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Effective plasma alfaxalone concentration to produce immobility in male neutered cats
Veterinary Anaesthesia and Analgesia 2018, xxx, 1e9
https://doi.org/10.1016/j.vaa.2017.10.006
RESEARCH PAPER
Effective plasma alfaxalone concentration to produce immobility in male neutered cats Bruno H Pypendopa, Kristine T Siaob,1, MG Ranasinghec & Kirby Pasloskec a
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of
California, Davis, CA, USA b
Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis,
CA, USA c
Jurox Pty Ltd, Rutherford, NSW, Australia
Correspondence: Bruno Pypendop, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, USA. E-mail: [email protected] 1
Dr. Siao's current address is 1848 24th Ave. San Francisco, CA 94122.
Abstract Objective To determine the effective plasma alfaxalone concentration for the production of immobility in cats. Study design Prospective up-and-down study. Animals Sixteen 1e2 year old male castrated research cats. Methods Cats were instrumented with catheters in a jugular and a medial saphenous vein. Alfaxalone was administered via the medial saphenous catheter, using a target-controlled infusion system. The infusion lasted for approximately 32 minutes. A noxious stimulus (tail clamp) was applied 30 minutes after starting the alfaxalone infusion, until the cat moved or 60 seconds had elapsed, whichever occurred first. The target alfaxalone concentration was set at 5 mg L1 in the first cat and increased or decreased by 1 mg L1 in subsequent cats, if the previous cat had moved or not moved in response to stimulation, respectively. This was continued until six independent crossovers (different responses in pairs of subsequent cats) had been observed. Blood samples were collected before alfaxalone administration, and 15 and 31 minutes after starting the administration, for the determination of plasma alfaxalone concentration using liquid chromatography/tandem mass spectrometry. The alfaxalone concentration yielding a probability of immobility in 50% (EC50), 95% (EC95) and 99% (EC99) of the population, and their respective 95% Wald confidence intervals were calculated.
Results The EC50, EC95 and EC99 for alfaxaloneinduced immobility were 3.7 (2.4e4.9), 6.2 (4.7e) and 7.6 (5.5e) mg L1, respectively. Conclusions and clinical relevance The effective plasma alfaxalone concentration for immobility in cats was determined. This value will help in the design of pharmacokinetic-based dosing regimens. Keywords alfaxalone, cats, pharmacology. Introduction Alfaxalone is an anesthetic neurosteroid. It is currently commercially available as a solution containing alfaxalone solubilized in hydroxypropylb-cyclodextrin, and is labeled in the United States, Canada, Europe, Korea, Japan, New Zealand, South Africa and Australia for induction and maintenance of anesthesia via intermittent intravenous (IV) boluses in dogs and cats. The pharmacokinetics of alfaxalone in cats have been characterized (Whittem et al. 2008); however, to the authors’ knowledge, the effective plasma concentration of alfaxalone has not been reported in either dogs or cats, making it difficult to use the available pharmacokinetic information to calculate adequate dosing for induction or maintenance of anesthesia, by intermittent IV boluses or IV infusion. The objective of this study was to estimate the effective plasma alfaxalone concentration in 50%, 95% and 99% of the cat population, resulting in immobility following noxious stimulation.
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
Materials and methods This study was approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Healthy, 1e2 years old, male, neutered cats were used. The health status of each cat was assessed by history and complete physical examination 2 days before, as well as on the day of the experiment. In addition, cats were observed daily for any signs of disease for 7 days before the experiment was initiated. Husbandry conditions for this institution have been previously described (Honkavaara et al. 2017). The cats were fed Laboratory Feline Diet 5003 (LabDiet, MO, USA) once daily. On the day prior to the experiment, each cat was briefly anesthetized with isoflurane in oxygen. The skin over a jugular vein and a medial saphenous vein was clipped and aseptically prepared using chlorhexidine and alcohol. A 20 gauge, 48 mm or a 19 gauge, 150 mm catheter was inserted in the jugular vein for collection of blood samples. A 20 gauge, 48 mm catheter was inserted in the medial saphenous vein for drug administration. Infusion plugs were placed on the catheters, and the catheters were flushed with heparinized 0.9% weight/volume (w/v) saline solution and secured to the skin with tape (medial saphenous) or suture (jugular). A light bandage was placed over the catheters and cats were allowed to recover from anesthesia. An Elizabethan collar was placed around the neck and cats were returned to their room. On the day of the experiment, the cat’s body weight was measured. A physical examination was performed, including the measurement of pulse rate (PR), respiratory rate (fR) and rectal body temperature (RT). The patency of the catheters was verified by injecting a small volume of heparinized saline solution. Alfaxalone (10 mg mL1) in hydroxypropylb-cyclodextrin was administered via the medial saphenous catheter, using a target-controlled infusion system. An alfaxalone preserved formulation was administered in the study. The alfaxalone formulation contained the preservatives ethanol (150 mg mL1), chlorocresol (1 mg mL1) and benzethonium chloride (0.2 mg mL1). The targetcontrolled infusion system consisted of a syringe pump (PHD2000; Harvard Apparatus, MA, USA) and computer program (Rugloop I; Demed, Belgium). The system rapidly loaded the central compartment to the target concentration by delivering a bolus calculated from [T] Vc, and maintained this concentration via a variable infusion rate updated every 10 seconds, 2
according to the following equation: R ¼ [T] Vc (k10 þ k12 ek21t), where R is the infusion rate; [T] is the target plasma concentration; Vc is the volume of the central compartment; k10, k12 and k21 are microrate constants and t is the infusion time. Vc, k10, k12 and k21 were 302 mL kg1, 0.101 minute1, 0.323 minute1 and 0.066 minute1, respectively. The latter values were obtained from the best fitting pharmacokinetic model obtained from 24 cats following IV bolus administration of the same alfaxalone formulation (RD0327) in a different study (Pasloske et al. 2018). Observations were made on whether any cat exhibited signs of pain or discomfort in response to the injection of the preserved alfaxalone formulation. The cat was placed on a heating pad. PR (palpation of the femoral pulse over 15 seconds), fR (observation of chest excursions over 15 seconds) and RT (electronic thermometer) were measured 15, 25 and 45 minutes after starting the drug administration. A noxious stimulus (clamping of the tail using a 20 cm Martin forceps closed to the first ratchet) was applied 30 minutes after starting the target-controlled infusion. The stimulus was continued until 1 minute had elapsed, or until movement was observed, whichever occurred first, and the response was recorded as positive (movement) or negative (lack of movement). Any movement of the limbs or head, except related to swallowing or coughing, was considered positive. Blood samples (2 mL) were collected prior to drug administration, 15 minutes after starting drug administration, and immediately after the noxious stimulus was delivered. Prior to sampling, blood (3 mL) was aspirated into a syringe containing a small amount of heparinized saline and later returned after the actual sample had been collected. Blood was transferred to tubes containing lithium heparin, placed on ice and centrifuged at 3901 g and 4 C for 10 minutes within 60 minutes of collection. The plasma was separated, transferred to cryotubes and frozen at e80 C until analyzed for alfaxalone concentration. Drug administration was discontinued following collection of the last blood sample, and the cat was allowed to recover under observation. Times from discontinuation of drug administration to head lift, return to sternal recumbency and ability to stand without assistance were recorded. The quality of anesthesia induction, the quality of anesthesia and the quality of recovery from anesthesia were subjectively assessed using a 100 mm visual analog scale (VAS). For quality of induction, 0 was defined as
© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e9
Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
worst possible induction (slow, excitement, paddling) and 100 as best possible induction (rapid, smooth). For quality of anesthesia, 0 was defined as no anesthesia or sedation and 100 as anesthesia for most of the infusion time (based on the presence of minimal jaw tone, absence of palpebral reflex, rotation of the eyes and response to tail clamping). For quality of recovery, 0 was defined as worst possible quality/ excessively long and 100 as best possible/fast. The scores were all assigned by the same investigator (BHP), who was not blinded to the target plasma alfaxalone concentration. The target plasma alfaxalone concentration was set at 5 mg L1 for the first cat. The target plasma alfaxalone concentration in subsequent cats was increased or decreased by 1 mg L1, according to the observation of a positive or negative response to noxious stimulation, respectively, in the previous cat. The study was continued until 20 cats had been used, or six independent crossover responses (i.e., different response in subsequent cats) had been observed, whichever occurred first. Cats were observed for signs of adverse effects such as vomiting, regurgitation, change in respiratory effort, seizures or convulsions, myoclonus and muscle rigidity until they had fully recovered from anesthesia. Alfaxalone analysis Alfaxalone concentration was measured in feline plasma using a validated liquid chromatography/ tandem mass spectrometry method. Ten microliters of acetonitrile and 150 mL of 1 mg mL1 aqueous solution of 11-hydroxyprogesterone as internal standard were added to the plasma samples (40 mL), and the samples were extracted using Waters Oasis HLB 30 mm mElution solid-phase extraction 96 well plates (Waters Corporation, MA, USA). The plate was preconditioned with methanol and water, the samples loaded and the wells washed with water followed by 15% methanol solution and then eluted with 75 mL of acetonitrile. The elute was diluted with 225 mL of water and analyzed for alfaxalone content using Agilent 1100 series liquid chromatography and 6410B triple-quadrupole mass detector operated on positive electrospray ionization mode (Agilent Technologies Inc., CA, USA). A calibration curve was generated using the calibration standard sample set prepared by spiking known amounts of alfaxalone and the internal standard in the blank (control) plasma. Twenty mL of each extracted plasma sample
was injected into a Phenomenex Kinetex PFP (2.6 mm, 50 2.1 mm high-performance liquid chromatography column; Phenomenex Inc., CA, USA) using a gradient elution with mobile phase consisting of 0.3 mM ammonium formate in 0.1% aqueous formic acid and 0.3 mM ammonium formate in 999:9:1 (v/v/v) acetonitrile:water:formic acid. The target mass ion fragment (m/z) 215 of parent ion 333 for analyte and 295 of parent ion 331 for the internal standard were used for the analysis. The alfaxalone concentration was determined by calculating the peak area ratio of the alfaxalone to 11hydroxyprogesterone. The lower limit of quantification (LLOQ) of alfaxalone in a plasma sample was 0.02 mg L1, whereas the upper limit of quantification was 10 mg L1. The lower limit of detection for the assay was 0.003 mg L1. The intra-assay coefficient of variation for samples at LLOQ was <10.2%. The minimum coefficient of determination (r2) of the standard curve was >0.99. Sample extraction recoveries were >89% for all concentration levels. The accuracy was deemed acceptable if the calculated concentration in calibration and quality control samples was within 100 ± 20% of the actual concentration at LLOQ, and 100 ± 15% of the actual concentration for all other concentrations. The precision was deemed acceptable if the individual relative standard deviation in replicate quality control samples was <20% at LLOQ and <15% for all other concentrations. All derived values were reported to three significant figures. The plasma alfaxalone concentration at the time of the measurement was considered to be the concentration measured in the last sample. Statistical analysis The probability of movement/immobility function was fitted to the plasma alfaxalone concentrationeresponse data using logistic regression. The alfaxalone concentration yielding a probability of immobility in 50% (EC50), 95% (EC95) and 99% (EC99) of the population, and their respective 95% Wald confidence intervals were calculated from the fitted function. Linear regression was used to examine the relationship between target and actual plasma alfaxalone concentration, and the Pearson productemoment correlation coefficient (r) was calculated and reported as r2. Normality of continuous data was assessed using the ShapiroeWilk test. Because the normality hypothesis was rejected for several data sets, nonparametric tests were used to
© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e9
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
analyze all data. The times to head lift, return to sternal recumbency and standing without assistance, and the VAS scores for quality of induction, anesthesia and recovery were compared between the 2 and 3 mg L1 target concentrations using the Wilcoxon test (not enough observations were available for statistical evaluation at the other target concentrations). The PR, fR and RT data were analyzed for the time effect within target concentration groups using Friedman’s test, followed where appropriate by Dunn’s test for comparisons to baseline data, and for the target concentration effect within the time points using the Wilcoxon test. Data are presented as median (range), except where otherwise indicated. The EC50, EC95 and EC99 are presented as estimated concentration (95% Wald confidence interval). Results Sixteen cats were administered alfaxalone in the study and assessed for response to a noxious stimulus. Six independent crossover responses (i.e., change in response in six pairs of successive cats, for a total of 12 cats) were obtained. The mean ± standard deviation body weight of the cats was 5.5 ± 0.7 kg. Target plasma alfaxalone concentrations were 1, 2, 3, 4 and 5 mg L1 in 1, 7, 6, 1 and 1 cats, respectively. Statistical analysis of PR, fR, RT, recovery time and VAS score data was therefore conducted on data from cats administered the 2 and 3 mg L1 target plasma alfaxalone concentrations only. Tracheal intubation was attempted in the first five cats, but not in subsequent cats owing to stimulated coughing. Actual
plasma alfaxalone concentrations [median (range) when measured in more than one individual at a target concentration] at 15 and 31 minutes were 1.25 and 1.19, 3.21 (2.92e4.48) and 3.56 (2.63e5.41), 5.5 (4.56e7.27) and 4.87 (4.54e5.28), 8.03 and 7.3, and 9.8 and 8.12 mg L1 for target plasma alfaxalone concentrations of 1, 2, 3, 4 and 5 mg L1, respectively (Fig. 1). The EC50, EC95 and EC99 for immobility were 3.7 (2.4e4.9), 6.2 (4.7e) and 7.6 (5.5e) mg L1, respectively (Fig. 2). The upper limit of the 95% Wald confidence interval could not be computed for EC95 and EC99 because of the shape of the concentrationeresponse relationship. There was a strong, linear association between target and actual plasma alfaxalone concentration (r2 ¼ 0.90, 0.85 and 0.85 for samples collected after 15 minutes of infusion, 31 minutes of infusion and all samples pooled, respectively). However, mean ± standard deviation actual plasma alfaxalone concentration was 181 ± 31% of target after 15 minutes of infusion, and 167 ± 34% of target after the noxious stimulation (approximately 31 minutes of infusion). The VAS score for quality of anesthesia was significantly higher in the cats administered the 3 mg L1 target plasma alfaxalone concentration than the 2 mg L1 target concentration (p ¼ 0.0268; Table 1). The lower quality scores were mainly attributed to persistence of muscle tone. No significant difference was found for VAS scores for quality of induction of anesthesia or quality of recovery.
Figure 1 Plasma alfaxalone concentration at 15 (closed circles) and 31 (open circles) minutes after starting the alfaxalone intravenous infusion in each cat. The number next to each closed circle is the target plasma alfaxalone concentration in that individual. 4
© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e9
Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
Figure 2 Logistic regression of the plasma alfaxalone concentrationeresponse data in 16 cats. The closed circles represent the response in each cat at the plasma alfaxalone concentration measured immediately after the noxious stimulation (approximately 32 minutes after starting the intravenous alfaxalone infusion). A response of 0 corresponds to movement, and a response of 1 to no movement observed during clamping of the tail for up to 60 seconds. The line represents the probability of immobility in response to noxious stimulation as a function of plasma alfaxalone concentration. The estimated EC50, EC95 and EC99 are shown, with their corresponding 95% Wald confidence interval. The upper limit of the confidence interval could not be computed for the EC95 and EC99 because of the shape of the concentrationeresponse relationship. EC50, alfaxalone concentration yielding a probability of immobility in 50% of the population; EC95, alfaxalone concentration yielding a probability of immobility in 95% of the population; EC99, alfaxalone concentration yielding a probability of immobility in 99% of the population.
Table 1 Median (range) visual analog scale (VAS) scores for quality of induction of anesthesia (VAS induction), quality of anesthesia (VAS anesthesia) and quality of recovery from anesthesia (VAS recovery) in 16 cats administered intravenous alfaxalone infusions targeting a plasma alfaxalone concentration of 1e5 mg L1 Target (mg L¡1)
n
VAS induction (mm)
VAS anesthesia (mm)
VAS recovery (mm)
1 2 3 4 5
1 7 6 1 1
22 33 (9e80) 80 (30e99) 91 89
23 68 (52e87) 87 (85e99)* 89 80
99 84 (73e100) 85 (65e86) 68 68
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration; VAS induction, 0 was worst possible induction (slow, excitement, paddling) and 100 was best possible induction (rapid, smooth); VAS anesthesia, 0 was no anesthesia or sedation and 100 was anesthesia for most of the infusion time (presence of minimal jaw tone, absence of palpebral reflex, rotation of the eyes); VAS recovery, 0 was worst possible quality/excessively long and 100 was best possible/fast. Values are presented as actual values rather than the median (range) for target concentrations administered to a single cat. *Significantly different from 2 mg L1 (p < 0.05).
Time from discontinuation of the infusion to sternal recumbency was significantly longer in the cats administered the 3 mg L1 target concentration compared with the 2 mg L1 target concentration (p ¼ 0.0260; Table 2). No significant difference was found for the times from discontinuation of the infusion to head lift or standing without assistance. The observation for time to standing without assistance was missing in the cat administered the 5 mg L1 target concentration (Table 2). A significant time effect was found for PR for both the 2 and 3 mg L1 target plasma alfaxalone concentrations (p ¼ 0.0024 and 0.0013, respectively; Table 3). PR was significantly lower at 15 and 25 minutes (i.e., during the administration of alfaxalone), but not at 45 minutes (after discontinuation of alfaxalone administration), compared with baseline, for both target plasma concentrations. PR was not significantly different between the two target plasma concentrations at any time point. fR was significantly lower at 15 minutes after starting the alfaxalone administration in the cats administered the 3 mg L1 target concentration compared with the
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al. Table 2 Median (range) time from discontinuation of alfaxalone administration to head lift, sternal recumbency and standing without assistance in 16 cats administered intravenous alfaxalone infusions targeting a plasma alfaxalone concentration of 1e5 mg L1 Target (mg L¡1)
n
Head lift (minutes)
Sternal recumbency (minutes)
Standing (minutes)
1 2 3 4 5
1 7 6 1 1
8.7 15.1 (3.9e33.9) 34.5 (12.3e42.3) 38.7 56.5
8.8 20.8 (9.4e62.2) 35.8 (31.4e44.3)* 40.6 57
9.1 21.8 (16.5e65.8) 37.9 (31.4e46.8) 48.4 Missing data
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration. Note that the value is presented rather than the median (range) for target concentrations administered to a single cat. *Significantly different from 2 mg L1 (p < 0.05).
Table 3 Median (range) pulse rate and respiratory rate prior to drug administration (T0) and 15 (T15), 25 (T25) and 45 (T45) minutes after starting an intravenous alfaxalone infusion targeting a plasma concentration of 1e5 mg L1 in 16 cats. The alfaxalone infusion was discontinued after approximately 32 minutes Target (mg L¡1)
n
Pulse rate (beats minute1) 1 1 2 7 3 6 4 1 5 1 Respiratory rate (breaths minute1) 1 1 2 7 3 6 4 1 5 1
Time points T0
T15
T25
T45
270 204 (140e270) 255 (180e270) 164 144
240 150 (128e180)* 166 (140e260)* 164 168
170 160 (124e180)* 178 (131e224)* 164 160
240 180 (136e240) 178 (142e250) 140 140
80 60 (24e110) 66 (50e100) 64 60
30 24 (18e28) 16 (10e24)*y 12 20
30 20 (14e20)* 16 (10e24)* 8 14
44 22 (18e40) 17 (12e60) 10 16
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration. Note that the value is presented rather than the median (range) for target concentrations administered to a single cat. *Significantly different from T0 (p < 0.05). y Significantly different from 2 mg L1 (p < 0.05).
2 mg L1 target concentration (p ¼ 0.0359; Table 3). A significant time effect was found for fR for both the 2 and 3 mg L1 target plasma alfaxalone concentrations (p ¼ 0.0006 and 0.0004, respectively; Table 3). fR was significantly lower at 25 minutes than at baseline with the 2 mg L1 target concentration, and at 15 and 25 minutes compared with baseline with the 3 mg L1 target concentration (Table 3). RT was 37e39.7 C (data from all cats and all time points pooled), and significant time or target plasma alfaxalone concentration effects were found for RT. No cat exhibited signs of pain or discomfort during administration of alfaxalone. Poor muscle relaxation, periodic paddling or twitching was observed in 10 cats during induction, and in three cats during anesthesia. In these cats, plasma concentrations at 15 minutes were 1.25e8.03 mg L1. Myoclonus was 6
observed in one cat during recovery of anesthesia; the plasma concentration at 31 minutes was 7.30 mg L1. No other adverse event was observed. Discussion The main result of this study is the characterization of the probability of immobility as a function of plasma alfaxalone concentration in cats. Knowledge of the shape of this probability curve allows the calculation of parameters of pharmacological and clinical relevance, such as the EC50, which is a commonly used index of drug potency, and EC95 or EC99, which represent desirable targets for clinical drug use. Combined with knowledge of drug pharmacokinetics, these parameters will help in calculating alfaxalone dosing in cats. For example, based on published pharmacokinetics, an IV constant rate infusion of
© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e9
Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
191 mg kg1 minute1 would be expected to produce immobility in response to noxious stimulation in 99% of cats once steady state is achieved (Whittem et al. 2008). This is similar to the rate required in clinical studies in cats (Beths et al. 2014; Schwarz et al. 2014; Campagna et al. 2015), although cats in these studies had received sedatives and opioids or non-steroidal anti-inflammatory drugs prior to alfaxalone, whereas alfaxalone only was used in the study reported here. It has been shown that four independent crossovers resulted in acceptable estimates of drug doses or concentrations, resulting in 50% probability of an allor-none event (Dixon 1965). Six independent crossovers were used to determine the number of subjects used in this study. This is based on a simulation study for the determination of the minimum alveolar concentration of inhalant anesthetics (i.e., the EC50 for inhalants) in humans, using the up-and-down method, suggesting that the accuracy of estimates of minimum alveolar concentration improves with an increasing number of crossovers until six; larger number of crossovers are predicted to minimally affect the accuracy of the estimate (Paul & Fisher 2001). While the principle that increasing the number of crossovers should, up to a point, improve the accuracy and precision of an estimate of the EC50 does equally apply to alfaxalone, it is unclear whether six is the optimal number, as the population variability in EC50 is likely different in alfaxalone in cats and inhalant anesthetics in humans. The quality of anesthesia, as defined in this study, appeared to increase with increasing plasma alfaxalone concentrations, which is not surprising as the lower concentrations would be expected to produce a light, possibly inadequate depth of anesthesia. Overall, the VAS scores for quality of induction and quality of anesthesia would be considered clinically good to excellent at plasma alfaxalone concentrations of 8 mg L1 and above, and 5 mg L1 and above, respectively. The VAS for quality of recovery suggests that recovery was deemed to be clinically good to excellent at plasma alfaxalone concentrations of 5 mg L1 and below. This was mainly related to recovery time being considered excessively long for a short infusion at higher plasma concentrations. However, these findings should be interpreted with caution owing to lack of blinding (see below), and the fact that concentrations higher than 8 mg L1 (for induction) and above 5 mg L1 (for recovery) were only recorded in two and six cats, respectively. It should also be noted that the 8 mg L1 for quality of
induction is based on a measurement after 15 minutes of infusion rather than at the time of induction; actual drug concentration at induction time is unknown. Alfaxalone appeared to affect time to return to sternal recumbency in a plasma concentrationdependent manner. While statistical significance was not reached for the other recovery times recorded in this study, it is likely that higher plasma concentrations at the time at which alfaxalone administration is discontinued would also affect them, as the higher concentrations would prolong the time needed to reach the concentration at which the recovery end point is observed. Alfaxalone appeared to affect PR and fR, although plasma concentration dependence was limited. These effects are in agreement with those previously reported in cats (Muir et al. 2009). Poor muscle relaxation, twitching and paddling were seen during induction or maintenance of anesthesia in a majority of cats. There was no obvious association with plasma alfaxalone concentration; these effects were seen with both high and low concentrations, and were inconsistent if a particular concentration is considered. Nevertheless, it should be noted that the majority of events was during induction of anesthesia, and as no plasma sample was collected until after 15 minutes of drug infusion, the actual plasma alfaxalone concentration around the time of induction is unknown. Myoclonus was observed in one cat during recovery, and in this cat, the plasma concentration immediately prior to discontinuing the alfaxalone administration was the second highest. No conclusion can be drawn, however, as this was a single incident. The results of this study should be interpreted in view of several limitations. 1) All cats were neutered males and it is unknown if sex influences the potency of alfaxalone. Unless the effect is very large, it would have been unlikely to be detectable with the study sample. Nevertheless, the effective concentrations reported may only apply to male neutered cats. In addition, the animals were young and healthy and it is likely that health status, and possibly age, affects the requirements for alfaxalone, as has been reported for other drugs in at least some species (Quasha et al. 1980; Larsson & Wahlstr€ om 1998; Patanwala et al. 2013; Zettervall et al. 2015). 2) The effective plasma concentrations reported are for alfaxalone alone, which is not representative of common clinical practice. The administration of sedative and/or analgesic drugs would be expected to lower the requirement for alfaxalone. 3) The formulation used
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
is not currently commercially available. While it differs from the available formulation only in the addition of preservatives and the two formulations were shown to be bioequivalent (Pasloske et al. 2018), pharmacodynamic differences cannot entirely be ruled out. 4) The end point used to determine the effective plasma alfaxalone concentration was immobility in response to a noxious stimulus. This end point has been commonly used for other anesthetic drugs, including inhalant anesthetics. However, it is possible that the potency for other effects (e.g., unconsciousness) is different, and the reported values should therefore only be considered relevant for producing immobility. 5) This study was conducted according to an up-and-down design. While this design is well suited for the determination of effective doses or concentrations, it results in different drug concentrations being used in individual subjects. In this study, each of the 1, 4 and 5 mg mL1 target concentrations was studied in only one cat, whereas alfaxalone 2 and 3 mg mL1 target concentrations were administered to seven and six cats, respectively. This resulted in limiting statistical analysis of possible time and target concentration effects on study variables to the data obtained from cats administered the 2 and 3 mg L1 target concentrations. 6) The actual plasma alfaxalone concentrations were considerably higher than the concentrations targeted. This does not influence the findings of this study, as the actual plasma concentrations were used for logistic regression, but suggests that the pharmacokinetic model used for the targetcontrolled infusions was poorly predictive of the disposition of alfaxalone in these cats. In particular, the model likely overestimated clearance and/or volume of distribution. The model was obtained by nonlinear regression of time-concentration data in 24 cats administered the same alfaxalone concentration (Pasloske et al. 2018). It is possible that the dose used in that study was inadequate for predicting the disposition following the amount of alfaxalone administered in this study. The pharmacokinetics of alfaxalone in cats have indeed been reported to be dose dependent (Whittem et al. 2008). Close examination of the timeeconcentration data in the 24 cats reveals that concentrations above the LOQ of the assay were measured for a longer time in a small subset of cats, and an additional, slower terminal phase is present in these subjects. Therefore, while the two-compartment model used for the target control infusions was the best overall fit for the 24 8
cats, it is possible that a three-compartment model is more predictive of the disposition of this drug. The slower elimination phase would result in a lower estimated clearance. 7) Monitoring of vital functions during this study was limited to PR, fR and RT to allow focus on assessing quality of induction of anesthesia, and of anesthesia itself, and collection of blood samples for plasma drug concentration measurement. The goal of this study was not to assess the effects of alfaxalone on cardiopulmonary function, and the significance of the changes in PR and fR is largely unknown owing to the lack of additional measurements (such as arterial blood pressure and partial pressure of carbon dioxide). In addition, the changes in RT would have been affected by the heating pad placed under the cats during the infusion of alfaxalone. 8) The ability of testing the dependence of the effects of alfaxalone on plasma concentration was limited by the fact that enough data were available for analysis only for the 2 and 3 mg L1 plasma concentrations, that is, a narrow concentration range, and that even for these plasma concentrations, the sample size was fairly small. 9) Some subjective assessments (VAS scores) were made by an individual who was not blinded to the target plasma alfaxalone concentration. This was due to logistical reasons, and because these assessments were considered secondary objectives. Nevertheless, the results should be interpreted with caution, as the lack of blinding prevents the control of bias and the VAS scoring system is a subjective measure of judgment. In conclusion, the EC50 EC95 and EC99 for alfaxalone-induced immobility were determined in cats. In combination with pharmacokinetic data, these values can be used to design pharmacokineticbased infusion regimens. Acknowledgements This work was supported by Jurox Pty Ltd (unrestricted gift). The authors thank Dr. Alessia Cenani, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, for technical assistance. Authors' contributions BHP: study design, data acquisition, data analysis, manuscript preparation; KTS: data acquisition, manuscript preparation; MGR: plasma alfaxalone concentration measurement, manuscript preparation; KP: data acquisition, manuscript preparation.
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Effective alfaxalone concentration in cats BH Pypendop et al.
Conflict of interest statement Alfaxalone was provided free of charge by Jurox Pty Ltd. Jurox Pty Ltd. funded the study and the plasma alfaxalone concentration analysis was conducted by the company. MGR and KP are employed by Jurox Pty Ltd. References Beths T, Touzot-Jourde G, Musk G, Pasloske K (2014) Clinical evaluation of alfaxalone to induce and maintain anaesthesia in cats undergoing neutering procedures. J Feline Med Surg 16, 609e615. Campagna I, Schwarz A, Keller S et al. (2015) Comparison of the effects of propofol or alfaxalone for anaesthesia induction and maintenance on respiration in cats. Vet Anaesth Analg 42, 484e492. Dixon WJ (1965) The up-and-down method for small samples. Am Stat Assoc J 60, 967e978. Honkavaara J, Pypendop B, Turunen H, Ilkiw J (2017) The effect of MK-467, a peripheral a2-adrenoceptor antagonist, on dexmedetomidine-induced sedation and bradycardia after intravenous administration in conscious cats. Vet Anaesth Analg 44, 42e51. Larsson JE, Wahlstr€ om G (1998) The influence of age and administration rate on the brain sensitivity to propofol in rats. Acta Anaesthesiol Scand 42, 987e994. Muir W, Lerche P, Wiese A et al. (2009) The cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in cats. Vet Anaesth Analg 36, 42e54.
Pasloske K, Ranasinghe MG, Sauer S, Hare J (2018) The bioequivalence of a single intravenous administration of the anesthetic alfaxalone in cyclodextrin versus alfaxalone in cyclodextrin plus preservatives in cats. J Vet Pharmacol Ther, in press. Patanwala AE, Christich AC, Jasiak KD et al. (2013) Agerelated differences in propofol dosing for procedural sedation in the emergency department. J Emerg Med 44, 823e828. Paul M, Fisher DM (2001) Are estimates of MAC reliable? Anesthesiology 95, 1362e1370. Quasha AL, Eger EI 2nd, Tinker JH (1980) Determination and applications of MAC. Anesthesiology 53, 315e334. Schwarz A, Kalchofner K, Palm J et al. (2014) Minimum infusion rate of alfaxalone for total intravenous anaesthesia after sedation with acepromazine or medetomidine in cats undergoing ovariohysterectomy. Vet Anaesth Analg 41, 480e490. Whittem T, Pasloske KS, Heit MC, Ranasinghe MG (2008) The pharmacokinetics and pharmacodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses. J Vet Pharmacol Ther 31, 571e579. Zettervall SL, Sirajuddin S, Akst S et al. (2015) Use of propofol as an induction agent in the acutely injured patient. Eur J Trauma Emerg Surg 41, 405e411. Received 1 June 2017; accepted 31 October 2017. Available online xxx
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https://doi.org/10.1016/j.vaa.2017.10.006
RESEARCH PAPER
Effective plasma alfaxalone concentration to produce immobility in male neutered cats Bruno H Pypendopa, Kristine T Siaob,1, MG Ranasinghec & Kirby Pasloskec a
Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of
California, Davis, CA, USA b
Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California, Davis,
CA, USA c
Jurox Pty Ltd, Rutherford, NSW, Australia
Correspondence: Bruno Pypendop, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, One Shields Avenue, Davis, CA 95616, USA. E-mail: [email protected] 1
Dr. Siao's current address is 1848 24th Ave. San Francisco, CA 94122.
Abstract Objective To determine the effective plasma alfaxalone concentration for the production of immobility in cats. Study design Prospective up-and-down study. Animals Sixteen 1e2 year old male castrated research cats. Methods Cats were instrumented with catheters in a jugular and a medial saphenous vein. Alfaxalone was administered via the medial saphenous catheter, using a target-controlled infusion system. The infusion lasted for approximately 32 minutes. A noxious stimulus (tail clamp) was applied 30 minutes after starting the alfaxalone infusion, until the cat moved or 60 seconds had elapsed, whichever occurred first. The target alfaxalone concentration was set at 5 mg L1 in the first cat and increased or decreased by 1 mg L1 in subsequent cats, if the previous cat had moved or not moved in response to stimulation, respectively. This was continued until six independent crossovers (different responses in pairs of subsequent cats) had been observed. Blood samples were collected before alfaxalone administration, and 15 and 31 minutes after starting the administration, for the determination of plasma alfaxalone concentration using liquid chromatography/tandem mass spectrometry. The alfaxalone concentration yielding a probability of immobility in 50% (EC50), 95% (EC95) and 99% (EC99) of the population, and their respective 95% Wald confidence intervals were calculated.
Results The EC50, EC95 and EC99 for alfaxaloneinduced immobility were 3.7 (2.4e4.9), 6.2 (4.7e) and 7.6 (5.5e) mg L1, respectively. Conclusions and clinical relevance The effective plasma alfaxalone concentration for immobility in cats was determined. This value will help in the design of pharmacokinetic-based dosing regimens. Keywords alfaxalone, cats, pharmacology. Introduction Alfaxalone is an anesthetic neurosteroid. It is currently commercially available as a solution containing alfaxalone solubilized in hydroxypropylb-cyclodextrin, and is labeled in the United States, Canada, Europe, Korea, Japan, New Zealand, South Africa and Australia for induction and maintenance of anesthesia via intermittent intravenous (IV) boluses in dogs and cats. The pharmacokinetics of alfaxalone in cats have been characterized (Whittem et al. 2008); however, to the authors’ knowledge, the effective plasma concentration of alfaxalone has not been reported in either dogs or cats, making it difficult to use the available pharmacokinetic information to calculate adequate dosing for induction or maintenance of anesthesia, by intermittent IV boluses or IV infusion. The objective of this study was to estimate the effective plasma alfaxalone concentration in 50%, 95% and 99% of the cat population, resulting in immobility following noxious stimulation.
1
Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
Materials and methods This study was approved by the Institutional Animal Care and Use Committee at the University of California, Davis. Healthy, 1e2 years old, male, neutered cats were used. The health status of each cat was assessed by history and complete physical examination 2 days before, as well as on the day of the experiment. In addition, cats were observed daily for any signs of disease for 7 days before the experiment was initiated. Husbandry conditions for this institution have been previously described (Honkavaara et al. 2017). The cats were fed Laboratory Feline Diet 5003 (LabDiet, MO, USA) once daily. On the day prior to the experiment, each cat was briefly anesthetized with isoflurane in oxygen. The skin over a jugular vein and a medial saphenous vein was clipped and aseptically prepared using chlorhexidine and alcohol. A 20 gauge, 48 mm or a 19 gauge, 150 mm catheter was inserted in the jugular vein for collection of blood samples. A 20 gauge, 48 mm catheter was inserted in the medial saphenous vein for drug administration. Infusion plugs were placed on the catheters, and the catheters were flushed with heparinized 0.9% weight/volume (w/v) saline solution and secured to the skin with tape (medial saphenous) or suture (jugular). A light bandage was placed over the catheters and cats were allowed to recover from anesthesia. An Elizabethan collar was placed around the neck and cats were returned to their room. On the day of the experiment, the cat’s body weight was measured. A physical examination was performed, including the measurement of pulse rate (PR), respiratory rate (fR) and rectal body temperature (RT). The patency of the catheters was verified by injecting a small volume of heparinized saline solution. Alfaxalone (10 mg mL1) in hydroxypropylb-cyclodextrin was administered via the medial saphenous catheter, using a target-controlled infusion system. An alfaxalone preserved formulation was administered in the study. The alfaxalone formulation contained the preservatives ethanol (150 mg mL1), chlorocresol (1 mg mL1) and benzethonium chloride (0.2 mg mL1). The targetcontrolled infusion system consisted of a syringe pump (PHD2000; Harvard Apparatus, MA, USA) and computer program (Rugloop I; Demed, Belgium). The system rapidly loaded the central compartment to the target concentration by delivering a bolus calculated from [T] Vc, and maintained this concentration via a variable infusion rate updated every 10 seconds, 2
according to the following equation: R ¼ [T] Vc (k10 þ k12 ek21t), where R is the infusion rate; [T] is the target plasma concentration; Vc is the volume of the central compartment; k10, k12 and k21 are microrate constants and t is the infusion time. Vc, k10, k12 and k21 were 302 mL kg1, 0.101 minute1, 0.323 minute1 and 0.066 minute1, respectively. The latter values were obtained from the best fitting pharmacokinetic model obtained from 24 cats following IV bolus administration of the same alfaxalone formulation (RD0327) in a different study (Pasloske et al. 2018). Observations were made on whether any cat exhibited signs of pain or discomfort in response to the injection of the preserved alfaxalone formulation. The cat was placed on a heating pad. PR (palpation of the femoral pulse over 15 seconds), fR (observation of chest excursions over 15 seconds) and RT (electronic thermometer) were measured 15, 25 and 45 minutes after starting the drug administration. A noxious stimulus (clamping of the tail using a 20 cm Martin forceps closed to the first ratchet) was applied 30 minutes after starting the target-controlled infusion. The stimulus was continued until 1 minute had elapsed, or until movement was observed, whichever occurred first, and the response was recorded as positive (movement) or negative (lack of movement). Any movement of the limbs or head, except related to swallowing or coughing, was considered positive. Blood samples (2 mL) were collected prior to drug administration, 15 minutes after starting drug administration, and immediately after the noxious stimulus was delivered. Prior to sampling, blood (3 mL) was aspirated into a syringe containing a small amount of heparinized saline and later returned after the actual sample had been collected. Blood was transferred to tubes containing lithium heparin, placed on ice and centrifuged at 3901 g and 4 C for 10 minutes within 60 minutes of collection. The plasma was separated, transferred to cryotubes and frozen at e80 C until analyzed for alfaxalone concentration. Drug administration was discontinued following collection of the last blood sample, and the cat was allowed to recover under observation. Times from discontinuation of drug administration to head lift, return to sternal recumbency and ability to stand without assistance were recorded. The quality of anesthesia induction, the quality of anesthesia and the quality of recovery from anesthesia were subjectively assessed using a 100 mm visual analog scale (VAS). For quality of induction, 0 was defined as
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Effective alfaxalone concentration in cats BH Pypendop et al.
worst possible induction (slow, excitement, paddling) and 100 as best possible induction (rapid, smooth). For quality of anesthesia, 0 was defined as no anesthesia or sedation and 100 as anesthesia for most of the infusion time (based on the presence of minimal jaw tone, absence of palpebral reflex, rotation of the eyes and response to tail clamping). For quality of recovery, 0 was defined as worst possible quality/ excessively long and 100 as best possible/fast. The scores were all assigned by the same investigator (BHP), who was not blinded to the target plasma alfaxalone concentration. The target plasma alfaxalone concentration was set at 5 mg L1 for the first cat. The target plasma alfaxalone concentration in subsequent cats was increased or decreased by 1 mg L1, according to the observation of a positive or negative response to noxious stimulation, respectively, in the previous cat. The study was continued until 20 cats had been used, or six independent crossover responses (i.e., different response in subsequent cats) had been observed, whichever occurred first. Cats were observed for signs of adverse effects such as vomiting, regurgitation, change in respiratory effort, seizures or convulsions, myoclonus and muscle rigidity until they had fully recovered from anesthesia. Alfaxalone analysis Alfaxalone concentration was measured in feline plasma using a validated liquid chromatography/ tandem mass spectrometry method. Ten microliters of acetonitrile and 150 mL of 1 mg mL1 aqueous solution of 11-hydroxyprogesterone as internal standard were added to the plasma samples (40 mL), and the samples were extracted using Waters Oasis HLB 30 mm mElution solid-phase extraction 96 well plates (Waters Corporation, MA, USA). The plate was preconditioned with methanol and water, the samples loaded and the wells washed with water followed by 15% methanol solution and then eluted with 75 mL of acetonitrile. The elute was diluted with 225 mL of water and analyzed for alfaxalone content using Agilent 1100 series liquid chromatography and 6410B triple-quadrupole mass detector operated on positive electrospray ionization mode (Agilent Technologies Inc., CA, USA). A calibration curve was generated using the calibration standard sample set prepared by spiking known amounts of alfaxalone and the internal standard in the blank (control) plasma. Twenty mL of each extracted plasma sample
was injected into a Phenomenex Kinetex PFP (2.6 mm, 50 2.1 mm high-performance liquid chromatography column; Phenomenex Inc., CA, USA) using a gradient elution with mobile phase consisting of 0.3 mM ammonium formate in 0.1% aqueous formic acid and 0.3 mM ammonium formate in 999:9:1 (v/v/v) acetonitrile:water:formic acid. The target mass ion fragment (m/z) 215 of parent ion 333 for analyte and 295 of parent ion 331 for the internal standard were used for the analysis. The alfaxalone concentration was determined by calculating the peak area ratio of the alfaxalone to 11hydroxyprogesterone. The lower limit of quantification (LLOQ) of alfaxalone in a plasma sample was 0.02 mg L1, whereas the upper limit of quantification was 10 mg L1. The lower limit of detection for the assay was 0.003 mg L1. The intra-assay coefficient of variation for samples at LLOQ was <10.2%. The minimum coefficient of determination (r2) of the standard curve was >0.99. Sample extraction recoveries were >89% for all concentration levels. The accuracy was deemed acceptable if the calculated concentration in calibration and quality control samples was within 100 ± 20% of the actual concentration at LLOQ, and 100 ± 15% of the actual concentration for all other concentrations. The precision was deemed acceptable if the individual relative standard deviation in replicate quality control samples was <20% at LLOQ and <15% for all other concentrations. All derived values were reported to three significant figures. The plasma alfaxalone concentration at the time of the measurement was considered to be the concentration measured in the last sample. Statistical analysis The probability of movement/immobility function was fitted to the plasma alfaxalone concentrationeresponse data using logistic regression. The alfaxalone concentration yielding a probability of immobility in 50% (EC50), 95% (EC95) and 99% (EC99) of the population, and their respective 95% Wald confidence intervals were calculated from the fitted function. Linear regression was used to examine the relationship between target and actual plasma alfaxalone concentration, and the Pearson productemoment correlation coefficient (r) was calculated and reported as r2. Normality of continuous data was assessed using the ShapiroeWilk test. Because the normality hypothesis was rejected for several data sets, nonparametric tests were used to
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Effective alfaxalone concentration in cats BH Pypendop et al.
analyze all data. The times to head lift, return to sternal recumbency and standing without assistance, and the VAS scores for quality of induction, anesthesia and recovery were compared between the 2 and 3 mg L1 target concentrations using the Wilcoxon test (not enough observations were available for statistical evaluation at the other target concentrations). The PR, fR and RT data were analyzed for the time effect within target concentration groups using Friedman’s test, followed where appropriate by Dunn’s test for comparisons to baseline data, and for the target concentration effect within the time points using the Wilcoxon test. Data are presented as median (range), except where otherwise indicated. The EC50, EC95 and EC99 are presented as estimated concentration (95% Wald confidence interval). Results Sixteen cats were administered alfaxalone in the study and assessed for response to a noxious stimulus. Six independent crossover responses (i.e., change in response in six pairs of successive cats, for a total of 12 cats) were obtained. The mean ± standard deviation body weight of the cats was 5.5 ± 0.7 kg. Target plasma alfaxalone concentrations were 1, 2, 3, 4 and 5 mg L1 in 1, 7, 6, 1 and 1 cats, respectively. Statistical analysis of PR, fR, RT, recovery time and VAS score data was therefore conducted on data from cats administered the 2 and 3 mg L1 target plasma alfaxalone concentrations only. Tracheal intubation was attempted in the first five cats, but not in subsequent cats owing to stimulated coughing. Actual
plasma alfaxalone concentrations [median (range) when measured in more than one individual at a target concentration] at 15 and 31 minutes were 1.25 and 1.19, 3.21 (2.92e4.48) and 3.56 (2.63e5.41), 5.5 (4.56e7.27) and 4.87 (4.54e5.28), 8.03 and 7.3, and 9.8 and 8.12 mg L1 for target plasma alfaxalone concentrations of 1, 2, 3, 4 and 5 mg L1, respectively (Fig. 1). The EC50, EC95 and EC99 for immobility were 3.7 (2.4e4.9), 6.2 (4.7e) and 7.6 (5.5e) mg L1, respectively (Fig. 2). The upper limit of the 95% Wald confidence interval could not be computed for EC95 and EC99 because of the shape of the concentrationeresponse relationship. There was a strong, linear association between target and actual plasma alfaxalone concentration (r2 ¼ 0.90, 0.85 and 0.85 for samples collected after 15 minutes of infusion, 31 minutes of infusion and all samples pooled, respectively). However, mean ± standard deviation actual plasma alfaxalone concentration was 181 ± 31% of target after 15 minutes of infusion, and 167 ± 34% of target after the noxious stimulation (approximately 31 minutes of infusion). The VAS score for quality of anesthesia was significantly higher in the cats administered the 3 mg L1 target plasma alfaxalone concentration than the 2 mg L1 target concentration (p ¼ 0.0268; Table 1). The lower quality scores were mainly attributed to persistence of muscle tone. No significant difference was found for VAS scores for quality of induction of anesthesia or quality of recovery.
Figure 1 Plasma alfaxalone concentration at 15 (closed circles) and 31 (open circles) minutes after starting the alfaxalone intravenous infusion in each cat. The number next to each closed circle is the target plasma alfaxalone concentration in that individual. 4
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Effective alfaxalone concentration in cats BH Pypendop et al.
Figure 2 Logistic regression of the plasma alfaxalone concentrationeresponse data in 16 cats. The closed circles represent the response in each cat at the plasma alfaxalone concentration measured immediately after the noxious stimulation (approximately 32 minutes after starting the intravenous alfaxalone infusion). A response of 0 corresponds to movement, and a response of 1 to no movement observed during clamping of the tail for up to 60 seconds. The line represents the probability of immobility in response to noxious stimulation as a function of plasma alfaxalone concentration. The estimated EC50, EC95 and EC99 are shown, with their corresponding 95% Wald confidence interval. The upper limit of the confidence interval could not be computed for the EC95 and EC99 because of the shape of the concentrationeresponse relationship. EC50, alfaxalone concentration yielding a probability of immobility in 50% of the population; EC95, alfaxalone concentration yielding a probability of immobility in 95% of the population; EC99, alfaxalone concentration yielding a probability of immobility in 99% of the population.
Table 1 Median (range) visual analog scale (VAS) scores for quality of induction of anesthesia (VAS induction), quality of anesthesia (VAS anesthesia) and quality of recovery from anesthesia (VAS recovery) in 16 cats administered intravenous alfaxalone infusions targeting a plasma alfaxalone concentration of 1e5 mg L1 Target (mg L¡1)
n
VAS induction (mm)
VAS anesthesia (mm)
VAS recovery (mm)
1 2 3 4 5
1 7 6 1 1
22 33 (9e80) 80 (30e99) 91 89
23 68 (52e87) 87 (85e99)* 89 80
99 84 (73e100) 85 (65e86) 68 68
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration; VAS induction, 0 was worst possible induction (slow, excitement, paddling) and 100 was best possible induction (rapid, smooth); VAS anesthesia, 0 was no anesthesia or sedation and 100 was anesthesia for most of the infusion time (presence of minimal jaw tone, absence of palpebral reflex, rotation of the eyes); VAS recovery, 0 was worst possible quality/excessively long and 100 was best possible/fast. Values are presented as actual values rather than the median (range) for target concentrations administered to a single cat. *Significantly different from 2 mg L1 (p < 0.05).
Time from discontinuation of the infusion to sternal recumbency was significantly longer in the cats administered the 3 mg L1 target concentration compared with the 2 mg L1 target concentration (p ¼ 0.0260; Table 2). No significant difference was found for the times from discontinuation of the infusion to head lift or standing without assistance. The observation for time to standing without assistance was missing in the cat administered the 5 mg L1 target concentration (Table 2). A significant time effect was found for PR for both the 2 and 3 mg L1 target plasma alfaxalone concentrations (p ¼ 0.0024 and 0.0013, respectively; Table 3). PR was significantly lower at 15 and 25 minutes (i.e., during the administration of alfaxalone), but not at 45 minutes (after discontinuation of alfaxalone administration), compared with baseline, for both target plasma concentrations. PR was not significantly different between the two target plasma concentrations at any time point. fR was significantly lower at 15 minutes after starting the alfaxalone administration in the cats administered the 3 mg L1 target concentration compared with the
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Effective alfaxalone concentration in cats BH Pypendop et al. Table 2 Median (range) time from discontinuation of alfaxalone administration to head lift, sternal recumbency and standing without assistance in 16 cats administered intravenous alfaxalone infusions targeting a plasma alfaxalone concentration of 1e5 mg L1 Target (mg L¡1)
n
Head lift (minutes)
Sternal recumbency (minutes)
Standing (minutes)
1 2 3 4 5
1 7 6 1 1
8.7 15.1 (3.9e33.9) 34.5 (12.3e42.3) 38.7 56.5
8.8 20.8 (9.4e62.2) 35.8 (31.4e44.3)* 40.6 57
9.1 21.8 (16.5e65.8) 37.9 (31.4e46.8) 48.4 Missing data
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration. Note that the value is presented rather than the median (range) for target concentrations administered to a single cat. *Significantly different from 2 mg L1 (p < 0.05).
Table 3 Median (range) pulse rate and respiratory rate prior to drug administration (T0) and 15 (T15), 25 (T25) and 45 (T45) minutes after starting an intravenous alfaxalone infusion targeting a plasma concentration of 1e5 mg L1 in 16 cats. The alfaxalone infusion was discontinued after approximately 32 minutes Target (mg L¡1)
n
Pulse rate (beats minute1) 1 1 2 7 3 6 4 1 5 1 Respiratory rate (breaths minute1) 1 1 2 7 3 6 4 1 5 1
Time points T0
T15
T25
T45
270 204 (140e270) 255 (180e270) 164 144
240 150 (128e180)* 166 (140e260)* 164 168
170 160 (124e180)* 178 (131e224)* 164 160
240 180 (136e240) 178 (142e250) 140 140
80 60 (24e110) 66 (50e100) 64 60
30 24 (18e28) 16 (10e24)*y 12 20
30 20 (14e20)* 16 (10e24)* 8 14
44 22 (18e40) 17 (12e60) 10 16
Target, target plasma alfaxalone concentration; n, number of cats administered the respective target plasma alfaxalone concentration. Note that the value is presented rather than the median (range) for target concentrations administered to a single cat. *Significantly different from T0 (p < 0.05). y Significantly different from 2 mg L1 (p < 0.05).
2 mg L1 target concentration (p ¼ 0.0359; Table 3). A significant time effect was found for fR for both the 2 and 3 mg L1 target plasma alfaxalone concentrations (p ¼ 0.0006 and 0.0004, respectively; Table 3). fR was significantly lower at 25 minutes than at baseline with the 2 mg L1 target concentration, and at 15 and 25 minutes compared with baseline with the 3 mg L1 target concentration (Table 3). RT was 37e39.7 C (data from all cats and all time points pooled), and significant time or target plasma alfaxalone concentration effects were found for RT. No cat exhibited signs of pain or discomfort during administration of alfaxalone. Poor muscle relaxation, periodic paddling or twitching was observed in 10 cats during induction, and in three cats during anesthesia. In these cats, plasma concentrations at 15 minutes were 1.25e8.03 mg L1. Myoclonus was 6
observed in one cat during recovery of anesthesia; the plasma concentration at 31 minutes was 7.30 mg L1. No other adverse event was observed. Discussion The main result of this study is the characterization of the probability of immobility as a function of plasma alfaxalone concentration in cats. Knowledge of the shape of this probability curve allows the calculation of parameters of pharmacological and clinical relevance, such as the EC50, which is a commonly used index of drug potency, and EC95 or EC99, which represent desirable targets for clinical drug use. Combined with knowledge of drug pharmacokinetics, these parameters will help in calculating alfaxalone dosing in cats. For example, based on published pharmacokinetics, an IV constant rate infusion of
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
191 mg kg1 minute1 would be expected to produce immobility in response to noxious stimulation in 99% of cats once steady state is achieved (Whittem et al. 2008). This is similar to the rate required in clinical studies in cats (Beths et al. 2014; Schwarz et al. 2014; Campagna et al. 2015), although cats in these studies had received sedatives and opioids or non-steroidal anti-inflammatory drugs prior to alfaxalone, whereas alfaxalone only was used in the study reported here. It has been shown that four independent crossovers resulted in acceptable estimates of drug doses or concentrations, resulting in 50% probability of an allor-none event (Dixon 1965). Six independent crossovers were used to determine the number of subjects used in this study. This is based on a simulation study for the determination of the minimum alveolar concentration of inhalant anesthetics (i.e., the EC50 for inhalants) in humans, using the up-and-down method, suggesting that the accuracy of estimates of minimum alveolar concentration improves with an increasing number of crossovers until six; larger number of crossovers are predicted to minimally affect the accuracy of the estimate (Paul & Fisher 2001). While the principle that increasing the number of crossovers should, up to a point, improve the accuracy and precision of an estimate of the EC50 does equally apply to alfaxalone, it is unclear whether six is the optimal number, as the population variability in EC50 is likely different in alfaxalone in cats and inhalant anesthetics in humans. The quality of anesthesia, as defined in this study, appeared to increase with increasing plasma alfaxalone concentrations, which is not surprising as the lower concentrations would be expected to produce a light, possibly inadequate depth of anesthesia. Overall, the VAS scores for quality of induction and quality of anesthesia would be considered clinically good to excellent at plasma alfaxalone concentrations of 8 mg L1 and above, and 5 mg L1 and above, respectively. The VAS for quality of recovery suggests that recovery was deemed to be clinically good to excellent at plasma alfaxalone concentrations of 5 mg L1 and below. This was mainly related to recovery time being considered excessively long for a short infusion at higher plasma concentrations. However, these findings should be interpreted with caution owing to lack of blinding (see below), and the fact that concentrations higher than 8 mg L1 (for induction) and above 5 mg L1 (for recovery) were only recorded in two and six cats, respectively. It should also be noted that the 8 mg L1 for quality of
induction is based on a measurement after 15 minutes of infusion rather than at the time of induction; actual drug concentration at induction time is unknown. Alfaxalone appeared to affect time to return to sternal recumbency in a plasma concentrationdependent manner. While statistical significance was not reached for the other recovery times recorded in this study, it is likely that higher plasma concentrations at the time at which alfaxalone administration is discontinued would also affect them, as the higher concentrations would prolong the time needed to reach the concentration at which the recovery end point is observed. Alfaxalone appeared to affect PR and fR, although plasma concentration dependence was limited. These effects are in agreement with those previously reported in cats (Muir et al. 2009). Poor muscle relaxation, twitching and paddling were seen during induction or maintenance of anesthesia in a majority of cats. There was no obvious association with plasma alfaxalone concentration; these effects were seen with both high and low concentrations, and were inconsistent if a particular concentration is considered. Nevertheless, it should be noted that the majority of events was during induction of anesthesia, and as no plasma sample was collected until after 15 minutes of drug infusion, the actual plasma alfaxalone concentration around the time of induction is unknown. Myoclonus was observed in one cat during recovery, and in this cat, the plasma concentration immediately prior to discontinuing the alfaxalone administration was the second highest. No conclusion can be drawn, however, as this was a single incident. The results of this study should be interpreted in view of several limitations. 1) All cats were neutered males and it is unknown if sex influences the potency of alfaxalone. Unless the effect is very large, it would have been unlikely to be detectable with the study sample. Nevertheless, the effective concentrations reported may only apply to male neutered cats. In addition, the animals were young and healthy and it is likely that health status, and possibly age, affects the requirements for alfaxalone, as has been reported for other drugs in at least some species (Quasha et al. 1980; Larsson & Wahlstr€ om 1998; Patanwala et al. 2013; Zettervall et al. 2015). 2) The effective plasma concentrations reported are for alfaxalone alone, which is not representative of common clinical practice. The administration of sedative and/or analgesic drugs would be expected to lower the requirement for alfaxalone. 3) The formulation used
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
is not currently commercially available. While it differs from the available formulation only in the addition of preservatives and the two formulations were shown to be bioequivalent (Pasloske et al. 2018), pharmacodynamic differences cannot entirely be ruled out. 4) The end point used to determine the effective plasma alfaxalone concentration was immobility in response to a noxious stimulus. This end point has been commonly used for other anesthetic drugs, including inhalant anesthetics. However, it is possible that the potency for other effects (e.g., unconsciousness) is different, and the reported values should therefore only be considered relevant for producing immobility. 5) This study was conducted according to an up-and-down design. While this design is well suited for the determination of effective doses or concentrations, it results in different drug concentrations being used in individual subjects. In this study, each of the 1, 4 and 5 mg mL1 target concentrations was studied in only one cat, whereas alfaxalone 2 and 3 mg mL1 target concentrations were administered to seven and six cats, respectively. This resulted in limiting statistical analysis of possible time and target concentration effects on study variables to the data obtained from cats administered the 2 and 3 mg L1 target concentrations. 6) The actual plasma alfaxalone concentrations were considerably higher than the concentrations targeted. This does not influence the findings of this study, as the actual plasma concentrations were used for logistic regression, but suggests that the pharmacokinetic model used for the targetcontrolled infusions was poorly predictive of the disposition of alfaxalone in these cats. In particular, the model likely overestimated clearance and/or volume of distribution. The model was obtained by nonlinear regression of time-concentration data in 24 cats administered the same alfaxalone concentration (Pasloske et al. 2018). It is possible that the dose used in that study was inadequate for predicting the disposition following the amount of alfaxalone administered in this study. The pharmacokinetics of alfaxalone in cats have indeed been reported to be dose dependent (Whittem et al. 2008). Close examination of the timeeconcentration data in the 24 cats reveals that concentrations above the LOQ of the assay were measured for a longer time in a small subset of cats, and an additional, slower terminal phase is present in these subjects. Therefore, while the two-compartment model used for the target control infusions was the best overall fit for the 24 8
cats, it is possible that a three-compartment model is more predictive of the disposition of this drug. The slower elimination phase would result in a lower estimated clearance. 7) Monitoring of vital functions during this study was limited to PR, fR and RT to allow focus on assessing quality of induction of anesthesia, and of anesthesia itself, and collection of blood samples for plasma drug concentration measurement. The goal of this study was not to assess the effects of alfaxalone on cardiopulmonary function, and the significance of the changes in PR and fR is largely unknown owing to the lack of additional measurements (such as arterial blood pressure and partial pressure of carbon dioxide). In addition, the changes in RT would have been affected by the heating pad placed under the cats during the infusion of alfaxalone. 8) The ability of testing the dependence of the effects of alfaxalone on plasma concentration was limited by the fact that enough data were available for analysis only for the 2 and 3 mg L1 plasma concentrations, that is, a narrow concentration range, and that even for these plasma concentrations, the sample size was fairly small. 9) Some subjective assessments (VAS scores) were made by an individual who was not blinded to the target plasma alfaxalone concentration. This was due to logistical reasons, and because these assessments were considered secondary objectives. Nevertheless, the results should be interpreted with caution, as the lack of blinding prevents the control of bias and the VAS scoring system is a subjective measure of judgment. In conclusion, the EC50 EC95 and EC99 for alfaxalone-induced immobility were determined in cats. In combination with pharmacokinetic data, these values can be used to design pharmacokineticbased infusion regimens. Acknowledgements This work was supported by Jurox Pty Ltd (unrestricted gift). The authors thank Dr. Alessia Cenani, Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, for technical assistance. Authors' contributions BHP: study design, data acquisition, data analysis, manuscript preparation; KTS: data acquisition, manuscript preparation; MGR: plasma alfaxalone concentration measurement, manuscript preparation; KP: data acquisition, manuscript preparation.
© 2017 Association of Veterinary Anaesthetists and American College of Veterinary Anesthesia and Analgesia. Published by Elsevier Ltd. All rights reserved., ▪, 1e9
Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006
Effective alfaxalone concentration in cats BH Pypendop et al.
Conflict of interest statement Alfaxalone was provided free of charge by Jurox Pty Ltd. Jurox Pty Ltd. funded the study and the plasma alfaxalone concentration analysis was conducted by the company. MGR and KP are employed by Jurox Pty Ltd. References Beths T, Touzot-Jourde G, Musk G, Pasloske K (2014) Clinical evaluation of alfaxalone to induce and maintain anaesthesia in cats undergoing neutering procedures. J Feline Med Surg 16, 609e615. Campagna I, Schwarz A, Keller S et al. (2015) Comparison of the effects of propofol or alfaxalone for anaesthesia induction and maintenance on respiration in cats. Vet Anaesth Analg 42, 484e492. Dixon WJ (1965) The up-and-down method for small samples. Am Stat Assoc J 60, 967e978. Honkavaara J, Pypendop B, Turunen H, Ilkiw J (2017) The effect of MK-467, a peripheral a2-adrenoceptor antagonist, on dexmedetomidine-induced sedation and bradycardia after intravenous administration in conscious cats. Vet Anaesth Analg 44, 42e51. Larsson JE, Wahlstr€ om G (1998) The influence of age and administration rate on the brain sensitivity to propofol in rats. Acta Anaesthesiol Scand 42, 987e994. Muir W, Lerche P, Wiese A et al. (2009) The cardiorespiratory and anesthetic effects of clinical and supraclinical doses of alfaxalone in cats. Vet Anaesth Analg 36, 42e54.
Pasloske K, Ranasinghe MG, Sauer S, Hare J (2018) The bioequivalence of a single intravenous administration of the anesthetic alfaxalone in cyclodextrin versus alfaxalone in cyclodextrin plus preservatives in cats. J Vet Pharmacol Ther, in press. Patanwala AE, Christich AC, Jasiak KD et al. (2013) Agerelated differences in propofol dosing for procedural sedation in the emergency department. J Emerg Med 44, 823e828. Paul M, Fisher DM (2001) Are estimates of MAC reliable? Anesthesiology 95, 1362e1370. Quasha AL, Eger EI 2nd, Tinker JH (1980) Determination and applications of MAC. Anesthesiology 53, 315e334. Schwarz A, Kalchofner K, Palm J et al. (2014) Minimum infusion rate of alfaxalone for total intravenous anaesthesia after sedation with acepromazine or medetomidine in cats undergoing ovariohysterectomy. Vet Anaesth Analg 41, 480e490. Whittem T, Pasloske KS, Heit MC, Ranasinghe MG (2008) The pharmacokinetics and pharmacodynamics of alfaxalone in cats after single and multiple intravenous administration of Alfaxan at clinical and supraclinical doses. J Vet Pharmacol Ther 31, 571e579. Zettervall SL, Sirajuddin S, Akst S et al. (2015) Use of propofol as an induction agent in the acutely injured patient. Eur J Trauma Emerg Surg 41, 405e411. Received 1 June 2017; accepted 31 October 2017. Available online xxx
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Please cite this article in press as: Pypendop BH, Siao KT, Ranasinghe MG et al. Effective plasma alfaxalone concentration to produce immobility in male neutered cats, Veterinary Anaesthesia and Analgesia (2018), https://doi.org/10.1016/ j.vaa.2017.10.006