TKPlate 1.0: An Open-access platform for toxicokinetic and toxicodynamic modelling of chemicals to implement new approach methodologies in chemical risk assessment.

This publication is linked to the following EFSA Supporting Publications articles: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8441/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8440/full, http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2023.EN-8437/full.

PBTK modeled perfluoroalkyl acid kinetics in zebrafish eleutheroembryos suggests impacts on bioconcentrations by chorion porosity dynamics.

The zebrafish eleutheroembryo (zfe) is widely used as a model to characterize the toxicity of chemicals. However, analytical methods are still missing to measure organ concentrations. Therefore, physiologically-based toxicokinetic (PBTK) modeling may overcome current limitations to help understand the relationship between toxic effects and internal exposure in various organs. A previous PBTK model has been updated to include the chorionic transport barrier and its permeabilization, hatching dynamics within a zfe population over development, and active mediated transport mechanisms. The zfe PBTK model has been calibrated using measured time-dependent internal concentrations of PFBA, PFHxS, PFOA, and PFOS in a zfe population and evaluated using external datasets from the literature. Calibration was successful with 96% of the predictions falling within a 2-fold range of the observed concentrations. The external dataset was correctly estimated with about 50% of the predictions falling within a factor of 3 of the observed data and 10% of the predictions are out of the 10-fold error. The calibrated model suggested that active mediated transport differs between PFAS with a sulfonic and carboxylic acid functional end groups. This PBTK model predicts well the fate of PFAS with various physicochemical properties in zfe. Therefore, this model may improve the use of zfe as an alternative model in toxicokinetic-toxicodynamic studies and help to refine and reduce zfe-based experiments, while giving insights into the internal kinetics of chemicals.

A PBPK model to evaluate zebrafish eleutheroembryos' actual exposure: bisphenol A and analogs' (AF, F, and S) case studies.

The zebrafish eleutheroembryo model is increasingly used to assess the toxicity and developmental adverse effects of xenobiotics. However, the actual exposure is seldom measured (poorly accessible), while a predictive model could estimate these concentrations. The predictions with a new eleutheroembryo physiologically based pharmacokinetic (PBPK) model have been evaluated using datasets obtained from literature data for several bisphenols. The model simulated the toxicokinetics of bisphenols A (BPA), AF, F, and S through the eleutheroembryo tissues while considering the body and organ growth. We further improved the predictions by adding dynamic flows through the embryo and/or its chorion, impact of experimental temperature, metabolic clearance, and saturation of the absorption by Bayesian calibration. The model structure was determined using the BPA dataset and generalized to the other bisphenols. This model revealed the central role of the chorion in the compound uptake in the first 48 h post-fertilization. The predictions for the BPA substitutes estimated by our PBPK model were compared to available toxicokinetics data for zebrafish embryos, and 63% and 88% of them were within a twofold and fivefold error intervals of the corresponding experimental values, respectively. This model provides a tool to design new eleutheroembryo assays and evaluate the actual exposure.

Baclofen self-poisoning: Is renal replacement therapy efficient in patient with normal kidney function?

AIMS: We aimed at assessing the effectiveness of renal replacement therapy in patients severely self-poisoned with baclofen and with normal kidney function. METHODS: A population pharmacokinetic model was built using analytical data extracted from 26 baclofen poisoning cases reported to a French Poison Centre: 8 patients underwent renal replacement therapy (RRT), 18 did not. In the RRT group, 2 patients suffered from kidney failure. Mechanical ventilation was required for 20 patients with normal kidney function; 15 were not treated by RRT and 5 were. Pharmacokinetic profiles of baclofen were measured in 28 patients and further modelled by a non-parametric approach (PMetrics®). The total data set was divided into a building data set (26 patients, 57 observations) and a validation set (2 external patients, 6 observations). Then, the estimated elimination half-life of baclofen and the duration of intubation were compared in patients with or without RRT using Wilcoxon-Mann-Whitney test. RESULTS: A model using three parameters plus a lag time and bioavailability was necessary to determine the pharmacokinetics of baclofen. Estimated elimination half-life in the 'RRT' group and the 'no RRT' group were respectively 3.1 [2.2-4.8] h (n = 6 patients) and 3.4 [1.4-5.5] h (n = 19 patients, p = 0.53). The median duration of intubation was not significantly different between groups (72 [48-72] h and 72 [24-96] h, respectively; p = 0.38). CONCLUSION: Renal replacement therapy did not appear to significantly increase baclofen clearance in patients without kidney failure.

In vivo comparison of the proangiogenic properties of chlordecone and three of its dechlorinated derivatives formed by in situ chemical reduction.

In situ chemical reduction (ISCR) has been identified as a possible way for the remediation of soils contaminated by chlordecone (CLD). Evidences provided by the literature indicate an association between the development of prostate cancer and CLD exposure (Multigner et al. 2010). In a previous in vitro study, we demonstrated that the two main dechlorinated CLD derivatives formed by ISCR, CLD-1Cl, and CLD-3Cl have lower cytotoxicity and proangiogenic properties than CLD itself (Legeay et al. 2017). By contrast, nothing is known on the in vivo proangiogenic effect of these dechlorinated derivatives. Based on in vitro data, the aims of this study were therefore to evaluate the in vivo influence of CLD and three of its dechlorinated metabolites in the control of neovascularization in a mice model of prostate cancer. The proangiogenic effect of CLD and three of its dechlorinated derivatives, CLD-1Cl, CLD-3Cl, and CLD-4Cl, was evaluated on a murine model of human prostate tumor (PC-3) treated, at two exposure levels: 33 µg/kg and 1.7 µg/kg respectively reflecting acute and chronic toxic exposure in human. The results of serum measurements show that, for the same ingested dose, the three metabolite concentrations were significantly lower than that of CLD. Dechlorination of CLD lead therefore to molecules that are biologically absorbed or metabolized, or both, faster than the parent molecule. Prostate tumor growth was lower in the groups treated by the three metabolites compared to the one treated by CLD. The vascularization measured on the tumor sections was inversely proportional to the rate of dechlorination, the treatment with CLD-4Cl showing no difference with control animals treated with only the vehicle oil used for all substances tested. We can therefore conclude that the proangiogenic effect of CLD is significantly decreased following the ISCR-resulting dechlorination. Further investigations are needed to elucidate the molecular mechanisms by which dechlorination of CLD reduces proangiogenic effects in prostate tumor.

Two dechlorinated chlordecone derivatives formed by in situ chemical reduction are devoid of genotoxicity and mutagenicity and have lower proangiogenic properties compared to the parent compound.

Chlordecone (CLD) is a chlorinated hydrocarbon insecticide, now classified as a persistent organic pollutant. Several studies have previously reported that chronic exposure to CLD leads to hepatotoxicity, neurotoxicity, raises early child development and pregnancy complications, and increases the risk of liver and prostate cancer. In situ chemical reduction (ISCR) has been identified as a possible way for the remediation of soils contaminated by CLD. In the present study, the objectives were (i) to evaluate the genotoxicity and the mutagenicity of two CLD metabolites formed by ISCR, CLD-5a-hydro, or CLD-5-hydro (5a- or 5- according to CAS nomenclature; CLD-1Cl) and tri-hydroCLD (CLD-3Cl), and (ii) to explore the angiogenic properties of these molecules. Mutagenicity and genotoxicity were investigated using the Ames's technique on Salmonella typhimurium and the in vitro micronucleus micromethod with TK6 human lymphoblastoid cells. The proangiogenic properties were evaluated on the in vitro capillary network formation of human primary endothelial cells. Like CLD, the dechlorinated derivatives of CLD studied were devoid of genotoxic and mutagenic activity. In the assay targeting angiogenic properties, significantly lower microvessel lengths formed by endothelial cells were observed for the CLD-3Cl-treated cells compared to the CLD-treated cells for two of the three tested concentrations. These results suggest that dechlorinated CLD derivatives are devoid of mutagenicity and genotoxicity and have lower proangiogenic properties than CLD.

Liquid chromatography-mass spectrometry methods for the intracellular determination of drugs and their metabolites: a focus on antiviral drugs.

Understanding the efficacy and/or toxicity of most drugs requires effective intracellular measurements of the drug and its metabolites. Nevertheless, the most common plasma marker of the biological effect of the drug is the area under the curve. Compared with drug determination in whole blood or urine, various difficulties occur in the development of analytical methods for intracellular measurements. We propose step-by-step guidelines to develop an analytical method exploring intracellular concentrations of antivirals and/or their metabolites. These guidelines are illustrated with the most sensitive liquid chromatography-mass spectrometry methods developed for human in vivo and in vitro studies. We summarize 18 studies that provided methods to explore intracellular concentrations of antivirals since 2002. To explore intracellular metabolites, two different approaches can be envisaged. The direct approach, most frequently using ion-pairing agents, is fast and requires only a small sample but is expensive. The indirect approach is the more widely used approach, but is cumbersome and time-consuming. In both cases, liquid chromatography-mass spectrometry has become the method of choice to determine intracellular drug concentrations with high sensitivity. These methods may increase our understanding of drug behavior in organisms. This is true for preclinical studies where the mechanism of action, the metabolism, and the toxicity of drugs are explored. It is also true for clinical applications when dose adjustment is needed and cannot rely on blood concentrations. Graphical Abstract Direct and indirect approaches to measure intracellular concentrations.

Models for drug absorption from the small intestine: where are we and where are we going?

The small intestine is a complex organ with movements, flora, mucus and flows. Despite this, the most widely used absorption models consider the organ a cylindrical monoepithelial tube. This review presents the recent evolution of models to take into consideration the complex nature of gut physiology. The most commonly encountered issues are ethical (in vivo models) and differences in drug transport as a result of a modified expression of drug transporters or metabolic enzymes compared with human (in vitro and in vivo models). Finally, this review discusses the way forward to reach an ideal equilibrium between reproducibility, predictability and efficiency for predicting permeability. The features of an ideal model are listed as a guideline for future development.

Multidrug resistance-associated protein 4 (MRP4) controls ganciclovir intracellular accumulation and contributes to ganciclovir-induced neutropenia in renal transplant patients.

Ganciclovir (GCV) is the cornerstone of cytomegalovirus prevention and treatment in transplant patients. It is associated with problematic adverse hematological effects in this population of immunosuppressed patients, which may lead to dose reduction thus favoring resistance. GCV crosses the membranes of cells, is activated by phosphorylation, and then stops the replication of viral DNA. Its intracellular accumulation might favor host DNA polymerase inhibition, hence toxicity. Following this hypothesis, we investigated the association between a selected panel of membrane transporter polymorphisms and the evolution of neutrophil counts in n=174 renal transplant recipients. An independent population of n=96 renal transplants served as a replication and experiments using HEK293T-transfected cells were performed to validate the clinical findings. In both cohorts, we found a variant in ABCC4 (rs11568658) associated with decreased neutrophil counts following valganciclovir (GCV prodrug) administration (exploratory cohort: ß±SD=-0.68±0.28, p=0.029; replication cohort: ß±SD=-0.84±0.29, p=0.0078). MRP4-expressing cells showed decreased GCV accumulation as compared to negative control cells (transfected with an empty vector) (-61%; p<0.0001). The efflux process was almost abolished in cells expressing MRP4 rs11568658 variant protein. Molecular dynamic simulations of GCV membrane crossing showed a preferred location of the drug just beneath the polar head group region, which supports its interaction with efflux transporters.

Plasma and intracellular exposure to ganciclovir in adult renal transplant recipients: is there an association with haematological toxicity?

OBJECTIVES: Ganciclovir is the most widely used treatment for cytomegalovirus infections. However, neutropenia is a frequent associated adverse effect leading to a decrease in the ganciclovir dose or discontinuation of the therapy, thereby favouring viral resistance. In the present study, the objectives were: (i) to describe the pharmacokinetics of blood and intracellular ganciclovir and its metabolites; and (ii) to explore the relationship between exposure to ganciclovir and/or its metabolites and evolution of the neutrophil count under treatment. METHODS: Pharmacokinetic profiles (pre-dose and 1, 2, 3 and 5 h after dosing) of ganciclovir and its metabolites were measured in 22 adult renal transplant patients and further modelled by a non-parametric approach (PMetrics(®)). The relationship between exposure indices to ganciclovir and the slope of the neutrophil count was investigated using multiple linear regression. RESULTS: A four-compartment open model was able to accurately describe ganciclovir and its intracellular forms. A significant association was found between intracellular ganciclovir triphosphate concentrations (AUC0-5) and the decrease in neutrophil count over the first 3 months of treatment (ß= -0.0019 ± 5 × 10(-4); P < 0.01). CONCLUSIONS: In this population of renal transplant patients, the decrease in neutrophil count, used as a surrogate marker of haematological toxicity, was associated with ganciclovir triphosphate accumulation in blood cells. Further studies are needed to test this biomarker as a predictive factor for toxicity.

Liquid chromatography tandem mass spectrometry quantitation of intracellular concentrations of ganciclovir and its phosphorylated forms.

Ganciclovir (GCV) is prescribed for cytomegalovirus infection which is a major issue in immunodepressed patients. It is however characterized by hematological toxicity. A better understanding of GCV concentration-effects relationships implies the measurement of intracellular forms. The objective of this study was to develop a method to measure GCV and its derivatives in cells. A four-stage procedure was developed with the following strategy: (1) to separate into different fractions the different intracellular forms of GCV (GCV itself and its phosphorylated forms) by solid-phase extraction (SPE) from blood cells, (2) to dephosphorylate the different phosphorylated forms into GCV, (3) to perform a second SPE to desalt samples and concentrate GCV, and (4) to measure GCV concentrations in the different extracts using a triple-quadrupole, linear ion trap mass spectrometer. Finally, the procedure was tested in 17 patients receiving GCV. From lysed cells, the different forms of GCV were fractionated, the phosphorylated forms were eluted with different KCl solutions, and the obtained fractions were treated with acid phosphatase to transform the phosphorylated metabolites back into GCV. The method was validated from 5 to 500 µg L(-1) with a limit of detection of 1 µg L(-1). The whole procedure was validated according to the US Food and Drug Administration guidelines and successfully applied in 17 patients receiving GCV. The method liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowing the measurement of GCV and its phosphorylated forms in blood cells was developed and can be used in developing clinical studies to explore the role of these biomarkers in the event of toxicity.