Methods
This study was performed in accordance with Project Licence PPL30/3156 issued under the Animal (Scientific) Procedures Act 2013 (EU Directive 2010/63/EU) and local ethics committee as part of a larger study investigating nociceptive withdrawal reflexes and diffuse noxious inhibitory controls. This study is reported in accordance with the ARRIVE 2.0 guidelines (du Sert et al., 2020).
Study design
The NC3Rs experimental design assistant (EDA) was used to design the study. The population PK model experiment was conducted in tandem with a larger study investigating nociceptive withdrawal reflexes (NWR) and the influence of anaesthesia on diffuse noxious inhibitory controls (DNIC) in the rat. This in vivo experimental system in rats replicates aspects of the human pain pathway (Le Bars et al., 1992, 2001; White et al., 2018). This study design facilitated the population PK model generating matched groups of male and female SD and Lewis rats undergoing nociceptive testing and DNIC studies. The motivation for undertaking the study was to interrogate the influence of the anaesthesia per se , which is essential prerequisite although oftentimes overlooked or ignored. This is particularly important in electrophysiology experiments and nociception paradigms conducted under anaesthesia, where the anaesthesia delivered can have a major influence on the reflexes being studied.
Animals
Sixteen adult (8-12 weeks) Lewis rats, 9 males (308 ± 49 g) and 7 females (222 ± 9 g) and 24 adult (9-12 weeks) SD rats, 8 males (422 ± 41 g) and 16 female (304 ± 15 g) (Charles River Laboratories, Margate, UK) were used.
Animals were housed in single sex groups of 4, in double layer ventilated cages, given access to food (Teklad 2018, Harlan) and tap water ad libitum and maintained on a 12-hour light/dark cycle. All cages had play tubes, bedding material and chew blocks for enrichment. All experiments started at 10:00 h each day.
General anaesthesia
The methods for instrumentation of animals used were identical to those previously described by (White et al., 2017). Rats were anaesthetised with isoflurane (3% for induction of anaesthesia, 1-1.5% during surgery) in nitrous oxide/oxygen (2:1) mixture. Lidocaine 2% (Lignol, Dechra, Shrewsbury, UK) 3 mg kg-1 was infiltrated subcutaneously prior to skin incision. Using aseptic techniques the left jugular vein was surgically cannulated using 0.63 mm O.D. polyethylene tubing (Fisher Scientific, Loughborough, UK) for administration of drugs and isotonic fluids. The left carotid artery was surgically cannulated using 1mm O.D. polyethylene tubing (Fisher Scientific, Loughborough, UK) to monitor arterial blood pressure and for sampling.
Monitoring anaesthesia
The hypnotic characteristics of the anaesthetic were evaluated by monitoring the paw withdrawal reflex in response to pinch, corneal reflex in response to light brushing, spontaneous blinking and gross purposeful movement and cardiopulmonary parameters. Arterial blood pressure was monitored by an arterial pressure transducer (SensoNor 840; SensoNor, Horten, Norway) and recorded using a PC running Spike2 software (CED Ltd, Cambridge, UK). Heart rate was recorded via two 25g needles inserted subcutaneously on the lateral sides of the thoracic wall. The ECG signal was amplified and used to trigger an instant rate meter (Neurolog NL253, Digitimer, Welwyn Garden City, UK) and again recorded using Spike2 software. Respiratory rate and effort were assessed by observing chest excursion and measuring end tidal carbon dioxide (CapStar 100, Linton, Diss, UK). Intermittent positive pressure ventilation (IPPV) was instigated (SAV04, Vetronic, Abbotskerwell, UK) in the face of hypoventilation to maintain normocapnia.
Infusion of alfaxalone
Infusion regimens of alfaxalone (Alfaxan®, Jurox, Malvern, UK) were administered to rats using a calibrated syringe driver (SP100iz, WPI, Hitchin, UK). All animals received a loading dose (1.67 mg kg-1 for 2.5 minutes) followed by a constant rate infusion (CRI). For all animals isoflurane and nitrous oxide were stopped 2.5 minutes after starting the alfaxalone infusion, but oxygen was supplied throughout the experiment. The male and female Lewis rats (n=16) were administered a 60-minute CRI (0.75 mg kg-1min-1) followed by a reduced CRI (0.57 mg kg-1 min-1) for the remainder of the experiment. The Sprague Dawley males (n=8) received a 0.75 mg kg-1 min-1 CRI throughout the experiment. The Sprague Dawley females received a 60-minute CRI (0.57 mg kg-1 min-1) followed by a reduced dose (0.42 mg kg-1 min-1) for the remainder of the experiment.
Sampling
Arterial blood was withdrawn from the carotid cannula into lithium heparin tubes and placed on ice. Blood samples (200 μl) were collected at baseline (prior to alfaxalone administration) and at standardised time points across the alfaxalone infusion period. Arterial blood gases, biochemistry and haematology parameters (pH, pCO2, pO2, bicarbonate, sodium, potassium, chloride, calcium, glucose, lactate and creatinine concentrations) were also measured (EPOC, Woodley Instrumentation, Bolton, Lancashire, UK). All rats received an equal volume of balanced electrolyte solution after blood sampling (Vetivex 11 (Hartmann’s), Dechra, Shrewsbury, UK). Samples were centrifuged (4000g for 10 minutes) within 30 minutes of collection. Plasma was harvested and stored at -20°C until determination of plasma alfaxalone concentration.
At the end of the experiments animals were euthanised by intravenous injection of pentobarbitone (pentobarbital, Ayrton Saunders Ltd, Runcorn, UK) followed by cervical dislocation (by a trained individual as required by UK Home Office regulations).
Sample analyses
Standard quantification (STD) curves for in vivo plasma samples were generated using authentic alfaxalone standard samples giving concentrations from 200 ng ml-1 to 40 μg ml-1 in addition to the use of quality controls (QC). Spiking solutions for standards and QCs were made from separate accurate weighing of drug compounds. The methanol standard curve and QCs were prepared by spiking 10 µL of a known concentration spike solution into a solution of 40 µL methanol + 100 µL methanol containing 3 µM of lansoprazole as internal standard + 50 µL of either male or female blank plasma (Charles River, Margate, UK). Alfaxalone in vivo plasma samples were prepared by adding 50 µL of the plasma samples + 50 µL methanol + 100 µL methanol containing 3 µM of lansoprazole as internal standard.
Samples, standards, and QCs were then vortexed, stored in a freezer at -20 °C overnight prior to centrifugation at 4000g for 20 minutes at 4 °C. The supernatant was then transferred into LC-MS vials for analysis and concentration determination. Finally, the STD curves were analysed at the beginning and end of the run to determine any variation or deterioration of LC/MS performance. The analytical methods were validated to ensure suitable precision and accuracy, lower limit of quantification (LLOQ), linearity, calibration range and selectivity.
The samples were analysed using a Micromass Quattro Premier mass spectrometer incorporating an Agilent 1100 HPLC. An Ascentis® C18 column (2.1 × 50 mm, 3 μm) (Sigma, UK) protected by a Phenomenex C18 guard cartridge (Phenomenex, UK) was used with the following LC conditions: Solvent A = 10% methanol, 90% water and 0.02% formic acid, Solvent B = 100% methanol and 0.02% formic acid, flow rate = 0.4 ml/min, column temperature = 60°C. LC gradient went from 70 % solvent A:30 % solvent B to 1 % solvent A:99 % solvent B over a 3 minute interval. The MS/MS method used electrospray positive mode with a 333.2 ˃315.2 and 297.2 transitions for the detection of alfaxalone. The lower limit of quantification (LLOQ) was 200 ng ml-1. Two separate LC/MS/MS runs were performed for the male and female samples, respectively.
Data and Statistical analysis
Pharmacokinetic analyses
Pharmacokinetic analyses were conducted using an IV infusion compartmental model for (a) individual Lewis rat data using dose per kg and (b) Lewis and Sprague Dawley population data using total dose. Both analyses used Phoenix® WinNonlin® version 8.3 software (Certara USA, Inc., Princeton, NJ). A 2-stage approach was applied to (a) which firstly involved the estimation of clearance (CL), half-life (T1/2), mean residence time (MRT) and steady-state volume of distribution (Vdss) for alfaxalone in each rat. Secondly, statistical tests were performed on pharmacokinetic parameters to determine any differences between male and female rats.
Compartmental non-linear mixed effects methods (NLME) models were applied to (b) using total dose given to each rat to determine whether body weight influenced PK parameters. Two populations were analysed: population 1 (28 rats) comprised the Lewis rat data with Sprague Dawley rat data described by White et al. (2017); population 2 (52 rats) comprised population 1 plus the additional Sprague Dawley rat data (Figure 1). Model residual error was based on a mixed ratio error model. An exponential random effect model was chosen to describe inter-individual variability i.e. parameter = typical parameter x exp(eta). Categorical covariates were implemented for sex (male = 0, female = 1) and strain (Lewis = 0, SD = 1) on the model parameters in a multiplicative exponential way. A continuous covariate for log of centralised body weight (LCBW) was applied in a multiplicative way. The model analysis started from the basic compartmental models without the covariates. Next, the contribution of the covariates on fixed effects and correlation on random effects to the PK parameters were assessed by a reduction in the objective function using stepwise forward inclusion. Selection of the best model was based on the lowest value of the Akaike and Bayesian Information Criteria (AIC and BIC), chi-square p-value based on the likelihood ratio test, visual inspection of the population predicted concentration versus the observed concentrations and the resulting conditional weighted residual errors. Finally, the best model was checked for robustness using a bootstrap resampling method. Monte Carlo simulations were used to determine a 95% confidence tolerance interval for the 5th and 95thpercentile of the population.
Statistical Analyses
Statistical tests were performed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA) version 9. The male and female log transformed Lewis rat pharmacokinetic parameters were compared using an unpaired, two tailed Student’s t-test (α = 0.05) and a p value of < 0.05 was considered significant. Data are reported as mean ± standard deviation (SD) unless stated otherwise. The male and female arterial blood pressure or plasma concentration data were compared at different time points using 2-way ANOVA with post hoc Sidak multiple comparison test.