Evaluation of the thermal antinociceptive effects of subcutaneous administration of butorphanol tartrate or butorphanol tartrate in a sustained-release poloxamer 407 gel formulation to orange-winged Amazon parrots (Amazona amazonica)

Delphine Laniesse 1Department of Pathobiology, Ontario Veterinary College, University of Guelph, ON N1G 2W1, Canada.
2Department of Clinical Studies, Ontario Veterinary College, University of Guelph, ON N1G 2W1, Canada.

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David Sanchez-Migallon Guzman 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Dale A. Smith 1Department of Pathobiology, Ontario Veterinary College, University of Guelph, ON N1G 2W1, Canada.

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Jamie M. Douglas 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Cornelia Mosley 4Anesthesiology and Pain Service, 404 Veterinary Emergency & Referral Hospital, Newmarket, ON L3Y 0B3, Canada.

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Hugues Beaufrère 2Department of Clinical Studies, Ontario Veterinary College, University of Guelph, ON N1G 2W1, Canada.

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Joanne Paul-Murphy 3Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

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Abstract

OBJECTIVE

To determine the thermal antinociceptive effects of butorphanol tartrate and butorphanol tartrate in a sustained-release 25% poloxamer 407 (P407) gel formulation (But-P407) in parrots.

ANIMALS

13 orange-winged Amazon parrots (Amazona amazonica).

PROCEDURES

First, butorphanol tartrate (5 mg/kg) or saline (0.9% NaCl) solution was administered IM to birds in a randomized complete crossover design. The temperature prompting a foot withdrawal response to a thermal stimulus (ie, the thermal threshold) was determined 30 minutes before (baseline) and at various points after treatment administration. Second, But-P407 (12.5 mg/kg) or P407 was administered SC in a similar crossover design. Thermal threshold was determined before and at various points after treatment administration. Third, But-P407 (12.5 mg/kg) or saline solution was administered SC and evaluated as in the second trial. Sedation was scored immediately before each time point in all 3 trials.

RESULTS

In the first trial, a significant increase in thermal threshold was noted 30 minutes after butorphanol tartrate (vs saline solution) administration. No sedation was noted. In the second and third trials, no significant difference was identified between results for But-P407 and those for either control treatment (saline solution or P407). Mild sedation was noted in the second trial following But-P407 administration.

CONCLUSIONS AND CLINICAL RELEVANCE

Results suggested a small but significant thermal antinociceptive effect of butorphanol tartrate lasting between 30 minutes and 1.5 hours in orange-winged Amazon parrots. No antinociceptive effect of butorphanol tartrate was demonstrated when delivered in P407. Further research is needed to evaluate the potential analgesic effects of But-P407.

Abstract

OBJECTIVE

To determine the thermal antinociceptive effects of butorphanol tartrate and butorphanol tartrate in a sustained-release 25% poloxamer 407 (P407) gel formulation (But-P407) in parrots.

ANIMALS

13 orange-winged Amazon parrots (Amazona amazonica).

PROCEDURES

First, butorphanol tartrate (5 mg/kg) or saline (0.9% NaCl) solution was administered IM to birds in a randomized complete crossover design. The temperature prompting a foot withdrawal response to a thermal stimulus (ie, the thermal threshold) was determined 30 minutes before (baseline) and at various points after treatment administration. Second, But-P407 (12.5 mg/kg) or P407 was administered SC in a similar crossover design. Thermal threshold was determined before and at various points after treatment administration. Third, But-P407 (12.5 mg/kg) or saline solution was administered SC and evaluated as in the second trial. Sedation was scored immediately before each time point in all 3 trials.

RESULTS

In the first trial, a significant increase in thermal threshold was noted 30 minutes after butorphanol tartrate (vs saline solution) administration. No sedation was noted. In the second and third trials, no significant difference was identified between results for But-P407 and those for either control treatment (saline solution or P407). Mild sedation was noted in the second trial following But-P407 administration.

CONCLUSIONS AND CLINICAL RELEVANCE

Results suggested a small but significant thermal antinociceptive effect of butorphanol tartrate lasting between 30 minutes and 1.5 hours in orange-winged Amazon parrots. No antinociceptive effect of butorphanol tartrate was demonstrated when delivered in P407. Further research is needed to evaluate the potential analgesic effects of But-P407.

The analgesic effects and duration of actions of various opioids differ by animal species and noxious stimulus.1–6 To investigate the antinociceptive effects of a given analgesic drug in a particular species, testing must be performed with an analgesiometric method. In birds, methods described in the literature include thermal, mechanical, chemical, and electric noxious stimuli. Withdrawal threshold as determined by use of thermal or electric stimuli has been evaluated for the objective assessment of modulation of nociception in African grey parrots (Psittacus erithacus),7 Japanese quail (Coturnix japonica),8 Hispaniolan Amazon parrots (Amazona ventralis),4 cockatiels (Nymphicus hollandicus),6,9 American kestrels (Falco sparverius),5,10,11 and broiler chickens (Gallus domesticus).12

Butorphanol, a κ-opioid receptor agonist and μ-opioid receptor antagonist, has been reported as the opioid drug of clinical choice in birds of the family Psittacidae because it is both affordable and readily available, with antinociceptive effects shown in Hispaniolan Amazon parrots,4,13 green-cheeked conures (Pyrrhura molinae),14 and African grey parrots.15 The plasma concentration of butorphanol above which an antinociceptive effect has been noted is estimated to be 55 ng/mL for electric stimuli and 88 ng/mL for thermal stimuli in Hispaniolan Amazon parrots receiving liposome-encapsulated butorphanol SC at a dose of 15 mg/kg.4 In a pharmacokinetic study16 involving the same species, plasma concentrations reaching these thresholds were attained following IM administration of butorphanol tartrate at 5 mg/kg and were expected to provide antinociception for 2 to 3 hours. However, to the authors' knowledge, data derived via an analgesiometric method have yet to be published to confirm this estimation. Repeated handling and injections are stressful to birds and may induce muscle pain and necrosis, which may exacerbate postoperative pain and discomfort. Conversely, appropriate analgesia speeds up recovery, improves prognosis, and decreases complication rates through improvements in healing, comfort, appetite, and attitude17; therefore, a definite need exists for a sustained-released opioid product for birds.

To extend the duration of butorphanol-induced analgesia, preparation and pharmacokinetics of a formulation of butorphanol tartrate mixed with P407, a thermosensitive hydrogel, were investigated.18,19 Butorphanol was slowly released from a 25% P407 base (But-P407) in vitro through a dialysis membrane.18 In a pharmacokinetic study19 involving Hispaniolan Amazon parrots and But-P407, butorphanol was well absorbed following SC administration at a dose of 12.5 mg/kg, and its absorption followed a pharmacokinetic profile compatible with a sustained-release drug. Plasma concentrations of butorphanol > 100 ng/mL were maintained for > 3 hours but < 8 hours.19 Pharmacodynamic studies of this formulation were considered necessary to confirm the degree and duration of analgesia and to provide recommendations for its clinical use in parrots and other avian species.

The overall objective of the study reported here was to determine the thermal antinociceptive effects of butorphanol tartrate and But-P407 in orange-winged Amazon parrots (Amazona amazonica) and identify potential secondary effects such as agitation or sedation and regurgitation. Our first hypothesis was that butorphanol tartrate administered IM would have thermal antinociceptive effects in this species. Our second hypothesis was that SC administration of But-P407 would result in thermal antinociceptive effects that would last significantly longer than effects achieved with butorphanol tartrate solution.

Materials and Methods

Animals

Thirteen orange-winged Amazon parrots (mean ± SD body weight, 434.1.6 ± 73.1 g) were used in 3 experimental trials. Birds that participated in the first 2 trials included two 2-year-old males, two 2-year-old females, three 13-year-old females, and three 13-year-old males. Birds that participated in the third study included one 16-year-old male, three 15-year-old males, two 15-year-old females, one 4-year-old male, two 4-year-old females, and 1 adult female of unknown age.

All parrots were part of a stable research colony and were housed individually in wire cages that measured 66 × 66 × 107 to 114 cm or 81 × 61 × 142 cm in a room maintained at 23°C, with a 12-hour light-dark cycle. Parrots had free access to a commercial pelleted dieta and fresh water. They had not received any medications for a period of at least 6 months before the first trial began and throughout the entire study period. All were confirmed via physical examination to be healthy before each trial began. The study protocol was approved by the University of Guelph Animal Care Committee and by the University of California-Davis Institutional Animal Care and Use Committee.

Preparation of But-P407

The But-P407 formulation was prepared by use of the cold method, as described elsewhere.18,20 The amount of P407 sufficient to yield a 25% gel (wt/wt) was added to previously refrigerated (5°C) butorphanol tartrateb in a sterile syringe case.c The syringe case, surrounded by ice packs, was placed on a vortex mixerd (at 3,200 rpm on “touch” mode) for 5 minutes until a homogeneous solution was obtained, after which the solution was refrigerated at 4°C for 10 to 15 minutes until only minimal bubbles were present at the surface of the solution. The solution was then passed through a 0.22-μm microfiltere directly into a sterile rubber-topped glass tube,f after which no more bubbles were seen. The solution was kept on ice until administered (within 2 hours after preparation). The calculated concentration of butorphanol in the formulation was 8.3 mg/mL.18 The same procedure for preparation of But-P407 was used for all 3 trials.

Experimental design

Three sequential trials were performed: comparison of the effects of butorphanol tartrate with those of saline (0.9% NaCl) solution (trial 1), comparison of the effects of But-P407 with those of control P407 solution (25% P407 only; trial 2), and comparison of the effects of But-P407 with those of saline solution (trial 3). Trials were separated by a 6-month interval. The same 10 parrots were used for trials 1 and 2; in trial 3, 7 of these 10 parrots were used as well as 3 new parrots substituted for 3 original ones that were unavailable.

A complete crossover experimental design was used for each trial. Parrots were randomized to 2 groups in each trial representing different orders of 2 treatments (n = 5/group). Because of the odd number of parrots per group, this randomization was unable to balance males with females or adults with juveniles.

In each trial, the first assigned treatment was administered, a 7-day washout period was observed, and then the alternate treatment was administered. In trial 1, treatments consisted of butorphanol tartrate (5 mg/kg) or an equivalent volume of saline solution administered IM in the left pectoral muscle. In trial 2, treatments consisted of But-P407 (12.5 mg/kg) or an equivalent volume of P407 administered SC in the left flank region. In trial 3, treatments consisted of But-P407 (12.5 mg/kg) or an equivalent volume of saline solution administered SC in the right flank region.

Antinociception testing procedure

Antinociception testing was performed with a previously reported thermal threshold method.3,5,6,9,11,13,21 Thermal thresholds were measured for each parrot by use of a test perch housed within a test box. The birds were acclimated to these test boxes for 4 weeks before trial 1 began. In trials 1 and 2, the test box measured 51 cm high, 14 cm wide, and 28 cm deep and contained a perch. The floor of the box was V shaped in design to discourage birds from standing on the bottom of the box. Trial 3 was carried out in a similar but newly designed testing box that measured 34 cm high, 13 cm wide, and 38 cm deep and contained a perch. Both types of test boxes had transparent front panels to allow remote monitoring of birds via video camera, and the remaining walls were opaque to reduce environmental stimulation. The room where antinociception testing occurred was maintained at 23°C, and white noise was broadcast to prevent sound-associated stimulation of the parrots.

The test perch used for trials 1 and 2 was designed to deliver a thermal stimulus to the plantar surface of the parrot's right foot. The temperature of the perch was increased from 30°C to 55°C at a rate of 0.43°C/s. The parrot was able to escape the stimulus by lifting its right foot from the perch. As soon as a foot lift was noted by the observer, the heating process was terminated. The thermal perch used in trials 1 and 2 involved a cooling system to provide rapid cooling, and the temperature of the perch decreased rapidly once heat was no longer applied. Birds were observed returning their feet to the perch within a few seconds afterward.

The test perch for trial 3 was designed to deliver a thermal stimulus to the plantar surface of the parrot's left foot. The temperature of the perch was increased from 35°C to 55°C at a rate of 0.4°C/s. Parrots could escape the thermal stimulus by lifting their left foot from the perch. As soon as a foot lift was noted by the observer or the maximum thermal threshold was reached, the heating process was terminated. Additionally, the thermal plate was rotated 180° after a foot lift was observed, as enabled by a design feature of this perch. Rotation of the thermal plate allowed birds to immediately return their feet to the perch. In all trials, an automatic cutoff temperature of 55°C was set to avoid potential soft tissue damage to the plantar surface of the foot. Overall, both types of test perches functioned similarly up to the point that the thermal stimulus was terminated, allowing comparisons among trials.

A video camera for remote monitoring was placed in front of the test box. A digital screen displaying the perch temperature could also be seen by the observer. Each parrot was observed throughout antinociception testing, and these observations were recorded. The same observer (DL for trials 1 and 2 and JMD for trial 3) assessed and managed all thermal thresholds within a given trial.

The thermal foot withdrawal threshold (referred to as thermal threshold) was characterized as the perch temperature at which the bird appeared to consciously lift its foot as a result of the thermal stimulus. A baseline value for each bird was determined at the beginning of each day's testing prior to treatment administration. Thermal thresholds were then assessed by administration of a single thermal challenge at 0.5, 1.5, 3, and 6 hours after treatment administration in trial 1; 1.5, 4, 8, and 12 hours after treatment administration in trial 2; and 0.75, 1.5, 3, 6, and 9 hours after treatment administration in trial 3.

Adverse effects and agitation-sedation scores

The parrots were kept in the testing room for the duration of the day's treatment and thermal testing session, which lasted approximately 6 to 13 hours. Between thermal challenges within a session, birds were individually housed in pet carriers (53 × 41 × 38 cm) with ad libitum access to food and water. Throughout the session, parrots were observed through the wire-mesh cage front and monitored for adverse effects, such as vomiting or regurgitation and evidence of agitation or sedation.19 After entering the test box, and before each thermal challenge, each parrot was observed for 1 to 2 minutes via remote video camera and assigned an agitation-sedation score on the basis of a modified version of a previously described system for use with Hispaniolan Amazon parrots (Appendix).21

Statistical analysis

Freely available statistical softwareg was used for data analyses. Longitudinal data analysis for each trial was performed by means of linear mixed modeling,h with thermal threshold as the outcome (dependent) variable; time, treatment (test treatment or control treatment), treatment order, bird sex, and all interactions among pairs of these variables as fixed effects; and bird as a random effect. Residual plots were used to assess linearity, homogeneity of variances, normality, and outliers. Quantile plots were also performed of the residuals by treatment group for normality assessment. Autocorrelation of the residuals over time was assessed with the autocorrelation function method. Type III ANOVA was performed on the fixed effects, and post hoc comparisons were performed with Tukey adjustment. Contrast statements were used to compare, within each time point, the 2 treatments within a trial with Bonferroni correction.

Agitation-sedation score data were compared between and within treatments by means of an ordinal logit mixed model,i with score (an ordinal variable) as the outcome variable; time, sex, thermal threshold, and interactions among pairs of these variables as fixed effects; and bird as a random variable. Residuals were evaluated graphically. Values of P < 0.05 were considered significant.

Results

Trial 1

One bird developed a superficial burn (ulceration and inflammation of the skin, with healthy deeper tissues) on the plantar surface of its right foot after the first experimental manipulation. The bird was treated for this burn and removed from the study at that point, before crossover to the control treatment; therefore, the data for this bird were available only for the assessment of the effect of butorphanol tartrate and not of saline solution (ie, the control treatment). Consequently, 10 birds contributed data on butorphanol tartrate and only 9 for the control treatment.

Results of comparisons between thermal thresholds (ie, foot withdrawal temperatures) achieved after IM administration of butorphanol tartrate (5 mg/kg) versus the control treatment were graphically displayed (Figure 1). Assumptions of the model were satisfied, with no evidence of autocorrelation of the residuals over time. Individual variability in response accounted for 46.6% of the total variability of the model, with an SD of 1.0°C. The total SD of the model was 1.4°C.

Figure 1—
Figure 1—

Mean thermal thresholds in 10 orange-winged Amazon parrots (Amazona amazonica) before (0 hours) and at various points after IM administration of butorphanol tartrate (5 mg/kg; black dots with dashed line) or an equivalent volume of saline (0.9% NaCl) solution (white dots with solid line). Error bars represent SEM. Data for saline solution represent only 9 parrots owing to 1 parrot being withdrawn from the study after the first treatment. *Mean value differs significantly (P < 0.05) between treatments at the indicated time point.

Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.543

Baseline values for the thermal threshold ranged from 37.9°C to 43.3°C for the 2 treatments. The baseline value had a significant (P = 0.002) effect on thermal threshold, with values increasing by a mean ± SD of 0.4 ± 0.1°C for each 1°C increase in the baseline value. The interaction between treatment and time was significant (P = 0.03). Comparisons with contrast statements revealed a significant (P = 0.003; α = 0.01 after Bonferroni correction) difference in thermal thresholds between the control treatment and butorphanol tartrate at 0.5 hours after drug administration that was of a mean ± SEM magnitude of 1.5 ± 0.5°C. No other comparisons yielded significant results. Agitation-sedation scores were not significantly different between the 2 treatments at each time point (all P > 0.05) and within each treatment over time (P = 0.99). None of the birds regurgitated.

Trial 2

Results of comparisons between thermal thresholds achieved after SC administration of But-P407 (12.5 mg/kg) versus P407 (ie, the control treatment) were graphically displayed (Figure 2) Assumptions of the model were satisfied, with no evidence of auto-correlation of the residuals over time. Individual variability in response accounted for 39.3% of the total variability of the model, with an SD of 0.9°C. The total SD of the model was 1.5°C.

Figure 2—
Figure 2—

Mean thermal thresholds in 10 orange-winged Amazon parrots before (0 hours) and at various points after SC administration of But-P407 (12.5 mg/kg; white dots with solid line) or an equivalent volume of P407 (black dots with dashed line). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.543

Baseline values for thermal threshold ranged from 38.6°C to 42.9°C for the 2 treatments. The baseline value had a significant (P < 0.001) effect on thermal threshold, with values increasing by a mean ± SEM of 0.5 ± 0.1°C for each 1°C increase in the baseline value. No significant effect on thermal threshold was identified for treatment, time, or their interaction. Parrots were more likely to appear sedated when But-P407 (vs P407) was administered, with 62% lower odds of having an increase in agitation-sedation score of 0.5 (proportional OR, 0.38; 95% CI, 0.14 to 0.98; P = 0.045). Time had no significant (P = 0.25) effect on agitation-sedation score. None of the birds regurgitated.

Trial 3

Results of comparisons between thermal thresholds achieved after SC administration of But-P407 (12.5 mg/kg) versus saline solution (ie, the control treatment) were graphically displayed (Figure 3) Assumptions of the model were satisfied, with no evidence of autocorrelation of the residuals over time. Individual variability in response accounted for 52.0% of the total variability of the model, and the total SD was 1.8°C. Baseline values for thermal threshold ranged from 48.5°C to 52.5°C for both treatments, and the baseline value was also identified as having a significant (P < 0.001) effect in the mixed model. However, the parameter estimate for the baseline value did not differ significantly from 1°C (mean ± SD, 0.9 ± 0.2°C), suggesting that a difference in the baseline value directly corresponded to a proportional difference in the thermal threshold in this trial (unlike in previous trials). No significant effect on thermal threshold was identified for treatment, time, or their interaction. Agitation-sedation scores did not change significantly (P = 0.60) from those at baseline after administration of But-P407. None of the birds regurgitated.

Figure 3—
Figure 3—

Mean thermal thresholds in 10 orange-winged Amazon parrots before (0 hours) and at various points after SC administration of But-P407 (12.5 mg/kg; black dots with dashed line) or an equivalent volume of saline solution (white dots with solid line). See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.543

Discussion

The results of the first trial of the present study suggested that butorphanol tartrate as administered (5 mg/kg, IM) had a small and brief antinociceptive effect in orange-winged Amazon parrots. This effect was detected only at 30 minutes after administration, suggesting the analgesic effect in this species persists for at least 30 minutes but < 90 minutes. The observed effect was shorter than expected given results of a pharmacokinetic study16 involving Hispaniolan Amazon parrots, in which mean butorphanol plasma concentrations remained > 100 ng/mL for 2 to 3 hours following IM administration of a 5-mg/kg dose of butorphanol tartrate.

In formulating our hypotheses for the present study, we assumed that Hispaniolan and orange-winged Amazon parrots would have similar pharmacokinetic profiles with butorphanol administration, which we have demonstrated in our laboratory (unpublished data). In an earlier pharmacodynamic study,4 Hispaniolan Amazon parrots receiving butorphanol IM at a lower dose (2 mg/kg) had thermal thresholds similar to parrots receiving saline solution, and no nociceptive effect was noted.

The brief observed duration of analgesic effect of butorphanol when administered IM at 5 mg/kg may also have been related to our assumption that the therapeutic threshold for plasma butorphanol concentration is 100 ng/mL. This threshold value was selected on the basis of the reported pharmacodynamics of liposome-encapsulated butorphanol in Hispaniolan Amazon parrots.4 Data from the pharmacodynamics study4 indicated that the nociceptive threshold of butorphanol was close to 55 ng/mL when parrots were exposed to an electrical stimulus and 88 ng/mL when parrots were exposed to a thermal stimulus. However, the target plasma concentration for thermal nociception may not be similar in orange-winged Amazon parrots. In the pharmacokinetic study16 involving Hispaniolan Amazon parrots given butorphanol IM at a dose of 5 mg/kg, maximum plasma concentration (653.42 ng/mL) occurred at the first measured time point (15 minutes); between 30 minutes and 1.5 hours after drug administration, the mean plasma concentration as graphically displayed decreased from approximately 450 to 150 ng/mL.

Given that the pharmacokinetic profile of But-P407 (25% P407 base in both the present study and in the previous study16) in these 2 species of Amazon parrots is similar, extrapolation of the plasma butorphanol concentration at the 30-minute time point when a thermal nociceptive effect was present in orange-winged Amazon parrots would suggest that the therapeutic threshold in this species is between 150 and 450 ng/mL. This extrapolation indicates that higher plasma butorphanol concentrations would be needed to provide a clinically relevant duration of analgesia. However, in mammalian species, butorphanol is associated with a ceiling effect at high doses, and administering the drug at higher doses is not guaranteed to provide a longer nociceptive effect.1 The higher therapeutic threshold extrapolated from our data would also be surprisingly different from that for Hispaniolan Amazon parrots. Although no direct comparison is possible, it is interesting to note that therapeutic thresholds for plasma butorphanol concentration are 45 ng/mL in cats (thermal and pressure stimulus)22 and 20 to 30 ng/mL in horses (superficial and visceral pain such as induced colic).23

No significant antinociceptive effect was detected in the orange-winged Amazon parrots of the present study after SC administration of But-P407 at a dose of 12.5 mg/kg. This surprising finding did not support our initial hypothesis that this formulation would not only provide analgesia but would also provide it for a longer period than standard butorphanol tartrate solution. The previously stated explanations for the lack of a nociceptive effect of butorphanol may be equally applicable to the findings for But-P407. In the pharmacokinetic study19 involving Hispaniolan Amazon parrots and But-P407 (also 25% P407 base), the mean ± SD maximum plasma butorphanol concentration was 452.3 ± 78 ng/mL at 1.3 hours after drug administration, and the minimum effective plasma concentration for orange-winged Amazon parrots could be higher than for Hispaniolan Amazon parrots, possibly higher than 400 ng/mL.19 It is also possible that the minimum effective plasma butorphanol concentration was not achieved in orange-winged Amazon parrots. Moreover, the time points for thermal threshold testing in trials 2 and 3 were different from those in trial 1, and the differences could have allowed an effect to be missed. Nevertheless, given the results of the pharmacokinetic study19 involving Hispaniolan Amazon parrots, we expected a significant difference between butorphanol and control treatments at the 0.75- and 1.5-hour time points.

In trial 3 of the present study, saline solution was used as the control treatment rather than P407 to investigate the possibility that the P407 itself has some analgesic effect. Results indicated this was not the case. Further evidence that P407 has no analgesic effect was found in an analgesiometric study24 involving rats (paw-pressure test) in which P407 was injected SC at the level of the sciatic nerve.

We also considered the possibility that P407 might interfere with the release or metabolism of butorphanol and thus render the poloxamer inadequate as a control vehicle. This possibility was considered unlikely because in vitro work18 has shown that the poloxamer does not prevent butorphanol release, as supported by findings of the pharmacokinetic study19 involving Hispaniolan Amazon parrots. The only reported secondary effects of P407 are alterations in lipid metabolism (increases in plasma concentrations of triglycerides, cholesterol, and very low-density lipoproteins) when given at higher doses than used in our study25–27; however, these secondary effects should not affect the metabolism of butorphanol. Also, although unlikely and speculative, the possibility cannot be ruled out that P407 interferes with the mechanism of action of butorphanol, perhaps at the opioid receptors, impairing the drug's ability to exert an analgesic effect. Nevertheless, the physicochemical structure and characteristics of P407, which are comparable to those of the native extracellular matrix,28 make this possibility unlikely.

In a similar manner, an error in the actual concentration of the But-P407 formulation was also considered as an explanation for the lack of an observed antinociceptive effect because the actual concentration of butorphanol was not calculated directly with analytic methods. This possibility appears unlikely given indirect evidence that this concentration was appropriate.18,19 Indeed, trials 1 and 2 of the present study yielded similar threshold results, making the possibility of a relevant error in the actual concentration of the drug even less likely. Nevertheless, we would encourage that direct measurements be performed in future studies of this or any other formulation.

Finally, as for other studies in which no significant effects of an intervention are identified, the possibility existed of type II error in the present study. However, we have used the same analgesiometric method in other research involving orange-winged Amazon parrots and found significant differences between another opioid treatment (hydromorphone hydrochloride) and a control treatment using a similar number of birds.j

No significant change in agitation-sedation scores was observed after administration of butorphanol tartrate alone, saline solution, or P407 in the study reported here. Sedation was noted when the parrots received But-P407 in trial 2 but not in trial 3, and the difference was most likely a result of different observers assigning scores in these 2 trials. Despite the establishment and use of this agitation-sedation scoring system,21 assessment of sedation remains subjective, particularly if the effects are mild. Although minimized during the study, stimulation of the birds during testing and movement in and out of the box likely resulted in some degree of underestimation of the sedative effects. Regardless of these limitations, it is important to note that no bird had signs of heavy sedation (ie, no score < −0.5) in any of the 3 trials.

Interestingly, values for baseline thermal threshold were substantially higher in trial 3 than in trials 1 and 2. The reasons for this difference remain unknown, but we postulate that it could have been attributable to the different thermal perches used. Although the rate at which the temperature increased was the same, the actual temperature (ie, the temperature at the contact surface) was measured differently by the sensors in these 2 pieces of equipment, accounting for this offset. Other variables, such as the blinded observers, may have contributed to some of that variability as well but were unlikely to be responsible for the entire difference between observed thermal thresholds. In more recent studies, we have used the thermal perch that was used in trial 3 and have obtained similar (eg, 48°C to 50°C) baseline values consistently.

Findings of the present study suggested that butorphanol tartrate has a small antinociceptive effect in orange-winged Amazon parrots when administered IM at a dose of 5 mg/kg. The duration of this effect lasted for at least 30 minutes but < 1.5 hours. No antinociceptive effect was observed for But-P407 administered SC at a dose of 12.5 mg/kg in 2 other trials. Whether these results truly reflected a poor analgesic effect of butorphanol or a confounding effect attributable to the poloxamer base or study design remains unknown. Further research is warranted into the antinociceptive effect of butorphanol in orange-winged Amazon parrots, including multidose studies and pharmacodynamic studies with other nociception test methods. Research is also necessary to determine whether the theoretical value of combining butorphanol tartrate and P407 to extend the duration of analgesia in avian patients has in vivo merit.

Acknowledgments

This manuscript represents a portion of a thesis submitted by Dr. Laniesse to the University of Guelph as partial fulfillment of the requirements for a Doctorate of Veterinary Science degree.

Funded by grants from the OVC Pet Trust and the University of Guelph Campbell Center for the Study of Animal Welfare as well as support from the Richard M. Schubot Parrot Wellness and Welfare Program at the University of California-Davis School of Veterinary Medicine. Dr. Laniesse was supported by the Tasha Award.

The authors thank Kristy Portillo and her team for their help setting up the experimental room.

ABBREVIATIONS

But-P407

Butorphanol tartrate in 25% poloxamer 407 gel

P407

Poloxamer 407

Footnotes

a.

Roudybush low-fat maintenance pellets, Roudybush Inc, Sacramento, Calif.

b.

Torbugesic, Fort Dodge Animal Health, Fort Dodge, Iowa.

c.

Monoject, Covidien, Mansfield, Mass.

d.

BenchMixer, Benchmark, Edison, NJ.

e.

Millex GP 0.22-μm filter unit, Millipore Express polyethersulfone membrane, Merck Millipore Ltd, Cork, Ireland.

f.

Monoject 3-mL glass red-top blood-collection tube, Sherwood Medical, St Louis, Mo.

g.

R: A language and environment for statistical computing, version 3.1-121, R Foundation for Statistical Computing, Vienna, Austria.

h.

nlme: Linear and nonlinear mixed effects models, version 3.1-103, Pinheiro J, Bates D, DebRoy S, et al. Available at: CRAN.R-project.org/package=nlme. Accessed Jan 1, 2019.

i.

ordinal: Regression models for ordinal, version 2012.09-11, Christensen RHB. Available at: CRAN.R-project.org/package=ordinal. Accessed Jan 1, 2019.

j.

Sanchez-Migallon Guzman D, Douglas J, Beaufrère H, et al. Thermal antinociceptive and agitation-sedation effects after intramuscular administration of hydromorphone hydrochloride in orange-winged Amazon parrots (Amazona amazonica) (abstr). Vet Anaesth Analg 2017;44:1262.e13.

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Appendix

Agitation-sedation scoring system used for orange-winged Amazon parrots (Amazona amazonica), as modified from a system described for Hispaniolan Amazon parrots (Amazona ventralis).21

ScoreDescription
2The bird tries to get off the perch
1The bird remains on the perch but constantly looks around
0.5The bird remains on the perch and intermittently looks around
0The bird remains on the perch, is calm, and does not look around but is extremely reactive to small movements that take place in front of the test box
−0.5The bird reacts mildly to small movements in front of the test box
−1The bird reacts mildly to large movements in front of the test box
−2The bird does not react to movements in front of the test box and only reacts when the back of the test box is opened
−3The bird is only responsive when touched
−1The bird is unresponsive to any visual or tactile stimulus
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