Pharmacodynamic effect of gabapentin on central nervous system in patients with chronic low back pain: a [99mTc]Tc-ECD SPECT study.

BACKGROUND: Gabapentin is an effective therapeutic alternative for chronic low back pain, indicated in several guidelines for treating neuropathic pain as first-line medication. This study aimed to describe the pharmacodynamics of gabapentin in the central nervous system of patients with chronic low back pain (CLBP) by using single-photon emission CT (SPECT) with [99mTc]Tc-ECD. METHODS: We selected 13 patients with CLBP due to lumbar disc herniation. They underwent SPECT before and after using gabapentin, compared with a SPECT database of healthy volunteers. A second analysis compared regional cerebral blood flow (rCBF) changes between responders and non-responders to gabapentin and the healthy controls. RESULTS: The mean age of patients was 41 years, and the mean pain intensity was 5.92 points, measured by the Numeric Rating Scale. After using gabapentin, SPECT showed an increase of rCBF in the bilateral anterior cingulate gyrus and a decrease of rCBF in periaqueductal gray matter. Non-responder patients with gabapentin showed a post-treatment decrease of rCBF in the paracentral lobule of the brain. CONCLUSIONS: A lack of improvement in some patients with gabapentin may be associated with an activated affective circuit of pain, evidenced by the increase of rCBF of the anterior cingulate cortex. A maladaptive brain state in chronic pain can explain the decrease of rCBF in the default mode network structures. Gabapentin acts directly or indirectly on neurons of periaqueductal gray substance by increasing the pain threshold and decreasing the rCBF of this structure.

Xerostomia and dysgeusia in the elderly: prevalence of and association with polypharmacy

Xerostomia is defined as the perception of dry mouth, and dysgeusia, as a change in taste. Both are common complaints in the elderly, especially among those making use of polypharmacy drug combinations. Aim: This study aimed to determine the prevalence of xerostomia and dysgeusia and to investigate their association with polypharmacy in the elderly. Methods: older people under follow-up at the Multidisciplinary Elderly Center of the University Hospital of Brasília were interviewed and asked about health problems, medications used, presence of xerostomia and dysgeusia. Descriptive statistics were used to determine the prevalence of the symptoms surveyed. The chi-square test was used to investigate the relationship between xerostomia and dysgeusia and polypharmacy. Secondary associations were performed using binomial logistic regression. Results: Ninety-six older people were evaluated and of these, 62.5% had xerostomia and 21.1%, had dysgeusia. The average number of medications used was 4±3 medications per individual. Polypharmacy was associated with xerostomia but not dysgeusia. It was possible to associate xerostomia with the use of antihypertensive drugs. Conclusion: Xerostomia was a frequent complaint among elderly people making use of polypharmacy, especially those using antihypertensives. Antihypertensives and antidepressants were used most drugs by the elderly and exhibited interactions with drugs most prescribed in Dentistry. Two contraindications were found between fluconazole and mirtazapine; and between erythromycin and simvastatin

Cohort study protocol of the Brazilian collaborative research network on COVID-19: strengthening WHO global data.

INTRODUCTION: With the COVID-19 pandemic, hospitals in low-income countries were faced with a triple challenge. First, a large number of patients required hospitalisation because of the infection's more severe symptoms. Second, there was a lack of systematic and broad testing policies for early identification of cases. Third, there were weaknesses in the integration of information systems, which led to the need to search for available information from the hospital information systems. Accordingly, it is also important to state that relevant aspects of COVID-19's natural history had not yet been fully clarified. The aim of this research protocol is to present the strategies of a Brazilian network of hospitals to perform systematised data collection on COVID-19 through the WHO platform. METHODS AND ANALYSIS: This is a multicentre project among Brazilian hospitals to provide data on COVID-19 through the WHO global platform, which integrates patient care information from different countries. From October 2020 to March 2021, a committee worked on defining a flowchart for this platform, specifying the variables of interest, data extraction standardisation and analysis. ETHICS AND DISSEMINATION: This protocol was approved by the Research Ethics Committee (CEP) of the Research Coordinating Center of Brazil (CEP of the Hospital Nossa Senhora da Conceicao), on 29 January 2021, under approval No. 4.515.519 and by the National Research Ethics Commission (CONEP), on 5 February 2021, under approval No. 4.526.456. The project results will be explained in WHO reports and published in international peer-reviewed journals, and summaries will be provided to the funders of the study.

Neurobiological substrates of chronic low back pain (CLBP): a brain [99mTc]Tc-ECD SPECT study.

BACKGROUND: Recent neuroimaging studies have demonstrated pathological mechanisms related to cerebral neuroplasticity in chronic low back pain (CLBP). Few studies have compared cerebral changes between patients with and without pain in the absence of an experimentally induced stimulus. We investigated the neurobiological substrates associated with chronic low back pain using [99mTc]Tc-ECD brain SPECT and correlated rCBF findings with the numeric rating scale (NRS) of pain and douleur neuropathique en 4 questions (DN4). Ten healthy control volunteers and fourteen patients with neuropathic CLBP due to lumbar disc herniation underwent cerebral SPECT scans. A quantitative comparison of rCBF findings between patients and controls was made using the Statistical Parametric Mapping (SPM), revealing clusters of voxels with a significant increase or decrease in rCBF. The intensity of CLBP was assessed by NRS and by DN4. RESULTS: The results demonstrated an rCBF increase in clusters A (occipital and posterior cingulate cortex) and B (right frontal) and a decrease in cluster C (superior parietal lobe and middle cingulate cortex). NRS scores were inversely and moderately correlated with the intensity of rCBF increase in cluster B, but not to rCBF changes in clusters A and C. DN4 scores did not correlate with rCBF changes in all three clusters. CONCLUSIONS: This study will be important for future therapeutic studies that aim to validate the association of rCBF findings with the pharmacokinetic and pharmacodynamic profiles of therapeutic challenges in pain.

Efficacy of House Dust Mite Sublingual Immunotherapy in Patients with Atopic Dermatitis: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Sensitization to house dust mites (HDMs) is frequent in patients with atopic dermatitis. OBJECTIVE: To investigate the efficacy of sublingual immunotherapy (SLIT) with Dermatophagoides pteronyssinus extract in patients with atopic dermatitis sensitized to HDM. METHODS: In this randomized, double-blind, placebo-controlled trial, we enrolled 91 patients 3 years or older, with SCORing Atopic Dermatitis (SCORAD) score greater than or equal to 15 and positive skin test result and/or IgE to D pteronyssinus. Patients were stratified according to age (<12 and ≥12 years) to receive HDM SLIT or placebo for 18 months. Primary outcome was a greater than or equal to 15-point decrease in SCORAD score. Secondary outcomes were decreases in SCORAD and objective SCORAD, Eczema Area and Severity Index, visual analog scale for symptoms, and pruritus scale scores; Investigator's Global Assessment 0/1; and decrease greater than or equal to 4 points in Dermatology Life Quality Index. Background therapy was maintained. RESULTS: A total of 66 patients completed the study (35 HDM SLIT, 31 placebo). After 18 months, 74.2% and 58% of patients in the HDM SLIT group and the placebo group, respectively, showed greater than or equal to 15-point decrease in SCORAD score (relative risk, 1.28; 95% CI, 0.89-1.83). Significant SCORAD score decreases from baseline of 55.6% and 34.5% in HDM SLIT and placebo groups (mean difference, 20.4; 95% CI, 3.89-37.3), significant objective SCORAD score decreases of 56.8% and 34.9% in HDM SLIT and placebo groups (mean difference, 21.3; 95% CI, 0.66-41.81), and more patients with Investigator's Global Assessment 0/1 in the HDM SLIT group as compared with the placebo group (14 of 35 vs 5 of 31; relative risk, 2.63; 95% CI, 1.09-6.39) were observed at 18 months. CONCLUSIONS: Our results suggest that HDM SLIT may be effective in HDM-sensitized patients as an add-on treatment for atopic dermatitis.

Sensitive LC-MS/MS Methods for Amphotericin B Analysis in Cerebrospinal Fluid, Plasma, Plasma Ultrafiltrate, and Urine: Application to Clinical Pharmacokinetics.

Neurocryptococcosis, a meningoencephalitis caused by Cryptococcus spp, is treated with amphotericin B (AmB) combined with fluconazole. The integrity of the brain-blood barrier and the composition of the cerebrospinal fluid (CSF) may change due to infectious and/or inflammatory diseases such as neurocryptococcosis allowing for the penetration of AmB into the central nervous system. The present study aimed to develop LC-MS/MS methods capable of quantifying AmB in CSF at any given time of the treatment in addition to plasma, plasma ultrafiltrate, with sensitivity compatible with the low concentrations of AmB reported in the CSF. The methods were successfully validated in the four matrices (25 µl, 5-1,000 ng ml-1 for plasma or urine; 100 µl, 0.625-250 ng ml-1 for plasma ultrafiltrate; 100 µl, 0.1-250 ng ml-1 for CSF) using protein precipitation. The methods were applied to investigate the pharmacokinetics of AmB following infusions of 100 mg every 24 h for 16 days administered as a lipid complex throughout the treatment of a neurocryptococcosis male patient. The methods allowed for a detailed description of the pharmacokinetic parameters in the assessed patient in the beginning (4th day) and end of the treatment with AmB (16th day), with total clearances of 7.21 and 4.25 L h-1, hepatic clearances of 7.15 and 4.22 L h-1, volumes of distribution of 302.94 and 206.89 L, and unbound fractions in plasma ranging from 2.26 to 3.25%. AmB was quantified in two CSF samples collected throughout the treatment with concentrations of 12.26 and 18.45 ng ml-1 on the 8th and 15th days of the treatment, respectively. The total concentration of AmB in plasma was 31 and 20 times higher than in CSF. The unbound concentration in plasma accounted for 77 and 44% of the respective concentrations in CSF. In conclusion, the present study described the most complete and sensitive method for AmB analysis in plasma, plasma ultrafiltrate, urine, and CSF applied to a clinical pharmacokinetic study following the administration of the drug as a lipid complex in one patient with neurocryptococcosis. The method can be applied to investigate the pharmacokinetics of AmB in CSF at any given time of the treatment.

Metabolizer phenotype prediction in different Peruvian ethnic groups through CYP2C9 polymorphisms.

OBJECTIVES: The CYP2C9 gene have three common alleles, CYP2C9*1, CYP2C9*2 and CYP2C9*3, associated with different homozygous (*1/*1, *2/*2 and *3/*3) and heterozygous (*1/*2 and *1/*3) genotypes, which in turn are related to extensive (gEM), intermediate (gIM) and poor (gPM) metabolizers. Likewise, the inter-ethnic variability was intimately associated with different drug metabolism. Therefore, the aim of the present study was predict the metabolizer phenotypes in different Peruvian ethnic groups from lowland (<2,500 m) and highland (>2,500 m). METHODS: TaqMan genotyping assays were performed in a group of 174 healthy unrelated Peruvian individuals. RESULTS: In this study, the allelic comparison between the three eco-regions showed that the CYP2C9*1 was the most common in Andean (96.32%); the *2 was the most frequent in Coast (7.45%, p<0.05). Regarding the *3 was the most common in Amazonian (6.25%, p<0.05). In a corroborative manner, the gEM was the most common in Andean (94.74%), the gIM in Coast (17.02%) and gPM in Amazonian (6.25%) populations. CONCLUSIONS: Our study provides a valuable source of information about to metabolizer phenotype drugs in different Peruvian ethnic groups. In this way, it could be established suitable genetic-dosage medicaments for various common diseases in these heterogenetic populations.

Translation, Cross-Cultural Adaptation, and Validation of the Portuguese Version of the Rotterdam Elderly Pain Observation Scale.

INTRODUCTION: This study reports on the translation, cultural adaptation, and validation of a Portuguese version of the Rotterdam Elderly Pain Observation Scale (REPOS), a Dutch scale to assess pain in patients who cannot communicate, with or without dementia. METHODS: This is a multicenter study in pain and neurological units involving Brazil (clinical phase) and the Netherlands (training phase). We performed a retrospective cross-sectional, 2-staged analysis, translating and culturally adapting the REPOS to a Portuguese version (REPOS-P) and evaluating its psychometric properties. Eight health professionals were trained to observe patients with low back pain. REPOS consists of 10 behavioral items scored as present or absent after a 2-min observation. The REPOS score of ≥3 in combination with the Numerical Rating Scale (NRS) of ≥4 indicated pain. The Content Validity Index (CVI) in all items and instructions showed CVI values at their maximum. According to the higher correlation coefficient found between NRS and REPOS-P, it may be suggested that there was an adequate convergent validity. RESULTS: The REPOS-P was administered to 80 patients with a mean age of 60 years (SD 11.5). Cronbach's alpha coefficient showed a moderate internal consistency of REPOS-P (α = 0.62), which is compatible with the original study of REPOS. All health professionals reached high levels of interrater agreement within a median of 10 weeks of training, assuring reproducibility. Cohen's kappa was 0.96 (SD 0.03), and the intraclass correlation coefficient was 0.98 (SD 0.02), showing high reliability of REPOS-P scores between the trainer (researcher) and the trainees (healthcare professionals). The Pearson correlation coefficient was 0.95 (95% confidence interval 0.94-0.97), showing a significant correlation between the total scores of REPOS-P and NRS. CONCLUSION: The REPOS-P was a valuable scale for assessing elderly patients with low back pain by different healthcare professionals. Short application time, ease of use, clear instructions, and the brief training required for application were essential characteristics of REPOS-P.

Nifedipine Does Not Alter the Pharmacokinetics of Venlafaxine Enantiomers in Healthy Subjects Phenotyped for CYP2D6, CYP2C19, and CYP3A.

Venlafaxine (VEN) is a P-glycoprotein (P-gp) substrate, and nifedipine has been described by in vitro and experimental studies as a P-gp inhibitor. The present study aimed to investigate whether nifedipine alters the kinetic disposition of VEN enantiomers and their metabolites in healthy subjects. A crossover study was conducted in 10 healthy subjects phenotyped as extensive metabolizers for cytochrome P450 (CYP) 2D6, CYP2C19, and CYP3A. In phase 1, the subjects received a single oral dose of 150 mg racemic VEN, and in phase 2, a single oral dose of 40 mg nifedipine was administered with the VEN treatment. Plasma concentrations of VEN enantiomers and their metabolites O-desmethylvenlafaxine and N, O- didesmethylvenlafaxine (ODV and DDV, respectively) were evaluated by liquid chromatography with tandem mass spectrometry up to 72 hours after drug administration. Phase 2 was compared with phase 1 using the 90% confidence interval (CI) of the ratio of geometric means for Cmax and area under the curve (AUC). AUC enantiomeric ratios S-(+)/R-(-) were evaluated within each and between phases using the Wilcoxon test (P ≤ .05). The kinetic disposition of VEN was enantioselective (phase 1) with VEN S-(+)/R-(-) AUC ratio median of 2.83 (AUC0-∞ , 526 vs 195 ng·h/mL). However, AUC median did not differ between enantiomers for the metabolites ODV (1971 vs 2226 ng·h/mL) and DDV (199 vs 151 ng·h/mL). The 90%CI of the ratio of geometric means showed that the phases are bioequivalent. A single oral dose of 40 mg nifedipine did not alter VEN enantiomer pharmacokinetics in healthy subjects.

Estimating resting energy expenditure from dual-energy X-ray absorptiometry: A cross-sectional study in healthy young adults.

OBJECTIVE: To qualify a 3C approach model of dual-energy X-ray absorptiometry (DXA) to estimate multicomponent resting energy expenditure (REE) referenced by indirect calorimetry (IC). METHODS: A sample of 155 college students, of both sexes (18-30 years old) was evaluated. Anthropometric measures, REE by IC, and whole-body DXA-scans were performed. The REE for each body component was determined after transforming the components from the molecular (DXA) to the organ tissue level. Bland-Altman and proportional bias analyses were used to verify agreement between REE measured (REEIC ) and estimated (REEDXA ). RESULTS: Statistically significant differences were found for all sex comparisons (P < .001), except for age (P = .950). Differences from the final sex-specific models' were not found between REEIC and REEDXA (P > .05). Men also presented greater expenditure (P < .001) in each component, except for adipose tissue. The plots confirmed the validity of the model for both sexes, with low difference values between the measured and estimated REE. The mean of the differences of REEIC and REEDXA showed heteroscedasticity of the data for men (P = .004). The same error tendency was not evident for women (P = .333). CONCLUSIONS: This 3C model, estimating REE from a multicomponent approach, allows a new application of DXA as tool for understanding intraindividual differences in terms of the mass of metabolically active tissue. Sex and populational differences should be taken in account. Consequently, we present qualified sex-specific DXA models that can be applied in different contexts such as health and sports, besides considering interpersonal differences in terms of energy expenditure.

Direct chiral LC-MS/MS analysis of fexofenadine enantiomers in plasma and urine with application in a maternal-fetal pharmacokinetic study.

This study shows the development and validation of two enantioselective LC-MS/MS methods for the determination of fexofenadine in biological matrices including the elution order determination. Plasma (200 µL) or urine (50 µL) aliquots were added to the internal standard solution [(S)-(-)-metoprolol] and extracted in the acid medium with chloroform. Resolution of the (R)-(+)- and (S)-(-)-fexofenadine enantiomers was performed in a Chirobiotic V column. The methods showed linearity at the range of 0.025-100 ng/mL plasma and 0.02-10 µg/mL urine for each fexofenadine enantiomer. These methods were applied to the maternal-fetal pharmacokinetics of fexofenadine enantiomers in plasma and urine of parturient women (n = 8) treated with a single oral 60 mg dose of racemic fexofenadine. Enantiomeric ratio in plasma (AUC0-∞(R)-(+)/(S)-(-)) was close to 1.5, nevertheless in urine was closed to unity. The transplacental transfer was approximately 18% for both fexofenadine enantiomers. The enantioselective methods can also be useful in future clinical studies of chiral discrimination of drug transporters.

Limited Sampling Modeling for Estimation of Phenotypic Metrics for CYP Enzymes and the ABCB1 Transporter Using a Cocktail Approach.

Plasma concentration data points (n = 2,640) from 16 healthy adults were used to develop and validate limited sampling strategies (LSS) for estimation of phenotypic metrics for CYP enzymes and the ABCB1 transporter, using a cocktail of subtherapeutic doses of the selective probes caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A), losartan (CYP2C9), omeprazole (CYP2C19), and fexofenadine (ABCB1). All-subsets linear regression modelling was applied to estimate the AUC0-12h for caffeine, fexofenadine, and midazolam, and the AUC0-12h ratio of metoprolol: α-OH metoprolol and omeprazole:5-OH omeprazole. LSS-derived metrics were compared with the parameters' 'best estimates' obtained by non-compartmental analysis using all plasma concentration data points. The correlation coefficient (R 2) was used to identify the LSS equations that provided the best fit for n timed plasma samples, and the jack-knife statistics was used as an additional validation procedure for the LSS models. Single time-point LSS models provided R 2 values greater than 0.95 (R 2 > 0.95) for the AUC0-12h ratio of metoprolol:α-OH metoprolol and omeprazole:5-OH omeprazole, whereas 2 time-point models were required for R 2 > 0.95 for the AUC0-12h of caffeine, fexofenadine, and midazolam. Increasing the number of sampling points to three led to minor increases in R 2 and/or the bias or prediction of the estimates. In conclusion, the LSS models provided accurate prediction of phenotypic indices for CYP1A2, CYP2C19, CYP2D6, CYP3A, and ABCB1, when using subtherapeutic doses of selective probes for these enzymes and transporter.

Pertussis epidemiological pattern and disease burden in Brazil: an analysis of national public health surveillance data.

Objective: We described pertussis epidemiological trends in Brazil between 2010 and 2015. We also assessed tetanus, diphtheria and acellular pertussis (Tdap) vaccine coverage among pregnant women from 2014, the year of the introduction of Tdap maternal immunization recommendation in Brazil, to 2016.Methods: Epidemiological data for incidence, prevalence, hospitalization, mortality, and maternal vaccination coverage were calculated based on the Brazilian public surveillance databases.Results: The epidemiological data analysis results showed that the pertussis average incidence rate (IR) was 2.19/100,000 inhabitants for all ages, with a peak in 2014 (4.03/100,000 inhabitants) and highest incidence in <1-year-old children (IR = 175.20/100,000). 97.6% of pertussis deaths (405/415) were in <1-year-old children. Maternal immunization coverage was 9.2% in 2014, 40.4% in 2015, and 33.8% in 2016.Conclusions: Pertussis incidence and pertussis-related deaths increased in Brazil from 2010 to 2014 and decreased in 2015. In the two years, 2015 and 2016 that followed the NIP recommendation, Tdap vaccination coverage of pregnant women was low and varying from region to region. More efforts and national plans would help increase awareness and maternal immunization coverage.

Hormonal status affects plasma exposure of tamoxifen and its main metabolites in tamoxifen-treated breast cancer patients.

BACKGROUND: Tamoxifen is considered a prodrug of its active metabolite endoxifen, which is dependent on the CYP2D6 and CYP3A enzymes. Tamoxifen pharmacokinetic variability influences endoxifen exposure and, consequently, its clinical outcome. This study investigated the impact of hormonal status on the pharmacokinetics of tamoxifen and its metabolites in TAM-treated breast cancer patients. METHODS: TAM-treated breast cancer patients (n = 40) previously believed to have CYP3A activity within the normal range based on oral midazolam and phenotyped as CYP2D6 normal metabolizers using oral metoprolol were divided into two groups according to premenopausal (n = 20; aged 35-50 years) or postmenopausal (n = 20; aged 60-79 years) status. All patients were treated with 20 mg/day tamoxifen for at least three months. Serial plasma samples were collected within the 24 h dose interval for analysis of unchanged tamoxifen, endoxifen, 4-hydroxytamoxifen and N-desmethyltamoxifen quantified by LC-MS/MS. CYP activities were assessed using midazolam apparent clearance (CYP3A) and the metoprolol/alfa-hydroxymetoprolol plasma metabolic ratio (CYP2D6). CYP3A4, CYP3A5 and CYP2D6 SNPs and copy number variation were investigated using TaqMan assays. RESULTS: Postmenopausal status increased steady-state plasma concentrations (Css) of tamoxifen (116.95 vs 201.23 ng/mL), endoxifen (8.01 vs 18.87 ng/mL), N-desmethyltamoxifen (485.16 vs 843.88 ng/mL) and 4-hydroxytamoxifen (2.67 vs 4.11 ng/mL). The final regression models included hormonal status as the only predictor for Css of tamoxifen [ß-coef ± SE, p-value (75.03 ± 17.71, p = 0.0001)] and 4-hydroxytamoxifen (1.7822 ± 0.4385, p = 0.0002), while endoxifen Css included hormonal status (8.578 ± 3.402, p = 0.02) and race (11.945 ± 2.836, p = 0.007). For N-desmethyltamoxifen Css, the final model was correlated with hormonal status (286.259 ± 76.766, p = 0.0007) and weight (- 8.585 ± 3.060, p = 0.008). CONCLUSION: The premenopausal status was associated with decreased endoxifen plasma concentrations by 135% compared to postmenopausal status. Thus, the endoxifen plasma concentrations should be monitored mainly in the premenopausal period to maintain plasma levels above the efficacy threshold value. TRIAL REGISTRATION: RBR-7tqc7k.

Geographic distribution of the 3435C>T polymorphism of the MDR1 gene in Peruvian populations.

Background The MDR1 gene presents several genetic polymorphisms with pharmacological implications. Therefore, the aim of the present study is to establish the genotype and allele frequencies of 3435C>T polymorphism of MDR1 gene into Peruvian populations (Coastal, Andean and Amazonian ecoregions), even considering the altitude (lowland <2500 m and highland >2500 m). Methods The polymorphism was analyzed by TaqMan genotyping assays in a group of 181 healthy unrelated Peruvian individuals. The comparison of genotype and allele frequencies of 3435C>T polymorphism was made with the Pearson test (X2), and, to calculate the genotype distributions, the Hardy-Weinberg equilibrium (HWE) was used. Results In all populations evaluated in this study, the genotype frequency distributions met HWE assumptions. The comparison between genotype and allele frequencies showed significant differences (p < 0.05), when the Andean, Coastal and Amazonian populations were compared. Also, significant differences (p < 0.05) were obtained when these populations were compared considering their altitudes. Likewise, in comparison with countries like USA, Finland, Nigeria and Kenya, the results showed significant differences (p < 0.05). Conclusions This investigation allowed us to establish the genotype and allele frequencies of 3435C>T polymorphism in different Peruvian populations, considering the geographic localization and even the altitude.

LC-MS/MS analysis of the plasma concentrations of a cocktail of 5 cytochrome P450 and P-glycoprotein probe substrates and their metabolites using subtherapeutic doses.

Drug transporters and CYP enzymes are important sources of pharmacokinetics (PK) variability in drug responses and can cause various pharmacological and toxicological consequences, leading to either toxicity or an insufficient pharmacological effect. In recent years, the cocktail approach was developed to determine in vivo CYP and transporters activities, but these approaches are somewhat limited. We described the development and validation of three sensitive and specific LC-MS/MS assays for the determination of P-gp and major human CYP isoenzyme activities following oral administration of a drug cocktail of subtherapeutic doses (lower than 10 times) of caffeine (CAF), omeprazole (OME), losartan (LOS), midazolam (MDZ), metoprolol (METO) and fexofenadine (FEX) in healthy volunteers. The three validated methods were selective for all tested analytes. No interference or matrix effect was observed for the mass transition and retention times for all compounds monitored. Additionally, assays were linear over a wide range, and limits of quantification varied between 0.01-5 ng/mL plasma. The coefficients of variation obtained in the precision studies and the inter- and intra-assay accuracies were less than 15%, guaranteeing the reproducibility and repeatability of the results. All substrates and metabolites were stable in plasma during freeze-thaw cycles. Three healthy volunteers were selected based on genotyping for CYP2C9, CYP2C19 and CYP2D6. One volunteer was genotyped as an extensive metabolizer (EM) for all tested CYP isoforms, one volunteer was genotyped as a poor metabolizer (PM) for the CYP2C9 isoform (CYP2C9*3/*3), and one volunteer was genotyped as a PM for the CYP2D6 isoform (CYP2D6*4/*4). The methods allowed the quantification of all analytes over the entire sampling period (12 h) in all studied genotypes. Thus, the analytical methods described here were sufficiently sensitive for use in low-dose pharmacokinetic studies.

Indicadores referentes à qualidade em centros da Rede Nacional de Pesquisa Clínica / Evaluation of quality-related indexes in National Clinical Research Network Centers

Modelo do estudo: Estudo transversal nos 32 centros participantes da Rede Nacional de Pesquisa Clínica (RNPC). Objetivos: Desenvolver e validar um questionário eletrônico capaz de medir o desempenho dos centros participantes da RNPC em uma auditoria de qualidade baseada nas Boas Práticas Clínicas (BPC). Avaliar a correlação do instrumento com o guia de inspeção em BPC disposto na Instrução Normativa nº 4, de 11/05/09 da ANVISA (IN4/ANVISA) e verificar, por meio do questionário desenvolvido, o desempenho dos centros participantes da RNPC, em relação ao atendimento a requisitos de um sistema de gestão da qualidade (SGQ). Métodos: Foi aplicado um questionário eletrônico, legitimado por uma validação de conteúdo. As questões foram agrupadas em três domínios, infraestrutura, estrutura organizacional e processos. Os dados foram pontuados de acordo com o cumprimento (1) ou não cumprimento (0) em BPC. As questões do questionário foram arbitrariamente correlacionadas aos itens da IN4, atribuído (0) ponto às questões conformes e (1) para não conforme. O índice de cumprimento para atendimento aos itens críticos e maiores (ICCM) foi calculado para cada domínio usando a equação ICCM = 1 * (Escore IN4) + valor total escore para dado domínio. As comparações foram feitas por teste de Kruskal-Wallis e teste de Spermann, considerando P valor < 0,05. Resultados: O instrumento representa aproximadamente 40% dos itens do roteiro de inspeção da IN4/ANVISA. A pontuação obtida com o questionário correlaciona-se significativamente ao escore correspondente à IN4/ANVISA (r=0,95, 0,89 a 0,98, IC95%, P<0.0001). A mediana do ICCM foi menor que 75% em todos os domínios estudados. Conclusões: O instrumento desenvolvido para medir o desempenho em BPC apresenta alta correlação com a IN4/ANVISA e poderia ser utilizado para auditoria interna de qualidade. A classificação para itens críticos e maiores definidas pela IN4/ANVISA mostrou-se muito restritiva e necessita ser adequada a realidade dos centros de pesquisa nacionais. (AU)

Study design: A transversal study that evaluated 32 Clinical research centers (CRC) of the National Clinical Research Network (Rede Nacional de Pesquisa Clínica/RNPC). Objectives: To develop and validate an electronic questionnaire to measure how CRC that are part of the RNPC performed in a quality audit based in Good Clinical Practice, to evaluate the correlation between our instrument and IN4/ANVISA guide and to access the quality of the CRC. Methods: An electronic questionnaire was applied and validated by contend valid index. The questions were divided into tree domains, infrastructure (I), organization structure (OS) and process (P). Data were scored according compliance (1) or non-compliance (0) with GCP. The questions of our questionnaires where arbitrary related to IN4/ANVISA guide and scored as non-conformity (1) or conformity (0). The indexes of compliance in critical and major items (ICCM) were calculated using the equation: ICCM = 1 * (IN4 score) + total score for the domain. The correlation of the two measurements above was done using Kruskal-Wallis and Spearman rank method, considering P <0.05. Results: The questionnaire represents about 40% of the IN$/ANVISA document. The score obtained with the questionnaire had a significant correlation to IN4/ ANVISA score (r=0.95, 0.89 to 0.98, CI95%, P<0.0001). The median ICCM was less than 75% for all domain studied. Conclusion: The instrument designed to measure performance GCP showed high correlation with IN4/ ANVISA guide and could be used to perform the internal quality audit of a CRC. The IN4/ANVISA guide showed to be very strict and might be necessary to review it and adequate to the reality of nationals centers. (AU)

Analysis of unbound plasma concentration of oxcarbazepine and the 10-hydroxycarbazepine enantiomers by liquid chromatography with tandem mass spectrometry in healthy volunteers.

This study describes the development and validation of a method for the analysis of unbound plasma concentrations of oxcarbazepine (OXC) and of the enantiomers of its active metabolite 10-hydroxycarbazepine (MHD) [S-(+)- and R-(-)-MHD] using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, the free fraction of the drug is described in healthy volunteers (n=12) after the oral administration of 300mg OXC/12h for 5days. Plasma aliquots of 200µL were submitted to ultrafiltration procedure and 50µL of the ultrafiltrate were extracted with a mixture of tert-butyl methyl ether:dichloromethane (2:1, v/v). OXC and the MHD enantiomers were separated on a OD-H chiral phase column. The method was linear in the range of 4.0-2.0µg/mL for OXC and of 20.0-6.0µg/mL plasma for the MHD enantiomers. The limit of quantification was 4ng for OXC and 20ng for each MHD enantiomer/mL plasma. The intra- and inter-day precision and inaccuracy were less than 15%. The free fraction at the time of peak plasma concentration of OXC was 0.27 for OXC, 0.37 for S-(+)-MHD and 0.42 for R-(-)-MHD. Enantioselectivity in the free fraction of MHD was observed, with a higher proportion of R-(-)-MHD compared to S-(+)-MHD.

The Diterpene Sclareol Vascular Effect in Normotensive and Hypertensive Rats / Efeito Vascular do Diterpeno Esclareol em Aortas Torácicas de Ratos Normotensos e Hipertensos

Abstract Background: The diterpene Sclareol has antimicrobial action, cytotoxic and cytostatic effects and anti-tumor activities. However, researches on the cardiovascular system are scarce. Objective: This study was designed to investigate the mechanisms involved in the Sclareol cardiovascular effect in normotensive and hypertensive rats. Methods: The arterial hypertension was promoted using 2-kidneys 1-clip model in rats. The effect of sclareol on blood pressure was performed by using three dose (10, 20 and 40 mg/kg). Cumulative dose-response curves for Sclareol were determined for endothelium-intact and endothelium-denuded aortic rings in presence or absence of L-NAME and ODQ. The NOx levels were measure in the plasma sample. Results: The Sclareol administration in vivo caused a significant reduction in blood pressure in both groups. In vitro the sclareol promoted relaxation in aorta, with endothelium, pre-contracted to Phe. The inhibitors of the nitric oxide synthase and soluble guanylate cyclase were as efficient as the removal of endothelium, in inhibiting the Sclareol-induced relaxation. Otherwise, it was no change of NOx. Also, for unknown reasons, the Sclareol is not selective for hypertensive animals. Conclusion: The diterpene Sclareol showed in vivo hypotensive and in-vitro vasodilator effects; The chemiluminescence plasmatic NO analysis showed no significant difference between groups and The Sclareol exhibit better effect on normotensive than hypertensive animals to reduce blood pressure. It is concluded that the diterpenes metabolites would be a promising source prototype for the development of new agents in the cardiovascular therapy.

Resumo Fundamento: O diterpeno Esclareol tem ação antimicrobiana, efeitos citotóxicos e citostáticos e atividades antitumorais. No entanto, pesquisas sobre o sistema cardiovascular são escassas. Objetivo: Este estudo foi desenvolvido para investigar os mecanismos envolvidos no efeito cardiovascular de Esclareol em ratos normotensos e hipertensos. Métodos: A hipertensão arterial foi promovida utilizando modelo de 2 clones de 1-clipe em ratos. O efeito do esclareol sobre a pressão arterial foi realizado utilizando três doses (10, 20 e 40 mg/kg). As curvas dose-resposta cumulativas para Esclareol foram determinadas para anéis aórticos endotélio-intactos e desprovidos de endotélio na presença ou ausência de L-NAME e ODQ. Os níveis de NOx foram medidos na amostra de plasma. Resultados: A administração de Esclareol in vivo causou uma redução significativa na pressão sanguínea em ambos os grupos. In vitro o esclareol promoveu relaxamento na aorta, com endotélio, pré-contraído a Phe. Os inibidores da óxido nítrico sintase e da guanilato ciclase solúvel foram tão eficientes quanto a remoção do endotélio, na inibição do relaxamento induzido por Esclareol. Por outra parte, não houve mudança de NOx. Além disso, por razões desconhecidas, o Sclareol não é seletivo para animais hipertensos. Conclusão: O diterpeno Esclareol apresentou efeitos hipotensores in vivo e vasodilatadores in vitro; A análise de NO plasmático por quimioluminescência não mostrou diferença significativa entre os grupos e O Esclareol exibe melhor efeito sobre os animais normotensos do que hipertensos para reduzir a pressão arterial. Conclui-se que os metabólitos de diterpenos seriam um protótipo de fonte promissora para o desenvolvimento de novos agentes na terapia cardiovascular.

The Diterpene Sclareol Vascular Effect in Normotensive and Hypertensive Rats.

BACKGROUND:: The diterpene Sclareol has antimicrobial action, cytotoxic and cytostatic effects and anti-tumor activities. However, researches on the cardiovascular system are scarce. OBJECTIVE:: This study was designed to investigate the mechanisms involved in the Sclareol cardiovascular effect in normotensive and hypertensive rats. METHODS:: The arterial hypertension was promoted using 2-kidneys 1-clip model in rats. The effect of sclareol on blood pressure was performed by using three dose (10, 20 and 40 mg/kg). Cumulative dose-response curves for Sclareol were determined for endothelium-intact and endothelium-denuded aortic rings in presence or absence of L-NAME and ODQ. The NOx levels were measure in the plasma sample. RESULTS:: The Sclareol administration in vivo caused a significant reduction in blood pressure in both groups. In vitro the sclareol promoted relaxation in aorta, with endothelium, pre-contracted to Phe. The inhibitors of the nitric oxide synthase and soluble guanylate cyclase were as efficient as the removal of endothelium, in inhibiting the Sclareol-induced relaxation. Otherwise, it was no change of NOx. Also, for unknown reasons, the Sclareol is not selective for hypertensive animals. CONCLUSION:: The diterpene Sclareol showed in vivo hypotensive and in-vitro vasodilator effects; The chemiluminescence plasmatic NO analysis showed no significant difference between groups and The Sclareol exhibit better effect on normotensive than hypertensive animals to reduce blood pressure. It is concluded that the diterpenes metabolites would be a promising source prototype for the development of new agents in the cardiovascular therapy. FUNDAMENTO:: O diterpeno Esclareol tem ação antimicrobiana, efeitos citotóxicos e citostáticos e atividades antitumorais. No entanto, pesquisas sobre o sistema cardiovascular são escassas. OBJETIVO:: Este estudo foi desenvolvido para investigar os mecanismos envolvidos no efeito cardiovascular de Esclareol em ratos normotensos e hipertensos. MÉTODOS:: A hipertensão arterial foi promovida utilizando modelo de 2 clones de 1-clipe em ratos. O efeito do esclareol sobre a pressão arterial foi realizado utilizando três doses (10, 20 e 40 mg/kg). As curvas dose-resposta cumulativas para Esclareol foram determinadas para anéis aórticos endotélio-intactos e desprovidos de endotélio na presença ou ausência de L-NAME e ODQ. Os níveis de NOx foram medidos na amostra de plasma. RESULTADOS:: A administração de Esclareol in vivo causou uma redução significativa na pressão sanguínea em ambos os grupos. In vitro o esclareol promoveu relaxamento na aorta, com endotélio, pré-contraído a Phe. Os inibidores da óxido nítrico sintase e da guanilato ciclase solúvel foram tão eficientes quanto a remoção do endotélio, na inibição do relaxamento induzido por Esclareol. Por outra parte, não houve mudança de NOx. Além disso, por razões desconhecidas, o Sclareol não é seletivo para animais hipertensos. CONCLUSÃO:: O diterpeno Esclareol apresentou efeitos hipotensores in vivo e vasodilatadores in vitro; A análise de NO plasmático por quimioluminescência não mostrou diferença significativa entre os grupos e O Esclareol exibe melhor efeito sobre os animais normotensos do que hipertensos para reduzir a pressão arterial. Conclui-se que os metabólitos de diterpenos seriam um protótipo de fonte promissora para o desenvolvimento de novos agentes na terapia cardiovascular.