Discussion
This study demonstrated a sex difference in alfaxalone PK parameters using a 2 stage deterministic approach for male and female Lewis rats. Sex differences were observed for both CL and Vd, where the former influences steady-state concentrations and the latter loading dose. As steady-state conditions are of the most interest only differences in CL will be discussed. Alfaxalone CL for female Lewis rats was significantly lower than for male animals. Separate studies by Visser et al. (2002) and Lau et al. (2013) have shown alfaxalone clearance to be 158 ± 29 ml min-1 kg-1 and 54.3 ± 6.8 ml min-1 kg-1 for male and female Wistar rats respectively, and White et al. (2017) also identified a sex difference for alfaxalone CL in SD rats. The stepping down of the infusion rates in the Lewis rats was for purposes of creating dynamic change towards the end of the experiment, however, no significant change in concentration was apparent. For this reason, the stepping down was omitted for the SD animals and the reduced CRI is in fact the maintenance CRI designed to achieve maintenance levels faster to replicate the male model.
NLME PK models offer the advantage of having a single model that describes a population and therefore shares data interpretation between animals unlike the standard deterministic PK approach. Moreover, sub populations such as the sex and strain can be described by co-variates that adjust the fixed effects (typical values). NLME models for populations 1 and 2 exemplify the sex difference in alfaxalone clearance where in both cases sex is a highly significant covariate. However, the deterministic estimated clearance parameter for Lewis rats for both sexes in the current study were in the lower range compared to the reported values by the Visser and Lau groups for Wistar rats as well as the reported values by White et al. for SD rats (White, et al. 2017). This is consistent with strain being a significant covariate on clearance in the population 1 NLME PK model.
However, the conclusion of the larger population 2 model suggests that LCBW is the more significant covariate source for clearance. This may be partly due to body weight having a wider range in population 2 compared to the narrower range in population 1 as the models were based on total dose. However, LCBW contains an allometric transformation for bodyweight and therefore Lewis rats, which have a lower bodyweight compared to age matched SD animals, have an enhanced lower clearance compared to SD animals. This is reflected in Table 4 where the estimated post hoc clearance in SD female rats is significantly higher than female Lewis rats when normalised per kg bodyweight. However, the allometric relationship between clearance and bodyweight is an inverse one, which is unusual. Alternatively, as there is a correlation between weight and strain, it may be the case that strain is the real reason for the difference, as was the case for model 1, and population 2 is biased by the majority SD data leading to LCBW being the most statistically significant co-variate.
The present study used arterial pressure measurements as a clinical biomarker for the PD investigation. Cardiovascular effects were chosen as a biomarker because alfaxalone exerts a dose dependent depression of the cardiorespiratory system i.e. a decrease in blood pressure (Khan et al., 2014; Muir et al., 2009; Sear, 1996) but to a lesser extent compared to other anaesthetics such as thiopental and propofol (Goodchild et al., 2015; Visser et al., 2002). As such, a dose reduction (33%) of alfaxalone near the end of infusion for Lewis rats was designed to observe a dynamic change in the cardiovascular effect to determine an IC50. However, there was no noticeable change in the cardiovascular response as a result of the 30% reduction phase. One explanation of the blood pressure discrepancies might be the female Lewis were most responsive being impacted by the higher alfaxalone plasma concentrations and this was manifest as a relative hypotension compared to the male animals where a stable blood pressure was maintained during anaesthesia. The population 1 NLME PK model was used to simulate a dosing regimen for female SD rats that would give a similar alfaxalone plasma profile to that of male SD whilst simultaneously minimising cardiopulmonary depression enhancing the profile particularly for prolonged anaesthesia.
There are some limitations in the present study for the PD measurements. All the rats had a sampling cannula secured in the left carotid artery which required surgery and the use of gaseous anaesthesia for instrumentation. Isoflurane can cause significant cardiovascular depression in a dose dependent manner (Yang, et al., 2014), as such, no arterial pressure baselines in conscious rats were available. Furthermore, after discontinuing the gases, most rats’ arterial pressure rebounded rapidly to a higher blood pressure. However, in view of the concurrent cessation of the volatile agent and a loading dose of alfaxalone infusion, it is not clear what the value of the real baseline is. Subsequent work evaluating the suitability of the alfaxalone model for surgical anaesthesia and compatibility with other adjuncts and analgesics is advised to move away from reliance on the volatile agents, thereby minimising their detrimental environmental impact.
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