The aim of the study was to determine the bioavailability of buprenorphine after subcutaneous (SC) and intramuscular (IM) injection in New Zealand White (NZW) rabbits.
In this prospective cross-over study, five female and five male NZW rabbits each received four treatments with buprenorphine (Vetergesic vet, 0.3 mg/mL, Orion Pharma, Danderyd, Sweden), with 2-week wash-out periods between treatments. The order in which the animal received the treatments was randomized (Excel, Microsoft cooperation, Redmond, WA, USA). The treatments were 0.05 mg/kg buprenorphine by the IV, IM and SC routes, and 0.1 mg/kg by the SC route (see supplement).
The rabbits originated from an SPF breeding colony (Lidköpings kaninfarm, Lidköping, Sweden) free from Clostridium piriforme, Encephalitozoon cuniculi, rabbit hemorrhagic disesase, rotavirus, Pasteurella spp. and Bordetella bronciseptica. They were aged 4–5 months, with a mean ± SD body weight of 3.1 ± 0.3 kg. The females were housed 2 and 3 together and the males singly, in pens of 3 m2. Aspen wood chips (Tapvei, Paekna, Estonia) and autoclaved straw (local farm, Uppsala, Sweden) were used for bedding, and the pens were cleaned weekly. The pens were furnished with combined shelters and resting shelves. The room temperature was 19 ± 2 °C, the light:dark cycle 8 h:16 h. The rabbits were fed a restricted amount of a pelleted diet (K1 special, Lantmännen, Stockholm) and had free access to autoclaved hay (local farm) and tap water. The rabbits were acclimatized for two weeks, during which members of the research team took turns in spending 15 min per day per pen to accustom the rabbits to the presence of the members and to handling. Body weight was recorded daily during a week before study begin and at each treatment. Before each treatment, the rabbits underwent clinical examination (visual inspection, auscultation of heart and lungs). Before study begin, blood was collected for hematology.
Drug administration and sampling
For placements of catheters (Venflon IV PVK, 22G, BD AB, Helsingborg, Sweden), the ears were treated with a local anesthetic cream (EMLA®, AstraZeneca, Södertälje, Sweden). After approximately 30 min, a catheter was placed in the central artery of one ear and on the day that buprenorphine was administered IV, one additional in the lateral ear vein of the contralateral ear. A 2 mL blood sample was collected from the arterial catheter before and at each sampling timepoint after administration of buprenorphine. Timepoints for sampling were 2 min (after IV administration only), 5, 10, 15, 30, 60, 90, 180, 360 and 480 min after injection. The time points were based on previous studies. Prior to blood collection, 0.2 mL of blood was discarded and after collection the catheter was flushed slowly with 2 mL of NaCl (Infusion solution, 9 mg/mL, B. Braun, Danderyd, Sweden) and 0.1 mL of heparinized saline (100 IU /mL, Heparin LEO 5000 IU/mL, LEO Pharma, Malmö, Sweden) deposited in the catheter. A maximum volume 7 mL blood/ kg was collected at each treatment. Depending on the weight of the rabbit, and the treatment, a random sample timepoint was sometimes excluded in order not to exceed the recommended maximum blood volume [15% of blood volume/14 d ]. Blood was collected in 2 mL serum tubes and left to coagulate at room temperature for at least 60 min. After centrifugation at 3000 g for 15 min, serum was separated and stored at minus 80 °C until analyzed. At the end of each treatment, 4 mg/kg carprofen (Norocarp vet, 50 mg / mL, N-Vet AB, Uppsala, Sweden) was administered SC for 24 h of post procedural analgesia. After the study end, the rabbis were re-used in a non-recovery anaesthesia study euthanized by injection of pentobarbital (Allfatal) IV.
Buprenorphine was quantified in rabbit plasma using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) using an Acquity UPLC coupled to a TQS micro tandem mass spectrometer (both Waters Corp, Milford, MA, USA) using an electrospray interface operating in the positive mode. To the plasma samples (250 μL), 50 μL of water, 50 μL of internal standard solution (buprenorphine-d4 40 ng/mL) and 250 μL of sodium carbonate buffer (0.05 M; pH 9.9) were added. Liquid-liquid extraction was performed to 3.0 mL of ter-butyl-methylether for 10 mins. The tubes were centrifuged and frozen at − 70 °C, after which the organic phases were poured into new tubes for evaporation to dryness under nitrogen at 50 °C. The residues were reconstituted in 100 μL of acetonitrile/water/formic acid (9/1/0.01, v/v). The extracts were transferred to vials for UHPLC-MS/MS injection. The column used was an Acquity UPLC BEH C18 (100 × 2.1 mm length x inner diameter, 1.7 μm particle size) from Waters Corp. The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Gradient elution was performed: initially 15% B for 0.50 min, linear increase to 90% B for 1.0 min, constant at 90% B for 0.50 min, back to 15% B in 0.10 min and constant at 15% B for 0.40 min. The total run time was 2.5 min. The flowrate was 0.40 mL/min and the injection volume was 3.0 μL.
The quantification was performed in the Selective Reaction Monitoring mode (SRM) with the transitions 468 > 396 for buprenorphine [M + H] + (collision energy 36 eV), and 472 > 400 for buprenorphine-d4 [M + H] + (collision energy 38 eV). For quantification, calibrator samples were prepared by spiking standard solutions of the analyte to blank plasma which were analyzed according to the method. The calibration curve was constructed by linear curve fitting using the chromatographic peak area ratio (analyte/internal standard) as a function of analyte concentration with a weighting factor of 1/x. The measurement interval was 0.01 ng/mL – 100 ng/mL. The precision and accuracy of the method were within the acceptance criteria .
For each animal, route and dose the concentration of buprenorphine were plotted vs. time. Different models and weighting factors were assessed by visual inspection of the curve fits and the residuals’ scatter plots, together with the accuracy of fit measures incorporated in the software (e.g. the Akaike criteria). A non-compartmental model was used to compare all routes. The maximal concentration of buprenorphine in serum (Cmax), tmax (time to reach Cmax), t½ (half-life) and AUC (area under the plasma concentration curve) were calculated with a non-compartmental model using the PK Solver add-in for Microsoft Office Excel. For IV administration also a 3-compartment model was used to calculate the maximal concentration at time 0/at the time for administration (C0).
The bioavailability (F%) for the SC and IM administration routes were calculated from the AUC0-t by using the equation:
$$ Fadm\ \left(\%\right)=100\ x\ \left( AUC adm\ x\ dose\ iv\right)/\left( AUC\ iv\ x\ dose\ adm\right) $$
InVivoStat (Version 4.0) was used for the sample size assessment . The number of rabbits used was selected based on a power calculation, with a significance level set at 5%, and a power of at least 80% to detect at least a 25% change from the area under the time-concentration curve (AUC0-t) after IV administration. Data from two animals were removed from the statistical analyses (#3 0.05 mg/kg SC and #6 0.05 mg/kg IM) because the concentration curves did not follow a normal pattern and the concentrations were in part so high than it is unlikely a normal biological variation. All data are included in the supplement data file.
Data were analyzed in two stages. In order to test the overall effect of the administration routes, the data from the 0.05 mg/kg groups were compared by two-way ANOVA with Route and sex as independent factors and animal and treatment day (of administration) as blocking factors (SAS/STAT software, Version 9.4 of the SAS System for Windows 10). This was followed by planned comparisons of the predicted means to compare the IV and IM routes back to SC. In the second stage the additional SC 0.1 mg/kg group was included to allow a comparison between the SC 0.05 mg/kg and SC 0.1 mg/kg groups.
Data are presented as mean ± SD. The level of statistical significance was set to p < 0.05.