Trial design and objectives
The study was a multicentre randomised blinded ‘positive controlled’ trial, and used a two-group parallel design. The main objective was to determine the effect of enteric coating on efficacy of a pancreatic enzyme supplement in the treatment of canine EPI. The studied complied with the University of Liverpool Guidelines on Animal Welfare and Experimentation, and was approved by the University of Liverpool Research Ethics Committee (RETH000328). Prior to enrolment, owners were informed as to the nature of the study and gave their informed consent in writing. At the end of the trial, all owners were asked to complete a trial feedback form to ensure that they were happy with trial conduct. As far as possible (for a trial in a veterinary species), the studied complied with the principles of Good Clinical Practice (GCP) [4
Cases were recruited between March 2009 and July 2011, when the target for enrolment had been reached. Dogs recently (within two weeks) diagnosed with EPI (i.e. serum trypsin-like immunoreactivity [TLI] less than the lower limit of the respective laboratory reference interval, typically 2.0-3.0 μg/L) were eligible for enrolment. Other eligibility criteria included absence of any concurrent disease that might affect diagnosis, response to treatment (especially body weight gain) or prognosis including concurrent cardiac disease, renal disease, hepatic disease, endocrine disease or neoplasia. Further, it was a requirement that routine haematological and serum biochemical analysis had been performed within 4
weeks of enrolment. Finally, cases could not have previously been treated with pancreatic enzyme supplementation.
Any first-opinion veterinary practice with a case fitting the inclusion criteria was eligible to request enrolment in the trial. When a request was made, one of the study observers (AM, PJN, AJG) discussed the study outline with the primary care veterinarian, who in turn discussed the trial with the owner of the eligible dog. Assuming that the practice remained interested, the owner and primary care veterinarian could decide whether or not the trial visits were conducted at the Small Animal Teaching Hospital (SATH), or at the practice of their primary care veterinarian. If owners and/or veterinarians preferred the latter, a detailed study pack was then posted (by next-day recorded delivery). This pack contained a detailed information brochure for the veterinarian, explaining their duties as a trial investigator, an owner information sheet, a consent form, a form to record details of all visits and client communication, and a 9-point body condition score (BCS) chart [5
]. In addition, the specific therapy, for the whole of the trial, was also provided (see below). For cases that were enrolled at the SATH, the owner information sheet and consent form were posted to the client prior to the first appointment.
Trial publicity and incentives
In order to maximise recruitment, the trial was advertised in a number of ways, including mailshots to local veterinary practices, advertising footnotes to referral letters (written by all clinicians within the SATH for the duration of the trial), letters submitted to the veterinary press (e.g. Veterinary Record), advertising by the trial sponsor when their company representatives visited first opinion practices, and sponsored continuing education meetings organised for first-opinion veterinarians. In addition, a footnote advertising the trial was added to the results report for any TLI test result diagnostic for EPI at a large commercial clinical pathology laboratory (NationWide Laboratories, Poulton-le-Fylde, UK). In all cases, veterinarians with potentially eligible cases were encouraged to contact the study observers for details.
For cases where the owner wished to attend the SATH for their appointments, the costs of travel were reimbursed. In addition, the study medication was provided free of charge. Finally, to encourage compliance, any costs for the contributing primary veterinarians were defrayed by a payment of £300 for every case that completed the trial where all study paperwork was returned in a timely manner. Study observers did not receive any incentives or remuneration for completing the trial.
Roles and responsibilities
For cases seen at the SATH, the study observers (AM, PJN, AJG) were responsible for liaising with the owners, performing the examination at each visit and modifying therapy when necessary. The respective primary care veterinarian was informed of case progress throughout by letter. In the cases not referred to SATH, the primary care veterinarian was responsible for examining and managing the dog, for liaising with the client, and for completing study paperwork. However, they could contact the study observers at any time if they had questions regarding case management.
Both the test treatment and control treatment were based upon a commercially-available porcine pancreatic enzyme extract with an enteric coating, designed to protect the active enzyme from acid digestion and ensure high concentrations reach the small intestine (Lypex, Vet Plus Ltd., Lytham, UK; 30,000 ph Eur U lipase, 18750 ph Eur U amylase, 1200 ph Eur U protease per capsule). This product is based on Pancreatin, a highly active, porcine-derived enzyme combination. After active ingredient extraction, the dried enzyme is then pelleted and contains no excipient. An enteric coating is then applied, which is added in a solvent-free polymethacrylic acid/ester process using a dispersion film former. The coated pellets range from 1.4-2.4
mm in size. The product was manufactured by Nordmark Arzneimittel GmbH & Co. KG (Uetersen, Germany), on behalf of VetPlus Ltd.
The test treatment was identical to the commercially available product; the control treatment was similar in all aspects, except that it lacked the enteric coating, and was not commercially available. However, the organoleptic properties were identical and both treatments were presented in similar plain packaging (see below), ensuring that test and control treatment could not be distinguished. Nordmark Arzneimittel purpose-formulated both treatments for the trial on 24/11/08, with an expiry date of 30/11/11. Efficacy was tested and confirmed and the product was certified to be free from microbial contamination. Sufficient treatment was manufactured for a total of 40 dogs (with 20 receiving the test treatment, and 20 receiving the control product) and, in order to budget for difference in dogs size, enough product was manufactured to last 2
months even for a large dog where dose increases were required at each visit (see below).
The same starting dose of enzyme was used for both treatments: one capsule per day was administered to dogs <10
kg, divided over two meals, whilst 2 capsules/day (1 capsule per meal) was given to dogs >10
kg. During administration, the gelatin capsule was opened and the product mixed well with the food immediately prior to feeding. In order to avoid the risk of any skin irritation, owners were instructed to wear gloves when handling the capsules.
Initial assessment and enrolment
During the initial assessment, a detailed medical history was taken, physical examination performed, body weight was measured, and body condition was scored [5
]. Information about the severity of clinical signs was then obtained from the owner, using a standardised system (Table ). This enabled each clinical sign (i.e. appetite, frequency of defecation, faecal consistency, vomiting, flatulence, borborygmus, coprophagia and attitude/activity), to be scored semi-quantitatively, in a manner similar to another clinical scoring system used for chronic enteropathy [6
The clinical signs scoring system used two assess efficacy of two enzyme treatments for canine exocrine pancreatic insufficiency
At this stage, the purpose of the trial was again discussed with the owner and, assuming that they were happy, they were asked to sign the study consent form. Thereafter, dogs were allocated a study number and the treatment dispensed. The owners were instructed on how to use the treatment and any specific questions that the owner had were answered at this stage.
Monitoring, treatment alterations and follow-up
A summary of the study protocol is given in Figure . Throughout the trial, dogs returned on a weekly basis for administration of subcutaneous cobalamin (see below). Detailed assessments were conducted on days 14, 28 and 56. At each visit, a physical examination was performed, body weight was measured (using the same electronic scales as for the first visit), and a body condition score was performed. In addition, clinical signs were again scored using the same questionnaire as for the initial visit. Compliance with administration of the treatment was confirmed and, if necessary, dosage alterations were made and additional therapy was added.
Figure 1 Summary of the trial design and inclusion of dogs. Forty dogs, with exocrine pancreatic insufficiency, were randomised to receive one of two enzyme treatments (test e.g. coated and control e.g. uncoated), and were re-examined at 3 follow-up visits (day (more ...)
In addition to the official re-evaluation visits, clients contacted either the attending veterinarian or study observers using telephone calls and, occasionally, e-mail updates.
Treatment alterations, use of additional therapy, and diet
The intention was that only the enzyme supplement and weekly cobalamin injections (see below) would be administered during the trial. However, in accordance with normal clinical practice, alterations in enzyme dose and the addition of other therapies were allowed if response to therapy was poor. Primary care veterinarians made all treatment changes, after discussing them with one of the study observers, and in accordance with a standardised protocol. In this regard if, at the first reassessment (14
days), clinical response was deemed to be insufficient (e.g. poor weight gain, lack of resolution of clinical signs), the dose of enzyme was doubled. A further enzyme dosage increases were allowed on days 28 and 56, if necessary.
Given that hypocobalaminaemia is a negative prognostic indicator in canine EPI [3
], and in order to ensure that this was not a confounding factor during the trial, weekly subcutaneous injections of cobalamin (at 20
μg/kg) were administered to all dogs. This treatment was given irrespective of whether hypocobalaminaemia was present in pre-trial serum biochemical results. No other treatment was initially allowed in any of the dogs. Additional therapies were avoided as far as possible but if, in the opinion of the attending veterinarian they were deemed to be necessary, then they could be added from third visit (week 4) onwards, again after discussion with the study observers. Sanctioned additional therapies included the use of either antibacterials (e.g. oxytetracycline at 10
h PO, or metronidazole at 10
h PO) or histamine-2-receptor antagonists (e.g. ranitidine at 2
mg/kg q12h PO).
In order to avoid any potential cofounding effect of a diet change on treatment response, all owners were instructed to continuing feeding their dog’s existing diet at the same level. In all cases, dogs were fed twice daily. Details of exact diets fed were not recorded.
Patient welfare, adverse events, early trial discontinuation, and euthanasia
Throughout the study, all efforts were made to safeguard the welfare of the dogs enrolled, and owners were free to withdraw at any stage. The attending veterinarian recorded details of all welfare matters, including protocol deviations, suspected adverse events, development of concurrent medical problems and euthanasia. In addition, they informed the study observers immediately to agree an appropriate course of action. If withdrawal from the trial proved to be necessary, the study observers recorded the reasons.
For adverse events where the treatment was suspected to be the cause, participation in the study was to be suspended immediately. Where it was thought to be unlikely that an adverse event was related to the treatment, the dog was allowed to continue with the trial, provided that the owners agreed. Participation could also be suspended if an enrolled dog developed an unrelated condition, whilst enrolled in the trial.
Where it became necessary to perform euthanasia (e.g. poor response to therapy, development of another medical disorder), the attending veterinary surgeon would perform this (using overdose of intravenous sodium pentobarbital), after obtaining written consent from the owner.
Sequence generation and allocation concealment
The 40 treatments (20 test and 20 control) were assigned a study number from 1 to 40, based upon a randomised sequence generated in Minitab (Minitab Inc, State College, PA, USA) by the trial statistician (PJC). Treatments were given in sequence according to ascending study number, and each group of four study numbers contained two of each treatment. This was to ensure that numbers remained approximately even throughout the course of the study in case, for whatever reason, it was not possible to recruit all cases. The trial statistician sent the numbered sequence to the treatment manufacturer, who assigned the respective treatments to numbered packaging before posting to the study observers. The treatments were stored away from light and at room temperature, until assigned. The study observers were responsible for allocating dogs to their respective treatment, which occurred in a sequential fashion every time that a new case was enrolled. Neither the study observers nor of any of the attending clinicians were aware of the sequence of treatments.
A two-stage blinding process was used; the first level ensured that, for the duration of the trial, all owners, attending veterinarians, and study observers were blinded as to what treatment any of the dogs were on. Identical packaging was used for all treatments, consisting of plain plastic pots containing the treatment itself, within plain outer cardboard. The only identifying mark was the study number. As mentioned above, the treatments themselves were identical, comprising granules contained within unmarked gelatin capsules, and organoleptic properties were identical, both before and after the gelatin capsules were opened.
Once all cases had been enrolled, and all dogs had completed the study, the second stage of blinding was then implemented. For this, the trial statistician passed the randomisation sequence to a separate investigator (DB) who was not involved in any other aspect of the trial. This investigator broke the code, randomly assigned a secondary study number to each dog, and assigned the dogs to two groups, named “A” and “B”. A coded spreadsheet of the trial data was then given to the trial statistician, who performed all statistical analyses without knowing which treatment was which. Only when all statistical analyses had been completed were the treatment identities revealed.
The primary outcome measure of interest was change in body weight. Secondary outcomes of interest included, change in severity of clinical signs, change in BCS, the dose of treatment used for each dog, and requirement for additional medications. For clinical signs, a composite score was created, by adding together the results of all clinical signs recorded in the questionnaire. All of these outcome measures were decided prior to commencement of the trial.
At the conceptualization stage of the study, the trial statistician (PJC) performed a sample size calculation using a statistical software package (Minitab). The primary outcome measure (percentage gain in body weight) was used and, based upon previous studies [7
], the expected mean (± standard deviation) change in body weight was 24 ±15.2%. A 1:1 test:control recruitment rate was assumed and, given that no previous clinical trials had been conducted in canine EPI, a clinically relevant difference in efficacy between treatments of 40% was decided. This figure was based upon the opinions of the study investigators. Calculations assumed that a power of 90% was required to identify this difference with a two-sided P
of <0.05. Based upon these criteria, it was determined that 20 animals per group would be required.
Data handling and statistics
Data were entered into an Excel spreadsheet (Microsoft inc.) and checked for errors. Statistical analysis used Minitab 16, STATA12 (Statacorp, College Station, TX, USA), and Stats Direct version 2.6.2 (Stats Direct Ltd., Altrincham, UK). Standard descriptive statistics were used to report baseline data (either median and range, or mean ± standard deviation). Baseline data comparisons were made with Fisher’s exact test (for proportions) or the Mann-Whitney test (for continuous variables). The level of statistical significance was set at P<0.05 for 2-sided analyses. Outcome data were analysed both on an intention to treat and per-protocol basis; where there was a discrepancy in results, the former were considered most important. In order to account for missing data in the intention to treat analyses, imputation was performed using the method of “Last Observation Carried-Forward”.
For the primary outcome measure, namely bodyweight, the study design involved repeated measurements of the same animal, and the effect of treatment on weight was, therefore, investigated using a mixed-effects linear regression model in STATA. The xtmixed command was used, animal identity was declared as a random effect and estimation was by Maximum Likelihood. The effect of treatment was assessed using a multivariable model, which included treatment group, the visit number and their interaction. Serum cobalamin concentrations were included in the form hypocobalaminaemic and normocobalaminaemic when results were less than and greater than the lower limit of the reference range, respectively. The statistical significance of variables in the model was examined using their Wald statistic or their effect on the deviance.
Enzyme dose and BCS data did not meet the requirements needed for parametric analysis, and were analysed with the signed ranks test (for time differences) and the Mann-Whitney test (for differences between groups at each time-point).
A number of required changes were made to study protocol at various stages, mainly because the rate of recruitment of cases was slower than expected. Firstly, the original plan was for all cases to be seen at the SATH; however, initial recruitment was slow and the major hurdle was found to be reluctance to travel. For this reason, compensation for client travel was introduced and administration by the first-opinion veterinarian was then allowed. Second, as based upon the power calculation, the initial intention was to recruit a total of 40 dogs (20 treatment and 20 controls). However, the slow recruitment meant that there were concerns that the treatments would exceed their expiry date, initial set for two years after product manufacture. As a result, two treatments were sacrificed (1 treatment, 1 control) and sent back to the manufacturer so that enzyme activity and microbial contamination could be retested, and enabling an extension to the expiry date to be granted. The first product was one from a dog that had been enrolled, only to withdraw soon after starting (Figure ). In order to maintain blinding, the trial statistician randomly selected the second product from the remaining treatments, by choosing a treatment opposite to the first sacrificed product.