Considerations for assessing the potential effects of antidiabetes drugs on cardiac ventricular repolarization: A report from the Cardiac Safety Research Consortium.

Thorough QT studies conducted according to the International Council on Harmonisation E14 guideline are required for new nonantiarrhythmic drugs to assess the potential to prolong ventricular repolarization. Special considerations may be needed for conducting such studies with antidiabetes drugs as changes in blood glucose and other physiologic parameters affected by antidiabetes drugs may prolong the QT interval and thus confound QT/corrected QT assessments. This review discusses potential mechanisms for QT/corrected QT interval prolongation with antidiabetes drugs and offers practical considerations for assessing antidiabetes drugs in thorough QT studies. This article represents collaborative discussions among key stakeholders from academia, industry, and regulatory agencies participating in the Cardiac Safety Research Consortium. It does not represent regulatory policy.

Development of cardiac safety translational tools for QT prolongation and torsade de pointes.

OBJECTIVE: A regulatory science priority at the Food and Drug Administration (FDA) is to promote the development of new innovative tools such as reliable and validated computational (in silico) models. This FDA Critical Path Initiative project involved the development of predictive clinical computational models for decision-support in CDER evaluations of QT/QTc interval prolongation and proarrhythmic potential for non-antiarrhythmic drugs. METHODS: Several classification models were built using predictive technologies of quantitative structure-activity relationship analysis using clinical in-house and public data on induction of QT prolongation and torsade de pointes (TdP) in humans. Specific models were geared toward prediction of high-risk drugs with attention to outcomes from thorough QT studies and TdP risk based on clinical in-house data. Models used were independent of non-clinical data or known molecular mechanisms. The positive predictive performance of the in silico models was validated using cross-validation and independent external validation test sets. RESULTS: Optimal performance was observed with high sensitivity (87%) and high specificity (88%) for predicting QT interval prolongation using in-house data, and 77% sensitivity in predicting drugs withdrawn from the market. Furthermore, the article describes alerting substructural features based on drugs tested in the clinical trials. CONCLUSIONS: The in silico models provide evidence of a structure-based explanation for these cardiac safety endpoints. The models will be made publically available and are under continual prospective external validation testing and updating at CDER using TQT study outcomes.

The evaluation and management of drug effects on cardiac conduction (PR and QRS intervals) in clinical development.

Recent advances in electrocardiographic monitoring and waveform analysis have significantly improved the ability to detect drug-induced changes in cardiac repolarization manifested as changes in the QT/corrected QT interval. These advances have also improved the ability to detect drug-induced changes in cardiac conduction. This White Paper summarizes current opinion, reached by consensus among experts at the Cardiac Safety Research Consortium, on the assessment of electrocardiogram-based safety measurements of the PR and QRS intervals, representing atrioventricular and ventricular conduction, respectively, during drug development.

Long-term consequences of drugs on the paediatric cardiovascular system.

Many pharmacological and toxicological actions of drugs in children cannot be fully predicted from adult clinical experience or from standard non-clinical toxicology studies. Numerous drugs have direct or indirect pharmacological effects on the heart and are prescribed for children of all ages. Toxicity or secondary effects may be immediate or delayed for years after drug exposure has ceased. Originally, the aim of this review was to compile information on the effect of specific drugs on the post-natal development of the cardiovascular system and to examine long-term follow-up of the use of cardio-active drugs in children. The limited database of published information caused the original question to evolve into an examination of the medical literature for three areas of information: (i) whether vulnerable developmental windows have been identified that reflect the substantial functional development that the cardiovascular system undergoes after birth; (ii) what is known about pharmacological perturbation of development; and (iii) what the likelihood is of drug exposure during childhood. We examined different scenarios for exposure including random, isolated exposure, conditions historically associated with adults, primary or secondary cardiac disease, psychiatric and neurological conditions, asthma, cancer and HIV. Except for random, isolated drug exposures, each category of possible exposure contained numerous drugs known to have either primary or secondary effects on the cardiovascular system or to influence factors associated with atherosclerosis. It is likely that a significant number of children will be prescribed drugs having either direct or indirect effects upon the immature cardiovascular system. A confounding factor is the simultaneous use of over-the-counter medications and herbal or nutraceutical preparations that a patient, parent or guardian does not mention to a prescribing physician. Metabolism is also important in assessing drug effects in children. Differences in body water : body fat ratio, age-related gastrointestinal absorption, distribution, excretion, renal function and drug metabolizing capabilities make it possible for children to have a different metabolite profile for a drug compared with adults. There is little examination of drug effects on the interdependent processes of cardiac maturation and less examination of metabolite effects. It is difficult to identify delayed toxicities in children as these adverse events may take years to manifest with many patients lost to follow-up. Clearly this is an area of study where intermediate endpoints and surrogate markers would be of great benefit. Pharmacogenomics may be useful in providing markers of increased risk or susceptibility. A perspective must be kept in balancing the possibility of a problem with the very real benefits that many children experience from the use of these pharmaceuticals.

Presynaptic AMPA and kainate receptors increase the size of GABAergic terminals and enhance GABA release.

In the developing cerebellum, NMDA receptors promote the maturation of axonal terminals of inhibitory interneurons. We compared the effects of AMPA/kainate receptor agonists in cultured cerebellar cells from GAD65-eGFP mice. Both AMPA and kainate augmented granule cell survival without affecting interneurons. The action of kainate was blocked by an AMPA but not by a NMDA receptor antagonist, suggesting AMPA receptor involvement. AMPA and kainate increased the size of the GABAergic terminals and the action of kainate was insensitive to NMDA blockers. Whole cell recordings in granule neurons revealed that chronic treatment for 5 days with kainate as well as NMDA decreased AMPA receptor expression while interneuronal kainate receptors were depressed by kainate treatment. Acute kainate application increased mIPSCs frequency in both granule neurons and interneurons and this effect was only partially blocked by an AMPA receptor antagonist. In contrast to what was reported for NMDA, chronic treatment with kainate induced a significant decrease of the basal mIPSCs frequency but increased the acute action of kainate on mIPSCs. Direct recordings from presynaptic GABAergic terminals suggest that AMPA and kainate receptors are present in developing GABAergic terminals and their activation affects the size of GABAergic terminals and spontaneous GABA release.

NMDA receptors contribute to the survival promoting effect of high potassium in cultured cerebellar granule cells.

The present report further analyzes the survival promoting effect of high potassium, a condition that mimics neural activity in cultured cerebellar granule cells, an excellent model to study trophic mechanisms induced by depolarization and trophic factors. We found that the survival promoting effect measured at 7 days in vitro (DIV 7) of depolarizing potassium concentrations (25 mM KCl), added at DIV2, is partially prevented by adding at DIV 2 the non-competitive NMDA blocker MK801 (10 microM). The concentration of MK801 used blocks completely the survival promoting effect of a supramaximal effective concentration of NMDA (100 microM). The addition at DIV 2 of anti-brain derived neurotrophic factor (anti-BDNF) antibody, failed to modify the effect of high potassium. The present report provides evidences that in cultured cerebellar granule cells, high potassium-induced survival promoting effect is due in part by the activation of NMDA receptors. The effect does not require the presence of BDNF.

NMDA receptors increase the size of GABAergic terminals and enhance GABA release.

In developing cerebellar interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors. We investigated the role of these receptors on differentiating basket/stellate cells in cerebellar cultures grown under conditions allowing functional synaptic transmission. Presynaptic GABAergic boutons were visualized either by GAD65 immunostaining or by using cells derived from GAD65-enhanced green fluorescent protein (eGFP) transgenic mice, in which cerebellar basket/stellate cells express eGFP. After the first week in culture, whole-cell recordings from granule cells reveal that acute application of NMDA increases miniature IPSC (mIPSC) frequency. Interestingly, after 2 weeks, the mIPSC frequency increases compared with the first week but is not modulated by NMDA. Furthermore, in cultures chronically treated with NMDA for 1 week, the size of the GABAergic boutons increases. This growth is paralleled by increased mIPSC frequency and the loss of NMDA sensitivity. Direct patch-clamp recording from these presynaptic terminals reveals single NMDA-activated channels, showing multiple conductance levels, and electronic propagation from the somatodendritic compartment. Our results demonstrate that NMDA receptors alter GABAergic synapses in developing cerebellar cultures by increasing the size of the terminal and spontaneous GABA release. These findings parallel changes in inhibitory synaptic efficacy seen in vivo in developing GABAergic interneurons of the molecular layer of the cerebellum.

Role of calcium and kinases on the neurotrophic effect induced by gamma-aminobutyric acid.

An increasing body of evidence supports a trophic action of gamma-aminobutyric acid (GABA) during nervous system development. The purported mediator of these trophic effects is a depolarizing response triggered by GABA, which elicits a calcium influx in immature CNS cells. This Mini-Review focuses on the neurotrophic role of neural activity and GABA and some of the most common intracellular cascades activated by depolarization and trophic factors. Several biological effects induced by GABA in the developing nervous system are reviewed, with particular emphasis on what is known about calcium-dependent neurotrophic effects induced by GABA and its intracellular mechanisms.

Evidence of oxidative stress in familial amyloidotic polyneuropathy type 1.

OBJECTIVE: To evaluate the oxidative state in patients with familial amyloidotic polyneuropathy type 1 (FAP1). DESIGN: From 3 unrelated families, patients with FAP1 carrying a transthyretin Met-30 mutation were studied. The diagnosis was confirmed by genetic analysis. Eleven of 21 patients carried the mutation; all were symptomatic and were clinically assessed using a clinical score. All of the patients were evaluated for copper-zinc superoxide dismutase type 1 activity in red blood cells using spectrophotometry. Plasma total reactive antioxidant potential was studied using a chemiluminescent method. The results were compared with those obtained from an age-matched control group. SETTING: A public and academic multidisciplinary research clinic. RESULTS: Six of the 11 FAP1-positive patients disclosed superoxide dismutase type 1 activity values greater than 55 U/mg of protein (upper control limit), whereas 9 of 10 patients in whom total reactive antioxidant potential was measured had values below the lower limit of the control group. No relationship was found between the levels of superoxide dismutase type 1 activity and the severity of the clinical involvement. CONCLUSIONS: Oxidative stress may be part of the mechanisms leading to tissue damage in patients with FAP1. The lack of correlation between the laboratory findings and the severity of clinical involvement may signal that oxidative processes are at work throughout the natural history of the disease.

GABA-induced neurite outgrowth of cerebellar granule cells is mediated by GABA(A) receptor activation, calcium influx and CaMKII and erk1/2 pathways.

During neuronal development, GABAA-mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1-1000 microm) induces an increase in the intracellular calcium concentration through the activation of GABA(A) receptors and voltage-gated calcium channels of the L-subtype. Perforated patch-clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to - 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium-dependent mechanism triggered by voltage-gated calcium channel activation. Specific blockers of calcium-calmodulin kinase II and mitogen-activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium-dependent kinases.

The modulation of mitochondrial nitric-oxide synthase activity in rat brain development.

Different mitochondrial nitric-oxide synthase (mtNOS) isoforms have been described in rat and mouse tissues, such as liver, thymus, skeletal muscle, and more recently, heart and brain. The modulation of these variants by thyroid status, hypoxia, or gene deficiency opens a broad spectrum of mtNOS-dependent tissue-specific functions. In this study, a new NOS variant is described in rat brain with an M(r) of 144 kDa and mainly localized in the inner mitochondrial membrane. During rat brain maturation, the expression and activity of mtNOS were maximal at the late embryonic stages and early postnatal days followed by a decreased expression in the adult stage (100 +/- 9 versus 19 +/- 2 pmol of [(3)H]citrulline/min/mg of protein, respectively). This temporal pattern was opposite to that of the cytosolic 157-kDa nNOS protein. Mitochondrial redox changes followed the variations in mtNOS activity: mtNOS-dependent production of hydrogen peroxide was maximal in newborns and decreased markedly in the adult stage, thus reflecting the production and utilization of mitochondrial matrix nitric oxide. Moreover, the activity of brain Mn-superoxide dismutase followed a developmental pattern similar to that of mtNOS. Cerebellar granular cells isolated from newborn rats and with high mtNOS activity exhibited maximal proliferation rates, which were decreased by modifying the levels of either hydrogen peroxide or nitric oxide. Altogether, these findings support the notion that a coordinated modulation of mtNOS and Mn-superoxide dismutase contributes to establish the rat brain redox status and participate in the normal physiology of brain development.

Contribution of Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase to neural activity-induced neurite outgrowth and survival of cerebellar granule cells.

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.

Inhibition by dopamine of the neurotransmission in the rat vas deferens

Se investigaron los efectos de agonistas y antagonistas alfa -adrenérgicos y dopaminérgicos sobre los receptores presinápticos de la porción prostática del conducto defrerente de rata. La variable estudiada fue el primer componente (250 ms) de la respuesta motora inducida por estimulación eléctrica de campo (pulso único). Con el objeto de bloquear los sitios de pérdida de las aminas, todos los experimentos se llevaron a cabo en presencia de cocaína 30 micronmol/l e hidrocortisona 28 micronmol/l. Asimismo, se empleó 1-propranolol 0.3 micronmol/l para bloquear los receptores ß -adrenérgiocs. Clonidina, noradrenalina (NA) y dopamina (DA) inhibieron la respuesta motora inducida por la aplicación de un pulso eléctrico. Este efecto fue, para los tres agonistas, dependiente de las concentraciones utilizadas. DA fue 10 y 10**4 veces menos potente que NA y clonidina respectivamente. El agonista selectivo D2, LY 141865, no logró inhibir la respuesta motora inclusive a una alta concentración (30 micronmol/l). Yohimbina (0.1, 0.3 y 1 micronmol/l) antagonizó en forma competitiva el efecto inhibitorio de clonidina, NA y DA, presentando valores similares de -log KB (7.57, 7.68 y 7.09 respectivamente). De manera análoga, idaxozán 0.03 micronmol/l bloqueó el efecto inhibitorio de DA con una potencia similar (- log KB = 7.81) a la de yohimbina. Por otra parte, pimozide 0.21 micronmol/l y Schering 23390 3 micronmol/l antagonizaron el efecto inhibitorio de DA, mostrando una potencia menor que los antagonistas alfa2 -adrenérgicos...

Inhibition by dopamine of the neurotransmission in the rat vas deferens

Se investigaron los efectos de agonistas y antagonistas alfa -adrenérgicos y dopaminérgicos sobre los receptores presinápticos de la porción prostática del conducto defrerente de rata. La variable estudiada fue el primer componente (250 ms) de la respuesta motora inducida por estimulación eléctrica de campo (pulso único). Con el objeto de bloquear los sitios de pérdida de las aminas, todos los experimentos se llevaron a cabo en presencia de cocaína 30 micronmol/l e hidrocortisona 28 micronmol/l. Asimismo, se empleó 1-propranolol 0.3 micronmol/l para bloquear los receptores ß -adrenérgiocs. Clonidina, noradrenalina (NA) y dopamina (DA) inhibieron la respuesta motora inducida por la aplicación de un pulso eléctrico. Este efecto fue, para los tres agonistas, dependiente de las concentraciones utilizadas. DA fue 10 y 10**4 veces menos potente que NA y clonidina respectivamente. El agonista selectivo D2, LY 141865, no logró inhibir la respuesta motora inclusive a una alta concentración (30 micronmol/l). Yohimbina (0.1, 0.3 y 1 micronmol/l) antagonizó en forma competitiva el efecto inhibitorio de clonidina, NA y DA, presentando valores similares de -log KB (7.57, 7.68 y 7.09 respectivamente). De manera análoga, idaxozán 0.03 micronmol/l bloqueó el efecto inhibitorio de DA con una potencia similar (- log KB = 7.81) a la de yohimbina. Por otra parte, pimozide 0.21 micronmol/l y Schering 23390 3 micronmol/l antagonizaron el efecto inhibitorio de DA, mostrando una potencia menor que los antagonistas alfa2 -adrenérgicos... (AU)