Introduction
Historically it was recommended that animals of the same strain, sex, age and weight should be used for biomedical investigations in order to reduce experimental variability (Lewis, 1960). Over sixty years later male animals still predominate in pre-clinical research; however, there is now extensive evidence of sex specific pharmacology. Indeed, substantial evidence shows that the stage of oestrous, and gonadal steroids can affect the response to drugs (Pham et al., 2001; Nakagawa et al., 2006; Salama and Bett, 2014). Translation of preclinical findings with novel molecular entities risks failure if the evidence is based on single sex research. Sex differences in pharmacology can no longer be ignored and a new paradigm is percolating through biomedical research with major funding agencies now requiring consideration of sex in all applications, and sex to be considered as a categorical variable in studies submitted for publication (Docherty et al., 2019).
Alfaxalone is a neuroactive steroid that modulates neurotransmission through interaction with a steroid recognition site on the GABAA receptor complex resulting in inhibition of neuronal excitability. This agent and similar molecules therefore have roles in anaesthesia, epilepsy, anxiety, insomnia, migraine, postpartum depression and drug dependence (Belelli et al., 2020). Alfaxalone is used to induce and maintain anaesthesia in a range of animal species and was used as an anaesthetic induction agent in humans but anaphylactoid reactions attributed to the polyethoxylated castor oil (Cremophor EL) vehicle made its use redundant. Subsequent formulations of alfaxalone incorporating a cyclodextrin have been devoid of the side effects and Alfaxan® (alfaxalone dissolved in 2-hydroxypropyl-β -cyclodextrin) is now registered for induction and maintenance of anaesthesia in dogs, cats, and rabbits. In biomedical research alfaxalone may offer some selective advantages over other anaesthetic combinations in terms of a wide safety margin, reflex suppression, cardiopulmonary depression, interaction with pain pathways/modulation and may also offer additional advantages in influencing CNS development and myelination (Rupprecht and Holsboer, 1999; Yawno et al., 2011; Shaw et al., 2021). To date human trials of alfaxalone (formulated with 13% 7-sulfobutyl ether β cyclodextrin) have been undertaken in healthy male volunteers (Goodchild et al., 2020).
Sex related differences in anaesthetic effects have been reported with old and new formulations of alfaxalone (Fink et al., 1982; Arenillas and Gomez de Segura, 2018). The cause of the sex difference was attributed to the presence of oestrogenic hormones potentiating the anaesthetic effects while others have suggested that differences of allopregnanolone concentration may increase the clinical sensitivity of alfaxalone in female rats. However, recent findings suggest that the sex differences observed for alfaxalone are pharmacokinetic (PK) dependent with female Sprague Dawley (SD) rats having a lower clearance than male SD rats (White et al., 2017).
The aim of this study was to investigate the influence of sex and strain (SD versus Lewis rats) on alfaxalone PK, anaesthetic and cardiovascular (CV) effects using standard deterministic and non-linear mixed effects methods (NLME) PK modelling. Furthermore, the resulting model was used to determine sex and strain specific dosing regimens for optimal anaesthesia.