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.