Drug-induced QTC prolongation dangerously underestimates proarrhythmic potential: lessons from terfenadine.

BACKGROUND: Terfenadine's proarrhythmia prompted market withdrawal; therapeutic antihistaminic concentration is less than 1 nM, whereas IC50 of IKr and INa exceed 200 nM. METHODS AND RESULTS: Rabbit hearts were perfused with terfenadine (1-10,000 nM; 10-450 minutes). A dosage of 1 nM tended to shorten action potential duration (APD60) (-30 ± 30.5 ms; n = 6); 10 nM (450 minutes) significantly prolonged APD60 (46 ± 11 ms; n = 6), but after 1 hour washout, APD60 further prolonged. Above 30 nM, APD60 shortening was followed by prolongation; net effect depended on exposure time (n = 33). In the µM range, cardiac wavelength (λ) shortened (APD60 shortened, conduction slowed; P < 0.05). Terfenadine induced triangulation, reverse use dependence, instability and dispersion of repolarization (TRIaD) at 1 to 1000 nM, increasing with concentration (450 minutes: 1 nM yielded 50% of hearts, 10 nM 100%) and exposure (100 nM: 10 minutes yielded 16%, 30 minutes 33%, 150 minutes 66%, 450 minutes 100%). TRIaD with APD prolongation preceded two Torsade de Pointes, with shortening seven ventricular tachycardia and five ventricular fibrillation. Terfenadine causes normally little QTc prolongation in patients and Food and Drug Administration records suggest that incidence of ventricular tachycardia/ventricular fibrillation exceeds Torsade de Pointes. CONCLUSION: For terfenadine, TRIaD predicts drug-induced proarrhythmia: with λ prolongation, Torsade de Pointes is preferred, otherwise ventricular tachycardia/ventricular fibrillation. APD/QTc alone is clearly inadequate for proarrhythmia evaluation.

Overexpression of endothelial nitric oxide synthase suppresses features of allergic asthma in mice.

BACKGROUND: Asthma is associated with airway hyperresponsiveness and enhanced T-cell number/activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma. METHODS: The contribution of eNOS on the development of asthmatic features was examined. We used transgenic mice that overexpress eNOS and measured characteristic features of allergic asthma after sensitisation and challenge of these mice with the allergen ovalbumin. RESULTS: eNOS overexpression resulted in both increased eNOS activity and NO production in the lungs. Isolated thoracic lymph nodes cells from eNOS overexpressing mice that have been sensitized and challenged with ovalbumin produced significantly less of the cytokines IFN-gamma, IL-5 and IL-10. No difference in serum IgE levels could be found. Further, there was a 50% reduction in the number of lymphocytes and eosinophils in the lung lavage fluid of these animals. Finally, airway hyperresponsiveness to methacholine was abolished in eNOS overexpressing mice. CONCLUSION: These findings demonstrate that eNOS overexpression attenuates both airway inflammation and airway hyperresponsiveness in a model of allergic asthma. We suggest that a delicate balance in the production of bioactive forms of NO derived from eNOS might be essential in the pathophysiology of asthma.

In search of a novel anti-HIV drug: multidisciplinary coordination in the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-dimethylphenyl]amino]-2- pyrimidinyl]amino]benzonitrile (R278474, rilpivirine).

Ideally, an anti-HIV drug should (1) be highly active against wild-type and mutant HIV without allowing breakthrough; (2) have high oral bioavailability and long elimination half-life, allowing once-daily oral treatment at low doses; (3) have minimal adverse effects; and (4) be easy to synthesize and formulate. R278474, a new diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitor (NNRTI), appears to meet these criteria and to be suitable for high compliance oral treatment of HIV-1 infection. The discovery of R278474 was the result of a coordinated multidisciplinary effort involving medicinal chemists, virologists, crystallographers, molecular modelers, toxicologists, analytical chemists, pharmacists, and many others.

In vitro effects of risperidone and 9-hydroxy-risperidone on human platelet function, plasma coagulation, and fibrinolysis.

BACKGROUND: Thrombotic events have been reported with the use of antipsychotic compounds, although the incidence, predisposing factors, and biological mechanisms associated with these events in psychiatric patients are subject to debate. OBJECTIVE: The in vitro actions of risperidone and its active metabolite 9-hydroxy-risperidone (9-OH-risperidone) on human platelet function, plasma coagulation, and fibrinolysis were examined to explore whether hematologic effects might be a mechanism for thrombotic events with these compounds. METHODS: Blood was donated by healthy white male subjects who were free of medications (particularly acetylsalicylic acid and nonsteroidal anti-inflammatory compounds). Platelet shape change and adhesion/aggregation reactions to risperidone and 9-OH-risperidone induced by adenosine diphosphate (ADP), collagen, epinephrine, and 5-hydroxytryptamine (5-HT) were tested in human platelet-rich plasma. Arachidonic acid metabolism was assessed in human platelets and rat aortic rings. Plasma coagulation was tested in human platelet-poor plasma. Fibrinolysis was measured in human whole blood. RESULTS: The 12 study subjects ranged in age from 20 to 40 years (median age 30 years). At concentrations of 1 x 10(-5) mol/L (approximately 4180 ng/mL), neither risperidone nor 9-OH-risperidone induced platelet shape change or aggregation, amplified reactions to ADP, or modified platelet adhesion/aggregation induced by collagen or ADP, but they did attenuate epinephrine-induced platelet aggregation (-50% in the case of 9-OH-risperidone; P < 0.05) and 5-HT-induced platelet aggregation (drug concentrations yielding 50% inhibition of 5-HT-induced platelet aggregation, 0.5 and 0.2 ng/mL, respectively). Cyclooxygenase, thromboxane A2 synthase, 12-lipoxygenase, prostacyclin synthase, plasma coagulation, and fibrinolysis were unaffected. CONCLUSIONS: Risperidone and 9-OH-risperidone reduced epinephrine- and 5-HT-induced human platelet aggregation but did not significantly alter other measures of platelet function, plasma coagulation, or fibrinolysis in vitro.

Differential responsiveness of proximal and distal parts of isolated guinea pig trachea.

This study addressed the question whether proximal and distal guinea pig tracheal segments respond differently to contractile agents. Using a perfused trachea set-up, histamine, KCl or the cyclo-oxygenase inhibitor, indomethacin, could be administered selectively to the mucosa (at the inside) or the serosa (at the outside) of the tracheal segments. Proximal parts contracted significantly more (40-60%) than distal parts when 1 mM histamine was administered to the mucosal or serosal side or when KCl (50 mM) was added to the serosal side. When histamine was administered to the mucosal side of epithelium-denuded segments, the contractions were twice as high in proximal than in distal parts (3057 vs. 1526 mg). Inhibition of tracheal cyclo-oxygenase with indomethacin at the mucosal side increased proximal and distal reactivity to mucosally administered histamine to the same extent. Serosal administration of indomethacin, however, increased histamine reactivity only in proximal segments (from 2690 to 5180 mg). In the latter segments, subsequent administration of histamine to the serosal side further increased the contraction, while serosal histamine in the absence of serosal indomethacin produced a relaxation (net difference of 4672 mg). In conclusion, the higher intrinsic contractility of proximal tracheal segments is counteracted by serosal cyclo-oxygenase products.

Drug-induced long QT in isolated rabbit Purkinje fibers: importance of action potential duration, triangulation and early afterdepolarizations.

In the present study, we investigated three drug-induced long-QT syndromes in isolated rabbit Purkinje fibers in order to identify the relationship of action potential duration (APD), triangulation of action potentials (APD(90)-APD(40)) and early afterdepolarizations. Isolated rabbit Purkinje fibers were superperfused in Tyrode solution with solvent, indapamide (1 x 10 (-4) M, an I(ks) blocker mimicking long QT1), dofetilide (1 x 10 (-9), 1 x 10 (-8) or 1 x 10 (-7) M, an I(kr) blocker mimicking long QT2) or anthopleurin (1 x 10 (-8) M, an inhibitor of the inactivation of the I(Na(+)) current mimicking long QT3) (n=8 per group) for 25 min, and stimulated at 1 Hz for 20 min and at 0.2 Hz for another 5 min. Indapamide did not change APD and triangulation or elicit early afterdepolarizations even in the presence of beta-adrenergic stimulation with isoproterenol. Dofetilide concentration-dependently prolonged APD(90), increased triangulation and elicited early afterdepolarizations. Anthopleurin markedly increased APD(90) as well as triangulation and elicited early afterdepolarizations. The induction of early afterdepolarizations by dofetilide and anthopleurin was associated with a prolongation of APD(90) or an increase in triangulation, but not with a change in APD(40). Moreover, the degree of the increase in the triangulation was larger than that of APD(90) in long QT2 (dofetilide-induced) and long QT3 (anthopleurin-induced) models in isolated rabbit Purkinje fibers. Our present study indicates that rabbit Purkinje fibers can be used as long QT2 (dofetilide-mimicking) and LQT3 (anthopleurin-mimicking) syndrome models, and confirms that drug-induced long QT1 (indapamide-mimicking) is absent. Our present study also shows the relationship between a prolongation of APD(90) or increase in triangulation and the induction of early afterdepolarizations with dofetilide (I(kr) blocker) and anthopleurin (I(Na) modulator) in isolated rabbit Purkinje fibers.

Ca2+ sensors modulate asthmatic symptoms in an allergic model for asthma.

We previously described two novel peptides, Ca2+-like peptide (CALP) 1 and CALP2, which interact with Ca2+-binding EF hand motifs, and therefore have the characteristics to define the role of the Ca2+-sensing regulatory protein calmodulin in asthma. In the present study, the effects of the calcium-like peptides were investigated in an animal model for allergic asthma. For that purpose, sensitized guinea pigs were intratracheally pretreated with CALP1 or CALP2. Thirty minutes later, the animals were challenged with aerosolized ovalbumin. Acute bronchoconstriction was measured as well as characteristic features of asthma 6 and 24 hours (h) after challenge. Neither CALP1 nor CALP2 prevented the anaphylactic response elicited by ovalbumin challenge. However, CALP1 pretreatment attenuated the influx of inflammatory cells in the lungs 6 h after challenge. Furthermore, radical production by these cells was diminished both 6 and 24 h after challenge. Moreover, CALP1 completely inhibited airway hyperresponsiveness in vitro 24 h after challenge. We conclude that CALP1, as a selective calmodulin agonist, inhibits the development of asthmatic features probably via the attenuation of mast cell degranulation and radical production. Specific modulation of calmodulin activity might therefore be a potential new target for the treatment of allergic asthma.

Review of the predictive value of the Langendorff heart model (Screenit system) in assessing the proarrhythmic potential of drugs.

Prolongation of the QTc interval of the electrocardiogram (ECG) is used as a surrogate marker for a rare, but life threatening, ventricular arrhythmia known as torsades de pointes (TdP). However, the clear link between QTc prolongation and the arrhythmogenic risk has not been demonstrated unequivocally. In the present review article, we examine (a) the current understanding of electrophysiological and pharmacological mechanisms linking changes in action potential (AP) properties with proarrhythmia and (b) the value of the isolated, paced Langendorff-perfused female rabbit heart model (Screenit system) in predicting the torsadogenic potential of drugs in man. The Screenit system records monophasic action potentials (MAPs) from which the following parameters are evaluated: action potential duration (APD), conduction, instability (indicative of beat to beat APD variability), triangulation (indicative of changes of Phase 3 repolarization), and reverse-use dependency (indicating that the APD is more prolonged at slow heart rates). So far, over 16,000 experiments have been conducted, including approximately 300 dedicated tests to evaluate, in a blinded manner, approximately 70 clinically used drugs. The drugs tested covered a wide range of compounds from various pharmacological and chemical classes with clinical torsadogenic propensity, as well as drugs without the latter effect in clinical settings. Overall, the Screenit system and its associated analysis classified the drugs based on their effects on AP morphology and conduction and additionally identified, in a qualitative manner, drugs clinically associated with TdP. Such an identification is based on the triangulation, reverse-use dependency, and instability of the AP, as well as on the direct indexes of proarrhythmia such as early afterdepolarization (EADs), ventricular tachycardia (VT), and ventricular fibrillation (VF). Overall, drugs that readily induce arrhythmia and/or EADs and/or causes triangulation, reverse-use dependency, and/or instability and/or a chaotic Poincaré plot in a range of concentrations likely to be achieved in man is likely to cause TdP in man, eventually. Only if none of these elements is present, at concentrations well exceeding the free therapeutic plasma concentration, can one expect that the drug will probably be devoid of torsadonenicity. Therefore, this in vitro model provides detailed information on the overall profile of drug-induced electrophysiological effects. In combination with other in vitro and in vivo repolarization assays and with pharmacokinetic data in man, it is a valuable tool to establish an integrated cardiovascular risk assessment of pharmaceutical compounds.

In vivo measurement of QT prolongation, dispersion and arrhythmogenesis: application to the preclinical cardiovascular safety pharmacology of a new chemical entity.

In addition to in silico and in vitro measurements, cardiac electrophysiology in experimental animals plays a decisive role in the selection of a potential 'cardio-safe' new chemical entity (NCE). The present synopsis critically reviews such in vivo techniques in experimental animals. In anaesthetized guinea-pigs, surface ECG recordings readily identify the typical effects of Class I to IV anti-arrhythmic compounds and of If blockers such as zatebradine on ECG intervals and morphology, but also of non-cardiovascular NCEs affecting cardiac electrical activity via ion channels or neurogenic mechanisms. QT/RR plots indicate that bradycardia is a dominant effect of IKr blockers (dual modulation by IKr of sinus node activity and ventricular repolarization). Nevertheless, correction of QT with Bazett's formula usually distinguishes between drug-induced heart rate reduction and real prolongation of ventricular repolarization (QTc). The anaesthetized guinea-pig model thus is a useful tool for first line in vivo testing of an NCE for effects on cardiac electrophysiology, in particular when combined with measurements of drug levels in plasma and heart tissues. In anaesthetized dogs, advanced ECG analyses identify drug-induced effects on atrial and ventricular intervals, on temporal and transmural dispersion of ventricular repolarization and on incidences of early after-depolarizations. This can be combined with complete haemodynamic, pulmonary and pharmacokinetic analyses in one preparation. However, compound doses/plasma levels needed for effects on ventricular repolarization in this model are substantially higher than those identified in guinea-pigs, at least for IKr blocking compounds. Therefore, we use this 'information-rich' canine model as a second line approach. In awake, trained and appropriately instrumented dogs, readings of surface ECG in combination with cardio-haemodynamic and behavioural assessments can be performed after the administration of an NCE via the expected therapeutic route, including oral medication. However, at higher doses the compound under scrutiny may induce overall behavioural side-effects, related to its primary pharmacological action, such as gastrokinetic repercussions or CNS-mediated sedation or excitation. Such primary pharmacological effects are bound to compromise the evaluation of real drug-induced changes on cardiac electrophysiology, readily identified by resource-friendly setups in smaller animals. Therefore, we use such paradigms as an imperative, final cardiovascular check-up, before a 'First in Man' administration of the NCE. In anaesthetized, methoxamine-challenged rabbits, arrhythmogenic effects of IKr blockers (torsades de pointes) and of dual channel INa/IKr blockers (conduction disturbances) are readily identified. Drug-induced QT dispersion rather than a 'simple' QTc prolongation determines the ventricular arrhythmogenic effect of IKr blockers. The latter effect also depends on the rate of drug delivery (plasma levels vs. heart level, equilibrium throughout the myocardium). Therefore, we use models sensitized for arrhythmogenesis to document further the profile of a comparatively 'cardio-safe' NCE. We conclude that the interpretation of an integrated profile of activity of an NCE on in vitro and in vivo cardiovascular parameters, in comparison with the characteristics of its primary pharmacology and target disease, determines its eventual selection via a scientific, rather than a 'checklist' or 'menu' approach to cardiovascular safety pharmacology. Appropriate tests in experimental animals play a key role in this process.

Preclinical cardiovascular safety evaluations of biologics: optimizing the approach and follow-up.

Standard therapeutic agents (STA) are usually relatively small and simple molecules, which are synthesized as highly pure and consistent molecules. However, their target specificity can be low so that there is a liability for side effects at non-targeted receptors, enzymes or ion channels. Alternatively, highly specific and targeted small molecules can elicit cardiovascular liabilities due to their target-based effects. As a result of their long existence in pharmaceutical practice, their safety evaluation is fairly well standardized and their potentially promiscuous, non-specific actions mandate broad evaluations. Biologics include a wide variety of products, ranging from relatively small synthesized polypeptides, which are also highly consistent, to very complex products, the composition of which may vary widely between production batches and sources. Biologics are usually highly specific for a single target, so that side effects at other targets are very rare. Their toxicities are more related to immune systems and to infection complications than to cardiovascular repercussions. The standard preclinical cardiac safety evaluations, derived from experience with STA, are frequently not appropriate or warranted for the evaluation of biologics. Indeed, because of the specificity of such biologic products, smaller test batteries than the ones needed for STA may be sufficient. However, because of the potential variability in composition of biologics between production batches and sources, evaluations of such products needs to be performed more frequently than for uniformly produced STA.

Short patent lives jeopardize drug and patient safety.

Exposure of patients to new medications carries potential safety hazards during widespread clinical practice. These are often detected only after a substantial period of use. Thus, there is considerable need for measures reducing drug-related morbidity and mortality, such as adequate, active postmarketing drug-safety surveillance systems with obligatory follow-up studies of suspected safety problems, or even an additional "Phase IV" safety study before marketing. However, drug development processes erode substantially into the useful patent life of a new drug. Therefore, we suggest that the potential benefits of patent life prolongation should be considered, under certain conditions for the sake of patient safety.

Detection of proarrhythmia in the female rabbit heart: blinded validation.

INTRODUCTION: Reliable detection of drug-induced proarrhythmia, especially the potential for polymorphic ventricular tachycardia, is of great importance in the development of new compounds that are safe for the heart and was evaluated in a blinded study. METHODS AND RESULTS: In 142 female rabbits, the monophasic action potential was used to determine intraventricular conduction, action potential duration (APD), triangulation (APD30 to APD90), reverse use-dependence, instability and presence of chaotic behavior, early afterdepolarizations, torsades de pointes (TdP), and ventricular fibrillation. In addition, 31 coded drugs were tested in a blinded fashion in another 150 hearts. Prototype cardiovascular agents [quinidine (IA), lidocaine (IB), flecainide (IC), propranolol (II), sotalol (IIIB), amiodarone (IIIAB) and verapamil (IV)] were correctly characterized in terms of their effects upon conduction and APD. Agents documented in clinical practice to have proarrhythmic potential (droperidol, sotalol, mibefradil, bepridil, lidoflazine, ketanserin, sertindole, terfenadine, haloperidol, astemizole, cisapride, ziprasidone, lubeluzole, dofetilide, quinidine, ibutilide) were identified as such. Pimozide is reported to rarely produce TdP and was also found to elicit Class III action with few adverse effects. Equally important, agents believed not to be proarrhythmic (two solvents, atenolol, propranolol, fenoximone, cetirizine, verapamil, sildenafil, lidocaine, diltiazem) were identified as having no proarrhythmic activity. CONCLUSION: The SCREENIT method properly characterized and quantified prototype cardiovascular drugs and correctly identified proarrhythmic noncardiovascular agents of various mechanisms, but it did not produce false-positive results.

Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323-333, 1999; Kelm M. Biochim Biophys Acta 1411: 273-289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158-28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285-L292, 1995). GSH and other thiols (1.0-10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels (P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH (P < 0.05). Preincubation of tracheas with GSH significantly (P < 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.

Specific modulation of calmodulin activity induces a dramatic production of superoxide by alveolar macrophages.

Airway inflammation is a characteristic feature in airway diseases such as asthma and chronic obstructive pulmonary disease. Oxidative stress, caused by the excessive production of reactive oxygen species by inflammatory cells like macrophages, eosinophils and neutrophils, is thought to be important in the complex pathogenesis of such airway diseases. The calcium-sensing regulatory protein calmodulin (CaM) binds and regulates different target enzymes and proteins, including calcium channels. In the present study, we investigated whether CaM, via the modulation of calcium channel function, influences [Ca(2+)](i) in pulmonary inflammatory cells, and consequently, modulates the production of reactive oxygen species by these cells. This was tested with a peptide termed calcium-like peptide 2 (CALP2), which was previously shown to regulate such channels. Specifically, radical production by purified broncho-alveolar lavage cells from guinea-pigs in response to CALP2 was measured. CALP2 was a strong activator of alveolar macrophages. In contrast, CALP2 was only a mild activator of neutrophils and did not induce radical production by eosinophils. The CALP2-induced radical production was mainly intracellular, and was completely blocked by the NADPH-oxidase inhibitor DPI, the superoxide inhibitor SOD and the CaM antagonist W7. Furthermore, the calcium channel blocker lanthanum partly inhibited the cellular activation by CALP2. We conclude that alveolar macrophages, but not neutrophils or eosinophils, can produce extremely high amounts of reactive oxygen species when stimulated via the calcium/CaM pathway. These results may contribute to new therapeutic strategies against oxidative stress in airway diseases.

Synthesis and biological evaluation of imidazol-2-one and 2-cyanoiminoimidazole derivatives: novel series of PDE4 inhibitors.

This communication describes the synthesis and in vitro PDE4 inhibitory activity of a novel series of imidazol-2-one and 2-cyanoiminoimidazole derivatives. The compounds described were also tested in in vivo models to evaluate their anti-inflammatory activity after topical administration as well as their gastro-intestinal side effects. Several compounds proved to be potent PDE4 inhibitors and some 2-cyanoiminoimidazoles showed less pronounced gastro-intestinal side effects than reference compounds but maintained anti-inflammatory activity after topical administration.