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OBJECTIVES: In the era of quality management in clinical laboratories, method validation can be a challenge without appropriate guidelines, such as in the field of pharmacogenetics. The present work describes a method validation for DNA extraction and CYP3A5*3 genotyping, which would meet ISO15189:2012 requirements. METHODS: DNA extraction was performed using a QIAamp DSP DNA Blood kit, DNA purity and concentration were determined using a Nanodrop, and the genotyping assay was a real-rime PCR using TaqMan reagents. Validation criteria were similar to those usually verified when validating methods in the analytical field: specificity, sensitivity, cross-over contamination, stability of reagents, robustness, lower and upper limits of detection, and between-run and within-run precisions. A comparison to alternate or reference methods was also performed (i.e. QiAamp kit versus DNA extractor and TaqMan genotyping versus Sanger sequencing). Each validation step is described from the pharmacogenetic point of view, as well as acceptance criteria for both DNA extraction [i.e. concentration relative SD (RSD) below 25%, verified purity, and no DNA in blank samples] and genotyping assay (i.e. specificity and diagnostic sensitivity, RSD of mean threshold cycle below 15%, no amplification in blank samples). RESULTS: Concerning CYP3A5 genotyping following a DNA extraction described as an example, validation criteria were met, allowing routine use of this analytical process. Cost estimation of the overall validation procedure was approximately 290 euros, concerning reagents and consumables. CONCLUSION: This work aims to provide a reference for method validation for pharmacogenetic analysis using real-time PCR to detect single nucleotide polymorphisms, in accordance with ISO15189:2012.
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Citocromo P-450 CYP3A , Farmacogenética , Alelos , DNA/genética , Humanos , Testes FarmacogenômicosRESUMO
BACKGROUND: This pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes. METHODS AND RESULTS: Radial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating. CONCLUSIONS: These results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations. Trial registration NCT02311075. Registered December 8, 2014.
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Diabetes Mellitus Tipo 2/sangue , Angiopatias Diabéticas/sangue , Eicosanoides/sangue , Hipertensão Essencial/sangue , Artéria Radial/metabolismo , Vasodilatação , Idoso , Biomarcadores/sangue , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/diagnóstico , Angiopatias Diabéticas/fisiopatologia , Epóxido Hidrolases/metabolismo , Hipertensão Essencial/diagnóstico , Hipertensão Essencial/fisiopatologia , Feminino , Humanos , Hipertermia Induzida , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Nitritos/sangue , Nitroglicerina/administração & dosagem , Artéria Radial/efeitos dos fármacos , Artéria Radial/fisiopatologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagemRESUMO
Point-of-care testing (POCT) is the analysis of patient specimens outside the clinical laboratory, near or at the site of patient care, usually performed by clinical staff without laboratory training, although it also encompasses patient self-monitoring. It is able to provide a rapid result near the patient and which can be acted upon immediately. The key driver is the concept that clinical decision making may be delayed when samples are sent to the clinical laboratory. Balanced against this are considerations of increased costs for purchase and maintenance of equipment, staff training, connectivity to the laboratory information system (LIS), quality control (QC) and external quality assurance (EQA) procedures, all required for accreditation under ISO 22870. The justification for POCT depends upon being able to demonstrate that a more timely result (shorter turnaround times (TATs)) is able to leverage a clinically important advantage in decision making compared with the central laboratory (CL). In the four decades since POCT was adapted for the self-monitoring of blood glucose levels by subjects with diabetes, numerous new POCT methodologies have become available, enabling the clinician to receive results and initiate treatment more rapidly. However, these instruments are often operated by staff not trained in laboratory medicine and hence are prone to errors in the analytical phase (as opposed to laboratory testing where the analytical phase has the least errors). In some environments, particularly remote rural settings, the CL may be at a considerable distance and timely availability of cardiac troponins and other analytes can triage referrals to the main centers, thus avoiding expensive unnecessary patient transportation costs. However, in the Emergency Department, availability of more rapid results with POCT does not always translate into shorter stays due to other barriers to implementation of care. In this review, we apply the principles of evidence-based laboratory medicine (EBLM) looking for high quality systematic reviews and meta-analyses, ideally underpinned by randomized controlled trials (RCTs), looking for evidence of whether POCT confers any advantage in clinical decision making in different scenarios.
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Tomada de Decisão Clínica , Medicina Baseada em Evidências , Testes Imediatos , Humanos , Fatores de TempoAssuntos
Klebsiella pneumoniae , Piperacilina , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infusões Intravenosas , Testes de Sensibilidade Microbiana , Ácido Penicilânico/farmacologia , Ácido Penicilânico/uso terapêutico , Piperacilina/farmacologia , Piperacilina/uso terapêutico , Combinação Piperacilina e TazobactamRESUMO
The banking of testicular tissue before highly gonadotoxic treatment is a prerequisite for the preservation of fertility in pre-pubertal boys not yet producing sperm. The aim of the current study is to evaluate the impact of a soaking temperature performed at -7 °C, -8 °C or -9 °C on the ability of frozen-thawed mouse spermatogonial stem cells (SSCs) to generate haploid germ cells after in vitro maturation. Testes of 6.5-day-old post-partum CD-1 mice were cryopreserved by using a controlled slow freezing protocol with soaking at -7 °C, -8 °C or -9 °C. Frozen-thawed pre-pubertal testicular tissues were cultured in vitro on agarose gel for 30 days. Histological evaluations were performed and flagellated late spermatids were counted after mechanical dissection of the cultured tissues. The differentiation of frozen SSCs into elongated spermatids was more efficient after treatment at -9 °C than at -7 °C and -8 °C. After dissection, flagellated late spermatids were observed by using Shorr staining. The number of flagellated late spermatids was significantly decreased after slow freezing when compared with a fresh tissue control. Therefore, the soaking temperature during slow freezing of pre-pubertal mouse testicular tissue might positively influence the course of in vitro spermatogenesis. Our slow freezing protocol with a soaking temperature at -9 °C was the optimal condition in terms of the achievement of in vitro spermatogenesis with a higher production of elongated spermatids, although the effectiveness of the maturation process was reduced compared with the fresh tissue control.
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Criopreservação , Congelamento , Maturidade Sexual , Espermatogênese , Testículo/fisiologia , Animais , Contagem de Células , Diferenciação Celular , Proliferação de Células , Epitélio/anatomia & histologia , Humanos , Células Intersticiais do Testículo/citologia , Masculino , Camundongos , Túbulos Seminíferos/anatomia & histologia , Células de Sertoli/citologia , Espermátides/citologia , Células-Tronco/citologia , Testosterona/metabolismo , Técnicas de Cultura de TecidosRESUMO
BACKGROUND: Little is known about clinical events occurring in older patients with type 2 diabetes mellitus according to their therapeutic modalities based on the prescription of insulin and/or oral antidiabetic drugs. OBJECTIVE: The aim of this study was to compare the complications of diabetes and geriatric alterations that occurred according to three therapeutic modalities prescribed over 5 years. METHODS: A total of 616 patients from the GERODIAB cohort (mean age 77.1 years) were divided into three groups: an insulin-only group (n = 200), a group receiving insulin and one or more oral antidiabetic drug (n = 169), and an oral antidiabetic drug group without insulin (n = 247). We compared the diabetic complications and geriatric alterations that occurred over 5 years in patients without these pre-existing complications. RESULTS: At inclusion, there was a significant difference between glycosylated hemoglobin values, and between the frequencies of most diabetic complications and geriatric alterations, with higher frequencies in the insulin group and lower frequencies in the oral antidiabetic drug group. At the end of the follow-up, there was still a significant difference between the mean glycosylated hemoglobin of the three groups (mean for all patients 7.4 ± 0.8%). The frequencies of new clinical events were high and they were generally higher in the insulin group. They were not significantly different between the three groups, with the exception of four events: heart failure, retinopathy, transfer to a nursing home (more frequent in the insulin group), and hypoglycemia (more frequent in the insulin + oral antidiabetic drug group). Some frequencies of the total diabetic complications (including complications at inclusion and at the follow-up) in the oral antidiabetic drug group were close to those in the insulin group, although only at inclusion. Mortality was higher in the insulin group and lower in the oral antidiabetic drug group. CONCLUSIONS: The increased frequency of hypoglycemia in the insulin + oral antidiabetic drug group raises doubts about the value of continuing a secretagogue drug when insulin is introduced. As the vast majority of patients were not yet receiving antidiabetic drugs with cardiovascular action, our results on heart failure could help in conducting specific studies on these drugs in older patients with type 2 diabetes.
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Complicações do Diabetes , Diabetes Mellitus Tipo 2 , Insuficiência Cardíaca , Hipoglicemia , Humanos , Idoso , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hemoglobinas Glicadas , Estudos Prospectivos , Seguimentos , Hipoglicemiantes/efeitos adversos , Complicações do Diabetes/induzido quimicamente , Complicações do Diabetes/tratamento farmacológico , Insulina/uso terapêutico , Hipoglicemia/induzido quimicamente , Hipoglicemia/epidemiologia , Insuficiência Cardíaca/tratamento farmacológico , GlicemiaRESUMO
Pharmacological treatments used for psychiatric disorders, such as clozapine, demonstrate large interindividual variability in terms of possible adverse effects and therapeutic benefit. This variability can be explained by multiple factors, including pharmacogenetic factors. Clozapine efficacy can be impacted by CYP polymorphisms. A growing body of literature on pharmacogenetics suggests the clinical benefit of concomitant use of clozapine and fluvoxamine to improve global pharmacotherapeutic management. This article reviews and discusses available clinical and pharmacological data and limitations of clozapine augmentation with fluvoxamine based on pharmacogenetic rationale and clinical experience. The aim is to provide an updated approach on how to use the pharmacological and pharmacogenetic profile to improve clozapine efficacy and tolerance in severely ill patients.
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Antipsicóticos , Clozapina , Transtornos Mentais , Psiquiatria , Antipsicóticos/efeitos adversos , Clozapina/efeitos adversos , Fluvoxamina/efeitos adversos , Humanos , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/genética , FarmacogenéticaRESUMO
Voriconazole is one of the most used antifungal azoles against pulmonary aspergillosis. Therapeutic drug monitoring (TDM) of the voriconazole concentration in plasma is recommended in clinical practice guidelines to prevent treatment failure and toxicity. The aim of this study was to evaluate the feasibility and utility of TDM of the voriconazole concentration in the sputum of patients treated for pulmonary aspergillosis. Fifty sputum and 31 plasma samples were analysed with high-performance tandem mass spectrometry (HPLC-MS/MS) in 24 patients included in the study. The voriconazole concentration was simultaneously assessed in the plasma and sputum in 22 samples. The correlation between the sputum and plasma levels was estimated with a univariate linear regression model, and the observed R2 was 0.86. We determined the following equation, Csputum = 0.45 (Cplasma) + 0.21, which could predict the voriconazole concentration in plasma from sputum. TDM of the voriconazole concentration in sputum is an easy, non-invasive and accurate method with which to evaluate voriconazole exposure in patients with pulmonary aspergillosis.
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Tissue plasminogen activator (t-PA) converts plasminogen into the serine protease plasmin, which in turn degrades fibrin clots. This study assessed whether an increase in shear stress is associated in humans in vivo with the release of t-PA in peripheral conduit arteries, the impact of high blood pressure and the role of NO and CYP450-derived epoxyeicosatrienoic acids (EETs). Local t-PA levels were quantified at baseline and during a sustained increase in radial artery wall shear stress induced by hand skin heating (from 34 to 44 °C) in a total of 25 subjects, among whom 8 were newly diagnosed essential hypertensive patients. The impact of the brachial infusion of NO synthase (L-NMMA) and CYP450 inhibitors (fluconazole) on t-PA release was assessed. The increase in shear stress induced by heating was associated with an increase in local t-PA release (from 3.0 ± 0.5 to 19.2 ± 5.5 ng/min, n = 25, P < 0.01). The magnitude of t-PA release was positively correlated with the increase in shear stress (r = 0.64, P < 0.001) and negatively correlated with mean blood pressure (r = -0.443, P = 0.027). These associations persisted after multiple adjustments for confounding factors. Finally, t-PA release was reduced by L-NMMA and to a larger extent by the combination of L-NMMA and fluconazole without a change in shear stress. The increase in wall shear stress in the peripheral conduit arteries induces a release of t-PA by a mechanism involving NO and EETs. The alteration of this response by high blood pressure may contribute to reducing the fibrinolytic potential and enhancing the risk of arterial thrombosis during exercise.
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Artérias , Endotélio Vascular , Hipertensão , Ativador de Plasminogênio Tecidual , Artérias/fisiopatologia , Endotélio Vascular/fisiologia , Humanos , Hipertensão/fisiopatologia , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
The human adrenal cortex is a complex organ which is composed of various cell types including not only steroidogenic cells but also mesenchymal cells, immunocompetent cells and neurons. Intermingling of these diverse cell populations favors cell-to-cell communication processes involving local release of numerous bioactive signals such as biogenic amines, cytokines and neuropeptides. The resulting paracrine interactions play an important role in the regulation of adrenocortical cell functions both in physiological and pathophysiological conditions. Especially, recent evidence indicates that adrenocortical cell microenvironment is involved in the pathogenesis of adrenal disorders associated with corticosteroid excess. The paracrine factors involved in these intraadrenal regulatory mechanisms may thus represent valuable targets for future pharmacological treatments of adrenal diseases.
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Corticosteroides/metabolismo , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Microambiente Celular , Citocinas/metabolismo , Humanos , Neuropeptídeos/metabolismo , Comunicação Parácrina , Transdução de SinaisRESUMO
Dabigatran, rivaroxaban, apixaban, edoxaban, and betrixaban are direct oral anticoagulants (DOACs). Their inter-individual variability in pharmacodynamics and pharmacokinetics (transport and metabolism) is high, and could result from genetic polymorphisms. As recommended by the French Network of Pharmacogenetics (RNPGx), the management of some treatments in cardiovascular diseases (as antiplatelet agents, oral vitamin K antagonists, and statins) can rely on genetic testing in order to improve healthcare by reducing therapeutic resistance or toxicity. This paper is a review of association studies between single nucleotide polymorphisms (SNPs) and systemic exposure variation of DOACs. Most of the results presented here have a lot to do with some SNPs of CES1 (rs2244613, rs8192935, and rs71647871) and ABCB1 (rs1128503, rs2032582, rs1045642, and rs4148738) genes, and dabigatran, rivaroxaban, and apixaban. Regarding edoxaban and betrixaban, as well as SNPs in the CYP3A4 and CYP3A5 genes, literature is scarce, and further studies are needed.
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Despite its drastic efficacy in resistant psychiatric disorders, clozapine remains rarely used in youth due to its side effects. Clozapine plasma level is determined through its metabolism involving several isoforms of cytochromes 450 (CYP450) family. Isoform CYP1A2 appears as a limiting enzyme involved in the metabolism of clozapine, while isoforms 2C19, 2D6, 3A4, and 3A5 also contribute in a minor way. Clozapine efficacy is limited by a significant inter-patient variability in exposure according to CYP's polymorphisms. Clozapine plasma levels may be increased with CYP inhibitors such as fluvoxamine. This drug is a potent enzymatic inhibitor of CYP1A2 and, to a lesser extent, of CYP3A4 and CYP2D6. Hence, in case of CYP's polymorphisms in youth, the use of fluvoxamine as add-on to clozapine could help in reaching clinical and biological efficacy and allowing lower clozapine dosage and a better tolerance profile as it has already been described in adults. We report four pediatric cases with severe psychiatric disorders underlying our experience with CYP polymorphism explorations and the use of fluvoxamine as add-on to clozapine. Our four patients clinically improved after the introduction of fluvoxamine, enhancing clozapine metabolism and therefore the clozapine plasma level within therapeutic range. Despite the interesting results of fluvoxamine, we report a severe issue of tolerance for one patient, emphasizing the need for caution regarding possible drug interactions when fluvoxamine is considered. Hence, we propose a detailed step-by-step multidisciplinary protocol.
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Aldosterone, produced by the adrenals and under the control of plasma angiotensin and potassium levels, regulates hydromineral homeostasis and blood pressure. Here we report that the neuropeptide substance P (SP) released by intraadrenal nerve fibres, stimulates aldosterone secretion via binding to neurokinin type 1 receptors (NK1R) expressed by aldosterone-producing adrenocortical cells. The action of SP is mediated by the extracellular signal-regulated kinase pathway and involves upregulation of steroidogenic enzymes. We also conducted a prospective proof-of-concept, double blind, placebo-controlled clinical trial aimed to investigate the impact of the NK1R antagonist aprepitant on aldosterone secretion in healthy male volunteers (EudraCT: 2008-003367-40, ClinicalTrial.gov: NCT00977223). Participants received during two 7-day treatment periods aprepitant (125 mg on the 1st day and 80 mg during the following days) or placebo in a random order at a 2-week interval. The primary endpoint was plasma aldosterone levels during posture test. Secondary endpoints included basal aldosterone alterations, plasma aldosterone variation during metoclopramide and hypoglycaemia tests, and basal and stimulated alterations of renin, cortisol and ACTH during the three different stimulatory tests. The safety of the treatment was assessed on the basis of serum transaminase measurements on days 4 and 7. All pre-specified endpoints were achieved. Aprepitant decreases aldosterone production by around 30% but does not influence the aldosterone response to upright posture. These results indicate that the autonomic nervous system exerts a direct stimulatory tone on mineralocorticoid synthesis through SP, and thus plays a role in the maintenance of hydromineral homeostasis. This regulatory mechanism may be involved in aldosterone excess syndromes.
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Aldosterona/sangue , Aprepitanto/farmacologia , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores da Neurocinina-1/metabolismo , Substância P/metabolismo , Adolescente , Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/metabolismo , Adulto , Aldosterona/metabolismo , Células Cultivadas , Humanos , Hipoglicemia/sangue , Masculino , Metoclopramida , Mineralocorticoides/biossíntese , Placebos/administração & dosagem , Estudo de Prova de Conceito , Estudos Prospectivos , Transaminases/sangue , Adulto JovemRESUMO
BACKGROUND AND AIMS: The mechanisms involved in endothelial dysfunction in humans during aging are largely unknown at the level of conduit arteries. We aimed to asses the role of NO and CYP450 epoxygenases-derived epoxyeicosatrienoic acids (EETs) in the regulation of endothelium-dependent flow-mediated dilatation of conduit arteries during aging. METHODS: Radial artery diameter and mean wall shear stress were determined by echotracking coupled with Doppler in 83 subjects (19-71 years old) during a sustained flow increase induced by hand skin heating, with the brachial infusion of saline or NO-synthase and cytochrome P450 epoxygenase inhibitors (L-NNMA and fluconazole respectively). Local blood sampling was performed for the quantification of NO metabolite nitrite and EETs. RESULTS: The magnitude of flow-mediated dilatation was independently and negatively correlated with age, baseline artery diameter and systolic blood pressure, and positively correlated with the increase in shear stress induced by heating. There was an increase in nitrite level during heating until the age of 35-40 years, which declined thereafter. However, the inhibitory effect of L-NMMA on flow-mediated dilatation progressively decreased during aging, demonstrating a decrease in functional NO availability. Moreover, aging progressively reduced the increase in EET level during heating as well as the inhibitory effect of fluconazole on flow-mediated dilatation. CONCLUSIONS: These results show that aging impairs the availability of EETs and NO and epoxyeicosatrienoic acids in peripheral conduit arteries, contributing to the development of endothelial dysfunction.
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Envelhecimento/sangue , Eicosanoides/sangue , Endotélio Vascular/metabolismo , Óxido Nítrico/metabolismo , Doença Arterial Periférica/sangue , Artéria Radial/metabolismo , Vasodilatação , Adulto , Fatores Etários , Idoso , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Endotélio Vascular/diagnóstico por imagem , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiopatologia , Feminino , Fluconazol/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitritos/sangue , Doença Arterial Periférica/diagnóstico por imagem , Doença Arterial Periférica/fisiopatologia , Artéria Radial/diagnóstico por imagem , Artéria Radial/efeitos dos fármacos , Artéria Radial/fisiopatologia , Ultrassonografia Doppler , Vasodilatação/efeitos dos fármacos , Adulto Jovem , ômega-N-Metilarginina/administração & dosagemRESUMO
Diabetes induces a decrease in the number and function of different pro-angiogenic cell types generically designated as putative endothelial progenitor cells (EPC), which encompasses cells from myeloid origin that act in a paracrine fashion to promote angiogenesis and putative "true" EPC that contribute to endothelial replacement. This not only compromises neovasculogenesis in ischemic tissues but also impairs, at an early stage, the reendotheliziation process at sites of injury, contributing to the development of endothelial dysfunction and cardiovascular complications. Hyperglycemia, insulin resistance and dyslipidemia promote putative EPC dysregulation by affecting the SDF-1/CXCR-4 and NO pathways and the p53/SIRT1/p66Shc axis that contribute to their mobilization, migration, homing and vasculogenic properties. To optimize the clinical management of patients with hypoglycemic agents, statins and renin-angiotensin system inhibitors, which display pleiotropic effects on putative EPC, is a first step to improve their number and angiogenic potential but specific strategies are needed. Among them, mobilizing therapies based on G-CSF, erythropoietin or CXCR-4 antagonism have been developed to increase putative EPC number to treat ischemic diseases with or without prior cell isolation and transplantation. Growth factors, genetic and pharmacological strategies are also evaluated to improve ex vivo cultured EPC function before transplantation. Moreover, pharmacological agents increasing in vivo the bioavailability of NO and other endothelial factors demonstrated beneficial effects on neovascularization in diabetic ischemic models but their effects on endothelial dysfunction remain poorly evaluated. More experiments are warranted to develop orally available drugs and specific agents targeting p66Shc to reverse putative EPC dysfunction in the expected goal of preventing endothelial dysfunction and diabetic cardiovascular complications.