A Pharmacokinetic and Efficacy Study of FDA-Indexed Pharmaceutical-Grade Extended-Release Buprenorphine and Compounded Sustained-Release Buprenorphine in African Green Monkeys (Chlorocebus aethiops)

Abstract

Compounded sustained-release buprenorphine (SRB) has historically been used at our institution for postoperative analgesia in African green monkeys (AGMs), but it is not FDA approved and lacks species-specific data. With the recent FDA indexing of pharmaceutical-grade extended-release buprenorphine (EXR; Ethiqa XR; Fidelis), we aimed to compare the pharmacokinetic (PK) profile using serial plasma sampling up to 96 hours and postoperative analgesic efficacy of EXR compared with SRB. A total of 10 AGMs (n = 5 per test article) received 0.2 mg/kg SC of either EXR or SRB to complete the PK study. For the efficacy study, 20 animals (n = 10 per test article) were monitored for 3 days postoperatively after EEG telemetry surgery with femoral artery catheterization for behavioral indicators of pain and adverse effects. Plasma buprenorphine levels were analyzed using liquid chromatography-tandem mass spectrometry. PK study observations included appetite, fecal output, and injection site reactions. Both formulations achieved plasma concentrations above the presumed therapeutic threshold (0.1 ng/mL) for up to 96 hours. SRB produced higher mean plasma levels at early time points (30 minutes, 4 hours), but the overall PK profiles were similar. Injection site reactions were minimal and resolved spontaneously. For the efficacy assessment, clinical observations were evaluated using a postoperative score sheet. No significant differences were observed in postoperative pain scores between groups. Both EXR and SRB at 0.2 mg/kg SC provided sustained therapeutic plasma levels up to 96 hours and were well tolerated in AGMs, with comparable efficacy in postoperative pain control. EXR offers a compliant and effective alternative to compounded SRB for analgesia in AGMs, supporting its use in laboratory settings and regulatory compliance with current guidelines.

Introduction

Rhesus monkeys (Macaca mulatta) have traditionally been the preferred nonhuman primate (NHP) model for behavioral and physiologic assessments in biomedical research.^1–3 However, in recent decades, African green monkeys (AGMs; Chlorocebus aethiops) have gained prominence as an alternative due to their increased availability, reduced cost, smaller size, and noncarrier status for herpes simian B virus.^4–6 Despite their growing use, limited published information exists on anesthesia and analgesia protocols specifically validated for AGMs.^7,8 Although some studies suggest AGMs may respond similarly both pharmacologically and behaviorally to the more commonly studied macaque, this presumed comparability warrants direct verification.^8–10

Buprenorphine is the most widely used opioid analgesic in NHPs, including AGMs.^11,12 As a partial agonist at the mu-opioid receptor and antagonist at the κ-opioid receptor, buprenorphine provides effective analgesia with a lower risk of respiratory depression than full opioid agonists.^13,14 Extended-release formulations further enhance animal welfare by reducing the need for repeated handling and dosing: an important consideration for AGMs, which are particularly sensitive to environmental and procedural stressors.¹⁵ In addition, buprenorphine’s ceiling effect on respiratory depression enhances its safety profile, making it well suited for use in primate models. However, no published studies have evaluated the pharmacokinetics, pharmacodynamics, safety, or efficacy of extended-release buprenorphine (EXR) or sustained-release buprenorphine (SRB) in AGMs. The therapeutic threshold of buprenorphine in AGMs remains undetermined.¹⁵ However, based on findings in dogs and humans, a concentration of 0.1 ng/mL is presumed to be a potentially effective therapeutic level.^15–19

Sustained-release buprenorphine (SRB; Wedgewood Pharmacy, Swedesboro, NJ) is a compounded, nonpharmaceutical-grade formulation containing 10 mg/mL buprenorphine in a patented biodegradable polymer matrix designed to provide sustained plasma levels for up to 72 hours following a single subcutaneous injection.²⁰ Injection-site reactions are the most reported adverse effect across species.^15,21 At our institution, SRB was previously used for postsurgical analgesia in AGMs. However, in March 2024, the FDA indexed a pharmaceutical-grade extended-release buprenorphine formulation, Ethiqa XR (EXR; Fidelis Animal Health, North Brunswick, NJ), for use in NHPs (specifically tested in macaques, baboons, and marmosets). EXR has demonstrated analgesic efficacy for up to 72 hours in multiple species, including captive rodents, ferrets, laboratory rabbits, and NHPs. Per label instructions, the time to reach estimated therapeutic blood levels after administration at 0.2 mg/kg SC is 15 minutes, and plasma levels greater than 0.1 ng/mL are maintained for 72 hours in NHPs.²² While data are available for species such as the common marmoset (Callithrix jacchus), owl monkey (Aotus spp.), rhesus monkey (Macaca mulatta), cynomolgus monkey (Macaca fascicularis), and baboon (Papio anubis), no such studies have been conducted in AGMs.^3,15,23–25 As a result, the continued use of compounded SRB, which lacks FDA approval or indexing, may be regarded as noncompliant with the Guide for the Care and Use of Laboratory Animals unless scientifically justified and approved by the IACUC. This regulatory change prompted our institution to transition to EXR to ensure compliance, which coincided with an ongoing telemetry implantation protocol in AGMs. This provided a unique opportunity to conduct a head-to-head comparison and validate EXR efficacy in AGMs, as this species was not included in the original FDA indexing studies. Therefore, the present study aimed to directly compare the pharmacokinetic profiles of EXR and SRB following subcutaneous administration at 0.2 mg/kg in AGMs.

In addition to pharmacokinetics, we evaluated postoperative analgesic efficacy using observational clinical scoring and physiologic parameters measured via telemetry. This dual assessment approach incorporated objective data, body temperature, and blood pressure, as well as subjective scoring criteria (alertness, body posture, head position, neurologic signs, pain, feeding, drinking, and vomiting) to support the comprehensive evaluation of pain management strategies.

The objective of this study was to evaluate and compare the pharmacokinetic profiles and analgesic efficacy of SRB and EXR in AGMs using validated mass spectrometry, postoperative score sheets, and telemetry-based physiologic data. We hypothesized that both formulations would yield comparable pharmacokinetic and analgesic profiles, consistent with findings reported in other NHP species.

Ethics Statement

The experimental protocol was approved by the IACUC at the United States Army Medical Research Institute of Chemical Defense, and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. The facility is accredited by AAALAC International and compliant with the Animal Welfare Act, Public Health Service Policy, and the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).^26–28

Materials and Methods

Experimental animals.

Twenty wild-caught, juvenile to adult male AGMs were obtained from Worldwide Primates (WWP; Miami, FL). Animals were estimated to be between 2 and 8 years old and weighed 4-8 kg. Before shipment, AGMs tested negative for simian T-lymphotropic virus type 1, cercopithecine herpesvirus 2, simian rotavirus A, simian immunodeficiency virus, measles, Bordetella bronchiseptica, Salmonella spp., Shigella spp., and Klebsiella pneumoniae (including rmpA and magA subtypes) and had no external parasites or fecal pathogens. They were confirmed to be tuberculosis-free via intradermal testing and TB serology. All AGMs were individually tattooed for identification purposes and determined to be clinically healthy based on CBC, serum chemistry, and initial physical examination.

All animals were housed individually in one-over-one, interconnecting, stainless steel 6.0-sq ft/0.56-m² enclosures featuring quick-select side panels and dividers that meet the USDA requirements and the Guide for the Care and Use of Laboratory Animals recommendations.^27,29 Although they were singly housed, they were within visual and auditory contact with conspecifics and were provided enrichment such as perches, hammocks, hanging and loose toys, and foraging devices. Animal rooms were maintained on full-spectrum lighting with a 12-hour light/12-hour dark cycle (lights on, 6:00 am to 6:00 pm), temperatures at 64-84 °F, and 30%-70% humidity with 100% conditioned air at 10-15 changes hourly. They were fed Purina LabDiet 5045 High Protein Monkey Diet (25% crude protein, 5% crude fat, and 6.5% crude fiber; Purina LabDiet, Richmond, IN) twice daily, along with daily fresh produce or foraging enrichment. Water was provided ad libitum from municipal city water that was purified on-site via reverse osmosis and chlorinated.

Sample size.

For the pharmacokinetic study, sample sizes of 5 AGMs per test article (10 total) were chosen based on a similar study in macaques. In that study,¹¹ buprenorphine plasma measurements in the range of 0.1-1 ng/mL had a SD, on average, of approximately 0.15 ng/mL. At all time points out to 96 hours, plasma levels remained at least 0.3 ng/mL above the presumed therapeutic threshold of 0.1 ng/mL. Assuming that AGMs will behave similarly, then s/δ = 0.15/0.3 = 0.5. For a one-sample t test (comparing measured plasma levels to the therapeutic threshold) with α = 0.05 and β = 0.10, an appropriate sample size per group is 5 animals. The average plasma buprenorphine levels at each time point could then be compared with the therapeutic threshold. Pharmacokinetic parameters such as C_max, T_max, clearance, and t_1/2 were calculated for both buprenorphine formulations.

For the efficacy study, we included all 20 animals participating in the broader protocol. This study represents a subanalysis of a 20-animal AGM protocol, rather than a standalone investigation. Since all 20 animals underwent identical postoperative monitoring and EEG telemetry data collection as components of the primary protocol, we analyzed the complete dataset regardless of which buprenorphine formulation each animal received.

Study design: pharmacokinetic analysis.

This study was a substudy of an ongoing research protocol that involved a total of 20 AGMs. All 20 animals did not arrive at the same time points. They were staggered over a 5-month period into 2 groups of 10. Between the time of the first 10 AGM cohort undergoing surgery and the next shipment of AGMs to arrive, we became aware that the FDA had indexed EXR for NHPs. Researchers at this institution were concerned that a switch from SRB to EXR in the middle of the protocol could cause confounding of experimental results. The research team requested a pharmacokinetic study be performed to alleviate their concerns. Before the institute’s 6-week quarantine period for the new cohort arriving, an amendment was approved for this substudy of EXR. Once the 10 new AGMs arrived, they underwent a stabilization period of approximately 3 weeks upon arrival before any study related to blood collection. The pharmacokinetic portion of this study was conducted during the fourth week of their quarantine. See Figure 1 for a visual representation of the timeline.

Figure 1.

Timeline: Visual Representation of Timeline. EXR, extended-release buprenorphine (Ethiqa XR); PK, pharmacokinetic; SRB, sustained-release buprenorphine.

The 10 newly arrived AGMs used for the pharmacokinetic study were randomly selected using a random number generator to receive a single subcutaneous injection of either 0.2 mg/kg of EXR or 0.2 mg/kg of SRB. To maintain methodological rigor in the context of a small sample size, no animals were excluded from the study. The research assistant administering the injections and monitoring for skin reactions was blinded to the test article. All AGMs were weighed the week before to ensure accurate drug dosing and had their abdomen and legs shaved to allow for evaluation of potential injection site reactions and ease of intravenous blood collection. The subcutaneous injection of the test article was administered on the left side of the ventral abdomen region. Both formulations of buprenorphine were administered to ketamine sedated AGMs (10 mg/kg IM; Ketaset; Zoetis Animal Health, Parsippany, NJ) using a 3-mL syringe (Nipro Medical, Miami, FL) with a 22-gauge, 1-inch needle (Terumo, Somerset, NJ). Drugs were administered in accordance with the manufacturer’s recommendation by tenting the skin and inserting the needle’s full length under the skin. The drug was injected slowly due to viscosity, and the needle was slowly withdrawn while pinching the skin for 5-10 seconds after needle withdrawal. To improve postinjection site monitoring, a surgical skin marker was used to draw a circle around the injection site, which was subsequently redrawn at each blood collection time point.

Blood sample collection.

Blood samples (approximately 1 mL) were collected at baseline, 30 minutes, 4, 8, 24, 48, 72, and 96 hours after test article administration, under ketamine sedation (5-10 mg/kg IM) for both buprenorphine formulations. Animals were fasted for 8-12 hours before sedation. An initial baseline blood sample was collected just before the administration (0 hour) of a single dose of the test article. AGMs were maintained under sedation with ketamine (10 mg/kg IM), and blood samples were collected 30 minutes later; animals were then allowed to recover. AGMs were resedated with ketamine (5 mg/kg IM) for blood samples taken at 4, 8, 24, 48, 72, and 96 hours after test article administration. Blood samples were collected from sedated animals using either the right or left saphenous vein using a heparinized 1-mL syringe with a 25-gauge, 5/8-inch needle (Terumo, Somerset, NJ). Heparinized blood samples were then immediately transferred into a cryovial and centrifuged for 10 minutes to obtain plasma. Plasma was stored at −80 °C until analysis was performed. A total of 80 plasma samples were collected.

Blood samples were analyzed using reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis was performed on an Agilent 1290 Infinity II LC system (Agilent Technologies, Santa Clara, CA) interfaced to a Sciex 6500+ QTrap triple quadrupole MS system (Sciex, Marlborough, MA) in positive electrospray ionization mode. Analyte concentrations were obtained using matrix-matched calibration and quality control standards with internal standardization. These standards were made using pooled-gender AGM plasma in sodium heparin. The entire analysis was performed on the same day. Solid phase extraction of the calibration curve (a blank, a zero calibrator, and 7 nonzero calibrators), quality control standards, and animal samples was performed in duplicate, and the replicates were analyzed in triplicate. Buprenorphine was quantifiable over the range of 0.1-50 ng/mL; the lower limit of quantitation (LLOQ) was 0.1 ng/mL. GraphPad Prism 10 software was used to prepare all graphical representations.

Study design: efficacy evaluation.

Before the FDA indexing of EXR for NHPs, 10 AGMs underwent surgical implantation of L11 telemetry transmitters (Data Sciences International [DSI], St. Paul, MN) using a flank approach along with femoral artery catheterization and EEG implants.³⁰ SRB was used as part of the pain control regimen. The FDA’s indexing of EXR occurred midway through the protocol, allowing comparison with the next 10 animals using EXR instead of SRB.

The day before surgery, all AGMs received a subcutaneous injection of Excede (20 mg/kg; 100 mg/mL; Zoetis Animal Health, Parsippany, NJ), and all food was removed from the cage before employees left for the evening. The day of surgery, AGMs were intramuscularly sedated in their home cage with ketamine (10 mg/kg, 100 mg/mL) and glycopyrrolate (0.005 mg/kg; 0.2 mg/mL; McKesson Medical-Surgical, Irving, TX) before being transported to the surgical prep suite where they were subcutaneously administered SRB (0.2 mg/kg; 10 mg/mL) before surgical prep began. After prepping for surgery, meloxicam (0.2 mg/kg; 5 mg/mL; Boehringer Ingelheim, Duluth, GA) was given subcutaneously immediately before moving the AGM into the surgical suite. The specific surgical technique is not described, as it is proprietary information of DSI, but generally, transmitters are normally placed in a pocket between the exterior and interior abdominal obliques. Telemetry implantation is routinely performed via a skin incision approximately 4 cm in length made lateral to the midline near the umbilicus. The aponeurosis between the rectus abdominis and external abdominal oblique muscles is located, and an incision approximately 3 cm in length is made. The external abdominal oblique is retracted, and a pocket is created. The antenna is tunneled through the inside of the pocket superficially. A skin incision is made over the femoral artery to place an arterial catheter. In general, EEG leads are placed by reflecting all muscle, connective tissue, and periosteum covering the skull with periosteal elevators in an area large enough to be able to place screws and loop the EEG lead. Bilateral burr holes are drilled in the skull. Screws are placed in holes perpendicularly to the skull. All exposed wire and screws are covered with dental glass ionomer. All muscle, subcutaneous, and skin incisions are closed via surgeons’ preference. All postsurgical AGMs were monitored and observed for abnormal behaviors by veterinary staff twice daily (8:00 am and 3:00 pm approximately) for 3 days following surgery. Meloxicam tablets (0.2 mg/kg; BioServ, Flemington, NJ), fiber gummies (Fiber Well; Vitafusion, Los Angeles, CA), and oral rehydrator bottles (BioServ, Flemington, NJ) were given orally once daily for 3 days following the surgery.

Approximately 4-5 months after the completion of surgery on the first 10 AGMs, the second 10 AGMs in the cohort completed their quarantine and underwent surgery. These 10 AGMs were the NHPs used in the pharmacokinetic study described above. Following the 45-day quarantine, they were deemed to be healthy and were released for surgery as described above. These 10 AGMs received EXR (0.2 mg/kg; 1.3 mg/mL) subcutaneously instead of SRB as the preoperative buprenorphine formulation at the time of surgical prep. All pre-, intra-, and postoperative procedures, medications, and postoperative monitoring score sheets were identical to those of the first cohort who were given SRB.

Outcome measures.

Pharmacokinetic analysis.

For the pharmacokinetic monitoring and observations, appetite, stool production, and injection site reactions were assessed for each of the 10 AGMs over the course of the 96-hour study. Injection site scores were based on evaluation for the presence of erythema, swelling, and pruritus (0: none; 1: minimal; 2: moderate; 3: severe) using digital palpation and visual inspection. To improve postinjection site monitoring, each AGM was shaved from the xyphoid process to the pelvis bilaterally, and a surgical skin marker was used to draw a circle around the injection site.

The primary outcome was to determine whether there was a difference between injection site reactions and pharmacokinetic measurements between the 2 buprenorphine formulations.

Efficacy evaluation.

For the analgesic efficacy component of the study, outcome measures were assessed using postoperative score sheets and physiologic telemetry data. The postoperative score sheet (Figure 2) evaluated alertness (0: awake/normal; 1: quiet but alert; 2: obtunded [decreased alertness, slowed responses, but responds to gentle prodding]; 3: stuporous [only response to painful stimuli or vigorous prodding]; 4: comatose [cannot be aroused]); body posture (0: normal/attempts to climb; 1: unstable when standing/sitting, weakness; 2: needs support, stumbling; 3: hunched but responsive; 4: hunched, unresponsive); head position (0: head erect; 1: head raised to stimulation; 3: raises head, but glazed or unfocused look; 4: unable to raise head); neurologic signs (0: normal; 1: mild [head tilt, circling, tremors, glazed look]; 2: moderate [stargazing]; 3: severe [head pressing]; 4: nonresponsive, paralysis, convulsions, nystagmus, opisthotonos); pain (0: no pain or distress observed; 1: mild [slow moving, but aware of surroundings]; 2: moderate [withdrawn, hunched, no interest in surroundings, increased respiratory effort]; 3: severe [vocalizations, immobility, or labored breathing]; 4: severe pain unresponsive to treatment); feeding (0: normal eating; 0.5: shows interest; 1: no eating); drinking (0: normal drinking; 0.5: shows interest; 1: no drinking); and vomiting (0: none; 1: drooling; 3: vomiting). In the postoperative score sheet (Figure 2), scores are summed for each observation with a maximum score of 33. A cumulative score of 0-6 is a category 0-1; no action is needed, continue to monitor. A cumulative score of 7-12 is a category 2; action is to address pain and/or incision, increase observations, and notify the principal investigator (PI) and veterinarian. A cumulative score of 13-19 is a category 3; action is to address pain and/or incision and evaluate for early euthanasia at the discretion of the attending veterinarian (AV) and PI. A cumulative score of 20-33 is a category 4; action is to euthanize under the direction of the AV and PI. All AGMs exhibited a cumulative score of zero (0) at baseline before surgery. Postoperative checks were conducted once after surgical recovery and then during morning and afternoon rounds for 3 days following surgery. The DSI telemetry system measured the following physiologic parameters: cortical EEG activity, blood pressure, heart rate, temperature, and motor activity inside the home cage.³⁰ The primary outcome was to determine whether there was a difference between EXR and SRB pain assessments during the postsurgical recovery time.

Figure 2.

Efficacy Study: Postoperative Score Sheet

Statistical methods.

Data analysis: pharmacokinetic.

At each time point for both formulations, the buprenorphine plasma level was tested against the therapeutic threshold of 0.1 ng/mL using a one-sample t test. The Shapiro-Wilk test was used to test the normality assumption. Mean levels for treatment were compared between groups for each time point using independent samples t tests. The Levene test for equality of variances was used to meet the analysis assumptions. The buprenorphine plasma level at each time point for each group met the assumption of normality. Sample size is 5 for each group at each time point. For the EXR group, time point 0.5 hour had 2 concentration values below the LLOQ (less than 0.1 ng/mL), and time point 96 hours had 1 value below the limit. The observed values were included in the analysis. SPSS was used for statistical analysis.

Pharmacokinetic analysis (Table 1) was performed on plasma concentration-time data obtained following subcutaneous administration of buprenorphine SRB or EXR. C_max and T_max were determined directly from the individual observed concentration-time data. Pharmacokinetic parameters (AUC and t_1/2) were derived using a model-independent (noncompartmental) approach.^31,32 AUC_0-TLast was determined using a linear up-log down trapezoidal rule. The area was extrapolated to infinity (AUC_0-∞) using the rate constant of the terminal elimination phase (λ_z), which was determined from the slope of the terminal log-linear portion of the concentration-time curve using a minimum of 3 measurable points after and including C_max. Furthermore, t_1/2 was calculated by dividing λ_z by the natural logarithm of 2. Apparent clearance (Cl/F) was calculated by dividing dose by AUC_0-∞, and apparent volume of distribution (Vz/F) was calculated by dividing the dose by the product of AUC_0-∞ and λ_z.

Table 1.

Pharmacokinetic Parameters: Buprenorphine Pharmacokinetics (average ± SD) after a Single Subcutaneous Injection of SRB or EXR in Male African Green Monkeys

Parameter	Buprenorphine SRB	Buprenorphine EXR
Cmax, ng/mL	1.5 ± 0.4	1.2 ± 0.3
Tmax, h	40 ± 10	40 ± 10
t1/2, h	40 ± 30	24 ± 9
Cl/F, L/h/kg	1.8 ± 0.3	2.7 ± 0.9
Vz/F, L/kg	90 ± 60	90 ± 30
AUC0-TLast, ng·h/mL	90 ± 20	70 ± 20
AUC0-∞, ng·h/mL	110 ± 20	80 ± 20

Abbreviations: AUC_0-∞, area under the curve from time zero to infinity; AUC_0-TLast, area under the concentration-time curve from time zero to the last measured concentration; Cl/F, apparent clearance; EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine; Vz/F, apparent volume of distribution.

Results

Pharmacokinetic analysis.

The plasma level was zero for all baseline (predrug administration) blood samples for both buprenorphine formulations. For EXR, 2 out of 5 AGMs were <0.1 ng/mL at 30 minutes postinjection but did reach >0.1 ng/mL by 4 hours postinjection. All 5 AGMs remained above 0.1 ng/mL between 4 and 72 hours postinjection. At 96 hours postinjection, one out of 5 AGMs fell below 0.1 ng/mL. For SRB, all 5 AGMs were above 0.1 ng/mL for all time points. Other than EXR at 30 minutes postinjection, all time points for each group had buprenorphine plasma levels significantly greater (P < 0.05) than the therapeutic threshold of 0.1 ng/mL. The average plasma buprenorphine concentration for EXR and SRB at each time point reached ≥0.1 ng/mL within 30 minutes and remained there to 96 hours as shown (Table 2) and plotted (Figure 3). Each comparison had at least 80% power to detect an effect size of 2 for the Cohen d (calculated as the difference between means divided by the pooled SD).

Table 2.

Pharmacokinetic Analysis: The Average Plasma Buprenorphine Concentrations over 96 h after Single Subcutaneous Injection of SRB or EXR

Time point, h	[Buprenorphine], ng/mL
	EXR (n = 5)	SRB (n = 5)
0	0 ± 0	0 ± 0
0.5	0.11 ± 0.05	0.47 ± 0.07
4	0.4 ± 0.2	0.9 ± 0.2
8	0.7 ± 0.2	0.8 ± 0.3
24	0.9 ± 0.2	1.2 ± 0.4
48	1.1 ± 0.3	1.4 ± 0.5
72	0.5 ± 0.2	0.68 ± 0.08
96	0.32 ± 0.05	0.39 ± 0.06

Values are expressed as average ± SD.

Abbreviations: EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine.

Figure 3.

Pharmacokinetic Analysis: The Average Plasma Buprenorphine Concentrations over 96 h after Single Subcutaneous Injection of SRB or EXR. Data are presented as mean ± SD. Boxed time points are displayed in insert. EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine.

Due to the small sample size, postinjection rating data, which set out to evaluate differences in erythema, swelling, pruritus, and stool presence/absence between EXR and SRB, lacked the statistical power to determine differences or equivalency between treatment groups. Furthermore, scores on the 0-3 scale for the presence of erythema or swelling lacked variation within and between groups. Nevertheless, in the interest of sharing pilot study data, response proportions for these rating data are shown (Table 3) and plotted (Figure 4). All AGMs remained healthy over the course of the pharmacokinetic study, apart from mild adverse effects in 4 animals. Both buprenorphine groups each had 2 animals with mild adverse effects. In the EXR group, one AGM showed mild erythema beginning at the 4-hour mark and resolved at the 48-hour mark. The second AGM showed mild erythema and mild pruritus beginning at the 48-hour mark and continuing past 96 hours. The SRB group had one AGM that showed moderate erythema beginning at the 4-hour mark and pruritus at the 24-hour mark. Erythema was resolved within 72 hours, while pruritus continued past 96 hours. The second animal demonstrated pruritus at the 24-hour mark that continued past 96 hours. No AGMs demonstrated excessive sedation or respiratory depression on cage-side observation. No significant changes were noted in behavior (no inappetence, bloating, constipation) at any time point. For both buprenorphine formulation groups, injection site scores (based on erythema and swelling) were highest at 8 hours after drug administration.

Table 3.

Compilation of Adverse Effects Scores

Symptom/group	Time points, h
	Baseline	0.5	4	8	24	48	72	96
Injecition site erythema
SRB	0	0	0.4	0.4	0.4	0.2	0	0
EXR	0	0	0.2	0.2	0.2	0.2	0	0
Injection site swelling
SRB	0	0	0	0	0	0	0	0
EXR	0	0	0	0	0	0	0.2	0.2
Injection site pruritus
SRB	0	0	0	0	0.4	0.2	0.4	0.4
EXR	0	0	0	0	0	0.6	0	0
Stool in cage
SRB	0	0	0.2	0.6	0	0.6	0	0
EXR	0	0	0	0.4	0	0.2	0	0

Pharmacokinetic analysis: proportion of animals with injection site symptoms posttest article administration from baseline to 96 h; n = 5 for each group. The table is based on the presence/absence of symptoms. For erythema and swelling, proportion was multiplied by pain score (0-3), allowing a maximum score of 3 for each group. Pruritus and defecation were yes/no responses, allowing a maximum score of 1 for each group. One animal exhibited moderate erythema, and all the other symptoms were mild. No severe symptoms were observed.

Abbreviations: EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine.

Figure 4.

Injection Adverse Effects: Proportions for All Injection Site Symptoms (Erythema, Swelling, Pruritus, and Defecation) Were Summed for Each Group to Create a Composite Score. EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine.

Efficacy study.

All postsurgical AGMs were monitored and observed for abnormal behaviors by veterinary staff twice daily for 3 days following surgery. Postoperative pain, assessed via cumulative pain scores (n = 10 per group), was analyzed using a linear mixed model for repeated measures. While significant changes in pain scores were observed over time, there were no significant overall differences between the treatment groups, nor was there a significant group-by-time interaction effect (Figure 5). Behavioral assessments further revealed the following: both groups had 6 animals that demonstrated a state of quiet alertness at least once during the observation period. However, signs of discomfort varied; mild pain was observed in 4 EXR animals and 7 SRB animals on at least one check, while abnormal posture was noted in 2 SRB animals. Regarding food and water intake, 2 animals in the EXR group displayed adipsia, and 4 displayed anorexia, at least once. All symptoms were observed within 24 hours postsurgery and resolved within 36 hours. All AGMs in both groups either returned to baseline or showed some interest in feeding and drinking by 72 hours. During one observation check, vomit was present in the cage of a single animal within the SRB group. The cage was found to be clear of vomit during the subsequent check.

Figure 5.

Efficacy Study: Summary of Postoperative (Post-OP) Scoresheet at Twice Daily Time Points over 6 Postoperative Health Checks (72 h). Data are presented as mean ± SD. EXR, extended-release buprenorphine (Ethiqa XR); SRB, sustained-release buprenorphine.

There were no significant adverse effects observed in either the EXR or SRB groups based on telemetry data. Due to user error, only temperature and blood pressure data were properly recorded via DSI telemetry for all AGMs during recovery. Motor activity within the home cage, EEG activity, and respirations were not recorded; therefore, these data were not reported. Body temperature and blood pressure for all AGMs in both groups were within normal biologic parameters.

Discussion

This study provides, to our knowledge, the first pharmacokinetic and efficacy data in AGMs on 2 extended-release formulations of buprenorphine, pharmaceutical-grade Ethiqa XR and Wedgewood’s compounded sustained-release buprenorphine. Both formulations achieved plasma concentrations above the presumed therapeutic threshold of 0.1 ng/mL for up to 96 hours, with no significant adverse clinical signs, thereby demonstrating sustained analgesic potential and good tolerability in this NHP model.

Pharmacokinetically, the 2 formulations exhibited broadly similar plasma profiles, although SRB achieved higher peak concentrations and more rapidly attained therapeutic levels at early time points (notably at 30 minutes and 4 hours postinjection). For EXR, there were 3 data points that were below the lower level of quantitation limit (<0.1 ng/mL) at time points of 30 minutes and 96 hours postinjection. This may reflect differences in formulation vehicle and release kinetics, as SRB includes a proprietary biodegradable polymer matrix, while EXR is an FDA-indexed formulation with previously documented pharmacokinetic profiles in other NHP species. Despite these differences, the mean residence time and duration of effective plasma concentrations were similar between groups, suggesting that EXR offers a viable and compliant alternative to SRB in AGMs. Importantly, both drugs were well tolerated, with only minor injection site reactions reported, including mild erythema and pruritus in a few individuals; these findings are consistent with reports in other species. Clinical observations also align with previously observed safety margins for buprenorphine including absence of respiratory depression.^{15,21,22,33,34} The absence of clinically relevant injection site inflammation or systemic adverse effects reinforces the suitability of both products, although EXR holds a regulatory advantage due to its FDA-indexed status.

Efficacy, as assessed through EEG-based telemetry and postoperative behavior scoring, showed no meaningful differences between the 2 treatment groups. The surgical procedure that the animals underwent in this study would be expected to result in moderate postoperative pain lasting up to 72 hours that can be alleviated by single agent or multimodal analgesia. The lack of abnormal physiologic findings or behaviors associated with significant pain in either group during recovery supports the conclusion that both EXR and SRB provided adequate analgesia following invasive EEG telemetry surgery and femoral artery catheterization. Although this portion of the study was underpowered with respect to detection of subtle behavioral differences, due to sample size constraints and limited telemetry data, the results support comparable clinical efficacy of EXR and SRB.

A key limitation of this study was the small sample size inherent to its substudy design, with resulting limited power to detect small differences, particularly in subjective outcome measures. In addition, incomplete telemetry recordings due to user error limited the full collection and interpretation of respiratory and activity data: important metrics for postoperative pain and sedation assessment. Nonetheless, the consistent pharmacokinetic profiles and absence of clinically relevant adverse events provide strong preliminary evidence supporting EXR’s use in this species.

The findings of this study hold direct implications for veterinary care and animal welfare in biomedical research involving AGMs. With EXR now FDA indexed for use in NHPs, institutions can comfortably phase out compounded SRB to align with current regulatory standards while maintaining effective analgesia. These findings expand the scarce literature on AGM pharmacology, a critical area of research given the species’ growing use as an alternative to macaques.

Future studies should further validate these findings in larger cohorts and explore additional behavioral and physiologic endpoints to refine postoperative pain assessments. Pharmacodynamic modeling may also clarify the relationship between plasma concentration and clinical analgesia in AGMs. Despite the constraints, this study represents a significant step forward in defining evidence-based analgesia protocols for a species with growing prominence in preclinical research.

Conflict of Interest

The Ethiqa XR used in this study was provided by Fidelis Animal Health.

Funding

This work was internally funded by the US Army Medical Research Institute of Chemical Defense.

Data availability

Data can be requested from the corresponding author.

Disclaimer

The views, opinions, and/or findings contained in this manuscript are those of the author and do not necessarily reflect the views of the Department of War (DoW) and should not be construed as an official DoW position, policy, or decision unless so designated by other documentation. No official endorsement should be made. Reference herein to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the U.S. Government. This research was supported in part by an appointment to the Department of War (DoW) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DoE) and the DoW. ORISE is managed by ORAU under DoE contract number DE-SC0014664. All opinions expressed in this paper are the author’s and do not necessarily reflect the policies and views of DoW, DoE, or ORAU/ORISE.