PBK modelling of topical application and characterisation of the uncertainty of Cmax estimate: A case study approach.

Combined with in vitro bioactivity data, physiologically based kinetic (PBK) models has increasing applications in next generation risk assessment for animal-free safety decision making. A tiered framework of building PBK models for such application has been developed with increasing complexity and refinements, as model parameters determined in silico, in vitro, and with human pharmacokinetic data become progressively available. PBK modelling has been widely applied for oral/intravenous administration, but less so on topically applied chemicals. Therefore, building PBK models for topical applications and characterizing their uncertainties in the tiered approach is critical to safety decision making. The purpose of this study was to assess the confidence of PBK modelling of topically applied chemicals following the tiered framework, using non-animal methods derived parameters. Prediction of maximum plasma concentration (Cmax) and area under the curve were compared to observed kinetics from published dermal clinical studies for five chemicals (diclofenac, salicylic acid, coumarin, nicotine, caffeine). A bespoke Bayesian statistical model was developed to describe the distributions of Cmax errors between the predicted and observed data. We showed a general trend that confidence in model predictions increases when more quality in vitro data, particularly those on hepatic clearance and dermal absorption, are available as model input. The overall fold error distributions are useful for characterizing model uncertainty. We concluded that by identifying and quantifying the uncertainties in the tiered approach, we can increase the confidence in using PBK modelling to help make safety decisions on topically applied chemicals in the absence of human pharmacokinetic data.

Predictive performance of next generation human physiologically based kinetic (PBK) models based on in vitro and in silico input data.

The goal of the present study was to assess the predictive performance of a generic human physiologically based kinetic (PBK) model based on in vitro and in silico input data and the effect of using different input approaches for chemical parameterization on those predictions. For this purpose, a dataset was created of 38,772 Cmax predictions for 44 compounds by applying different combinations of in vitro and in silico approaches for chemical parameterization, and these predicted Cmax values were compared to reported in vivo data. Best results were achieved when the hepatic clearance was parameterized based on in vitro (i.e., hepatocytes or liver S9) measured intrinsic clearance values, the method of Rodgers and Rowland for calculating tissue:plasma partition coefficients, and the method of Lobell and Sivarajah for calculating the fraction unbound in plasma. With these parameters, the median Cmax values of 34 out of the 44 compounds were predicted within 5-fold of the observed Cmax, and the Cmax values of 19 compounds were predicted within 2-fold. The median Cmax values of 10 compounds were more than 5-fold overestimated. Underestimations (> 5-fold) did not occur. A comparison of the current generic PBK model structure with chemical-specific PBK models available in literature was made to identify possible kinetic processes not included in the generic PBK model that might explain the overestimations. Overall, the results provide crucial insights into the predictive performance of PBK models based on in vitro and in silico input and the influence of different input approaches on the model predictions.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

In Silico Simulation of Simultaneous Percutaneous Absorption and Xenobiotic Metabolism: Model Development and a Case Study on Aromatic Amines.

PURPOSE: To advance physiologically-based pharmacokinetic modelling of xenobiotic metabolism by integrating metabolic kinetics with percutaneous absorption. METHOD: Kinetic rate equations were proposed to describe the metabolism of a network of reaction pathways following topical exposure and incorporated into the diffusion-partition equations of both xenobiotics and metabolites. The published ex vivo case study of aromatic amines was simulated. Diffusion and partition properties of xenobiotics and subsequent metabolites were determined using physiologically-based quantitative structure property relationships. Kinetic parameters of metabolic reactions were best fitted from published experimental data. RESULTS: For aromatic amines, the integrated transdermal permeation and metabolism model produced data closely matched by experimental results following limited parameter fitting of metabolism rate constants and vehicle:water partition coefficients. The simulation was able to produce dynamic concentration data for all the dermal layers, as well as the vehicle and receptor fluid. CONCLUSION: This mechanistic model advances the dermal in silico functionality. It provides improved quantitative spatial and temporal insight into exposure of xenobiotics, enabling the isolation of governing features of skin. It contributes to accurate modelling of concentrations of xenobiotics reaching systemic circulation and additional metabolite concentrations. This is vital for development of both pharmaceuticals and cosmetics.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Application of physiologically based kinetic (PBK) modelling in the next generation risk assessment of dermally applied consumer products.

Next Generation Risk Assessment (NGRA) is a procedure that integrates new approach methodologies (NAMs) to assure safety of a product without generating data from animal testing. One of the major challenges in the application of NGRA to consumer products is how to extrapolate from the in vitro points of departure (PoDs) to the human exposure level associated with product use. To bridge the gap, physiologically based kinetic (PBK) modelling is routinely used to predict systemic exposure (Cmax or AUC) from external exposures. A novel framework was developed for assessing the exposure of new ingredients in dermally applied products based on the construction of PBK models describing consumer habits and practices, formulation type, and ADME (absorption, distribution, metabolism and excretion) properties exclusively obtained from NAMs. This framework aims to quantify and reduce the uncertainty in predictions and is closely related to the risk assessment process (i.e., is the margin of safety sufficient to cover the uncertainties in the extrapolation between the in vitro and in vivo toxicodynamics and toxicokinetics?). Coumarin, caffeine, and sulforaphane in four product types (kitchen cleaner liquid, face cream, shampoo, and body lotion) were selected to exemplify how this framework could be used in practise. Our work shows initial levels of the framework provide a conservative estimate of Cmax in most cases which can be refined using sensitivity analysis to inform the choice of follow-up in vitro experiments. These case studies show the framework can increase confidence in use of PBK predictions for safety assessment.

Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol.

OECD test guideline 428 compliant protocol using human skin was used to test the penetration of 56 cosmetic-relevant chemicals. The penetration of finite doses (10 µL/cm2 ) of chemicals was measured over 24 hours. The dermal delivery (DD) (amount in the epidermis, dermis and receptor fluid [RF]) ranged between 0.03 ± 0.02 and 72.61 ± 8.89 µg/cm2 . The DD of seven chemicals was comparable with in vivo values. The DD was mainly accounted for by the amount in the RF, although there were some exceptions, particularly of low DD chemicals. While there was some variability due to cell outliers and donor variation, the overall reproducibility was very good. As six chemicals had to be applied in 100% ethanol due to low aqueous solubility, we compared the penetration of four chemicals with similar physicochemical properties applied in ethanol and phosphate-buffered saline. Of these, the DD of hydrocortisone was the same in both solvents, while the DD of propylparaben, geraniol and benzophenone was lower in ethanol. Some chemicals displayed an infinite dose kinetic profile; whereas, the cumulative absorption of others into the RF reflected the finite dosing profile, possibly due to chemical volatility, total absorption, chemical precipitation through vehicle evaporation or protein binding (or a combination of these). These investigations provide a substantial and consistent set of skin penetration data that can help improve the understanding of skin penetration, as well as improve the prediction capacity of in silico skin penetration models.

Mathematical modelling of a liver hollow fibre bioreactor.

A mathematical model has been developed to assist with the development of a hollow fibre bioreactor (HFB) for hepatotoxicity testing of xenobiotics; specifically, to inform the HFB operating set-up, interpret data from HFB outputs and aid in optimizing HFB design to mimic certain hepatic physiological conditions. Additionally, the mathematical model has been used to identify the key HFB and compound parameters that will affect xenobiotic clearance. The analysis of this model has produced novel results that allow the operating set-up to be calculated, and predictions of compound clearance to be generated. The mathematical model predicts the inlet oxygen concentration and volumetric flow rate that gives a physiological oxygen gradient in the HFB to mimic a liver sinusoid. It has also been used to predict the concentration gradients and clearance of a test drug and paradigm hepatotoxin, paracetamol (APAP). The effect of altering the HFB dimensions and fibre properties on APAP clearance under the condition of a physiological oxygen gradient is analysed. These theoretical predictions can be used to design the most appropriate experimental set up and data analysis to quantitatively compare the functionality of cell types that are cultured within the HFB to those in other systems.

Development of a Two-Dimensional Model for Predicting Transdermal Permeation with the Follicular Pathway: Demonstration with a Caffeine Study.

PURPOSE: The development of a new two-dimensional (2D) model to predict follicular permeation, with integration into a recently reported multi-scale model of transdermal permeation is presented. METHODS: The follicular pathway is modelled by diffusion in sebum. The mass transfer and partition properties of solutes in lipid, corneocytes, viable dermis, dermis and systemic circulation are calculated as reported previously [Pharm Res 33 (2016) 1602]. The mass transfer and partition properties in sebum are collected from existing literature. None of the model input parameters was fit to the clinical data with which the model prediction is compared. RESULTS: The integrated model has been applied to predict the published clinical data of transdermal permeation of caffeine. The relative importance of the follicular pathway is analysed. Good agreement of the model prediction with the clinical data has been obtained. The simulation confirms that for caffeine the follicular route is important; the maximum bioavailable concentration of caffeine in systemic circulation with open hair follicles is predicted to be 20% higher than that when hair follicles are blocked. CONCLUSIONS: The follicular pathway contributes to not only short time fast penetration, but also the overall systemic bioavailability. With such in silico model, useful information can be obtained for caffeine disposition and localised delivery in lipid, corneocytes, viable dermis, dermis and the hair follicle. Such detailed information is difficult to obtain experimentally.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Combined mathematical modelling and experimentation to predict polymersome uptake by oral cancer cells.

This study is motivated by understanding and controlling the key physical properties underlying internalisation of nano drug delivery. We consider the internalisation of specific nanometre size delivery vehicles, comprised of self-assembling amphiphilic block copolymers, called polymersomes that have the potential to specifically deliver anticancer therapeutics to tumour cells. The possible benefits of targeted polymersome drug delivery include reduced off-target toxic effects in healthy tissue and increased drug uptake by diseased tissue. Through a combination of in vitro experimentation and mathematical modelling, we develop a validated model of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway, incorporating receptor binding, clustering and recycling. The model predicts how the characteristics of receptor targeting, and the size and concentration of polymersomes alter uptake by tumour cells. The number of receptors per cell was identified as being the dominant mechanism accounting for the difference between cell types in polymersome uptake rate. FROM THE CLINICAL EDITOR: This article reports on a validated model developed through a combination of in vitro experimentation and mathematical modeling of nanoparticle uptake by cells via the clathrin-mediated endocytotic pathway. The model incorporates receptor binding, clustering, and recycling and predicts how the characteristics of receptor targeting, the size and concentration alter polymersome uptake by cancer cells.

The evolution of covert, silent infection as a parasite strategy.

Many parasites and pathogens cause silent/covert infections in addition to the more obvious infectious disease-causing pathology. Here, we consider how assumptions concerning superinfection, protection and seasonal host birth and transmission rates affect the evolution of such covert infections as a parasite strategy. Regardless of whether there is vertical infection or effects on sterility, overt infection is always disadvantageous in relatively constant host populations unless it provides protection from superinfection. If covert infections are protective, all individuals will enter the covert stage if there is enough vertical transmission, and revert to overt infections after a 'latent' period (susceptible, exposed, infected epidemiology). Seasonal variation in transmission rates selects for non-protective covert infections in relatively long-lived hosts with low birth rates typical of many mammals. Variable host population density caused by seasonal birth rates may also select for covert transmission, but in this case it is most likely in short-lived fecund hosts. The covert infections of some insects may therefore be explained by their outbreak population dynamics. However, our models consistently predict proportions of covert infection, which are lower than some of those observed in nature. Higher proportions of covert infection may occur if there is a direct link between covert infection and overt transmission success, the covert infection is protective or the covert state is the result of suppression by the host. Relatively low proportions of covert transmission may, however, be explained as a parasite strategy when transmission opportunities vary.