Plasma pharmacokinetics and pharmacodynamics of alfaxalone in neonatal foals after an intravenous bolus of alfaxalone following premedication with butorphanol tartrate

Veterinary Anaesthesia and Analgesia, 2012

doi:10.1111/j.1467-2995.2012.00734.x

RESEARCH PAPER

Plasma pharmacokinetics and pharmacodynamics of alfaxalone in neonatal foals after an intravenous bolus of alfaxalone following premedication with butorphanol tartrate Wendy Goodwin*, Helen Keates*, Kirby Pasloske , Martin Pearson*, Ben Sauer  & Millagahamada G Ranasinghe  *School of Veterinary Science, University of Queensland, Gatton, Qld, Australia  Jurox Pty Ltd, Rutherford, NSW, Australia

Correspondence: Wendy A Goodwin, Veterinary Medical Centre, University of Queensland, Gatton, Qld 4343, Australia. E-mail: [email protected]

Abstract Objective To determine the pharmacokinetics and pharmacodynamics of the neurosteroid anaesthetic, alfaxalone, in neonatal foals after a single intravenous (IV) injection of alfaxalone following premedication with butorphanol tartrate. Study design Prospective experimental study. Animals Five clinically healthy Australian Stock Horse foals of mean ± SD age of 12 ± 3 days and weighing 67.3 ± 12.4 kg. Methods Foals were premedicated with butorphanol (0.05 mg kg)1 IV) and anaesthesia was induced 10 minutes later by IV injection with alfaxalone 3 mg kg)1. Cardiorespiratory variables (pulse rate, respiratory rate, direct arterial blood pressure, arterial blood gases) and clinical signs of anaesthetic depth were evaluated throughout anaesthesia. Venous blood samples were collected at strategic time points and alfaxalone plasma concentrations were assayed using liquid chromatography-mass spectrometry (LC/MS) and analysed by noncompartmental pharmacokinetic analysis. Results The harmonic, mean ± SD plasma elimination half life (t½) for alfaxalone was

22.8 ± 5.2 minutes. The observed mean plasma clearance (Clp) and volume of distribution (Vd) were 19.9 ± 5.9 mL minute kg)1 and 0.6 ± 0.2 L kg)1, respectively. Overall, the quality of the anaesthetic inductions and recoveries was good and most monitored physiological variables were clinically acceptable in all foals, although some foals became hypoxaemic for a short period following recumbency. The mean durations of anaesthesia from induction to first movement and from induction to standing were 18.7 ± 7 and 37.2 ± 4.7 minutes, respectively. Conclusions The anaesthetic protocol used provided a predictable and consistent plane of anaesthesia in the five foals studied, with minimal cardiovascular depression. In foals, as in the adult horse, alfaxalone has a short elimination half life. Clinical relevance Alfaxalone appears to be an adequate anaesthetic induction agent in foals and the pharmacokinetics suggest that, with continuous infusion, it might be suitable to provide more prolonged anaesthesia. Oxygen supplementation is recommended. Keywords alfaxalone, anaesthesia, foal, pharmacodynamics, pharmacokinetics.

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Alfaxalone anaesthesia in the foal W Goodwin et al.

Introduction )1



Alfaxan , alfaxalone 10 mg mL in 2-hydroxypropyl-beta-cyclodextrin (HCPD), is a neurosteroidal anaesthetic agent currently registered in a number of countries for intravenous (IV) induction and maintenance of anaesthesia in dogs and cats. Alfaxalone (3a-hydroxy-5a-pregnane-11, 20-dione) is a water-insoluble molecule which interacts with the gamma aminobutyric acid (GABA)A receptors and produces anaesthesia and muscle relaxation. Alfaxalone has been shown in the dog, cat and horse, to have a number of properties that make it desirable for use as an induction agent or short term anaesthetic agent and as a maintenance agent administered by incremental dosing or a continuous infusion (Ambros et al. 2008; Whittem et al. 2008; Leece et al. 2009; Goodwin et al. 2011). In the horse and pony alfaxalone has been used for induction of anaesthesia and has provided safe, short term anaesthesia with rapid and uncomplicated recoveries (Pearson et al. 2006; Leece et al. 2009; Goodwin et al. 2011; Kloppel & Leece 2011). A prospective observational epidemiological multicentre study (Johnston et al. 2002) found that foals <4 weeks of age had a higher risk of dying during the perioperative period compared to older animals. One of the possible explanations for this is that neonatal foals (£1 month) and to some extent juveniles (1–3 months) are physiologically and metabolically different from mature horses and neonates of other species (Robertson 2005). Most of these age-related differences in foals are attributed to differences in drug absorption and disposition that consequently affect plasma drug concentrations and the amount of drug present at the receptor site (Baggot & Short 1984). Therefore it is not appropriate to use adult dose rates or to extrapolate across species to calculate dose rates when anaesthetising foals. Special consideration needs to be given to the foals’ unique physiology. The aim of this work was to study the pharmacokinetic and pharmacodynamic parameters of the HPCD-based formulation of alfaxalone in the neonatal foal after an IV bolus of 3 mg kg)1 following premedication with butorphanol tartrate, and to determine the suitability of this dose for anaesthetic induction and short term maintenance of anaesthesia. Materials and methods This study was performed with the approval of the University of Queensland Production and Compan2

ion Animals Ethics Committee (SVS/470/07/RIRDC).

Animals Five (two female, three male) Australian Stock Horse foals aged mean ± SD 12 ± 3 days and weighing 67.3 ± 12.4 kg from the University of Queensland Gatton Campus commercial breeding herd were used in the study. Foals were considered healthy on the morning of the study based on results of a physical examination and on haematology and biochemistry profiles. Mares and foals were housed in a free-range situation and stabled the morning of the study. Mares were fed lucerne hay and foals allowed to suck ad libitum until induction of anaesthesia. Mares and foals were stabled for up to 24 hours after induction of anaesthesia and returned back to the herd after a physical examination.

Test agent The commercial formulation of alfaxalone (Alfaxan, Jurox Pty Ltd, New South Wales, Australia) was used in the study.

Study protocol The skin overlying both jugular veins was clipped and surgically prepared. Following deposition of 20 mg of lidocaine (Lignomav; Mavlab Pty Ltd, Queensland, Australia) at the site of catheter placement, a two inch 16 gauge catheter was placed in the left jugular vein and a 2 inch 18 gauge catheter placed in the right jugular vein (Surflo; Terumo Corporation, Phillippines). The left jugular catheter was used for venous blood collection and the right jugular catheter used for drug administration. Heart rate (HR) was measured by stethascope, and respiratory rate (fR) counted. Foals were premedicated with butorphanol tartrate 0.05 mg kg)1 IV (Torbugesic, Fort Dodge, New South Wales, Australia). After 10 minutes alfaxalone 3 mg kg)1 was administered IV over a period of 60 seconds. Once recumbent, foals were placed in right lateral recumbency and their trachea intubated with a size 14 mm endotracheal tube. Oxygen was supplied at 6 L minute)1 by insufflation into the endotracheal tube. A 1¼ inch 22 gauge catheter (Surflo; Terumo Corporation, Phillippines) was placed in the left dorsal metatarsal artery for arterial blood pressure

 2012 The Authors. Veterinary Anaesthesia and Analgesia  2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

Alfaxalone anaesthesia in the foal W Goodwin et al.

monitoring and blood collection for blood gas measurement. Cardiorespiratory variables and clinical signs of anaesthesia were assessed every five minutes throughout anaesthesia. Pulse rate (PR) and arterial haemoglobin oxygen saturation (SaO2) were evaluated using a pulse oximeter with the probe placed on the tongue (Nellcor Oximax; Coviden-Nellcor, MA) and fR was calculated by counting thoracic wall excursions. Pulse quality, mucous membrane colour and capillary refill time were assessed subjectively. A Cardiocap II (Datex-Ohmeda, Finland) was used for continuous monitoring of ECG, HR and arterial blood pressure. The pressure transducer was zeroed at the height of the heart. Arterial blood was taken anaerobically at 5, 10, 20 and 30 minutes post anaesthetic induction, and analysed for blood gases values and pH using a Vetstat Analyser (Idexx Laboratories, ME). Venous blood glucose was measured prior to premedication and at 5, 10, 30 and 120 minutes post anaesthetic induction using a glucometer (Accu-Check Go, Roche Diagnostics GmbH, Germany). Nystagmus, palpebral reflex and muscle and anal tone were evaluated as signs of anaesthetic depth. The time intervals were recorded from the end of the aflaxalone injection to when the foal first began to move, lifted its head, was extubated (when swallowing and chewing) achieved sternal recumbency, stood and was returned to the mare. All foals stood with assistance and were supported until they were no longer ataxic. Time recording for the pharmacokinetic schedule was started at the end of alfaxalone administration. At each collection time, 5 mL of venous blood was collected into a lithium heparin tube following a 3 mL blood discard. The catheter was then flushed with 3 mL of heparinized saline. Blood samples were collected just before alfaxalone administration and at 2, 4, 6, 10, 15, 20, 30, 45, 60, 120, 240, 360 and 480 minutes thereafter. Samples were immediately stored at 5 C and within 8 hours of sampling the tubes were centrifuged (280 g, 5 minutes). The plasma was harvested and stored at )70 C. Determination of plasma alfaxalone concentrations The analytical phase was conducted at Jurox Pty Ltd, Australia. The plasma samples were extracted using Waters Oasis HLB solid phase extraction (SPE) cartridges (1 cc 10 mg)1) with 11-hydroxy

progesterone as the internal standard. The extracted equine plasma samples (100 lL) were analysed for alfaxalone content using Agilent 1100 series LC-MS system with electrospray ion trap mass detector. Calibration of the LC-MS was achieved by spiking known amounts of alfaxalone into extracted blank equine plasma. Ten microlitre of each extracted and spiked plasma samples were injected into a Phenomenex Luna C18 (2) (Phenomenex; CA) (3 l, 3.0 · 50 mm) column using 1:1 acetonitrile water mobile phase with 1 mM ammonium acetate buffer. Alfaxalone concentration was determined by calculating the peak area ratio of the alfaxalone to 11-hydroxy progesterone. Both un-extracted and extracted samples were stored at )70 C. The lower limit of quantification (LLOQ), i.e. the lowest alfaxalone concentration point of the standard curve, for this assay was 0.02 mg L)1 of alfaxalone in extracted foal plasma. The intra-assay coefficient of variation for samples at LLOQ was <20%. The limit of detection (LOD), i.e the lowest alfaxalone concentration that the measuring device can detect, for the assay was 0.007 mg L)1 and the target mass ion fragment (m/z) was 333. All test and standard samples were analysed over a 24 hour period in the same analytical run. Two linear standard curves were employed with ranges covering 0.02–1 mg L)1 and 0.8–5 mg L)1 respectively. The minimum coefficient of determination (r2) for both curves was 0.99. Both accuracy and precision were deemed acceptable if their individual residual standard deviation (RSD) was ± <20% at LLOQ or ± <15% for all other concentrations. Pharmacokinetic analysis Pharmacokinetic analysis of data was performed using non-compartmental methods (Model: NCA 201) with uniform weighting of the data in WinNonlin Version 5.3 (Pharsight Co., CA). Equal weighting was applied to all data points within each individual foal alfaxalone concentration over time data set. A non-compartmental pharmacokinetic method was used for each plasma concentration-time plot for each foal. Actual doses and time points for each horse were used in the modelling. The area under the curve from the time of administration to the last quantifiable time point (AUC0-Tlast) was calculated with the linear trapezoidal rule (Gibaldi & Perrier 1982). The terminal slope (k) was calculated for each individual data

 2012 The Authors. Veterinary Anaesthesia and Analgesia  2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

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Alfaxalone anaesthesia in the foal W Goodwin et al.

set by linear regression using the function of best fit. The total AUC was calculated from the time of administration to infinity (AUC0-a) and represents the sum of AUC0-Tlast plus the area under the extrapolated curve. The percentage AUC extrapolated from the last quantifiable time point (AUCextrap%) was calculated as the area under the extrapolated curve divided by AUC0-a multiplied by 100 percent. The plasma clearance (Clp), volume of distribution area (VD) and elimination half-life (t1/2elim) were calculated using standard noncompartmental formulae. The initial plasma concentration C0 (i.e. t = 0 min) was back extrapolated from the terminal slope (k) calculation of each individual foal while observed maximum plasma concentration (Cmax) and the time to maximum plasma concentration (Tmax) was taken directly from each individual foal’s plasma alfaxalone versus time data set. Statistical Analysis Continuous data and pharmacokinetic parameters are reported as means, standard deviations and ranges calculated in Microsoft Office Excel 2007 (Microsoft Corporation, WA, USA). Heart rate, fR and venous blood glucose before premedication and then at designated time points after induction were compared using repeated measures ANOVA (level of significance set at p < 0.05) with Dunnett’s multiple comparison test. Variables were first tested for normality using the Kolmogorov–Smirnov test and transformed (Y = rank(Y)) as necessary to satisfy this assumption. All calculations were performed using Graphpad Prism version 5.02 (GraphPad Software Inc, CA, USA).

Results Pharmacodynamics The mean times from induction with alfaxalone (3 mg kg)1) to when foals first exhibited generalised movement, lifted their head and were extubated were 18.7 ± 7, 23.6 ± 5.6 and 24.6 ± 5.4 minutes, respectively. The mean recovery times from induction to achieving sternal recumbency and standing were 34.9 ± 5.6 and 37.2 ± 4.7 minutes. Foals were returned to the mare at 49.1 ± 1.2 minutes. Induction of anaesthesia was smooth with all foals becoming recumbent approximately three quarters way through the 60 second injection. Endotracheal intubation was performed without difficulty within the first 5 minutes post induction and no swallowing was noted. Additionally, no muscle rigidity or paddling was observed, although two foals displayed very mild generalised muscle twitching for approximately 30 seconds immediately following the alfaxalone injection. Palpebral movements and nystagmus were variable in onset and duration and did not tend to be associated with purposeful movement. No adverse outcomes associated with alfaxalone were observed in the study. Heart rate did not differ significantly from baseline values and no cardiac arrhythmias were detected. No baseline values were available for arterial blood pressure comparison, however during anaesthesia mean arterial pressure was maintained above 70 mmHg at all observed time points (Table 1). Respiratory rate decreased significantly after anaesthetic induction, however no post induction apnoea was observed. Three foals exhibited moderate to severe hypoxaemia 5 minutes

Table 1 Monitored anaesthetic variables in five neonatal foals after intravenous administration alfaxalone 3 mg kg)1 and following premedication with butorphanol

Variable

Baseline

5

Blood glucose (mmol L)1) Respiratory rate (breaths minute)1) Heart rate (beat minute)1) SpO2 (%) Mean arterial pressure (mmHg) Systolic arterial pressure (mmHg) Diastolic arterial pressure (mmHg)

7.1 (0.6) 40 (25) 99 (4) – – – –

– 13 110 97 86 117 67

(1)b (27) (3) (22) (37) (20)

10

15

5.5 (0.7) 15 (6)b 97 (15) 97 (2) 72 (15) 99 (23) 57 (14)

– 15 103 97 76 97 64

(5)b (23) (3) (14) (22) (16)

20

25

5.2 (1.5)a 18 (6)b 117 (26) 96 (5) 74 (10) 92 (6) 58 (10)

– 18 115 98 75 95 61

(8)b (28) (2) (16) (14) (17)

30

120

5.4 (1.2)a – –

9.5 (0.8)a – –

– – –

– – –

Results are expressed as mean (SD). Values with different letters in columns are significantly different from baseline (prior to premedication values) (p < 0.05).

4

 2012 The Authors. Veterinary Anaesthesia and Analgesia  2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

Alfaxalone anaesthesia in the foal W Goodwin et al.

ondary peak in alfaxalone plasma levels was seen at 120 minutes in two foals (Fig. 1).

after anaesthetic induction (partial pressure of arterial oxygen (PaO2) of 11.7, 9.1, and 5.5 kPa or 88, 68 and 41 mmHg respectively). Mean values for blood glucose and pulse oximetry is shown in Table 1 and arterial pH, blood gas values and bicarbonate are shown in Table 2.

Discussion Pharmacodynamics Alfaxalone 3 mg kg)1 IV administered to foals following premedication with butorphanol provided a smooth induction of anaesthesia with good intubation conditions. Although foals were not surgically stimulated, the depth and duration of anaesthesia was judged to be suitable for brief procedures or for transition to inhalational anaesthesia. This finding is similar to studies in adult horses and ponies administered 1 mg kg)1 IV alfaxalone following premedication and co-induction with xylazine and

Pharmacokinetics The harmonic, mean ± SD t1/2elim for alfaxalone was 22.8 ± 5.2 minutes. The observed Clp and Vd were 19.9 ± 5.9 mL minute)1 kg)1 and 0.6 ± 0.2 L kg)1, respectively (Table 3). The Cmax of alfaxalone occurred two minutes (Tmax) after the end of administration and was 5.54 ± 0.42 mg L)1. Alfaxalone was quantifiable in all foals at 120 minutes but only two out of five foals at 240 minutes. A minor secTable 2 Blood gas values in five neonatal foals after administration of intravenous alfaxalone 3 mg kg)1 following premedication with butorphanol

Minutes after induction of anaesthesia

Variable

Arterial pH PaCO2 kPa mmHg PaO2 kPa mmHg HCO3 (mmol/L)

5

10

20

30

7.4 (0.04)

7.4 (0.05)

7.43 (0.04)

7.2 (0.9) 54 (7)

6.9 (1.1) 52 (8)

6.4 (0.5) 48 (4)

5.9 (0.4) 44 (3)

10.7 (3.6) 80 (27) 30.4 (1.4)

19.3 (6.3) 145 (47) 30 (1.4)

20 (7.9) 150 (59) 29.1 (0.6)

15.6 (5.7) 117 (43) 28.7 (1.8)

7.5 (0.01)

Results are expressed as mean (SD).

Table 3 Main pharmacokinetic parameters of alfaxalone estimated by non-compartmental analysis after an intravenous bolus administration of alfaxalone at 3 mg kg)1 in 5 neonatal foals which had been premedicated with butorphanol

Variable

Units

Foal 1

Foal 2

Foal 3

Foal 4

Foal 5

Mean (SD)

C0 Cmax Tmax AUC(0-LLOQ) AUC(0-¥) AUC(% extrap) VD Clp t½ elim MRT inf

mg L)1 mg L)1 minutes minutes mg L)1 minutes mg L)1 % L kg)1 mL minute)1 kg)1 minutes minutes

5.71 5.25 2 119.6 135 11.4 0.58 22.2 18.1 27.8

6.17 6.17 2 169.7 175.4 3.2 0.68 17.1 27.4 52.2

5.23 5.23 2 101.3 103.1 1.7 0.42 29.1 10.1 17.2

5.29 5.29 2 174.3 179.9 3.1 0.84 16.7 35 55.5

5.78 5.78 2 171.3 208.9 18 0.48 14.4 23.3 63.3

5.64 5.54 2 147.2 160.5 7.5 0.60 19.9 22.8 43.2

(0.39) (0.42) (34.2) (41.5) (7.0) (0.2) (5.9) (5.2) (19.7)

Initial plasma concentration (Co), Observed maximum plasma concentration (Cmax), Time to maximum plasma concentration (Tmax), Area under the curve from administration to last quantifiable time point (AUC0-LLOQ), Area under the curve from administration to infinity (AUC0-¥), Percentage area under the curve extrapolated from last quantifiable time point (AUC%extrap), Volume of distribution (VD), Plasma clearance (Clp), Elimination half life (t½ elim), Mean residence time to infinity (MRT inf).  2012 The Authors. Veterinary Anaesthesia and Analgesia  2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

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Plasma concentration (mg L–1)

Alfaxalone anaesthesia in the foal W Goodwin et al.

10.0

1.0

0.1

0

60

120

180

240

Time (minutes)

Figure 1 Mean (SD) plasma alfaxalone concentration over time in five neonatal foals after administration of alfaxalone 3 mg kg)1 following premedication with butorphanol.

guaifenesin (Pearson et al. 2006), acepromazine, xylazine and guaifenesin (Goodwin et al. 2011) or romifidine and diazepam (Leece et al. 2009; Kloppel & Leece 2011). The studies in these heavily premedicated adult horses reported similar anaesthetic conditions and recovery times with a lower dose of alfaxalone (1 mg kg)1). However, the situation is not directly comparable as inclusion of alpha2 adrenergic agonists and centrally acting muscle relaxants in the adult horse anaesthetic regimen almost certainly accounts for some of the reduction in dose rate compared with the foal. In this study, premedication with butorphanol provided satisfactory conditions for induction of anaesthesia. An alpha2 adrenergic receptor agonist such as xylazine was not used in the study as, although it provides analgesia and sedation, it has been shown to reduce heart rate in foals by 20–30% (Carter et al. 1990). This is clinically relevant as cardiac output in the foal is heart rate dependent (Robertson 2005). In contrast, Arguedas et al. (2008) found that butorphanol administered to foals at 0.05 mg kg)1 IV produced no significant changes in heart rate over time whilst at the same time it provided analgesia and sedation. Benzodiazepines were also excluded from the premedication regimen as pharmacodynamic interaction with neuroactive steroids has been reported (Harrison & Simmonds 1984; Visser et al. 2003). Baseline heart and pulse rates recorded in this study were similar to those reported in the literature (Lombard et al. 1984; Carter et al. 1990). As in the adult horse, alfaxalone produced no significant changes in PR over time and no changes in the ECG, such as arrhythmias or bradycardia, were noted (Leece et al. 2009; Goodwin et al. 2011; Kloppel & Leece 2011). Although no pre-anaesthetic measurements of direct arterial blood pressures were available for in-study comparison, recorded values were comparable to indirect mea6

surements reported in foals anaesthetised with inhalant anaesthesia (Steffey et al. 1991) or with an infusion of propofol (Matthews et al. 1995). Although blood glucose concentrations differed significantly from baseline values at 20, 30 and 120 minutes, all values were within normal limits and comparable to those reported in healthy unsedated foals and foals sedated with xylazine (Bauer et al. 1984; Robertson et al. 1990). Baseline fRs also were similar to other reported values in neonatal foals (Stewart et al. 1984; Koterba et al. 1995). Although fRs decreased, following induction of anaesthesia, they remained within the range of values for anaesthetised foals (Hubbell & Muir 2009) and apnoea was not reported. Values for arterial PaO2, partial pressure of carbon dioxide (PaCO2) and pH in standing healthy foals receiving similar supplemental oxygen flows (approximately 100 mL kg)1 minute)1) via a unilateral nasopharyngeal catheter have been reported as 23.4 ± 1.9 kPa (175 ± 15 mmHg), 6.7 ± 0.3 kPa (51 ± 2 mmHg) and 7.42 ± 0.01 respectively (Wong et al. 2010). The foals in this study exhibited hypoxaemia, mean ± SD PaO2 being 10.7 ± 3.6 kPa (80 ± 27 mmHg), the lowest reaching 5.5 kPa (41 mmHg), during the first five minutes of anaesthesia and some hypercapnia at five and ten minute time points was noted. As with most anaesthetics, respiratory depression has been reported for alfaxalone (Ambros et al. 2008; Whittem et al. 2008). The hypoxaemia at the five minute time point may be a result of some foals breathing room air due to the time taken to intubate and provide oxygen supplementation. Unfortunately, precise times for endotracheal intubation and the commencement of oxygen supplementation are not available. Thus it is possible that hypoxaemia might have been avoided by providing oxygen supplementation prior to induction of anaesthesia. Another explanation is the possibility of a mismatch between ventilation and perfusion of lung regions resulting in

 2012 The Authors. Veterinary Anaesthesia and Analgesia  2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists

Alfaxalone anaesthesia in the foal W Goodwin et al.

impaired arterial oxygenation. The decrease in PaO2 at the 30 minute reading (15.6 kPa, 117 mmHg) compared to the 20 minute reading (20 kPa, 150 mmHg) can be attributed to some foals no longer receiving oxygen supplementation as they had been extubated and were breathing air. Pharmacokinetics There were a number of differences between the pharmacokinetic results of alfaxalone administered at 3 mg kg)1 in neonatal foals and the pharmacokinetic results in adult horses administered alfaxalone at 1 mg kg)1 following premedication with acepromazine, xylazine and guaifenisen (Goodwin et al. 2011). The Vd of alfaxalone was greater in these adult horses (1.6 L kg)1) compared with the neonatal foals (0.6 L kg)1) in this study. It is generally expected that foals will have a higher Vd than adult horses as, compared to adults, the relative extracellular fluid volume is higher in foals up to 24 weeks of age (Baggot & Short 1984; Spensley et al. 1987). Norman et al. (1997) reported an agerelated increase in the volume of distribution of diazepam administered IV to foals. They proposed that as diazepam is a highly lipophilic molecule, the increase in Vd most likely reflected an increase in fat-to-water ratio in the body. As alfaxalone is also a highly lipophilic molecule it is possible that the same hypothesis applies to foals in this study. The Clp of alfaxalone in neonatal foals (19.9 mL minute)1 kg)1) was comparatively lower than that reported by Goodwin et al. (2011) for adult horses (37.1 mL minute)1 kg)1). This is consistent with other studies that have found reduced clearance of several drugs in neonates and increased clearance with age (Adamson et al. 1991; Crisman et al. 1996; Norman et al. 1997; Bucki et al. 2004). Norman et al. (1997) reported that the hepatic oxidative capacity for diazepam is lower in 4-day old foals than in older foals and adult horses and suggested that this may account for increasing diazepam Clp values with increasing age. As alfaxalone is a steroid molecule with a chemical structure similar to progesterone and in dogs and cats is eliminated primarily by hepatic metabolism, it is possible that comparatively less hepatic enzyme activity may account for the longer alfaxalone Clp in foals compared with adults (Pasloske 2006). The terminal half life of alfaxalone was shorter in these foals (22.8 minutes) than adult horses (33.4 minutes) (Goodwin et al. 2011). The terminal

half life is a hybrid parameter that is influenced by both clearance and volume of distribution according to the following equation (Toutain & BousquetMelou 2004);   0:693 t1=2 ¼ vD Cl In these foals the change in VD was proportionately lower than the change in Clp and therefore the t½ was shorter in comparison with the adult horse. A minor secondary peak in plasma alfaxalone levels was noted in two foals between 60 and 120 minutes after anaesthetic induction. This effect has previously been observed with alfaxalone in the dog, cat and horse (Ferre et al. 2006; Whittem et al. 2008; Goodwin et al. 2011) and maybe to be due to the metabolite 3b-OH-alfaxalone being assayed as alfaxalone as both molecules have virtually the same weight and retention time. However, this is only speculation since the 3b-OH-alfaxalone will not separate under the conditions of the assay used in this study. The cause of the peak may also be due to sequestration and subsequent elution of the drug from lungs and muscle as discussed by Goodwin et al. (2011). In conclusion, IV premedication of the foal with butorphanol 0.05 mg kg)1 followed 10 minutes later by anaesthetic induction with alfaxalone at 3 mg kg)1 IV, provided a stable and predictable level of anaesthesia. In this study, in order to achieve a comparable depth of anaesthesia to that seen in more heavily premedicated adult horses, the foal appears to have a larger dose requirement for alfaxalone. However, as in the adult horse, alfaxalone it is rapidly cleared from the plasma of foals. Some evidence of hypoxaemia was seen soon after recumbency, so oxygen supplementation is recommended, preferably commencing before induction of anaesthesia. Acknowledgements The authors wish to thank Jurox Pty Ltd (Rutherford, NSW, Australia) for supplying the Alfaxan and performing the plasma alfaxalone analysis: Rural Industries Research and Development Corporation (Australia) and Idexx Laboratories (Australia) for aiding in funding of the project. References Adamson PJ, Wilson WD, Baggot JD et al. (1991) Influence of age on the disposition kinetics of chloramphenicol in equine neonates. Am J Vet Res 52, 426–431.

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