Pharmacokinetics of Extended-Release Buprenorphine and Clinical Efficacy for Postoperative Pain Management in the Domestic Ferret (Mustela putorius furo)
Buprenorphine hydrochloride (Bup-HCl) is a common injectable opioid analgesic. In ferrets, Bup-HCl must be administered every 8 to 12 h to maintain clinical efficacy. Extended-release analgesics offer multiple advantages, including reduced handling and injection frequency, improved compliance, and increased protection from end-of-dose failure. Although efficacy of extended-release buprenorphine formulations has been demonstrated in other species, their use in the domestic ferret has not been investigated. In this study, we evaluated the pharmacokinetics of a compounded polymeric formulation of buprenorphine (Bup-ER) and a pharmaceutical-grade, FDA-indexed liposomal suspension (Bup-XR). Two doses each of Bup-ER (0.12 and 0.2 mg/kg) and Bup-XR (0.2 and 0.6 mg/kg SC) were administered to young adult female ferrets and plasma concentrations were measured between 0 and 96 h (n = 4 animals per timepoint). All doses of both drugs achieved therapeutic plasma levels by 30 min. Furthermore, high-dose Bup-XR maintained therapeutic levels for 72 h, followed by high-dose Bup-ER (less than 48 h), low-dose Bup-XR (24 h), and low-dose Bup-ER (less than 24 h). In this study, we also developed a pain scoring system and utilized this to compare analgesic efficacy between single high-dose Bup-XR (0.6 mg/kg SC) and a standard postoperative course of Bup-HCl (0.02 mg/kg SC every 10 to 12 h for 8 doses) after ovariohysterectomy. Ferrets receiving Bup-XR had significantly lower respiratory rate and posture scores in the first 24 h postoperatively than did those that received Bup-HCl and were less likely to react to palpation of the surgical incision. Of note, ferrets that received high-dose Bup-ER had a significantly higher incidence of injection site reactions than ferrets that received Bup-HCl (P = 0.0137). This study demonstrates that a single dose of Bup-XR (0.6 mg/kg SC) is a safe and effective analgesic in female ferrets, with a duration of action up to 72 h and minimal side effects, offering a refinement to analgesia in this species.Abstract
Introduction
The domestic ferret (Mustela putorius furo) is a common pet and research animal model, with an estimated 500,000 ferrets across 326,000 households in the United States2 and approximately 12,000 ferrets used for research in the United States annually.71 As both pets and research models, ferrets may undergo procedures or experience diseases that require analgesic therapy. For instance, ovariohysterectomy (OHE; spay) is a common procedure for female ferrets, as intact female ferrets that do not mate may remain in prolonged estrus with potentially severe clinical consequences, up to and including death32 Ferrets also serve as an invaluable model for inflammatory airway disease, infectious respiratory disease, and neurobehavioral studies.4,15,49,56,57 Infection with highly virulent influenza strains results in clinical illness and discomfort in ferrets,4 while neurobehavioral studies may require surgical implants.20 Researchers have the responsibility to recognize and alleviate pain in laboratory animals, as mandated by federal laws and regulations.42 Not only can pain compromise animal welfare, but it may also confound research outcomes.48,65 Accordingly, the availability of systemic analgesic options for effective pain management is important in ferrets in both research and companion animal settings.
The value of preemptive analgesia (the administration of analgesics before the painful stimulus) has been demonstrated in multiple animal species and with different classes of systemic and local anesthetics.14,27,38,54,58,78 The goals of preemptive analgesia are to improve the quality of anesthesia, reduce acute postoperative pain, prevent central sensitization of pain pathways that can lead to development of maladaptive pain syndromes, and reduce the development of chronic pain. Preemptive analgesia is, thus, the standard of care in many research and companion animal settings. Given that surgical procedures can vary in duration, an extended-release opioid that provides preemptive analgesia and remains effective into the postoperative period is desirable. Buprenorphine is an opioid commonly used to provide preemptive and postoperative analgesia in ferrets72 and has been suggested as a refinement to mitigate discomfort without altering inflammatory pathways in ferret influenza models.41 Injectable buprenorphine hydrochloride (Bup-HCl) has a duration of action between 6 and 12 h in ferrets.72,77 Extended-release buprenorphine formulations have been shown to be effective in multiple research animal species, including mice,7,26 rats,10,19 guinea pigs,80 rabbits,12 macaques,29,44 common marmosets,18 cats,8 dogs,45 swine,68 and sheep.75 Extended-release opioid formulations allow for less frequent dosing, thus promoting animal welfare by reducing the number of injections and restraint episodes, and improving handler compliance. In addition, extended-release drugs reduce fluctuations in drug concentration associated with repeated dosing, instead maintaining prolonged steady-state blood levels. However, there are no studies evaluating the duration of action or the clinical efficacy of extended-release buprenorphine formulations in ferrets. Establishment of a pharmacokinetic (PK) time to onset and duration of action are key in ensuring appropriate analgesia and avoiding end-of-dose failure (pain that occurs when an analgesic wears off before the next dose is administered).
One concern regarding the use of extended-release drugs is the prolongation of negative side effects without the ability to titrate, withdraw, or easily reverse the drug, should adverse events be observed. For instance, dose-dependent sedation has been reported following administration of Bup-HCl in ferrets, and thus titration to effect is recommended.72 Interestingly, Fitz et al.18 reported that although common marmosets are more sensitive to the effects of Bup-HCl than other primates, extended-release buprenorphine resulted in less ataxia and sedation compared with Bup-HCl, suggesting that the side effects of extended-release drugs cannot always be extrapolated from corresponding regular-release formulations. Evaluation of the safety of extended-release buprenorphine in domestic ferrets is therefore also necessary before its use can be recommended.
A compounded, polymer-encapsulated formulation of extended-release buprenorphine (Bup-ER) has been implemented at our institution in both large animals and small rodents given its documented efficacy for providing analgesia between 48 and 72 h depending on the species. Meanwhile, a newer, FDA-indexed liposomal suspension of extended-release buprenorphine (Bup-XR) has become available, with efficacy lasting up to 72 h in mice and rats.36,43,69 In this study, we evaluated the PK of 2 doses of Bup-ER and Bup-XR in female ferrets to determine a dose that sustains a therapeutic plasma concentration (TPC). We then assessed clinical efficacy of Bup-XR compared with Bup-HCl, using a pain scoring system developed in this study.
Pain recognition in animals can be challenging and varies by species.39 Parameters that can be used to assess pain can be classified as physiologic (such as heart rate, respiratory rate, temperature, and body weight), biochemical (such as cortisol or the presence of a stress leukogram), or behavioral. Changes in behavior are often the first indicators of pain in animals,24 but behavior can often be overlooked or misinterpreted, particularly when investigators are unfamiliar with normal species-specific behaviors.39 Assessing animals using an ethogram or clinical scoring system can be helpful in identifying changes in spontaneous behavior that may be correlated with stress or pain.70 In ferrets, behaviors consistent with pain include lethargy, decreased mobility, squinting, hunching, teeth grinding, and decreased appetite.39 Grimace scales are useful in identifying pain in ferrets52,73 and in other research animal species.16,34,61 The ferret grimace scale evaluates the degree of orbital tightening, nose bulging, cheek bulging, ear changes, and whisker retraction, with orbital tightening having high sensitivity and specificity when used to evaluate postoperative pain.52 Despite its utility, that scale does not incorporate behavioral assessments such as posture and mobility, vocalization, teeth grinding, or reaction to incisional palpation, although these signs are important when assessing postoperative pain. In this study, we developed a clinical scoring system for postoperative pain in ferrets, validated using the current standard of care for analgesia in ferrets at our institution: Bup-HCl at a dose of 0.02 mg/kg SC, administered every 8 to 12 h.77 We used this scoring system to examine the safety and analgesic efficacy of Bup-XR compared with Bup-HCl following OHE.
Materials and Methods
Ethical approval.
All animal work was approved by the Animal Care and Use Committee at Johns Hopkins University.
Animals and housing.
Twenty-four 9-wk-old female wild-type (sable) domestic ferrets (Marshall BioResources, North Rose, NY) were purchased as 2 cohorts (12 ferrets per cohort). Ferrets were pair-housed in accordance with federal regulations and the Guide for the Care and Use of Laboratory Animals in an AAALAC-accredited facility. The housing room was maintained at positive pressure, 30% to 70% relative humidity, 17 to 21 °C [63 to 70 °F]), and a 12-h light/12-h dark cycle. Ferrets had free access to food (Marshall premium ferret diet; Marshall BioResources) and reverse osmosis water via an automated watering system. Stainless-steel housing cages (Allentown Caging Equipment, Allentown, NJ) were spot cleaned daily and changed every other day. Environmental enrichment included one large corrugated plastic tube, 2 rubber chew toys (KONG Company, Golden, CO), and a metal bell, and ferrets were provided chicken-flavored treats (Marshall BioResources) twice weekly.
Buprenorphine formulations.
Three buprenorphine formulations were evaluated: Bup-HCl (Par Pharmaceutical, Chestnut Ridge, NY), liposomal Bup-XR (Ethiqa XR™; Fidelis Animal Health, North Brunswick, NJ), and polymer-encapsulated Bup-ER (manufactured as buprenorphine SR-LAB by ZooPharm (Laramie, WY) at the time of study (now buprenorphine ER-LAB by Wedgewood Pharmacy, Swedesboro, NJ)).
Study timeline.
The study timeline and animal numbers are shown in Figure 1A. Ferrets from each cohort underwent 2 PK studies and one clinical efficacy study. All study phases were separated by washout periods of at least 2 wk.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
Ferrets in cohort 1 (n = 12) received a single injection of low-dose Bup-XR (0.2 mg/kg SC), and a PK study was performed. Following a washout period of 2 wk, ferrets in cohort 1 then underwent OHE and received a course of Bup-HCl (0.02 mg/kg SC every 12 h) to validate the clinical scoring system. Following review of initial Bup-XR PK data and approximately 8 wk after OHE, we conducted a second PK study in 4 ferrets from cohort 1 using a single injection of high-dose Bup-XR (0.6 mg/kg SC).
Ferrets in cohort 2 (n = 12) received a single injection of low-dose Bup-ER (0.12 mg/kg SC) and a PK study was performed. Following a 2-wk washout period, ferrets received a single injection of high-dose Bup-ER (0.2 mg/kg SC) and another PK study was performed. After approximately 4 wk, ferrets in cohort 2 underwent OHE to assess clinical efficacy of extended-release buprenorphine for postoperative pain management. Ferrets in cohort 2 were initially randomly assigned to receive either a single subcutaneous injection of high-dose Bup-XR (n = 6) or high-dose Bup-ER (n = 6) for postoperative analgesia. Due to a lack of prior similar studies in ferrets, the minimum sample size of 6 was selected based on a resource equation approach to sample size calculation for repeated-measures ANOVA.1 However, due to multiple observations of injection site reactions (ISRs) in ferrets receiving Bup-ER (Figure 1B), the Bup-ER efficacy study was terminated early, and all other ferrets received Bup-XR. Ultimately, 10 ferrets received Bup-XR and 2 ferrets received Bup-ER during the clinical efficacy study. One ferret received an incomplete dose of Bup-XR and was thus withdrawn from the study prior to clinical scoring. An additional ferret that received Bup-XR developed a small incisional hernia and underwent revision surgery 3 d postoperatively, and was thus withdrawn from the pain scoring assessment; however, this individual was still observed for ISRs. One animal that received Bup-ER postoperatively required rescue analgesia, as described below.
PK studies.
The fur over the injection site (between the shoulder blades) was shaved with electric clippers to enable observation of the injection site and ensure complete and consistent dose delivery, as well as observe for development of any skin lesions at the injection site. Each ferret received a single subcutaneous injection of their assigned dose of Bup-ER or Bup-XR. At each sampling timepoint, ferrets were sedated with an intramuscular injection of ketamine (5 mg/kg), and cranial vena cava blood collection (approximately 200 µL) was performed. For all drug groups, each timepoint included blood samples from 4 ferrets, consistent with statistical literature and prior studies evaluating buprenorphine PK in other species.11,26,29,35,50 PK sampling timepoints for individual animals are outlined in Table S1. Samples from the low-dose Bup-XR group (n = 12 animals) were collected at 0.25, 0.5, 1, 4, 8, 24, 48, 72, and 96 h. Due to an unforeseen reduction in animal availability and to remain in compliance with regulations on the volume and frequency of blood sampling, samples from the high-dose Bup-XR group (n = 4 animals) were collected at 0.5, 24, 48, 72, and 96 h. Samples from both Bup-ER groups (n = 12 animals per group) were collected at 0.25, 0.5, 1, 8, 24, 48, 72, and 96 h. Baseline blood samples (n = 16 animals) were collected up to 48 h prior to drug administration for calibration of the assay, with buprenorphine concentrations of all baseline samples being below the lower limit of quantification (LLOQ). All injections and blood collections were performed by the same veterinarian.
The buprenorphine plasma concentration assay was performed at Samford University. After initial validation of a surrogate matrix against ferret plasma, buprenorphine standard curves (0.2 to 200 ng/mL) were prepared using mouse plasma. Ferret plasma samples and standards were analyzed using HPLC-MS/MS and Analyst 1.6.2 software (Applied Biosystems, Foster City, CA) as previously described.36 In the Bup-XR assays, the LLOQ was 0.05 ng/mL. In the Bup-ER assays, the LLOQ was 0.1 ng/mL.
Clinical efficacy study.
Between 12 and 16 wk of age, ferrets underwent anesthesia, performed by a veterinarian or skilled veterinary technician, and OHE, performed using a standardized surgical protocol by veterinarians with similar surgical skill level. Maropitant (1 mg/kg SC) was administered on the morning of surgery. Anesthesia was induced via intramuscular injection of ketamine (5 mg/kg), hydromorphone (0.1 mg/kg), and midazolam (0.5 mg/kg) and maintained with isoflurane delivered in 100% oxygen via an endotracheal tube, titrated to effect. Cefazolin (22 mg/kg) was administered preoperatively through a peripheral intravenous catheter. Using an aseptic technique, a 3- to 4-cm midline skin incision was made in the middle third of the abdomen between the umbilicus and pubis. A stab incision was made in the linea alba, and the incision was lengthened using forceps and a scalpel blade. The left uterine horn was located and exteriorized. The left ovary was located and a hemostat was placed on the proper ligament of the ovary. Two circumferential ligatures were placed around the ovarian pedicle using 3-0 Biosyn (Covidien, Minneapolis, MN). The pedicle was transected distal to the ligatures, inspected for appropriate hemostasis, and replaced into the abdomen. The left uterine horn was followed to the body of the uterus. The right ovary was identified and the ovarian pedicle was ligated and transected as above. The broad ligament of the uterus was bluntly dissected and the body of the uterus was exposed. Two circumferential ligatures were placed around the body of the uterus using 3-0 Biosyn (Medtronic, Minneapolis, MN) and the uterus was transected distal to the ligatures. After ensuring hemostasis, the linea alba and subcutaneous layers were closed with 3-0 Biosyn in a simple continuous pattern and the skin was closed with 4-0 Biosyn in a subcuticular pattern. The average surgical time was 45 min.
Postoperatively (approximately 1.5 h following hydromorphone) ferrets received a dose of either 1) Bup-HCl (0.02 mg/kg SC) (n = 12), 2) high-dose Bup-XR (0.6 mg/kg SC) (n = 9), or 3) high-dose Bup-ER (0.2 mg/kg SC) (n = 2). Ferrets in the Bup-HCl group received Bup-HCl every 10 to 12 h for a total of 8 doses, as a standard postoperative analgesic regimen to validate the clinical scoring system for sensitivity and interobserver agreement. All ferrets received maropitant (1 mg/kg SC) every 24 h for 2 doses postoperatively. One ferret that received high-dose Bup-ER was assessed to be painful by the facility veterinarian in the evening of postoperative day 1 and was administered 0.6 mg/kg Bup-XR SC for rescue analgesia; this animal was therefore withdrawn from clinical scoring. No additional medications were administered to any animals. Postoperative injections were administered by a veterinarian or skilled veterinary technician.
In the postoperative period, ferrets were observed twice daily for clinical evidence of pain using a scoring system (Figure 2) developed using previously described indicators of pain and distress in ferrets52,58 and other domestic animals.3,5,6,30,33,39,47,51 Scoring was performed by up to 3 observers (2 veterinarians and one veterinary technician) per scoring timepoint, with 78% (84/108) of scores having multiple observers in cohort 1 and 96% (69/72) of scores having multiple observers in cohort 2. Each observer scored all parameters except for rectal temperature, which was performed only once per timepoint per animal. Observers scored within 30 min of each other and did not discuss their scores. Baseline preoperative scoring was performed the evening prior to surgery. The first postoperative score was performed in the afternoon on the day of surgery, approximately 3.5 h after buprenorphine administration. For 3 d thereafter, clinical scoring was performed twice daily. Clinical score timing was determined according to the cohort receiving Bup-HCl, with morning scoring performed 30 min before Bup-HCl was given and afternoon scoring performed approximately 6 h later; scoring times were then kept consistent across cohorts.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
Statistical analysis.
Interobserver agreement was evaluated for 10 clinical scoring parameters: resting respiratory rate; orbital tightening; posture; vocalization; teeth grinding; incisional erythema; incisional swelling; incisional dehiscence; incisional discharge; and reaction to incisional palpation. At each data collection timepoint with more than one observer, each parameter was classified as ‘in agreement’ (if scores were the same between observers) or ‘in disagreement’ (if scores differed between observers). Interobserver variation was calculated by averaging the number of parameters in agreement across all timepoints and dividing by the total number of parameters. For timepoints with more than one observer, each animal’s average clinical score was calculated for each parameter and used for downstream analysis.
To compare the clinical efficacy of a standard postoperative analgesic regimen of Bup-HCl (n = 12) to single high-dose Bup-XR (n = 8), Prism 9.5.1 (GraphPad Software, San Diego CA) was used to run repeated measures ANOVA with Geisser–Greenhouse correction and Sidak multiple comparisons tests to evaluate the effects of opioid, time, and individual subject on scores for resting respiratory rate, orbital tightening, posture, incisional erythema, incisional swelling, and reaction to incisional palpation, as well as subcutaneous ISR. A mixed effects model was used to evaluate the effects of opioid and time on rectal temperature (°F and score). Contingency tables and Fisher exact tests were used to compare proportions of development of ISRs after opioid administration. The significance level used throughout the study was α = 0.05.
For each set of PK data, the area under the curve (AUC) was calculated, and a one-phase decay nonlinear fit model was fit to each graph to determine the half-life and elimination rate constant (K). The following equation was used to calculate the predicted peak drug concentration (C0): C = C0 × e−Kt.
Results
In the PK study, all extended-release formulations and doses evaluated reached TPC (0.5 ng/mL) by 30 min postadministration (Figure 3). Low-dose Bup-XR, low-dose Bup-ER, and high-dose Bup-ER achieved TPC by 15 min postadministration, while high-dose Bup-XR reached TPC by 30 min postadministration, the first PK timepoint measured for this group. High-dose Bup-XR maintained TPC for the longest period of time (72 h in all animals), followed by high-dose Bup-ER (less than 48 h), low-dose Bup-XR (24 h), and low-dose Bup-ER (less than 24 h).
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
The elimination constant and half-life were dose-dependent and were comparable between the 2 extended-release formulations (Table 1). For Bup-ER, the measured Cmax was similar to the calculated maximum drug concentration (C0) and was dose-dependent. For Bup-XR, Cmax was lower than C0, indicating that our sampling timepoints likely missed the true peak plasma concentration in both Bup-XR dosing groups. The calculated peak plasma concentration (C0) was higher for low-dose Bup-XR (11.7 ng/mL) than for low-dose Bup-ER (3.71 ng/mL), and higher for high-dose Bup-XR (26.16 ng/mL) than for high-dose Bup-ER (10.01 ng/mL). In both dosing categories, the AUC was higher for Bup-XR compared with Bup-ER. The AUC for low-dose Bup-XR was 2-fold higher than that of low-dose Bup-ER, and 1.6-fold higher for high-dose Bup-XR compared with high-dose Bup-ER.
| Bup-XR | Bup-ER | ||||
|---|---|---|---|---|---|
| PK parameter | Unit | 0.2 mg/kg | 0.6 mg/kg | 0.12 mg/kg | 0.2 mg/kg |
| K | 1/h | 0.1827 | 0.04745 | 0.1854 | 0.04877 |
| t1/2 | h | 3.793 | 14.61 | 3.739 | 14.21 |
| Cmax | ng/mL | 5.84 | 8.59 | 3.12 | 9.58 |
| C0 | ng/mL | 11.71 | 26.16 | 3.714 | 10.01 |
| AUC | h·ng/mL | 73.79 | 321.0 | 36.90 | 196.2 |
| LLOQ | ng/mL | 0.05 | 0.05 | 0.1 | 0.1 |
AUC, area under the concentration–time curve; Cmax, measured mean maximum concentration; C0, calculated mean maximum concentration; K, elimination rate constant; LLOQ, lower limit of quantification of the buprenorphine assay; t1/2, half-life.
During the clinical efficacy study, the average interobserver agreement across clinical scoring parameters was 86% (8.6/10) in the first cohort and 88% (8.8/10) in the second cohort. When comparing Bup-XR to Bup-HCl for the management of postoperative pain, ferrets receiving Bup-XR had significantly lower average resting respiratory rate scores (P < 0.0001) and posture scores (P < 0.0001) compared with ferrets receiving Bup-HCl (Figure 4). There was no significant effect of opioid choice on orbital tightening score (P = 0.3585) or average rectal temperature (P = 0.3794). There was no significant effect of individual subject on average scores of resting respiratory rate (P = 0.2556), orbital tightening (P = 0.0933), or posture (P = 0.5143). Average respiratory rate score (P < 0.0001), orbital tightening score (P < 0.0001), posture score (P < 0.0001), and average rectal temperature (P < 0.0001) decreased over time in all ferrets. Incisional erythema score (P = 0.2646) and incisional swelling score (P = 0.2943) were not significantly different between opioid groups, but both varied significantly with time (P < 0.0001) and scores were dependent on individual subject (P = 0.0011) (Figure 5). There was a significant overall effect of opioid choice on reaction to incisional palpation (P = 0.0021) on repeated measures ANOVA, but no significant difference between individual timepoints on multiple comparisons testing. No vocalization, teeth grinding, or incisional discharge was observed in any animals. All ferrets recovered uneventfully from all sedation and anesthetic events during the study, and no adverse effects attributable to opioid administration, such as sedation, ventilatory depression, ileus, anorexia, nausea, or vomiting, were observed in any cohort.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
ISRs were observed to develop within 24 to 72 h of administration of high-dose Bup-ER during both study phases (Figure 1B). During the PK studies, ISR occurred in 50% (6/12, cohort 2) of ferrets after high-dose Bup-ER, and in 0% (0/4, cohort 1) of ferrets after high-dose Bup-XR. Fine needle aspirate of an ISR was performed in one animal by the facility veterinarian for clinical diagnostic purposes, and cytology was consistent with a sterile abscess (Figure 6). During the clinical efficacy study, 2 ferrets with a prior history of ISR went on to receive high-dose Bup-ER and ISRs recurred in both individuals. Due to numerous observations of ISRs in animals that received Bup-ER, we elected to stop evaluating Bup-ER and all remaining animals in the clinical efficacy study received high-dose Bup-XR. Three ferrets with a prior history of ISRs received high-dose Bup-XR without ISR recurrence. Two ferrets with no prior history of ISRs did develop ISRs after high-dose Bup-XR administration (Figure 1B). All ISRs clinically resolved during the washout periods between study phases, and no discomfort on regional palpation was observed during examination in any animals.
Citation: Journal of the American Association for Laboratory Animal Science 63, 5; 10.30802/AALAS-JAALAS-24-000011
Ferrets that received high-dose Bup-ER had a significantly higher incidence of ISR (50%, 6/12, cohort 2) compared with ferrets that received Bup-HCl (0%, 0/12, cohort 1) (P = 0.0137). There was no statistically significant difference in the incidence of ISRs between ferrets that received high-dose Bup-XR (22%, 2/9, cohort 2) compared with ferrets that received Bup-HCl (0%, 0/12, cohort 1) (P = 0.0935). As expected, there was a significant effect of individual subject on ISR formation for animals during the cohort 2 Bup-XR clinical efficacy study (P < 0.0001), with ferrets that had an ISR on one day more likely to still have an ISR on subsequent days of postoperatively scoring. When considering injections of each drug dose and formulation, rather than individual animals, the rate of ISR development was 57.4% (8/14) after injections of high-dose Bup-ER, 15.1% (2/13) after injections of high-dose Bup-XR, and 0% (0/12 per formulation) after low-dose Bup-ER, low-dose Bup-XR, or Bup-HCl.
Discussion
This study demonstrates that both extended-release buprenorphine formulations provided prolonged analgesia in domestic ferrets, with high-dose Bup-XR providing the longest duration of action (72 h), followed by high-dose Bup-ER (less than 48 h). Despite the ability of high-dose Bup-ER to achieve and maintain TPC, we found that injection of high-dose Bup-ER was associated with more instances of ISR development, as discussed below. Thus, in our opinion, Bup-ER is suboptimal for use in ferrets. We also developed a pain scoring system and used it to assess the clinical efficacy of Bup-XR for postoperative pain management following OHE in comparison to a standard course of regular-release Bup-HCl. Clinical scores from animals that received high-dose Bup-XR corresponded to appropriate analgesic efficacy for 3 d postoperatively, consistent with the duration of action determined in the PK studies. Orbital tightening and incisional reaction scores were similar between the 2 treatment groups, demonstrating that high-dose Bup-XR provided adequate analgesia compared with the standard of care. In addition, ferrets receiving high-dose Bup-XR had significantly lower respiratory rate and posture scores, consistent with lower postoperative pain levels, compared with Bup-HCl, suggesting that high-dose Bup-XR may even provide improved analgesia over the standard of care. Overall, a single dose of Bup-XR (0.6 mg/kg SC) is a safe and effective analgesic option in young, otherwise healthy female ferrets and provides advantages over repeated dosing of Bup-HCl, including improved analgesia, a duration of action up to 72 h, and few side effects. These findings also align with the duration of action of Bup-XR previously established in mice and rats.36
In the literature, the reported or assumed TPC of buprenorphine varies widely between species, from 0.1 ng/mL in macaques,59 0.1 to 0.5 ng/mL in rabbits,12,46 0.6 ng/mL in dogs,31 and over 2.0 ng/mL in cats.66 Only one study has evaluated the PK of buprenorphine in ferrets: Katzenbach et al.28 reported plasma levels of 0.2 ng/mL at 12 h after administration of buprenorphine (0.04 mg/kg IM), with a mean maximum concentration of 6.96 ng/mL achieved 9 min after administration, and mean half-life of 219.1 min (3.6 h). Given that buprenorphine appears to have clinical efficacy in ferrets for 6 to 12 h, and considering the available literature, we used a TPC of 0.5 ng/mL for this study. The clinical analgesic efficacy observed in our study supports the relevance of a TPC of 0.5 ng/mL in ferrets.
In our PK study, all formulations reached TPC by the first timepoint evaluated: 15 min for low-dose Bup-XR, low-dose Bup-ER, and high-dose Bup-ER, and 30 min for high-dose Bup-XR. Based on the decay function for high-dose Bup-XR and our PK results from the low-dose Bup-XR group, high-dose Bup-XR likely also reaches TPC by 15 min postadministration. Our results also showed that half-life was dose-dependent, with low-dose Bup-XR and low-dose Bup-ER having shorter half-lives (3.793 and 3.739 h, respectively), compared with high-dose Bup-XR (14.61 h) and high-dose Bup-ER (14.21 h), consistent with properties of other extended-release drugs. For instance, a dose-dependent increase in half-life has been observed in a liposomal formulation of gentamicin sulfate in mice and rats.64 A drug’s half-life is a function of its absorption, distribution, metabolism, and elimination. Extended-release drugs prolong half-life through several mechanisms, including protecting the drug from degradation, targeting the drug to specific sites, and increasing the volume of distribution by establishing a depot within tissues.37,55,79 Despite the similar half-lives between low-dose Bup-XR and low-dose Bup-ER, and between high-dose Bup-XR and high-dose Bup-ER, Bup-XR remained above TPC for longer than Bup-ER in both dose categories. This difference in duration of action is likely due to Bup-XR achieving higher peak plasma concentrations than Bup-ER at both dosing regimens.
While we report Cmax for each group in Table 1, our results indicate that sampling timepoints for both Bup-XR groups missed the true peak plasma concentration, as indicated by differences between Cmax and C0. Conversely, Cmax closely approximated C0 for both Bup-ER doses, suggesting that we sampled close to the true peak in those groups. Comparisons in C0 between groups are, thus, more appropriate than direct comparisons of Cmax. Sampling frequency is a clear challenge with performing a PK study in small mammals. Frequent venipuncture can be technically challenging, and maximum plasma volumes that can be safely collected from each individual animal must be considered alongside minimum plasma volumes required for drug assays. Given these limitations in sampling frequency, the decay curve is valuable in supplementing measured timepoints and calculating peak plasma concentration. Similarly, because only 4 animals were available to perform the high-dose Bup-XR PK assay, we maintained the sample size of n = 4 animals per timepoint but selected fewer timepoints to evaluate for this group. PK data from the low-dose Bup-XR study indicated that the drug rapidly exceeded TPC by a large margin; we thus performed one early timepoint to confirm the onset of action of high-dose Bup-XR, and then prioritized later sampling timepoints to determine its duration of action. There are certainly drawbacks in using such a model, and future studies could use more animals to allow for more sampling timepoints. In addition, while it would not address the limitation on sampling volumes, implantation of an indwelling jugular vein catheter or vascular access port could also potentially be employed to reduce invasiveness and facilitate increased sampling frequency, potentially without the need for sedation.
In this study, we also developed and validated a clinical scoring system for ferrets, with particular utility for assessing pain following abdominal surgery. In designing this scoring system, we incorporated physiologic measures of respiratory rate and temperature, as well as behavioral measures, with a focus on assessment of not only the ferret’s face, such as demonstrated in the established ferret grimace scale,52 but also its posture, mobility, and reaction to incisional palpation. While clinical pain scoring systems that incorporate more of these features have been established for other species, such as cats, dogs, rabbits, and rats,3,5,6,29,51 our study presents, to our knowledge, the first such postoperative pain scoring system for domestic ferrets. We designed our scoring system with the goal of rapid, sensitive, and accurate detection of pain and distress, ease of use with minimal personnel training, consistency of results among different observers, avoidance of invasive sampling or specialized equipment, and preservation of social housing conditions. Additional monitoring, such as food intake and fecal output, could also be valuable for assessing pain in this species; however, social housing limits the quantitative assessment of these variables. To prioritize animal welfare, we elected to forgo a control group that would not receive any postoperative analgesia.
We validated our pain scoring system in a cohort of ferrets receiving Bup-HCl in a standard dosing regimen following OHE. Significant decreases over time were observed in scores for average respiratory rate, orbital tightening, posture, and average rectal temperature for all ferrets, consistent with gradual temporal resolution of postoperative discomfort. No animals met criteria for administration of rescue analgesia per evaluation by the facility veterinarian. Our scoring system is thus accurate and sensitive enough to detect behavioral and physiologic changes associated with normal postoperative recovery in female ferrets. The system was further validated with the strong interobserver agreement for both cohorts. We then applied this validated pain scoring system to compare the clinical efficacy of a single injection of Bup-XR to a course of Bup-HCl. Ferrets in both groups remained comfortable for 4 d postoperatively. In addition, we observed significantly lower average resting respiratory rate and posture scores in ferrets that received Bup-XR during the early postoperative period. Taken together, these findings suggest that Bup-XR is safe and effective in ferrets, offers utility in reducing the number of injections required to maintain effective analgesia during the postoperative period, thus simplifying administration on the part of the handler and reducing discomfort for the animal, and also provides a clinical advantage in improved early pain control compared with Bup-HCl. We also observed a significant effect of drug choice on reactions to palpation of the surgical incision when considering the overall time-course of the study, with no reactions observed in any animals receiving Bup-XR; however, no significant differences between the 2 groups were apparent at individual timepoints on multiple comparisons testing. We attribute this finding to the low incidence of incision palpation reaction overall. Buprenorphine is not expected to have anti-inflammatory properties, and therefore it is not surprising that there were no significant differences in incisional erythema or swelling between Bup-HCl and Bup-XR groups. However, the fact that these scores remained consistent between the 2 cohorts indicates that surgical technique and tissue trauma were similar in both groups, and rules out complications such as incisional infection that could add to postoperative discomfort, thus increasing our confidence in comparing pain scoring data across these cohorts.
This scoring system has only been validated for use in otherwise healthy female ferrets following a routine abdominal surgery. Future studies should be performed to validate its use in ferrets of other sexes and ages and for other types of surgical procedures. Based on the average cumulative pain scores in our study, we expect that for female ferrets with a normal postoperative recovery from OHE, the cumulative score should be less than or equal to 3 on the evening of surgery, less than or equal to 2 on postoperative days 1 to 2, and less than 2 thereafter. We recommend considering rescue analgesia if an animal’s cumulative score is greater than or equal to 3 starting on postoperative day 1, or if an animal receives a score of 2 in any single category in section 1 or section 2 of the scoring system (respiratory rate, orbital tightening, posture, vocalization, or teeth grinding). The results of observation of general attitude, rectal temperature measurement, and reaction to incisional palpation are not absolute indications for rescue analgesia but should be considered in the context of the complete clinical picture, including the ferret’s cumulative score, trend of scores over time, the extent and invasiveness of the surgery, concurrent medications, and other clinical signs.
Skin lesions have been documented following administration of sustained-release drugs, including extended-release opioids, in other animal species.11,19,21–23,44,63 In prior studies, ISRs have more often been described to occur with polymeric formulations compared to newer liposomal formulations; however, this may also reflect the much wider use of polymeric drugs in research animals thus far. In our study, we observed ISRs in ferrets in both high-dose Bup-ER and high-dose Bup-XR groups, but not in ferrets that received either of the low-dose formulations or Bup-HCl. High-dose Bup-ER was associated with a significantly higher incidence of ISRs (6/12) compared with Bup-HCl (0/12), while there was no significant difference in ISR development between high-dose Bup-XR (2/9) compared with Bup-HCl (0/12). However, because external factors resulted in a reduction in group number for the high-dose Bup-XR PK study, this study lacks power to compare the incidence of ISRs between independent animals that received either high-dose Bup-XR (0/4) or high-dose Bup-ER (6/12). A follow-up study with more animals would be necessary to directly compare the risk of ISR development between the 2 extended-release buprenorphine formulations. When considering each individual injection, 57.1% of high-dose Bup-ER injections were followed by ISR development, compared with 15.4% of high-dose Bup-XR injections. While all ISRs clinically resolved in the 2- to 4-wk washout periods between study phases, we cannot entirely rule out the presence of persistent underlying reaction and/or sensitization from prior injections in the same location. For example, 2 ferrets that developed ISRs after high-dose Bup-ER in the PK study developed ISRs again approximately 4 wk later after receiving high-dose Bup-ER in the clinical efficacy study. Sensitization could also account for the development of ISRs in response to high-dose Bup-XR; this is a liposomal formulation that is not typically expected to cause these reactions and the only reactions observed occurred in animals that had previously received Bup-ER. Overall, ISR development is likely multifactorial.
This study only examined PK in healthy, young, intact female ferrets, which represents a limitation in extending the study conclusions to other demographics. Female ferrets were chosen for several reasons. Female ferrets are induced ovulators, requiring copulation to trigger ovulation. In the absence of mating, intact female ferrets remain in prolonged estrus, resulting in hyperestrogenism. This can cause bone marrow suppression and aplastic anemia, leading to anorexia, lethargy, collapse, hemorrhage, or death.32 OHE is therefore a common procedure for female ferrets that will not be bred and is routinely recommended by facility veterinarians at our institution. An indication for surgery was therefore already present in this cohort of animals, which were ordered for an unrelated neurobehavioral study, allowing us to reduce animal numbers while accomplishing goals for both studies. Our veterinarians have significant experience performing OHE in ferrets using a standard surgical protocol, allowing us to reduce potentially confounding variables. Assessing animals of other sexes or reproductive statuses would have required the purchase of additional animals and/or an additional surgical procedure. It has been well documented in humans and animals that sex and reproductive status can affect pain tolerance, metabolism, and drug PK.17,40,53,60,67,74,76 While no studies have evaluated the effect of sex or reproductive status on pain tolerance in ferrets, a few studies have examined drug metabolism in this species, with male ferrets having higher concentrations of hepatic cytochrome P-450 enzymes25 and more rapid PK of meloxicam compared with female ferrets.9 Metabolism and pain tolerance may also be affected by age and health status. These factors should be considered before extending the study conclusions to male ferrets, other age groups, or animals with comorbidities, and further research is warranted to assess the PK profile and clinical efficacy of Bup-XR in other demographics.
While it would have been optimal to align study timelines between both cohorts, unforeseen circumstances in scheduling and operating room availability required modification of the study timeline. As a result, cohort 1 underwent the first PK study (low-dose Bup-XR) before OHE, and the second PK study (high-dose Bup-XR) after OHE. Conversely, cohort 2 underwent both PK studies (low-dose Bup-ER and high dose Bup-ER) before OHE. Due to the unknown effects of reproductive status on drug metabolism in ferrets, we should be cautious in making direct comparisons between PK data from the high-dose Bup-XR group, which used recently spayed ferrets, and other groups, which used intact female ferrets. However, our study results still contribute key information to our understanding of extended-release buprenorphine use in this species. All ferrets in this study were young, no signs of estrus were observed in any animals on frequent observations throughout the study period, and all ferrets were spayed between 12 and 16 wk of age; we thus consider it unlikely that ferrets were in estrus during the study. In addition, while we lack PK data from high-dose Bup-XR in intact female ferrets, the duration of action determined in the clinical efficacy study aligned with the PK-predicted duration of action.
The doses for Bup-ER (0.12 and 0.2 mg/kg) were selected based on the dosage of extended-release buprenorphine in domestic cats,8,13 given the similarity in dosing of Bup-HCl between cats and ferrets.28,62 While no studies in the literature document a dose of Bup-XR for ferrets, studies in mice suggest that Bup-XR can be safely dosed at approximately 3-fold the dose of Bup-ER.43,69 Applying this ratio to the selected high Bup-ER dose (0.2 mg/kg), we set the high-dose of Bup-XR at 0.6 mg/kg in ferrets. Given the potential risks of sedation, respiratory depression, and cardiovascular depression associated with opioid use, we selected a lower initial dose (0.2 mg/kg) for safety assessment before proceeding with evaluation of the higher dose. Because no prior studies had demonstrated the safety or efficacy of extended-release opioids in ferrets, we also elected not to administer the extended-release formulations in this study until after surgery, due to concerns for inadequate intraoperative analgesia and/or negative side effects. Furthermore, we elected not to administer regular-release buprenorphine as a preemptive analgesic because this drug lasts up to 12 h in ferrets, meaning that the preemptive analgesic could confound the results of clinical scoring in the Bup-XR group. To avoid this confounding factor while ensuring appropriate intraoperative analgesia, we administered hydromorphone, a pure µ-opioid receptor agonist. While hydromorphone is preferable for intraoperative analgesia over a partial µ-opioid receptor agonist such as buprenorphine, hydromorphone is also highly emetogenic, causes sedation, and has a short duration of action in the ferret, making it largely impractical as a postoperative analgesic in this species. By administering preoperative hydromorphone and postoperative extended-release buprenorphine, our protocol offered sustained analgesic coverage. Referring to previously established PK data,28 we designed our study such that hydromorphone would wear off by the first postoperative scoring timepoint, and thus would not confound assessment of the analgesic efficacy of buprenorphine. Furthermore, buprenorphine has higher affinity for the µ-opioid receptor compared with hydromorphone, and thus would also be expected to displace any residual hydromorphone.
Our study demonstrates that Bup-ER and Bup-XR provide sustained analgesia for nearly 48 and 72 h, respectively, in female domestic ferrets. Bup-XR provided effective analgesia in the postoperative period, with clinically evident benefits over Bup-HCl based on our pain scoring system. With considerations for formulation-specific side effects, Bup-XR offers a refinement to analgesia in female ferrets with benefits to both companion animal practice and research.
Supplementary Materials
Study timeline. (A) Each cohort of 12 ferrets underwent 2 PK studies and one efficacy study, with all study phases separated by a washout period of at least 2 wk. For the PK studies, ferrets in cohort 1 received low-dose Bup-XR (0.2 mg/kg SC) and high-dose Bup-XR (0.6 mg/kg SC), and ferrets in cohort 2 received low-dose Bup-ER (0.12 mg/kg SC) and high-dose Bup-ER (0.2 mg/kg SC). For the clinical efficacy study, each cohort underwent ovariohysterectomy, followed by administration of an opioid regimen and clinical scoring. All ferrets in cohort 1 (n = 12) received Bup-HCl (0.02 mg/kg SC every 10 to 12 h for 8 doses) for postoperative analgesia. Ferrets in cohort 2 received either a single injection of high-dose Bup-XR (0.6 mg/kg SC; n = 9) or high-dose Bup-ER (0.2 mg/kg SC; n = 2). One ferret originally assigned to the Bup-XR group received an incomplete dose and was thus withdrawn from the study prior to clinical scoring. (B) Injection site reactions (ISRs) observed in ferrets in cohort 2. ‘Y’ indicates the presence of ISR; ‘N’ indicates the absence of ISR; and ‘W’ indicates one ferret that received an incomplete dose of Bup-XR, and was thus withdrawn from the efficacy study.
Postoperative pain scoring system for ferrets. After administration of postoperative analgesia, the cumulative score is expected to be less than or equal to 3 on the evening of surgery, less than or equal to 2 on postoperative days 1 to 2, and less than 2 thereafter. Consider rescue analgesia if an animal’s cumulative score is 3 or higher starting on postoperative day 1, or if an animal receives a score of 2 in any single category in section 1 or section 2. Changes in the general attitude, rectal temperature, and reaction to incisional palpation are not absolute indications for rescue analgesia, but should be considered in the context of the complete clinical picture.
Time compared with buprenorphine plasma concentration (ng/mL) of 2 sustained-release formulations. The therapeutic level (0.5 ng/mL) is indicated by the dotted line. The dashed line indicates the one-phase decay function fitted to each data set. (A) Low-dose Bup-XR achieved therapeutic plasma concentrations (TPC) by 15 min postadministration and remained above the therapeutic level for 24 h. (B) High-dose Bup-XR achieved TPC by 30 min postadministration (the first timepoint assessed) and remained at or above the therapeutic level for 72 h. (C) Low-dose Bup-ER achieved TPC by 15 min postadministration but mean drug concentration dropped below the therapeutic level by 24 h. (D) High-dose Bup-ER achieved TPC by 15 min postadministration and dropped below therapeutic levels by 48 h.
Comparison of pain scores between ferrets receiving subcutaneous Bup-HCl (0.02 mg/kg) or a single dose of Bup-XR (0.6 mg/kg) for management of postoperative pain. Scores from all observers were averaged at each timepoint, and data were analyzed by repeated measures ANOVA (A–C) or a mixed effects model (D). (A) Ferrets receiving Bup-XR had significantly lower average resting respiratory rate scores compared with ferrets receiving Bup-HCl (P < 0.0001), with no significant effect of individual subject (P = 0.2556). Resting respiratory rate score also decreased significantly over time in both groups (P < 0.0001). (B) Average orbital tightening score decreased significantly over time in all ferrets (P < 0.0001). There was no effect of opioid choice (P = 0.3585) or of individual subject (P = 0.0933) on orbital tightening score. (C) Ferrets receiving Bup-XR had significantly lower average posture score compared with ferrets receiving Bup-HCl (P < 0.0001), with no significant effect of individual subject (P = 0.5143). Posture score also decreased significantly over time in both groups (P < 0.0001). (D) There was no effect of opioid choice on rectal temperature (°F) (P = 0.3794) or rectal temperature score (P = 0.1804). Rectal temperature significantly decreased over time in all ferrets (P < 0.0001). POD, postoperative day.
Comparison of incisional scores between ferrets receiving subcutaneous Bup-HCl (0.02 mg/kg) or a single dose of Bup-XR (0.6 mg/kg) for management of postoperative pain. Scores from all observers were averaged at each timepoint, and data were analyzed by repeated measures ANOVA. (A) Incisional erythema score was not significantly different between opioid groups (P = 0.2646) but varied significantly between individual ferrets (P = 0.0011) and with time (P < 0.0001). (B) Incisional swelling score was not significantly different between opioid groups (P = 0.2943) but varied significantly between individual ferrets (P < 0.0001) and with time (P < 0.0001). (C) There was a significant overall effect of opioid on reaction to incisional palpation (P = 0.0021) on repeated measures ANOVA, but no significant difference between individual timepoints on multiple comparisons testing.
Representative images of injection site reaction (ISR) following SC injection of high-dose Bup-ER. Hematoxylin and eosin staining of a fine needle aspirate sample revealed a mixed inflammatory cell population without bacteria or degenerative changes, consistent with a sterile abscess.
Contributor Notes
This article contains supplemental materials online.