Sedation Characteristics of Intranasal Alfaxalone in Adult Yucatan Swine

Abstract

Compared with intravenous and intramuscular methods, intranasal administration of sedatives is a less invasive and nonpainful technique. In this prospective, randomized, crossover study, we evaluated the sedative characteristics of 2 doses (1 and 2 mg/kg) of alfaxalone administered intranasally to 7 adult Yucatan swine. We compared sedation scores before and after administration of alfaxalone and between groups by using a composite sedation scoring system (range, 0 to 12, with 12 being the highest level of sedation)). Pigs were randomly assigned to receive 2 doses of intranasal alfaxalone (1 mg/kg [A1]); 2 mg/kg [A2]) as 2 separate events in a crossover design with a 60-d washout period. Categories scored were posture, palpebral droop, uninhibited behavior, drowsiness, and acceptance of anesthetic facemask. Sedation scores were collected before sedation was administered and then every 3 min for 30 min afterward. Instilled volumes (mean ± 1 SD) were 5.7 ± 0.5 and 11.3 ± 0.8 mL for A1 and A2, respectively. Both alfaxalone doses produced significant increases in sedation scores compared with baseline. Median sedation scores for A1 (6; range, 4–12) were not different from those for A2 (6; range, 6 to 12). Intranasal administration of alfaxalone as the sole sedative agent increased sedation scores from baseline, achieving peak sedation at 6 to 9 min after instillation of A2. However, sedation scores were similar between the 2 groups, and neither dose produced sufficient sedation to facilitate handling or the performance of any clinical procedures. Given the concentration of alfaxalone solution currently available, volume is the major limiting factor regarding testing higher doses of this drug for its use as a sole sedative agent in swine.

Achieving physical restraint for clinical procedures in swine has historically been challenging.¹¹ In this species, stress induced by handling can lead to severe pathophysiologic consequences, such as hyperthermia, respiratory distress, and decreased meat quality in production subjects.^1,18,21 Stress has also been shown to be detrimental to overall animal welfare.¹ Intramuscular injection of sedatives and anesthetics is useful and widely used in swine, due to its practicality.¹⁴ However, this method can cause pain and anxiety upon injection and has the potential to damage muscle groups destined for the meat chain. Therefore, investigation of alternative routes of administration for sedative agents is warranted.

Compared with intravenous and intramuscular techniques, intranasal administration of sedatives is noninvasive and nonpainful.⁴ Absorption through the intranasal route is prompt because the blood–brain barrier is bypassed due to direct nose-to-brain transport via the olfactory and trigeminal neural pathways.¹⁷ Intranasal administration of compounds, alone or in combination, has been used to achieve tranquilization and sedation in several species, including humans.^8,16,20

In swine, intranasal midazolam has been widely used for tranquilization and sedation, and its sedation properties have been investigated at different dosages.¹² Midazolam is a benzodiazepine, acting on the GABA_A receptor by potentiating the effect of γ-aminobutyric acid at the receptor site.¹³ In contrast, alfaxalone is a neurosteroidal compound, acting directly as an agonist to the GABA_A receptor to produce dose-dependent sedation, anesthesia, and muscle relaxation.⁵ In several countries, alfaxalone is available in a 10-mg/mL formulation containing 2-hydroxy propyl β-cyclodextrin as a carrier (Alfaxan, Jurox, North Kansas City, MO). The effects of intravenous, intramuscular, and intratesticular alfaxalone in swine have been described.^6,7,10 To our knowledge, information regarding the sedative characteristics of intranasal alfaxalone administered as a sole agent to swine is currently unavailable.

In this study, we evaluated the sedative characteristics of 2 doses (1 and 2 mg/kg) of alfaxalone administered intranasally to adult Yucatan swine. Sedation scores before and after the administration of alfaxalone were compared within subjects and between groups by using a composite sedation scoring system. Specifically, the tested null hypotheses were that sedation scores after the administration of alfaxalone at the 2 dosages would be unchanged compared with those recorded at baseline before intranasal administration and that subjects given 2 mg/kg of alfaxalone would achieve the same sedation scores as those that received 1 mg/kg.

Materials and Methods

Animals and husbandry.

Adult Yucatan castrated male swine (Sus scrofa domestic; tuberculosis-free, brucellosis-free, premises negative for porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus, and pseudorabies-free; n = 7; age, [mean ± 1 SD], 504 ± 24 d; weight, 56 ± 2 kg; Exemplar Genetics, Flandreau, SD) were included in a prospective, randomized, crossover study, with each subject serving as its own control. According to a type I error of 5%, power of 80%, and an effect size of 1.2 obtained from a one-tailed Wilcoxon signed-rank test of preliminary data, a total sample size of 7 was required. Randomization of subjects and drug sequence was achieved by using a randomization tool (Excel, Microsoft, Redmond, WA). Ethical approval from the Louisiana State University IACUC was obtained (protocol no. 14-077). All pigs were current on standard vaccinations (Mycoplasma hyopneumoniae Bacterin, Schering Plough, Memphis, TN; Circumvent PCV-M G2, Intervet, Omaha, NE; Ingelvac ERY-ALC, Boehringer Ingelheim, St Joseph, MO; MaxiGuard, Addison Biologic Laboratory, Fayette, MI; Enterisol SC-54, Boehringer Ingelheim; Maxivac Excel 5.0, Merck, Omaha, NE; and Espisure-One, Zoetis, Parsippany, NJ). The pigs were cared for according to the Guide for the Care and Use of Laboratory Animals.⁹ They were housed with 4 or 5 other pigs in pens (3.1 × 2.1 m) with concrete flooring covered by 2 to 3 in. of pine shavings (S and S Farms, Franklinton, LA) and acclimated for 2 mo before the trial began. Pigs were provided with chains and toys for enrichment and a plastic housing shed within the pen for shelter. A pelleted miniature swine diet (Mazuri, Land O'Lakes, Saint Paul, MN) was provided twice daily, with water available ad libitum.

For about 30 d prior to the study's start, pigs were trained to elevate their rostrum to touch a target and were conditioned to the person administering the drug. On each day before the trial, pigs were deemed healthy on the basis of physical examination and were assigned a body condition score based on previous literature.³ Subjects were excluded from the trial if they had abnormalities on physical examination or a history of vomiting or diarrhea within the previous 48 h. They were fasted for 12 h before the trial day, with water available ad libitum throughout the study period. For administration of alfaxalone, pigs were moved for a maximum of 2 h during the trial to individual pens adjacent to their mates, allowing them to maintain auditory, visual, and physical contact through a metal screen. On 2 separate occasions, and with a washout period of at least 60 d, each pig received 1 mg/kg (A1) or 2 mg/kg (A2) of alfaxalone (10 mg/mL; Alfaxan, Jurox, North Kansas City, MO) intranasally. Tested doses were established by considering the limitations imposed by the volume to be delivered, due to the concentration of the commercially available alfaxalone formulation. By using the slip tip of a disposable 12-mL syringe (Monoject, Covidien, Dublin, Ireland), half of the total volume of alfaxalone calculated for each subject was instilled in each nostril. When necessary, moderate physical restraint was performed by temporarily lifting both thoracic limbs. The subject's head was maintained elevated for 2 to 4 s after administration. Pigs were observed continuously during the sedation period and monitored visually for signs of excessive sedation such as apnea and unconsciousness.

Sedation scoring.

The 6-item sedation scoring system used in this study was extrapolated from a previously published system¹² to fit the current study's objectives. Subjects were evaluated in their pens for posture (score of 0, standing; 1, lying down with back up; 2, reclining), palpebral droop (0, eyes wide open; 1, moderate droop; 2, marked droop), uninhibited behavior (0, retreats at approach of human hand; 1, moderate retreat; 2, absence of retreat and permits head touch), drowsiness (0, steady on feet; 1, beginning to stagger; 2, staggering), grunting (0, high frequency; 1, moderate frequency; 2, none), and acceptance of an anesthetic face mask (0, difficult; 1, accepts mask after mild retreat; 2, easy acceptance) before administration of alfaxalone (baseline) and at 3-min intervals for 30 min afterward. The lowest and highest scores possible were 0 and 12, respectively. The same investigator, who was blind to the dose of alfaxalone administered, performed the sedation scoring throughout the trial. When the subject became recumbent, ease of arousal from sedation was tested by touching the pig's flank. If this stimulus did not produce arousal, a noxious stimulus consisting of a skin prick from a 21-gauge, 1-in. hypodermic needle was applied, and the response was recorded. Collection of physiologic data such as heart rate, respiratory rate, and blood pressure was intentionally not performed to avoid excitement and arousal from sedation. All trials were videorecorded.

Statistical analysis.

Statistical analysis was performed by using Prism 8 (GraphPad Software, San Diego, CA). Sedation scores were treated as nonparametric data, and they are reported as median and range. The Friedman test followed by Dunn posthoc testing was used to compare baseline sedation scores with the maximal score achieved during the 30-min evaluation period. The Wilcoxon signed-rank test was used to compare sedation scores between A1 and A2 at each time point and within each category. A P value less than 0.05 was considered statistically significant.

Results

Administered volumes (mean ± 1 SD) were 5.7 ± 0.5 and 11.3 ± 0.8 mL for A1 and A2, respectively. In 2 events in the A2 group, about 1.5 mL was lost during instillation and replaced with an identical volume of alfaxalone via the same route. Commonly observed behavioral changes were widening of the distance between thoracic limbs, head drop, and decreased exploratory and motor activity. Subjects were easily aroused when stimulated by touch or an accidental loud noise. Only one subject (A1 group) achieved lateral recumbence, yet remained readily arousable by the application of noxious stimulus. No other procedures were performed during this study.

Sedation scores are plotted in Figure 1. Baseline sedation scores did not differ between groups, although one subject presented different baseline scores on the 2 days of the trial. Both of the alfaxalone doses (A1 and A2) produced significant increases (P = 0.0432 and 0.0066, respectively) in sedation scores compared with baseline. Peak sedation scores (Table 1) for A1 (median, 6; range, 4 to 12) were not significantly lower than for A2 (median, 6; range, 6 to 12; P > 0.9999); however, scores at 3, 6, and 9 min were significantly higher although for A2 than A1 (P = 0.0469, 0.0156, and 0.0469, respectively) and represented the time window of the peak in sedation scores in A2. Both groups had a single subject (different animals) that achieved the maximal sedation score possible (i.e., 12). The administration of 2 mg/kg alfaxalone produced significantly less grunting (P < 0.0001) and uninhibited behavior (P = 0.0037) than did 1 mg/kg.

Figure 1.

Boxplot showing sedation scores before (0) and at 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30 min after the administration of intranasal alfaxalone at 1 mg/kg (A1) and 2 mg/kg (A2) to 7 Yucatan pigs in a cross-over trial.

Table 1.

Highest sedation scores recorded throughout the 30-min scoring period after intranasal administration of alfaxalone at 1 mg/kg (A1) or 2 mg/kg (A2)

Pig	Baseline	A1	A2
1	4	5	12
2	4	6	6
3	0	8	8
4	4	5	9
5	4	4	6
6	3	12	6
7	4	6	6

Discussion

In this study, we evaluated the effects of alfaxalone administered at 1 and 2 mg/kg intranasally to adult Yucatan pigs. Our results indicate that, in this cohort of subjects, intranasal alfaxalone increased sedation scores from baseline, independent of the dose used. We also showed that the 2-mg/kg dose is associated with a narrow window of peak sedation that occurs between 3 and 9 min after administration when using 2 mg/kg. However, neither dose produced a consistent and sufficient level of sedation to permit the performance of any noninvasive procedure involving touch and manipulation of the pigs.

The intranasal route for the administration of sedatives and other drugs has been widely investigated in human and veterinary medicine. The nasal mucosa is highly vascularized, therefore providing a large surface for absorption.⁴ Furthermore, rapid onset of action, direct delivery to the CNS, and the lack of first-pass metabolism make this route attractive for the administration of sedative agents.^4,19 In pigs, tranquilization is successfully achieved by intranasal administration of 0.2 mg/kg midazolam¹² and is usually followed by a combination of sedatives to achieve more profound sedation for performing clinical procedures while minimizing stress from handling. One previous study¹² used a much smaller volume of midazolam (around 1.5 mL) compared with the volumes we used here, which reached 12.6 mL in some animals, because our subjects were adult pigs, whereas those that received intranasal midazolam were 7.5 wk of age.¹² This difference is clinically important: larger volumes are more difficult to deliver intranasally, because some of the dose can be lost or swallowed during administration and because dose delivery requires a relatively long time compared with intramuscular injection. In 2 of our subjects in the A2 group, around 1.5 mL were lost during administration, thus demonstrating the difficulty we encountered administering 2 mg/kg alfaxalone intranasally. While designing this experiment, we considered using an atomizer (MAD Nasal Intranasal Mucosal Atomization Device, Teleflex, Morrisville, NC) to distribute the drug into microdroplets. Ultimately, we decided against using this device given our previous experience of administering large drug volumes through this type of atomizer and the excessively long time needed to atomize such volumes.

Sedation characteristics due to intranasal administration of alfaxalone have been studied in terrapins, mallard ducks, and rabbits.^2,15 Terrapins received 5 mg/kg of alfaxalone intranasally via the use of micropipettes,² whereas mallard ducks received 24 mg/kg of a combination of alfaxalone and alphadolone.¹⁵ Although monitored physiologic parameters did not change after administration, sedation was considered null in both species, with no observed loss of monitored reflexes, which is consistent with the lack of clinically useful sedation that we observed in our pigs. This failure to achieve sedation might be due to the physicochemical properties of the drug (molecular weight, hydrophobicity, solubility), the nasal physiology of swine, and intra- and interindividual variability.⁴ Currently, no studies have investigated the pharmacokinetics of alfaxalone administered intranasally to any species, so we can only speculate on the bioavailability of this drug through the intranasal route.

Our current study is the first to evaluate intranasal administration of alfaxalone as the sole agent to sedate Yucatan pigs. The doses evaluated in this study were established in consideration of the limitations imposed by the volume to be delivered, due to the concentration of the commercially available formulation. Intratesticular administration of alfaxalone (4, 6, or 8 mg/kg) in combination with lidocaine (1.5 to 2.5 mg/kg) has been used to perform orchiectomies and in combination with diazepam administered through intramuscular injection in piglets younger than 8 wk. The investigators found that combinations with lidocaine and with diazepam produced deep sedation with variable results on obtaining lateral recumbency^6,7 and a recovery time (standing) of about 30 min.⁷ The alfaxalone doses used for intramuscular and intratesticular administration were much higher than those tested in the current study, thus corroborating the idea that doses delivered here were too low to produce sedation in pigs. Shaking, twitching, and paddling occurred after both intramuscular and intratesticular administration of alfaxalone in pigs,^6,7 but these phenomena did not present in any of our subjects.

The behavioral changes observed in this group of pigs after intranasal alfaxalone were mainly postural, with widening of the distance between thoracic limbs to increase stability, head drop, and decreased exploratory and motor activity. Grunting and retreating were the 2 categories that were most affected by the dose used. In addition, subjects were easily and readily arousable when stimulated by touch or accidental loud noises. These findings are consistent with results from sedation with intranasal alfaxalone in terrapins and mallard ducks, as previously mentioned.^2,15 Only one subject, which had received 1 mg/kg alfaxalone, displayed lateral recumbency. After the evaluator touched the pig's flank and obtained only mild and transient grunting, the skin of the tip of the ear was pricked by using a hypodermic needle. At this point, the subject suddenly aroused and moved quickly away from the operator. The authors of the sedation scoring system¹² discussed the possibility that it did not adequately capture the behavioral variations produced by alfaxalone. We share this opinion, given that our scoring system was extrapolated from the previous study,¹² in which it was used to evaluate sedation from intranasal midazolam. Ideally, the results of the current study should be used to improve the scoring system for evaluating alfaxalone in pigs.

The peak in sedation scores after A2 was achieved between 3 and 9 min. However, even the maximal sedation obtained with alfaxalone as a sole, intranasal sedative was absolutely insufficient to perform any noninvasive clinical procedure, and we therefore do not recommend its use in this species for these purposes. Adding another sedative such as diazepam or dexmedetomidine may produce more effective and prolonged sedation and likely would promote easier manipulation of the subjects for noninvasive procedures, such as physical examination and intravenous catheter placement.

The main limitation of this study is the low doses of alfaxalone that were used. This limitation was imposed by the fact that the commercially available solution of alfaxalone is not sufficiently concentrated to allow for intranasal instillation of smaller volumes. Yucatan pigs are used mainly for biomedical research, and they are different from standard commercial pigs in regard to final adult weight. Yucatan pigs usually do not exceed 140 lb (63.5 kg), which would mandate a drug volume of about 12 mL if 2 mg/kg alfaxalone was used. This volume, in our experience, is difficult to administer intranasally because careful and spill-free instillation requires a relatively long time (about 15 s), during which subjects might move their rostrum, chew, snort, and possibly move away from the operator. This constraint is an important source of error with this technique, because the drug can exit the nostrils or remain in the nasopharynx without reaching the nasal mucosa for absorption. This complication would further lower the dose of alfaxalone that was actually administered. This possibility coupled with the overall insufficient dose used may have caused the relative lack of sedation that we observed in this study. Finally, this study lacks a placebo group or a no-treatment control group. However, given that no description of the sedative properties of intranasal alfaxalone was available at the time of the experiment, the main goal of our study was to acquire information as groundwork for further research, and the use of a placebo or control group was outside the scope of this investigation.

In summary, intranasal alfaxalone as a sole sedative agent in pigs increased sedation scores from baseline, with no significant differences between tested doses. Neither dose produced adequate sedation to allow for handling, easy performance of any noninvasive clinical procedure, or the reduction of stress. Given the concentration of the currently available alfaxalone solution, volume is the major limiting factor in testing higher doses of this drug as a sole sedative agent administered intranasally. The combination of alfaxalone with other sedative agents may provide effective premedication options through the intranasal route.