In vivo/in vitro investigation of alphaxalone PK/PD sex differences in rats and other species

Abstract

Alphaxalone is an anaesthetic agent from the neurosteroids class and is currently licensed for cats, dogs and rabbits, while it is also being considered for human use. However, it has been reported that alphaxalone has PK/PD sex differences in rats. The cause of this difference is most likely to be sex related PK differences as has been observed by White, et al., (2017). In general, the primary cause of PK differences tends to be related to sex-based differences in drug metabolism which is usually a reflection of sex differences in the expression of liver metabolism enzymes. This is most pronounced in rodents but has also been observed in humans, rabbits, dogs and monkey. The aim of this study was to investigate the source of alphaxalone PK/PD sex differences in rats and whether this translates to dog, rabbit, monkey and human. An in vivo population PK analysis and in vitro investigation of alphaxalone metabolism were conducted in rats to determine sex specific optimal dosing and the major metabolism pathways for alphaxalone, respectively. Thus, an in vivo rat comparison study for alphaxalone was conducted which consisted of two groups using a total of 56 rats (17 male and 23 female) with either Lewis (n=16) or Sprague Dawley (n=24) strains. Alphaxalone PK parameters in rats were determined via deterministic PK models and showed female rats to have significantly lower alphaxalone clearance in comparison to male rats (36.8±20 and 98.3±32 mL/min/kg respectively). This led female rats to have higher alphaxalone plasma exposure resulting in cardiovascular inhibition. A population PK model was developed for alphaxalone in male and female rats for both strains. Initially, strain and sex were the most significant covariates influencing the population typical PK parameter values. However, it was later shown that sex and body weight were most significant with a larger population set. Furthermore, Monte Carlo simulations were performed to address the variability in alphaxalone exposure in rats among the population. Analysis showed Abstract that SD female rats need a 45% reduction in alphaxalone infusion rate in order to have equivalent plasma concentrations to SD male rats. Furthermore, alphaxalone was investigated in vitro using fresh rat hepatocytes in male (n=9) and female (n=6). The sex bias was also observed in vitro as male intrinsic clearance for disappearance of alphaxalone was approximately 5-fold larger than female rats. In addition, the male rats had significantly larger Vmax compared to female (135±54 versus 12±6 nmol/min), whereas Km was only marginally larger in male (170±106 versus 64±85 µM). Furthermore, the extrapolated in vivo hepatic clearance (ClH) compared to the estimated clearance from the in vivo study were in close agreement. In addition, alphaxalone metabolite and metabolism pathway identification in rat showed the identification of two distinct alphaxalone metabolites (Ketoalphaxalone and beta-alphaxalone) and the observation of an unidentified third alphaxalone (M3) metabolite and sex differences for the rate of metabolite formation were demonstrated. Using the knowledge acquired for alphaxalone metabolite formation in rats a mathematical kinetic model was created that described the metabolic pathway and estimated the metabolism conversion rate constants. This allowed the magnitude of sex differences to be determined for alphaxalone in rats, human, dog, rabbit, and monkey. Rats showed the largest difference in alphaxalone in vitro clearance between the sexes as well as sex differences in the alphaxalone metabolism kinetic rates and direction of the pathways. While the other species did not show clear sex differences in alphaxalone in vitro clearance each species showed different preferences in the direction of alphaxalo