Buprenorphine is a partial μ-opioid receptor agonist derived from thebaine and has been used in cats,1,2 dogs,3,4 and foals5 for antinociception because it provides postoperative analgesia for 6 to 8 hours. Buprenorphine is a potent analgesic despite its biochemical role as a partial agonist of the μ-opioid receptor.6 Buprenorphine is approved by the FDA only for use in humans, except for a long-acting (24-hour) buprenorphine formulation approved for use in cats. Despite this fact, pharmacokinetic and pharmacodynamic studies of buprenorphine have been conducted for many species, including dogs,7 cats,8 horses,9,10 mice,11 sheep,12 goats,13 and humans,14 which has revealed slow but long-lasting biphasic receptor binding. Adverse effects, including respiratory depression, bradycardia, and sedation, are consistent with those of other opioids. Buprenorphine can have excitatory effects when administered to horses at doses ≥ 0.005 mg/kg.10
An SR formulation of buprenorphine has been introduced for use in veterinary medicine and provides antinociception for up to 72 hours in rats with experimentally induced pain15,16 and in cats after ovariohysterectomy.17 For this SR formulation, buprenorphine is slowly released from a liquid polymer matrix, which provides effective long-lasting analgesia while minimizing the frequency of stress-inducing drug administration.16,18
Limited information is available regarding the use of buprenorphine in camelids, a species in which NSAIDs are primarily used for pain management. However, there are circumstances in which an alternative to NSAID usage could be advantageous (eg, patients with evidence of renal dysfunction, suspected ulceration of the third stomach compartment, or other forms of gastrointestinal tract illness). An SR formulation of buprenorphine could provide analgesia for alpacas when used on an outpatient basis (which would be especially useful for owners who cannot perform IV or SC injections and cannot give oral medications to their animals because of the temperament of the alpacas) or perhaps for an alpaca recovering after oral surgery. An abstracta that provided a pharmacokinetic analysis after administration of low doses of buprenorphine (0.005 mg/kg, IV and IM) to alpacas revealed good bioavailability and detectable plasma concentrations consistent with therapeutic ranges in other species.8,12,19 The purpose of the study reported here was to determine the pharmacokinetics after IV and SC administration of buprenorphine and SC administration of SR buprenorphine and determine sedative and antinociceptive effects in adult alpacas. It was hypothesized that the standard formulation of buprenorphine administered IV and SC and the SR formulation of buprenorphine administered SC would result in analgesia with minimal sedation in alpacas. It was also hypothesized that analgesia resulting from administration of SR buprenorphine would be of longer duration than that provided by administration of the standard formulation of buprenorphine.
Materials and Methods
Alpacas
Six alpacas (3 males and 3 females; mean ± SD age, 5.3 ± 4.2 years; mean body weight, 62.7 ± 14.5 kg) were included in the study. Prior to inclusion in the study, a physical examination, CBC, serum biochemical analysis, and fecal analysis were performed to ensure the alpacas were healthy. All hematologic and biochemical values were within reference ranges for camelids used by the Clinical Pathology laboratory at The Ohio State University College of Veterinary Medicine. Fecal samples had negative results when tested for parasites. Alpacas were housed in stalls in single-sex groups in The Ohio State University Veterinary Medical Center. Alpacas were provided a diet of hay; water was available ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of The Ohio State University.
Experimental design
Alpacas were sedated by administration of xylazine hydrochlorideb (0.6 mg/kg, IM), and catheters were aseptically placed in both jugular veins. Fiber over the right and left jugular veins was clipped, and the skin was aseptically prepared. Lidocaine hydrochloride was injected SC at each catheter insertion site (20 mg/site) to desensitize the skin. For male alpacas, a stab incision with a No. 15 blade was made through the desensitized skin over each jugular vein. For all alpacas, an 18-gauge, 5.0-cm catheterc was placed in the right jugular vein for buprenorphine administration, and a 16-gauge, 8.2-cm catheterd was placed in the left jugular vein for collection of venous blood samples that were used to determine plasma concentrations of buprenorphine and buprenorphine metabolites. The catheters were filled with heparinized saline (0.9% NaCl) solution, a cap was placed on each catheter, and catheters were covered with an elastic bandage to protect them until initiation of the experiments.
A minimum of 12 hours after catheters were placed in the alpacas, buprenorphine was administered. Each alpaca received 3 treatments; there was a 14-day washout period between successive treatments. An FDA-approved formulation of buprenorphine hydrochloridee (0.02 mg/kg, IV and SC) and an FDA-indexed SR formulation of buprenorphinef (0.12 mg/kg, SC) were administered by use of a randomized crossover design. The dose of the SR formulation was based on a recommendation provided by the compounding pharmacy that manufactured the drug.20 This dose was extrapolated from the dose for dogs and cats and was calculated as the total dose a patient would receive if the drug were administered at a dosage of 0.02 mg/kg every 12 hours for 72 hours. Buprenorphine was administered IV through the catheter in the right jugular vein followed by injection of 10 mL of heparinized saline solution. Buprenorphine and buprenorphine SR were administered SC in the area over the ribs dorsal to the elbow joint.
Collection of blood samples
A 10-mL aliquot of blood was aspirated from the catheter in the left jugular vein at each blood collection point. A separate syringe then was used to collect a 7-mL blood sample for analysis, which was immediately transferred to a sodium heparin blood collection tube. Finally, the catheter was flushed with 10 mL of heparinized saline solution. The investigator collecting the blood samples was unaware of the buprenorphine formulation or route of administration for each alpaca.
Samples of venous blood (n = 21) were collected before (time 0) and 3, 6, 12, 18, 30, 45, 60, and 90 minutes and 3, 4.5, 8, 12, 24, 36, 42, 48, 54, 60, 72, and 96 hours after each drug administration. These time points were chosen to allow creation of the best pharmacokinetic profiles for evaluation. Blood samples were placed on ice and centrifuged for 10 minutes at 2,000 × g within 1 hour after collection. Plasma was harvested and frozen at −80°C until assayed.
Analysis of plasma concentrations of buprenorphine and buprenorphine metabolites
Plasma concentrations of buprenorphine and its metabolites, norbuprenorphine, buprenorphine-3-glucuronide, and norbuprenorphine-3-glucuronide, were determined by use of a liquid chromatography-triple-quadrupole mass spectrometerg operated in the positive electrospray ion mode with selective reaction monitoring. Chromatographic separation was achieved by use of a linear gradient with mobile phase A (3mM ammonium formate with 0.005% formic acid in water) and mobile phase B (3mM ammonium formate with 0.005% formic acid in 75% methanol). A C18 columnh (2.1 × 50 mm; 3.5 μm) with a C18 5-μm guard column were used. The system incorporated a column heater, which was maintained at 50°C during the analyses. The gradient used was 35% mobile phase B for 0.2 minutes, linear increase to 100% mobile phase B over a 3-minute period, hold for 1 minute, return to 35% mobile phase B over a 0.1-minute period, and reequilibrate for 1.9 minutes. Retention times for buprenorphine, norbuprenorphine, buprenorphine-glucuronide, norbuprenorphine-glucuronide, buprenorphine d4, and norbuprenorphine d3 were 3.4, 2.34, 2.37, 1.1, 3.4, and 2.34 minutes, respectively. Transitions were summarized (Appendix). Spray voltage was set at 5,000 V. Sheath gas, ion sweep, and auxiliary gas pressures were set to 40 arbitrary units, 2 arbitrary units, and 10 arbitrary units, respectively. Capillary temperature was set at 350°C, and a skimmer offset of −10 V was used.
Recovery of buprenorphine and buprenorphine metabolites from plasma was determined over the range of all calibrators used (0, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1,000 ng/mL). Quality control samples of 0.5, 1, 5, 50, and 500 ng/mL were included in each assay. Internal standards (buprenorphine d4i and norbuprenorphine d3j) were used to validate the assay and to control for sample recovery. Ratio of the peak area of analyte to buprenorphine d4 or to norbuprenorphine d3 was used to quantitate sample concentrations.
For each calibrator, 100 μL of plasma was placed into a tube, followed by 10 μL of internal standard (1,000 ng/mL) and 10 μL of 50% mobile phase B. Tubes were then mixed in a vortex device, and protein in the samples was precipitated by adding 1 mL of cold (4°C) acetonitrile. Samples were centrifuged at 20,000 × g for 10 minutes. Each supernatant was decanted into a separate borosilicate glass tube and dried under a stream of nitrogen gas. Dried samples were reconstituted in 100 μL of 15% mobile phase B. Reconstituted samples, calibrators, and quality control samples were then mixed in a vortex device and centrifuged for 10 minutes at 20,000 × g at 4°C. An aliquot (80 μL) of each sample, standard, and quality control sample was placed into individual autosampler vials for injection. Fifteen microliters of sample, calibrator, or quality control sample was injected onto the chromatograph. Samples that yielded a concentration greater than the upper limit of quantitation were diluted and reanalyzed. Analysis of buprenorphine, norbuprenorphine, and their glucuronide conjugates was validated by examining the consistency of results (within-run and between-run), correlation coefficient of the calibration curves, and extraction efficiency of the assay.
Pharmacokinetic analysis
Plasma concentration-versus-time data for each alpaca were subjected to noncompartmental analysis by use of a computer software programk and default values with data weighted as 1/C, where C is the actual plasma buprenorphine concentration. Noncompartmental analysis was conducted by use of default parameters, with several exceptions. First, samples with concentrations below the LLOQ were assigned a concentration of 0 ng/mL if before Tmax and as a missing value if after Tmax. Consecutive samples with concentrations below the LLOQ were handled in a similar manner. The maximum observed concentration after IV administration was designated as Cmax. Values for Cmax and the corresponding Tmax were obtained directly from the concentration-versus-time curves for each alpaca after SC administration.
The value for λz was estimated by use of log-linear regression of time versus the natural logarithm of the concentration. These slopes incorporated at least 3 terminal data points of plasma concentration-versus-time data. The terminal phase t1/2 was calculated as (ln 2)/λz. The AUC to the last measured time point was calculated by use of the log-linear trapezoidal rule and was extrapolated to infinity as the area from time 0 to the last measured time point plus (the concentration at the last measured time point divided by λz). Volume of distribution based on the terminal phase after IV administration was determined as IV dose/(λz•AUC0-∞), and steady-state volume of distribution after IV administration was calculated as (IV dose/AUC0-∞)•MRT from time 0 extrapolated to infinity. Total clearance of buprenorphine after IV administration was estimated as drug dose/AUC0-∞. The AUMC from time 0 extrapolated to infinity was calculated by use of the product of plasma concentration × time × time. The MRT after IV administration was estimated as MRT = AUMC/AUC. Mean absorption time was calculated as the difference between MRT for SC administration and MRT for IV administration for each alpaca.
Plasma concentration-versus-time data for SC administration were analyzed in a similar manner. Values for Cmax and Tmax were obtained directly from the individual plasma concentration-versus-time curves; λz, terminal phase t1/2, AUC at the last measured time point, AUC0-∞, AUMC from time 0 extrapolated to infinity, and MRT from time 0 extrapolated to infinity were calculated as described for IV administration. Percentage of AUC extrapolated to infinity after SC administration was determined as indicated for IV administration. Fractional absorption of buprenorphine after SC administration was calculated as the ratio of AUC after SC administration/AUC after IV administration. Pharmacokinetic parameters were tabulated for each alpaca by route of administration and reported as mean ± SD.
Pharmacodynamic measurements
Quality of sedation at each blood collection time point was scored by use of a sedation scoring system previously used in camelids.21 Heart and respiratory rates were measured at each blood collection time point. A mechanical stimulating devicel was manually applied to the abaxial surface of the left antebrachium to evaluate mechanical nociception.22 Mechanical threshold was defined as the total force applied to a limb before a positive response was detected. Application of the mechanical stimulating device was terminated when 5 kg of force was applied and no movement or response was detected. When no response was elicited, the maximum value of 5 kg of force was recorded. Thermal threshold testing involved application of a heat-generating sourcem to the skin of the flank. The device consisted of a halogen bulb (5 V; 50 W) housed in a metal cylinder (height, 3 cm; diameter, 5.7 cm). Heat intensity was increased in a steady linear manner (approx 2 W/s). Thermal threshold was defined as the interval from illumination of the heat lamp until purposeful movement of an alpaca. A positive response to the stimuli included turning the head toward the stimulus, moving away from the stimulus, deliberate lifting of the limb, or vocalizing. When no response was elicited, the thermal stimulus was terminated after 25 seconds.
A description of behavior was recorded during assessment of sedation. All sedation scores and behavior observations were assessed by the same observer, who was unaware of the treatment administered to each alpaca. Sedation and behavior were assessed first, followed by heart rate, respiratory rate, response to a mechanical stimulus, and, finally, response to a thermal stimulus. Blood samples were collected after assessments were performed to minimize any effects on subjective and cardiorespiratory variables.
Statistical analysis
Pharmacokinetic values were determined by noncompartmental analysis of data performed by use of a computer program.k Method inaccuracy was expressed as relative SD at each concentration, whereas method precision was the source of variation between and within groups of calibration standards determined on 4 days and by calculation of the coefficient of variation. Physiologic data (heart rate, respiratory rate, and thermal and mechanical thresholds) were normally distributed, as determined by results for a Kolmogorov-Smirnov test, and were reported as mean ± SD. A repeated-measures 1-way ANOVA was performed on the physiologic data for each treatment, with a Dunnett post hoc test. Time to onset of sedation and time to peak sedation were compared with Wilcoxon rank sum tests, and results were reported as median and range. Values of P < 0.05 were considered significant.
Results
Pharmacokinetic analysis
Correlation coefficients for the calibration curves of buprenorphine, norbuprenorphine, and buprenorphine glucuronide were 99.0%, 99.9%, and 99.7%, respectively. Selectivity of the method was indicated by injection of samples of alpaca plasma that were not spiked with analyte or internal standard but that were subjected to the same extraction method as described for standards, quality control samples, and alpaca plasma (unknown) samples (Figure 1). The major peak for the blank samples was smaller and associated with later elution than were the peaks for the analytes or internal standards. One sample obtained from an alpaca 45 minutes after IV injection of buprenorphine was compared with a low standard (0.03 ng/mL). This comparison revealed a peak elution, with fragments that had the same m/z ratio as the standards. A mixture of each analyte and internal standard were injected into the mass spectrometer, and individual peaks were overlaid into a single chromatogram (Figure 2). Because the internal standards were deuterated (+3 or +4), their retention times were similar to those for the calibrators.
Chromatographs of samples of blank alpaca plasma (A), alpaca plasma spiked with buprenorphine (0.2 ng/mL; B), and alpaca plasma obtained from an alpaca 45 minutes after IV injection of buprenorphine (0.02 mg/kg; C). Time 0 = Time of injection of the sample into the mass spectrometer.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Chromatographs of samples of blank alpaca plasma (A), alpaca plasma spiked with buprenorphine (0.2 ng/mL; B), and alpaca plasma obtained from an alpaca 45 minutes after IV injection of buprenorphine (0.02 mg/kg; C). Time 0 = Time of injection of the sample into the mass spectrometer.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Chromatographs of samples of blank alpaca plasma (A), alpaca plasma spiked with buprenorphine (0.2 ng/mL; B), and alpaca plasma obtained from an alpaca 45 minutes after IV injection of buprenorphine (0.02 mg/kg; C). Time 0 = Time of injection of the sample into the mass spectrometer.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Chromatograph depicting the elution profile for buprenorphine and buprenorphine metabolites. A mixture of each analyte and internal standard was injected into the mass spectrometer, and individual peaks were overlaid into a single chromatogram. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Chromatograph depicting the elution profile for buprenorphine and buprenorphine metabolites. A mixture of each analyte and internal standard was injected into the mass spectrometer, and individual peaks were overlaid into a single chromatogram. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Chromatograph depicting the elution profile for buprenorphine and buprenorphine metabolites. A mixture of each analyte and internal standard was injected into the mass spectrometer, and individual peaks were overlaid into a single chromatogram. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Mean ± SD recovery of buprenorphine, norbuprenorphine, and buprenorphine glucuronide were 97.0 ± 8.0%, 98.5 ± 3.3%, and 98.8 ± 4.5%, respectively, over the range of concentrations tested. Within-run accuracy of the method over the range of concentrations tested (0.5, 1, 5, 50, and 500 ng/mL; n = 6 runs) was 97.0 ± 8.5% for buprenorphine. Mean within-run assay precision for buprenorphine was 6.5 ± 3.8%. Within-run accuracy and precision of the method for norbuprenorphine over the range of concentrations tested (1, 5, 50, and 500 ng/mL; n = 6 runs) were 98.5 ± 3.3% and 12.2 ± 4.7%, respectively. Within-run accuracy and precision for buprenorphine glucuronide over the range of concentrations tested (0.2, 0.5, 1, 5, 50, and 500 ng/mL; n = 6 runs) were 98.8 ± 4.4% and 9.2 ± 1.3%, respectively. Between-run accuracy and precision for buprenorphine were 92.0 ± 2.8% and 14.1 ± 9.6%, respectively, and for norbuprenorphine were 85.4 ± 5.5% and 10.8 ± 5.1%, respectively. Limits of quantitation for each analyte were 0.5 ng/mL for buprenorphine, 1 ng/mL for norbuprenorphine, and 0.2 ng/mL for buprenorphine glucuronide. Plasma concentration-versus-time profiles were plotted for both IV and SC administration of buprenorphine (Figure 3).
Mean ± SD plasma concentration of buprenorphine after IV (A) and SC (B) administration of buprenorphine (0.02 mg/kg) to each of 6 healthy alpacas. Time 0 = Immediately before buprenorphine administration. There was a 14-day washout period between subsequent treatments. Notice that the axis scales differ between panels.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Mean ± SD plasma concentration of buprenorphine after IV (A) and SC (B) administration of buprenorphine (0.02 mg/kg) to each of 6 healthy alpacas. Time 0 = Immediately before buprenorphine administration. There was a 14-day washout period between subsequent treatments. Notice that the axis scales differ between panels.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Mean ± SD plasma concentration of buprenorphine after IV (A) and SC (B) administration of buprenorphine (0.02 mg/kg) to each of 6 healthy alpacas. Time 0 = Immediately before buprenorphine administration. There was a 14-day washout period between subsequent treatments. Notice that the axis scales differ between panels.
Citation: American Journal of Veterinary Research 78, 3; 10.2460/ajvr.78.3.321
Plasma concentrations of buprenorphine declined rapidly after administration. After IV administration, the Cmax of buprenorphine was 11.60 ± 4.50 ng/mL, whereas mean Cmax after SC administration was 1.95 ± 0.80 ng/mL (Table 1). After SC administration, Tmax was 0.60 ± 0.17 hours. Harmonic mean t1/2 of the elimination phase after IV and SC administration was 1 hour (range, 0.7 to 1.4 hours) and 0.98 hours (range, 0.33 to 8.20 hours), respectively. The MRT for buprenorphine after SC administration was approximately 2.3 hours longer than that after IV administration, which reflected slower absorption. Mean total clearance of buprenorphine from alpaca plasma after IV administration was 3.00 ± 0.33 L/h/kg. Volume of distribution at steady state and volume of distribution based on the terminal phase were 3.8 ± 1.0 L/kg and 4.5 ± 0.8 L/kg, respectively. Buprenorphine glucuronide was the only metabolite detected, which was evident at approximately 6 minutes after IV administration.
Mean ± SD values for pharmacokinetic parameters after IV and SC administration* of buprenorphine (0.02 mg/kg) to each of 6 healthy alpacas.
| Parameter | IV | SC |
|---|---|---|
| λz (h−1) | 0.69 ± 0.17 | 0.70 ± 0.72 |
| Terminal phase t1/2 (h)† | 1.00 (0.70 to 1.40) | 0.98 (0.33 to 8.20) |
| Tmax (h) | — | 0.60 ± 0.17 |
| C0 (ng/mL) | 11.60 ± 4.50 | — |
| Cmax (ng/mL) | — | 1.95 ± 0.80 |
| AUC0-∞ (h•ng/mL) | 6.7 ± 0.7 | 4.5 ± 2.7 |
| AUCextrap (%) | 5.7 ± 1.0 | 17.0 ± 7.6 |
| AUMC0–∞ (h•h•ng/mL) | 8.5 ± 2.5 | 25.7 ± 38.0 |
| MRT (h) | 1.3 ± 0.3 | 3.6 ± 3.7 |
| MAT (h) | — | 2.6 ± 3.4 |
| Bioavailability (%) | — | 64 ± 28 |
| Vdss (L/kg) | 3.8 ± 1.0 | — |
| Vdt (L/kg) | 4.5 ± 0.8 | — |
| Clearance (L/h/kg) | 3.00 ± 0.33 | — |
There was a 14-day washout period between subsequent treatments.
Reported as harmonic mean (range).
AUCextrap = Percentage of AUC0–∞ extrapolated. AUMC0–∞ = The AUMC from time 0 extrapolated to infinity. C0 = Plasma concentration extrapolated to time 0. MAT = Mean absorption time. Vdss = Volume of distribution at steady state. Vdt = Volume of distribution during the terminal phase. — = Not applicable.
Plasma concentrations of buprenorphine were detectable in 2 of 6 alpacas at 8 hours after SC administration. Buprenorphine was detectable in the plasma sample of 1 alpaca at 36 hours after SC administration. Mean ± SD systemic bioavailability of buprenorphine after SC administration to alpacas was 64 ± 28%.
Plasma concentrations after SC administration of SR buprenorphine reached a mean ± SD peak of 0.59 ± 0.24 ng/mL in 2 alpacas at 45 minutes. At all other time points after SC administration of the SR formulation for all other alpacas, plasma concentrations of buprenorphine were below the LLOQ. A pharmacokinetic profile could not be determined for SC administration of SR buprenorphine.
Pharmacodynamic analysis
Heart rate and respiratory rate did not change over time with route of administration and were not different among treatments (Table 2). No significant differences were found among treatments for peak sedation score or time to peak sedation. Median peak sedation score after IV administration of buprenorphine (2; range, 0 to 4) was not significantly higher than after SC administration of buprenorphine (1; range, 0 to 2) or SC administration of SR buprenorphine (1; range, 0 to 2). Median time to onset of sedation was 3 minutes (range, 3 to 30 minutes) after IV administration of buprenorphine, 6 minutes (range, 3 to 12 minutes) after SC administration of buprenorphine, and 12 minutes (range, 6 to 18 minutes) after SC administration of SR buprenorphine. One alpaca did not have signs of sedation at any time after SC administration of SR buprenorphine. Time to peak sedation was 15 minutes (range, 3 to 90 minutes) after IV administration of buprenorphine, 6 minutes (range, 3 to 90 minutes) after SC administration of buprenorphine, and 30 minutes (range, 12 to 90 minutes) after SC administration of SR buprenorphine. There was no significant difference in time to onset of sedation or time to peak sedation among treatments.
Mean ± SD values for cardiopulmonary variables in 6 healthy alpacas after administration* of buprenorphine (0.02 mg/kg, IV and SC) or SR buprenorphine (0.12 mg/kg, SC).
| Heart rate (beats/min) | Respiratory rate (breaths/min) | |||||
|---|---|---|---|---|---|---|
| Time | IV | SC | SR SC | IV | SC | SR SC |
| 0 min | 51 ± 7 | 53 ± 6 | 57 ± 8 | 22 ± 9 | 33 ± 28 | 34 ± 7 |
| 3 min | 45 ± 7 | 52 ± 8 | 58 ± 9 | 23 ± 6 | 35 ± 18 | 35 ± 6 |
| 6 min | 48 ± 12 | 53 ± 7 | 52 ± 8 | 22 ± 6 | 39 ± 20 | 37 ± 13 |
| 12 min | 51 ± 13 | 54 ± 7 | 48 ± 9 | 24 ± 4 | 36 ± 24 | 35 ± 14 |
| 18 min | 42 ± 4 | 49 ± 5 | 47 ± 7 | 19 ± 3 | 31 ± 18 | 32 ± 12 |
| 30 min | 45 ± 10 | 51 ± 2 | 49 ± 12 | 23 ± 7 | 33 ± 18 | 29 ± 15 |
| 45 min | 52 ± 8 | 47 ± 5 | 46 ± 8 | 24 ± 5 | 30 ± 14 | 26 ± 7 |
| 1 h | 49 ± 4 | 51 ± 8 | 47 ± 7 | 22 ± 7 | 35 ± 22 | 21 ± 5 |
| 1.5 h | 51 ± 10 | 51 ± 8 | 48 ± 5 | 24 ± 5 | 31 ± 18 | 21 ± 5 |
| 3 h | 52 ± 10 | 47 ± 5 | 49 ± 10 | 22 ± 3 | 24 ± 9 | 23 ± 7 |
| 4.5 h | 51 ± 8 | 52 ± 8 | 53 ± 12 | 21 ± 6 | 29 ± 18 | 22 ± 7 |
| 8 h | 52 ± 13 | 54 ± 9 | 47 ± 10 | 20 ± 7 | 25 ± 13 | 18 ± 4 |
| 12 h | 49 ± 8 | 49 ± 6 | 47 ± 7 | 19 ± 6 | 24 ± 14 | 19 ± 6 |
| 24 h | 52 ± 11 | 49 ± 7 | 51 ± 10 | 21 ± 9 | 23 ± 12 | 21 ± 3 |
| 36 h | 48 ± 9 | 50 ± 7 | 53 ± 10 | 19 ± 7 | 23 ± 15 | 21 ± 3 |
| 42 h | 55 ± 7 | 51 ± 8 | 50 ± 7 | 19 ± 7 | 21 ± 10 | 17 ± 3 |
| 48 h | 57 ± 13 | 51 ± 8 | 52 ± 12 | 21 ± 8 | 25 ± 13 | 17 ± 5 |
| 54 h | 56 ± 4 | 51 ± 7 | 50 ± 12 | 20 ± 5 | 23 ± 12 | 18 ± 3 |
| 60 h | 51 ± 5 | 48 ± 7 | 48 ± 9 | 18 ± 6 | 23 ± 11 | 15 ± 3 |
| 72 h | 49 ± 6 | 53 ± 9 | 53 ± 10 | 25 ± 18 | 27 ± 11 | 19 ± 3 |
| 96 h | 50 ± 7 | 55 ± 9 | 54 ± 8 | 21 ± 9 | 32 ± 11 | 19 ± 3 |
Time 0 = Immediately before buprenorphine administration.
See Table 1 for remainder of key.
Analysis of thermal and mechanical antinociceptive thresholds revealed no significant differences among or within treatments (Table 3). Threshold variations were large, and there were no consistent patterns over time among or within treatments.
Mean ± SD values for mechanical and thermal thresholds in 6 healthy alpacas after administration* of buprenorphine (0.02 mg/kg, IV and SC) or SR buprenorphine (0.12 mg/kg, SC).
| Mechanical threshold (kg/cm2) | Thermal threshold (minutes) | |||||
|---|---|---|---|---|---|---|
| Time | IV | SC | SR SC | IV | SC | SR SC |
| 0 min | 0.13 ± 0.13 | 0.14 ± 0.11 | 0.62 ± 1.14 | 9.6 ± 5.4 | 7.5 ± 3.7 | 13.5 ± 7.6 |
| 3 min | 0.41 ± 0.52 | 1.19 ± 1.83 | 0.24 ± 0.16 | 14.7 ± 8.0 | 15.3 ± 8.7 | 9.9 ± 7.7 |
| 6 min | 0.16 ± 0.13 | 0.11 ± 0.09 | 0.11 ± 0.10 | 14.2 ± 6.8 | 13.1 ± 4.7 | 8.5 ± 5.8 |
| 12 min | 1.82 ± 4.02 | 0.61 ± 1.28 | 0.13 ± 0.10 | 11.3 ± 7.8 | 13.8 ± 7.2 | 13.0 ± 6.5 |
| 18 min | 1.91 ± 3.98 | 0.35 ± 0.53 | 0.24 ± 0.15 | 9.4 ± 3.5 | 12.8 ± 7.2 | 10.7 ± 7.4 |
| 30 min | 2.28 ± 3.88 | 0.15 ± 0.18 | 0.20 ± 0.24 | 9.5 ± 6.1 | 7.4 ± 4.1 | 9.9 ± 5.7 |
| 45 min | 0.35 ± 0.47 | 0.23 ± 0.31 | 0.26 ± 0.22 | 11.6 ± 8.0 | 11.0 ± 5.4 | 14.3 ± 7.3 |
| 1 h | 0.56 ± 0.44 | 0.32 ± 0.50 | 0.34 ± 0.31 | 10.8 ± 7.2 | 11.6 ± 9.8 | 12.2 ± 5.9 |
| 1.5 h | 1.87 ± 3.99 | 1.25 ± 2.82 | 0.51 ± 0.40 | 12.5 ± 3.7 | 8.8 ± 4.1 | 13.2 ± 7.1 |
| 3 h | 0.46 ± 0.53 | 0.35 ± 0.51 | 0.24 ± 0.19 | 9.1 ± 4.2 | 11.2 ± 6.9 | 9.9 ± 8.2 |
| 4.5 h | 0.38 ± 0.57 | 0.52 ± 1.01 | 0.21 ± 0.16 | 10.0 ± 4.3 | 10.5 ± 6.1 | 11.1 ± 8.1 |
| 8 h | 0.47 ± 0.55 | 0.44 ± 0.42 | 0.44 ± 0.41 | 14.1 ± 6.8 | 15.2 ± 6.6 | 12.1 ± 7.6 |
| 12 h | 0.26 ± 0.27 | 1.13 ± 1.04 | 0.46 ± 0.45 | 12.2 ± 4.0 | 11.6 ± 6.1 | 8.2 ± 4.9 |
| 24 h | 0.79 ± 1.12 | 0.50 ± 0.55 | 0.21 ± 0.16 | 11.7 ± 3.2 | 10.5 ± 4.9 | 6.6 ± 1.9 |
| 36 h | 0.61 ± 0.97 | 0.36 ± 0.46 | 0.39 ± 0.46 | 12.7 ± 5.7 | 12.7 ± 7.4 | 12.3 ± 6.3 |
| 42 h | 0.66 ± 0.97 | 0.54 ± 1.11 | 0.35 ± 0.49 | 16.2 ± 7.0 | 13.7 ± 7.2 | 11.1 ± 5.6 |
| 48 h | 0.90 ± 0.95 | 0.50 ± 0.90 | 0.35 ± 0.52 | 13.8 ± 5.6 | 13.3 ± 7.5 | 10.9 ± 6.0 |
| 54 h | 0.74 ± 1.01 | 0.25 ± 0.30 | 0.26 ± 0.36 | 14.8 ± 6.8 | 12.2 ± 4.8 | 14.7 ± 8.3 |
| 60 h | 0.7 ± 0.94 | 0.68 ± 1.00 | 0.32 ± 0.37 | 10.5 ± 6.9 | 12.5 ± 5.3 | 11.5 ± 6.8 |
| 72 h | 0.60 ± 0.80 | 0.76 ± 0.91 | 0.22 ± 0.18 | 13.0 ± 9.2 | 10.6 ± 6.4 | 8.3 ± 5.4 |
| 96 h | 0.40 ± 0.78 | 0.57 ± 0.99 | 0.33 ± 0.52 | 13.2 ± 3.5 | 10.3 ± 6.1 | 11.4 ± 6.5 |
See Tables l and 2 for key.
Discussion
The Cmax for plasma buprenorphine concentrations after IV and SC administration was 11.60 ng/mL and 1.95 ng/mL, respectively. Plasma concentrations of buprenorphine were > 0.7 ng/mL for 1.30 ± 0.24 hours after IV administration and 1.30 ± 0.25 hours after SC administration. Plasma concentrations > 0.7 ng/mL are considered therapeutic in other animal species8,12 and humans19,23; however, we did not detect a significant relationship between plasma concentration and antinociception.
Mean buprenorphine bioavailability after SC administration was 64%, which is comparable with that reported for horses10 (65% after IM administration). Overall, buprenorphine in alpacas appeared to have a higher clearance and shorter t1/2 than in dogs,24,25 cats,2 or horses.10 Pharmacokinetic values after IV administration of buprenorphine to the alpacas of the study reported here were similar to those reported elsewherea and suggested that the disposition of buprenorphine in alpacas is most similar to that of sheep12 and goats.13 In the present study, only a glucuronide metabolite was detectable after IV administration. This is in contrast to metabolism in dogs25,26 and humans,27,28 in which norbuprenorphine metabolites are also frequently detected. Moderate bioavailability and subsequent rapid clearance of buprenorphine and potentially its metabolites in alpacas may have been a factor for the inability to consistently detect metabolites.
The fact that plasma concentrations were above the LLOQ in only 2 alpacas after SC administration of SR buprenorphine prevented the estimation of a pharmacokinetic profile of this drug formulation in alpacas. Possible explanations for plasma concentrations below the detectable limit were an inadequate dose and differences in absorption rates and disposition among species. At a dose of 1.2 mg/kg to rats, plasma concentrations were detectable for 72 hours after SC administration of the SR formulation,16 and when administered to dogs at 0.2 mg/kg, plasma concentrations remained above 0.6 ng/mL for approximately 5 days.29 Although inconsistent or inaccurate drug concentrations of the compounded SR formulation were a possibility, the pharmaceutical company ensured accuracy by assaying all drugs before shipment. However, this did not exclude the potential for loss of stability during shipping and transit.
Obvious sedation (sedation score, > 0) was observed in all alpacas for all treatments, except for 1 alpaca after SC administration of SR buprenorphine. Peak sedation scores were lower after SC administration of buprenorphine than after IV administration. As expected, the measured effects of buprenorphine after IV administration were more profound than those detected after SC administration of buprenorphine or SR buprenorphine.
Alpacas were obviously sedated starting at 3 minutes after IV administration of buprenorphine. All alpacas when receiving buprenorphine IV had behavioral changes in the form of vocalization, hyperactivity, or increased responsiveness to stimuli. Two alpacas became recumbent (one within 12 minutes and the other within 60 minutes). Neck-bending (dorsiflexion) was evident in 2 alpacas. Although most alpacas resumed pretreatment behavior within 24 hours after buprenorphine administration, some alpacas remained dysphoric at all time points. Others had signs of sedation, resumed pretreatment behavior, and then appeared sedated again at a later point during the study.
One alpaca became recumbent 90 minutes after SC administration of buprenorphine and developed a cardiac arrhythmia (heard during auscultation) suggestive of a respiratory sinus arrhythmia. However, an ECG was not performed to confirm this. Respiratory sinus arrhythmia can be a normal finding in healthy conscious alpacas30 but was not previously detected in any of the alpacas in the study reported here. Behavioral changes were inconsistent for this treatment. Some alpacas initially became more docile, which was followed by vocalization and wandering around the pen, whereas some alpacas became more prone to abnormal behavior, such as kicking, stomping, and biting.
Buprenorphine is highly lipophilic. It reportedly can have a delayed onset of action (relative to plasma concentrations) in cats8 and sheep.12 This hysteresis has been attributed to the long biophase equilibrium and slow receptor dissociation kinetics of buprenorphine.15,31 However, in the study reported here, IV and SC administration of buprenorphine to alpacas appeared to result in a rapid onset of action.
Mild sedation was detected in 5 of 6 alpacas after SC administration of SR buprenorphine. The alpaca that never appeared to be sedated was 1 of 2 alpacas with detectable plasma concentrations. Two alpacas had signs of mild sedation until a much later time period than was observed after either IV or SC administration of buprenorphine. One of the alpacas with a prolonged duration of sedation had detectable plasma concentrations, whereas the other alpaca with a prolonged duration of sedation had plasma concentrations below the LLOQ at all time points. This treatment resulted in the fewest behavioral changes. The alpacas appeared calmer or unaffected, with only occasional periods of vocalization and agitation. One alpaca appeared to be more affected than the others; it developed mydriasis, carried hay in its mouth but did not eat it, and wandered around the stall. It was difficult to correlate the noticeable behavioral changes after SC administration of SR buprenorphine with undetectable plasma concentrations in most of the alpacas. It is possible that alpacas are extremely sensitive to the effects of opioids, and a low plasma concentration could have been responsible for the effects seen in the alpacas of the present study.
Despite reaching a plasma concentration that should be considered adequate to provide analgesia, there were no changes in heart rate, respiratory rate, or mechanical or thermal thresholds after drug administration. The pharmocodynamic measurements may have been confounded by the fact that many alpacas became agitated and had increased locomotor activity. This response is similar to findings for horses32 and goats.13 This is in contrast to the effect of buprenorphine in cats, in which long-lasting analgesia and a euphoric state is not usually accompanied by dysphoric behavior.33
Limitations of the present study included the fact that the sedation scale used did not account for opioid-induced excitement and behavioral changes. In addition, it was difficult to accurately assess antinociception thresholds in alpacas because of their temperaments and resistance to manipulation. Although use of xylazine in alpacas has not been evaluated, its short t1/2 in other species would suggest that the xylazine administered to facilitate catheter placement should not have interfered with our assessments. At times in this study, some alpacas appeared extremely sedated and dull, but they would react instantly to mechanical or thermal stimulation. At other times, the thresholds were much higher than expected, given the apparently normal mentation of the alpacas. As such, it was difficult to differentiate between the effect of buprenorphine and the temperament of each of the alpacas. The use of buprenorphine in alpacas with clinically evident pain may yield different results for antinociceptive and behavioral influences of the drug, and further investigations are recommended.
Acknowledgments
Supported by the Alpaca Research Foundation.
The authors thank Dr. Joseph Lozier, Carl O'Brien, Amanda Cardenas, Devin Heilman, and Daniel Davidson for technical assistance.
ABBREVIATIONS
| AUC | Area under the plasma concentration-versus-time curve |
| AUC0-∞ | Area under the plasma concentration-versus-time curve from time 0 extrapolated to infinity |
| AUMC | Area under the first moment of the plasma concentration-versus-time curve |
| Cmax | Maximum observed concentration |
| λz | First-order rate constant associated with the terminal portion of the plasma concentration-versus-time curve |
| LLOQ | Lower limit of quantification |
| MRT | Mean residence time |
| SR | Sustained release |
| Tmax | Time of the maximum observed concentration |
| tl/2 | Half-life |
Footnotes
Hanselmann R, Mosley CI, Mosley CM, et al. Pharmacokinetics of buprenorphine in alpacas (Lama pacos) after intravenous and intramuscular administration (abstr), in Proceedings. 10th World Cong Vet Anaesth 2009;61.
Xylazine, Vedco Inc, St Joseph, Mo.
Surflo catheter, Terumo Medical Corp, Elkton, Md.
Angiocath, Parke, Davis & Co, Sandy, Utah.
Buprenex, Reckitt Benckiser, Hull, Yorkshire, England.
Buprenorphine sustained release, ZooPharm, Fort Collins, Colo.
TSQ Vantage triple-stage quadrupole mass spectrometer, ThermoFisher Scientific, San Jose, Calif.
Agilent Zorbax extend-C18, Agilent Technologies, Santa Clara, Calif.
Buprenorphine-d4 (Cerulliant), Sigma-Aldrich Co, St Louis, Mo.
Norbuprenorphine-d3 (Cerulliant), Sigma-Aldrich Co, St Louis, Mo.
Phoenix, WinNonlin version 5.2, Pharsight Corp, St Louis, Mo.
Wagner Force One model FDIX, Wagner Instruments, Greenwich, Conn.
Tail flick analgesiometer, Columbus Instruments International Corp, Columbus, Ohio.
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Appendix
Settings used for chromatographic analysis of analytes and internal standards by use of liquid chromatography-triple-quadrupole tandem mass spectrometry.
| Compound | ESI mode | SRM transition* | Collision energy | Tube lens |
|---|---|---|---|---|
| Buprenorphine | Positive | 468.29→414.08/396.04 | 32/28 | 120/120 |
| Norbuprenorphine | Positive | 414.21→396.10/340.00 | 23 | 125 |
| Buprenorphine glucuronide | Positive | 644.32→468.13 | 33 | 138 |
| Norbuprenorphine glucuronide | Positive | 590.30→414.10 | 29 | 140 |
| Buprenorphine d4† | Positive | 472.30→400.00 | 28 | 114 |
| Norbuprenorphine d3† | Positive | 417.12→343.00 | 23 | 125 |
Precursor m/z→product m/z.
Internal standard.
ESI = Electrospray ion. SRM = Selective reaction monitoring.
