Prediction of the dose range for adverse neurological effects of amiodarone in patients from an in vitro toxicity test by in vitro–in vivo extrapolation

Amiodarone is an antiarrhythmic agent inducing adverse effects on the nervous system, among others. We applied physiologically based pharmacokinetic (PBPK) modeling combined with benchmark dose modeling to predict, based on published in vitro data, the in vivo dose of amiodarone which may lead to adverse neurological effects in patients. We performed in vitro-in vivo extrapolation (IVIVE) from concentrations measured in the cell lysate of a rat brain 3D cell model using a validated human PBPK model. Among the observed in vitro effects, inhibition of choline acetyl transferase (ChAT) was selected as a marker for neurotoxicity. By reverse dosimetry, we transformed the in vitro concentration-effect relationship into in vivo effective human doses, using the calculated in vitro area under the curve (AUC) of amiodarone as the pharmacokinetic metric. The upper benchmark dose (BMDU) was calculated and compared with clinical doses eliciting neurological adverse effects in patients. The AUCs in the in vitro brain cell culture after 14-day repeated dosing of nominal concentration equal to 1.25 and 2.5 mu M amiodarone were 1.00 and 1.99 mu g*h/mL, respectively. The BMDU was 385.4 mg for intravenous converted to 593 mg for oral application using the bioavailability factor of 0.65 as reported in the literature. The predicted dose compares well with neurotoxic doses in patients supporting the hypothesis that impaired ChAT activity may be related to the molecular/cellular mechanisms of amiodarone neurotoxicity. Our study shows that predicting effects from in vitro data together with IVIVE can be used at the initial stage for the evaluation of potential adverse drug reactions and safety assessment in humans

In Vitro–In Vivo Extrapolation by Physiologically Based Kinetic Modeling: Experience With Three Case Studies and Lessons Learned

Physiologically based kinetic (PBK) modeling has been increasingly used since the beginning of the 21st century to support dose selection to be used in preclinical and clinical safety studies in the pharmaceutical sector. For chemical safety assessment, the use of PBK has also found interest, however, to a smaller extent, although an internationally agreed document was published already in 2010 (IPCS/WHO), but at that time, PBK modeling was based mostly on in vivo data as the example in the IPCS/WHO document indicates. Recently, the OECD has published a guidance document which set standards on how to characterize, validate, and report PBK models for regulatory purposes. In the past few years, we gained experience on using in vitro data for performing quantitative in vitro–in vivo extrapolation (QIVIVE), in which biokinetic data play a crucial role to obtain a realistic estimation of human exposure. In addition, pharmaco-/toxicodynamic aspects have been introduced into the approach. Here, three examples with different drugs/chemicals are described, in which different approaches have been applied. The lessons we learned from the exercise are as follows: 1) in vitro conditions should be considered and compared to the in vivo situation, particularly for protein binding; 2) in vitro inhibition of metabolizing enzymes by the formed metabolites should be taken into consideration; and 3) it is important to extrapolate from the in vitro measured intracellular concentration and not from the nominal concentration to the tissue/organ concentration to come up with an appropriate QIVIVE for the relevant adverse effects

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress

International audience; High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14 days. CsA was quantified in supernatants and cellular lysates by LC-MS/MS for kinetic modeling. There was a rapid cellular uptake and accumulation of CsA, with a non-linear relationship between intracellular and applied concentrations. CsA at 15 µM induced mitochondrial disturbances and activation of the Nrf2-oxidative-damage and the unfolded protein-response pathways. All three omic streams provided complementary information, especially pertaining to Nrf2 and ATF4 activation. No stress induction was detected with 5 µM CsA; however, both concentrations resulted in a maximal secretion of cyclophilin B. The study demonstrates for the first time that CsA-induced stress is not directly linked to its primary pharmacology. In addition we demonstrate the power of integrated omics for the elucidation of signaling cascades brought about by compound induced cell stress

Biokinetics in repeated-dosing in vitro drug toxicity studies

The aim of the EU FP7 Predict-IV project was to improve the predictivity of in vitro assays for unwanted effects of drugs after repeated dosing. The project assessed the added benefit of integrating long-lived in vitro organotypic cell systems with 'omics' technologies and in silico modelling, including systems biology and pharmacokinetic assessments. RPTEC/TERT1 kidney cells, primary rat and human hepatocytes, HepaRG liver cells and 2D and 3D primary brain cultures were dosed daily or every other day for 14days to a selection of drugs varying in their mechanism of pharmacological action. Since concentration-effect relationships not only depend on the activity of the drug or the sensitivity of the target, but also on the distribution of compounds in the in vitro system, the concentration of a selection of drugs in cells, microtitre plate plastic and medium was measured over time. Results, reviewed in this paper, indicate that lipophilic drugs bind significantly to plastic labware. A few drugs, including less lipophilic drugs, bind to cell-attachment matrices. Chemicals that reach high concentrations in cells, including cyclosporin A and amiodarone, significantly accumulate over time after repeated dosing, partly explaining their increased toxicity after repeated dosing, compared to a single dose

Prediction of the dose range for adverse neurological effects of amiodarone in patients from an in vitro toxicity test by in vitro–in vivo extrapolation

Amiodarone is an antiarrhythmic agent inducing adverse effects on the nervous system, among others. We applied physiologically based pharmacokinetic (PBPK) modeling combined with benchmark dose modeling to predict, based on published in vitro data, the in vivo dose of amiodarone which may lead to adverse neurological effects in patients. We performed in vitro-in vivo extrapolation (IVIVE) from concentrations measured in the cell lysate of a rat brain 3D cell model using a validated human PBPK model. Among the observed in vitro effects, inhibition of choline acetyl transferase (ChAT) was selected as a marker for neurotoxicity. By reverse dosimetry, we transformed the in vitro concentration-effect relationship into in vivo effective human doses, using the calculated in vitro area under the curve (AUC) of amiodarone as the pharmacokinetic metric. The upper benchmark dose (BMDU) was calculated and compared with clinical doses eliciting neurological adverse effects in patients. The AUCs in the in vitro brain cell culture after 14-day repeated dosing of nominal concentration equal to 1.25 and 2.5 µM amiodarone were 1.00 and 1.99 µg*h/mL, respectively. The BMDU was 385.4 mg for intravenous converted to 593 mg for oral application using the bioavailability factor of 0.65 as reported in the literature. The predicted dose compares well with neurotoxic doses in patients supporting the hypothesis that impaired ChAT activity may be related to the molecular/cellular mechanisms of amiodarone neurotoxicity. Our study shows that predicting effects from in vitro data together with IVIVE can be used at the initial stage for the evaluation of potential adverse drug reactions and safety assessment in humans

Amiodarone biokinetics, the formation of its major metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14 days, Amiodarone major oxidative metabolite (mono-Ndesethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure

Human Variability in Carboxylesterases and carboxylesterase-related Uncertainty Factors for Chemical Risk Assessment

International audienceCarboxylesterases (CES) are an important class of enzymes involved in the hydrolysis of a range of chemicals and show large inter-individual variability in vitro. An extensive literature search was performed to identify in vivo probe substrates for CES1 and CES2 together with their protein content and enzymatic activity. Human pharmacokinetic (PK) data on Cmax, clearance, and AUC were extracted from 89 publications and Bayesian meta-analysis was performed using a hierarchical model to derive CES-related variability distributions and related uncertainty factors (UF). The CES-related variability indicated that 97.5% of healthy adults are covered by the kinetic default UF (3.16), except for clopidogrel and dabigatran etexilate. Clopidogrel is metabolised for a small amount by the polymorphic CYP2C19, which can have an impact on the overall pharmacokinetics, while the variability seen for dabigatran etexilate might be due to differences in the absorption, since this can be influenced by food intake. The overall CES-related variability was moderate to high in vivo (<CV 50%), which might be due to possible polymorphism in the enzyme but also to the small sample size available per chemical. The presented CES-related variability can be used in combination with in vitro data to derive pathway-specific distributions

In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments.

International audienceThe limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis. The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation

Inter-ethnic differences in CYP3A4 metabolism: A Bayesian meta-analysis for the refinement of uncertainty factors in chemical risk assessment

CYP3A4 is the major human cytochrome P450 isoform responsible for the metabolism of more than 50% of known xenobiotics. Here, inter-ethnic differences in CYP3A4 metabolism have been investigated through a systematic review of pharmacokinetic data for 15 CYP3A4 probe substrates and parameters reflecting acute (Cmax, oral route) and chronic exposure (clearance and area under the plasma concentration-time curve, oral and intravenous route). All data were extracted in a structured database and meta-analyses were performed using a hierarchical Bayesian model in the R freeware to derive parameter, route and population-specific variability distributions for CYP3A4 metabolism. Two different approaches were applied. 1) Inter-individual differences were quantified using North American healthy adults as a reference group to compare with European, Asian, Middle East, and South-American healthy adults and with ethnicity, elderly, children and neonates. 2) Intra-ethnic–specific variability distributions were derived without comparing to a reference group. Overall, subgroup-specific distributions for CYP3A4-variability provided the basis to derive CYP3A4-related uncertainty factors (UF) to cover 95th or 97.5th centiles of the population and were compared with the human default toxicokinetic UF (3.16). The results indicate that CYP3A4-related UFs in healthy adults were higher for chronic oral exposures (2.5–3.0, UF95 and UF97.5, 10 compounds) than for intravenous exposures (1.7–1.8, 2 compounds). All UFs were within the default TK UF. These distributions allow for: 1) the application of CYP3A4-related UFs in the risk assessment of compounds for which in vitro CYP3A4 metabolism evidence is available without the need for animal data; 2) the integration of CYP3A4-related variability distributions with in vitro metabolism data into physiologically based kinetic (PBK) models for quantitative in vitro to in vivo extrapolation (QIVIVE) and 3) the estimation of UFs in chemical risk assessment using variability distributions of metabolism