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Evaluation of thermal antinociceptive effects and pharmacokinetics after intramuscular

administration of butorphanol tartrate to American kestrels (Falco sparverius)

David Sanchez-Migallon Guzman, LV, MS; Tracy L. Drazenovich, DVM; Butch KuKanich, DVM, PhD; Glenn H. Olsen, DVM, PhD; Neil H. Willits, PhD;

Joanne R. Paul-Murphy, DVM

Objective—To evaluate antinociceptive effects and pharmacokinetics of butorphanol tar-trate after IM administration to American kestrels (Falco sparverius).Animals—Fifteen 2- to 3-year-old American kestrels (6 males and 9 females). Procedures—Butorphanol (1, 3, and 6 mg/kg) and saline (0.9% NaCl) solution were ad-ministered IM to birds in a crossover experimental design. Agitation-sedation scores and foot withdrawal response to a thermal stimulus were determined 30 to 60 minutes before (baseline) and 0.5, 1.5, 3, and 6 hours after treatment. For the pharmacokinetic analysis, butorphanol (6 mg/kg, IM) was administered in the pectoral muscles of each of 12 birds. Results—In male kestrels, butorphanol did not significantly increase thermal thresholds for foot withdrawal, compared with results for saline solution administration. However, at 1.5 hours after administration of 6 mg of butorphanol/kg, the thermal threshold was signifi-cantly decreased, compared with the baseline value. Foot withdrawal threshold for female kestrels after butorphanol administration did not differ significantly from that after saline solution administration. However, compared with the baseline value, withdrawal threshold was significantly increased for 1 mg/kg at 0.5 and 6 hours, 3 mg/kg at 6 hours, and 6 mg/kg at 3 hours. There were no significant differences in mean sedation-agitation scores, except for males at 1.5 hours after administration of 6 mg/kg. Conclusion and Clinical Relevance—Butorphanol did not cause thermal antinociception suggestive of analgesia in American kestrels. Sex-dependent responses were identified. Further studies are needed to evaluate the analgesic effects of butorphanol in raptors. (Am J Vet Res 2014;75:11–18)

Received June 13, 2013.Accepted September 4, 2013.From the Department of Veterinary Medicine and Epidemiol-

ogy, School of Veterinary Medicine (Sanchez-Migallon Guzman, Drazenovich, Paul-Murphy), and the Department of Statistics, Col-lege of Letters and Science (Willits), University of California-Davis, Davis, CA, 95616; the Department of Anatomy and Physiology, Col-lege of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (KuKanich); and the United States Geological Survey, Patux-ent Wildlife Research Center, 12100 Beech Forest Rd, Ste 4039, Laurel, MD 20708 (Olsen).

Supported by the Morris Animal Foundation (grant No. D10ZO-305).The authors thank Dr. Bruno H. Pypendop for technical assistance.Address correspondence to Dr. Sanchez-Migallon Guzman (guzman@

ucdavis.edu)

Raptors are frequently brought to veterinarians at wildlife rehabilitation centers, zoological institu-

tions, or private clinical practices because of conditions that require analgesia. Traumatic injury is the most common reason that raptors are brought to wildlife centers, and 58.2% to 82% of these raptors are affect-ed by collisions or traumatic wounds and fractures.1–6 However, despite advances in analgesia for birds, there is limited information regarding dose response and dos-

ing interval for analgesic drugs in raptors, and veteri-narians have been compelled to extrapolate from stud-ies conducted in other species.

Opioids are a diverse group of drugs that bind revers-ibly to specific receptors in the CNS and peripheral nervous system and modify the transmission and perception of a noxious stimulus in numerous vertebrate species.7 Opioid drugs are used for their analgesic properties, acting on the µ-, κ- and δ-opioid receptors as well as the orphan opioid-like receptor.8 The action of opioid drugs on these recep-tors activates a G-protein, which leads to a reduction in the transmission of nerve impulses and inhibition of neuro- transmitter release.9 Research on the distribution, quan-tity, and functionality of each opioid receptor type in birds has been limited.10–12 Butorphanol tartrate and nalbuphine hydrochloride, a κ-opioid receptor agonist and µ-opioid receptor antagonist, are the opioid drugs currently recom-mended for the management of acute pain and to provide preemptive analgesia in psittacine birds,13–16 but their analgesic properties have not been evaluated in raptors. Fentanyl, a µ-opioid receptor agonist, administered as a continuous rate infusion in red-tailed hawks (Buteo jamai-censis) decreases isoflurane minimum anesthetic concen-

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12 AJVR, Vol 75, No. 1, January 2014

tration, which suggests that fentanyl could have analgesic properties in this species.17 Hydromorphone, a µ-opioid receptor agonist, has been investigated in American kes-trels (Falco sparverius), and it was found that it provides dose-responsive thermal antinociception, which suggests that hydromorphone would provide analgesia in this spe-cies.18 Other investigators evaluated the pharmacokinetics of butorphanol in red-tailed hawks and great-horned owls (Bubo virginianus )19 and tramadol in bald eagles (Haliaee-tus leucocephalus)20 and red-tailed hawks,21 but the an-algesic efficacy of these drugs was not investigated. The pharmacokinetics of butorphanol has also been evaluated in Hispaniolan Amazon parrots (Amazona ventralis)22 and domestic chickens (Gallus domesticus).23

Antinociceptive analgesimetry is one of the sim-plest and least invasive methods that can be used to quantify analgesic effects.24,25 The use of a thermal noxious stimulus for evaluating analgesia has been validated for several domesticated mammalian species, including cats, dogs, rabbits, and rats.25 This method has also been used in chickens,25 several species of par-rots,14–16,26,27 and American kestrels.18 To our knowledge, there have been no studies conducted to investigate the antinociceptive effects of butorphanol in raptors. The purpose of the study reported here was to determine the antinociceptive effects, sedation-agitation effects, and pharmacokinetics of butorphanol tartrate after IM administration to American kestrels. Our hypoth-esis was that butorphanol tartrate would cause signifi-cant dose-dependent increases in thermal thresholds and sedative effects in American kestrels and have a pharmacokinetic profile similar to that in other species of birds.

Materials and Methods

The study comprised 2 phases. The first phase in-volved evaluation of thermal antinociception after ad-ministration of butorphanol tartrate, and the second phase involved a pharmacokinetic analysis after butor-phanol administration.

THERMAL ANTINOCICEPTION

Animals—Fifteen 2- to 3-year-old American kes-trels (9 females and 6 males) were used in the experi-ment. Mean ± SD body weight was 108.4 ± 6.4 g. All kestrels were captive-bred birds and were healthy as determined by results of physical examinations per-formed before and during the experiment. Eleven of the 15 kestrels had been included in a study18 conducted to evaluate the thermal antinociceptive effects of hydro-morphone hydrochloride; that study was completed 3 weeks before the start of the experiments reported here.

Kestrels were maintained in three 2.5 X 2.5 X 3.2-m rooms; perches were spaced throughout each room. Kestrels were maintained on a cycle of 12 hours of light and 12 hours of darkness. They were fed frozen-thawed, medium-sized mice and provided water ad libitum. The experimental protocol was approved by the Institution-al Animal Care and Use Committee of the University of California-Davis. Because the experiments involved a species for which the effects (eg, antinociceptive ef-fects, duration of action, and interindividual variabil-

ity) of other common analgesics were not definitively known, the use of a positive control group in place of a negative control group28 was not feasible for the evalu-ation of antinociceptive effects and duration of action of butorphanol.

Experimental design—A within-subject, complete crossover experimental design was used. Each kestrel received 4 treatments, which were administered IM in the left pectoral muscle. Birds received butorphanol tartratea (1, 3, and 6 mg/kg; 10 mg/mL of solution) and saline (0.9% NaCl) solution (0.33 mL/kg; con-trol treatment). Randomization for the order of treat-ments for each kestrel was determined by an integer set generator.b There was a washout period of 14 days between subsequent treatments (ie, periods).

Antinociception testing procedures—Thermal foot withdrawal threshold was determined for all kes-trels by use of a test box equipped with a test perch. The test box was 52.1 cm high, 10.2 cm wide, and 34.3 cm deep. The perch was placed 7 cm from the front of the box and 18.4 cm from the bottom of the box. The bot-tom of the box had a V shape to discourage birds from standing on it. There were small holes on top of the box for ventilation. The test box had dark sides to inhibit a bird from viewing its surroundings or observers and a clear front that allowed observers to monitor real-time behavioral responses by use of a remote video camera. Two weeks prior to the experiment, each kestrel was allowed to acclimate to the test chamber for a full day.

The test perch was designed to deliver a thermal stimulus to the right plantar surface of a bird’s foot; thermal microchips rapidly changed the temperature of the perch.26 The thermal stimulus generated by the thermoelectric modules ranged from 29° to 55°C and resulted in a rapid increase in perch temperature (rate of temperature increase, 0.3°C/s). The cutoff temper-ature was 55°C to avoid tissue damage to the plantar surface of a bird’s foot. A bird could escape the brief noxious thermal stimulus by lifting the foot, and the foot could then be placed back on the perch within 2 or 3 seconds after the withdrawal response because the temperature of the perch decreased rapidly (rate of tem-perature decrease, 0.3°C/s).

A thermal threshold withdrawal response was de-fined as the perch temperature at the time of a foot withdrawal response. A separate baseline thermal with-drawal threshold value was determined for each period by a single measurement obtained 30 to 60 minutes before treatment administration. Time of treatment administration was designated as time 0. Thermal foot withdrawal threshold was determined by a single mea-surement at 0.5, 1.5, 3, and 6 hours after IM admin-istration of butorphanol or saline solution. All ther-mal thresholds were determined by a single observer (TLD); the observer was not aware of the treatment administered to each kestrel. The observer initiated the thermoelectric module and then observed and recorded the kestrels with the video camera.

Agitation-sedation score and adverse effects—All birds were placed in the test box 5 minutes prior to thermal testing and observed to determine their behav-ior. An agitation-sedation score was assigned by use of

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a scoring system based on a parrot agitation-sedation scoring system29 modified for kestrel behavior (Appen-dix). During the 7 hours of data collection on each test day, kestrels remained in the same test room. Between testing times, birds were placed in transport carriers (23 X 30.5 X 43 cm); each carrier contained a perch-ing brick. Thus, the observer was able to continuously monitor the birds for any adverse effects, including vomiting and diarrhea, throughout the testing period.

Statistical analysis—The difference between withdrawal temperature at any given time point after treatment administration and the baseline withdrawal threshold temperature for each bird in each period was calculated. A repeated-measures ANOVA was used, with fixed effects of dose, time, sex, period, and all associated interactions. Correlation within birds over time within a period was modeled with a spatial power structure. Analysis of residuals resulting from the fitted model revealed that they were acceptably normally dis-tributed and had no evidence of heteroscedasticity. The least squares mean of changes in withdrawal temperature was obtained from values generated by the fitted model. Pairwise comparisons of the least squares mean for the various treatments, both within each time point and over all time points, were performed with the post hoc least significant difference method. Values were considered sig-nificant at P < 0.05. The sedation-agitation scores were analyzed in the same manner. All data were analyzed with commercially available software.c

PHARMACOKINETIC ANALYSIS

Animals—Twelve 3-year-old American kestrels (6 females and 6 males) were used. Mean ± SD body weight was 114.2 ± 6.0 g. The kestrels were a subgroup of the birds used for the thermal antinociception testing and were healthy as determined on the basis of results of physical examinations performed before and during the experiments. Kestrels were maintained in the same conditions as described previously. The experimental protocol for this portion of the study was approved by the Institutional Animal Care and Use Committee of the University of California-Davis.

Experimental design—Kestrels were assigned to 3 groups (A, B, and C). Each group comprised 4 birds (2 males and 2 females). Each kestrel was manually re-strained and received 6 mg of butorphanol tartrate/kg, IM, in the left pectoral muscle. Birds were manually re-strained for collection of blood samples. Samples were collected at predetermined time points after butorpha-nol administration (group A, 5 minutes, 1 hour, and 3 hours; group B; 0.25, 1.5, and 9 hours; and group C, 0.5, 2, and 6 hours). The birds were housed in transport carriers throughout the sample collection period.

Blood samples (0.3 mL/sample) were collected from a jugular vein or medial metatarsal vein into hepa-rin-lithium microtainer tubes and placed in ice-packed containers. Within 1 hour after collection, samples were centrifuged at 3,500 X g for 6 minutes. Plasma was harvested and stored at –80ºC until analysis.

Measurement of plasma butorphanol concen-trations—Plasma concentrations of butorphanol were

determined with liquid chromatographyd and triple quadrupole mass spectrometry.e Qualifying ions moni-tored had an m/z of 328.27 for butorphanol and 337.14 for the internal standard (ie, fentanyl). Quantifying ions had an m/z of 157.20 for butorphanol and 105.3 for fentanyl. The mobile phase consisted of acetonitrile and 0.1% formic acid. The mobile phase gradient was 95% formic acid from 0 to 0.5 minutes, 95% formic acid to 65% formic acid from 0.5 to 1.5 minutes, 65% formic acid from 1.5 to 6.5 minutes, and 65% formic acid to 95% formic acid from 6.5 to 7 minutes. Separation was achieved with a C18 columnf at 40ºC.

Sample processing consisted of liquid extraction. Plasma (0.05 mL) was mixed with 100 µL of the inter-nal standard (fentanyl; 250 ng/mL in 2% ammonium hydroxide) and 1 mL of methyl tertiary-butyl ether. The solution was mixed in a vortexer for 10 seconds and then centrifuged at 10,000 X g for 5 minutes. The up-per organic layer was transferred to a culture tube and evaporated in a 40ºC water bath under an air stream for 10 minutes. Samples and standards were reconstituted with 0.2 mL of 50% methanol, mixed in a vortexer for 5 seconds, and transferred to an injection vial. Injection volume was 25 µL. Plasma standard curves were creat-ed in canine plasma and were linear from 5 to 1,000 ng/mL. Plasma standard curves were accepted if the cor-relation coefficient was ≥ 0.99 and the predicted values were within 15% of the actual values. Accuracy of the assay was determined in kestrel plasma. Accuracy was 97.3%, and the coefficient of variation was 13.7% for 3 replicates at each of 3 concentrations (5, 50, and 500 ng/mL).

Pharmacokinetic analysis—Plasma concentrations were analyzed with naïve pooling of data and a 2-com-partment open model with an absorption phase and bi-phasic elimination phase. Weighting (1/actual plasma concentration) was used.g The pharmacokinetic mod-el was chosen on the basis of visual inspection of the goodness of fit and residuals.

Results

Thermal antinociception—Baseline tempera-ture for thermal foot withdrawal ranged from 35.9° to 47.3°C. The within-bird SD for the 6-hour period after administration of saline solution ranged from 0.65° to 1.65°C. There were no significant (P = 0.815) changes in thermal threshold over time after administration of saline solution. There was no significant effect attribut-able to dose (P = 0.366), time (P = 0.082), or period (P = 0.097); however, there was a significant effect of sex (P = 0.002) and the dose-by-time interaction (P = 0.025). Because of the significant effect of sex, results were analyzed separately for males and females.

At all doses of butorphanol administered to female kestrels, the thermal foot withdrawal threshold did not change significantly (P = 0.392), compared with the val-ue for the saline solution, but withdrawal threshold was significantly increased after butorphanol administration, compared with baseline values, for 1 mg/kg at 0.5 hours (P = 0.027) and 6 hours (P = 0.011), 3 mg/kg at 6 hours (P = 0.031), and 6 mg/kg at 3 hours (P = 0.034). For all doses of butorphanol administered to male kestrels, the thermal

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14 AJVR, Vol 75, No. 1, January 2014

foot withdrawal threshold did not change significantly, compared with the threshold after administration of sa-line solution; however, compared with the baseline value, the withdrawal threshold was significantly (P = 0.008) de-creased 1.5 hours after administration of butorphanol at 6 mg/kg (Figures 1 and 2; Table 1).

We did not detect significant effects on sedation-agitation for either sex at any dose of butorphanol, ex-cept male kestrels had a significantly higher sedation-agitation score 1.5 hours after administration of butor-phanol at 6 mg/kg (Table 2).

Pharmacokinetic analysis—Plasma concentrations over time and pharmacokinetic parameters of butor-phanol tartrate administered to American kestrels were determined (Figure 2; Table 3). Plasma butorphanol concentrations were below the limit of detection (5 ng/mL) in 1 of 4 birds at 6 hours and in all 4 birds at 9 hours. Mean plasma concentrations were > 100 ng/mL for approximately 2 hours after IM administration of butorphanol.

Discussion

Butorphanol tartrate administered IM at 1, 3, and 6 mg/kg caused sex-dependent responses in kestrels, which resulted in increased thermal thresholds in fe-males and decreased thermal thresholds in males. To the authors’ knowledge, this is the first report of sex dif-ferences for opioid drugs in birds. Changes in thermal nociception in females were suggestive of mild analge-sia at specific time points, whereas the changes in males suggested mild hyperesthesia to the thermal stimulus or mild hyperalgesia as well as mild agitation at higher doses. An explanation that must be considered for the fact there was not a significant difference from results for the saline solution is a type 1 error. The significant

Butorphanol SalineSex Time solution 1 mg/kg 3 mg/kg 6 mg/kg

Male (n = 6) 0.5 −1.362 0.568 −0.098 −0.328 1.5 −0.002 −0.552 −0.258 −2.328*† 3.0 −0.222 −0.452 0.282 −0.348 6.0 −0.922 −0.652 0.122 −1.568

Female (n = 9) 0.5 0.889* 0.860* 0.105 0.134 1.5 0.145 0.260 0.583 0.390 3.0 0.189 0.693 0.194 0.812* 6.0 0.489 0.993* 0.850* 0.112

Baseline values were obtained 30 to 60 minutes before injec-tions; time of treatment administration was designated as time 0. There was a washout period of 14 days between subsequent treat-ments. The SE for males was 0.749 for saline solution, 0.798 for 1 mg/kg, 0.750 for 3 mg/kg, and 0.774 for 6 mg/kg. The SE for females was 0.384 for saline solution, 0.384 for 1 mg/kg, 0.389 for 3 mg/kg, and 0.378 for 6 mg/kg.

*Within a sex within a column, value differs significantly (P < 0.05) from the baseline value. †Within a sex within a row, value dif-fers significantly (P < 0.05) from the value for the control treatment.

Table 1—Least squares mean changes in thermal thresholds from baseline values in 15 American kestrels (Falco sparverius) after IM administration of saline (0.9% NaCl) solution (control treatment) or butorphanol tartrate at 1, 3, and 6 mg/kg.

Figure 1—Estimated mean change in thermal threshold from baseline values in 6 male (A) and 9 female (B) American kes-trels (Falco sparverius) after IM administration of saline (0.9% NaCl) solution (diamonds with solid line; control treatment) and butorphanol tartrate at 1 mg/kg (squares with dashed line), 3 mg/kg (triangles with dashed-and-dotted line), and 6 mg/kg (squares with dotted line). Baseline values were obtained 30 to 60 min-utes before injections; time of treatment administration was designated as time 0. There was a washout period of 14 days between subsequent treatments. *Value differs significantly (P < 0.05) from the baseline value for 6 mg/kg in panel A and for 1 mg/kg at 0.5 and 6 hours, 3 mg/kg at 6 hours, and 6 mg/kg at 3 hours in panel B. †Within a time point, value for 6 mg/kg differs significantly (P < 0.05) from the value for the control treatment.

Figure 2—Plasma concentrations of butorphanol after IM admin-istration of 6 mg/kg in the pectoral muscles of 12 American kes-trels (6 females [black circles] and 6 males [white circles]). The birds were assigned to 3 groups (A, B, and C); each group com-prised 4 birds (2 males and 2 females). Blood samples were col-lected at predetermined times after drug administration (group A, 5 minutes, 1 hour, and 3 hours; group B, 0.25, 1.5, and 9 hours; and group C, 0.5, 2, and 6 hours).

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time-by-dose interaction and small magnitude of the change in thermal thresholds preclude broad clinical application. On the basis of results of the present study, butorphanol tartrate at the doses used may not provide effective analgesia in American kestrels. Alternatively, the method used may not have been sensitive enough to assess the effects of butorphanol.

The thermal nociceptive response has been used to study several opioids and doses in psittacine spe-cies14,15,23,26,29–33 and raptors.18 Nociception is caused by a thermal stimulus activating thermal receptors. Ther-mal receptors in birds are associated with afferent Aδ and C fibers, which transmit nociceptive information to different areas of the midbrain and forebrain by ascend-ing spinal pathways.34–36 The use of a thermal stimu-lus to affect the natural perching behavior of raptors is a noninvasive method for evaluation of nociceptive thresholds and modulation of nociceptive thresholds by analgesic treatments. The within-kestrel SD over the

6-hour period after the administration of saline solution ranged from 0.65° to 1.65°C. This was similar to results of another study18 in American kestrels and substan-tially smaller than the within-parrot SD over a 6-hour period after IM administration of saline solution.16 The sample size (n = 15) was larger than that in a similar study18 in American kestrels,0 which was conducted to evaluate the effectiveness of hydromorphone, similar to or larger than that in other nociception studies with psittacine species performed by our research group,37 and larger than that in several studies in dogs38,39 and cats.40–43 Lack of a significant period effect and lack of significant changes in thermal threshold during the 6 hours after the administration of saline solution (ex-cept in the females at 0.5 hours) validated the use of the control group and baseline values for the analysis.

Results of the present study differ from those in other studies13–15,44–46 with psittacine species, in which butorphanol and nalbuphine were recommended as the opioid drugs for the management of acute pain. The bu-torphanol doses used in the present study were based on doses recommended for psittacine species.14,15,22 In the experience of one of the authors (JRPM), electri-cal stimulus thresholds in Hispaniolan Amazon parrots were significantly increased throughout the 90-minute study period after IM administration of 3 mg of butor-phanol/kg. However, it is unclear whether the results of thermal and electrical stimuli can be directly compared. All methods for assessment of nociception have their limitations. Thermal stimuli also activate temperature-sensitive neurons; thus, the response may be to warmth rather than pain, and we would not expect this to be affected by an opioid. Electrical stimuli activate all neu-rons (touch, motor, temperature, autonomic, and pain).

Other factors can also affect results of studies. The distribution of nociceptors on the feet may differ among species of birds; thus, different responses may be obtained to a stimulus. The sensitivity of the noci-ceptors may also differ, which could result in differ-ent responses independent of the drug administered. Differences in the thickness of the sole of a bird’s foot could also influence the results. The difference in re-sponse to butorphanol for psittacine birds, compared with the response for kestrels, might be attributable to a variety of factors such as species differences in drug metabolism, protein binding, or peripheral thermal re-ceptors or dissimilarities in the quantity, distribution, or function of κ- and µ-opioid receptors in the limbic system (subcortical and spinal levels).

Individual variation in the antinociceptive effects of opioids has been detected in many species and appears to be multifactorial, including genotype, sex, age, type of noxious stimulus, receptor, and relative efficacy of the agent.47,48 In particular, there are marked differences between sexes in rodents and nonhuman primates after injection of low-efficacy opioids such as butorphanol, with the drug being more potent and effective in males than females.49 Despite the relatively small number of kestrels of each sex in the present study, there was a significant difference between males and females in the response to the thermal stimulus and treatments. Males generally had a decrease in the thermal threshold for both the control and butorphanol treatments, with

Butorphanol SalineSex Time solution 1 mg/kg 3 mg/kg 6 mg/kg

Male (n = 6) 0.5 0.987 1.171 0.994 1.010 1.5 0.987 1.088 0.994 1.224† 3.0 0.987 1.088 1.161 1.010 6.0 0.987 1.005 1.077 1.010Female (n = 9) 0.5 1.060 1.011 1.033 1.063 1.5 0.782 0.844 1.089 0.952 3.0 1.060 0.788 0.978 1.063 6.0 1.004 1.066 0.978 1.174

For males, the SE was 0.089 for saline solution, 0.083 for 1 mg/kg, 0.082 for 3 mg/kg, and 0.076 for 6 mg/kg. For females, the SE was 0.117 for saline solution, 0.117 for 1 mg/kg, 0.119 for 3 mg/kg, and 0.116 for 6 mg/kg.

See Table 1 for remainder of key.

Table 2—Least squares mean changes in sedation-agitation scores in 15 American kestrels after IM administration of saline solution (control treatment) or butorphanol tartrate at 1, 3, and 6 mg/kg.

Parameter Value

A (ng/mL) 3,460B (ng/mL) 155K01 (/h) 3.27α (/h) 2.46β (/h) 0.47AUC0–∞ (h•ng/mL) 631α t1/2 (h) 0.28β t1/2 (h) 1.48Volcc/F (L/kg) 6.06CL/F (mL/min/kg) 159Volpc/F (L/kg) 5.18Tmax (h) 0.38Cmax (ng/mL) 445

A = α intercept. α = Rate constant for the distribution phase. α t1/2 = Half-life of the distribution phase. AUC0–∞ = Area under the concentration-time curve extrapolated to infinity. B = β intercept. β = Rate constant for the elimination phase. β t1/2 = Half-life of the terminal phase. CL/F = Clearance per fraction of the dose absorbed. Cmax = Maximum plasma concentration. K01 = Absorption rate constant. Tmax = Time to maximum plasma concentration. Volcc/F = Volume of distribu-tion of the central compartment per fraction of the dose absorbed. Volpc/F = Volume of distribution of the peripheral compartment per

Table 3—Pharmacokinetic parameters derived from naïve pool-ing of data in a 2-compartment pharmacokinetic analysis after IM administration of butorphanol tartrate at 6 mg/kg to 12 American kestrels.

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hyperalgesia in the males for 6 mg/kg at 1.5 hours, where-as there was an increase in the thermal threshold in the females. The difference between males and females for a low-efficacy opioid such as butorphanol suggests a fun-damental difference in signal transduction mechanisms or receptor density between males and females.49

The pharmacokinetic method used in the present study was a naïve pooling of drug concentrations from multiple birds, which was necessitated because the small size of the birds precluded blood collection from each kes-trel at all time points. A limitation of this method is that it does not allow measurement of variation in calculated pharmacokinetic parameters because pooled concentra-tions are analyzed as though they have been derived from a single bird.50 However, strong interindividual variation in the pharmacokinetics and duration of the effects of bu-torphanol has been reported in other species.51,52

Butorphanol tartrate administered IM at 6 mg/kg rapidly resulted in high plasma concentrations (maxi-mum concentration, 444.68 ng/mL at 0.38 hours) in American kestrels. Plasma concentrations of butorpha-nol after IM administration decreased rapidly over time. The termination half-life for butorphanol in American kestrels that we calculated for the present study (1.48 hours) was comparable to that reported for red-tailed hawks and great horned owls19 injected IM with a dose of 0.5 mg/kg (0.93 and 1.78 hours, respectively) or chickens23 injected IV with a dose of 2 mg/kg (1.19 hours), but it was longer than that in Hispaniolan Ama-zon parrots22 injected IM with a dose of 5 mg/kg (0.51 hours). Clearance per fraction of the dose absorbed was also higher for the present study (158.59 mL/min/kg) than that reported after IV administration in red-tailed hawks and great horned owls19 (58.03 and 26.1 mL/min/kg, respectively). The higher clearance per fraction of the dose absorbed in American kestrels may have been attributable to greater metabolism, which occurs by hepatic hydroxylation in other species,53 or to a lower bioavailability after IM administration. It may be more likely to truly be a slower clearance because the terminal half-life was much longer in the kestrels, but evaluation after IV administration is needed to confirm the true clearance.

In kestrels, IM administration of 6 mg of butor-phanol/kg resulted in plasma concentrations ≥ 100 ng/mL for 2 hours in 3 of 4 birds. Plasma concentrations of butorphanol and metabolites > 80 ng/mL reportedly provide analgesia in Hispaniolan Amazon parrots.15 However, a direct relationship between plasma drug concentrations and antinociceptive effects could not be inferred from the study15 of Hispaniolan Amazon par-rots because the assay measured concentrations of bu-torphanol and its metabolites together, and some of the metabolites may not be effective analgesics. The plasma concentrations in the present study would be sufficient to provide analgesia in other species, so caution should be used when plasma concentration is used alone to predict analgesia. Antinociceptive effects are likely de-termined by the concentration at the receptor, which lags behind the plasma concentration.54 Furthermore, the affinity of a drug for the receptors, the quantity and distribution of the receptors, and the interactions with other receptors might also determine the effect.

Adverse effects observed in the present study were hyperesthesia or hyperalgesia and agitation. Opioid-induced hyperesthesia or hyperalgesia was seen in male kestrels (6 mg/kg at 1.5 hours). Hyperesthesia is an increased sensitivity to sensa-tion. Therefore, the response may not necessarily in-dicate an increase in pain sensitivity. Opioid-induced hyperalgesia is defined as a state of nociceptive sen-sitization caused by exposure to opioids.55 The con-dition is characterized by a paradoxical response whereby a patient receiving opioids for the treatment of pain could actually become more sensitive to cer-tain painful stimuli.55 Opioid-induced hyperalgesia is associated with upregulation of the compensatory pronociceptive pathways.56 Acute receptor desensi-tization via uncoupling of the receptor from G-pro-teins, upregulation of the cAMP pathway, activation of the N-methyl-D-aspartate receptor system, and de-scending facilitation have been proposed as potential mechanisms underlying opioid-induced hyperalge-sia.57 Opioid-induced hyperalgesia occurs in several distinct settings and is characterized on the basis of the opioid dose administered and the pattern of ad-ministration. Most of the studies on opioid-induced hyperalgesia have been performed during ongoing (maintenance) therapy or withdrawal from opioids, but another study56 has revealed opioid-induced hyperalgesia during administration of extremely high or extremely low doses of opioids.56 Factors determining the net effect after administration of κ-opioid receptor agonists are complex and incom-pletely understood, and they likely include the site of drug administration and the nociceptive method used for testing. Contrary to the biphasic, analgesic-hyperal-gesic temporal response observed after administration of µ-opioid receptor agonists, the response elicited by κ-opioid receptor agonists appears to be monophasic (ie, it is either analgesic or hyperalgesic).56

Sedation was not observed in individual birds in the present study, but the male kestrels appeared agi-tated at 1.5 hours after administration of 6 mg of butor-phanol/kg. The sedation-agitation scoring in the study involved evaluation of the birds after they were placed in the perching box and may not have fully reflected the sedative effects of this drug in another setting.

The cardiorespiratory effects of butorphanol were not evaluated in the study reported here. However, in red-tailed hawks and great horned owls administered 0.5 mg/kg, IV and IM, the decreases in heart rate and respiratory rate were not considered clinically rel-evant.19 In that study,19 only minor sedative effects of short duration were detected in some birds. Butorpha-nol at a dose of 2 mg/kg administered IM did not cause significant changes in anesthetic and cardiopulmonary variables in Hispaniolan Amazon parrots anesthetized with sevoflurane.58 There were no other adverse effects detected during that study. Results of the present study supported the contention that butorphanol appears to be safe for use in American kestrels, but further stud-ies to evaluate the antinociceptive, sedation-agitation, cardiorespiratory, and thermoregulatory effects of bu-torphanol in American kestrels and other raptor species are needed.

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Butorphanol tartrate administered IM at 1, 3, and 6 mg/kg did not cause significant increases in thermal thresholds or sedative effects in American kestrels. Instead, it caused hyperesthesia or hyperalgesia and agitation in males receiving 6 mg/kg. These findings suggested that butorphanol may not provide effective analgesia in American kestrels. Additional studies with other types of stimulation, formulations, doses, routes of administration, and testing times are needed to fully evaluate the antinociceptive and adverse effects of bu-torphanol in American kestrels and other species of birds and their relevance for clinical application.

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Score Description

+3 Bird does not stay on the perch and constantly flies off. +2 Bird intermittently flies off the perch but returns to the perch on its own. +1 Bird stays on the perch but constantly looks around. 0 Bird stays on the perch, is calm, and does not look around but is reactive to movement in front of the test box. –1 Bird stays on the perch, is calm, and has only sluggish response to movement in front of the test box. –2 Bird does not react to movement in front of the test box and only reacts if the back of the box is opened and a hand is inserted into the box. –3 Bird is only responsive when touched. –4 Bird is nonresponsive to a visual or tactile stimulus.

Adapted from Geelen S, Sanchez-Migallon Guzman D, Souza MJ, et al. Antinociceptive effects of tramadol hydrochloride after intravenous administration to Hispaniolan Amazon parrots (Amazona ventralis). Am J Vet Res 2013;74:201–206. Reprinted with permission.

Appendix

Agitation-sedation scores used to assess behavioral effects of butorphanol tartrate administered to American kestrels (Falco sparverius).

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