Pharmacological blockage of the AHR-CYP1A1 axis: a call for in vivo evidence.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that can be activated by structurally diverse compounds arising from the environment and the microbiota and host metabolism. Expanding evidence has been shown that the modulation of the canonical pathway of AHR occurs during several chronic diseases and that its abrogation might be of clinical interest for metabolic and inflammatory pathological processes. However, most of the evidence on the pharmacological abrogation of the AHR-CYP1A1 axis has been reported in vitro, and therefore, guidance for in vivo studies is needed. In this review, we cover the state-of-the-art of the pharmacodynamic and pharmacokinetic properties of AHR antagonists and CYP1A1 inhibitors in different in vivo rodent (mouse or rat) models of disease. This review will serve as a road map for those researchers embracing this emerging therapeutic area targeting the AHR. Moreover, it is a timely opportunity as the first AHR antagonists have recently entered the clinical stage of drug development.

Investigator Initiated Clinical Trials (IICTs): A Systematic Search in Registries to Compare the Czech Republic and Portugal in Terms of Funding Policies and Scientific Outcomes.

OBJECTIVES: Clinical trials provide one of the highest levels of evidence to support medical practice. Investigator initiated clinical trials (IICTs) answer relevant questions in clinical practice that may not be addressed by industry. For the first time, two European Countries are compared in terms of IICTs, respective funders and publications, envisaging to inspire others to use similar indicators to assess clinical research outcomes. METHODS: A retrospective systematic search of registered IICTs from 2004 to 2017, using four clinical trials registries was carried out in two European countries with similar population, GDP, HDI and medical schools but with different governmental models to fund clinical research. Each IICT was screened for sponsors, funders, type of intervention and associated publications, once completed. RESULTS: IICTs involving the Czech Republic and Portugal were n = 439 (42% with hospitals as sponsors) and n = 328 (47% with universities as sponsors), respectively. The Czech Republic and Portuguese funding agencies supported respectively 61 and 27 IICTs. Among these, trials with medicinal products represent 52% in Czech Republic and 4% in Portugal. In the first, a higher percentage of IICTs' publications in high impact factor journals with national investigators as authors was observed, when compared to Portugal (75% vs 15%). CONCLUSION: The better performance in clinical research by Czech Republic might be related to the existence of specific and periodic funding for clinical research, although further data are still needed to confirm this relationship. In upcoming years, the indicators used herein might be useful to tracking clinical research outcomes in these and other European countries.

European survey on national training activities in clinical research.

BACKGROUND: Investigator-initiated clinical studies (IITs) are crucial to generate reliable evidence that answers questions of day-to-day clinical practice. Many challenges make IITs a complex endeavour, for example, IITs often need to be multinational in order to recruit a sufficient number of patients. Recent studies highlighted that well-trained study personnel are a major factor to conduct such complex IITs successfully. As of today, however, no overview of the European training activities, requirements and career options for clinical study personnel exists. METHODS: To fill this knowledge gap, a survey was performed in all 11 member and observer countries of the European Clinical Research Infrastructure Network (ECRIN), using a standardised questionnaire. Three rounds of data collection were performed to maximize completeness and comparability of the received answers. The survey aimed to describe the landscape of academic training opportunities, to facilitate the exchange of expertise and experience among countries and to identify new fields of action. RESULTS: The survey found that training for Good Clinical Practice (GCP) and investigator training is offered in all but one country. A specific training for study nurses or study coordinators is also either provided or planned in ten out of eleven countries. A majority of countries train in monitoring and clinical pharmacovigilance and offer specific training for principal investigators but only few countries also train operators of clinical research organisations (CRO) or provide training for methodology and quality management systems (QMS). Minimal requirements for study-specific functions cover GCP in ten countries. Only three countries issued no requirements or recommendations regarding the continuous training of study personnel. Yet, only four countries developed a national strategy for training in clinical research and the career options for clinical researchers are still limited in the majority of countries. CONCLUSIONS: There is a substantial and impressive investment in training and education of clinical research in the individual ECRIN countries. But so far, a systematic approach for (top-down) strategic and overarching considerations and cross-network exchange is missing. Exchange of available curricula and sets of core competencies between countries could be a starting point for improving the situation.

Essential competencies in prescribing: A first european cross-sectional study among 895 final-year medical students.

European medical students should have acquired adequate prescribing competencies before graduation, but it is not known whether this is the case. In this international multicenter study, we evaluated the essential knowledge, skills, and attitudes in clinical pharmacology and therapeutics (CPT) of final-year medical students across Europe. In a cross-sectional design, 26 medical schools from 17 European countries were asked to administer a standardized assessment and questionnaire to 50 final-year students. Although there were differences between schools, our results show an overall lack of essential prescribing competencies among final-year students in Europe. Students had a poor knowledge of drug interactions and contraindications, and chose inappropriate therapies for common diseases or made prescribing errors. Our results suggest that undergraduate teaching in CPT is inadequate in many European schools, leading to incompetent prescribers and potentially unsafe patient care. A European core curriculum with clear learning outcomes and assessments should be urgently developed.

Insulin resistance is associated with tissue-specific regulation of HIF-1α and HIF-2α during mild chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a feature of obstructive sleep apnea (OSA). Whereas clinical studies have demonstrated the association between OSA and insulin resistance, the molecular mechanisms behind it are still unknown. Herein we investigated the effect of mild CIH on insulin sensitivity and we evaluated the changes in insulin and HIF signaling pathways that occur in CIH-induced insulin resistance. We showed that mild CIH obtained by 5/6 hypoxic (5%O2) cycles/h, 10.5h/day during 28 and 35 days increased arterial blood pressure. Insulin resistance and insulinemia increased with CIH duration, being significantly different after 35 days of CIH. Thirty-five days of CIH decreased insulin receptor expression and phosphorylation in skeletal muscle and adipose tissue, but not in the liver. Conversely, Glut2 expression increased in the liver of CIH-animals. Thirty-five days of CIH up-regulated HIF-1α in the liver and down-regulated HIF-1α and HIF-2α in skeletal muscle. We concluded that the effect of CIH on insulin sensitivity and signaling is time-dependent and is associated with changes in HIF signaling in insulin-sensitive tissues.

Investigator-initiated clinical trials conducted by the Portuguese Clinical Research Infrastructure Network (PtCRIN).

Interventional clinical studies can provide the highest levels of evidence and generate significant results on specific investigational medicinal products or medical devices. In order to have powerful studies, attain unquestionable results and make significant discoveries, the number of patients enrolled must be high. Therefore, multinational, randomised clinical trials are necessary. The multicentre, multinational recruitment of subjects in investigator-initiated clinical trials (IICTs) increases their logistical burden, justifying the need for specific infrastructures to ease implementation. Herein, we provide for the first time an overview of the facts and figures concerning IICTs, existing infrastructures' capacity for interventional clinical research, and scientific performance of investigators in a European country, Portugal. We aim to highlight the relevance and need for investing in European infrastructures such as the European Clinical Research Infrastructure Network (ECRIN) for multinational IICTs. A public, non-profit organisation, ECRIN facilitates the conduct of multinational clinical trials in Europe by coordinating scientific partners and their networks, and providing advice, management services and tools to enhance collaboration. Currently in Portugal, few multinational randomised IICTs are coordinated by national investigators. This is most likely due to the lack of human resources dedicated to clinical trials in clinical research centres (CRCs) as well as the scarcity of professional academic clinical trial units (CTUs) providing logistics and management services at non-profit rates. With the data shown, we expect to trigger the development of similar studies in other European countries and stress the impact of government support for IICTs.

N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo.

BACKGROUND AND PURPOSE: The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. EXPERIMENTAL APPROACH: The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg(-1)) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. KEY RESULTS: An N-terminal valine haemoglobin adduct derived from an α,ß-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. CONCLUSIONS AND IMPLICATIONS: This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,ß-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations.

Hypoxic intensity: a determinant for the contribution of ATP and adenosine to the genesis of carotid body chemosensory activity.

Excitatory effects of adenosine and ATP on carotid body (CB) chemoreception have been previously described. Our hypothesis is that both ATP and adenosine are the key neurotransmitters responsible for the hypoxic chemotransmission in the CB sensory synapse, their relative contribution depending on the intensity of hypoxic challenge. To test this hypothesis we measured carotid sinus nerve (CSN) activity in response to moderate and intense hypoxic stimuli (7 and 0% O(2)) in the absence and in the presence of adenosine and ATP receptor antagonists. Additionally, we quantified the release of adenosine and ATP in normoxia (21% O(2)) and in response to hypoxias of different intensities (10, 5, and 2% O(2)) to study the release pathways. We found that ZM241385, an A(2) antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 30.8 and 72.5%, respectively. Suramin, a P(2)X antagonist, decreased the CSN discharges evoked by 0 and 7% O(2) by 64.3 and 17.1%, respectively. Simultaneous application of both antagonists strongly inhibited CSN discharges elicited by both hypoxic intensities. ATP release by CB increased in parallel to hypoxia intensity while adenosine release increased preferably in response to mild hypoxia. We have also found that the lower the O(2) levels are, the higher is the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia, while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high-intensity hypoxia.

Effect of efavirenz on high-density lipoprotein antioxidant properties in HIV-infected patients.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: * In previous work, we showed a long-term and concentration-dependent beneficial effect of the non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) on high-density lipoproteins (HDL) in human immunodeficiency virus (HIV)-infected patients. * Furthermore, it has been suggested that instead of the current practice of only measuring HDL-chelesterol values, the evaluation of HDL function, namely its antioxidant properties, might be an improved tool for identifying subjects at increased risk for cardiovascular events. * Paraoxonase-1 (PON-1) is an enzyme associated with HDL that is responsible for HDL antioxidant function. WHAT THIS STUDY ADDS: * In the present work, we studied the effect of EFV on the activity of PON-1 and showed, for the first time, that EFV-based antiretroviral therapy is associated with a better antioxidant function, i.e. with a higher PON-1 activity. AIMS: A long-term and concentration-dependent beneficial effect of efavirenz (EFV) on cholesterol associated with high-density lipoprotein (HDL-c) in human immunodeficiency virus (HIV)-infected patients has been documented. Furthermore, it has been suggested that, instead of the current practice of only measuring HDL-c values, the evaluation of HDL quality might be an improved tool for identifying subjects at increased risk of cardiovascular events. Paraoxonase-1 (PON-1) is an enzyme associated with HDL that is involved in the onset of cardiovascular disease and responsible for HDL antioxidant function. The aim of the present study was to investigate the effect of EFV on the circulating activity of PON-1 in HIV-infected patients. METHODS: The patients included were adults with a documented HIV-1 infection, nontreated or treated with antiretroviral regimens including EFV 600 mg once daily as first therapeutic regimen for at least 3 months. The influence of treatment with EFV, HDL-c and CD4 cell count on PON-1 activity was analysed. RESULTS: HIV-infected White patients treated with EFV had higher PON-1 activity [77.35 U l(-1) (65.66, 89.04)] (P < 0.05) and higher PON-1 activity : HDL-c ratio [1.88 (1.49, 2.28)] (P < 0.01) than untreated patients. PON-1 activity was higher in Black patients (P < 0.001) and in patients with a CD4 cell count >500 cells ml(-1) (P= 0.0120). CONCLUSIONS: EFV-based antiretroviral regimens are associated with HDL particles with a better antioxidant function, i.e. with a higher PON-1 activity. The PON-1 activity of Black patients is higher than that found in Whites regardless of treatment. Ethnicity should be taken into consideration when studying drug effects on PON-1 activity.

Adenosine in peripheral chemoreception: new insights into a historically overlooked molecule--invited article.

In the present article we review in a concise manner the literature on the general biology of adenosine signalling. In the first section we describe briefly the historical aspects of adenosine research. In the second section is presented the biochemical characteristics of this nucleoside, namely its metabolism and regulation, and its physiological actions. In the third section we have succinctly described the role of adenosine and its metabolism in hypoxia. The final section is devoted to the role of adenosine in chemoreception in the carotid body, providing a review of the literature on the presence of adenosine receptors in the carotid body; on the effects of adenosine at presynaptic level in carotid body chemoreceptor cells, as well as, its metabolism and regulation; and at postsynaptic level in carotid sinus nerve activity. Additionally, a review on the effects of adenosine in ventilation was done. This review discusses evidence for a key role of adenosine in the hypoxic response of carotid body and emphasizes new research likely to be important in the future.

The A(2B)-D(2) receptor interaction that controls carotid body catecholamines release locates between the last two steps of hypoxic transduction cascade.

We have recently demonstrated that adenosine controls the release of catecholamines (CA) from carotid body (CB) acting on A(2B) receptors. Here, we have investigated the hypothesis that this control is exerted via an interaction between adenosine A(2B) and dopamine D(2) receptors present in chemoreceptor cells and if it is, the location of this interaction on the CB hypoxic transduction cascade. Experiments were performed in vitro in CB from 3 months rats. The effect of adenosine A(2B) and dopamine D(2) receptor agonists applied alone or conjunctly, was studied on the basal and evoked release (10% O(2) and ionomycin) of CA from CB. We have observed that the inhibitory action of propylnorapomorphine, a D(2) selective agonist, on the normoxic and 10%O(2)-evoked release of CA was abolished by NECA, an A(2) agonist, meaning that an interaction between the D(2) and A(2B) receptors controls the release of CA from CB. Further, propylnorapomorphine inhibits the release of CA evoked by ionomycin, being this effect totally reversed by NECA. The present results provide direct pharmacological evidence that A(2B) and D(2) receptors interact to modulate the release of CA from rat CB between the steps of Ca(2+) entry and increase in intracellular free Ca(2+), and the activation of exocytosis and neurotransmitter release, of the stimulus-secretion coupling process.

Bicarbonate-regulated soluble adenylyl cyclase (sAC) mRNA expression and activity in peripheral chemoreceptors.

UNLABELLED: Peripheral arterial chemoreceptors in the carotid body (CB) are modulated by pH/CO(2). Soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate ions (HCO(3)). Because CO(2)/HCO(3) mediates depolarization in chemoreceptors, we hypothesized that sAC mRNA would be expressed in the CB, and its expression and function would be regulated by CO(2)/HCO(3).Sprague-Dawley rats at postnatal days 16-17 were used to compare sAC mRNA gene expression between CB and non-chemosensitive tissues: superior cervical (SCG), petrosal (PG) and nodose ganglia (NG) by quantitative real time-PCR. Rat sAC gene expression was standardized to the expression of GAPDH (housekeeping gene) and the data were analyzed with the Pfaffl method. Gene and protein expression, and sAC regulation in the testis was used as a positive control. To determine the regulation of sAC mRNA expression and activity, all tissues were exposed to increasing concentrations of bicarbonate (0, 24, 44 mM, titrated with CO(2) and maintained a constant pH of 7.40). RESULTS: sAC mRNA expression was between 2-11% of CB expression in the SCG, PG and NG. Furthermore, only in the CB did HCO(3) upregulate sAC gene expression and increase cAMP levels. CONCLUSION: sAC mRNA and protein expression is present in peripheral arterial chemoreceptors and non-chemoreceptors. In the CB, CO(2)/HCO(3) not only activated sAC but also regulated its expression, suggesting that sAC may be involved in the regulation of cAMP levels in response to hyper/hypocapnia.

Does ageing modify ventilatory responses to dopamine in anaesthetised rats breathing spontaneously?

Dopamine has been widely used in humans in the management of cardiocirculatory shock, and its inhibitory effect on ventilation has received particular attention in clinical situations more prevalent in the elderly. Dopamine has been extensively studied at the carotid body in adult animals but little is known in aged animals. We investigated the ventilatory responses caused by dopamine in 3 and 24 months old rats.Cumulative intracarotid bolus injections of dopamine were performed in anaesthetised and vagotomised rats, in the absence and in the presence of i.v. infusions of domperidone (23.5-1175 nmol Kg(-1) min(-1)). Airflow (V), tidal volume (V(T)), respiratory rate (f), arterial blood pressure and heart rate were monitored and respiratory minute volume (V(E)) calculated. Basal values of V(E) were lower in 24 months rats (322.9+/-18.8 mL Kg(-1) min(-1)) than in 3 months old rats (442.5+/-24.2 mL Kg(-1) min(-1)), mainly due to reductions in V(T). The dose-dependent decreases caused by dopamine (3-100 nmol) in V(T), f and V(E), were totally prevented by section of the carotid sinus nerve and were not modified by ageing. The maximal % antagonism of the inhibitory effect of dopamine on V(E) caused by domperidone was similar in both 3 (74.6+/-2.7) and 24 (70.7+/-0.8) months old rats. Domperidone alone, increased basal V(E) by 59.6+/-16.6 mL min(-1) Kg(-1), and by 11.8+/-1.2 mL min(-1) Kg(-1), respectively in 3 and 24 months old rats (p<0.01).The inhibitory basal tonus caused by dopamine in ventilation was reduced in aged rats, although the decrease in V(E) caused by its exogenous administration remained unchanged.

The study of steady magnetic fields associated with primary and secondary ST shift in ischaemic rabbit hearts.

The study of injury potentials associated with DC currents that generate the primary or secondary ST shifts during cardiac ischaemia is possible only through the invasive technique of the DC electrogram. Clinical surface ECG recordings are AC coupled and cannot be used. This paper reports the use of non-invasive and unshielded magnetocardiographic measurements to evaluate the DC injury currents associated with ST shifts during coronary artery occlusions in the isolated rabbit heart. The effect on the magnetic ST shift is studied under different ischaemic conditions including regional ischaemia, global ischaemia, global ischaemia following long periods of regional ischaemia, regional ischaemia after repeated episodes of reversible global ischaemia, and bilateral regional ischaemia. Recording of DC magnetic fields allows the characterization of primary and secondary ST displacement for each induced ischaemic condition. Our measurements show that the ST shift starts earlier when inducing ischaemia in hearts previously subjected to ischaemic episodes than in hearts where the ischaemia was produced for the first time.

Detection of reentry currents in atrial flutter by magnetocardiography.

We discuss the use of magnetocardiography to detect reentry currents in cardiac flutter and fibrillation. The magnetic field produced by induced atrial flutter was measured in isolated rabbit hearts. A moving dipole model is proposed to treat the experimental data and to locate the reentry path.

Adenosine and the bradycardiac response to vagus nerve stimulation in rats.

The effects of intracardiac infusions of adenosine on the changes in heart rate (HR), electrocardiogram (ECG) and arterial blood pressure (BP) induced by both vagal stimulation and exogenous acetylcholine (ACh) were studied in anesthetized rats. Adenosine inhibited the bradycardia induced by vagal nerve stimulation, an effect antagonized by theophylline, decreased the elongation caused by vagal stimulation of the R-R intervals of the ECG, and caused a small but consistent decrease in the hypotensive effect of vagus nerve stimulation. At the dose that reduced the bradycardiac responses to vagal stimulation, adenosine enhanced the bradycardiac effect of exogenous ACh, increased R-R intervals and the number of P waves not followed by the ECG and had little or no effect on the inhibition induced by ACh on BP. The effects of adenosine on the bradycardiac responses to vagal nerve stimulation or to ACh administration were similar in both non-reserpinized and reserpinized animals. These results suggest that exogenous adenosine can modify the vagal influences on the heart by exerting pre-junctional inhibition of the vagus nerve and post-junctional enhancement of the ACh actions, and that the adrenergic system does not contribute to these effects of adenosine.

Adenosine deaminase and adenosine uptake inhibitions facilitate ventilation in rats.

The effects of intracarotid (i.c.) infusions of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and of the adenosine uptake blocker, dipyridamole on spontaneous ventilation were studied in rats anaesthetized with sodium pentobarbitone. Both EHNA and dipyridamole mimicked the excitatory effect of adenosine on respiration increasing in a dose-dependent manner respiratory ventilation determined as increases in tidal volume (VT), respiratory frequency (f) and minute volume (VE). These excitatory effects were abolished after section of the carotid sinus nerves. The excitatory effect of EHNA on respiration was prevented by adenosine deaminase and antagonized by 1,3-dipropyl-8(p-sulfophenyl)xanthine (DPSPX). DPSPX also antagonized the excitatory effect of dipyridamole on respiration. Both EHNA and dipyridamole in doses virtually devoid of effect on respiration potentiated the excitatory effect of exogenous adenosine on respiration. Two different effects on respiration were observed during i.c. infusions of cumulative doses of DPSPX: one inhibitory, not present in glomectomized animals and another, excitatory, present in both glomectomized and non-glomectomized animals. It is concluded that endogenous adenosine could be involved in respiration mediated through carotid body chemoreceptors and that the nucleoside is inactivated at this level by deamination and uptake.

Inhibition by 1,3-dipropyl-8(p-sulfophenyl)xanthine of the respiratory stimulation induced by common carotid occlusion in rats.

The effects of 1,3-dipropyl-8(p-sulfophenyl)xanthine (DPSPX) and enprofylline on the respiratory responses to bilateral occlusions of the common carotid arteries (CCO) were studied in rats anesthetized with sodium pentobarbitone and breathing spontaneously. CCO during periods of 5, 10 and 15 s caused increases in tidal volume (VT) and respiratory frequency (f) dependent on duration of the occlusions, these effects were markedly reduced after section of the carotid sinus nerves. Transient increases in systemic arterial blood pressure (BP) associated with the ventilatory effects of CCO were also observed. Intracarotid infusions of DPSPX (100 nmol/min, for 3 min) but not of enprofylline (100 nmol/min, for 3 min) decreased the respiratory stimulation induced by CCO without modifying significantly the increases in BP. It is concluded that adenosine may be involved in the ventilatory responses to CCO that are mediated by carotid body chemoreceptors.