Animals and housing
Twenty-four, non-naïve healthy Beagle dogs, bred for research purposes (Marshall BioResources, North Rose, NY), were divided into 2 treatment groups (n = 6 males and 6 females/group). Dogs ranged from 13.3 to 13.8 months of age at the study start. Body weights ranged from 6.5 to 9.4 kg for females and 10.0 to 12.6 kg for males at randomization. Dogs were housed individually in stainless steel cages with controlled temperature (18 to 29 °C), relative humidity ranging from 30 to 70% and a photoperiod of 12 h of light alternating with 12 h of darkness. All dogs were under the care of a licensed veterinarian. Water was provided ad libitum (see below for information about feeding). At the end of the study (following weighing on Day 4), all dogs were returned to the facility colony.
The study tested capromorelin flavored oral solution (ENTYCE®) with 30 mg/ml of capromorelin compared to a matched placebo flavored oral solution treatment (which contained all the ingredients of the formulation without capromorelin) administered for 4 days. Dogs were randomized into two groups, with Group 1 receiving placebo (0.1 ml/kg) and Group 2 receiving 3.0 mg/kg ENTYCE®. Both groups were treated once a day at approximately 9 AM each day. The first day of dosing was considered Day 0. The placebo and test drug were administered by a syringe placed in the corner of the mouth. The Day 0 weight was used for dose calculations.
Dogs were observed at least twice daily and any clinical or behavioral observations were recorded. A physical examination of each dog was completed on Days -14, -2, 0 and on Day 4 at the time of the final body weight measurement. Serum chemistry and hematology (CBC) screening to confirm the health status of each dog prior to enrollment was conducted on Day -2 and repeated on Day 3, approximately 4 to 5 h following administration of the last dose and within 1 h after removal of food, to evaluate any change over the 4 day treatment period. Body weights were recorded prior to feeding on Days -14, -2, 0 and 4.
Feeding and evaluation of food consumption
Dogs were fed a 25.44% protein nutritionally complete and balanced dietFootnote 1 once daily. The dogs were placed on a time-restricted feeding period beginning 14 days prior to the start of the study. At approximately 10 AM (±30 min) following an overnight fast, dogs were offered 600 grams of food (i.e., twice their normal ration) for a total of 3 h (± 10 min), at which time any remaining food was removed. Food was weighed prior to and after food offering. Food consumption was recorded daily from Day -14 through Day 3.
Individuals making any observations on the condition of the dogs, including physical examinations, and any individuals recording data on food consumption and body weight were masked to treatment group.
For food consumption, the baseline value was defined as the mean of the values collected on Day -3, Day -2 and Day -1. The treatment period was the mean of the values collected from Day 0 through Day 3 (4 days) and the percent change was calculated between baseline average and the treatment period average. For body weight, the baseline value was the value obtained at Day 0. Percent changes from baseline were calculated at Day 4, the treatment period for body weight.
Descriptive statistics (number of subjects, mean, standard deviation, standard error of the mean, minimum, median and maximum values) were presented for food consumption percent change at each day measured and body weight percent change, for the treatment period, and baseline. All tests of significance were 2-sided and performed at alpha = 0.05. The SAS® software system,Footnote 2 Version 9.2, was used for all calculations.
Analysis of variance (ANOVA) modeling was utilized to evaluate differences between the treated and placebo groups. The ANOVA model contained terms for treatment, sex and the interaction of treatment by sex. When the interaction term was statistically significant then the treatment group comparisons were carried out for each sex separately. Otherwise, the treatment group comparisons were carried out across the pooled sexes.
In addition, the treatment groups were compared by permutation testing using the SAS® procedure MULTTEST. When, in the ANOVA described above, either the treatment by sex interaction or the sex main effect was significant, the treatment groups were compared separately for each sex. Otherwise, the treatment groups were compared across the pooled sexes. Also, within treatment group, comparisons were assessed by the one-sample t-test or Wilcoxon signed rank test, as appropriate, to evaluate the percent change from baseline for body weight and food consumption. The Shapiro-Wilk test for normality was conducted, at the 0.05 significance level, to determine the appropriateness of the one-sample t-test. When statistically significant, the Wilcoxon signed rank test was conducted.