Serum Pharmacokinetics of a Highly Concentrated Buprenorphine Formulation in Older Female Sprague–Dawley Rats (Rattus norvegicus)
Laboratory rats (Rattus norvegicus) are common animal models used in biomedical, psychological, and toxicological research. Their long-established research use has driven the progressive refinement of experimental techniques so that associated pain/distress may be ameliorated. One of these refinements is the use of opioids to provide analgesia. Buprenorphine, a partial mu-opioid agonist with high affinity for mu receptors, is commonly used for rodents, as the longer duration of action compared with morphine reduces the need for direct handling during administration of supplemental doses. While conventional buprenorphine (CB) requires dosing two to four times per day to provide sufficient pain control in many mammalian species, a novel, highly concentrated formulation of buprenorphine (HCB; Simbadol) is the first FDA-approved, veterinary-specific opioid labeled for every 24-hour dosing in cats (Felis catus). We hypothesized that, at the labeled feline dose of 0.24 mg/kg SC, HCB would achieve buprenorphine serum concentrations ≥1 ng/mL in older adult female Sprague–Dawley rats for at least 12 to 24 hours. Mean peak serum concentrations of 13.79 ± 6.76 ng/mL occurred 0.5 hour after administration. Twelve hours postadministration, the mean serum concentration was 2.12 ± 0.59 ng/mL with all treated rats maintaining individual serum concentrations well above 1 ng/mL. Twenty-four hours postadministration, the mean serum concentration was 1.02 ± 0.33 ng/mL with 4 of 6 rats maintaining individual serum concentrations of greater than or equal to 0.99 ng/mL. With the exception of a minor, focal injection site reaction in one animal, none of the other known side effects of opioid administration in rats were observed. These results support that administration of HCB at 0.24 mg/kg SC to older adult female SD rats produces serum buprenorphine concentrations consistent with analgesia for at least 12 hours and for up to 24 hours in some rats.
Introduction
Laboratory rats (Rattus norvegicus) have played an important role in many scientific discoveries. Second only to mice (Mus musculus), their diverse use in many avenues of research has established the rat as an important animal model for various experimental purposes. Of the outbred rat stocks available, Sprague–Dawley (SD) rats are the most commonly used in biomedical research and thus are the subject of this study.1–3 Developed in 1925, SD rats are known for their docile behavior, ease of handling, and excellent reproductive capabilities, among other positive characteristics that make this stock popular for use across all biomedical disciplines. Surgical technique modeling, toxicology, neuroscience, carcinogenesis, and aging-related conditions are some of the most common research uses in which the need for species- and study-appropriate analgesia may arise. Identifying the ideal analgesia regimen for rats, however, can prove challenging. Considerations specific to the animal, such as normal compared with abnormal behaviors in the rat ethogram and restraint/handling stressors, must be taken into account. Considerations specific to the analgesic being used, such as duration of action, concurrent medication contraindications, and known adverse effects, must also be taken into account. In addition, potential confounding effects of therapeutics on research data must be considered before use of a specific medication regimen.
At this time, there is currently no singular universal guideline used to definitively determine if rodents are in pain.4–7 As such, the inherent challenges in assessing pain in prey species, specifically rats and other rodents, are multifactorial. As a prey specie, it is within their species-specific behaviors for rats to mask signs of pain and distress. Their stoic behaviors even in the face of moderate to severe pain can confound assessments where affected animals may continue to display normal behaviors in their ethogram, such as fastidious grooming and nest-building.4,8–13 Rats also vocalize at ultrasonic ranges thatcan be associated with aversive, agonistic, or painful stimuli which are outside of the human range of hearing.14–17 Rats are also nocturnal, thus they are most active in the latter portion of their light:dark cycle,4,14–16 typicallly outside of working hours for vivarium staff, thereby complicating activity and pain assessments. In fact, the very addition of human assessors during cageside evaluations or during direct handling can incite a prey response consistent with stoic behavior in contrast to more overt signs of pain that may be displayed without an assessor present.4,8–13 All of these factors, in addition to the varying causes and degrees of painful stimuli, variances in individual animal responses to such stimuli, and the inherent subjectivity arising from individual assessor evaluations, support the need for effective analgesia.
Of the many available opioids for veterinary use, buprenorphine is the preferred opioid of choice for rats and other rodents. A synthetic, primary partial mu-opioid agonist with secondary κ- and δ-opioid antagonist effects, buprenorphine is considered 30 times as potent as the naturally occurring opioid morphine18 and has a reported minimum therapeutic plasma concentration level in rats is 1.0 ng/mL.19–23 Despite having partial versus full agonistic action with the mu-opioid receptors, the high affinity of buprenorphine means substantially prolonged binding to the receptors and, thus, prolonged duration of action. In fact, parenteral administration of the conventional formulation (conventional buprenorphine [CB]) has a reported duration of action up to 12 hours for rats.18,24–28 Importantly, the prolonged duration of action reduces the need for multiple administration events, thus decreasing the stressors associated with handling/restraint. An additional advantage of CB is the wide margin of safety with a multitude of published doses and routes of administration offered for rats, including 0.01 to 0.05 mg/kg SC or IV every 8 to 12 hours (5-fold span between low and high doses)16,18,23; 0.02 to 0.5 mg/kg SC, IV, or IP every 6 to 12 hours (25-fold span between low and high doses)25,26; 0.1 to 0.25 mg/kg PO every 8 to 12 hours (2.5-fold span between low and high doses)18,23; and 0.02 mg/mL in drinking water.23,29
Despite these potential advantages, there are still important considerations regarding the use of buprenorphine in rats and other species. The greater dose range of CB can mean potential breakthroughs of unalleviated pain when dosing is not administered appropriately, such as when a lower dose is chosen at decreased administration frequencies. A challenge with CB, and thus all of its analogs, is the dose-dependent ceiling effect: at higher doses of the medication, opioid receptor antagonist properties begin to dominate, thus decreasing analgesic capability.18,30,31 The aforementioned stoic behavior of rats and other prey animals can also confound the choice of analgesic as well as the choice of doses for administration. An important adverse effect of buprenorphine administration is the increased incidence of pica, where rats treated with the medication will begin consuming nonnutritive substances such as bedding and enrichment material and which can potentially lead to impaction and gastrointestinal obstruction.16,30–34 Of note, the general adverse effects of opioid administration in other species are also well-conserved when applied to rats, as reduced food intake, ileus, sedation, bradycardia, and respiratory depression have been reported in the species.16,30,35,36 Thus, assessing food and water intake and fecal and urine output as physiologic parameters of pain can be challenging: the very same treatments for ameliorating pain can actually drive clinical signs consistent with pain. Rats are also prone to injection site reactions in which the adjuvant, vehicle of administration, or active compound/ingredient can incite local-to-systemic effects in the treated animal.37–39 Furthermore, while CB dosing has been thought to last up to 12 hours in rats (twice daily; see above), recent studies19 have demonstrated that more frequent dosing of the medication is required to maintain adequate levels of analgesia in this species, such as once every 6 to 8 hours subcutaneously or intraperitoneally (3 times daily to 4 times daily).
Most injectable CB formulations for parenteral use in veterinary medicine are dispensed in 1-mL single-dose vials or ampules. Both require that they be discarded after puncture and opening, respectively, for a single dose and/or single patient use regardless of volume depletion (usually within 24 hours, though this can vary based on manufacturer guidelines).40–42 Because rats and other rodents are relatively small and, thus, typically require lower volumes of buprenorphine per dose,43 it is not uncommon for institutions to consistently discard mostly full vials or ampules. This results in the need for planning and executing the discard of partially full vials of controlled substances. With regard to the single-dose glass ampules, another logistical concern is the required use of filter needles before administration to capture any residual glass shards in solution, which is common practice in human medical facilities.44–46 In addition to the additional cost of purchasing such supplies, additional personnel and time are required to complete the necessary tasks related to CB administration and discard.
The development of a compounded, sustained-release formulation of the opioid (Buprenorphine Base Lab in Polymer, formerly Buprenorphine SR-Lab; Wedgewood Pharmacy, Swedesboro, NJ), which is delivered within a biodegradable polymer system in a compatible organic solvent, offers an intriguing possibility for analgesia in rats.47 In previous studies and published information from the manufacturer, this formulation has been shown to provide dose-dependent, extended periods of postoperative analgesia for rats that have undergone plantar skin and underlying muscle incisions35,47 and skin incisions and unicortical tibial bone surgical defects.20,47 Sustained-release buprenorphine (SRB) provides pain management for 48 to 72 hours with one subcutaneous injection.16,47 Following previous studies20,48 that examined use of SRB in mice and rats and which demonstrated localized skin irritation over injection sites, the manufacturers of SRB created a lab animal-specific formulation with a unique adjuvant to decrease the incidence of such reactions.47
However, despite its prolonged duration of action and potential for use in rats, it can be a challenge to obtain the medication due to campus policies and strict state-specific laws governing compounded controlled substances.49–52 Such is the case at our institution, where campus-wide policies prevent the use of SRB through regulations involving its status as a compounded, non-FDA-approved and non-FDA-indexed medication and exclusive procurement from an out-of-state source.
Most recently, an extended-release, pharmaceutical-grade formulation of buprenorphine (Ethiqa XR; Fidelis Animal Health, North Brunswick, NJ) was introduced as an alternative to both CB and SRB. Extended-release buprenorphine is an FDA-indexed drug labeled for the control of postprocedural pain in mice, rats, ferrets, and nonhuman primates.53 This formulation is viscous, lipid bound, and is suspended in medium-chain triglyceride oil within multidose vials and with a recommended shelf life of 90 days after first puncture53. After a single subcutaneous injection of extended-release buprenorphine (XRB) at the manufacturer’s recommended dosage (0.65 mg/kg), therapeutic drug concentrations of buprenorphine were maintained for 72 hours in rats.53 In addition, target animal safety studies in rats revealed no adverse effects at subcutaneous doses 2, 6, and 10 times the labeled dose; and clinical trials showed no substantial adverse effects related to hematology, chemistry, and histopathologic parameters in the species53. In fact, recent studies examining the use of XRB in rats showed that at both 1 and 2 times the manufacturer’s recommended dosage, adult male and female SD rats maintained plasma levels of buprenorphine consistent with analgesia for up to 72 hours54 as well as the ability of XRB to attenuate mechanical hypersensitivity for up to 72 hours in male SD rats after plantar surface hind paw incision.55 Similar to SRB, the extended duration of action of XRB as compared with CB results in decreased overall administration frequency, thereby also reducing handling-related stressors.
Another recently developed buprenorphine formulation that may address some of the same concerns is a novel, highly concentrated analog (Simbadol; Zoetis Animal Health, Parsippany, NJ). Highly concentrated buprenorphine (HCB) is a clear, injectable, nonviscous liquid mixture containing a higher concentration of buprenorphine than CB, SRB, and XRB and is admixed with anhydrous dextrose, methylparaben, propylparaben, sodium acetate trihydrate, glacial acetic acid, anhydrous ethanol, water, and hydrochloric acid and/or sodium hydroxide for pH adjustment.56 Stored at temperatures up to 25 °C (77 °F) and protected from light and excessive heat above 40 °C (104 °C), HCB has a shelf life of 56 days after initial puncture of the 10-mL multidose vial.56 HCB is one of the very few opioid medications that is FDA approved and specifically labeled for use in a veterinary species, in this case, the domestic cat (Felis domesticus). Labeled for SC administration, a single injection of the labeled feline dosage (0.24 mg/kg) was shown to provide up to 24 hours of analgesia in cats that, in respective experimental groups, underwent soft tissue and orthopedic surgical procedures.56,57 Manufacturer recommendations include administering the first dose 1 hour before surgery, with 2 additional doses given once daily for a total of 3 days of treatment with HCB.56
The potential use of HCB in rats is the focus of this and 3 recent studies.22,32,33 Previously, self-injurious behavior (SIB), food intake, fecal output, and hind limb withdrawal from thermal stimuli in rats were studied after administration of HCB at 0.075, 0.15, and 0.30 mg/kg SC. The highest dose showed the most evidence of thermal hypoalgesia, but SIB was observed at all three dosages for up to 8 hours after initial administration.32 A follow-up study33 comparing the same high dose of HCB (0.30 mg/kg SC) with CB (0.05 mg/kg SC) and SRB (1.2 mg/kg SC) resulted in SIB being observed with all treatment groups, most prominent in rats treated with HCB and SRB, and evidence of increased pica in rats treated with SRB. Most recently, a study22 evaluating HCB in young, 6- to 8 week-old, male and female SD rats suggested that HCB given at a higher 0.5 mg/kg SC dosage provided therapeutic plasma concentrations for 12 to 24 hours after initial dosing and provided analgesic efficacy for a similar 12 hours after initial dose.
Although all 3 prior studies assessed the potential for hypoalgesia and adverse effects after administration of HCB, we believe our study to be the first to elucidate the serum pharmacokinetics and gross safety profile of the medication in older, adult female rats when given at the labeled dose. We hypothesize that at the labeled feline dose of 0.24 mg/kg SC, serum concentrations of buprenorphine will be maintained for at least 12 to 24 hours at the purported therapeutic level of greater than or equal to 1.0 ng/mL in older, adult female SD rats, thus offering an alternative to CB, one that has a longer duration of action and reduced dosing frequency.
Materials and Methods
Animals.
Six adult approximately 6- to 7- month-old female SD rats (Rattus norvegicus; Charles River Laboratories, Wilmington, MA) were used for these studies. The experimental protocol was approved by the IACUC at the University of California, Davis, and the work was conductined in a USDA-registered and AAALAC International-accredited facility. The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals and the American Association for Laboratory Animal Science Position Statements entitled “Humane Care and Use of Laboratory Animals,” “Alleviating Pain and Distress,” “Scientific Basis for Regulation of Animal Care and Use,” “Determining Laboratory Animal Housing Standards,” and “Standards for Assessing the Quality of Laboratory Rodents.”58–63 All rats were healthy upon arrival on our campus, and were maintained in a conventional, medium-health status, nonbarrier vivarium on a 12-hour light:12-hour dark cycle. Indirect surveillance monitoring revealed that colony animals were negative for rat coronavirus, sialodacryoadenitis virus, Sendai virus, pneumonia virus of mice, Kilham rat virus, Toolan H1 parvovirus, rat parvovirus, reovirus 3, lymphocytic choriomeningitis virus, Theiler murine encephalomyelitis virus, cilia-associated respiratory bacillus, Hantavirus, Mycoplasma pulmonis, Mycoplasma arthritidis, ectoparasites, and endoparasites. While the sentinels were culture-positive for Pasteurella pneumotropica (now classified as Rodentibacter spp.), this organism is usually not associated with disease in immunocompetent animals.15
All animals were transferred from a routine teaching protocol where acepromazine (0.5 to 1.0 mg/kg SC or IP) with or without midazolam (0.2 to 2.0 mg/kg SC or IM) was administered as needed for sedation to instruct veterinary students on handling and physical examination of rats. After their initial protocol use and before transfer to this experimental protocol, the animals underwent a 2-month washout period with only minimal handling conducted during routine husbandry practices and no additional pharmaceutical administration. Animals were pair housed on corncob bedding in Super Rat 1400 Micro-Isolator Systems caging (Lab Products, Aberdeen, MD) on a Super Rat 1400 individually ventilated rack (Lab Products, Aberdeen, MD). Animals were fed ad libitum 2018 Teklad global 18% protein rodent diets (Inotiv, West Lafayette, IN) and provided Hydropac pouches (Lab Products, Aberdeen, MD) containing water that was ultrafiltered (10-, 1.2-, and 0.2-μm filtered), treated with 3-ppm sodium hypochlorite, and then allowed to stand/dissipate for 7 days. Rats were provided Enviropaks (W.F. Fisher and Son, Somerville, NJ) as well as small nylon bones (Animal Specialties and Provisions, Quakertown, PA) for enrichment.
Study design.
Physical examinations of all animals completed before the current protocol use identified no abnormalities or evidence of illness. The dorsum of each animal was shaved from approximately the cranial intrascapular to the flank regions for visualization of potential injection site reactions. A baseline weight was collected for each animal (range: 294 to 306 g; mean and standard deviation 298 ± 5 g) and was used to calculate the respective doses of HCB. A baseline, preinjection blood sample (approximately 250 μL) was collected from each animal using either a 22-gauge × 1-inch or 25-gauge × 5/8-inch needle and venipuncture of the lateral saphenous vein with nonsterile collection into a serum separator blood tube (BD Microtainer; Becton Dickinson, Franklin Lakes, NJ). After collection, manual pressure hemostasis was applied with gauze directly over the venipuncture site for 10 to 15 seconds. Using the published feline dosage of 0.24 mg/kg for a 1.8 mg/mL concentration vial, the dose for each animal was determined to be 0.04 mL. Sterile technique was used to withdraw the required volumes from the multidose vial of HCB. Because of the small volume requiring precise administration and concern for substantial hub loss of a controlled substance using larger syringes, U-100 insulin syringes were used to administer 4 units (equivalent to 0.04 mL) of HCB into each animal. Using small, rectangular towels for conventional towel restraint (towel-wrap method), injections were administered in numerical order coinciding with their assigned animal ID numbers and into the subcutaneous tissue well within the margins of the shaved areas for each animal. Injection sites were outlined lightly with a permanent marker for follow-up observations. Blood was then collected from each respective animal at the same volume using the same aforementioned technique (in one or both saphenous veins to maximize volume collection) in numerical animal order for a total of 9 additional time points: 0.5, 1.5, 5, 7, 12, 24, 48, 72, and 96 hours after HCB injection. After each blood collection time point, the samples were allowed to clot. Each sample was then centrifuged at 9.5 × 1,000 rpm for 10 min at ambient room temperature, and serum supernatant was immediately transferred into 0.5-mL Eppendorf Safe-Lock Tubes (MilliporeSigma, Burlington, MA) and stored at −80 °C until assay completion.
At each blood collection time point, cageside health assessments were completed that consisted of general condition and rat-specific behavior evaluations such as engagement with enrichment, ambulation, and exploratory behaviors. In addition to these observations, recorded health assessments were completed once to twice daily during days 1 to 5 of the project where mentation, hydration status, appetite, and fecal and urine output were qualitatively assessed for each pair of cohoused animals: the 10 blood collection time points all fell within these first 5 days, thus these assessments were made concurrent with handling for venipuncture. The same recorded health assessments were completed once daily on days 6 to 12 of the project. Including the baseline measurement collected before HCB injection, body weights for all animals were collected once daily on days 1 to 7 with an end-project weight collected on day 12 of the study (11 days after HCB injection). Injection sites were monitored during each recorded assessment.
Serum sample analysis.
For analysis, buprenorphine working solutions were prepared by dilution of the 1-mg/mL stock solution (Cerilliant, Round Rock, TX) with methanol to concentrations of 0.001, 0.01, 0.1, 1, and 10 ng/µL. Serum calibrators were prepared by dilution of the working standard solutions with drug-free rat serum to concentrations ranging from 1 to 40 ng/mL. Calibration curves and negative control samples were prepared fresh for each quantitative assay. In addition, quality control samples (drug-free rat serum fortified with analyte at 3 concentrations within the standard curve) were included with each sample set as an additional check of accuracy.
Before analysis, 0.05 mL serum was diluted with 2.0 mL 0.1 M pH 6 phosphate buffer and 0.1 mL water containing the d4-buprenorphine internal standard (40 ng/mL; Cerilliant, Round Rock, TX). All samples were vortexed gently to mix and subjected to solid phase extraction using C18UC columns 200 mg/3 mL (UCT). In brief, the columns were conditioned with 2.5 mL of methanol and 3 mL of water. The samples were loaded onto the column, and a minimum of 2 min was allowed for samples to pass through the column. The columns were rinsed with 2 mL of 50% methanol in water before eluting with 2.5 mL methanol. Samples were dried under nitrogen, dissolved in 100 μL of 10% acetonitrile (ACN) in water with 0.2% formic acid, and 40 μL was injected into the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system.
The concentration of buprenorphine was measured in serum by LC-MS/MS using positive electrospray ionization (ESI[+]). Quantitative analysis was performed on a TSQ Vantage triple quadrupole mass spectrometer (ThermoFisher Scientific, Waltham, MA) having LC-10ADvp liquid chromatography systems (Shimadzu Scientific Instruments, Columbia, MD). The spray voltage was 3,500 V, and the sheath and auxiliary gas were 45 and 30, respectively (arbitrary units). Product masses and collision energies of each analyte were optimized by infusing the analytes into the mass spectrometer. Chromatography employed an ACE 3 C18 10 cm × 2.1 mm 3 μm column (Mac-Mod Analytical, Chadds Ford, PA) and a linear gradient of ACN in water with a constant 0.2% formic acid at a flow rate of 0.35 mL/min. The initial ACN concentration was held at 10% for 0.33 min, ramped to 95% over 4.17 min, and held at that concentration for 0.17 min before reequilibrating for 3.8 min at initial conditions.
Detection and quantification were conducted using selective reaction monitoring of initial precursor ion for buprenorphine (mass-to-charge ratio [m/z]: 468.3) and the internal standard d4-buprenorphine (m/z: 472.3). The response for the product ions for buprenorphine (m/z: 101.0 and 186.9) and the internal standard (m/z: 100.9 and 186.9) were plotted and peaks at the proper retention time integrated using Quanbrowser software (ThermoFisher Scientific, Waltham, MA). Quanbrowser software was used to generate calibration curves and quantitate analytes in all samples by linear regression analysis. A weighting factor of 1/× was used for all calibration curves.
The response for buprenorphine was linear and gave correlation coefficients of 0.99 or better. Accuracy was 101% for 1.5 ng/mL, 109% for 5 ng/mL, and 108% for 40 ng/mL. Precision was 13% for 1.5 ng/mL, 13% for 5 ng/mL, and 3% for 40 ng/mL. The technique was optimized to provide a limit of quantitation of 1 ng/mL and a limit of detection of approximately 0.5 ng/mL for buprenorphine.
Pharmacokinetic analysis.
Pharmacokinetic parameters were estimated using compartmental analysis in population mode using the NLME component (Phoenix WinNonlin v8.0; Certara, Radnor, PA). Data were analyzed using both a 2- and 3-compartment models, and several error structures were assessed. The models were run with the first-order conditional estimation-extended least squares engine. Goodness of fit and the appropriate error term were selected based on visual analysis of observed compared with predicted concentration graphs and residual plots as well as coefficient of variation, Akaike information criterion, and CV% of parameter estimates. A 2-compartment extravascular model (Cp = Ae − αt + Be − βt + Ce − ka × t) with clearance parameterization and an additive weighting factor gave the best fit to buprenorphine concentration data points. Secondary parameters calculated include distribution (α) and elimination (β) half-lives, and the AUC. Pharmacokinetic parameters are reported as population mean.
Results
Pharmacokinetics.
The individual serum concentrations of all rats at each of the 10 time points are shown in Table 1. At 0.5 hour after HCB administration, the variance of individual animal serum concentrations is immediately evident: serum concentrations at this first time point showed the highest magnitude standard deviation of all time points at ±6.76 ng/mL (range: 7.44 to 24.4 ng/mL; mean: 13.79 ng/mL). The variance in serum concentrations and thus standard deviation continued to decrease in magnitude with each proceeding time point, however. Peak serum concentrations of buprenorphine occurred 0.5 hour after administration of HCB in 3/6 rats (range 7.44 to 24.4 ng/mL; mean and standard deviation: 17.28 ± 8.80 ng/mL), whereas the remaining 3/6 rats showed peak serum concentrations of the drug at the next time point, 1.5 hours after administration (range: 10.05 to 12.8 ng/mL; mean and standard deviation: 11.6 ± 1.15 ng/mL). At 12 hours after dosing (Figure 1), serum concentrations in all rats showed levels greater than 1 ng/mL. At 24 hours after dosing (Figure 1), serum concentrations in 3/6 rats showed levels greater than 1 ng/mL with a fourth rat showing a serum concentration level of 0.99 ng/mL; the remaining 2 rats had serum concentration levels less than 0.99 ng/mL (range: 0.61 to 1.58 ng/mL; mean and standard deviation: 1.02 ± 0.33 ng/mL). Forty-eight hours after dosing, the same 4 rats with serum concentrations of buprenorphine greater than or equal to 0.99 ng/mL showed a concentration mean and standard deviation of 0.58 ± 0.04 ng/mL at this time point (range: 0.55 to 0.63 ng/mL; see Table 2); the remaining 2 rats had no detectable serum buprenorphine at this time point. At 72 and 96 hours post-HCB administration, respectively, no serum buprenorphine was detectable in any animal. Overall, buprenorphine serum concentrations decreased as a function of time after the peak concentration of the drug was reached. A summary of the pharmacokinetic parameters for buprenorphine in HCB at a dose of 0.24 mg/kg SC for all 6 rats is noted in Table 2.
| Time (h) | Rat 1 | Rat 2 | Rat 3 | Rat 4 | Rat 5 | Rat 6 | Mean | Standard deviation |
|---|---|---|---|---|---|---|---|---|
| 0 | ND | ND | ND | ND | ND | ND | ND | ND |
| 0.5 | 9.85 | 11.0 | 20.0 | 24.4 | 10.1 | 7.44 | 13.79 | 6.76 |
| 1.5 | 10.05 | 12.8 | 17.1 | 15.9 | 11.5 | 6.74 | 12.35 | 3.81 |
| 5 | 3.63 | 4.40 | 5.01 | 5.41 | 4.77 | 3.11 | 4.39 | 0.87 |
| 7 | 2.94 | 5.10 | 4.86 | 4.20 | 3.95 | 3.19 | 4.04 | 0.87 |
| 12 | 1.80 | 3.14 | 2.38 | 1.44 | 2.12 | 1.86 | 2.12 | 0.59 |
| 24 | 0.99 | 1.58 | 0.61 | 0.78 | 1.03 | 1.13 | 1.02 | 0.33 |
| 48 | 0.63 | 0.59 | ND | ND | 0.57 | 0.55 | 0.58 | 0.04 |
| 72 | ND | ND | ND | ND | ND | ND | ND | ND |
| 96 | ND | ND | ND | ND | ND | ND | ND | ND |
For this cohort, the minimum mean serum concentration of buprenorphine consistent with analgesia in rats is highlighted (12 and 24 h postadministration of HCB). No detectable (ND) levels of buprenorphine are noted for all animals at 72 and 96 h postadministration of HCB.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-124
| Values | |
|---|---|
| Cmax (ng/mL) | 15.2 ± 6.48 |
| Tmax (h) | 0.857 ± 0.177 |
| Alpha (1/h) | 1.20 ± 0.635 |
| Beta (1/h) | 0.076 ± 0.047 |
| Alpha HL (h) | 0.806 ± 0.545 |
| Beta HL (h) | 11.5 ± 5.08 |
| AUClast (ng × h/mL) | 104.6 ± 17.2 |
Pharmacokinetic parameters were determined using compartmental analysis. Alpha and Beta, slopes for the modeled equation; Alpha HL, phase 1 half-life; Beta HL, phase 2 half-life; AUClast, area under the curve until the last time point.
Animal health.
All animals remained clinically healthy throughout the course of the study with the exception of a minor, pinpoint injection site reaction in 1 rat. The lesion began as a focal area of mild erythema directly over the SC injection site on the right flank of the animal on day 4 of the study, 3 days after HCB injection (Figure 2). The lesion remained relatively static but was most prominent on day 11 of the study, 10 days after HCB injection (Figure 3), and it progressed to a 0.25 to 0.5 mm diameter pinpoint, dry, erythemic crust. The animal remained on study with medical monitoring initiated, but no pharmaceutical treatment was provided (including rescue analgesia) because of the relatively mild nature of the lesion and the animal remaining bright, alert, responsive, and clinically normal. No evidence of SIB was observed. Three days after medical monitoring was initiated (13 days after HCB injection), the lesion had fully resolved with no scabbing or erythema grossly appreciable. Another minor adverse effect observed in our animals was mild erythema and bruising at the venipuncture site. The erythema and bruising were consistent with repeat venipuncture and did not progress clinically further than small, focal areas of hyperemia and ecchymoses over the proximal and distal portions of the lateral saphenous vein, which were all resolved by the end of animal health monitoring (day 12); no hematomas were identified in any animal during the course of the study.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-124
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-24-124
Animals were all observed to be consistently eating well, both chow and higher value nutritional enrichment, with only minor signs of stress such as very mild porphyrin staining over the face and neck, again mostly resolved by day 12. In addition, end-of-study body weights collected on day 12 (11 days after HCB administration) showed that 3/6 rats gained between 0.3% and 3.7% above their respective baseline weights (range: 1 to 11 g; mean and standard deviation: 6.33 ± 5.03 g); 2/3 of these animals maintained serum concentrations of buprenorphine greater than or equal to 0.99 ng/mL. In comparison, the remaining 3/6 rats lost between 3.9% and 5.6% of their respective baseline body weights (range: 12 to 17 g; mean and standard deviation: 4.67 ± 1.15 g); 2/3 of these animals maintained serum concentrations of buprenorphine greater than 1 ng/mL 24 hours after HCB administration. At our institution, IACUC guidelines consider a 20% or more decrease in baseline body weight as a humane endpoint criterion that requires euthanasia64: none of the 3 rats with measurable weight loss met these criteria, and all 6 animals maintained a normal body condition score of 3 to 3.53 out of 5 for the course of the study.
Discussion
The results of this study suggest that older adult female SD rats are able to maintain serum concentration levels of buprenorphine consistent with therapeutic analgesia (as noted in prior studies20,22,33) for at least 12 hours after a single SC injection of HCB at a dosage of 0.24 mg/kg. These results support an approach to analgesia in rats that can potentially reduce dosing frequency by 50%, from up to 4 times daily with CB to twice daily with HCB. In addition, only minimal, clinically insignificant adverse effects on animals were observed, including as a mild injection site reaction (1/6 rats) and weight loss (3/6 rats). Since weight trends for the 6 animals were not tracked before initiation of the study, downstream weight loss may be a function of normal trends for the individual animals. While 2 animals did not maintain buprenorphine serum concentration levels greater than or equal to 0.99 to 1 ng/mL at the 24 hours time point, the overall mean concentration of this cohort (1.02 ng/mL) exceeds the reported minimum therapeutic drug level for buprenorphine (1 ng/mL), suggesting an even longer duration of action than 12 hours for most older adult female SD rats. At this same dosage, all rats showed clearance of the drug with levels less than 1 ng/mL at 48 hours and no detectable serum levels of the compound at 72 and 96 hours. It should be noted, however, that by study design, serum concentrations were not measured between 24 and 48 hours when levels could have remained greater than or equal to 1 ng/mL. Despite this, the mean serum concentration of buprenorphine for the cohort suggests that older adult female SD rats receiving HCB at 0.24 mg/kg SC should be dosed at least twice daily (once every 12 hours) to maintain appropriate levels of the drug consistent with therapeutic analgesia. Furthermore, while our results support the recent study showing that younger SD rats maintain plasma concentrations consistent with analgesia for at least 12 hours,22 our study specifically demonstrated that older female SD rats also maintain serum concentrations consistent with analgesia for 12 hours even when HCB is given at a comparatively lower dosage (0.24 mg/kg SC as compared with 0.5 mg/kg SC). These results suggest additional refinements with the use of HCB stemming from differing dosing regimens: a lower dosage results in a lower volume of drug given.
Despite the overall decreases in serum concentration over time for all animals (Table 1), there is no apparent correlation between earlier or later onset of peak buprenorphine concentrations and metabolism of the active compound to below or above the effective analgesic benchmark of greater than or equal to 1 ng/mL, respectively. Of the 3/6 rats measured to have peak serum concentrations at the first time point after baseline (0.5 hour; compared with the remaining rats showing peak concentrations at 1.5 hours, the second time point), one of these rats still had 1.13 ng/mL of buprenorphine in serum 24 hours after initial HCB administration in spite of the peak concentration appreciated earlier in the experimental timeline. Differences in individual animal metabolic and absorption rates, as evidenced by the individual animal serum concentrations measured for each time point, and that are, in general, disproportionately higher in smaller animals such as rats,16 may have contributed to these variances in serum buprenorphine concentrations over time. Further investigation is warranted to evaluate the pharmacokinetics of HCB in older rats when administered at varying dosages such as those greater than the labeled feline dose (greater than 0.24 mg/kg SC).
Further studies may elucidate the safety profile of HCB in rats at higher dosages, as well as evaluating the possibility of increased durations of action, the latter of which may occur as a result of higher dosages driving persistent serum levels of buprenorphine consistent with analgesia over a longer period of time (greater than 24 hours) as was seen in a study from our institution involving rhesus macaques (Macaca mulatta).52 Two of 6 rats were also found to have mild increases in serum buprenorphine concentrations both occurring from the 5-hour to the 7-hour time point (Table 1). While it is unclear if this is due to instrument/reading errors of the measurement devices or human errors or true accumulations in serum concentrations over singular points in time, the relative increases are mild (+0.08 and +0.70 ng/mL) and did not appear to impact the proceeding serum concentration measurements.
There are adverse effects associated with the SC route of administration in rats, specifically with buprenorphine.20,54,55 A notable adverse effect of opioid administration is the development of local macroscopic and histologic injection site lesions ranging from erythema and crusting/scabbing to granulomatous inflammation, such as those that have been observed directly over the injection site, specifically with HCB-related analogues SRB20 as well as XRB.54,55 Apparently similar in appearance to 1/6 rats in our cohort, the dermal reactions to buprenorphine are often focal, present as erythema and crusting, but otherwise do not incite a systemic inflammatory response and are often not severe in clinical presentation.20 We considered submission for gross necropsy and histopathologic analyses of the affected rat, but ultimately decided to maintain the animal in the colony due to the relatively minor nature of the lesion we expected to heal unremarkably.
Another notable adverse effect of opioid administration in rats is SIB. SIB in rats is a multifactorial expression of stereotypic behavior, which can range from focused, increased interest over a focal body part by sniffing or licking to actual self-biting or even cage-biting (metal lid toppers for food and water).32,33,65 Arising from a multitude of potential exogenous causes such as stress and anxiety, the specific neurobiological cause has been linked to dopaminergic stimulation,32,33,65,66 including mu-opioid activation with morphine, which has been shown to enhance stereotypic behavior in this species.32,67–69 The aforementioned effects-focused HCB study in rats suggests that the same mu-opioid receptor activation occurred when the drug was administered, as all 3 experimental doses were significantly associated with SIB. In that study, 2 doses (0.075 and 0.15 mg/kg) were lower and 1 dose was higher (0.30 mg/kg) than our experimental dose (0.24 mg/kg), and SIB was observed in their experimental cohort up to 8 hours after injection. In contrast, we found that a dose of 0.24 mg/kg did not result in any grossly observable signs of SIB such as dermal lesions, fur loss, or abnormal dentition from biting the metal lid toppers in any of the 6 animals. The focal dermal lesion observed in one rat was, by appearance and character, most consistent with an injection site reaction (Figures 1 and 2). It has been suggested that SIB may be reduced with the presence of bedding or other nonnutritive material positively influencing pica behavior, which, in turn, weakens the driven, stereotypic SIB behaviors.32 As none of our animals exhibited SIB during the course of the study, this may have been a key difference, as our animals were housed in conventional shoebox-style rat caging with corncob bedding as compared with their study in which wire-bottom caging was used with no bedding. Furthermore, our rats were pair housed with environmental enrichment, which likely mitigated any inherent stressors from being singly housed. Since anxiogenic compounds and external factors like environmental stress drive SIB in rats,70,71 we believe that pair-housing allowed for ample conspecific interactions that may have modulated the clinical appearance of SIB. Finally, our rats were transferred from a teaching protocol where they were handled twice weekly for approximately 2 month for the purposes of teaching veterinary students manual and mechanical restraint in addition to physical exam techniques. This, in addition to routine animal care practices and the provision of high-value nutritional rewards after experimental handling, may have all contributed to acclimatization and decreased stress and anxiety. Cumulatively, this may have prevented the breakthrough of any observable SIB. As a follow-up study using pair-housed animals and kaolin clay to measure pica behaviors showed that rats still displayed SIB after injection with CB, HCB, and SRB formulations,33 additional studies are indicated to evaluate strain-specific differences in SIB expression, the influence of experimental factors such as positive reinforcement and operant conditioning with high value nutritional or play rewards (that is, rat tickling)72–74 and/or varying degrees and frequencies of handling and their respective effects on SIB following HCB administration.
As previously mentioned, other notable adverse effects of opioid administration in rats are those related to the gastrointestinal tract such as pica and functional obstruction from ileus. In our study, we empirically observed a few rats chewing on crinkle paper enrichment with small amounts of corncob bedding pellets also observed on or near their mouths and teeth; these were observed almost immediately postadministration of HCB, after returning the animals to their respective home cages. However, no appreciable swallowing nor actual intake of said environmental implements was directly observed, and these behaviors subsided after these singular observations at time point 0: no additional chewing of enrichment or bedding was seen with subsequent handling events for blood collection nor throughout the course of the study when health monitoring was performed. In very much the same way that pica in rats can be induced through motion sickness, nutritional deficiencies, and other illness-related stressors,75–79 we suspect that this observed behavior in our experimental cohort may have arisen due to the general stress of, cumulatively, manual and mechanical restraint using the towel-wrap method and the discomfort of percutaneous injection of HCB. While the animals did receive prior SC injections of sedative from their original protocol, the 2-month washout period between that unrelated teaching protocol and our experimental use may have been substantial enough to cause a recrudescence of generalized neophobia to our restraint technique for HCB administration but not severe enough to incite persistent or true pica. Fortunately, since the pica behavior was not persistent, no adverse clinical effects, such as hyporexia or mechanical obstruction, were seen, as appetite and fecal output remained normal throughout the course of the study; and these same clinical findings also ruled out clinical ileus in our cohort of animals. Although pica has been specifically reported to be a side effect of CB80 and SRB,33 it is also an important consideration when administering HCB to this species, as buprenorphine remains the active opioid compound in all 3 formulations. In fact, following administration of XRB, the manufacturers specifically recommend housing rats on paper or other soft bedding as compared with wood chip-type bedding to prevent ingestion of the latter, which can lead to lethal obstruction53; pica was, however, not observed in rats of a recent XRB study54. Future studies with HCB and other opioids, including those expanding on the current study, should include gross necropsy analyses of all animals in each cohort to characterize the presence of any nonnutritive substances within the stomach and gastrointestinal tract consistent with pica. The risk of pica with buprenorphine administration highlights the importance of animal health observations focused on food intake/appetite and fecal output. However, because these can be confounded by side effects of opioid administration, observations should be married with assessments of overall rat activity and mentation in the cage.
In addition to the refinement of reduced dosing frequency, the use of longer-acting analgesics in rats is also advantageous with regard to other aspects of an animal care program. Decreasing the need for multiple restraint events for medicament administration directly decreases the risk of workplace injuries intrinsically related to handling of rats. since reduced handling means a reduced risk of animal bites, scratches, and therefore exposure to potentially zoonotic pathogens, such as rat bite fever.16,81 In addition, reduced exposure of personnel to allergens is a possible outcome due to the overall reduction in handling of the animals. Rat urine proteins, fur, and skin dander have been identified as common causes of laboratory animal worker hypersensitivity,16,82–85 and this includes inanimate objects that proallergenic particles come into contact with such as bedding. Allergies typically develop within 2 years of working with animals,16,84 and the risk of allergy development increases with individuals who receive relatively larger exposure “doses” of rat allergens.83,85 Logically, this would apply to personnel who are in greater and more consistent/constant contact with the animals: during daily husbandry management, cage cleaning, and handling animals multiple times a day for medicament administration as is needed for those animals receiving CB analgesia compared with other longer-acting preparations.
Reduction in the redosing frequency of analgesic by using HCB in rats can also have positive fiscal and other downstream program benefits. The inherent nature of any animal care program is one in which multiple roles are involved in the care and use of animals, and wages are a direct expense to a research institution.86,87 From a workforce management perspective, payroll costs might be somewhat reduced by as a result of reduced treatment dosing. For example, potentially decreasing buprenorphine analgesia administration by a factor of 2 in rats from 4 times daily to twice daily dosing results in a reduced workload. This can lead to institutional cost savings, such as that associated with overtime pay and mileage required to complete analgesic treatments. Likewise, the time and resources that may have been used for multiple visits to vivaria can, instead, be redirected to other veterinary care or research-related activities that can further enrich an animal care program. These potential benefits can also be extrapolated to laboratory staff, as well.
At our institution, we recharge the cost of analgesic administration to our principal investigators (PIs) when (1) an experimentally induced condition occurs that is considered painful (that is, surgery, unexpected adverse effects, etc), and/or (2) veterinary care team members provide the actual treatment for affected animals.88,89 At the time of the current study, the cost of HCB to our PIs was 70% higher per mL than CB. However, the need for multiple doses (up to 4 times daily) for the latter compared with the possible twice daily dosing for rats for the former can actually mean a significant cost reduction to PIs when HCB is used. Using the mean weight of our experimental group of adult female SD rats (298 g) and applying both the (1) CB high dose and recommended frequencies of administration and comparing to (2) our experimental HCB dose at twice daily administration, it would actually cost the PIs at our institution 24% more per dose to administer CB 4 times daily than it would to administer HCB at the experimental dose twice daily. Although the cost of purchasing HCB for our institution is initially grossly higher than CB, the use of HCB over time can reduce and even reverse the cost disparity between the 2 buprenorphine formulations. This can occur, in part, because HCB is 6 times more concentrated than most CB formulations (1.8 mg/mL compared with 0.3 mg/mL, respectively), resulting in a slower depletion from the source vial with smaller volumes needing to be administered. This is also potentiated by its longer shelf life, as well (56-days multidose vial compared with a single-use vial, respectively).
Our study focused on the most commonly used laboratory animal rat model (SD), a specific age group (adult rats), and sex (female). Possible ways to expand upon our study would include incorporating other ages of rats as well as male rats of comparable age (older adult, 6 to 7 month old) to characterize any age-dependent or hormone-dependent influences on metabolism of buprenorphine which, in turn, can affect duration of action of HCB as in other opioids or other medications such as anesthetics.34,36 Likewise, expansion of our study to include other outbred and inbred lines of rats is indicated to analyze stock-dependent and strain-dependent effects, respectively, on buprenorphine metabolism and duration of action, as metabolism of opioids and other compounds in rats has shown variability based on stock and strain type.30,34,90 In addition, while we have characterized the pharmacokinetics of HCB in rats, the analgesic efficacy of this specific buprenorphine formulation has yet to be studied in the species using surgical or other models that induce pain.
While the concentration of buprenorphine consistent with analgesia was measured in a previous study using plasma,20 there is yet no current evidence to demonstrate appreciable differences for drug pharmacokinetics studies using serum compared with plasma analytes.91 However, as some other parameters such as metabolites and biochemistry values can vary in terms of serum compared with plasma concentrations in rats and other species,92,93 an additional aspect of future studies might include screening for any appreciable concentration differences, especially with respect to any strain, age, or sex-specific variability when applied to surgical or other pain models.
At our institution, most rats and other rodents used in studies with a survival surgical component receive buprenorphine analgesia for a total of 2 to 3 days (day of surgery and 48 hours postprocedure) and then as needed based on both scientific and veterinary discretion.94 This means that with CB administration and typical dosing guidelines,18,25,95 rats must be restrained at least 3 to 4 times daily to provide this modality of analgesia, which may ameliorate any associated pain, but might inadvertently drive stress in manipulated animals. CB is also used to treat nonsurgical instances of pain, whether experimentally induced, spontaneous, or medically driven, where the animals are also administered this medication with the same frequency, thus also driving handling-associated stressors as a consequence of analgesic administration. By potentially incorporating HCB and other longer-acting analogues into an analgesic regimen, the concern for restraint stress is mitigated and analgesia is optimized, thereby advancing the principles of animal welfare and protecting scientific data from the confounders of pain and distress.
Semilog Scale Graph of the Mean Serum Concentrations of Buprenorphine in ng/mL in 6 Rats Over 48 h (2 d) After Receiving a Singular Dose of HCB at 0.24 mg/kg SC (Time Point 0). The minimum effective plasma concentration of buprenorphine in rats is noted with a solid line at 1 ng/mL and the 24 h postadministration time point is circled. The remaining 72- and 96-h time points (3 and 4 d post-HCB injection, respectively) are not shown as serum concentrations of the drug were not detectable in any animal at those given time points.
Focal, Mild Erythema Over the Injection Site found in a Single Rat on Day 4 of the Study (3 d After HCB Injection). Lateral right dorsum, flank area of affected rat. Minor, moderately well-circumscribed focal area of erythema with skin intact (circle).
Injection Site of Rat, Day 11 of Study (10 d After HCB Injection). Lateral right dorsum, flank area of affected rat. Lesion has been lightly encircled with blue permanent marker. Pinpoint, dry erythemic crust developed directly over injection site (arrowhead).
Contributor Notes