Studies have demonstrated that buprenorphine, a front line drug for veterinary analgesia, may alleviate symptoms of chronic pain. A cage side observation protocol was used to record behavioral signs in a mouse clinical trial of extended release buprenorphine. A retrospective review of the observations for signs of pain and stress revealed that mice given a fivefold overdose of buprenorphine (16.25 mg/kg) showed lethargy and facial signs associated with stress. However, similar signs were observed in the drug-free control mice as early as Day 3 of single-cage housing. This appears to be the first report of cage effects in a clinical trial for a veterinary drug.
Centers for veterinary medicine at the Food and Drug Administration (FDA) in the US and the European Medicines Agency provide guidance documents for clinical trials to evaluate the safety and efficacy of new veterinary pharmaceuticals [
We conducted TAS trials to examine the safety of a lipid-bound buprenorphine analgesic in mice [
Our daily clinical observations generated reports for each mouse in the trials. As the drug was intended for postsurgical analgesia, the observations recorded physical signs and behaviors that are consensus indicators of pain and distress. In addition to observations focused on the surgical site, our studies collected approximately 20,000 data points regarding the behavior of mice treated with drug and their drug-negative controls. Because the trials involved a large dose of an opiate, we expected to measure modest and transient signs of nausea, lethargy, and stress, which are common side effects of opiate therapy in animals and humans. These signs were observed in mice treated with 16.25 mg/kg of drug, a fivefold excess of the intended dose, and will be reported elsewhere. Surprisingly, an analysis of the data from the control mice demonstrated significant signs of lethargy and stress in the drug-free animals. The signs increased in the days following surgery. Thus, we believe that the behaviors can be attributed to the stress of social isolation and provide evidence that the lonely mouse phenomenon may play a previously unexpected role in similar short-term drug trials.
Studies were approved by University Institutional Animal Care and Use Committee (IACUC). The IACUC protocol complies with the National Research Council’s Guide for the Care and Use of Laboratory Animals and fulfills the requirements of the Association for the Assessment and Accreditation of Laboratory Animal Care, International Program. The study was conducted at University Department of Molecular and Comparative Pathobiology. Health surveillance and detection of pathogen contaminants were conducted by a soiled-bedding sentinel system. Male and female BALB/cAnNCrl mice (6–8 weeks old; weighing 20–22 g) were obtained from Charles River Laboratories (Wilmington, MA). Mice were inspected for general health conditions before being housed at a population of 4-5 mice per cage in Smart Bio-Pak cages (Allentown, NJ) with Tek-Fresh bedding (Harlan, Madison, WI) and were allowed free access to Teklad Global Rodent Diet chow (Harlan, Madison, WI) and defined water. Mice housed 3-4 per cage were held for approximately 10 days prior to the start of the experiment. Mice were weighed prior to assignment to the drug or control groups to ensure the weight of each mouse was within 10% of the average group weight at the start of the study. Throughout the duration of the study, mice were housed individually. Cages were changed daily to prevent buprenorphine redosing by coprophagy.
The study design was based on TAS guidelines for assessing the safety of veterinary pharmaceutical products [
Eight male and eight female mice per group were used in Trial 1 comparing a control (0.0 mg/kg) and 5-fold dose (16.25 mg/kg) challenge. Eight male and eight female mice per group were used in Trial 2 comparing control and 5-fold doses, repeated at three 4-day intervals. As shown in Table
Experimental design displaying the number of daily observations and harvest schedule for the single dose (Trial 1) and repeat dose (Trial 2) studies.
Trial day | |||||||||
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Single dose: Trial 1 | |||||||||
Number of mice | Days 1 | 2 | 3 | 4 | 5 | ||||
Per dose 16♂, 16♀ | 32 | ||||||||
per harvest | 16 | 16 | |||||||
Charts AM | 32 | 16 | 16 | ||||||
Charts PM | 32 | 16 | 16 | 16 | |||||
Repeat dose: Trial 2 | |||||||||
Number of mice | Days 1 | 2 to 3 | 4 | 5 | 6 | 7 | 8 | 9 to 11 | 12 |
Per dose 16♂, 16♀ | 32 | 32 | 16 | ||||||
Per harvest | 16 | 16 | |||||||
Charts AM | 32 | 32 | 32 | 32 | 16 | 16 | 16 | ||
Charts PM | 32 | 32 | 32 | 32 | 16 | 16 | 16 | 16 |
The cholesterol-buprenorphine drug powder was supplied by Animalgesic Laboratories Inc. (Millersville MD). The drug powder contained USP (United States Pharmacopeia) grade buprenorphine HCl (Noramco, Wilmington DE), cholesterol, and glycerol tristearate, (Sigma, St Louis MO). Drug preparations were verified for purity and content by AAI Pharma (Wilmington NC). Negative control, drug-free powder was prepared by tumble blending a mixture of cholesterol and glycerol tristearate (96/4, w/w) for 48 hours at 5°C. Injectable suspensions of drug powder and the control were prepared by suspending 80 mg of powder per mL of medium chain triglyceride (MCT) oil (Miglyol 812, from Sasol, Hamburg Germany) followed by brief shaking to make a homogeneous suspension. Suspensions were generally prepared within 1-2 days of use and stored at 2–8°C. A single (1x) dose consisted of a 0.05 mL drug suspension. One mL syringes with 1 inch 20 gauge needles were used to inject suspensions (described below) of cholesterol-triglyceride-buprenorphine powder and the negative control, cholesterol-triglyceride control powder. The surgical procedure was based on the procedure used to implant Alzet miniosmotic pumps in mice and rats. A video of the subcutaneous implantation procedure, which is briefly described below, is available at the Alzet website [
Mice were given intraperitoneal (IP) anesthesia with a solution containing 8.4 mg/mL (65 mg/kg) ketamine, 0.84 mg/mL (0.65 mg/kg) xylazine, and 4.75% ethanol in saline. The dose of anesthesia was 0.15 mL/20 g mouse. When anesthesia was established, approximately 1 cm2 of middorsal skin was shaved, washed with ethanol, and then coated with povidone-iodine solution. Mice were transferred to a procedural table that was cleaned with 70% ethanol solution and covered with a clean disposable towel. A sterile disposable number 10 blade was used to make a 4-5 mm incision through the skin only. Bleeding, if any, was controlled with sterile gauze and light pressure. Sterile forceps were used to separate the skin and to create approximately a 2 × 4 cm subcutaneous pocket. The skin was then apposed and stapled with 9 mm Autoclips (Kent Scientific, Torrington CT). After the skin was stapled, mice were injected with either the drug or control suspension (0.25 mL/mouse) into the interscapular subcutis. All mice in the study were treated to this “sham surgical procedure.” After the procedure, mice were moved to a holding cage. This cage contained a 37°C heating pad covered with a clean disposable towel. When the mouse regained consciousness, as demonstrated by movement and the absence of signs of distress, which included but were not limited to abnormal paw movements, efforts to scratch the incision site, and cowering, each mouse was placed individually into a clean cage.
The two trials had a different number of observation days: 4 days in Trial 1 and 5 days in Trial 2. Behavioral data per mouse were collected in the morning, between 8-9 am, and evenings, between 5-6 pm. The cage side observations were conducted and recorded by the same female veterinarian, blind to the treatment groups. To quantify this data, FDA validated observation forms were used. The forms were designed for the entry of numerical grading of the extent to which pain or distress was present across nine parameters: mouse respiration, nasal/skin appearance, fur appearance, motor activity, ocular activity (closed or open eyes), behavior suggesting stress (i.e., aggressiveness), presence of tremors, and surgical site erythema, edema, or infection (swelling, pus, and exudate). Ratings were made on scales ranging from 1 to 3 or from 1 to 6, depending on the parameter. Higher numbers indicated more severe signs of pain/distress. The ocular score was recorded using a scale of 1 to 5: (1) no observed abnormalities (NOA); (2) squinting; (3) eyes closed; (4) crusty secretions; and (5) porphyrin stain. The motor activity was recorded using a scale from 1 to 4: (1) NOA; (2) rapid darting; (3) hunched/lethargic; and (4) hunched/motionless. In addition, a “yes/no” score was given for an assessment of the general condition of each mouse and its cage. Space on the forms was also available for comment. Observations of the following were recorded twice daily: motor activity, ocular signs, fur appearance, and general conditions. The daily observation form for each mouse hereinafter is referred to as the mouse chart.
Outcome of the experiments on ocular and motor actives were described as ordinal scores. Data were summarized as frequency and percentage. The possible natural variability of the ocular score and motor activity with respect to trial, gender, time (morning versus afternoon), and day were assessed using data from the control group. Chi-square test was used for group comparisons and treatment comparison between the drug and the control. Subgroup comparisons were performed due to statistically significant confounding factors which were identified using the control data. All
The 272 charts from the drug-free control male and female mice in Trial 1 and in Trial 2 were analyzed to examine whether elements in the experimental design, including single-cage housing, were potentially confounding factors. The analyses of the control data revealed a significant difference in the observation of ocular scores between the two trials. As shown in Table
Variability of ocular scores for drug-free (Control) animals in Trials 1 and 2.
Ocular score | Trial 1 | Trial 2 |
---|---|---|
NOA: | 82 (85.4) | 104 (72.2) |
Eyes closed: number (%) | 13 (13.6) | 40 (27.8) |
Crusty secretions: number (%) | 1 (1.0) | 0 (0) |
Significant gender difference in motor activity of drug-free (control) animals.
Motor score: | Male (%) | Female (%) |
---|---|---|
NOA: number (%) | 85 (70.8) | 63 (52.5) |
Rapid, darting: number (%) | 13 (10.8) | 36 (30.0) |
Hunched, lethargic: number (%) | 2 (1.7) | 4 (3.3) |
Hunched, motionless: number (%) | 20 (16.7) | 17 (14.2) |
Frequency and percentage distributions of signs of stress increase in drug-free (control) animals over a 5-day time period.
Ocular score | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 |
---|---|---|---|---|---|
NOA: number (%) | 26 (81.3) | 55 (85.9) | 45 (80.4) | 38 (79.2) | 22 (55.0) |
Eyes closed: number (%) | 6 (18.7) | 9 (14.1) | 11 (19.6) | 9 (18.7) | 18 (45.0) |
Crusty secretions: number (%) | 0 (0.0) | 0 (0.0) | (0.0) | 1 (2.1) | 0 (0.0) |
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Motor score | Day 1 | Day 2 | Day 3 | Day 4 | Day 5 |
| |||||
NOA: number (%) | 17 (53.2) | 46 (71.9) | 35 (62.5) | 31 (64.6) | 19 (47.5) |
Rapid, darting: number (%) | 11 (34.4) | 9 (14.1) | 11 (19.6) | 10 (20.8) | 8 (20.0) |
Hunched, lethargic: number (%) | 3 (9.4) | 1 (1.6) | 2 (3.6) | 0 (0.0) | 0 (0.0) |
Hunched, motionless: number (%) | 1 (3.1) | 8 (12.5) | 8 (14.3) | 7 (14.6) | 13 (32.5) |
The present study demonstrates a potentially confounding variable in studies using TAS protocols for assessing drug safety in mice. Single-cage studies can introduce a lonely mouse syndrome. Stress avoidance should be optimized in every animal model. However, lonely mouse models have provided tools to investigate behavioral phenomenon including autonomic stress and negative emotional states. Recent studies by Furuhashi and Sakamoto and by Matthews et al. [
The cage side observation tool used in the present study appears to provide a sensitive and specific tool to detect mouse stress. More research is needed to confirm that our results demonstrating behavioral studies using a numerical scoring system across five independent variables can provide an early diagnosis for stress in mice:
M. Guarnieri has a significant financial interest in Animalgesic Labs.
Funding for this research was supplied by the Maryland Biotechnology Center Biotechnology Development Awards, Maryland Industrial Partnerships (MIPS), and Animalgesic Labs.