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.

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.

Bilastine: an environmental risk assessment.

CONTEXT: Bilastine is a new oral selective, non-sedating histamine H1 antagonist for the symptomatic treatment of allergic rhinoconjunctivitis and urticaria. The European Medicines Agency requires an Environmental Risk Assessment (ERA) for all novel medicines for human use. OBJECTIVE: To calculate the bilastine predicted environmental concentration in surface water (PECsw; phase I ERA), and to determine the effects of bilastine on aquatic systems (phase II [tier A]). MATERIALS AND METHODS: Bilastine PECsw was calculated using the maximum daily dosage (20 mg), assuming that all administered bilastine was released into the aquatic environment. A persistence, bioaccumulation and toxicity assessment was conducted using the log Kow from the molecular structure. In phase II (tier A), a ready biodegradability test was performed, and bilastine's potential toxicity to various aquatic and sediment-dwelling micro-organisms was evaluated. RESULTS: Bilastine PECSW was calculated as 0.1 µg L(-1), and the compound was not readily biodegradable. Bilastine had no significant effects on Chironomus riparius midges, or on the respiration rate of activated sludge. For green algae, the bilastine no observed effect concentration (NOEC) was 22 mg L(-1); bilastine had no effect on zebra fish development, or on the reproduction rate of daphnids. DISCUSSION: Bilastine NOEC values against zebra fish, algae, daphnids, and aerobic organisms in activated sludge were at least 130 000-fold greater than the calculated PECSW value. CONCLUSION: No environmental concerns exist from bilastine use in patients with allergic rhinoconjunctivitis or urticaria.

Investigation of cytotoxic and genotoxic effects of the antihistaminic drug, loratadine, on human lymphocytes.

CONTEXT: Loratadine (LOR) is a new generation antihistamine used in the treatment of allergic disorders. OBJECTIVE: The aim of this study was to evaluate the cytogenotoxic effect of LOR on human peripheral blood lymphocytes. MATERIALS AND METHODS: We investigated the genotoxic effect of this drug in cultured human peripheral blood lymphocytes using sister chromatid exchange (SCE), chromosomal aberrations (CA) and micronucleus (MN) assay in culture conditions. Proliferation index (PI), mitotic index (MI) and nuclear division index (NDI) were also calculated to determine the cytotoxic/cytostatic effect. Cultures were treated with LOR at three concentrations (5, 15 and 25 µg/ml) for 48 h. RESULTS: Although the MI significantly decreased at the higher concentrations (15 and 25 µg/ml) compared with negative (solvent) control, LOR indicated weaker cytotoxic potential in PI and NDI values at all the tested concentrations. LOR increased the frequencies of SCE, CA and MN in all lymphocyte cultures. However, significant increase was observed in MN at the medium and highest doses (15 and 25 µg/ml) and in CA at the medium dose (15 µg/ml) compared with negative (solvent) control culture. Our results indicate that LOR has cytotoxic and genotoxic effects on human peripheral blood lymphocyte cultures. DISCUSSION: Although most of previously findings have shown that LOR does not reflect genotoxicity, our results indicated that it may be a genotoxic drug. CONCLUSION: More studies are necessary to elucidate the relationship between cytotoxic, genotoxic and apoptotic effects, and to make a possible risk assessment in patients receiving therapy with this drug.

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.

Simultaneous gene expression signature of heart and peripheral blood mononuclear cells in astemizole-treated rats.

We investigated the effects of astemizole, a second-generation antihistamine, on the heart and peripheral blood mononuclear cells (PBMCs) and identified the early markers of its cardiotoxicity using gene expression profiling. Astemizole causes torsades de pointes, which is a type of ventricular tachycardia. We administered astemizole (dosage: 20, 60 mg/kg) to male Sprague-Dawley rats, using an oral gavage. Cardiac tissue and PBMCs were collected from the rats 4 h after treatment. Gene expression profiles were obtained using an Affymetrix GeneChip. The most deregulated genes were associated with energy metabolism pathways and calcium ion homeostasis in the heart of astemizole-treated rats. The most altered genes in the PBMCs were those involved in developmental processes and cardiotoxicity. Genes related to the response to oxidative stress, reactive oxygen species, heat shock proteins, hypoxia, immunity, and inflammation were also deregulated in the heart and PBMCs. These data provide further insight into the genetic pathways affected by astemizole. In addition, the simultaneously deregulated genes identified herein may be further studied. It will be interesting to find out whether single genes or certain sets of these genes could finally serve as biomarkers for cardiotoxicity of astemizole or other similar antihistamine drugs.

Improved methodology for identifying the teratogenic potential in early drug development of hERG channel blocking drugs.

Drugs blocking the potassium current IKr of the heart (via hERG channel-inhibition) have the potential to cause hypoxia-related teratogenic effects. However, this activity may be missed in conventional teratology studies because repeat dosing may cause resorptions. The aim of the present study was to investigate an alternative protocol to reveal the teratogenic potential of IKr-blocking drugs. The IKr blocker astemizole, given as a single dose (80 mg/kg) on gestation day (GD) 13 to pregnant rats caused digital defects. In whole rat embryo culture (2h) on GD 13, astemizole caused a decrease in embryonic heart rate at 20 nM, and arrhythmias at 200-400 nM. Cetirizine, without IKr-blocking properties, did not affect the rat embryonic heart in vitro. The present study shows that single dose testing on sensitive days of development, together with whole embryo culture, can be a useful methodology to better characterize the teratogenic potential of IKr-blocking drugs.

Temporal variability of QT interval and changes in T wave morphology in dogs as markers of the clinical risk of drug-induced proarrhythmia.

INTRODUCTION: The aim of this experiment was to establish the usefulness of assessing temporal variability of the QT interval and changes in the T wave morphology in dogs as markers of pro-arrhythmic risk in humans. For this purpose, the electrocardiographic effects of astemizole and cisapride, two well known pro-arrhythmic drugs in humans, were assessed in dogs. METHODS: Astemizole was administered intravenously at single doses of 1 and 3 mg/kg whilst cisapride was administered orally at doses of 1.5 and 6 mg/kg. Electrocardiograms (ECG) were recorded before and after treatment. From each ECG tracing, QT intervals were recorded over 100 beats for calculation of the mean and standard deviation (SD) of QT and mean corrected QT (QTc). The coefficient of variation of QT (CV=SD/mean) was calculated as an indicator of QT temporal variability. The changes in T wave morphology were assessed in precordial lead CV5RL. RESULTS: Astemizole increased both the QTc interval and the CV of QT. Increases in these parameters also occurred after cisapride, but were less marked than after astemizole. In addition, both compounds produced a notching of the T wave, consisting of the presence of two peaks. DISCUSSION: The effects of astemizole and cisapride on the CV of QT and their propensity to induce of T wave notching are consistent with the blocking of I(Kr) channels and indicate, respectively, an increase in temporal variability of cardiac repolarization and an increased heterogeneity of the repolarization of cardiac cells across the myocardium. These changes are key triggers of arrhythmic events and are thus consistent with the pro-arrhythmic properties of these drugs. This study therefore indicates that the evaluation of CV of QT and T wave morphology in dogs may help in predicting drug-induced pro-arrhythmic risk in humans.

Circadian time-dependent differences in murine tolerance to the antihistaminic agent loratadine.

Loratadine is a second-generation histamine H(1)-receptor antagonist used in the treatment of allergic diseases. The aim of the study was to assess whether lethal toxicity and motor incoordination (neurotoxicity) of loratadine is circadian rhythm-dependent. A total of 210 male Swiss mice, aged 10 wk, were synchronized for 3 wk to 12 h light (rest span)/12 h dark (activity span). The drug was administered per os. The choice of the sublethal (TD(50) = 82 mg/kg body weight) and the lethal (LD(50) = 4 g/kg body weight) dosage was based on preliminary studies. Each of these two doses was administered to comparable groups of animals at six different circadian time points (1, 5, 9, 13, 17, and 21 Hours After Light Onset [HALO]). The survival duration was dosing time-dependent (chi(2) = 16.96; p < 0.001). Drug dosing at 17 HALO resulted in best (67%) survival rate; whereas, dosing at 9 HALO resulted in poorest (21%) survival rate. Cosinor analyses (with a trial period tau = 24 h) validated a statistically significant circadian rhythm in survival rate (p < 0.04) with an acrophase (peak time Ø of best tolerance to loratadine) being at 17.5 HALO +/- 4.65 h. Troughs of motor incoordination were located at the administration times of 5 and 17 HALO (60% and 32% of animals affected, respectively), whereas peaks were located at 9 and 21 HALO (87% and 68% of animals affected, respectively). The 24 h mean of the motor incoordination was 61%, the mean proportion of animals affected by the treatment for the six different circadian times studies. The extent of this neurotoxic effect varied as a function of loratadine dosing time (p < 0.001). A statistically significant ultradian component rhythm (p < 0.01) with a trial period tau = 12 h was also validated. The obtained results show that the dosing time of loratadine at the mid-activity (dark) span seems to be optimal, since it corresponds to the longest (21 vs. 12 days) survival span and to least neurotoxicity.

[Circadian rhythms in the toxic effects of the histamine antagonist cetirizine in mice]. / Rythmes circadiens des effets toxiques d'un antihistaminique H1: la cétirizine chez la souris.

UNLABELLED: Cetirizine is a second generation histamine H(1) receptor antagonist used to provide symptomatic relief of allergic signs caused by histamine release. The aim of the study was to learn whether the survival and the motor incoordination (ataxia) side effect of cetirizine administration is dosing time-dependent. MATERIALS AND METHODS: A total of 240 male Swiss mice, 10 weeks of age were synchronized for 3 weeks by 12 h light (rest span)/12 h dark (activity span). Different doses of cetirizine were administered orally at fixed times during the day to determine both the sublethal (TD(50)) and lethal (LD(50)) doses, which were, respectively, 55 +/- 0.35 and 750 +/- 0.40 mg/kg. In the chronotoxicologic study a single dose of cetirizine (DL(50)) was administered to comparable groups of animals at six different circadian stages [1, 5, 9, 13, 17, 21 h after light onset (HALO)]. RESULTS: The survival was statistically significant dosing time-dependent (chi(2) = 16.73; P < 0.001). Drug dosing at 17 HALO resulted in 83.3% survival rate whereas drug dosing at 5 HALO was only 23.25%. Cosinor analysis revealed a statistically significant circadian (period approximately 24 h) rhythmic component in survival. Lowest (20%) and highest (88%) ataxia occurred when cetirizine was administered, respectively, at 17 and 5 HALO. Cosinor analysis revealed a statistically significant circadian (period approximately 24 h) rhythmic component in ataxia. CONCLUSION: Our results reveal that the best safety is shown when cetirizine is administered in the middle of the dark (activity) span of the mice, since it produces some side effects: ataxia and hyperthermia. Taking into account of the hour administration of cetirizine, improves treatment efficacy and permit the best control of allergic diseases.

Norpiperidine imidazoazepines as a new class of potent, selective, and nonsedative H1 antihistamines.

Clinical doses of available H(1) antihistamines are limited mainly by sedative side effects. However, higher doses are often required to obtain optimal therapeutic activity, especially in dermatology. We report the synthesis of three norpiperidine imidazoazepines representative of a new class of selective and nonsedating H(1) antihistamines. The compounds were at least as potent as cetirizine and loratadine as measured by H(1) receptor binding affinity, by protection against compound 48/80- and histamine-induced lethality in rats and guinea pigs, respectively, and by skin reaction tests in rats, guinea pigs, and dogs. The compounds, in particular 3a, were less prone than the reference compounds to penetrate the brain and to occupy central H(1) receptors, suggesting absence of sedative side effects. In vitro and in vivo cardiovascular safety tests showed that 3a had no intrinsic potential to prolong ventricular repolarization or induce cardiac arrhythmias. Compound 3a has been selected for further clinical development, mainly for application in dermatology.