Pharmacokinetic Evaluation of Topical Long-Acting Buprenorphine in New Zealand White Rabbits (Oryctolagus cuniculus)

Abstract

Rabbits are commonly used as surgical models, thus requiring analgesics for painful procedures and optimal animal welfare. Buprenorphine, a partial µ opioid, is commercially available in various concentrations and sustained-release formulations and has historically been used as an analgesic in rabbits. A topical long-acting buprenorphine formulation (Zorbium, Bup-TP) has been approved for analgesic use in cats but has not yet been evaluated in rabbits. The present study evaluated the plasma concentrations and pharmacokinetic parameters of Bup-TP in New Zealand white rabbits (Oryctolagus cuniculus). Healthy adult male (n = 4) and female (n = 4) New Zealand white rabbits were used in a randomized crossover design and received a single high (7 mg/kg) and low (3 mg/kg) dose of Bup-TP. In this study, Bup-TP achieved a plasma blood concentration >0.25 ng/mL starting at 0.5 hours after dosing that was maintained up to 72 hours after dosing in adult New Zealand white rabbits. Compared with baseline, fecal and urinary output were reduced for an average of 3.5 days after dosing; food consumption was reduced for an average of 10 days after dosing. All resolved with time and supportive care. No lesions were grossly visible on any rabbit at site of application. Bup-TP may be an effective, long-lasting, and noninvasive method of providing analgesia in rabbits. Future study is recommended to optimize dosing and procedural analgesic efficacy.

Introduction

Rabbits are a commonly used laboratory animal species, often preferred for their economical size, docile nature, and ease of breeding.¹ According to the USDA’s annual report for animal usage in 2023, >125,000 rabbits were used in research studies.² Despite their common use in research, pain management in this species can be difficult to achieve. Each existing mode of analgesia each has its own drawbacks, including short duration of effect, need for frequent redosing, and potential adverse effects, such as weight loss, anorexia, decreased fecal output, and gastrointestinal stasis. Thus, there is a need for alternative modes of analgesia that are easier to apply and provide therapeutic analgesic efficacy for an extended period with minimal adverse effects.

Currently, analgesic options used in rabbits include non-steroidal anti-inflammatory agents (NSAIDs) such as carprofen and meloxicam, local anesthetics such as lidocaine, and opioids including buprenorphine. Buprenorphine is a high-affinity partial µ opioid agonist and δ and κ antagonist.^3–5 In veterinary patients, buprenorphine has a long half-life compared with other opioids such as morphine and fentanyl, allowing for a longer duration of action and potentially less frequent need for redosing.^6–8 As a partial agonist, buprenorphine plateaus into a ceiling effect, limiting adverse effects such as respiratory depression. Because buprenorphine has a high affinity for the µ opioid receptor, binding tightly and dissociating slowly,⁴ clinical effects of buprenorphine are not necessarily correlative with plasma concentrations.^6,7

Numerous studies have evaluated various buprenorphine formulations in rabbits, including buprenorphine HCl, buprenorphine sustained release, and buprenorphine high concentration, with commonly used dosages of 0.01 to 0.05 mg/kg. In all cases, dosing occurred every 6 to 12 hours, and adverse effects included decreased food and water intake, decreased fecal output, and delayed gastrointestinal transit.^9–13 However, a single high dose of buprenorphine (0.1 mg/kg IM) showed no adverse effect on gastrointestinal motility.¹⁴ Buprenorphine caused a similar decrease in fecal output as meloxicam and fentanyl patches, but returned to baseline faster than fentanyl.^15,16

Several studies have been conducted to evaluate the efficacy of buprenorphine as an analgesic in rabbits following surgeries of varying invasiveness, such as jugular catheter placement,⁹ ovariohysterectomy,¹⁵ and orthopedic procedures.^16,17 In all cases, fecal output and some form of a rabbit grimace/behavior or activity scale were used to assess postoperative pain.

A transdermal buprenorphine patch, Buprederm, was found to be effective for 72 hours in New Zealand white rabbits. Peak plasma levels occurred between 1 and 24 hours with plasma concentration (0.5 to 1 ng/mL) maintained for 72 hours. This patch, applied to the dorsum, required the fur to be shaved and the skin to be rinsed after removal. Skin irritation was assessed, and the patch was found to be nonirritating.¹⁸

Thus, a need arises for a sustained-release buprenorphine formulation that is easy to apply and is long-lasting, resulting in less frequent redosing while potentially reducing stress from handling and prolonging of adverse effects. Bup-TP is a long-acting transdermal buprenorphine solution and an FDA-approved analgesic (Zorbium; Elanco Animal Health, Greenfield, IN, NADA 141-547) for use in cats.¹⁹ When applied to cats once transdermally as labeled, Bup-TP provides analgesia within 2 hours with continual release of buprenorphine through skin into the systemic circulation each day for 4 days.²⁰ This long-acting buprenorphine formulation could offer multiple practical solutions both for pet rabbits in the clinic and in a laboratory setting, including less handling and therefore reduced stress on the animal, avoidance of potentially painful injections, convenience for the clinician and the client, and decreased need with dispensing or administering multiple doses of controlled substance.

Recent studies evaluated the pharmacokinetics and efficacy of Bup-TP in mice, demonstrating absorption of Bup-TP and maintenance of serum drug concentration above the purported minimum therapeutic threshold in mice (1 ng/mL) for up to 96 hours.^21,22 Another study evaluating Bup-TP for analgesic efficacy in rats via thermal footpad nociceptive testing found comparable pain relief in comparison with injectable subcutaneous long-acting buprenorphine.²³ These results show that Bup-TP could be an effective analgesic in rabbits, with many practical and welfare-related benefits along with improved analgesic outcomes secondary to sustained therapeutic plasma levels, depending on the surgical model and/or needed duration of analgesia.

To our knowledge, there is currently no published pharmacokinetic data regarding Bup-TP in rabbits or potential adverse effects. In the current study, we assessed the plasma concentrations and pharmacokinetic parameters of a long-acting, transdermally absorbed formulation of buprenorphine in the New Zealand white rabbit up to 72 hours after dosing. The objective of this study was to determine pharmacokinetic trends of Bup-TP in healthy adult New Zealand white rabbits. We also assessed expected adverse effects including reduced fecal/urinary output and food consumption.

Materials and Methods

Animals.

Female and male New Zealand white rabbits (Oryctolagus cuniculus; age, 7 to 14 months; weight, 3.0 to 4.01 kg) were purchased from Inotiv (West Lafayette, IN) and housed individually (Euro Rabbit; Allentown Caging Equipment, Allentown, NJ) in a facility accredited by AAALAC International. The facility had a 12-hours light/12-hours dark cycle, temperature of 70 ± 2 °F (21.1 ± 1.0 °C), and average humidity of 50% (range, 15% to 80%). Prior to shipping, all rabbits were vendor verified to be free of reovirus, lymphocytic choriomeningitis virus, parainfluenza viruses types 1 and 5, rotavirus, rabbit hemorrhagic disease virus, Bordetella bronchiseptica, Helicobacter spp., Lawsonia spp., Pasteurella spp., Salmonella spp., Treponema spp., Tyzzer disease pathogen, CAR bacillus, Cheyletiella parasitovorax, Leporacarus gibbus, Psoroptes cuniculi, other ectoparasites, Passalurus ambiguus, other helminths, Eimeria spp., Eimeria stiedae, other intestinal protozoa, and Encephalitozoon cuniculi prior to shipping.

Rabbits were physically examined on arrival and found to be free of clinical signs of disease. They were then allowed to acclimate for 7 days prior to the start of the study. Rabbits were fed a commercial pelleted rabbit diet (5326 Laboratory Rabbit Diet HF; LabDiet, Richmond, IN) and provided potable municipal water via a Lixit watering device ad libitum throughout the project. Each rabbit received ∼50 g of timothy hay daily. Fresh fruit or vegetable enrichment was provided once weekly. Paper cage pan liners were changed regularly when soiled. All work conducted in this study was compliant with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. All study procedures were approved by the University of Michigan’s IACUC.

Treatment groups.

A randomized crossover design was used to evaluate plasma levels, food consumption, and fecal output in 2 treatment groups, that is, low (3 mg/kg) dose and high (7 mg/kg) dose. Rabbits were randomized according to treatment dose (including receiving high or low dose first) and sex using Microsoft Excel. Individuals conducting the study and data analysis were not blinded and were aware of treatment group assignments at all stages of the study. A priori inclusion criteria included sex and weight range (3 to 4 kg) of each rabbit. Each rabbit (n = 4 male and n = 4 female) received a low Bup-TP (3 mg/kg) and high Bup-TP (7 mg/kg) treatment with a 20-day washout period in between treatments: these 2 data collection periods were denoted as treatment period 1 and treatment period 2. In treatment period 1, some animals received a low dose, and some received a high dose. In treatment period 2, each animal received whatever dose they did not receive in treatment period 1. Doses were based on previously established dosing in cats (range 2.7 to 6.7 mg/kg) and allometrically scaled dosing in mice (9 to 18 mg/kg).^19–21 Treatment group doses of Bup-TP were dispensed from the FDA-approved topical buprenorphine product (Zorbium, 20 mg/mL; Elanco Animal Health, Greenfield, IN) manufactured in 0.4- and 1-mL applicators. Treatments were applied topically to parted hair over the dorsal cervical skin of each rabbit prior to beginning blood collection. The hair was not clipped prior to administration. The application site was allowed to dry for a minimum of 30 min.

Blood collection.

Lidocaine/prilocaine cream (EMLA, lidocaine 2.5% + prilocaine 2.5%; Actavis, Parsippany-Troy Hills, NJ) was applied topically to the dorsal portion of auricular pinnae and allowed to sit for at least 15 to 30 min prior to blood collection. Each sample collected consisted of a volume of 200 µL. Blood was collected at 0.75, 1.5, 3, 4, 8, 24, 48, and 72 hours (average time ranges) following administration of Bup-TP. Baseline samples prior to dosing (0 hour) were collected from one male and one female rabbit. The serial blood collection schedule was selected based on previously published pharmacokinetic studies.^9,21,22 A nominal time range was used due to delays caused by attempts to acquire blood samples on 8 rabbits simultaneously despite staggering the dosing start time. As some samples were not collected at exactly the same time per early time points, an average time range was used. Daily time points were collected within 60 min of stated time. A 22- to 23-gauge needle was used to access the marginal ear vein or central auricular artery, and a Microvette lithium heparin micro tube (Kent Scientific, Torrington, CT) was used for sample collection at each time interval.

Supportive care and other observations.

Food intake was recorded via a daily written food log, with baseline normal appetite determined as consumption prior to beginning treatment. After treatment, animals were monitored daily for food consumption, fecal output, and urinary output. Water in crocks, fresh produce, and dietary supplemental gel (DietGel Criticare; ClearH₂O, Westbrook, ME) were provided daily in addition to standard rabbit chow, timothy hay, and water via a Lixit watering device. If animals were not eating independently or producing feces by day 4 after dosing, daily oral supplementation of a nutritionally complete feeding formula (Critical Care Herbivore; Oxbow Animal Health, Papillion, NE) and a laxative powder polyethylene glycol 3350 mixture (MiraLAX; Bayer Healthcare, Leverkusen, Germany) were provided. Fecal output (volume) was scored visually according to a previously published scoring system (see Figure 1).¹⁰ Urinary output was assessed on a binary basis, that is, present or not present. Cage pan lining was replaced once daily to accurately assess daily output. Scoring for fecal and urinary output was not performed in a blinded manner.

Figure 1.

System Used for Fecal Output Scoring in Rabbits. From Weaver et al¹⁰.

Plasma sample preparation and LC-MS/MS methodology.

Plasma samples were submitted to the Pharmacokinetic and Mass Spectrometry Core (Bioanalysis and LC-MS, College of Pharmacy, University of Michigan, Ann Arbor, MI) and were analyzed on the same day in a single batch for quantification of buprenorphine concentration. A liquid LC-MS/MS method was developed for the quantification of buprenorphine using an AB SCIEX QTRAP 4000 mass spectrometer operated in positive electrospray ionization mode. Chromatographic separation was achieved on an XBridge C18 column (5 cm × 2.1 mm inside diameter, 3.5 µm) with a gradient elution using mobile phase A (0.1% formic acid in purified deionized water) and mobile phase B (0.1% formic acid in acetonitrile) at a flow rate of 0.4 mL/min. The injection volume was 10 µL, and the total run time was 5.6 min. The gradient program was as follows: 95% (phase A) at 0.5 min, 5% (phase A) at 1.5 min, held until 3.5 min, returned to 95% (phase A) at 3.6 min, and re-equilibrated until 5.6 min. Multiple reaction monitoring was used with the following transitions: m/z 468.20 → 414.3 for buprenorphine and m/z 472.20 → 414.3 for the internal standard (buprenorphine-d4). The decluttering potential, collision energy, collision exit potential, and electrode potential were optimized as follows: 89.0, 44.8, 5.6, and 9.6 V for buprenorphine, and 99.0, 41.0, 30.0, and 8.0 V for the internal standard, respectively. Data acquisition and quantification were performed using Analyst software, with calibration curves constructed by plotting the peak area ratios of buprenorphine to the internal standard against known concentrations. The analytical curve was constructed using 8 non-zero standards with buprenorphine concentration ranging from 0.25 to 250 ng/mL in the blank male and female rabbit plasma, respectively. The linear regression analysis of buprenorphine was performed by plotting the peak area ratio (y) against the buprenorphine concentrations (x) in ng/mL. The accuracy and precision were evaluated at 3 concentration levels (1, 100, and 250 ng/mL) with 2 individual replicates at each concentration. At least 50% of quality controls at each level were within 15% of their nominal concentration. Quantification levels (limit of quantification [LOQ]) for buprenorphine were ≥0.25 ng/mL.

Data and statistical analysis.

A noncompartmental analysis was used to analyze pharmacokinetic data (PKanalix 2024R1, Simulations Plus, doi: 10.5281/zenodo.11401684). All sample values were included in the analysis. Plasma concentrations below the LOQ after T_max were adjusted as LOQ/2 per pharmacokinetic software parameters.²⁴ The linear trapezoidal rule was used for the AUC calculation. The following pharmacokinetic parameters were estimated: C_max, T_max, t_1/2 obtained from ln(2)/lambda z (first-order rate constant estimated by linear regression of plasma concentration–time curve at terminal time points), AUC from time of dosing to last time point, AUC from time 0 extrapolated to infinity, and percent AUC extrapolated. All statistical analyses were conducted with NCSS statistical software and GraphPad Prism (GraphPad Software, La Jolla, CA). Statistical sampling of pharmacokinetic data was not performed. Numbers of days to resume baseline food consumption and fecal/urinary output were compared between treatment groups of dose, sex, and data collection time period. If data passed D’Agostino and Pearson or Anderson–Darling normality tests, 2-way ANOVA or paired t tests were performed; if data failed, a Mann–Whitney U Test was performed. A P value <0.05 was considered statistically significant.

Results

All animals tolerated application of Bup-TP to the target site. Some animals exhibited weight shifting and attempts to groom immediately following application, but no signs of distress were otherwise noted.

Pharmacokinetics.

Mean plasma concentrations ± SD per group and time period are listed in Table 1 and Figure 2. Outlier or high plasma concentrations relative to samples at the same time point and dose were confirmed via review of quality control procedures, sample processing, individual sample variation, or potential needle injection variance. A noncompartmental analysis was performed for comparison with samples grouped per treatment (see Table 2).

Table 1.

Buprenorphine Plasma Concentrations per Time (n = 4 Males and 4 Females NZW Rabbits per Group)

Dose (mg/kg)	Time (h)	Median	Mean	SD	SE
3	0.75	5.5	11.3	14.3	5.4
	1.5	5.2	12.7	22.3	7.9
	3	8.7	15.9	15.7	5.5
	4	6.5	55.8	91.8	32.5
	8	4.1	4.6	3.6	1.3
	24	3.6	21.7	45.8	16.2
	48	1.2	24.2	63.4	22.4
	72	0.8	6.0	14.3	5.1
7	0.75	4.9	4.9	3.4	1.2
	1.5	6.7	7.4	4.4	1.6
	3	13.5	18.1	15.3	5.4
	4	14.5	34	47.4	16.8
	8	9.4	15.3	14.5	5.1
	24	4.7	15.6	20.7	7.3
	48	2.5	8.1	16.6	5.9
	72	3.0	42.6	100.4	35.5

Figure 2.

Mean ± SD Plasma Concentrations per Time Point. Plasma Concentrations Are Plotted on a Semilogarithmic Graph.

Table 2.

Pharmacokinetic Noncompartmental Analysis Parameters (n = 4 Male and 4 Female Rabbits per Dose Group)

Dose (mg/kg)	Parameter	Median	Mean	SD
3	AUC0-inf (h⋅ng/mL)	36,102	83,401	109,313
	AUC0-inf, pred (h⋅ng/mL)	36,194	80,477	105,076
	% Extrapolated AUC	6.5	11.1	14.0
	% Extrapolated AUCpred	3.5	7.2	8.4
	AUC0-last (h⋅ng/mL)	32,147	78,641	104,964
	Cmax (ng/mL)	54.4	93.7	87.8
	t1/2 (h)	23.7	21.9	10.5
	Tmax (h)	3	8.3	16.1
7	AUC0-inf (h⋅ng/mL)	64,405	133,532	195,342
	AUC0-inf, pred (h⋅ng/mL)	37,327	81,318	105,883
	% Extrapolated AUC	16.6	24.8	27.1
	% Extrapolated AUCpred	7.4	8.1	6.5
	AUC0-last (h⋅ng/mL)	33,470	77,527	106,381
	Cmax (ng/mL)	31.7	65.7	94.0
	t1/2 (h)	21.6	23.7	11.8
	Tmax (h)	14.3	28.6	31.3

Abbreviations: AUC_last, AUC from time of dosing to last time point; AUC_0-inf, AUC from time 0 extrapolated to infinity; Pred = predicted; % Extrapolated AUC = AUC_last-inf/AUC_0-inf; % Extrapolated AUC_Pred = % Extrapolated AUC from last predicted plasma concentration value to infinity.

Fecal and urinary output.

Fecal and urinary output were evaluated up to 13 days after dosing. Fecal output scoring was performed using a previously established visual scoring system.¹⁰ Mean fecal output scores for 3 mg/kg compared with 7 mg/kg over time are shown in Figure 3. The overall median number of days after dosing to resume normal fecal/urinary output was 3.5 days. Median baseline urinary output score was 1 (0, not present; 1, present). On day 1 after dosing, the median urinary output score was 0.5. The median urinary output score returned to 1 (normal) on days 2 and 3 after dosing. The longest duration that any individual rabbit took to urinate after dosing was 3 days. Days to return to normal urinary output are combined with days to return to normal fecal output in Figure 4. There was a notable statistically significant difference between data collection time periods regarding number of days to resume normal fecal/urinary output (P = 0.0002), with treatment period 1 taking almost twice as many days to recover compared with treatment period 2 (mean = 5.12 days compared with mean = 2.62 days, respectively) (see Figure 4). There were no statistically significant differences between dose or sex.

Figure 3.

Mean ± SD Fecal Output Scores for Both Low and High Doses for All Animals. Day 0 Denotes Baseline Value. ***, P < 0.001.

Figure 4.

Numbers of Days After Dosing Needed to Resume Both Urinary Output and Baseline Fecal Output (Combined) Between Groups: Data Collection Time Period, Sex, and Dose.

Food consumption.

Normal appetite was determined as the amount of food each individual animal consumed daily prior to dosing. The overall median number of days to resume normal appetite was 10 (range 3 to 19 days). The median number of days after dosing time to resume normal appetite was 6 (range 3 to 16 days) and 10.5 (range 2 to 19 days) for the 3 and 7 mg/kg treatment groups, respectively. There was a statistically significant difference between data collection time periods with regard to the number of days to resume normal food consumption (P = 0.025), with treatment period 1 taking almost twice as many days to recover compared with treatment period 2 (mean = 10.87 days compared with mean = 5.5 days, respectively) (see Figure 5). There were no statistically significant differences between dose or sex.

Figure 5.

Number of Days After Dosing Needed to Resume Baseline Appetite Between Groups: Data Collection Time Period, Sex, and Dose. *, P < 0.05.

Discussion

Plasma buprenorphine concentration results observed in this study demonstrated that the topical formulation of buprenorphine, Bup-TP, achieved mean plasma concentration of buprenorphine >0.25 ng/mL up to 72 hours after dosing.

Only 2 of 16 samples were below the LOQ of 0.25 ng/mL at the 72 hours time point. Both samples were from male rabbits in the 7 mg/kg group yet had demonstrated plasma concentrations above the LOQ at the same time point with the previous 3 mg/kg treatment. This may be due to variations in absorption despite application in the same dorsal cervical area after the washout period. Large variances in plasma buprenorphine concentrations were observed in both male and female rabbits at several time points. Such wide buprenorphine plasma concentration disparities compared with other animals within the same treatment and time point have been previously reported in cats.¹⁹ In addition, we have observed similar large-scale variations in plasma buprenorphine concentrations in other species administered Bup-TP (data not shown). In such instances, the plasma concentrations were confirmed by re-assay and review of LC-MS/MS methodology and instrument quality control. Transdermal delivery of small molecules via passive diffusion requires the dissolution or release of the active pharmaceutical ingredient from the formulation, partitioning into, and diffusion through, the stratum corneum, and diffusion into the dermis with uptake into systemic circulation via the skin capillary network. The rate-limiting process of absorption through the cornified stratum corneum occurs via diffusion through keratinocyte lipid bilayers (transcellular) or through the intracellular ceramide/cholesterol lipid matrix and tight junctions.^25–27 Permeation or chemical enhancers such as ethanol, fatty acids, liposomes, and surfactants can be tailored to promote permeability and absorption via alterations in the lipid matrix and intercellular proteins.²⁸ Alternatively, transdermal absorption may occur through an appendageal route via follicular and adnexal glands.²⁷ Due to complexity of drug diffusion and permeation through the stratum corneum, it is not surprising that wide variations in buprenorphine plasma concentrations may be observed within subjects, between subjects, and at different time points. Such deviations in plasma concentrations could be due to variations in absorption across the stratum corneum (transcellular, intracellular, and appendageal), changes in local drug concentration gradients, alterations in capillary blood flow, depot formation within the stratum corneum with subsequent release, or alterations in stratum corneum thickness between subjects.²⁸ Variations in plasma concentrations or spontaneous spikes could be partially due to enterohepatic cycling, which has been noted in studies using buprenorphine in rabbits⁹ and in rats.²⁹ Furthermore, given the evaporative concentrated delivery system of Bup-TP, it is possible that larger depots of drug may pass through the stratum corneum at different intervals and concentrations depending on how the stratum corneum is disrupted.

In this study, serial serum samples were collected from the marginal ear vein and central auricular artery, as suggested by multiple studies.^30–32 A possibility for wide variations in plasma concentrations at selected time points may be due to the source of serum sampling or an unexpected interaction with topically applied lidocaine/prilocaine cream, as these may share a common microsomal enzymatic metabolic pathway of elimination. Due to diffusion gradients, there may be discrepancies between plasma concentration values depending on sampling site and the site of administration. Arterial blood sampling is considered to be the most accurate for pharmacokinetic studies, as drug concentration in these vessels show drug delivery to the body where it exerts its effect.³³ The highly variable plasma concentrations from the present study could be due to a mix of sampling sites: serum was sampled primarily from the marginal ear vein, but some samples were also collected from the central auricular artery. These were not differentiated during collection in the present study, but future studies should make note of arterial compared with venous samples.

Given that repeated blood sampling from the ears likely contributed to animal discomfort, other alternative serial serum sample collections were considered, such as jugular blood collection with manual restraint.³⁴ However, due to a concern for stress caused to the animals, the need for additional restraint, and the rabbits’ temperaments, this technique was not used. Other indwelling catheter techniques were considered and attempted, including short-term cannulation of auricular vessels, but these are not typically intended to dwell for long periods of time^35,36 and often clot after initial collection. A peripherally inserted central catheter³⁷ as well as a modified Seldinger percutaneously placed jugular catheter with ultrasound guidance were attempted multiple times in our laboratory, but proved technically challenging and were ultimately unsuccessful. With further practice, these techniques may have been effective, but they did not fit the small scope and time restraints of this project. Surgically placed vascular access buttons³⁸ and jugular catheters⁹ are other published methods for long-term blood collection. Although not employed in our initial study, our laboratory did successfully surgically place jugular catheters in a subsequent pilot study (results not shown).

Limitations to the pharmacokinetic aim of this study included underestimating the length of the absorption period and need for sampling at longer time intervals (for example, 96 to 128 hours) to optimize predictions for terminal absorption or elimination, as the AUC from time 0 extrapolated to infinity is linked to the terminal half-life and slope of the plasma concentration–time curve. Higher buprenorphine plasma concentrations at late time points observed in the 7 mg/kg treatment group may have led to an overestimation of the AUC from time of dosing to last time point and AUC from time 0 extrapolated to infinity parameters. Transdermal applications are known for ‘flip-flop’ kinetics where the rate of elimination is much greater than the rate of absorption, and thus longer sampling times are recommended for future studies.^19,39,40 Using additional animals per group to account for spontaneous variations in plasma concentrations due to the process of diffusion and absorption within the stratum corneum may have reduced variations in pharmacokinetic parameters.

While a definitive therapeutic plasma concentration has not been established for buprenorphine in rabbits, previous publications have employed a range varying between 0.1 and 1.0 ng/mL.^9,16,18,41 Additional pharmacodynamic efficacy studies paired with pharmacokinetic data should be pursued to best establish or correlate a minimum analgesic threshold depending on surgical procedure.

Adverse effects seen in cats dosed with Bup-TP included mydriasis within 4 hours after dosing, and increased rectal temperature through 168 hours after dosing.²¹ Mydriasis and mild to moderate hyperthermia are responses to opioids that are unique to cats.^8,42,43 As these adverse effects to opioids are not known to occur in rabbits, these were not assessed in the current study.

While one study has shown that buprenorphine administration to rabbits results in worse adverse effects,¹¹ most have found no significant difference when compared with other modalities of analgesia, including NSAIDs and fentanyl.^10,15,16,44 Use of long-acting buprenorphine can exacerbate adverse effects, though several studies demonstrated that a single dose of long-acting buprenorphine formulations maintained analgesia for a longer period of time,^9,16,17 but also reducing fecal output to a greater degree compared with regular buprenorphine.⁹ While a single high dose of buprenorphine has not been found to result in adverse effects in healthy adult rabbits,¹⁴ a single dose of sustained-release buprenorphine was shown to result in adverse effects.⁹ These adverse effects include decreased fecal production, decreased food and water consumption,^9–13 and increased gastrointestinal transit time.^12,13 All animals in the current study demonstrated commonly reported adverse effects from buprenorphine application within 24 hours of dosing, including hyporexia and decreased fecal and urinary output. If animals were not eating on their own by day 4 after dosing, oral supplementation with nutritional support and laxatives was provided. This may have affected time to recover normal fecal output and appetite. Some animals took 3 days to urinate after dosing, suggesting decreased water consumption. As animals received water ad lib through a Lixit watering device, water intake was not monitored.

All animals were dosed with both a high dose (7 mg/kg) and a low dose (3 mg/kg), with a 3-week washout period in between to allow them to recover from adverse effects and eliminate buprenorphine from the system. The high dose did not result in a faster increase of blood plasma concentration compared with the low dose. The high dose did result in overall slightly longer times for animals to regain normal appetite and fecal/urinary output, but this increase was not statistically significant. The high dose is used in cats weighing 6.6 to 16.5 lb. In contrast, most of the rabbits used in the present study were ≤3.0 kg (6.6 lb), at the lower end of this range. Because there was no added benefit with an increased dose of 7 mg/kg compared with 3 mg/kg, the high dose is not recommended in 3.0 kg New Zealand white rabbits. However, efficacy studies are still needed.

Animals were not handled by the laboratory prior to the first dose and data collection beyond initial physical examination and acclimation time of about 1 week. Statistically, more days were needed to recover back to baseline fecal/urinary output and food consumption following the first data collection time period (treatment period 1), regardless of dose concentration and sex. This may be explained by the animals acclimating to the dosing and sample collection procedures following the second data collection time period (treatment period 2), including human handling, restraint boxes, and more frequent human proximity when viewing for health checks. In addition, human procedures may have been streamlined from the first round of data collection to the second, refining serial blood collection technique and general efficiency of workflow. Thus, these findings may highlight the importance of acclimation of animals prior to study.

Sedative effects were difficult to accurately assess, as the rabbits were constantly handled and placed in and out of caging and restraint devices during dosing and early time point blood collection. Animals may also have become more comfortable/acclimated with moving around in the cage later in the day (4 hour time point and later) compared with initial and more frequent sampling at earlier time points. In addition, rabbits are a prey animal species and may be more alert in the immediate presence of people, especially non-acclimated animals. Many studies evaluating pain/sedation in rabbits do so at a distance,⁴⁵ evaluate recorded video footage,^46–49 or even use telemetry to record vital signs.¹⁰ In addition to sedation and pain scoring, efficacy testing of Bup-TP application could be performed, such as with thermal analgesiometry⁵⁰ or a surgical model.^9,10,17,48

Alternative application methods may be considered for future pharmacokinetic studies. Given that skin thickness can vary, application of Bup-TP to other parts of the body, such as the ear pinnae, could affect absorption. Care was taken to apply Bup-TP to the cervical region just cranial to the intrascapular region and allowed to dry for 30 min, yet it is possible that the formulation may have been licked off and possibly ingested by the animal before full absorption into the skin. Placing a cone or collar may prevent rabbits from grooming/licking the application site. No grossly visible adverse skin reactions were seen at the application site in this study, but further investigation and histologic evaluation may be warranted.

Conclusion

According to our findings, a topical formulation of buprenorphine, Bup-TP, achieved a plasma blood concentration >0.25 ng/mL, starting at 0.5 hours after dosing, that was maintained up to 72 hours in adult New Zealand white rabbits. Due to depot formation within the stratum corneum, Bup-TP can be used as an extended-release therapy for prolonged analgesia. Adverse effects such as decreased appetite and fecal production were associated with Bup-TP, but typically resolved within 7 to 14 days with supportive care. Depending on the nature of the study or surgical procedure that may cause pain to a rabbit, the adverse effects may outweigh the benefits that come with a topically administered, long-lasting buprenorphine formulation. Further study on efficacy, such as Von Frey testing and observational grimace scaling for rabbits, is needed to assess practical use as a surgical analgesic in rabbits.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This work was internally funded. For pharmacokinetic and MS core assays, research reported in this study was supported by the National Cancer Institute under Grant P30 CA046592.

Generalizability/Translation

Finding of this study may be applied to general rabbit medicine and rabbit surgery as a refinement to analgesia. Furthermore, findings from this study can be used to tailor additional research in rabbits and other species to optimize dosing and administration of Bup-TP in other species.

Protocol Registration

A protocol was not registered prior to the study.

Data Access

Data can be supplied with permission.