Randomized Open-Label Clinical Trial Comparing Prednisolone and Cyclosporine With a Nonrandomized Active Control for Treating Presumed Chronic Pancreatitis in Cats

2.1 Trial Design

Three-week open-label randomized clinical trial comparing prednisolone and cyclosporine with a nonrandomized active control for the treatment of presumed chronic pancreatitis in cats. The study consisted of two screening steps and two scheduled reevaluations. The second screening step was used as pretreatment baseline for the immunosuppressive treatment groups. Reevaluations were scheduled on Days 10 and 21 of the treatment trial. Figure 1 depicts the study timeline and the main outcomes assessed.

2.2 Study Population and Eligibility (First Screening Step)

Cats were recruited from veterinarians in the United States who submitted testing of serum fPLI concentration as part of their patient's diagnostic evaluation. A first screening serum fPLI concentration > 10 μg/L (reference interval [RI], ≤ 4.4 μg/L) along with serum cobalamin concentration > 400 ng/L (RI, 290–1500 ng/L) and serum folate concentration within the RI (9.7–21.6 μg/L) were used as search criteria in the laboratory database to identify potential candidates for enrollment. The rationale for setting the first screening fPLI criterion at 10 μg/L is based on our empirical observation that patients with higher serum fPLI concentrations are more likely to continue to have increased serum fPLI concentrations when compared to those cats with a serum fPLI concentration that is only mildly increased. The inclusion of normal cobalamin and folate concentrations in the search criteria was intended to help decrease the chance of enrolling cats with concurrent intestinal diseases. The cutoff for serum cobalamin concentration was chosen because previous work showed that some cats with serum cobalamin concentrations in the lower end of the RI had increased serum methylmalonic acid concentrations, suggesting cobalamin deficiency on a cellular level [5, 6].

Additional medical histories and diagnostic tests were requested from veterinarians with patients who met the search criteria to confirm eligibility. Cats of any age, breed, and sex were eligible for enrollment. Criteria for eligibility consisted of (1) diagnosed by the primary veterinarian as having pancreatitis for > 2 weeks based on clinical evaluation (e.g., medical history and physical examination); (2) no diet changes within 2 weeks, but if this was the only violation of criteria, cats were allowed to be enrolled 2 weeks after the diet change; (3) no treatment with any medications that might cause pancreatitis (e.g., phenobarbital, organophosphates, calcium); and (4) no recent (within 3 months) or current use of corticosteroids or other immunosuppressants. Concurrent disease was not an exclusion factor, but patients who were stable and had fewer concurrent diseases were prioritized for recruitment.

2.3 Treatment Assignment and the Second Screening Step

For cats that met the first screening criteria and were considered eligible, the owner and the attending veterinarian were given the choice to enroll the cat into either the control or the treatment group. A choice was given, instead of being based on randomization, mainly for ethical reasons, understanding that the balance of measured and unmeasured covariates cannot be ensured between the control group and the two treatment groups.

Cats that were enrolled in the treatment group were randomly assigned to receive prednisolone or cyclosporine (described in Section 2.5). Randomization was performed before the second screening step, and results were revealed to the care team immediately to avoid delaying potentially beneficial immunosuppressive treatment after blood collection at baseline. If the second screening serum fPLI concentration was < 5.4 μg/L, the cat was excluded from the study. The second screening fPLI criterion was set at 5.4 μg/L because the study followed the previous Spec fPL cutoff for diagnosis of pancreatitis [7] and was conducted before the cutoff was adjusted to ≥ 8.8 μg/L [8].

2.4 Sample Size Determination

Sample size was not calculated because no studies have documented the crucial variables regarding the effects of symptomatic treatment alone, prednisolone, or cyclosporine on serum fPLI concentration or the CAI in cats with pancreatitis. A desirable difference in clinically meaningful outcomes for pancreatitis in cats has not been determined. Therefore, we aimed to enroll a reasonable number of cats in hopes that doing so would lead us to the ideal sample size for future studies. Hence, the number of cats per group was empirically determined to be 15. To account for the number of cats that might be excluded at the second screening step or die or be euthanized because of their illness, five additional cats were requested for each group. Both first and second screening serum fPLI concentrations were initially set at > 20 μg/L following a previous study [9]; during this period, two cats were enrolled but later excluded after failing to meet the second screening fPLI criterion. The screening fPLI criteria were revised (detailed in Sections 2.2 and 2.3), and only 58 cats enrolled with these criteria were analyzed and discussed in the study.

2.5 Intervention Regimen

Cats in the control group only received symptomatic treatment as needed at the discretion of the attending veterinarian, and these treatments were not necessarily started at baseline. Cats in the prednisolone group received prednisolone at 2 mg/kg PO q12h for the first 5 days and were then transitioned to 1 mg/kg PO q12h for the remainder of the 16 days. Cats in the cyclosporine group received cyclosporine at 5 mg/kg PO q24h for 21 days.

For ethical reasons, all groups were allowed to receive symptomatic treatments or treatments for concurrent diseases as needed at the discretion of the attending veterinarian, understanding that doing so could confound clinical outcome assessment. Owners were required to keep the current diets unchanged throughout the trial period. No additional anti-inflammatory or immunosuppressive treatments were allowed during the study period.

2.6 Outcome Assessments and Assays

Serum fPLI concentration and CAI were used as outcomes for the evaluation of treatment effects. Outcomes were assessed at baseline, Day 10, and Day 21 of the study period. All visits were conducted at the patient's local hospital with the attending veterinarians. Serum fPLI concentrations were measured using a commercially available ELISA Kit (Spec fPL, IDEXX Laboratories, Westbrook, ME, USA) [8]. The CAI, modified from the feline chronic enteropathy activity index [10], was the sum of scores for seven clinical variables that often are related to the clinical presentation of cats with pancreatitis, which was recorded by the attending veterinarian at each visit. These consisted of activity, appetite, weight loss, vomiting, icterus, abdominal pain, and diarrhea. Each clinical variable was assessed with an ordinal value of 0, 1, 2, or 3; a higher score corresponds to a more severe clinical sign. Definition of score and corresponding severity for each clinical variable is shown in Table S1.

Complete blood counts (CBCs) were submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory. Serum biochemistry profiles were automatically analyzed (AU 480 chemistry analyzer, Beckman Coulter, Indianapolis, Indiana); serum cobalamin and folate concentrations were measured by automated competitive binding chemiluminescence immunoassay (IMMULITE 2000 XPi, Siemens Healthineers, Erlangen, Germany), and serum feline trypsin-like immunoreactivity (fTLI) concentrations were measured by radioimmunoassay [11] at the Gastrointestinal Laboratory, Texas A&M University.

2.7 Statistical Analyses

Spearman correlations were used to evaluate associations between changes in serum fPLI concentration and days between the first and second screening tests. Relevant clinical signs that developed within the last 3 months before baseline and demographics at baseline were described using descriptive statistics. Binomial testing was used to determine if the proportion of male cats in total or in each group was different from 50%. Normality was evaluated using Shapiro–Wilk tests. Differences in age, body condition score (BCS), serum fPLI concentration, and CAI were compared using Kruskal–Wallis tests, followed by Dunn's multiple comparison tests.

To account for the baseline clinical presentations in the regression model, principal components derived from the baseline clinical variables were used instead of individual baseline clinical variables or the baseline CAI, because (1) baseline clinical variables showed little variability across subjects and (2) sums of ordinal data (e.g., CAI) are not meaningful statistics because of unequal intervals between categories. That is, adding ordinal values together does not accurately represent the data. The significance of the principal component analysis (PCA) was analyzed [12], and only the first principal component was significant at the 5% level, which captures only 42% of the variability in the data. Therefore, the first three principal components that together explained at least 80% of variability were extracted to perform principal component regression [13].

Treatment effects on serum fPLI concentration and CAI were estimated with linear mixed-effects models using different baseline adjustment methods; the corresponding response types were (1) fPLI_reevaluation: serum fPLI concentration on Days 10 and 21, (2) ∆fPLI = fPLI_reevaluation − fPLI_baseline, (3) %∆fPLI = ∆fPLI/fPLI_baseline × 100%, (4) CAI_reevaluation: CAI on Days 10 and 21, and (5) ∆CAI = CAI_reevaluation − CAI_baseline (Table 1; fPLI_baseline: baseline serum fPLI concentration; CAI_baseline: baseline CAI). Standard errors for regression coefficients were estimated using the bootstrap method because of the moderate sample size [14]. The point estimates and 95% confidence intervals (CIs) for treatment effects comparing cyclosporine and control ($$$ \hat{\upbeta} $$$₁), prednisolone and control ($$$ \hat{\upbeta} $$$₂), and cyclosporine and prednisolone ($$$ \hat{\upbeta} $$$₁ − $$$ \hat{\upbeta} $$$₂), as well as for other covariates ($$$ \hat{\upbeta} $$$_3–7), were reported for each baseline adjustment method. Seven of the 48 cats had a CAI_baseline = 0 despite the clinical history of chronic GI signs. Therefore, the percentage change in CAI was not evaluated as a response type.

Repeated measures of correlation between serum fPLI concentration and the CAI of three visits (baseline, Day 10, and Day 21) were examined using the R package rmcorr [15].

Statistical analyses were performed in R (version 4.4.2, R Core Team (2024), R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism (version 9.0, GraphPad Software, Boston, MA, USA). Statistical significance was set at p < 0.05.

2.8 Ethics Statement

The study was approved by the Clinical Research Review Committee at Texas A&M University (IACUC 2017-0416 CA and IACUC 2020-0317 CA). Informed owner consent forms were obtained from each owner.

3.1 Recruitment Statistics

Figure 2 summarizes the recruitment statistics. Of the 58 enrolled cats, 48 (83%) cats had serum fPLI concentration ≥ 5.4 μg/L at baseline and hence continued to meet the inclusion criteria to stay in the trial, with 16 cats in the control group, 15 cats in the prednisolone group, and 17 cats in the cyclosporine group. Out of 48 cats, 1 cat in the control group missed a serum sample for one of the visits and the CAI for both reevaluation visits. Otherwise, all 48 cats finished the study without reported protocol deviations, and all were analyzed.

3.2 Changes in Serum fPLI Concentrations and Time Between First and Second Screening

For 58 enrolled cats, the median time between the first and second screening was 24 days (range, 6–52 days). The median change in serum fPLI concentration was −5.8 μg/L (range, −195.5 to 156.4 μg/L). The median percentage change in serum fPLI concentration was −31.2% (range, −97.8 to 506.3%). Time was not correlated with either type of change in serum fPLI concentration (p = 0.14 and 0.11, respectively; Figure S1).

3.3 Clinical Signs Within the Last 3 Months Before Baseline Visit (Day 0, Second Screening)

Overall, the most commonly reported signs in the 3 months before the baseline visit were weight loss, vomiting, anorexia, and lethargy (Table 2).

3.4 Baseline Demographics

Table 3 summarizes the baseline demographics. Most cats were domestic shorthair cats. Overall, the proportion of males was higher than females (p = 0.03); the proportion of males was higher in the cyclosporine group (p = 0.01), but the proportions of males and females were not significantly different in the control or the prednisolone group (p = 0.80 and 0.61, respectively). Cats were generally older (median, 13.25 years). Body condition score was different between groups (p = 0.004); BCS in the control group was higher than in the prednisolone (adjusted p = 0.02) or cyclosporine (adjusted p = 0.01) groups, but was not different between the prednisolone and the cyclosporine groups (adjusted p = 0.67). The fPLI_baseline was not different between groups (p = 0.18), but the CAI_baseline was different between groups (p = 0.01). The CAI_baseline in the control group was lower than in the prednisolone (p = 0.02) or the cyclosporine (p = 0.03) groups, but was not different between the prednisolone and the cyclosporine groups (p > 0.99). Seven of the 48 cats had a CAI_baseline of 0: 5 cats in the control group and 1 cat each in the prednisolone and the cyclosporine groups, respectively.

The CBC, biochemistry profile, serum cobalamin, folate, fPLI, and fTLI concentrations at each time point, comorbidities, and available ultrasonography reports are summarized in Tables S2–S8. The number of cats that received symptomatic and other treatments near the time of baseline is summarized in Table S9.

3.5 Serum fPLI Concentrations Over Time and Treatment Effects

Serum fPLI concentration over time and changes in serum fPLI concentration between time points are shown in Figures 3 and 4, with corresponding descriptive statistics in Tables S10 and S11, respectively. Table 4 summarizes the number of cats in the selected ranges of serum fPLI concentration over three visits. Tables 5 and 6 summarize the point estimates and 95% CI for the treatment effect and other covariates on serum fPLI concentrations with respective baseline adjustment methods. Trends for each patient over time and percentage changes in serum fPLI concentrations between time points are shown in Figures S2 and S3, respectively.

Treatment effect on serum ∆fPLI was significant when comparing cyclosporine and control ($$$ \hat{\upbeta} $$$₁, Table 5), indicating that cyclosporine was associated with a larger decrease in serum fPLI concentration compared with control. When fPLI_reevaluation was used as the response, the treatment effect was not significant (95% CI, −20.9 to 1.9 μg/L), but the upper limit was close to 0.

Treatment effect comparing prednisolone and control ($$$ \hat{\upbeta} $$$₂, Table 5) was significant when either fPLI_reevaluation or ∆fPLI was evaluated. These results indicate that prednisolone treatment was associated with a higher serum fPLI concentration at the time of reevaluation and a larger increase in serum fPLI concentrations compared with control.

Treatment effect comparing cyclosporine and prednisolone ($$$ \hat{\upbeta} $$$₁ − $$$ \hat{\upbeta} $$$₂, Table 5) was significant regardless of the baseline adjustment method used. All treatment effects indicate that cyclosporine was associated with a decrease and a larger decrease in serum fPLI concentrations compared with prednisolone.

The fPLI_baseline ($$$ \hat{\upbeta} $$$₇) was significantly associated with fPLI_reevaluation. Regardless of the baseline adjustment methods used, time ($$$ \hat{\upbeta} $$$₃) and principal components derived from the baseline clinical variables ($$$ \hat{\upbeta} $$$_4–6) were not significant (Table 6).

3.6 CAI, Clinical Variables Over Time, and Treatment Effects

Figure 5 shows the CAI and each clinical variable over time. Figure S4 shows the changes in CAI between time points. Prednisolone was associated with a decrease and larger decrease in CAI, compared with the control ($$$ \hat{\upbeta} $$$₂) or the cyclosporine group ($$$ \hat{\upbeta} $$$₁ − $$$ \hat{\upbeta} $$$₂). The treatment effect was not significant when comparing the cyclosporine and the control group ($$$ \hat{\upbeta} $$$₁; Table 5). Regardless of the baseline adjustment used, principal components derived from the baseline clinical variables ($$$ \hat{\upbeta} $$$_4–6) were significant, but time ($$$ \hat{\upbeta} $$$₃) and fPLI_baseline ($$$ \hat{\upbeta} $$$₇) were not (Table 6).

3.7 Correlation Between Serum fPLI Concentration and CAI

Serum fPLI concentrations and the CAI were not correlated, with a repeated measures correlation at 0.03 (p = 0.79; 95% CI, −0.15 to 0.17).