The Effect of Capromorelin and Extended-Release Buprenorphine on Body Weight in Guinea Pigs (Cavia porcellus)

Abstract

Guinea pigs (Cavia porcellus) are widely used in biomedical research and are sensitive to stress-induced anorexia and weight loss. Buprenorphine extended release (XR) is commonly used for analgesia in laboratory animals but has been associated with decreased appetite and weight loss. Capromorelin, a ghrelin receptor agonist, is FDA-approved to stimulate appetite in dogs and cats. However, its utility in mitigating opioid-associated weight loss in guinea pigs has not been evaluated. This study’s primary objective was to evaluate the effects of capromorelin on body weight in guinea pigs, both independently and in conjunction with buprenorphine XR administration, and whether oral capromorelin mitigates the weight loss associated with buprenorphine administration. A secondary objective was to evaluate habituation to voluntary consumption of an oral delivery puree and continued consumption of the puree with capromorelin added. Forty-eight female guinea pigs were randomized into 4 groups (n = 12/group) in a 2 × 2 factorial design. Animals were acclimated for 5 days to receive oral treatments, and all received either oral capromorelin (5 mg/kg) or sham treatment daily for 3 days, combined with either a single subcutaneous dose of buprenorphine XR (0.48 mg/kg) or saline. Body weights were recorded days −7 through −3 and days 0 through 5. All animals accepted oral treatments without the need for oral gavage. Guinea pigs receiving buprenorphine XR experienced significant weight loss compared with controls and were significantly less likely to return to baseline weight. Capromorelin treatment did not significantly affect weight change, and no interaction between capromorelin and buprenorphine XR was observed. Therefore, capromorelin at 5 mg/kg once a day for 3 days did not mitigate weight loss associated with buprenorphine XR administration in guinea pigs. These findings emphasize the need for species-specific evaluation of appetite stimulants and support the value of extended monitoring following administration of long-acting analgesics.

Introduction

Guinea pigs (Cavia porcellus) are a valuable animal model used in biomedical research to study respiratory disease, metabolic disease, infectious disease, toxicology, and reproduction.¹ Therefore, it is vital to limit confounding variables that may limit interpretation from results using guinea pigs with in vivo research. Body weight is a standard and sensitive indicator of health status in laboratory animals. Weight loss is a key indicator of health problems, and understanding the factors that contribute to natural variation in body weight is critical for monitoring an animal’s condition. Natural weight changes can occur in guinea pigs, as they are fastidious eaters and can easily lose their appetite with changes in their environment.² In addition, seasonal changes (e.g., photoperiod) and cold exposure can cause natural weight fluctuations.³ However, these variables are easily regulated in animal care facilities where daylight hours are strictly regulated, and temperatures are tightly controlled. Common interventions that may cause stress to guinea pigs in a research setting include induced disease, implementation of a medication or treatment, surgery, and changes in husbandry practices, among numerous others.^4–6 Changes in body weight can alter physiology and behavior and may inadvertently alter research outcomes.⁷ Although guinea pigs are usually fed ad libitum, natural or induced stressors can cause them to alter their food consumption.⁸ The current standard of care for anorexia and weight loss in guinea pigs includes fluid therapy, nutritional support, analgesia, and thermal support, as needed.⁹ Prokinetic therapies, including metoclopramide, cisapride, capromorelin, mirtazapine, and ranitidine, although not necessarily first-line treatments, have been employed to varying success to enhance gastrointestinal motility and restore normal digestive function in hind-gut fermenters.^9–12 Given the severity of anorexia in guinea pigs and its associated risks, it is imperative in both clinical and research settings to evaluate new pharmacologic options as they become available, thereby expanding the range of therapeutic strategies available to prevent morbidity and mortality in this vulnerable species. A body-weight reduction of >20% in many laboratory animals is often used as an endpoint to prompt humane euthanasia, potentially leading to early termination before study end.⁷

The use of appetite stimulants, such as capromorelin and mirtazapine, could help maintain weight during events that could reduce weight. Capromorelin is a centrally penetrant ghrelin receptor agonist. Ghrelin, produced mainly in the gastric fundus and body with additional secretion from the pancreas, influences appetite regulation and food intake through systemic neuroendocrine pathways.^13,14 Studies in mice and rats using capromorelin and other ghrelin receptor agonists have principally focused on the short-term effects of capromorelin on gastrointestinal emptying times.^15–18 Studies using intraperitoneally injected guinea pig–specific ghrelin increased food intake and blood glucose level.^14,19 In guinea pigs, intraperitoneal ghrelin does not induce a direct mechanical response in gastrointestinal smooth muscle in vitro. Instead, its effects are mediated through a capsaicin-sensitive vago-vagal reflex involving efferent cholinergic neurons.^14,19 Ghrelin activates a subset of myenteric neurons in the guinea pig jejunum, and there is morphologic evidence for the presence of ghrelin and ghrelin receptors in the myenteric plexus of the guinea pig ileum.^20,21 The use of capromorelin has been explored in several other species, including mice, rabbits, dogs, cats, chickens, pigeons, four-toed hedgehogs, and nonhuman primates (NHPs), for its effects on food intake, gastrointestinal motility, and body weight.^10,22–29 Recently, capromorelin was approved by the FDA for appetite stimulation in dogs and for the management of weight loss in cats with chronic kidney disease, and it is now available commercially under the product names Entyce (Elanco, Indianapolis, IN) and Elura (Elanco, Indianapolis, IN), respectively.³⁰ They are both labeled for daily dosing, with the effectiveness of Entyce not studied beyond 4 days of treatment, and Elura was evaluated for 56 days.^31,32

In contrast to capromorelin, mirtazapine is a presynaptic α₂-adrenergic receptor antagonist originally developed as an antidepressant in humans, where side effects of weight gain and appetite stimulation were observed.^33,34 These effects are thought to result from blockade of the 5-HT₁B, 5-HT₂, and 5-HT₃ serotonin receptors, which normally regulate peptides involved in appetite suppression and nausea induction.^33,34 In veterinary medicine, mirtazapine has been widely investigated as an appetite stimulant. In cats, both oral and transdermal formulations significantly increase food intake, and in dogs the drug is widely used to counteract anorexia, with evidence of accelerated gastric emptying and enhanced colonic transit.^34–36 Transdermal mirtazapine has also improved hyporexia in macaques and increased feed intake and body weight in rabbits.^10,37,38 A recent article published after the start of this study highlighted the utilization of mirtazapine in guinea pigs with initial pharmacokinetics and dosing to help treat inappetence and decreased gut motility.¹¹ For this study, capromorelin was selected based on previous literature success and ease of administration from the liquid formulation of Entyce.

Providing analgesia to research animals is important for the welfare of the animals, minimizing pain and suffering, adhering to ethical and legal standards, and improving research outcomes. A commonly used analgesic in laboratory rodents is buprenorphine, which acts as a partial agonist at the μ-opioid receptor and as an antagonist at the κ-opioid receptor.³⁹ Buprenorphine has been estimated to be ∼25- to 30-fold more potent than morphine, while its high receptor affinity allows it to displace other opioids while maintaining analgesia, and it is generally associated with fewer adverse effects than many alternative opioid agents.³⁹ Documented side effects of buprenorphine in guinea pigs include reduced food and water consumption, measurable decreases in body weight (sometimes approaching 10% with extended-release [XR] formulations), and lowered activity levels in the postoperative period.^39,40 A key benefit of longer-acting buprenorphine formulations is reduced animal handling, thereby decreasing stress. Prior studies evaluating a buprenorphine XR preparation (Animalgesic Laboratories, Millersville, MD) in guinea pigs demonstrated effective analgesia at a dose of 0.48 mg/kg administered subcutaneously, with a duration of action lasting ∼48 hours.^40,41 Buprenorphine XR (Ethiqa XR; Fidelis Animal Health, North Brunswick, NJ) is FDA indexed for captive rodents, ferrets, laboratory rabbits, and NHPs, has a good margin of safety at 0.48 mg/kg, and is effective for at least 72 hours.⁴² Reported adverse effects of Ethiqa XR include decreased body weight and fecal output, as well as increased passive behaviors (e.g., eyes closed, squinting, subtle body movement, incomplete movement).^41–43 As buprenorphine is a commonly prescribed analgesic for numerous small mammalian species used in research settings, it would be useful to have a method of mitigating weight loss due to buprenorphine. Methods to mitigate weight loss due to buprenorphine have been used in other studies.^17,22,44 The utility of capromorelin to mitigate weight loss in guinea pigs has not been investigated before this study, as supported by negative database searches in PubMed, Scopus, Agricola (EBSCOhost), and Embase (Elsevier) with no date restrictions or other filters applied.

Traditional oral gavage in guinea pigs poses significant welfare and technical challenges. These include the need for physical restraint, the risk of aspiration or esophageal injury, stress-induced alterations in physiology and behavior, and the potential requirement for anesthesia, all of which may confound study outcomes and raise ethical concerns.^4,45,46 Oral dosing in guinea pigs is difficult due to their pharyngeal anatomy, which is composed of an elongated soft palate that covers the back of the throat, leaving only the small palatal ostium for access to the trachea and esophagus.² There are refined gavage tools that may still result in procedural complications and stress responses.⁴⁷ Voluntary oral dosing has been shown in other rodent models to enhance animal welfare, reduce procedural stress, and support more reproducible pharmacologic outcomes.^22,48–55 There is limited published evidence for voluntary oral dosing in guinea pigs. By adapting this refinement to this study, the investigators sought to leverage a welfare-friendly and effective method of drug administration that minimized the need for restraint, anesthesia, or oral gavage for the delivery method for capromorelin and sham treatments.

The objectives of this study were to evaluate the effects of capromorelin on body weight in guinea pigs, both independently and in conjunction with buprenorphine XR administration, and whether oral capromorelin mitigates weight loss associated with buprenorphine. It is hypothesized that capromorelin administration can mitigate weight loss associated with buprenorphine treatment. A secondary objective was that guinea pigs would habituate to voluntary consumption of an oral delivery puree and would continue consuming the puree with capromorelin added.

Materials and Methods

All work described in this study was reviewed and approved by the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) IACUC. The USAMRIID is fully accredited by AAALAC International, and all animal research conducted complies with the Animal Welfare Act, Public Health Service Policy for the Humane Care and Use of Laboratory Animals, and other federal statutes and regulations relating to animals in research. Animals were housed in accordance to the Guide for the Care and Use of Laboratory Animals.⁵

Animals and housing.

Forty-eight female Dunkin-Hartley guinea pigs (Charles River Laboratories, Montreal, QC, Canada) were used in this study. Animals were transferred from other IACUC-approved research protocols in which they were used as control animals for a vaccine trial, which concluded 8 months before the start of this study, and small-molecule therapeutics, which concluded 2 months before the start of this study. Animals were identified via microchip and weighed (scale, model EX4202; Ohaus, Parsippany, NJ) before randomization. All guinea pigs had negative screening at the vendor for a range of pathogens, including murine respirovirus (Sendai), pneumonia virus of mice, reovirus, lymphocytic choriomeningitis virus, guinea pig adenovirus, Bordetella bronchiseptica, Helicobacter spp. including H. bilis and H. hepaticus, Klebsiella pneumoniae, Klebsiella oxytoca, Mycoplasma pulmonis, Pasteurella multocida, Rodentibacter pneumotropicus, Rodentibacter heylii, Pseudomonas aeruginosa, Salmonella species, Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus pneumoniae, Streptococcus zooepidemicus, beta Streptococcus spp., Clostridium piliforme, visible ectoparasites, enteric protozoa, endoparasites, and Eimeria cuniculi. USAMRIID predominantly uses female guinea pigs, as compared with males, for research projects due to their relatively calm demeanor, which is advantageous in long-term experiments that house animals together. The guinea pigs used in this study had body weights of 962 ± 245 g and ranged from 7 to 12 months of age.

Guinea pigs were group-housed (3 per cage) in open-top stainless-steel cages (Allentown, Allentown, NJ) with pressed cellulose (ALPHA-dri, Shepherd Specialty Papers, Watertown, TN), under controlled temperature (74.5 ± 5 °F [23.6 ± 2.8 °C]), humidity (30%-70%), and a 12-hour light/12-hour dark cycle. All animals had ad libitum access to standard guinea pig chow (Teklad 2040; Inotiv, West Lafayette, IN) and water via an automated watering system (Edstrom Industries, Waterford, WI). Dietary enrichment included 35-50 g of Timothy hay (product no. S1012; Bio-Serv, Flemington, NJ) daily and fresh fruits (e.g. ¼ apple per animal) or vegetables (e.g. 30 mL of carrots per animal) twice a week on alternating days.

Study design.

Forty-eight female guinea pigs were randomized into 1 of 4 treatment-intervention groups. Intervention (I) was defined as subcutaneously administered buprenorphine XR (B; Ethiqa XR) or saline (S; saline, control). Treatment (T) was defined as either orally administered capromorelin (superscript C; Entyce) or nothing added (superscript N, control).

The intervention-treatment groups were as follows: group B^C is I = buprenorphine XR + T = capromorelin; group B^N is I = buprenorphine XR + T = nothing added; group S^N is I = saline + T = nothing added; and group S^C is I = saline + T = capromorelin.

Guinea pigs in intervention groups B^N and B^C received one dose of buprenorphine XR SQ injected in the area between the neck and shoulders or the scruff. Groups S^N and S^C received a saline injection subcutaneously once as a control. The saline was given at the same volume and with the same gauge needle as used to administer buprenorphine XR to group B^N and B^C guinea pigs. The personnel administering the intervention were not blinded as to which intervention was given and did not perform any other procedures for the protocol. Treatments were given on days 0, 1, and 2, ∼24 ± 2 hours apart. All guinea pigs were habituated to daily weighing and to consume 2 mL of unmedicated vegetable puree (carrot baby food from Gerber Products, Fremont, MI) from a needleless Luer lock 3-mL syringe for 5 days (days −7 to −3), with a 2-day period prior to treatment/interventions on day 0. The vegetable puree was the delivery vehicle for capromorelin treatment (groups S^C and B^C) or as a control treatment (groups S^N and B^N). Figure 1 is provided as a reference for the timeline of the study.

Figure 1.

Procedure Timeline for Day −7 through Day 5.

Habituation and administration procedures.

From days −7 to −3, individual guinea pigs were habituated to daily weighing ∼24 ± 2 hours apart. During this 5-day period, after obtaining daily weight, they were habituated to voluntarily consume unmedicated, vegetable puree, herein referred to as oral delivery puree. Technicians offered the oral delivery puree to the guinea pig without restraint. If this was not successful, the guinea pigs were gently restrained by hand, and the syringe tip was placed into the mouth of the guinea pig, and the plunger was slowly depressed until the guinea pig consumed all the oral delivery puree. Success of consumption was set at consuming at least 1.5 mL of the 2 mL of the oral delivery puree. Guinea pigs that did not voluntarily ingest the oral delivery puree were to receive the dose via oral gavage with light anesthesia from isoflurane. The guinea pigs had a scheduled 2 days (days −2 and −1) of rest before initiating interventions and treatments.

Interventions.

On day 0, all groups were weighed prior to receiving their intervention. Groups B^N and B^C received an intervention of buprenorphine XR subcutaneously at 0.48 mg/kg. Groups S^N and S^C received a saline dose at the same volume and with the same gauge needle as the buprenorphine XR dose. Weights collected on day −3 were used to calculate intervention doses. The personnel administering the interventions were not blinded.

Treatments.

The guinea pigs in all groups received their treatments on days 0, 1, and 2, ∼24 ± 2 hours apart. The capromorelin dose of 5 mg/kg for groups B^C and S^C was extrapolated from a study in rats,¹⁵ and the dosing frequency of once a day was taken from a study in rabbits and label indications for the capromorelin formulation.^10,15,31 Capromorelin (Entyce; Elanco, Greenfield, IN) at 5 mg/kg was mixed with the oral delivery puree daily with calculations based on the previous day’s weight collection. Animals in the “nothing added” groups (B^N and S^N) recieved the oral puree alone at equal volume. The oral treatments (capromorelin or nothing added) were given once daily for 3 consecutive days (days 0-2). Animals were encouraged to take the oral delivery puree voluntarily. If needed, the animals were restrained by hand and the syringe was placed in the cheek and the puree slowly administered. If the oral gavage was needed, 16-gauge, 3-in. curved gavage needles (Fine Science Tools, Foster City, CA) were available for use. Animals could be anesthetized with light isoflurane anesthesia, if needed, to facilitate gavage. The personnel administering the treatments were blinded regarding the treatment groups, and those who administered the interventions on day 0 did not administer treatments or conduct postintervention/posttreatment evaluations.

Evaluations.

On day 0, welfare assessments were conducted 4 times (Figure 1), including 10-12 hours postintervention, due to the potential delayed effects of buprenorphine XR, which reaches peak plasma levels up to 8 hours postadministration.⁴¹ On day 1, 2 assessments were completed: the standard 24-hour check and a 30-hour postinjection check. All animals were observed at least once daily (days −7 to 5). Observations included appearance, behavior, fecal output, hydration status, and food intake, documented using standardized scoring logs. Each guinea pig’s appearance and behavior were scored from 0 to 3 with following scoring used: appearance score is normal = 0; reduced grooming, but clear eyes and nostrils = 1; dull/rough coat with or without nasal ocular discharge = 2; piloerection, hunched, soiled fur = 3; behavior score is normal = 0; slower, alert but subdued = 1; less alert, but mobile, isolated from others = 2; immobile, prostrate, unresponsive to simulation = 3. Animals receiving a clinical score of 1-2 for appearance or behavior were scheduled for follow-up evaluation by the primary investigator (PI) within 1 day, whereas scores of ≥3 triggered immediate veterinary evaluation. Any specific adverse signs, including diarrhea, hypersalivation, lethargy, and decreased fecal output, obtundation, or passive behavior, if observed, were to be documented.^24,32,41,43

Statistical analysis.

The study was a 2 × 2 factorial design with treatment and intervention as factors. The 2 treatment levels were capromorelin or nothing added. The 2 intervention levels were buprenorphine XR or saline. Interactions between the treatment effect and intervention effect were examined, with specific pairwise comparisons of interest as follows: (1) saline + nothing added (group S^N) compared with buprenorphine XR + nothing added (group B^N); (2) saline + nothing added (group S^N) compared with saline + capromorelin (group S^C); (3) buprenorphine XR + nothing added (group B^N) compared with buprenorphine XR + capromorelin (group B^C); and (4) saline + capromorelin (group S^C) compared with buprenorphine XR + capromorelin (group B^C).

Sample size.

A power analysis of the primary outcome measure of weight was conducted using PASS 14 (NCSS, Kaysville, UT) prior to the start of the study for the specific pairwise comparisons of interest. A factorial design with 2 factors, each with 2 levels, resulting in 4 possible combinations, was used to test whether there are differences among the levels of the factors. Terms were to be tested using a linear model analysis of variance F test with a type I error rate (α) of 0.05. With a sample size of 12 subjects per treatment combination, and a total number of 48 subjects, this design achieved ≥78% power to detect an effect size of 0.417 for each factor and interaction effect. Animals were randomized without replacement using SAS Proc Plan (SAS 9.4; SAS, Cary, NC) into 1 of 4 intervention/treatment groups balanced by intervention/treatment and weight. The statistician who performed randomization was not blinded to the intervention and treatment groups or weight. Overall, the statistician, primary investigator, and a team of one veterinarian and one veterinary technician who injected the animals were made aware of the randomization and group allocation for intervention of buprenorphine XR/saline injections. Only the primary investigator knew the treatment of capromorelin in the oral delivery puree.

Statistical methods.

Mixed model analysis of variance was performed using the GLIMMIX procedure of SAS version 9.4 (SAS Institute, Cary, NC) to examine the effects of treatment, intervention, and interaction thereof on daily posttreatment animal weights (primary outcome measure). The model included the random effects of guinea pigs and day, as well as the covariant of baseline weight, which was defined as the animal’s average of the weights taken over the 5 habituation days and the weight taken on day 0 prior to treatment. Because the time points between posttreatment weight observations were uniform, an autoregressive covariance structure was used. Orthogonal contrasts were used to test the interaction of treatment and intervention as previously specified in the discussion of study design. The Kenward and Roger method⁵⁶ was used to calculate degrees of freedom. Model assumptions were checked using the Shapiro-Wilk normality test⁵⁷ and by visual inspection of residual and fitted value plots. Descriptive statistics are presented as means, SDs, and minimum and maximum observed values.

Fisher exact tests⁵⁸ and Pearson χ² tests⁵⁹ were used to examine differences in the proportion of animals returning to baseline weight after initiation of treatment. Kaplan-Meier⁶⁰ failure analysis was performed using the LIFETEST procedure of SAS to examine the effects of treatment, intervention, and interaction thereof on the time in days required to return to or exceed (RoE) baseline weight after initiation of treatment. The event of interest was recording of weight equal to or greater than the animal’s average of the weights taken over the 5 habituation days and the weight taken on day 0 prior to treatment. Animals failing to RoE baseline weight were right-censored. Log-rank tests⁶¹ were used to compare the cumulative failure distributions. Descriptive statistics are presented as median time to RoE baseline weight, means and SEs of time to RoE baseline weight, number and percentage of animals RoE baseline weight, and number and percentage of animals failing to RoE baseline weight (censored).

The level of significance was set at P < 0.05. All tests were 2-tailed. No adjustments for multiple comparisons were made. Missing values were treated as missing at random, and values were not imputed. The total number of animals used for analysis was 48. The statistician performing the analysis was not blinded.

Results

Study population.

The weights obtained during habituation (days −7 through −3) and the day of treatment initiation (day 0), prior to intervention or treatment, were used as the baseline weights by which to evaluate treatment outcome. Guinea pigs lost an average of 3.3% of body weight from day −7 (999.0673 ± 116.265 g) to day −5 (965.8567 ± 116.8545 g). However, by day 0 they had begun to reverse the weight loss and had, on average, no more than 2% lower body weight (979.2773 ± 114.1177 g) over the habituation period. This initial weight loss during the habituation period was significant (P < 0.001). In addition, the rate of change over the 7 days period did not differ by intervention (P = 0.2600) or treatment assignment (P = 0.4723).

There were no statistically significant differences in baseline weight between treatment assignment (P = 0.6988), intervention assignment (P = 0.2612), or any intervention-by-treatment combinations (P = 0.3340). During the daily observations, no adverse signs were noted. However, 2 guinea pigs (one from group B^N and one from group B^C) were found deceased on what would have been days 7 and 8, respectively, had the protocol extended beyond the defined monitoring period of 6 days. Weights were not obtained when the animals were found deceased, and both guinea pigs were found at a body condition score of 4/5. Their weights from days −7 through 5 were included in the statistical analysis.

Changes in weight.

Main effects of treatment and intervention.

An analysis of treatment and intervention effects over the period starting 1 day (day 1) after initiation of treatment/intervention administration and continuing until the study concluded 5 days postintervention (day 5) showed no difference between treatments (P_{days 1-5} = 0.5757) but did show a significant difference between interventions. Guinea pigs receiving buprenorphine XR (groups B^N and B^C) showed a statistically significant decrease in body weight (>11.9% reduction by day 5; group B^N: day 0 = 976.014 g (±134.039 g), day 5 = 856.569 g (±119.525 g); group B^C: day 0 = 1,021.231 g (±75.986 g), day 5 = 894.351 g (±68.572 g) as compared with controls (<1% reduction by day 5) (groups B^N and B^C; P_{days 1-5} < 0.0001) (Figure 2). In addition, the intervention effect was significant on each of the individual days when each day was analyzed separately (P_{day 1} < 0.0001, P_{day 2} < 0.0001, P_{day 3} < 0.0001, P_{day 4} < 0.0001, P_{day 5} < 0.0001).

Figure 2.

Mean Percent Change in Body Weight from Baseline Weight for Each Group. Groups B^N and B^C lost more weight than did their respective controls (S^N and S^C) over days 1-5 (P < 0.0001 for both comparisons). When each day was analyzed separately, significant differences were seen between group B^N and group S^N for days 1-5 (P_{day 1} = 0.0179; P < 0.0001 for each day 2 through 5), and between group B^C and S^C for days 1-5 (P < 0.0001 for each day 1 through 5).

Interaction effect.

An analysis of the interaction between treatment and intervention over the period starting 1 day (day 1) after initiation of treatment/intervention administration and continuing until the study concluded 5 days postintervention (day 5) showed no significant interactive effect, indicating that weight change did not differ between treatments (capromorelin/nothing added) within each intervention group (P_{days 1-5} = 0.7727). When each day was analyzed separately, the interaction effect resulted in a significant decrease in group weights on day 1 (P_{day 1} = 0.0460) after the treatments/interventions were initiated, but not on any other individual days.

Pairwise group comparisons.

Pairwise comparisons of specific groups confirmed the pronounced effect of intervention on weight change. Among guinea pigs treated with oral delivery puree with nothing added, those that were given buprenorphine XR (group B^N) lost significantly more weight than did the guinea pigs that were given the oral delivery puree with nothing added (group S^N) over the period from day 1 to day 5 (P_{days 1-5} < 0.0001), as well as on each of the individual days when each day was analyzed separately (P_{day 1} = 0.0179, P_{day 2} < 0.0001, P_{day 3} < 0.0001, P_{day 4} < 0.0001, P_{day 5} < 0.0001). Similarly, among guinea pigs given capromorelin, those that were given buprenorphine XR (group B^C) lost significantly more weight than did capromorelin guinea pigs given saline control (group S^C) over the period from day 1 to day 5 (P_{days 1-5} < 0.0001), as well as on each of the individual days when each day was analyzed separately (Figure 3; P_{day 1} < 0.0001, P_{day 2} < 0.0001, P_{day 3} < 0.0001, P_{day 4} < 0.0001, P_{day 5} < 0.0001). Among guinea pigs given buprenorphine XR, no significant differences were seen between those given oral delivery puree with nothing added and those given capromorelin over the period from day 1 to day 5 (P_{days 1-5} = 0.8490) or any of the individual days. Among guinea pigs given saline control, no significant differences were seen between those given oral delivery puree with nothing added and those given capromorelin over the period from day 1 to day 5 (P_{days 1-5} = 0.5510) or any of the individual days.

Figure 3.

Group Body Weight, in Grams, by Day from Baseline (Days −7 to −3 and Day 0) and after Interventions/Treatments (Days 1-5). Boxplots represent the median and IQR, the marker represents the mean, and the range is denoted by the whiskers.

RoE baseline weight.

Main effects of treatment and intervention.

An analysis of intervention on the proportion of guinea pig RoE baseline weight by postintervention day 4 showed a significantly higher proportion RoE baseline weight in guinea pigs administered saline (58.3%; 14 out of 24) as compared with those given buprenorphine XR (0%; P < 0.0001). The proportion of guinea pig RoE baseline weight was the same in guinea pigs given oral delivery puree with nothing added and those given capromorelin (29.2%; 7 out of 24).

Pairwise group comparisons.

Among guinea pigs in treatment groups with nothing added, group S^N had a significantly higher proportion of guinea pig (58.3%; 7 out of 12) RoE baseline weight as compared with those given buprenorphine XR (group B^N) (0%; P = 0.0046). Among guinea pigs given capromorelin, those that were given saline control (group S^C) showed a significantly higher proportion (58.3%; 7 out of 12) RoE baseline weight as compared with those given buprenorphine XR (group B^C) (0%) (P = 0.0046). Among guinea pigs in group B, no significant differences in proportion were seen between those given oral delivery puree with nothing added (B^N; 0%) and those given capromorelin (B^C) (0%; P = 1.0). Among guinea pigs in group S, no significant differences were seen between those given oral delivery puree with nothing added (S^N; 58.3%; 7 out of 12) and those given capromorelin (S^C; 58.3%; 7 out of 12) (P = 1.0). Animals that RoE baseline weight did so evenly over the 5 days of monitoring. On days 1 and 5, 28.6% (4 out of 14 animals) RoE to baseline weight on each day. Over days 2-4, 14.3% (2 out of 14 animals) RoE baseline weight on each of the days (Figure 4).

Figure 4.

Kaplan-Meier Graph Demonstrating the Time, in Days, to RoE Baseline Weight for Each Group. Significant differences in rate of RoE baseline weight were seen between groups B^N and S^N (P = 0.0033) and between groups B^C and S^C (P = 0.0010). Interaction effect between groups representing body weight in grams over the 6-day monitoring is shown.

Success of voluntary oral consumption.

During the 5-day habituation phase (days −7 through −3), the guinea pigs demonstrated progressive improvement in voluntary oral consumption of the oral delivery puree. Some guinea pigs consumed all of the oral delivery puree on day −7, while others missed >50% of the oral delivery puree. However, by day −5 and −4, 40 out of 48 (83.3%) guinea pigs were consistently consuming the full volume of the oral delivery puree. By the last day of habituation (day −3), 45 out of 48 (93.75%) were consuming all of the oral delivery puree. The remaining 3 guinea pigs, which did not consume all of the oral delivery puree, only missed a small amount (<0.5 mL), which was within the success rate of consumption. Following the habituation phase, 100% of guinea pigs voluntarily ingested their assigned treatment solutions during the active dosing period (days 0-2). No guinea pigs required oral gavage during the treatment period.

Discussion

This study evaluated the impact of enterally administered capromorelin (5 mg/kg) on body weight in guinea pigs, both alone and following buprenorphine XR (0.48 mg/kg) administration. Buprenorphine XR significantly (B^N+C; P_{days 1-5} < 0.0001) reduced body weight in guinea pigs relative to saline controls. This effect persisted on posttreatment days 1-5, confirming previous reports that opioids such as buprenorphine can negatively impact appetite and weight in small mammals.^{40,41,43,44,62} Animals treated with buprenorphine XR, regardless of oral treatment, experienced >11.9% reductions in body weight and failed to RoE baseline weight over the 5-day observation period. Capromorelin did not significantly impact body weight in saline controls (group S^C), and the administration of capromorelin failed to mitigate weight loss caused by buprenorphine XR (group B^C). No interaction was detected between capromorelin and buprenorphine XR. Animals in group B^C exhibited similar weight trajectories and failure to regain baseline weight as their counterparts in group B^N, with no significant difference in either daily weight data or cumulative RoE baseline rates. These findings are notable for several reasons. First, while ghrelin receptor agonists such as capromorelin have been successfully employed to stimulate appetite and weight gain in other species, including dogs, cats, and NHPs, this appears not to translate to guinea pigs at the dose of 5 mg/kg once per day.^10,22–29 These data corroborate previous research suggesting that the mechanisms of ghrelin signaling in guinea pigs differ significantly from other rodents and carnivores.^14,19–21 Ghrelin’s appetite-stimulating effects in guinea pigs may require specific pathways involving vagal afferents or cholinergic activation, which may not be adequately stimulated by once daily orally administered capromorelin, especially under the suppressive physiologic effects of buprenorphine.^14,19–21 Second, the absence of an observable benefit from capromorelin underscores the challenge of pharmacologically mitigating opioid-induced anorexia. The physiologic basis for buprenorphine-related appetite suppression likely involves multiple mechanisms, including central opioid receptor activity, delayed gastric emptying, and reduced locomotor behavior.^34,35,51,58 Therefore, capromorelin, a selective ghrelin agonist, may be insufficient alone to override this multifactorial anorectic effect.

A notable strength of this study was the 100% voluntary oral consumption of the oral delivery puree among all guinea pigs during the treatment period. This finding demonstrates that a 5-day structured habituation is sufficient to train guinea pigs to voluntarily consume an oral delivery puree. The habituation period allowed animals to become familiar with the oral delivery method using a syringe, thereby eliminating the need for gavage and reducing potential stress-related confounders. The success of this approach demonstrates the value of pretreatment training protocols to improve voluntary compliance, animal welfare, and consistency of drug administration in guinea pig research models.

Several limitations of this study merit discussion. The capromorelin dose of 5 mg/kg once daily was derived from extrapolated data in other species, as no prior pharmacokinetic or pharmacodynamic data are available for guinea pigs. It is possible that the selected dose, route, or frequency was suboptimal for this species. Previous studies in rabbits, another hindgut fermenting species, demonstrated that the once daily dosing of capromorelin at either 4 or 8 mg/kg was not sufficient to increase fecal output or food intake.¹⁰ In addition, the study duration was limited to 6 days; longer observation may be necessary to detect delayed recovery or compensatory weight gain in treatment groups. In addition to the limitations noted above, 2 guinea pigs (1 from group B^N and 1 from group B^C) were found deceased on what would have been days 7 and 8, respectively, had the protocol extended beyond the defined monitoring period of 6 days. Although weights were not obtained, their body condition scores (4/5 for both) were similar to the rest of the animals. Necropsies were performed on the deceased animals, and no definitive cause of death could be identified. While both animals received buprenorphine XR during the study, their deaths occurred after the observation window had concluded and were not temporally associated with acute intervention or treatment administration. Although causality cannot be established, these findings underscore the importance of considering extended posttreatment monitoring in future studies involving sustained-release analgesics.

Future research should explore dose-escalation and increased frequency of dosing studies of capromorelin in guinea pigs, including pharmacokinetic analysis, and consider alternative appetite-stimulating agents such as mirtazapine and/or classes of drugs such as benzodiazepines.^10,11 Evaluating combinations of dietary, environmental, and pharmacologic interventions may also yield improved outcomes. Finally, exploring strain- or sex-specific responses to both buprenorphine and capromorelin could provide further insights into inter-animal variability and individualized care.

In conclusion, this study confirms that buprenorphine XR causes significant weight loss in female guinea pigs up to 5 days after administration. Capromorelin, while effective in other species, did not prevent or mitigate buprenorphine-associated weight loss in this model when dosed at 5 mg/kg once daily. A 5-day habituation period for voluntary consumption allowed the animals to become familiar with the oral delivery method, thereby eliminating the need for gavage and, therefore, reduced potential stress-related confounders. These findings highlight the need for species-specific validation of supportive care practices in laboratory animals and emphasize the importance of proactive weight monitoring following opioid administration. In addition, the success of voluntary consumption demonstrates the value of pretreatment training protocols to improve voluntary compliance, animal welfare, and consistency of drug administration in guinea pig research models.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This work was internally funded.