablets (Zambon, Spain), following single-dose administration in 26 healthy volunteers. The study had an open, randomized, crossover design with a 7-day wash-out. Data from 24 subjects were used for the pharmacokinetic (PK) analysis. Racemic tramadol and racemic O-demethyltramadol (M1) were assayed in plasma using a liquid chromatography/tandem mass spectrometry method. Primary PK parameters estimated were AUC(0-t), AUC(0-infinity), C(max), C(24 h), and T(max). Results were compared using an ANOVA, and the residual variability thereby obtained was used to construct the classical 90% confidence intervals. The parametric Schuirmann's test was also performed. T(max) was analyzed by a nonparametric approach. For both racemic tramadol and racemic O-demethyltramadol, the ANOVA showed a statistically significant formulation effect. Significantly higher values were obtained for Tramadol Contramid o.d. for all PK parameters, except for T(1/2). For Tramadol Contramid o.d., mean tramadol plasma levels were maintained at a plateau level above 200 ng/ml from 4 to 16 h after dose, while for the reference formulation, that level was sustained from 4 to only 6 h. Consistent results for both formulations were obtained for the metabolite. At the end of the dosing interval, plasma tramadol and O-demethyltramadol concentrations were 39% and 49% higher, respectively, for Tramadol Contramid o.d. than those for Zytram (p < 0.0001). Tramadol Contramid o.d. could be considered suprabioavailable to Zytram o.d.

Validation of an automated liquid chromatographic method for omeprazole in human plasma using on-line solid-phase extraction.

An automated system using on-line solid-phase extraction and HPLC with UV detection has been validated in order to determine omeprazole in human plasma. The extraction was carried out using C18 cartridges. After washing, omeprazole was eluted from the cartridge with mobile phase onto an Inertsil ODS-2 column. The developed method was selective and linear for drug concentrations ranging between 5 and 500 ng ml(-1). The recovery of omeprazole ranged from 88.1 to 101.5%, and the limit of quantitation (LOQ) was 5 ng ml(-1). The intraday accuracy ranged from 93.1 to 106.2% and the interday accuracy varied from 95.4 to 105.1%. For the LOQ, good values of precision (8.7 and 17.5% for intraday and interday, respectively) were also obtained. This automated system has been applied to determine omeprazole in human plasma samples from bioequivalence studies.

Absorption, distribution and excretion of [14C]-Lesopitron after single and repeated administration in rats and dogs.

Disposition of [14C]-lesopitron was investigated in male rats and dogs after single and repeated oral administration. Radioactivity was rapidly and efficiently absorbed from gastrointestinal tract following oral administration. After 7 days, the radioactivity was mainly excreted into feces via bile. The cumulative urinary and fecal excretion was 99% and 75% of the administered dose in rats and dogs, respectively. [14C]-Lesopitron was widely distributed in rats, with the highest concentrations in liver and kidney, while the concentration in brain was similar to that in plasma. Radioactivity in most tissues decreased essentially in parallel with that in plasma. In rats, plasma levels of [14C]-lesopitron radioactivity achieved steady state on day 2 of repeated administration. The distribution pattern obtained after 7 consecutive daily oral doses was similar to that in the single-dose study. At 72 h after the last administration, tissue radioactivity was fully eliminated and no accumulation occurred. After i.v. administration in rats and dogs, plasma concentrations of lesopitron decreased biphasically with an apparent elimination half-life of 100 min. The absolute bioavailability of lesopitron was about 10%, suggesting an important first-pass effect. In rats, the lesopitron plasma concentrations were similar to those obtained for its metabolites (5-hydroxylesopitron and PmP), whereas in dogs, the PmP plasma concentrations were higher than those for lesopitron and 5-hydroxylesopitron.

Automated high-performance liquid chromatographic assay for lesopitron, a novel anxiolytic, in human plasma using on-line solid-phase extraction.

A totally automated liquid chromatographic assay method based on a Prospekt solid-phase extraction unit was developed for the analysis of lesopitron and its metabolite 5-hydroxylesopitron in human plasma. On-line solid-phase extraction of lesopitron, 5-hydroxylesopitron and its internal standard in human plasma was carried out using C2 cartridges. After washing, the test substances were eluted with mobile phase onto an ODS-2 Inertsil column and measured by fluorescence detection. The total time for one analysis was 25 min. The method developed was selective and linear in the concentration range from 1 to 40 ng/ml for both parent drug and metabolite. Recovery of lesopitron and 5-hydroxylesopitron were higher than 80% and the quantification limits were 1 ng/ml for both compounds. Coefficients of variation obtained for precision parameters were all below 14.5% and 13.9% for parent drug and metabolite, respectively. Good values of accuracy were also obtained.