Analysis of reproducibility and robustness of a human microfluidic four-cell liver acinus microphysiology system (LAMPS).

A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 µM), Troglitazone (150 µM), and caffeine (600 µM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 µM), Tolcapone (88 µM), Trovafloxacin (150 µM with or without 1 µg/mL lipopolysaccharide), Troglitazone (28 µM), Rosiglitazone (0.8 µM), Pioglitazone (3 µM), and caffeine (600 µM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.

Evaluation of Possible Proarrhythmic Potency: Comparison of the Effect of Dofetilide, Cisapride, Sotalol, Terfenadine, and Verapamil on hERG and Native IKr Currents and on Cardiac Action Potential.

The proarrhythmic potency of drugs is usually attributed to the IKr current block. During safety pharmacology testing analysis of IKr in cardiomyocytes was replaced by human ether-a-go-go-related gene (hERG) test using automated patch-clamp systems in stable transfected cell lines. Aim of this study was to compare the effect of proarrhythmic compounds on hERG and IKr currents and on cardiac action potential. The hERG current was measured by using both automated and manual patch-clamp methods on HEK293 cells. The native ion currents (IKr, INaL, ICaL) were recorded from rabbit ventricular myocytes by manual patch-clamp technique. Action potentials in rabbit ventricular muscle and undiseased human donor hearts were studied by conventional microelectrode technique. Dofetilide, cisapride, sotalol, terfenadine, and verapamil blocked hERG channels at 37°C with an IC50 of 7 nM, 18 nM, 343 µM, 165 nM, and 214 nM, respectively. Using manual patch-clamp, the IC50 values of sotalol and terfenadine were 78 µM and 31 nM, respectively. The IC50 values calculated from IKr measurements at 37°C were 13 nM, 26 nM, 52 µM, 54 nM, and 268 nM, respectively. Cisapride, dofetilide, and sotalol excessively lengthened, terfenadine, and verapamil did not influence the action potential duration. Terfenadine significantly inhibited INaL and moderately ICaL, verapamil blocked only ICaL. Automated hERG assays may over/underestimate proarrhythmic risk. Manual patch-clamp has substantially higher sensitivity to certain drugs. Action potential studies are also required to analyze complex multichannel effects. Therefore, manual patch-clamp and action potential experiments should be a part of preclinical safety tests.

Investigation of the effect of hepatic metabolism on off-target cardiotoxicity in a multi-organ human-on-a-chip system.

Regulation of cosmetic testing and poor predictivity of preclinical drug studies has spurred efforts to develop new methods for systemic toxicity. Current in vitro assays do not fully represent physiology, often lacking xenobiotic metabolism. Functional human multi-organ systems containing iPSC derived cardiomyocytes and primary hepatocytes were maintained under flow using a low-volume pumpless system in a serum-free medium. The functional readouts for contractile force and electrical conductivity enabled the non-invasive study of cardiac function. The presence of the hepatocytes in the system induced cardiotoxic effects from cyclophosphamide and reduced them for terfenadine due to drug metabolism, as expected from each compound's pharmacology. A computational fluid dynamics simulation enabled the prediction of terfenadine-fexofenadine pharmacokinetics, which was validated by HPLC-MS. This in vitro platform recapitulates primary aspects of the in vivo crosstalk between heart and liver and enables pharmacological studies, involving both organs in a single in vitro platform. The system enables non-invasive readouts of cardiotoxicity of drugs and their metabolites. Hepatotoxicity can also be evaluated by biomarker analysis and change in metabolic function. Integration of metabolic function in toxicology models can improve adverse effects prediction in preclinical studies and this system could also be used for chronic studies as well.

Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.

INTRODUCTION: Multi-electrode array (MEA) systems and human induced pluripotent stem (iPS) cell-derived cardiomyocytes are frequently used to characterize the electrophysiological effects of drug candidates for the prediction of QT prolongation and proarrhythmic potential. However, the optimal experimental conditions for obtaining reliable experimental data, such as high-pass filter (HPF) frequency and cell plating density, remain to be determined. METHODS: Extracellular field potentials (FPs) were recorded from iPS cell-derived cardiomyocyte sheets by using the MED64 and MEA2100 multi-electrode array systems. Effects of HPF frequency (0.1 or 1Hz) on FP duration (FPD) were assessed in the presence and absence of moxifloxacin, terfenadine, and aspirin. The influence of cell density on FP characteristics recorded through a 0.1-Hz HPF was examined. The relationship between FP and action potential (AP) was elucidated by simultaneous recording of FP and AP using a membrane potential dye. RESULTS: Many of the FP waveforms recorded through a 1-Hz HPF were markedly deformed and appeared differentiated compared with those recorded through a 0.1-Hz HPF. The concentration-response curves for FPD in the presence of terfenadine reached a steady state at concentrations of 0.1 and 0.3µM when a 0.1-Hz HPF was used. In contrast, FPD decreased at a concentration of 0.3µM with a characteristic bell-shaped concentration-response curve when a 1-Hz HPF was used. The amplitude of the first and second peaks in the FP waveform increased with increasing cell plating density. The second peak of the FP waveform roughly coincided with AP signal at 50% repolarization, and the negative deflection at the second peak of the FP waveform in the presence of E-4031 corresponded to early afterdepolarization and triggered activity. DISCUSSION: FP can be used to assess the QT prolongation and proarrhythmic potential of drug candidates; however, experimental conditions such as HPF frequency are important for obtaining reliable data.

Zebrafish as a new model for phenotype-based screening of positive inotropic agents.

The zebrafish-based assay is a widely used animal model system for cardiovascular research. In this study, we investigated the cardiac defects caused by terfenadine and tested the pharmacological response of digoxin to zebrafish with cardiac defects. The study suggested that zebrafish could be a suitable model for phenotype-based screening and evaluation of positive inotropic agents. This phenotype-based heart failure zebrafish model system was then utilized in in-house library screen. Some positive inotropic compound was discovered, which could attenuate the cardiac defects by increasing the flow velocity of caudal artery blood.

H1-antihistamines exacerbate high-fat diet-induced hepatic steatosis in wild-type but not in apolipoprotein E knockout mice.

We examined the effects of two over-the-counter H1-antihistamines on the progression of fatty liver disease in male C57Bl/6 wild-type and apolipoprotein E (ApoE)-/- mice. Mice were fed a high-fat diet (HFD) for 3 mo, together with administration of either cetirizine (4 mg/kg body wt) or fexofenadine (40 mg/kg body wt) in drinking water. Antihistamine treatments increased body weight gain, gonadal fat deposition, liver weight, and hepatic steatosis in wild-type mice but not in ApoE-/- mice. Lobular inflammation, acute inflammation, and necrosis were not affected by H1-antihistamines in either genotype. Serum biomarkers of liver injury tended to increase in antihistamine-treated wild-type mice. Serum level of glucose was increased by fexofenadine, whereas lipase was increased by cetirizine. H1-antihistamines reduced the mRNA expression of ApoE and carbohydrate response element-binding protein in wild-type mice, without altering the mRNA expression of sterol regulatory element-binding protein 1c, fatty acid synthase, or ApoB100, in either genotype. Fexofenadine increased both triglycerides and cholesterol ester, whereas cetirizine increased only cholesterol ester in liver, with a concomitant decrease in serum triglycerides by both antihistamines in wild-type mice. Antihistamines increased hepatic levels of conjugated bile acids in wild-type mice, with the effect being significant in fexofenadine-treated animals. The increase was associated with changes in the expression of organic anion transport polypeptide 1b2 and bile salt export pump. These results suggest that H1-antihistamines increase the progression of fatty liver disease in wild-type mice, and there seems to be an association between the severity of disease, presence of ApoE, and increase in hepatic bile acid levels.

Human cardiotoxic drugs delivered by soaking and microinjection induce cardiovascular toxicity in zebrafish.

Cardiovascular toxicity is a major challenge for the pharmaceutical industry and predictive screening models to identify and eliminate pharmaceuticals with the potential to cause cardiovascular toxicity in humans are urgently needed. In this study, taking advantage of the transparency of larval zebrafish, Danio rerio, we assessed cardiovascular toxicity of seven known human cardiotoxic drugs (aspirin, clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride) and two non-cardiovascular toxicity drugs (gentamicin sulphate and tetracycline hydrochloride) in zebrafish using six specific phenotypic endpoints: heart rate, heart rhythm, pericardial edema, circulation, hemorrhage and thrombosis. All the tested drugs were delivered into zebrafish by direct soaking and yolk sac microinjection, respectively, and cardiovascular toxicity was quantitatively or qualitatively assessed at 4 and 24 h post drug treatment. The results showed that aspirin accelerated the zebrafish heart rate (tachycardia), whereas clomipramine hydrochloride, cyclophosphamide, nimodipine, quinidine, terfenadine and verapamil hydrochloride induced bradycardia. Quinidine and terfenadine also caused atrioventricular (AV) block. Nimodipine treatment resulted in atrial arrest with much slower but regular ventricular heart beating. All the tested human cardiotoxic drugs also induced pericardial edema and circulatory disturbance in zebrafish. There was no sign of cardiovascular toxicity in zebrafish treated with non-cardiotoxic drugs gentamicin sulphate and tetracycline hydrochloride. The overall prediction success rate for cardiotoxic drugs and non-cardiotoxic drugs in zebrafish were 100% (9/9) as compared with human results, suggesting that zebrafish is an excellent animal model for rapid in vivo cardiovascular toxicity screening. The procedures we developed in this report for assessing cardiovascular toxicity in zebrafish were suitable for drugs delivered by either soaking or microinjection.

Optimization of the adult zebrafish ECG method for assessment of drug-induced QTc prolongation.

INTRODUCTION: Recent studies have shown the utility of adult zebrafish ECG (electrocardiogram) in assessing drug-induced QTc prolongation. While the method has significant advantages over current ECG animal models including ethical issues, low compound requirement and expense, adoption of the method into drug discovery programs has been hampered by specific limitations. The limitations include the inability to determine the exact dose of test compound administered, and potential effects due to variables such as flow rate of oral perfusion and immobilization method. We describe a refined method for the reproducible recording of the adult zebrafish ECG and illustrate its application in investigating drug-induced QTc prolongation using the histamine receptor antagonist Terfenadine as a test drug. METHOD: We chose to perform parenteral administration of test drug instead of perfusion on the basis of mg per kg body weight of adult zebrafish. Acclimatization and immobilization methods were optimized to avoid ECG artifacts due to sudden environmental changes. We further modified the formula for QT correction and ensured reproducible recording of stable ECGs. Various concentrations of Terfenadine were used and the resultant proarrhythmic effects were analyzed as compared to the baseline and untreated controls. RESULTS: Normal, stable and reproducible ECGs were recorded in all zebrafish. Terfenadine at the rate of 0.1mg/kg body weight was found to be the NOAEL. We found an excellent correlation between known QTc effects in humans and those observed in adult zebrafish at all concentrations. All Terfenadine-induced proarrhythmic effects observed in zebrafish were dose and time dependent. DISCUSSION: We report a refined method for reproducible recording of stable zebrafish ECGs to facilitate its routine application in preclinical investigation of QTc-prolonging drugs with reliable estimation of NOAEL. Our study is of relevance to the development and use of alternate animal models in drug discovery.

Evaluation of the genotoxicity and cytotoxicity of fexofenadine in cultured human peripheral blood lymphocytes.

Fexofenadine (FXF) is a new non-sedating antihistamine used in the treatment of seasonal allergic rhinitis and chronic idiopathic urticaria. Studies on FXF genotoxicity and cytotoxicity in cultured human peripheral blood lymphocytes have not been reported so far. Therefore, the present study is the first report investigating the genotoxic and cytotoxic effects of FXF in cultured human peripheral blood lymphocytes in vitro. Cultures were treated with FXF at three concentrations (50, 100 and 150 µg/ml) for 24 and 48 h. Endpoints analyzed included: mitotic index (MI), nuclear division index (NDI), chromosomal aberrations (CA) and micronucleus (MN) assay. Mitomycin C (MMC) was used as a positive control. The results of CA and MN assays showed that FXF was not genotoxic at all the concentrations tested, meanwhile MI and NDI results showed dose-dependent decrease and significant differences were found for at least one concentration. In conclusion, the results of this study suggest that FXF has a cytotoxic effect but not genotoxic effect on human peripheral blood lymphocyte cultures. Further cytogenetic studies, especially about the cell cycle kinetics of FXF are required to elucidate the decreases in dividing cells, and biomonitoring studies should also be conducted with patients receiving therapy with this drug.

Evaluation of terfenadine and ketoconazole-induced QT prolongation in conscious telemetered guinea pigs.

Terfenadine and ketoconazole are the most widely used positive reference agents in non-clinical cardiac repolarization safety studies. The aim of the present study was to evaluate the effects of terfenadine, ketoconazole and their combination on QT prolongation using conscious guinea pigs. Conscious telemetered guinea pigs were orally administered terfenadine (50 mg/kg), ketoconazole (200 mg/kg) or a combination of the two, and effects on QT were recorded using a telemetry system. The QT correction was carried out with Bazett's formula to eliminate confounding effect of HR. Neither terfenadine nor ketoconazole produced any effect on the RR and QT intervals, QRS complex or heart rate (HR). However, a combination of terfenadine and ketoconazole significantly prolonged the RR and QT intervals and decreased HR in a time-dependent manner. This study demonstrated that the combination of terfenadine and ketoconazole produces QT prolongation in conscious telemetered guinea pigs.

Interferences of the autonomic nervous system with drug induced QT prolongation: a point to consider in non-clinical safety studies.

INTRODUCTION: QT interval assessment by telemetry has become one of the most useful models in testing strategies adopted for detection of drug induced QT prolongation in non-clinical safety pharmacology studies. This study reports experimental data showing that the autonomic nervous system might influence drug induced QT prolongation. METHODS: Animals were instrumented with telemetric transmitters and epicardial ECG leads. Effects on QT interval of reference drugs such as thioridazine and terfenadine were analysed with different approaches, the Holzgrefe's probabilistic method, the QT shift method and an individual analysis of beat-to-beat QT/RR pair distribution visualised as points-cloud. RESULTS: Two cases of unexpected absence of QT interval prolongation are reported with thioridazine and terfenadine in conscious beagle dogs under conditions of concomitant tachycardia. The pro-arrhythmic properties of these two molecules were unmasked by co-treatment with sympatholytic agents, atenolol and clonidine respectively suggesting that sympathetic activation and/or parasympathetic withdrawal might impair a drug induced QT prolongation. DISCUSSION: The apparent absence of changes in the QT interval due to novel drug candidates should be interpreted cautiously under conditions of concomitant tachycardia or elevated heart rate levels in non-clinical safety studies.

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio) embryos.

BACKGROUND: Zebrafish (Danio rerio), due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis. RESULTS: The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05). In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p < 0.01). Moreover, there was a significant increase of the rhythmicity index (p < 0.01), which was supported by an increase in beat-to-beat interval variability (p < 0.01) of treated embryos as shown by Poincare plot. CONCLUSION: The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this method can potentially be applied to cardiotoxicity screening assay.

Comparison of the QT interval response during sinus and paced rhythm in conscious and anesthetized beagle dogs.

INTRODUCTION: The aim of the present study was to compare sensitivity in detecting the drug-induced QT interval prolongation in three dog models: conscious telemetered at sinus rhythm and conscious and anesthetized dogs during atrial pacing. The test substances used represent different chemical classes with different pharmacological and pharmacokinetic profiles. METHOD: Dofetilide and moxifloxacin were tested in all models, whereas cisapride and terfenadine were tested in the conscious telemetered and paced models. All substances were given as two consecutive 1.5-h intravenous infusions (infusions 1 and 2). The individual concentration-time courses of dofetilide, moxifloxacin, and cisapride were linked to the drug-induced effects on the QT interval and described with a pharmacokinetic-pharmacodynamic model to obtain an estimate of the unbound plasma concentrations at steady state that give a 10- and 20-ms drug-induced QT interval prolongation (CE10ms and CE20ms). RESULTS: In the conscious telemetered, conscious paced, and anesthetized dog models, the mean CE10ms values were 1.4, 4.0, and 2.5 nM for dofetilide and 1300, 1800, and 12,200 nM for moxifloxacin. For cisapride, the CE10ms values were 8.0 and 4.4 nM in the conscious telemetered and conscious paced dog models. The drug-induced QT interval prolongation during the last 30 min of infusions 1 and 2 was comparable in the conscious models, but smaller in the anesthetized dog model. Terfenadine displayed a marked delay in onset of response, which could only be detected by the extended ECG recording. DISCUSSION: All dog models investigated detected QT interval prolongation after administration of the investigated test substances with similar sensitivity, except for a lower sensitivity in the anesthetized dogs following moxifloxacin administration. The conscious telemetered dog model was favorable, mainly due to the extended continuous ECG recording, which facilitated detection and quantification of delayed temporal differences between systemic exposure and drug-induced QT interval prolongation.

QT prolongation in guinea pigs for preliminary screening of torsadogenicity of drugs and drug-candidates. II.

Experimental approaches on anaesthetised guinea pigs have been shown recently to be satisfactorily predictive of the torsadogenic risk of drugs. This work aimed at obtaining additional data, for a further understanding of the reliability and/or the limits of this model. Clonidine (non-torsadogenic in humans) induced a lengthening of the ECG parameter of RR in anaesthetised guinea pigs, without any corresponding increase of QT (corrected by the algorithms of Bazett and Fridericia). Thus, 'QT correct' prolonging effects produced by drugs torsadogenic in humans, on the guinea pig model are primarily due to inhibition of cardiac repolarisation. The corresponding RR prolongation is a consequence (not the cause) of this primary effect. Astemizole, haloperidol and terfenadine, torsadogenic in humans, produced in Langendorff perfused guinea pig hearts a prolongation of the QT interval. Chlorprotixene (non-torsadogenic) did not produce any significant effect on QT. These results are fully consistent with previous observations in anaesthetised guinea pigs. In Langendorff perfused hearts, pentobarbital does not affect cardiac repolarisation and does not potentiate the QT-prolonging effect of astemizole. Together with the findings reported by many authors, these data suggest that ECG recording in anaesthetised guinea pigs is a reliable model for cardiac safety studies evaluating the influence of drugs on the repolarisation process.

Dimethyl lithospermate B, an extract of Danshen, suppresses arrhythmogenesis associated with the Brugada syndrome.

BACKGROUND: Dimethyl lithospermate B (dmLSB) is an extract of Danshen, a traditional Chinese herbal remedy, which slows inactivation of INa, leading to increased inward current during the early phases of the action potential (AP). We hypothesized that this action would be antiarrhythmic in the setting of Brugada syndrome. METHODS AND RESULTS: The Brugada syndrome phenotype was created in canine arterially perfused right ventricular wedge preparations with the use of either terfenadine or verapamil to inhibit INa and ICa or pinacidil to activate IK-ATP. AP recordings were simultaneously recorded from epicardial and endocardial sites together with an ECG. Terfenadine, verapamil, and pinacidil each induced all-or-none repolarization at some epicardial sites but not others, leading to ST-segment elevation as well as an increase in both epicardial and transmural dispersions of repolarization (EDR and TDR, respectively) from 12.9+/-9.6 to 107.0+/-54.8 ms and from 22.4+/-8.1 to 82.2+/-37.4 ms, respectively (P<0.05; n=9). Under these conditions, phase 2 reentry developed as the epicardial AP dome propagated from sites where it was maintained to sites at which it was lost, generating closely coupled extrasystoles and ventricular tachycardia and fibrillation. Addition of dmLSB (10 micromol/L) to the coronary perfusate restored the epicardial AP dome, reduced EDR and TDR to 12.4+/-18.1 and 24.4+/-26.7 ms, respectively (P<0.05; n=9), and abolished phase 2 reentry-induced extrasystoles and ventricular tachycardia and fibrillation in 9 of 9 preparations. CONCLUSIONS: Our data suggest that dmLSB is effective in eliminating the arrhythmogenic substrate responsible for the Brugada syndrome and that it deserves further study as a pharmacological adjunct to implanted cardioverter/defibrillator usage.

Effects of cyamemazine on hERG, INa, ICa, Ito, Isus and IK1 channel currents, and on the QTc interval in guinea pigs.

The antipsychotic and anxiolytic phenothiazine, cyamemazine, was investigated for its effects on the hERG (human ether-à-go-go related gene) channel expressed in HEK 293 cells and on native INa, ICa, Ito, Isus, or IK1 of human atrial myocytes. Moreover, cyamemazine and terfenadine were compared for their effects on the QT interval in anesthetized guinea pigs. Cyamemazine reduced hERG current amplitude with an IC50 value of 470 nM. Cyamemazine 1 microM failed to significantly affect INa, Ito, Isus, or IK1 amplitudes and slightly decreased ICa (18%). For comparison, haloperidol (30 nM) and olanzapine (300 nM) reduced hERG current amplitude by 44.2+/-3.9% and 49.7+/-4.2%, respectively. The cardiac safety ratio of cyamemazine, calculated from the IC50/receptor affinity ratios, is 81 and 313 against dopamine D2 receptors and 5-HT2A receptors, respectively. In guinea pigs, QT and QTcBazett were not significantly modified by intravenous cyamemazine when compared to the effects produced by the vehicle. Conversely, terfenadine (5 mg/kg iv) increased significantly QTcBazett (+58 ms), QTcFrediricia (+83 ms) and QTcVan de Water (+78 ms). In conclusion, cyamemazine concentrations required to inhibit hERG current exceed substantially those necessary to achieve therapeutic activity in humans. Moreover, cyamemazine, in contrast to terfenadine, does not delay cardiac repolarization in the anesthetized guinea pig. These non-clinical findings confirm the excellent cardiac safety records of cyamemazine during its 30 years of extensive therapeutic use.

A novel predictive pharmacokinetic/pharmacodynamic model of repolarization prolongation derived from the effects of terfenadine, cisapride and E-4031 in the conscious chronic av node--ablated, His bundle-paced dog.

INTRODUCTION: Terfenadine, cisapride, and E-4031, three drugs that prolong ventricular repolarization, were selected to evaluate the sensitivity of the conscious chronic atrioventricular node--ablated, His bundle-paced Dog for defining drug induced cardiac repolarization prolongation. A novel predictive pharmacokinetic/pharmacodynamic model of repolarization prolongation was generated from these data. METHODS: Three male beagle dogs underwent radiofrequency AV nodal ablation, and placement of a His bundle-pacing lead and programmable pacemaker under anesthesia. Each dog was restrained in a sling for a series of increasing dose infusions of each drug while maintained at a constant heart rate of 80 beats/min. RT interval, a surrogate for QT interval in His bundle-paced dogs, was recorded throughout the experiment. RESULTS: E-4031 induced a statistically significant RT prolongation at the highest three doses. Cisapride resulted in a dose-dependent increase in RT interval, which was statistically significant at the two highest doses. Terfenadine induced a dose-dependent RT interval prolongation with a statistically significant change occurring only at the highest dose. The relationship between drug concentration and RT interval change was described by a sigmoid E(max) model with an effect site. Maximum RT change (E(max)), free drug concentration at half of the maximum effect (EC(50)), and free drug concentration associated with a 10 ms RT prolongation (EC(10 ms)) were estimated. A linear correlation between EC(10 ms) and HERG IC(50) values was identified. DISCUSSION: The conscious dog with His bundle-pacing detects delayed cardiac repolarization related to I(Kr) inhibition, and detects repolarization change induced by drugs with activity at multiple ion channels. A clinically relevant sensitivity and a linear correlation with in vitro HERG data make the conscious His bundle-paced dog a valuable tool for detecting repolarization effect of new chemical entities.

RNA synthesis-dependent potentiation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated toxicity by antihistamine terfenadine in cultured rat cerebellar neurons.

We have studied the effects of terfenadine on neurotoxicity and elevation of free cytoplasmic Ca2+ levels upon stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured cerebellar neurons. Pre-exposure to terfenadine (5 microM, 5 h) significantly increased neuronal death following specific stimulation of receptors by 100 microM AMPA or by subtoxic concentrations of domoate (8 microM), stimuli that are non-toxic when applied to terfenadine-untreated sister cultures. Terfenadine potentiation was prevented by the transcription inhibitor actinomycin D and was significantly ameliorated by histamine (1 mM). In terfenadine-treated neurons, AMPA increased [Ca2+](i) by approximately five fold, while AMPA induced no significant increase in [Ca2+](i) in the absence of terfenadine. Terfenadine reduced neuronal steady-state concentrations of [Ca2+](i) by approximately 75%. Our results suggest a role for histamine H1 receptors and intracellular calcium in the modulation of the excitotoxic response via AMPA receptors.

Photomutagenic properties of terfenadine as revealed by a stepwise photostability, phototoxicity and photomutagenicity testing approach.

Administration of the second-generation antihistamine, terfenadine, is sometimes associated with photosensitivity and other skin reactions. To obtain information on its photoreactivity, we used a stepwise experimental approach involving tests for photostability, phototoxicity (PT) (mouse fibroblast cell line [3T3] neutral red uptake [NRU] test) and photomutagenicity (with standard Ames salmonella tester strains TA98, TA100 and TA102). Terfenadine was not phototoxic to cultured mammalian cells under the conditions used (i.e. 5000/161 mJ cm(-2) UVA-UVB). Natural sunlight and UV radiations caused considerable drug decomposition and formation of several photoproducts. Addition of the irradiated terfenadine solution (i.e. a mixture of photoproducts) to the tester did not significantly increase background mutation frequency. Irradiation of terfenadine coplated with the TA102 strain induced a clear-cut photomutagenic response, the magnitude of which was dependent upon the precursor compound concentration and the UV dose (212/7 to 339/11 mJ cm(-2) UVA-UVB). These findings demonstrate that in vitro terfenadine is photomutagenic in absence of PT. Further in vitro and in vivo studies are therefore needed to provide an adequate safety assessment of the photochemical genotoxicity--carcinogenicity potential of terfenadine. In the meantime, patients should be advised to avoid excessive exposure to sunlight.