Pharmacokinetics and Adverse Effects of a Long-Acting Transdermal Buprenorphine Formulation in Rats (Rattus norvegicus)
Rats regularly undergo surgical procedures that may result in pain. Alleviation of unnecessary pain is an ethical and regulatory responsibility. Buprenorphine is an opioid analgesic commonly used in rats and requires dosing every 6 to 8 h to be effective. Frequent administration is time consuming and may increase stress, post-surgical pain, and dehiscence in rats, making the use of long-acting formulations an attractive alternative. A transdermal buprenorphine solution (TBS), FDA approved for use in felines, is commercially available and effective for up to 96 h. We hypothesize that a single dose of TBS in rats will result in clinically relevant plasma buprenorphine concentrations (greater than 1 ng/mL) for up to 96 h. To test this, 39 rats were randomly assigned to the following treatment groups: low dose (LD; 5 mg/kg; n = 6 females, 6 males), high dose (HD; 10 mg/kg; n = 6 females, 6 males), and vehicle control (CON; n = 7 females, 8 males). TBS or anhydrous ethanol (CON) were topically applied. Blood was collected at 4, 24, 72, 96, and 168 h postadministration, and buprenorphine concentrations were determined via HPLC-MS. To quantitatively assess adverse effects, daily fecal output, food intake, and body weight were measured, and observations of hematuria and skin lesions were documented. Plasma buprenorphine concentrations exceeded 1 ng/mL in all TBS rats at 4, 24, 48, and 72 h. No rats experienced serious adverse effects or developed gross lesions at the application site. The HD group had decreased fecal output compared with CON. Both TBS groups had reduced weight gain compared with CON. These results suggest that TBS dosed at 5 to 10 mg/kg could provide analgesia for up to 3 d in rats, and administering a lower dose mitigates some adverse effects.
Introduction
Rats regularly undergo surgical procedures both as experimental models in research and as companion animals with surgically treated conditions. Any surgical procedure has the potential to result in pain which is often managed with pharmacological interventions. Besides being an ethical and regulatory responsibility, alleviation of unnecessary pain in research animals is imperative for generating reliable and reproducible data. Pain is a stressor3,4 that can have significant impacts on an animal’s physiologic state if left unmanaged. Untreated pain can increase tissue healing time,5 alter heart rate,6 influence endocrine regulation,5 reduce immune function,5 and cause changes in rodent behavior such as reduced food and water intake, reduced sleep,4 and a reduction in social activities, (grooming,7 nest building,7 and/or burrowing8).
Buprenorphine is a partial mu-agonist opioid commonly used for the management of postsurgical pain in rats and many other laboratory animal species. Despite being the longest-acting opioid used in rodent pain management, the standard buprenorphine formulation (Bup-HCL) requires repeat dosing every 6 to 8 h to maintain an analgesic effect.1 This necessitates the handling of rats 3 to 4 times daily during the postoperative period, increasing restraint-associated stress while potentially exacerbating surgical pain from tension placed on incisions during restraint. This repeat application of tension to the surgical site, in conjunction with delayed healing secondary to stress,10–12 may amplify the risk of dehiscence. This frequency of administration is time consuming for researchers and introduces many opportunities for errors in dosing and logging the use of controlled drugs, especially when working with large numbers of animals.
Buprenorphine is a highly effective analgesic in rats with a large margin of safety, considering that the intravenous LD50 is well over a hundred times greater than dosages used clinically.13 However, its administration in rats is associated with pica,14,15 decreased weight gain,9 decreased food intake, reduced fecal output, sedation, and excessive grooming that may result in skin lesions.16 Extended-release formulations of buprenorphine may mitigate or eliminate these adverse effects. Administration of extended release buprenorphine formulations before plantar incision in rats did not result in these complications, with sedation being the only observed adverse effect.17 Another study18 found that the only adverse event in rats receiving extended-release buprenorphine after surgery was excessive grooming of the front limbs, attributed to nausea behavior, with no instances of pica or weight loss.
Currently, only 2 formulations of extended-release buprenorphine are available for use in rodents: buprenorphine XR and buprenorphine base lab in polymer (Bup-lab), previously termed sustained-release buprenorphine (Bup-SR). Both have barriers to access and use. Bup-lab is only available as a compounded product, is not pharmaceutical-grade, and is not FDA approved or indexed for use in rats.19,20 Buprenorphine XR is FDA approved for use in mice, rats, and ferrets, so it can be ordered by investigators with a research Drug Enforcment Agency license. However, in the US, the price per dose of buprenorphine XR in rats is more than twice that of Bup-lab.21 Furthermore, both buprenorphine XR and Bup-lab have been associated with skin lesions at the injection site in rodents.22–24 These reactions could preclude their use in studies investigating inflammatory mediators. A transdermal buprenorphine would theoretically eliminate this risk of injection site lesions. A needleless delivery system would also be beneficial in infectious disease research, where the use of sharps should be minimized.
A long-acting transdermal buprenorphine solution (TBS) has been approved by the FDA for use in felines. TBS comes in single-dose applicator tubes containing 0.4 or 1.0 mL of 20 mg/mL TBS. The solution is applied 1 to 2 h before surgery and has been demonstrated to be safe and effective for managing postoperative pain for up to 96 h in cats enrolled in a phase 3 clinical study.2 In a study25 investigating the pharmacokinetics of TBS in cats, the mean buprenorphine plasma concentration for all dosage groups remained at clinically relevant levels for 72 h, and in the moderate- and high-dose groups, through 168 h. Since gaining FDA approval in 2022, there has been an interest among laboratory animal medicine professionals in exploring the utility of TBS in research species. Studies have been published demonstrating buprenorphine plasma concentrations above 1 ng/mL for 72 h or more in mice following administration of TBS.26,27 The plasma concentration of 1 ng/mL threshold of buprenorphine was assumed to be efficacious in rodents28,29 and was estimated based on the minimum concentration that produced analgesia in humans,28 and it has since been confirmed to reduce thermal sensitivity in rats.30 Although TBS administration has been shown to attenuate thermal sensitivity in rats with no lethal effects,31 to the authors’ knowledge, there are no published studies of the pharmacokinetics in rats.
The goals of this study are to evaluate the pharmacokinetics of TBS in rats; estimate the duration that plasma concentrations exceed 1 ng/mL, the assumed therapeutic threshold for buprenorphine in rodents; and quantitatively assess common adverse effects associated with TBS in rats. Specific adverse effects evaluated included the impact of TBS administration on body weight, food intake, fecal output, and grooming, since these changes in these parameters have been documented in rats following the administration of buprenorphine.9,16 Incidence of hematuria was also assessed due to anecdotal accounts of hematuria occurring in multiple rats following administration of TBS and reports in the literature32 of rats developing hematuria after buprenorphine dosing. We hypothesized that a single dose of TBS in rats would result in clinically relevant plasma buprenorphine concentrations (>1 ng/mL) in high (10 mg/kg)-dose and low-dose (5 mg/kg) groups for up to 96 h. In addition, we hypothesized that the high-dose group would have significantly reduced fecal production, food intake, and weight gain, and an increased incidence of hematuria and skin lesions from overgrooming compared with the low-dose and control group.
Methods
Animals.
Male and female Sprague–Dawley rats (20 males and 23 females; Rattus norvegicus; Crl:CD[SD] IGS; Charles River) generated from healthy, unmanipulated adult rats bred in-house or donated were used in this study. Rats were 8 to 15 wk old, with females weighing 150 to 300 (average = 212.3 ± 7.7) g and males weighing 250 to 400 (average = 313.1 ± 13.2) g at the start of the study. Rats were free of pinworms (Syphacia muris, Syphacia obvelata, Aspiculuris tetraptera), fur mites (Myocoptes, Radfordia, Myobia), rat coronavirus, rat Theiler virus, Kilham rat virus, rat parvovirus, Toolan H1 virus, rat minute virus, Sendai virus, pneumonia virus of mice, and Mycoplasma pulmonis, as monitored by soiled-bedding sentinels and quarterly exhaust air dust testing. Rats were housed in IVCs (21.3 cm [high] × 34.6 cm [long] × 39.6 cm [wide]; GR900; Tecniplast) on ALPHA-dri bedding (1/4 in., irradiated; ALPHA-dri; Lab Supply) with red tunnel tubes (K3325; Red, Certified, Rat Tunnel; Bio-Serv) and autoclaved cardboard glove boxes as enrichment and nesting material. The room had a 12:12-h light:dark cycle (lights on at 0700; lights off at 1900) at 70 to 74 °F (21.1 to 23.3 °C) and 30% to 70% relative humidity. Rats were provided food (5V0F; Select Rodent 50 IF/6F Auto; LabDiet) and reverse osmosis-filtered water ad libitum. All experiments were approved by the University of North Carolina at Chapel Hill IACUC. All rats were treated in accordance with the Guide for the Care and Use of Laboratory Animals3 in an AAALAC-accredited facility.
Experimental design.
A pilot study was conducted to inform the selection of a low and high dose of TBS to be administered for the main study. In addition, a pilot was necessary to ensure no lethal effects were observed at the tested dosages and that sufficient blood volume could be collected at each time point using the described venipuncture methods. For the pilot study, 15 rats (9 females, 6 males) were randomly assigned to the following TBS (Zorbium; Elanco)-dose groups: 5 mg/kg (n = 3), 7.5 mg/kg (n = 3), 10 mg/kg (n = 3), 12.5 mg/kg (n = 3), and 15 mg/kg (n = 3) using randomizer software. Rats were temporarily single housed for the duration of data collection. Body weight and food intake were measured daily beginning on the day of dosing (D0) and continuing through 7 d postadministration (D7). All groups were examined for skin lesions and blood was collected at the following time points: 4, 24, 48, 72, 96, and 168 h. Time points for plasma concentration analysis were selected based on similar time points in other publications investigating pharmacokinetics of long-acting buprenorphine formulations in rodents.17,26,33
For the main study, 28 rats (14 females, 14 males) were randomly assigned by sex to 1 of the following treatment groups: low dose (LD; 5 mg/kg; n = 12 [6 females, 6 males]), high dose (HD; 10 mg/kg; n = 12 [6 females, 6 males]), and vehicle control (CON; n = 4 [2 females, 2 males]). An additional 11 rats (5 females, 6 males) used in the pilot study were enrolled as controls in the main study. These rats met age (8 to 15 wk) and weight (female: 150 to 300 g; male: 250 to 400 g) inclusion criteria and underwent a 3-wk washout period before use in the main study. Group size was chosen based on n per time point in previously published studies investigating plasma levels of buprenorphine after administration of injectable long-acting formulations in rodents.24,33–35 The 5- and 10-mg/kg doses were selected based on data from the pilot study showing adequate plasma concentrations at these doses, with 5 mg/kg being the lowest dose evaluated. The vehicle control was anhydrous ethanol (Decon Laboratories) because it is the solvent used in TBS.25 Rats were acclimated to individual housing for 3 d (D-5, -4, and -3) before the collection of data. Baseline measurements were recorded beginning 2 d before dosing, with the last measurement being taken the morning of treatment (D-2, -1, and 0) (Figure 1). Baseline body weight was calculated by averaging 3 measurements (D-2, -1, and 0). Food intake and fecal output data represent cumulative food consumed and grams defecated within the 24 h period before each time point. Initial food weight and removal of all fecal pellets from cages occurred D-2, with first baseline data collection being D-1, resulting in 2 measurements (D-1 and 0) to be averaged for calculation of baseline. Multiple baseline measurements were averaged to reduce the effect of day-to-day variation in eating, drinking, and defecation habits on baseline values. Rats were examined for skin lesions affecting the limbs or dorsal neck and underwent hematuria screening once before dosing (D-1) and at each study time point. Body weight, food intake, and fecal output were measured daily through D7 postdosing. Blood was collected from TBS groups at: 4, 24, 48, 72, 96, and 168 h. In a subcohort of 16 rats (LD: n = 6; HD: n = 6; and CON: n = 4,) body weights were measured weekly between D16 and D44 postdosing.
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
Clinical outcome measures.
Body weights, food intake, and fecal output were measured in grams using a scale with 0.01 g of sensitivity (APX-4001; Denver Instrument). Daily food intake was calculated by subtracting the weight of remaining pellets in the food hopper from the previous day’s weight, thereby determining grams consumed per day. Any food pellet pieces found within the cage were added to the remaining pellets before weighing. Daily fecal output was quantified by sifting through the bedding, collecting, and weighing all fecal pellets over a 24-h period. ALPHA-dri bedding was used to facilitate fecal pellet collection. Hematuria screening was performed by placing rats in an empty cage lined with white paper towel and leaving them undisturbed for 7 to 10 min. The same paper towel was used to wipe the bottom and sides of the cage and then inspected for urination. Urination was classified as having no hematuria (if clear to dark yellow), hematuria (if orange to red tinted), or unable to evaluate (if no urine was present). To evaluate application site skin lesions and those consistent with overgrooming of the limbs, the skin along the dorsal neck base was examined for erythema, excoriations, or scabbing. The limbs were examined similarly, with the addition of evaluating for swelling and lameness. Lesions affecting the limbs were attributed to overgrooming, while dorsal neck skin abnormalities were considered a reaction to TBS application. Overgrooming and application site lesion data was recorded as the presence or absence of a lesion. Histopathologic analysis and scoring of the severity of skin lesions were not performed.
Buprenorphine administration.
TBS or 0.75 mL/kg of CON (equivalent volume to 15 mg/kg of TBS) was applied to the skin at the dorsal base of the neck using a 200-μL micropipette. The administration volume of CON was selected based on the highest TBS dose tested and shown to have no lethal adverse effects during the pilot study. Hair was parted to access skin for administration but not removed. Immediately after dosing, rats were placed in an observation cage for 7 to 10 min for informal monitoring of excessive grooming of the application site and hematuria screening. No measures were taken to prevent grooming while the product dried.
Plasma buprenorphine concentration.
Approximately 300 μL of whole blood was collected from TBS groups at 6 time points: 4, 24, 48, 72, 96, and 168 h. Blood was not taken from the CON group. Blood was collected from the tail vein using a tail clip technique (4, 24, 48, and 72 h) or lateral tail vein venipuncture (96 and 168 h), although 1 female LD rat underwent lateral tail vein venipuncture in addition at 48 and 72 h due to insufficient volume collection via tail clip. For the tail clip, rats were warmed for 60 to 90 s under a heat lamp before removing 1 to 2 mm of tail tissue from the tail tip, using a No. 10 scalpel blade. For subsequent collections, the same procedure was followed except only the scab was removed from the tail tip to avoid amputating additional tissue. For lateral tail vein venipuncture, rats were briefly anesthetized with 2% to 4% isoflurane via inhalation, and a 25-g needle was placed into the vein, establishing blood flow. TBS groups underwent lateral tail vein venipuncture for time points 96 and 168 h to reduce trauma to the tail tip associated with the tail clip. At each time point, 100 to 400 μL of whole blood per rat was collected into EDTA tubes (Microvette 500 K3 EDTA; Sarstedt). Blood was centrifuged at 3.5-k RPM for 12 min (Eppendorf 5452 MiniSpin; Eppendorf) to separate plasma. Plasma was transferred to microtubes (Eppendorf Safe-Lock Tubes; Eppendorf) and stored at −80 °C for future analysis.
For HPLC MS/MS analysis, samples were run in 3 analytical batches (1 batch per day). Buprenorphine standard spiking solutions were prepared in 50:50 deionized water:acetonitrile to give concentrations in plasma ranging from 0.2 to 200 ng/mL. The buprenorphine limit of detection for the method was 0.05 ng/mL. The buprenorphine plasma samples and standards (100 μL) were fortified with internal standard (50 ng/mL terfenadine). Acetonitrile (1 mL) was added to precipitate the plasma proteins, and the mixture was vortexed (approximately 10 s) and centrifuged at 15,000 × g for 5 min. The organic layer was transferred to a clean test tube and evaporated to dryness under nitrogen in a water bath set at 50 °C. The samples were reconstituted in dilution solvent (50/50 5 mM ammonium acetate/acetonitrile) and analyzed by HPLC MS/MS. Matrix-matched standards and quality control (QC) samples were prepared using blank control plasma. QCs were prepared at 3 concentrations (0.5, 5, and 50 ng/mL) in triplicate and ran with each batch of samples. QCs had less than 15% variability per set on each of the 3 d of sample analysis.
Chromatographic separation of buprenorphine and the internal standard from the plasma matrix was achieved using a Shimadzu HPLC system consisting of 2 Shimadzu LC20-AD pumps, an SIL20-AC HT autosampler, and a DGU-20A3-3channel in-line degasser and controller with a 100 × 2-mm Luna C18 reverse phase column (Phenomenex) at ambient temperature. The mobile phase consisted of 5 mM ammonium acetate and acetonitrile, each fortified with 0.1% formic acid. The compounds were analyzed using a gradient elution profile in which mobile phase B was held at 30% for 1 min, then increased to 80% over 4 min, held at 80% for 0.5 min, then returned to 30% and equilibrated for 2.5 min. Mass detection was carried out with an Applied Biosystems 5500 QTRAP triple quadrupole ion trap mass spectrometer equipped with an electrospray ionization source operated at a potential of 5 kV at 450 °C operating in the MRM mode. Data were collected using Analyst 1.6.2 (Applied Biosystems). The following mass transitions of the compounds were monitored: buprenorphine (m/z: 468.4 to 396.1) and terfenadine (m/z: 472.4 to 436.2).
Statistical analysis.
Daily percent change from baseline for fecal output, food intake, and body weight was calculated for individual rats by dividing the weight of feces defecated, food consumed, and body weight at time points D1 to D7 by the corresponding baseline values. Individual values were averaged to determine group mean percent change from baseline in body weight (g), food intake (g/day), and fecal output (g/day) for each time point, and overall average change was determined by calculating the mean of data from D1 to D7 for each group. The overall average at each time point is reported as group mean ± SEM. Differences between groups in plasma buprenorphine concentration, body weight, food intake, and fecal output were analyzed using a linear mixed model. The model accounted for multiple observations within each rat and included all possible interactions (time, sex, and group). Model-based linear contrasts were performed to make the following comparisons with and without sex for each clinical outcome: LD compared with CON, HD compared with CON, and LD compared with HD (SAS Proc GLM; SAS Institute). A P value less than 0.05 was considered the threshold for significance for all comparisons. Assumptions of normality appeared to be met based on visual assessment of quantile-quantile plots. Pharmacokinetic analysis was performed using a naïve pooled-data approach, wherein the group mean plasma buprenorphine concentrations at each time point were used to estimate pharmacokinetic parameters using noncompartmental analysis using PKSolver 2.0. Parameter estimates are reported with SD.
Results
Pilot study.
No rats dosed with TBS experienced lethal complications related to drug administration for the duration of the study. One rat, from the 15-mg/kg TBS group, was euthanized before the 2-h time point due to a tail injury unrelated to blood collection. No rats developed skin lesions at the TBS application site. Two rats, 1 from the 7.5-mg/kg and 1 from the 10-mg/kg TBS group, developed mild erythema and swelling of 1 front paw that resolved within 5 and 3 d, respectively.
For the pilot study, a total of 85 plasma samples were analyzed for buprenorphine concentration. For the 5-, 7.5-, 10-, and 12.5-mg/kg dose groups, 3 plasma samples were analyzed per time point. Five plasma samples were missing for the 15-mg/kg group (1 from the 24-, 48-, 72-, 96-, and 168-h time points). The mean plasma concentration for all dose groups (5, 7.5, 10, 12.5, 15 mg/kg) exceeded the assumed therapeutic threshold of 1 ng/mL, at 4, 24, 48, 72, and 96 h after TBS administration (Table 1).
| Pilot: mean plasma buprenorphine concentration (ng/mL) | |||||
|---|---|---|---|---|---|
| 5 mg/kg (n = 3) |
7.5 mg/kg (n = 3) |
10 mg/kg (n = 3) |
12.5 mg/kg (n = 3) |
15 mg/kg (n = 2) |
|
| 4 h | 33 ± 10 | 13 ± 4.6 | 16 ± 10 | 19 ± 9.4 | 52 ± 10 |
| 24 h | 31 ± 50 | 29 ± 38 | 120 ± 120 | 110 ± 61 | 33 ± 30 |
| 48 h | 19 ± 16 | 13 ± 12 | 46 ± 26 | 27 ± 6.7 | 53 ± 11 |
| 72 h | 11 ± 9.5 | 14 ± 12 | 25 ± 23 | 12 ± 16 | 75 ± 7.6 |
| 96 h | 3.7 ± 4.0 | 5.3 ± 4.4 | 8.0 ± 6.2 | 55 ± 7.9 | 18 ± 7.9 |
| 168 h | 1.6 ± 1.5 | 0.9 ± 0.8 | 1.5 ± 0.7 | 3.4 ± 3.4 | 0.6 ± 0.3 |
Buprenorphine concentrations above 1 ng/mL in plasma are assumed to produce an analgesic effect. The TBS doses of 5 and 10 mg/kg were selected based on these data. Data are presented as group mean ± SD.
Main study.
Group mean percent change from baseline as an overall average and at each time point for each clinical outcome are presented in Table S1. All group comparisons of body weight, food intake, and fecal output with corresponding P values are presented in Table S2.
Body weight.
A difference was detected in the overall average percent change in body weight for the LD and HD groups compared with the CON group, with both TBS groups exhibiting −4.26 ± 0.52% and −4.13 ± 0.52% weight gain relative to CON, respectively (all P < 0.0001, Figure 2A) However, no difference in overall average weight change was found between the TBS groups (P = 0.8117). Differences in percent change in body weight were observed based on time points (Figure 2C). On D1, the LD group had 1.54 ± 0.72% greater weight gain compared with the CON group (P = 0.0326). From D2 to D7, both TBS groups had lower percent weight increases compared with the CON group (all P < 0.05, Table S2). No differences in weight gain between the LD and HD groups were observed at any time point (all P > 0.14). When evaluating sex (Figure 2B), both male and female LD and HD groups showed reduced weight gain compared with their sex-matched CON group (all P < 0.01, Table S2). Among males, the LD group had 1.71 ± 0.77% increased weight gain compared with the HD group (P = 0.0276). Conversely, in females, the LD group had lower weight gain (−1.98 ± 0.77%) compared with the HD group (P = 0.0113).
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
Food intake.
Both TBS groups had reduced food intake (LD: −16.42 ± 5.08%; HD: −24.02 ± 5.08%) compared with the CON group (all P < 0.005, Figure 3A) when comparing average change from baseline for duration of data collection. There was no difference between TBS groups in average change in food consumption (P = 0.1564). On D6 and D7, there were no differences in food intake between groups (all P > 0.08, Figure 3C). However, on D1, 2, 4, and 5 both TBS groups had decreased intake relative to the CON group (all P < 0.05), with the HD group also having reduced intake on D3 compared with the CON group (−24.01 ± 6.37%, P = 0.0002) and LD group (LD compared with HD = 14.85 ± 6.71%, P = 0.0273). No differences in food intake between TBS groups were detected at any other time point (all P > 0.08). When evaluating sex (Figure 3B), the male HD, female HD, and female LD groups exhibited significantly reduced food intake compared with their sex-matched CON groups (all P < 0.05, Table S2) There was no difference between the LD and HD groups for either sex (females: P = 0.8385: males: P = 0.0818).
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
Fecal output.
The HD group had −16.48 ± 7.12% reduced fecal output relative to the CON group (P = 0.0215) when comparing the average change from baseline (Figure 4A). No differences in overall average fecal output were detected between the LD and CON groups (P = 0.7695) or the TBS groups (P = 0.0560). However, differences in daily fecal output were appreciated when analyzed by time point (Figure 4C). The HD group had reduced output compared with the LD (LD compared with HD = 42.34 ± 10.25%, P < 0.0001) and CON (−60.50 ± 9.74%, P < 0.0001) groups at D1. In addition, at D2 the LD group had reduced output relative to the CON group (19.71 ± 9.61%, P = 0.0409) and on D4-D5 the HD group had decreased defecation compared with the CON group (−18.96 ± 9.61%, P = 0.0492; −12.69 ± 9.61%, P = 0.0265). When evaluating sex effects on average fecal output (Figure 4B), the male HD group had reduced output compared with the male LD (LD males compared with HD males = 21.86 ± 10.61%, P = 0.0405) and CON (−20.01 ± 9.93%, P = 0.0450) groups. There was no difference in average fecal output between the female TBS and CON groups (all P > 0.20).
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
Health observations.
None of the rats developed skin irritation at the treatment application site or experienced lethal complications during data collection (Table 2). Hematuria was recorded in a total of 6 rats, which included 1 each from the CON and LD groups and 4 belonging to the HD group. No rat exhibited hematuria at more than 1 time point. Skin lesions consistent with overgrooming developed in 1 CON rat, 4 LD rats, and 4 HD rats. Overgrooming lesions were mild, always superficial, never impacted mobility, and fully resolved within 1 to 4 d without the need for treatment.
| Group and incidence of clinical signs | ||||||
|---|---|---|---|---|---|---|
| LD (n = 12) | HD (n = 12) | CON (n = 12) | ||||
| Hematuria | 1 (8.3%) | 4 (33.3%) | 1 (6.7%) | |||
| Skin lesions | 4 (33.3%) | 4 (33.3%) | 1 (6.7%) | |||
| LD F (n = 6) | LD M (n = 6) | HD F (n = 6) | HD M (n = 6) | CON F (n = 6) | CON M (n = 6) | |
| Hematuria | 1 (16.7%) | 0 (0.00%) | 1 (16.7%) | 3 (50.0%) | 1 (14.3%) | 0 (0.0%) |
| Skin lesions | 2 (33.3%) | 2 (33.3%) | 3 (50.0%) | 1 (16.7%) | 0 (0.0%) | 1 (12.5%) |
Reported as number of rats affected with percent of group affected in parentheses. Statistical comparisons between groups for this data were not performed. F, female; M, male.
Buprenorphine concentrations and pharmacokinetic analysis.
A total of 144 plasma samples were analyzed representing 12 (6 female + 6 male) rats per dose group per time point. All TBS rats (n = 24) had buprenorphine plasma concentrations exceeding 1 ng/mL at 4, 24, 48, and 72 h postadministration. Half of the rats (8 HD + 4 LD) had plasma concentrations above the threshold (i.e. 1 ng/mL) at 96 h. No LD group rats had plasma buprenorphine concentrations above 1 ng/mL at 168 h, while 3 of 12 (2 males, 1 female) rats in the HD group exceeded 1 ng/mL at 168 h after application. Mean plasma concentrations by group at each time point are shown in Figure 5 and Table 3. There were no significant differences between groups in buprenorphine plasma concentration at any time point when comparing HD to LD and HD males to LD males.The LD female group had significantly increased concentration relative to HD females at 24 h only (P = 0.0180, Table S3).
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
| Estimated pharmacokinetic parameters | ||||||
|---|---|---|---|---|---|---|
| LD F (n = 6) | LD M (n = 6) | HD F (n = 6) | HD M (n = 5) | |||
| ke (h−1) | 0.022 ± 0.006 | 0.029 ± 0.005 | 0.024 ± 0.007 | 0.026 ± 0.009 | ||
| t1/2 (h) | 30.9 ± 11.3 | 23.9 ± 5.8 | 28.4 ± 9.4 | 29.7 ± 12.2 | ||
| AUC0-last (ng/mL·h) | 1,554 ± 1,697 | 1,366 ± 851 | 1,262 ± 491 | 2,250 ± 1,736 | ||
| AUC0-∞ (ng/mL·h) | 1,567 ± 1,704 | 1,380 ± 847 | 1,286 ± 482 | 2,310 ± 1,713 | ||
| Cmax (ng/mL) | 51.3 ± 75.1 | 45.7 ± 53.6 | 31.9 ± 76.7 | 72.6 ± 51.3 | ||
| Tmax (h) | 24 | 4 | 4 | 4 | ||
| MRT0-∞ (h) | 29.0 ± 6.8 | 31.9 ± 7.1 | 41.3 ± 8.7 | 33.9 ± 4.6 | ||
| Mean plasma buprenorphinencentration (ng/mL) | ||||||
| 4 h | 24 h | 48 h | 72 h | 96 h | 168 h | |
| LD (n = 12) | 32.7 ± 11.5 | 32.7 ± 16.6 | 7.4 ± 2.2 | 4.8 ± 1.4 | 0.9 ± 0.2 | 0.4 ± 0.04 |
| Females (n = 6) | 19.6 ± 4.9 | 50.1 ± 32.5 | 4.0 ± 1.1 | 3.2 ± 0.8 | 0.7 ± 0.1 | 0.3 ± 0.1 |
| Males (n = 6) | 45.8 ± 22.1 | 15.3 ± 6.1 | 10.9 ± 3.9 | 6.3 ± 2.5 | 1.1 ± 0.3 | 0.4 ± 0.1 |
| HD (n = 12) | 52.3 ± 19.9 | 24.8 ± 8.0 | 7.9 ± 1.6 | 14.5 ± 5.9 | 1.3 ± 0.2 | 0.8 ± 0.2 |
| Females (n = 6) | 31.9 ± 11.0 | 15.2 ± 3.5 | 8.0 ± 2.9 | 10.8 ± 3.3 | 1.5 ± 0.4 | 0.6 ± 0.1 |
| Males (n = 6) | 72.7 ± 38.2 | 34.4 ± 15.2 | 7.9 ± 1.6 | 18.2 ± 11.6 | 1.1 ± 0.1 | 1.0 ± 0.4 |
Pharmacokinetic parameters are presented with ± SD, and mean plasma buprenorphine concentrations are shown with ± SEM.
One male rat in the HD group was excluded from the calculation of the pharmacokinetic parameters due to being identified as an outlier by the Dixon outlier test. The maximum concentration measured (Cmax) for the female LD (n = 6) and HD (n = 6) groups was 51.3 ± 75.1 ng/mL and 31.9 ± 76.7 ng/mL, respectively, and for the male LD (n = 6) and HD (n = 5) groups was 45.7 ± 53.6 ng/mL and 72.6 ± 51.3 ng/mL, respectively. The observed time to peak drug concentration (Tmax) was 24 h for the female LD group and 4 h for all other groups. There were no detected differences in any calculated pharmacokinetic parameters (elimination constant [ke], t1/2, AUC, Cmax, and mean response from time 0 to infinity [MRT0–∞]) between groups. Additional estimated pharmacokinetics are summarized in Table 3.
Discussion
This study demonstrated that plasma buprenorphine concentrations assumed to be therapeutically relevant are reached within 4 h and maintained for at least 72 h, following the application of TBS at doses of 5 and 10 mg/kg in Sprague–Dawley rats, regardless of sex. Although no severe adverse effects were observed, TBS administration resulted in decreased weight gain, food intake, fecal output, and a higher incidence of hematuria and overgrooming skin lesions relative to CON groups.
Our findings suggest administration of a lower dose of TBS (i.e., 5 mg/kg) can mitigate many of these adverse effects. Overall, the LD group had a similar fecal output to the CON group, fewer days of reduced food intake than the HD group, and fewer occurrences of hematuria compared with the HD group. Similarly, findings in other studies support that dose reduction may ameliorate impacts on fecal output and food intake36 and lessen the frequency of self-injurious behavior.36,37 All adverse effects attributed to TBS administration, except impact on weight gain, were transient with differences between TBS groups and CON groups resolving within the 7-d study period. Resolution of these effects, excluding reduced weight gain, coincided with when the plasma buprenorphine concentrations fell below 1 ng/mL for 50% or more of rats in the LD and HD groups after the 72- and 96-h time points, respectively. Peak severity of adverse effects also correlates with the timing of peak plasma buprenorphine concentrations. The lowest food intake and fecal output occurred on D1 and Tmax for LD females and for all other groups was 24 and 4 h, respectively.
Unlike the other adverse effects, the reduction in weight gain relative to CON observed in the TBS groups did not resolve by D7, suggesting TBS administration may have a long-lasting impact on growth rate when administered in young rats. Most rats used in biomedical research are between the ages of 8 to 12 wk.38 We elected to use 8- to 15-wk-old Sprague–Dawley rats for this study to assess TBS as an analgesic option in the stock and age of rats most likely to undergo a research-related surgical procedure. Rats are in a phase of rapid growth for the first 24 wk of life.39 Juvenile rats were shown to have a lasting reduction in weight gain and bone elongation rate compared with controls, after just 3 d of fasting.40 The reduction in food intake experienced by the TBS groups, during this critical time of growth, may have impacted bone elongation rates resulting in growth inhibition and the observed reduced weight gain. The fasted rats had recovered similar bone elongation rates and body weight to controls by 3 and 4 wk, respectively.40 Once the potential lasting impact on weight gain following TBS application was appreciated, the remaining rats of the final cohort were maintained for longer observation, and body weight was recorded weekly from 16 to 44 d postadministration (Table S4). The small size of the cohort prohibits statistical analysis; however, the data suggest that TBS-dosed groups have similar weight to sex-matched control groups by 23 d, similar to the recovery described in the fasted rats in the previously referenced study.
Differences between male and female TBS groups’ pharmacokinetic parameters were observed with the LD female group having delayed peak plasma concentration relative to all other groups. The observed Tmax for LD females occurred at 24 h, rather than at 4 h. Peak plasma concentration being at a later time point (24 h) is consistent with the finding that LD females had a higher plasma buprenorphine concentration than HD females at 24 h (P = 0.0180). In another study,24 the opposite sex-based differences were observed following parenteral administration of buprenorphine XR in rats, with LD females having earlier Tmax and larger volume of distribution (Vd) than males. Our findings further support the impact of sex on buprenorphine metabolism, with additional differences based on the administration route.
In addition, there was marked individual variation in plasma concentrations within groups at each time point. Interindividual differences in the rate of absorption are a known characteristic of transdermal opioid formulations in people and are attributed to disparities in thickness of the stratum corneum, skin hydration, skin injuries or diseases, and variation in body temperature between individuals.41 Application of transdermal buprenorphine patches in dogs,42 Yorkshire pigs,43 and Göttingen minipigs44 has similarly resulted in variation in plasma concentration between individuals. Sprague–Dawley rats are an outbred stock, so variation in factors affecting transdermal absorption is expected. Due to this inherent variability related to the dosing route and distribution of time points, absorption half-life was not calculated.
Although the duration of anesthesia was never longer than 5 min, altered blood flow to the liver during anesthesia could impact buprenorphine metabolism.45 Anesthesia could also alter the perfusion of the skin and subcutaneous tissues, impacting the absorption of transdermal formulations. However, this is expected to have had minimal impact in this study since anesthesia was brief (less than 5 min) and only performed at the 96- and 168-h time points. In addition, isoflurane anesthesia was used and has been shown to have no significant effect on skin perfusion in mice.46 Variation between groups due to anesthetic effects was limited by subjecting all TBS rats to identical anesthesia regimens and frequency, with the exception of 1 LD female who required anesthesia for blood collection purposes at 2 additional time points. Another factor contributing to variability in this study was that rats were not prevented from grooming following TBS application, so likely received a small portion of the dose via transmucosal absorption during grooming. TBS label instructions recommend a 30-min drying time in cats before allowing contact or grooming of the application site. Restricting grooming in rats for that amount of time is not practical in a research setting, which is why no measures were taken to restrain or distract rats during the TBS drying period in this study. Although not directly assessed, we found that TBS appeared dried after 10 min (Figure 6). The single-use packaging of TBS requires cutting open the crimped end of the tube to access the product for dosing of multiple animals or transfer to a storage container. Since ethanol is the vehicle in TBS, care must be taken to ensure evaporation is minimized during dosing and/or transfer of TBS as loss of ethanol could alter buprenorphine concentration.
Citation: Journal of the American Association for Laboratory Animal Science 64, 3; 10.30802/AALAS-JAALAS-24-136
The limitations of this study include lack of plasma buprenorphine measurements before 4 h postadministration and between 4 and 24 h, limitations of hematuria detection and fecal output quantification methods, single-housing of rats, small sample size, and absence of a blood collection sham control. A study27 describing TBS use in mice has shown buprenorphine levels greater than 1 ng/mL in several undosed cage mates, suggesting transmucosal exposure can occur during grooming. If rats absorb TBS during grooming, especially before the product drying, there could be an earlier peak in plasma buprenorphine concentrations that went undetected. Adding an earlier time point, and time points between 4 and 24 h for plasma buprenorphine quantification, would allow more accurate calculation of the true Tmax and provide information for estimating the onset of analgesic effects. Given this limitation, the observed difference in Tmax between sexes may have been an artifact. The method of hematuria detection limited the ability to determine true prevalence because data could not be collected from rats that did not urinate within the 10-min observation period, and the presence of blood in urine was determined visually. Porphyrin could be deposited and mixed with urine, falsely giving the appearance of hematuria. This is a potential explanation for the observation of hematuria in a CON rat. Urinary tract inflammation, unrelated to experimental manipulations, could also result in hematuria. However, hematuria was previously reported in rats following repeated high doses of Bup-HCL,32 supporting that the observations of hematuria seen in treated rats during this study were accurate and associated with buprenorphine administration.
Although the bedding used in this study is less absorbent than corncob bedding,47 it still resulted in desiccation of fecal pellets, falsely reducing the weight of the pellets. In addition, there can be variation between rats in daily fecal moisture content and fecal output, independent of experimental manipulations. To control for variation between rats and moisture loss of pellets, all rats received the same bedding, and group comparisons were made based on the group mean percent change from baseline, rather than directly comparing grams per day defecated. Although this method of analysis accounts for inherent differences between rats, it cannot fully negate the possibility of day-to-day variation in output within individual rats. Nausea is not the only cause of overgrooming in rats. Rats are known to pathologically overgroom due to the stress of social isolation.48 Stress is the likely cause of excessive grooming lesions seen in the CON rat and could have contributed to lesions observed in treated rats, further highlighting the importance of having a control group for comparison.
Small group size precludes quantitative analysis of normality, requiring visual assessment of data distribution, an inherently more subjective method. In addition, small group size combined with large interindividual variability in plasma buprenorphine concentration may have prevented the detection of significant differences in pharmacokinetic parameters between groups. Finally, pain associated with tail snip may have contributed to differences seen between the CON and TBS groups. Tail snip results in minor, but permanent, tissue damage to the tail tip, so we elected against performing sample collection from CON rats since only TBS group samples would be analyzed. Not sampling CON rats also enabled the reuse of rats that had previously undergone tail snip collection during the pilot experiments. For studies involving repeat blood collection, options that do not result in permanent tissue damage, such as dorsal pedal sampling, could be used in all experimental and control groups. The addition of a sham control for venipuncture may reduce the influence of collection stress or pain. The loss of tissue from the tail tip due to sample collection was also the reason that rats used in the pilot could only be assigned to the control groups in the main study since blood was not collected from CON rats.
Overall, this study suggests that TBS is a viable option for providing analgesic coverage for 72 h or more in rats. In the US, TBS is generally less expensive than other long-acting formulations used in rodents, application was simple and could be performed in awake animals, and measured adverse effects were mild and most transient. The adverse effects observed in this study (i.e., reduced weight gain, food intake, and fecal production) are common adverse effects of buprenorphine, not unique to transdermal formulations.9,37,49 Because these negative impacts are associated with other forms of buprenorphine, TBS should continue to be explored as an alternative to standard buprenorphine. Regardless of formulation, investigators should consider these adverse effects when using models that require multimodal analgesia. Although this study demonstrates presumed adequate plasma buprenorphine concentration, the analgesic efficacy should be directly evaluated in a surgical pain model before endorsing routine use of TBS in rats. This is especially critical since there are limited studies directly demonstrating that plasma buprenorphine concentrations above 1 ng/mL in rats do result in an analgesic effect. Future studies could assess the thermal and mechanical antinociceptive ability of TBS, the presence and severity of facial grimace, and effects on locomotion in rats postlaparotomy to ensure the doses and plasma concentrations described in this paper correlate with the expected level and length of clinical efficacy. According to a recent publication, rats given the same dose of TBS as the HD group (10 mg/kg) in this study had a reduction in thermal sensitivity relative to controls only at 1 and 8 h postadministration,31 further demonstrating the need for additional evaluation of efficacy. The same clinical outcomes (i.e., weight gain, food intake, fecal output, and lesions from overgrooming) could be assessed to determine if there is a difference in the frequency and severity of adverse effects when pain is present. A study50 involving a surgical model of traumatic brain injury demonstrated increased weight gain in the rats treated with buprenorphine compared with those that were not given buprenorphine after surgery, suggesting that weight loss, and other adverse effects, may not be observed in the context of pain. Finally, although few rats exhibited hematuria, confirmation and clinical significance of hematuria following TBS dosing should be investigated as this could be indicative of renal toxicity.
Supplementary Materials
Table S1. Average change from baseline in body weight, food intake, and fecal output for study duration (D1-D7) and at each time point. Reported as group mean with SEM.
Table S2. Group comparisons of overall average change from baseline in body weight, food intake, and fecal output for study duration and at each time point. Presented as difference between group means with SEM. Group mean values are shown in table
Table S3. Group comparisons of plasma buprenorphine concentrations at each time point with SEM. LD females had greater plasma buprenorphine concentration than HD females at 24 h (P = 0.0180). There were no differrences between groups otherwise.
Table S4. Change from baseline body weight for a cohort of 16 rats was recorded weekly for an additional 37 days beyond the end of data collection for the main study. Data for long-term weight tracking is presented here as group mean change from baseline in percent with SEM.
Visual representation of experiment timeline and data collected for each time point for the main study. Rats were acclimated to housing changes D-5, D-4, and D-3 before initiation of baseline data collection. Control groups did not undergo blood collection.
Change from baseline body weight averaged across all time points (A and B) and at each time point (C). Data are presented as group mean ± SEM. (A) The TBS groups had reduced overall average weight gain compared with CON (§, P ≤ 0.0001). (B) Similarly, female and male TBS groups had lower average weight gain compared with sex-matched CON groups (†, P ≤ 0.01; §, P ≤ 0.0001). The HD female and LD male groups had greater weight gain than the LD F and HD M groups respectively (*, P ≤ 0.05). (C) When analyzed by time point, D2 to D7 CON had greater weight gain than the TBS groups (*, P ≤ 0.05; §, P ≤ 0.0001). In addition to the significant differences shown, CON had reduced weight gain compared with the LD group on D1 (P = 0.0326).
Change from baseline food intake averaged across all time points (A and B) and at each time point (C). Data are presented as group mean ± SEM. (A) TBS groups had reduced overall average food intake compared with CON (†, P ≤ 0.01). (B) When considering sex effects, both female TBS groups had lower average food intake relative to female CON (†, P ≤ 0.01) while only the HD males had reduced average intake compared with CON males (‡, P ≤ 0.001). (C) For comparisons at each time point, CON had increased food intake relative to both TBS groups on D1 to D2 and D4 to D5 (*, P ≤ 0.05; §, P ≤ 0.0001). On D3, only HD had reduced intake (*, P ≤ 0.05).
Change from baseline fecal output averaged across all time points (A and B) and at each time point (C). Data are presented as group mean ± SEM. (A) HD had decreased overall average fecal output compared with CON (*, P ≤ 0.05). (B) When analyzed by sex, the HD males had reduced output compared with LD and CON males (*, P ≤ 0.05). There were no differences in fecal output between female groups. (C) Analysis of each time point showed reduced output for the HD group compared with all other groups on D1 (§, P ≤ 0.0001). In addition, CON had increased fecal output compared with LD on D2 (P = 0.0409) and HD on D4 to D5 (P = 0.0492, P = 0.0265).
Mean plasma buprenorphine concentration after administration of TBS at doses of 5 and 10 mg/kg. This data is from the main study and is presented as group mean ± SEM, with (A) and without (B) consideration for sex. The dashed line at 1 ng/mL indicates the assumed threshold for analgesic efficacy in rats. Both dose groups had a mean plasma buprenorphine concentration above the threshold at all time points through 72 h, regardless of sex. (A) There was no difference between dose groups at any time point. (B) LD females had higher plasma buprenorphine levels than HD females at 24 h (P = 0.0180). There were no differences between groups at any other time point.
Appearance of the TBS application site (A) seconds after administration of a 5-g/kg dose and (B) 10 min later, demonstrating that the product visibly dries within 10 min.
Contributor Notes
This article contains supplemental materials online.