Fenofibrate in the management of AbdoMinal aortic anEurysm (FAME): study protocol for a randomised controlled trial

Fenofibrate in the management of AbdoMinal aortic anEurysm (FAME) is a multicentre, randomised, double-blind, placebo-controlled clinical trial to assess the effect of orally administered therapy with fenofibrate on key pathological markers of AAA in patients undergoing open AAA repair. The trial will be conducted at four sites in Australia: The Royal Brisbane and Women’s Hospital, Brisbane; The Holy Spirit Northside Private Hospital, Brisbane; The Townsville Hospital, Townsville and The Mater Hospital, Townsville. The trial will be reported according to the Standard Protocol Items: Recommendations for Intervention Trials (SPIRIT) (see Additional files 1 and 2). Only research personnel who are directly involved in the recruitment and data collection aspect of the study will have access to patients’ personal details. All Case Report Forms (CRFs), source documentation and samples will be stored de-identified where personal information has been removed and coded with a study number.

The FAME trial will include participants recruited from specialist vascular outpatient clinics who have an asymptomatic infrarenal AAA with a maximum orthogonal diameter ≥50 mm. FAME will not include individuals who require emergency or urgent AAA repair due to the requirement for a minimum 2 weeks of treatment with trial medication prior to surgery. Furthermore, participants will only be included if it is determined that they have a high likelihood of treatment compliance according to the treating physician or local study coordinator. Additional exclusion criteria include current treatment with fibrates, known contraindications to fenofibrate treatment and previous aortic surgery. A full list of inclusion and exclusion criteria is given in Table 1.

The overall design of the FAME trial is shown in Fig. 1. At the initial visit, potential participants booked for an elective open repair of an AAA will be assessed against the eligibility criteria (Table 1) and, if appropriate, informed consent will be obtained. Individuals will undergo a medical examination, resting blood pressure and heart rate assessments, and collection of blood samples for measurement of full blood count (haemoglobin, white cell count, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils), urea and electrolytes (sodium, potassium, creatinine, estimated glomerular filtration rate, urea, chloride, bicarbonate), liver function tests (Albumin, total bilirubin, alanine transaminase, aspartate aminotransferase, gamma-glutamyl transpeptidase, lactate dehydrogenase), fasting lipids (cholesterol, triglyceride, HDL cholesterol, low-density lipoprotein (LDL) cholesterol), fasting glucose and C-reactive protein. Serum, plasma and whole blood will also be collected for the later assessment of circulating concentrations of protein (such as cytokines) and genetic (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) markers. Investigational blood samples will be collected into the following tubes: 2 × 5-mL SST, 2 × 4-mL EDTA tubes, 1 × 4-mL sodium citrate tube and 1 × 2.5-mL PaxGene tube. Blood samples will be processed according to site-specific standard operating procedures (SOPs) and shipped to the study centre in Townsville. Eligible participants will be randomised to receive 145 mg fenofibrate or placebo, administered once a day for a minimum of 2 weeks directly prior to surgery, in a parallel-group design. Randomisation to fenofibrate or placebo will be stratified by study centre. Random allocation sequences will be computer-generated by a statistician and provided to each study centre's clinical trial pharmacist, ensuring both investigators and participants are blinded to drug assignment. Trial medication will be allocated and dispensed by the local study centre’s clinical trials pharmacist. Allocation concealment will be achieved by using identical packaging of the intervention and placebo. In the case of an emergency situation where breaking of the group allocation blinding would be required, the local study centre clinical trial pharmacist will be contacted. To facilitate compliance, participants will be provided with the phone number of the local study coordinator with instructions to contact them in the event of possible medication-related problems or consideration of discontinuation. In this event arrangements will be made for the participant to be reviewed by the study physician to ascertain whether discontinuation is required.

On the day of surgery, further blood samples will be collected (as per initial visit). Adverse and clinical events will be recorded, changes to usual medication noted, and compliance with the study drug regimen analysed by capsule counting. During open surgery, biopsies will be taken from the following sites: (1) subcutaneous fat at the incision site, (2) periaortic fat near the AAA, (3) AAA neck, (4) AAA body (opposite the inferior mesenteric artery) and (5) AAA thrombus. To preserve RNA and protein integrity, tissue samples will be collected into liquid nitrogen immediately upon harvest for subsequent genetic and protein analysis. An additional AAA body sample will be collected and immediately stored in 10% (v/v) paraformaldehyde and wax-embedded for immunohistochemical analysis. All collection of tissues will be performed according to site-specific SOPs and shipped to the study centre in Townsville.

Outcome assessment will be performed at the study centre in Townsville on the tissue and blood samples collected. All outcome assessment will be conducted by scientists blinded to the treatment allocation of the participants.

To assess AAA-wall macrophage number, serial cryostat sections 7 μm thick will be cut from each AAA wall sample for subsequent macrophage staining, as previously described [19]. All samples will be stained simultaneously using identical reagents and incubation times. Serial frozen sections will be air-dried, fixed in acetone for 10 min at −20 °C, air-dried and rehydrated with phosphate-buffered saline (PBS) before being incubated in 3% H₂O₂/0.1% sodium azide/PBS to block endogenous peroxidase. For macrophage detection, sections will be blocked in 2% (v/v) normal goat serum in PBS followed by staining using pan-macrophage antibody (Abcam) and goat anti-rat HRP (Chemicon). An IgG (Sigma) will be used as isotype control. Slides will be incubated in the peroxidase substrate 3,3’-diaminobenzidine (ImmPACT DAB, Vector Laboratories), counterstained in Mayer’s haematoxylin, dehydrated, cleared in xylene and mounted in Depex mounting medium. Stained sections will be photographed using a Leica BMLB microscope fitted with a SPOTTM CCD Camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and digital images captured to a PC supported with SPOT32TM software (version 2.1.2; Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Identical exposure times and settings will be used for sections. Image analysis will be performed on digital tiff images using Adobe Photoshop CS6 Extended software. For each section, the total tissue area and area of macrophage staining will be measured using the ‘Selection Tool’ and ‘Record Measurements’ functions. Macrophage staining will be expressed as macrophage number and also as a percentage of total tissue area.

AAA-wall OPN will be determined using an enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. For determination by ELISA, protein will be extracted from individual frozen AAA wall biopsies by homogenising in buffer (10 mM cacodylic acid, 60 mM L-arginine, 0.25% (v/v) Triton x-100 in PBS, pH 7.2) and centrifuging at 18,000 × g at 4 °C for 20 min. Supernatant protein will be quantified by the Bradford technique (Protein Assay, Bio-Rad, Hercules, CA, USA). OPN concentration will be measured by ELISA (Quantikine, R&D Systems for OPN) and expressed as pg/mg of protein. Excellent reproducibility of similar assays has previously been reported [39]. For determination by immunohistochemistry, slides will be incubated in 2% (v/v) normal goat serum (Vector Laboratories) in PBS and endogenous avidin and biotin-blocked using an Avidin/Biotin blocking kit (Vector Laboratories), then 2 μg/mL rabbit anti-human OPN (Immuno Biological Laboratories), biotinylated goat anti-rabbit IgG (Vector Laboratories) and Vectastain Elite ABC-HRP. Rabbit IgG (Vector Laboratories) will be used as isotype control antibody.

Serum OPN concentration will be measured using blood collected from participants following an overnight fast. Serum will be stored at −80 °C until later batch assessment using ELISA according to the manufacturer’s instructions, and expressed as ng/mL (R&D Systems). This assay has previously demonstrated excellent intra- and inter-assay reproducibility [39].

Additionally, inflammatory cells (neutrophils, B- and T-lymphocytes), MMPs and proinflammatory cytokines will be assessed by immunohistochemistry and ELISA as previously described [40, 41]. Circulating concentrations of other AAA biomarkers, including osteoprotegerin, resistin, D-dimer and fasting lipids, will be assessed by ELISA and automated assays as previously described [31, 36‐38]. Whole genome microarrays and real-time polymerase chain reaction will be used to examine gene expression levels based on findings from ongoing expression arrays and biomarker studies.

Estimated outcomes for the control group are based on assessments performed in human AAA biopsies or blood samples from previous studies [28, 32, 42‐44]. Estimated effect sizes for fenofibrate therapy are based on previous rodent and human studies, and the consideration of outcomes likely to be required for clinical efficacy [19, 28, 32, 42‐44]. To significantly influence the natural history of aortic destruction, it is estimated that a reduction in all primary outcomes assessed within AAA biopsies of at least 50% is likely to be required. In a rodent model, 4 weeks’ treatment with fenofibrate reduced aortic OPN concentration and macrophage infiltration by a median of 95% and 70%, respectively, suggesting that this treatment effect size is realistic [19]. For serum OPN, it has previously been reported that a fibrate reduced circulating OPN concentration by approximately 40% after 4 weeks of treatment [32]. Based on these data, the estimated mean values for control and treatment groups were calculated and are given in Table 2. Sample sizes were calculated using GPower3.1, based on a t test as the statistical analysis test. Since there are three primary outcomes, alpha was set at .017, adjusted from .05 for multiple testing. Sample size was estimated based on a power of 80% and equal numbers of participants in each treatment arm. As a result, 20 participants receiving fenofibrate and 20 participants receiving placebo are required. Assuming a dropout rate of 5%, a total of 42 participants will be required.

Participant safety will be assessed prior to the administration of the medication and at the end of the study period. At the initial visit, a consultation with a physician will occur, during which the participant will be informed about known side effects including symptoms of abdominal/back pain, chest pain, and renal and liver dysfunction. Pathology tests consisting of full blood count, fasting lipids, glucose, inflammation markers, liver and renal function will be performed. The participant will undergo a physical assessment which will include blood pressure and heart rate measurements that will be reviewed by a physician along with the results of pathology tests prior to randomisation. At the final visit, pathology tests as per the initial visit will be performed and reviewed by a physician. Any adverse event will be reported to the coordinating centre and carefully monitored throughout the study. Serious adverse events (SAEs) will be defined as death, requirement for inpatient hospital treatment and persistent or significant disability. All SAEs will be reported by the site principle investigator to the HREC and reviewed by the chief principle investigator, where a decision regarding withdrawal of trial medication will be made. Where a decision to withdraw trial medication is made, participants will be encouraged to remain on the study protocol. Previous studies suggest that fenofibrate is a relatively safe medication [45, 46]. Participants who are concurrently on warfarin will have two additional safety assessments after randomisation. Current routine care for patients on warfarin involves ongoing measurement of International Normalised Ratio (INR) levels, which in turn dictates the dose of medication required to manage clotting without increasing the risk of excessive bleeding. Participants will be instructed to have their INR concentrations assessed via their usual system at 3–5 days and again at 14–21 days post first dose so that warfarin dosage may be adjusted accordingly.

Trial documentation including protocols, SOPs and CRFs will be shared electronically with participating study centres. Protocol amendments will be submitted to the Royal Brisbane and Women’s Hospital HREC and local site research governance offices and disseminated to the relevant parties at each study site. Data recorded on printed CRFs will be scanned to the study centre in Townsville where it will be entered centrally and examined for data quality. This will allow confirmation of entry criteria and collection of set entry and outcome data. Examples of important baseline data which will be collected include age, gender, presence of diabetes and/or dyslipidaemia, concurrent medications and maximum aortic diameter. At completion of the trial the database will be checked for errors and data confirmed with source documentation where required. Analysis of primary and secondary endpoints will be based on intention-to-treat at the time of randomisation. All participants who meet the eligibility criteria, provide written informed consent and are enrolled in the study will be included in the primary analysis, regardless of adherence to medication allocation.

To identify potential confounders, collected clinical and demographic data will be compared between groups via univariate statistics. The distribution of all continuous data variables will be assessed for normality using the Kolgorov-Smirnov test. Normally distributed continuous variables will be compared between test groups via t test; non-normally continuous distributed variables will be compared between groups using the Mann-Whitney U test. Nominal data will be compared using the chi-squared test. Characteristics showing a p value < 0.100 on univariate tests will be considered as potential confounders and will be entered as covariates in subsequent binary logistic regression models assessing the association of each of the outcome measures with treatment allocation. Following binary logistic regression, the association of all covariates with treatment allocation will be reported as odds ratios and 95% confidence intervals. For all analyses, p values <0.05 will be considered to be significant. Data will be published in a peer-reviewed journal with copies of the paper available to participants if required.

At the time of submission, recruitment was ongoing.