Next Generation Risk Assessment of the Anti-Androgen Flutamide Including the Contribution of Its Active Metabolite Hydroxyflutamide

Abstract

In next generation risk assessment (NGRA), non-animal approaches are used to quantify the chemical concentrations required to trigger bioactivity responses, in order to assure safe levels of human exposure. A limitation of many in vitro bioactivity assays, which are used in an NGRA context as new approach methodologies (NAMs), is that toxicokinetics, including biotransformation, are not adequately captured. The present study aimed to include, as a proof of principle, the bioactivity of the metabolite hydroxyflutamide (HF) in an NGRA approach to evaluate the safety of the anti-androgen flutamide (FLU), using the AR-CALUX assay to derive the NAM point of departure (PoD). The NGRA approach applied also included PBK modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE). The PBK model describing FLU and HF kinetics in humans was developed using GastroPlus™ and validated against human pharmacokinetic data. PBK model-facilitated QIVIVE was performed to translate the in vitro AR-CALUX derived concentration-response data to a corresponding in vivo dose-response curve for the anti-androgenicity of FLU, excluding and including the activity of HF (-HF and +HF, respectively). The in vivo benchmark dose 5% lower confidence limits (BMDL05) derived from the predicted in vivo dose-response curves for FLU, revealed a 440-fold lower BMDL05 when taking the bioactivity of HF into account. Subsequent comparison of the predicted BMDL05 values to the human therapeutic doses and historical animal derived PoDs, revealed that PBK modelling-facilitated QIVIVE that includes the bioactivity of the active metabolite is protective and provides a more appropriate PoD to assure human safety via NGRA, whereas excluding this would potentially result in an underestimation of the risk of FLU exposure in humans.

Next generation risk assessment of human exposure to estrogens using safe comparator compound values based on in vitro bioactivity assays

“…The present study focusing on estrogenicity and a previous study focusing on anti-androgenicity (van Tongeren et al 2022 ) showed that the DCR approach can offer a relatively quick analysis on the safety of a defined exposure scenario regarding biological endpoints of which corresponding in vitro bioactivity assays are available. In NGRA, a tiered workflow could be followed when an exposure to a (novel) compound is to be evaluated.…”

“…When using the three in vitro bioactivity assays in the DCR approach, this contribution to the estrogenicity may not be captured so that the observed in vitro toxicity of a parent compound may underestimate the toxicity in the human body. This issue can be overcome by using PBK models describing the kinetics of a parent compound and its respective relevant metabolites in humans enabling the prediction of the corresponding combined internal concentrations in parent compound equivalents (Mendez-Catala et al 2021 ; van Tongeren et al 2022 ; Wang et al 2020 ). Furthermore, co-incubation with liver S9 fraction in the in vitro bioactivity assays (Mollergues et al 2017 ) offers the opportunity to evaluate whether a compound will be converted to hepatic metabolites and whether they would be more potent to the corresponding biological target.…”

Next generation risk assessment of androgens and estrogens

“…When using the three in vitro bioactivity assays in the DCR approach, this contribution to the estrogenicity may not be captured so that the observed in vitro toxicity of a parent compound may underestimate the toxicity in the human body. This issue can be overcome by using PBK models describing the kinetics of a parent compound and its respective relevant metabolites in humans enabling the prediction of the corresponding combined internal concentrations in parent compound equivalents (Mendez-Catala et al, 2021;van Tongeren et al, 2022;Q. Wang et al, 2020).…”

“…Following physiologically based kinetic (PBK) modelling-based quantitative in vitro to in vivo extrapolation (QIVIVE) (Fabian et al, 2019;Louisse et al, 2017;Punt et al, 2019;Rietjens et al, 2011;Wetmore et al, 2015;Yoon et al, 2012), the in vitro concentration-responses of T and DHT in the presence of hepatic biotransformation can be transformed to the corresponding doseresponses for the androgenic effects in humans, to set the PoD. Another method to reflect hepatic biotransformation and thus the contribution of relevant metabolites to the in vivo bioactivity of the parent compound is using a toxic equivalency factor (TEF) approach in PBK modelling-based (QIVIVE) of the parent compound (van Tongeren et al, 2022). Other advantages of the two-chamber co-culture system with human liver and reporter cells is its applicability as a high-throughput screening tool of (anti)androgenic compounds, giving insight into both toxicokinetics and toxicodynamics.…”