Pharmacokinetics of Oral and Subcutaneous Carprofen in Common Marmosets (Callithrix jacchus)

Animals.

Three adult marmosets (3.1 ± 1.6 y old [mean ± SD]) were used to complete this study (one female originally from Texas Biomedical Research Institute, and one female and one male born at the Salk Institute for Biologic Studies). They were part of a marmoset breeding colony housed at the Salk Institute for Biologic Studies, an AAALAC-accredited facility. Historically, the females had been treated for Giardia (testing negative posttreatment with tinidazole) and received once a month Estrumate (cloprostenol) injections for birth control. The male had experienced a distal tail amputation and had undergone sperm collection in the past. Pathogens such as Giardia and Clostridium difficile cycle through the colony and are treated when causing clinical disease.

The study was performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act as Amended. 2008. 7 USC §2131–2156, and United States Public Health Service policy.^3,24,47 All procedures performed were approved by the Salk Institutional Animal Care and Use Committee. All marmosets in this study were socially housed as either male/female or male/male pairs. They were deemed healthy based on routine physical examination (including body condition scoring, bloodwork [CBC and full chemistry panel], an intradermal tuberculosis test, and fecal culture and analysis for ova and parasites) performed by a veterinarian semiannually prior to enrollment. The marmosets were maintained on a 12-h light/12-h dark cycle, temperature range of 80 ± 2 °F (23.3 ± 1.0 °C), with a relative humidity range of 30% to 70%. Enclosures (2 marmoset Britz and 1 in-house custom-built enclosure) contained perches, nest boxes, hammocks, and hanging toys. The diet consisted of commercial biscuits soaked lightly in orange-flavored Tang (Kraft Heinz, Chicago, IL) fed in the morning (LabDiet New World Primate Diet 5040; Land O’Lakes, Inc, Richmond, IN). The evening meal consisted of a soft canned diet (ZuPreem, Premium Nutritional Products, Mission, KS) supplemented with fresh food items including fruits or vegetables and a protein source (egg, cottage cheese, garbanzo beans) once daily. Enrichment included acacia gum, foraging boxes, and mealworms. Animals were provided reverse osmosis water ad libitum from water bottles. Staff observed animals at least twice daily and individual health records were maintained.

Acclimation.

To decrease the potential stress associated with study procedures, animals were acclimated to transport, capture, handling, and venipuncture restraint position prior to initiating the study. Animals were acclimated to capture into the transport cage with positive reinforcement using a high food item of choice (marshmallow, banana chips, pudding, marshmallow fluff). Once the animal voluntarily entered the transport cage they were acclimated to restraint and appropriate venipuncture handling position, also with positive reinforcement. Acclimation was performed in daily 30- to 40-min sessions for a minimum of 5 continuous days prior to study initiation. Acclimation was repeated prior to use in the next analgesic group.

Study design and drug administration.

A crossover design was used such that each of the 3 animals received all 4 treatment groups. The 3 animals were randomized into each group, that is, low dose orally, high dose orally, low dose subcutaneously, and high dose subcutaneously, prior to study initiation. Once an experimental group was determined, prior to administration of the drug, each marmoset was weighed to calculate an accurate dose. Animals received the drug while conscious and restrained by a handler. For subcutaneous administration of carprofen (Rimadyl 50 mg/mL; Zoetis, Kalamazoo, MI), animals received either a single 2 or 4 mg/kg SC injection between shoulder blades. The interscapular region was shaved prior to subcutaneous drug administration for visual monitoring of potential injection site reactions.

Oral carprofen formulation was composed of flavor tablets (Rimadyl 50 mg/mL; Zoetis, Kalamazoo, MI) reconstituted in deionized water. Animals received either a high dose (4 mg/kg) or low dose (2 mg/kg) of reconstituted carprofen by mouth at a given time point. They then had their blood drawn for pharmacokinetic analysis as described below. All animals had a minimum 10-d washout period before use in the next randomized, analgesic treatment. No animals were used in the same analgesic group twice. The low and high doses chosen were based on suggested published doses,^22,37 and what has historically been used at our institution.

Sample collection.

Blood samples (approximately 0.2 to 0.4 mL) were collected using either via saphenous or femoral venipuncture into heparinized BD Microtainer blood collection tubes (BD, Inc.; Franklin Lakes, NJ). Blood samples were collected from all analgesic groups immediately prior to analgesic administration at a baseline time point (0 h) and then subsequently at time points 1, 2, 4, 6, 12, and 24 h after drug administration. For saphenous blood collection, one person manually restrained the animal and provided positive reinforcement treats such as marshmallow, banana chips, pudding, or marshmallow fluff, while a second person performed phlebotomy from the saphenous vein. All blood samples were collected via the saphenous vein, except the 12-h time point, to rest the saphenous vein after the first 5 blood collections. During the 12-h time point, animals were anesthetized with isoflurane (3% to 4% in 1.5 L/min oxygen for induction, 1% to 3% for maintenance) via face mask, and blood collection was performed via femoral venipuncture. Anesthesia was used to safely immobilize the animal for the procedure. Animals were provided atropine (Hikma Pharmaceuticals, Berkeley, NJ) at 0.02 mg/kg IM to control tracheobronchial secretions. Blood collection volume amounts (0.2 to 0.8 mL) were replenished as a lactated Ringer solution fluid (Covetrus; Portland, ME), which was administered subcutaneously at every other blood draw. The marmosets were returned to a transfer cage between sample collection time points. Transfer cage location was maintained within the same light cycle, temperature range, and relative humidity as animal holding rooms. Animals were allowed to return to their home cage after the 24-h blood collection. Blood samples were quickly processed after collection by centrifugation (10,000 × g) for 10 min, and plasma was separated and stored in cryogenic tubes at –80 °C until analysis.

Observations.

All marmosets were observed at each blood collection time point within the transfer cage during the study and for up to 24 h after within their holding cage. Marmosets were evaluated for general wellbeing and for potential adverse effects after drug administration. Temperature, pulse, respiration, mentation, urination, defecation, and food and water intake were noted daily, and any abnormalities were recorded.

Sample analysis.

Carprofen plasma concentrations were measured by liquid chromatography–MS. Deuterated carprofen was added as an internal standard to 5- to 20-μL aliquots of plasma samples. Samples were extracted with 400 μL of an acetonitrile/methanol mixture (1:1, v/v). The samples were vortexed and centrifuged (20,000 × g, 10 min at 4 °C). The supernatants were transferred to another tube and evaporated, and the extracts were reconstituted in 50 μL of water/methanol (1:1, v/v) and transferred to an autosampler vial for liquid chromatography–MS analysis.

Liquid chromatography–MS analysis was performed on a Dionex UltiMate 3000 LC system (Thermo Fisher Scientific; Waltham, MA) coupled to a TSQ Quantiva mass spectrometer (Thermo Fisher Scientific; Waltham, MA). A Kinetex (Phenomenex; Torrance, CA) C18 column (2.6 μm, 150 × 2.1 mm) was used. Solvent A consisted of 0.1% formic acid in water, and solvent B was 0.1% formic acid in methanol. The gradient was increased linearly from 0% to 100% in solvent B in 12 min, held at 100% in solvent B from 12 to 18 min, returned to 0% in solvent B, and held at 0% in solvent B until 25 min. MS analyses were performed using electrospray ionization in a positive mode, with spay voltages of 3.5 kV, ion transfer tube temperature of 325 °C, and vaporizer temperature of 275 °C. Sheath, auxiliary, and sweep gases were 15, 10, and 1, respectively. Multiple reaction monitoring was performed for carprofen (274.1 > 167.1, 274.1 > 193.1, 274.1 > 228.1) and d3-carprofen (277.1 > 170.1, 274.1 > 196.1, 274.1 > 231.1) as an internal standard. Skyline³⁴ was used to extract and quantitate peak areas. Carprofen concentrations were determined from the peak area ratios of the analyte relative to its internal standard and compared with the calibration curve generated from the analysis of human plasma spiked with known concentrations of the analyte and its internal standard.

Data analysis.

Statistic and noncompartmental pharmacokinetic analysis were performed by using the PK package⁶¹ in R (version 4.2.0), with code available on GitHub. C_max and T_max were determined based on direct observation of concentration–time data. The observed AUC (AUC 0–time of last measurable concentration [T_last]) was calculated using the log-linear trapezoidal rule, and the AUC to infinity (AUC 0-inf) was calculated assuming exponential decay of the last 3 time points. The T_last was decided at the time of the study design as 24 h and was the same for each marmoset. The last measurable concentration was directly observed at T_last. The other parameters are functions of the AUC and dosage. Mean resident time was calculated by dividing area under the first moment curve by AUC 0-inf. Terminal half-life was calculated by multiplying mean resident time by the natural log. Total clearance was calculated by dividing the dose by AUC 0-inf. The volume of the distribution at steady state was calculated by multiplying the clearance by the mean residence time. Bioavailability issues are unlikely to have influenced clearance and volume of distribution, and this was an assumption made during statistical analysis in our study, because carprofen is almost 100% bioavailable after oral dosing and after subcutaneous administration in other species.^{11,12,53,55,59} Oral and subcutaneous carprofen exposure has been deemed to be bioequivalent, with comparable absorption under steady-state conditions, despite differing peak plasma concentrations.⁵⁵

Observations.

All marmosets remained healthy throughout the entirety of the study. Adverse effects were temporary and resolved without intervention. Adverse effects included hyporexia, skin lesions, and weight loss. Up to 10% weight loss from initial body weight was noted during the 24-h study period. Weight loss was attributed to stress of handling and anesthesia. Table 1 provides baseline body weights and weights at completion of each 24-h cycle after carprofen administration (following the 24-h blood collection). Weights returned to baseline by the next study time point in all but the young female, which was noted to have mild progressive weight loss of 6.14% from baseline weight during the study period.

Skin lesions (Figure 1) developed after subcutaneous injections of carprofen. Lesions were seen in 4 of the 6 subcutaneously injected sites within 12 h. They occurred in the young male with the 2 and 4 mg/kg dose, in the young female with the 4 mg/kg dose, and in the old female with the 2 mg/kg dose. Lesions were less than 0.5 cm in circumference, superficial, and mild, with surrounding erythema with no obvious swelling, pain, or discharge present. All skin lesions resolved within 10 d.

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Pharmacokinetic analysis.

Plasma concentrations of 2 carprofen doses and 2 administration routes were determined in 3 marmosets over a 24-h period (Figure 2). Baseline blood samples at time 0 demonstrate no detectable carprofen in the plasma after washout periods.

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Plasma concentrations after administration of 2 mg/kg carprofen showed a rapid and robust increase (Figure 2). The peak plasma concentration when administered orally was calculated to be 3170.37 ± 1.51 ng/mL and achieved at 1.26 ± 1.49 h after administration. For the subcutaneous route, peak plasma concentration of 5,414.43 ± 1.43 ng/mL was calculated and achieved at 1 ± 0 h after administration. The plasma concentrations declined by 24 h after administration to 359.83 ± 1.27 ng/mL for the oral route and 649.39 ± 1.51 ng/mL for the subcutaneous route. After 4 mg/kg carprofen administration either orally or subcutaneously, there was a similar rapid and robust increase in the plasma concentration (Figure 2). For the oral route, a peak plasma concentration of 8,005.74 ± 1.33 ng/mL was calculated and achieved at 1 ± 0 h after administration. For the subcutaneous route, the peak plasma concentration was calculated to be 7,011.61 ± 1.27 ng/mL and was achieved at 1.26 ± 1.49 h after administration. After 24 h the plasma concentrations declined to 903.33 ± 1.21 ng/mL for the oral route and to 1,106.49 ± 1.34 ng/mL for the subcutaneous route. T_max at 24 h did not differ significantly in regard to route or dose. Bioavailability for 2 mg/kg PO compared with subcutaneous dosing was calculated to be 55.28%. Comparatively, the 4 mg/kg PO compared with the subcutaneous dosing was calculated to be 94.44%. The bioavailability difference between 2 mg/kg compared with 4 mg/kg is likely to be due to nonlinear pharmacokinetics, and the bioavailability of carprofen appears to be dose-dependent.

Table 2 provides the complete pharmacokinetic parameters for both low-dose (2 mg/kg) and high-dose (4 mg/kg) carprofen administered orally and subcutaneously.