Augusto Filipe

The effects of chronic stress on hippocampal adult neurogenesis and dendritic plasticity are reversed by selective MAO-A inhibition.

There is accumulating evidence that adult neurogenesis and dendritic plasticity in the hippocampus are neuroplastic phenomena, highly sensitive to the effects of chronic stress and treatment with most classes of antidepressant drugs, being involved in the onset and recovery from depression. However, the effects of antidepressants that act through the selective inhibition of monoamine oxidase subtype A (MAO-A) in these phenomena are still largely unknown. In the present study, adult neurogenesis and neuronal morphology were examined in the hippocampus of rats exposed to chronic mild stress (CMS) and treated with the selective reversible MAO-A inhibitor (RIMA) drug, pirlindole and the selective serotonin reuptake inhibitor (SSRI), fluoxetine. The results provide the first demonstration that selective MAO-A inhibition with pirlindole is able to revert the behavioural effects of stress exposure while promoting hippocampal adult neurogenesis and rescuing the stress-induced dendritic atrophy of granule neurons.

Polyphenols journey through blood-brain barrier towards neuronal protection.

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Comparative bioavailability of two immediate release tablets of enalapril/hydrochlorothiazide in healthy volunteers

SummaryA bioequivalence study of two oral formulations of 20/12.5 mg tablets of enalapril/hydrochlorothiazide was carried out in 20 healthy male volunteers according to a single dose, two-sequence, crossover randomized design. One washout period of nine days was observed between the two periods. Multiple samples were collected over 96 hours post-dosing. Bioavailability was evaluated on the basis of plasma concentrations of enalapril and its main active metabolite, enalaprilat and hydrochlorothiazide. Plasma samples were assayed for enalapril, enalaprilat and hydrochlorothiazide using a selective and sensitive high-performance liquid chromatography method with mass spectrometry detection (LC-MS).The pharmacokinetic parameter values of Cmax and tmax were obtained directly from plasma data, ke was estimated by log-linear regression, and AUC was calculated by trapezoidal rule. Different statistical tests were performed on the basis of untransformed and log-transformed data and the overall residual variance from ANOVA. Assuming the accepted tolerance intervals, a β-error of 20% and 90% confidence intervals (α=0.10), all the generally accepted tests (Schuirmann test and Wilcoxon-Tukey and Hauschke nonparametric tests) showed that the formulations can be considered as bioequivalent with respect to the extent of absorption, given by the AUC0−∞ and with respect to rate of absorption as assessed by Cmax and tmax.

Results of a single-center, single-dose, randomized-sequence, open-label, two-way crossover bioequivalence study of two formulations of valsartan 160-mg tablets in healthy volunteers under fasting conditions

BACKGROUND Valsartan is a nonpeptide, orally active angiotensin II type 1 receptor blocker used to treat hypertension alone or in combination with other antihypertensive agents. OBJECTIVE The aim of this study was to compare the relative bioavailability of a new valsartan 160-mg formulation (ie, test drug) and that of a reference formulation so that bioequivalence could be assessed, as required by European regulatory authorities for the marketing of a generic product. METHODS This was a single-center, single-dose, randomized-sequence, open-label, 2-way crossover study with a minimum washout period of 7 days; drug was administered to healthy volunteers under fasting conditions. Blood samples were collected up to 36 hours postadministration, and valsartan levels were gauged from plasma by reverse liquid chromatography and tandem mass spectrometry detection (ie, the LC-MS/MS method). Pharmacokinetic parameters were calculated from valsartan concentration data using noncompartmental analysis. AUC(last), AUC(infinity), and C(max) were analyzed. The 90% CIs of the ratios of the test-versus-reference pharmacokinetic parameters (AUC(last), AUC(infinity), and C(max)) were obtained by ANOVA on ln-transformed data. The 90% CIs were required to be within 80.00% to 125.00% of the 90% CI to meet the criteria for bioequivalence. Tolerability was monitored using physical examination (including vital-sign measurements) and ECG performed at screening, as well as laboratory analysis, including biochemistry tests, hematology tests, and urinalysis, which were performed at screening and during the study period. RESULTS Thirty-eight white (90.5%), 2 black (4.8%), and 2 Hispanic subjects (4.8%) enrolled in the study; the sample included a total of 27 men and 15 women. The mean (SD) age was 37 (11) years and mean weight was 65.4 (7.6) kg. The 90% CI values for pharmacokinetic measurements were as follows: AUC(last), 94.45% to 118.59%; AUC(infinity), 93.58% to 116.51%; and C(max), 93.61% to 122.02%. Thus, they were all within the predefined 80.00% to 125.00% range. Thirty-six postadministration adverse events were reported; the most common was blood pressure decrease. A decrease of blood pressure was experienced by 6 subjects (14.6%) after the administration of the test formulation, and by 5 subjects (12.5%) after the administration of the reference formulation. Thirty-three of these adverse events were graded as mild and 3 as moderate; 11 were judged as probably related, 12 as possibly related, 3 as remotely related, and 10 as unrelated to the study medication. CONCLUSIONS In this open-label study of healthy volunteers, the test and reference formulations of valsartan 160 mg met the European regulatory definition of bioequivalence, based on the rate and extent of absorption of a single dose under fasting conditions. Both formulations were well tolerated.

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

INTRODUCTION Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. METHODS In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. RESULTS Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. DISCUSSION The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates.

Pirlindole in the Treatment of Depression

AbstractBackground: Depressive disorders are common health problems. Both preclinical and clinical studies have shown that pirlindole, a tetracyclic compound, is suitable for the management of depression; however, a systematic review is needed to accurately select randomized controlled trials (RCTs) for a meta-analysis that will provide more consistent and accurate results regarding the efficacy and tolerability of the drug. Objectives: To evaluate the efficacy and frequency of adverse events with pirlindole in comparison with active comparators (monoamine oxidase inhibitors [MAOIs], tricyclic antidepressants, tetracyclic antidepressants, and selective serotonin reuptake inhibitors [SSRIs]) for the treatment of major depression. Methods: Data were searched through MEDLINE (via PubMed), EMBASE, the Cochrane Central Register of Controlled Trials and a manual search through the sponsor’s available archives (1966 to 30 August 2010). The metaanalysis was performed using the Mantel-Haenszel technique and analysing data through Comprehensive Meta-Analysis software version 1.0.23. Studies were included if they were RCTs evaluating the efficacy and number of reported adverse events with pirlindole in comparison with active comparators for the treatment of major depression in adults. Placebo-controlled trials were excluded to minimize study heterogeneity. Results: This systematic review included ten published articles and one nonpublished report corresponding to a total of 13 clinical trials in the adult population. Two RCTs were excluded from the meta-analysis because the comparator was placebo. Two more studies were excluded, one because randomization could not be confirmed and the other because it described follow-up data on patients from a study that had already been included in the meta-analysis. Therefore, only nine RCTs were included in the meta-analysis. No differences were found between pirlindole and its active comparators with regard to the percentage of patients whose clinical condition improved by 50% according to the Hamilton Depression Rating Scale (HDRS) [odds ratio (OR) 1.52; 95% confidence interval [CI] 0.92, 2.51; p = 0.11] and Hamilton Anxiety Rating Scale (HARS) [OR 1.15; 95% CI 0.69, 1.90; p = 0.59]. With regard to the improvements in HDRS and HARS, the results were favourable for patients treated with pirlindole (depression: absolute value 0.18; 95% CI −0.01, 0.37; p = 0.06; anxiety: absolute value 0.26; 95% CI 0.03, 0.48; p = 0.03). Conclusion: This systematic review and meta-analysis showed that all RCTs included reported efficacy outcomes for pirlindole comparable to those of its comparators, and that pirlindole was significantly better in terms of reducing anxiety symptoms. However, the analysis of these results should take into account the quality of the original included articles, which had a mean Jadad trial quality score of 3.7 (out of 5). Therefore, further clinical trials should be conducted to evaluate the benefits of pirlindole.

Single-dose randomized, open-label, 2-way crossover bioequivalence study of clopidogrel 75 mg tablet in healthy volunteers under fasting conditions.

AIM This study aimed to assess the bioequivalence of 2 formulations of 75 mg clopidogrel hydrogen sulphate film-coated tablet, under fasting conditions. SUBJECTS AND METHODS 64 healthy subjects, age ranging from 19 to 55 years, were enrolled in a single-centre, randomized, single-dose, open-label, 2-way crossover study, with a minimum washout period of 7 days. Plasma samples were collected up to 24 h post dosing. Clopidogrel and clopidogrel carboxylic acid levels were determined by reverse-phase high-performance chromatography coupled to tandem mass spectrometry detection, LC-MS-MS method. Pharmacokinetic parameters used for bioequivalence assessment were the AUClast (area under the concentration-time curve from time zero to time of last observed nonzero concentration) and the Cmax (maximum observed concentration). These parameters were determined from the clopidogrel concentration data using non-compartmental analysis as well for clopidogrel carboxylic acid concentration data. RESULTS The 90% CI (90% confidence intervals), obtained by analysis of variance (ANOVA) were within the predefined ranges (80.00 - 125.00%) for both analytes. CONCLUSION Bioequivalence between test and formulations, under fasting conditions, was concluded both in terms of rate and extent of absorption.

Bioequivalence evaluation of three different oral formulations of ciprofloxacin in healthy volunteers

SummaryTwo bioequivalence studies were performed in twenty four healthy male volunteers with the objective of comparing the bioavailability of three different oral formulations of ciprofloxacin as immediate release tablets 250, 500 and 750 mg (test formulations) with a reference formulation at 500 and 750 mg strengths forms.In study 1, the subjects were enrolled in a single-dose, open-label, 3-period, crossover randomised study, designed to compare the bioavailability of two test formulations of ciprofloxacin (A and B) as 250 and 500 mg tablets, compared to the reference formulation (C), as 500 mg tablets.In study 2, the same 24-subjects were included in a single-dose, open-label, 2-period, crossover randomised study, designed to compare the bioavailability of one test formulation of ciprofloxacin (A) as compared to the reference formulation (B), both products as 750 mg tablets. In both studies multiple blood samples were collected over 24 hours post-dosing. One washout period of six days was observed between the periods. Plasma was harvested and assayed for ciprofloxacin using a selective and sensitive high-performance liquid chromatography (HPLC) method with UV detection.The pharmacokinetic parameter values of Cmax and tmax were obtained directly from plasma data, ke was estimated by log-linear regression, and AUC was calculated by trapezoidal rule. Different statistical tests were performed on the basis of untransformed and log-transformed data and the overall residual variance from ANOVA. Assuming the accepted tolerance intervals, a β-error of 20% and 90% confidence intervals (α=0.10) of all the generally accepted tests (Westlake, Schuirmann test and Wilcoxon-Tukey nonparametric tests) showed that the formulations can be considered as bioequivalent with respect to the extent of absorption, given by the AUC0-∞ and with respect to rate of absorption as assessed by Cmax and tmax.

Bioequivalence study of two different film-coated tablet formulations of losartan-hydrochlorothiazide in healthy volunteers.

The study was conducted in order to assess the bioequivalence of two film-coated formulations containing 100 mg of losartan (CAS 124750-99-8) and 12.5 mg of hydrochlorothiazide (CAS 58-93-5). Seventy-three healthy subjects were enrolled in a randomised, single-dose, open-label, two-way crossover study, with a minimum washout period of 7 days. A total of 21 blood samples were collected up to 36 h post-dosing. Losartan, losartan carboxy acid and hydrochlorothiazide levels were determined by liquid chromatography with tandem mass detection (lower limit of quantification: 1.01 ng/mL for hydrochlorothiazide, 2.02 ng/mL for losartan and 2.51 ng/mL for losartan carboxy acid). Pharmacokinetic parameters used for bioequivalence assessment (AUC(0-t) and Cmax as primary and AUC(0-inf) as secondary pharmacokinetic parameters) were determined from the losartan and hydrochlorothiazide concentration data using non-compartmental analysis. Data from losartan carboxy acid was reported and presented as supportive data. The 90% confidence intervals (obtained by ANOVA) for losartan were 97.05-118.48% for Cmax 100.76-106.10% for AUC(0-t) and 100.80-106.10% for AUC(0-inf) whereas for hydrochlorothiazide the 90% confidence intervals obtained were 103.94-115.33% for Cmax, 101.97-109.61% for AUC(0-t) and 101.77-109.02% for AUC(0-inf), and for losartan carboxy acid the intervals obtained were 98.31-107.82% for Cmax, 97.89-104.30% for AUC(0-t) and 98.06-104.30% for AUC(0-inf). All the 90% confidence intervals obtained for all the parameters assessed were within the predefined ranges (80-125%). Based on these results, it can be concluded that the evaluated formulations are bioequivalent in terms of rate and extent of absorption.

Bioequivalence study of two different tablet formulations of donepezil using truncated areas under the curve. A single-center, single-dose, randomized, open-label, 2-way crossover study under fasting conditions.

BACKGROUND Donepezil hydrochloride (CAS 120014-06-4) is a piperidine-based, reversible inhibitor of the enzyme acetylcholinesterase (AChE). It is postulated to exert its therapeutic effect by enhancing cholinergic function. This is accomplished by increasing the concentration of acetylcholine (ACh) through reversible inhibition of its hydrolysis by AChE. OBJECTIVE The aim of this study was to assess the bioequivalence of a new donepezil 10 mg formulation (test formulation) vs. the reference product, as required by European regulatory authorities for the marketing of a generic product. Additionally, the applicability of the truncated area under the plasma concentration curve (AUC) approach to this drug and under these test conditions was determined. METHODS This was a single center, randomized, single-dose, open-label, 2-way crossover study in healthy volunteers under fasting conditions. Plasma samples were collected up to 288 h post-dosing and (+)-donepezil and (-)-donepezil plasma levels were determined by reverse liquid chromatography and by tandem mass spectrometry detection (ie, the LCMS/MS method). Pharmacokinetic parameters were calculated using non-compartmental analysis. Area under the concentration-time curve from time zero to the time of the last non-zero concentration (AUC(last)) and maximum observed concentration (C(max)) were the main evaluation criteria, while area under the concentration-time curve from time zero to infinity (AUC(inf)) was also analyzed for additional information. For the assessment of the applicability of the truncated AUC approach, AUCs truncated at 24, 48, 72, 96, 144, 192, 240, and 288 h were calculated. All of the abovementioned pharmacokinetic parameters were analyzed using 90% geometric confidence interval of the ratio (T/R) of least-squares means from the ANOVA of the In-transformed parameter. Tolerability was monitored using physical examination, including vital sign measurements and laboratory analysis. RESULTS According to the classical approach, the 90% geometric confidence intervals obtained by analysis of variance for AUC(last), C(max) and AUC(inf) were within the predefined ranges (80.00-125.00%) for both analytes. Truncated AUCs were also in all cases within the predefined ranges for acceptance of bioequivalence. CONCLUSION Bioequivalence between test and reference formulations, both in terms of rate and extension of absorption, under fasting conditions, was concluded according to European guidelines. Both formulations were well tolerated. The conclusion of bioequivalence was also supported using the truncated AUCs approach.