Results
Alfaxalone pharmacodynamics for Lewis rats
Arterial blood pressure measurements for the Lewis rats during anaesthesia are presented in Figure 2. All rats showed an initial short-lived decrease in mean arterial blood pressure (MAP), heart rate and respiratory rate because of concomitant administration of isoflurane and alfaxalone. Within 5 minutes following discontinuation of isoflurane, all rats demonstrated an increase in blood pressure from the baseline readings obtained during anaesthesia with the gaseous volatile agent. Blood pressure (mean, systolic, diastolic), heart rate and respiratory rate at baseline were not significantly different between male and female rats under isoflurane anaesthesia. Heart rates remained stable during alfaxalone anaesthesia and there was no significant difference between the sexes at any time points. Systolic, mean and diastolic arterial pressures all increased from baseline under isoflurane anaesthesia. The MAP remained elevated until 220 minutes for males before declining, while females showed early signs of a MAP decrease from between 40-75 minutes. Significant differences in MAP were detected between the sexes at 120 minutes (p < 0.05).
All Lewis rats required IPPV as judged by apnoea, or a rise in end tidal carbon dioxide coupled with a decrease in respiratory rate and effort at some point and for different time periods. Blood gas parameters and biochemistry values are presented in Table 1. There were no significant differences between sexes for all parameters.
Hypnotic effect
The plane of anaesthesia was continually evaluated by serial cardiopulmonary measurements, blood gas analysis and reflex responses. Subjective evaluation of this hypnotic effect of the alfaxalone in all Lewis rats was excellent.
Deterministic individual alfaxalone PK for Lewis rats
A 1-compartment infusion model was shown to have the best fit to the individual Lewis rat PK data according to AIC. Figure 3 compares the male versus female Lewis rat PK data and model fit (curves) for each rat. Plasma concentrations of alfaxalone during maintenance CRI were greater in the females compared to male rats. PK parameters obtained from the model fit are shown in Table 2. Logarithmic transformed pharmacokinetic parameters were shown to be significantly different between the male and female rats. Mean CL of alfaxalone for male rats was more than twice that of female rats and with a higher Vdss for the latter, resulted in an almost 5 fold longer half-life in female rats compared to males.
NLME PK model for alfaxalone (population 1)
The most parsimonious NLME model obtained for population 1 was a 1-compartment model with random effects included on all parameters and no correlation (diagonal omega matrix). Goodness of model fit can be found in the supplementary data file. The covariates for sex and rat strain had the most significant influence on clearance (CL) and rat strain having the most significant influence on volume of distribution (Vd). The CL and Vd for individual rats (expressed per total body weight) within the population model are described as follows:
CL = CLTV * e(-0.841 * sex covariate)* e(0.478 * strain covariate) * e(CL eta) Equation 1
Vd = VdTV * e(-0.0237 * strain covariate) * e(Vd eta) Equation 2
Where CLTV and VdTV are the typical values (fixed effect) for Clearance (25.2 ml min-1) and volume of distribution (0.57 L) within population 1. These fixed effects are adjusted by the covariates (0 for male and Lewis, 1 for female and Sprague Dawley) to give an adjusted typical value for each group. CL eta and Vd eta represent the random effects, such as inter-individual variability, in the population for clearance and volume of distribution. Sex and strain outputted adjusted typical values (per total bodyweight) and post hoc PK parameters (per kg bodyweight) for the most parsimonious NMLE model are shown in Table 3. These PK parameters were encompassed by the 2.5 and 97.5% confidence intervals of the bootstrap resampling analysis indicating a robust model. Mean arterial pressure plotted against plasma alphaxalone concentration for male Lewis and female Lewis rats is depicted in Figure 4 (A and B). However, MAP decreases for female Lewis rats (B) were evident when alfaxalone concentration exceeded approximately 20 µg ml-1.
Adjusted alfaxalone infusion regimen for female SD rats
The NLME PK model for alfaxalone using population 1 was used to design an adjusted alfaxalone infusion regimen for female SD rats that matches male SD plasma concentrations minimising cardiopulmonary depression but maintaining an appropriate hypnotic effect.
Alfaxalone pharmacodynamics for SD male and females
There was no statistical difference between dose adjusted female and male SD rats mean arterial blood pressure (Figure 5).
IPPV was required for 9/16 female SD, and 5/8 male SD rats. Blood gas parameters and biochemistry values are presented in Table 1. There were no significant differences between sexes or strains for all parameters.
Hypnotic effect
The plane of anaesthesia was deemed inadequate for injection of capsaicin as part of the antinociception study for two female SD rats during the final reduced phase of the CRI in view of a very faint sluggish paw withdrawal and spontaneous blinking. No rats demonstrated gross purposeful movement or required a change in the infusion rate to improve the plane of anaesthesia however surgical anaesthesia was not an outcome measure of the model.
Alfaxalone pharmacokinetics for SD males and females
Figure 6 shows the measured alfaxalone plasma concentrations versus time for male and female SD rats using the adjusted regimen for the latter along with the simulated median, upper and lower 95% confidence intervals using the NMLE PK model. Plasma concentrations were similar with no statistical difference between male and female SD rats during the plateau phase.
Figure 7 shows no relationship between MAP and alfaxalone concentration for male SD (A) and dose adjusted female SD (B) rats.
NLME pharmacokinetic model for alfaxalone (population 2)
The most parsimonious NLME model obtained for population 2 was again a 1-compartment model with random effects included on all parameters and no correlation (diagonal omega matrix). Goodness of model fit can be found in the supplementary data file. The covariates for LCBW and sex had the most significant influence on alfaxalone clearance. Similar to the model for population 1, rat strain had the most significant influence on Vd. The CL and Vd for individual rats (expressed for total body weight) within the population model are described as follows:
CL = CLTV * (1 + LCBW * 3.64) * e(-0.43 * sex covariate) * e(CL eta) Equation 3
Vd = VdTV * e(-0.692 * strain covariate) * e(Vd eta) Equation 4
where CLTV and VdTV are the typical values for Clearance (35.2 ml min-1) and volume of distribution (0.51 L) within population 2. These fixed effects are adjusted by the covariates (0 for male and Lewis, 1 for female and Sprague Dawley and LCBW) to give an adjusted typical value for each group. Sex and strain outputted adjusted typical values and post hoc PK parameters (normalised per kg bodyweight) for the most parsimonious NMLE model are shown in Table 4. These PK parameters were encompassed by the 2.5 and 97.5% confidence intervals of the bootstrap resampling analysis indicating a robust model.