ABSTRACT
Rationale
Patients with a Fontan circulation suffer from progressive multiorgan dysfunction, yet central biochemical drivers remain poorly defined. Our recent work exploring metabolomic analyses have identified- elevated circulating bile acids (BAs) in adult Fontan patients compared with healthy controls. Elevated BAs, especially secondary and hydrophobic ones produced by the gut microbiome were found to correlate with worse exercise capacity, greater frailty, and impaired hemodynamics.
Primary hypothesis
Bile acid accumulation may contribute to Fontan pathophysiology. Colesevelam, a nonabsorbed bile acid sequestrant, offers a potential targeted therapy to reduce BA levels and interrupt this disease pathway.
Design
The MYSTIC trial is a prospective, randomized, double-blind, placebo-controlled cross-over pilot study in 25 adult Fontan patients (with 25 age- and sex-matched healthy controls for baseline comparisons) to evaluate the safety, tolerability, and efficacy of colesevelam in lowering plasma BA levels. Primary outcomes include safety/tolerability and change in total plasma BA levels. Secondary outcomes include changes in noninvasive hemodynamics, gut microbiome composition, fecal bile acid excretion, and biochemical profiles.
Sites
Single center.
Estimated enrollment
25 patients and 25 healthy subjects.
Enrollment dates
September 2025 to August 2027.
Trial Registration
NCT06197763 This manuscript describes the rationale and design of the MYSTIC study, which to our knowledge is the first interventional trial targeting a Fontan-specific metabolic derangement. The results will inform the feasibility of BA sequestration therapy in Fontan patients and guide future larger studies aimed at improving outcomes in this growing high-risk population.
Background
The Fontan procedure, a palliative surgical correction for complex single-ventricle congenital heart defects, has dramatically improved survival for thousands of patients over the past half-century. However, the unique Fontan circulation results in chronic systemic venous hypertension, low cardiac output, and lack of pulsatile pulmonary flow. Over time, these hemodynamic conditions lead to multisystemic complications including ventricular dysfunction, liver fibrosis, plastic bronchitis, protein-losing enteropathy, and renal dysfunction. Fontan patients often exhibit reduced exercise capacity, increased frailty, and diminished quality of life as they age. A 2019 American Heart Association scientific statement on Fontan circulation highlighted “an important unmet need in the medical community for a more complete understanding of the pathophysiology, origins, and mechanisms of the development of end-organ consequences . ” In particular, identifying circulating biomarkers or biochemical derangements that drive Fontan failure has been a priority, as traditional heart failure markers (eg, BNP) are often not descriptive of Fontan circulatory dysfunction despite significant disease.
Recent research has begun to fill this knowledge gap by using-omics approaches to profile Fontan patients. Our recent metabolomic work comparing Fontan with matched healthy subjects demonstrated that adult Fontan patients in that cohort had significantly worse exercise performance, reduced skeletal muscle mass, excess frailty, and impaired hemodynamics, both at rest and with exercise augmentation. Notably, metabolomic analysis revealed marked elevations in circulating bile acids (BAs) in Fontan patients compared to controls; many of these were secondary BAs, product of gut microbial metabolism of primary BAs. Equally important, specific elevated BAs showed strong negative correlations with clinical metrics—higher BA levels were associated with lower skeletal muscle mass, greater frailty, worse exercise capacity, and impaired hemodynamic measurements. These findings were novel, as others have described BAs to be elevated in Fontan circulation but have not implicated them as biomarkers. ,
The discovery of elevated BAs in Fontan patients has suggested a new pathophysiological link in Fontan circulation. Bile acids are signaling molecules known to interact with cardiomyocytes, vascular cells, and the liver. Excess bile acids, especially hydrophobic secondary BAs like deoxycholic acid can have direct toxic effects. Experimental evidence indicates that elevated BAs may induce mitochondrial dysfunction in cardiomyocytes, impairing ATP production and resulting in reduced contractility and relaxation. Such BA-induced cardiomyopathy could contribute to the diastolic dysfunction and diminished cardiac output reserve observed in many Fontan patients. Moreover, patients with chronic heart failure were noted to have elevated secondary to primary bile acid ratio. Elevated bile acids can also promote hepatic injury: hydrophobic BAs are cytotoxic to hepatocytes, triggering apoptosis and activation of hepatic stellate cells that produce fibrosis. , This mechanism aligns with the progressive Fontan-associated liver disease (FALD) seen in long-term Fontan survivors, where chronic liver congestion and now possibly BA-related toxicity drive fibrosis and cirrhosis. Moreover, BAs are also nephrotoxic and may have a role in Fontan associated progressive renal dysfunction. Collectively, these insights paint a picture in which excess bile acids act as multisystem antagonists in Fontan circulation.
Given this background, we identified bile acid sequestration as a promising and targeted therapeutic strategy to mitigate the metabolic derangements observed in Fontan patients. Bile acid sequestrants are nonabsorbed resins that bind bile acids in the intestine, interrupting their enterohepatic recirculation and promoting fecal BA excretion. They are clinically approved for hyperlipidemia and have well-documented safety profiles. In the context of Fontan physiology, a bile acid sequestrant could potentially decrease systemic BA levels and thereby mitigate the downstream toxic effects. Additionally, by altering BA availability in the gut, sequestrants can induce shifts in gut microbiome composition, since bile acids modulate microbial growth. One such sequestrant, colesevelam, is particularly attractive for study. Colesevelam is a water-insoluble polymer that binds bile acids with high capacity and is generally better tolerated than older sequestrants like cholestyramine, with fewer gastrointestinal side effects. Importantly, colesevelam is virtually unabsorbed (<0.1% systemic absorption), making it safe for use in a variety of populations. Common side effects of colesevelam are gastrointestinal (constipation, bloating), which are usually mild and manageable.
Prior to conducting large-scale definitive efficacy trials, a pilot study is essential to assess the feasibility, safety, and biochemical impact of this therapy in Fontan patients. Here, we present the rationale and design of the MYSTIC trial (IMpact of therapY using coleSevelam Treatment reducing bile acids in patients with fonTan cIrCulation), a Phase II pilot study designed to evaluate the safety and efficacy of colesevelam in adult Fontan patients, with a focus on its ability to lower plasma bile acid levels and favorably modulate associated pathophysiologic markers.
Methods
Study design
The MYSTIC trial is a single-center, prospective, randomized, double-blind, placebo-controlled cross-over study designed to evaluate the effects of bile acid sequestration in adult Fontan patients. The study is registered as a Phase II pilot trial (investigator-initiated) and will be conducted at St. Boniface Hospital, Winnipeg, Canada. All participants will provide written informed consent prior to enrollment. The local Research and Ethics Board has approved the study [HS25540 (B2022:052)]. The study has been registered on the ClinicalTrials.gov (NCT06197763).
Study participants
Fontan participants
Inclusion criteria: Adults (≥18 years old) with a history of Fontan palliation are eligible for the trial. Both male and female Fontan patients are eligible; women of childbearing potential must agree to use effective contraception during the study (given the unknown effects of colesevelam in pregnancy, though it is not systemically absorbed). Participants should be in a relatively stable condition, defined as New York Heart Association class I–II symptoms and with no hospitalizations in the past 3 months. Each participant must be able to understand and provide informed consent.
Exclusion criteria: We will exclude any Fontan patient who has either received or under work up to receive a heart transplant. Patients with contraindications or allergy to colesevelam will be excluded. As colesevelam can affect absorption of fat-soluble vitamins, patients with baseline coagulopathy or vitamin K deficiency will be excluded. We will also exclude individuals with severe liver dysfunction or renal failure requiring dialysis, as these conditions could confound outcome measures or safety.
Healthy control
Inclusion/Exclusion: Healthy control participants must be adults (≥18) with no history of any significant medical conditions. Controls should not be on any medications that could alter bile acid metabolism such as fibrates, bile sequestrants, or probiotics/antibiotics affecting gut flora. They should match the Fontan group’s age (±3 years) and sex distribution as closely as possible, and to achieve approximate matching, we will recruit them after identifying the Fontan cohort characteristics. Controls who are pregnant or nursing will be excluded. The control group will not participate in the randomized cross-over intervention; rather, their data will provide a reference range for various measurements and help distinguish which abnormalities are specific to Fontan physiology. Controls will attend a single study visit for baseline assessments. The study flow is described in Figure 1 .
MYSTIC trial design .
ACHD, adult congenital heart disease; BA, bile acids; CBC, complete blood count, LFT, liver function test; NICaS, non-invasive cardiac system.
Recruitment: Fontan patients will be recruited from the Manitoba Adult Congenital Heart (MACH) clinic at St. Boniface Hospital. The investigative team will work with- cardiologists to identify eligible Fontan patients during routine visits. Interested patients will be provided with study information and consent forms. Healthy controls will be recruited via posted advertisements and word-of-mouth among hospital staff and the local community. Recruitment for controls will occur after most Fontan patients have been enrolled to allow appropriate matching. We aim to enroll 25 Fontan participants and 25 controls.
Study flow: Following baseline assessment, Fontan participants will be randomized to one of the sequences as described here:
Sequence A: colesevelam (6 weeks) → washout (8 weeks) → placebo (6 weeks)
Sequence B: placebo (6 weeks) → washout (8 weeks) → colesevelam (6 weeks)
Randomization is blinded. The 8-week washout is designed to eliminate carryover and allow re-establishment of baseline bile acid and microbiome status.
Randomization will be performed by an authorized member of the research team using a secure, Excel-based system for randomization ensuring allocation concealment.
Intervention and study drug
Colesevelam: The active study drug is colesevelam hydrochloride. We chose a dosing regimen of 3.75 grams per day, which corresponds to 3 tablets (each tablet is 625 mg) twice a day, preferably with food. This dosing is within the typical range used for hyperlipidemia and has demonstrated significant bile acid binding effects. A 6-week treatment duration was selected based on prior pharmacodynamic data indicating that colesevelam can reduce circulating bile acids and induce measurable shifts in gut microbiota within a few weeks. This time frame is considered sufficient to assess short-term safety, tolerability, and biochemical response, aligning with the objectives of this pilot study. Colesevelam tablets will be provided in bottles labeled only with a random kit number. They are advised to swallow tablets whole and not crush or chew them. If a dose is missed, it can be taken later the same day, but double dosing at once is discouraged.
Placebo: The placebo is an inert microcrystalline cellulose pill manufactured to be visually identical to the colesevelam tablets. Placebo tablets will also be taken as 3 tablets twice daily with meals during the placebo phase. Both colesevelam and placebo are packaged in identical bottles. The hospital research pharmacy will prepare sequentially numbered drug kits according to the randomization scheme (each kit containing either active drug or placebo for the 6-week course). Allocation is concealed from participants and investigators.
Compliance: All participants will be encouraged to be compliant after randomization into the trial. Compliance will be encouraged through 2 weekly reminders phone calls and tablet counts. Participants will keep a daily log (diary) of treatment ingestion and be asked to return unused tablets for counting. Participants will continue their standard medications for Fontan management (eg, diuretics, ACE inhibitors, beta-blockers) during the trial.
Washout period: An 8-week washout interval separates the 2 treatment phases. This duration was deemed sufficient for gut microbiome to normalize after colesevelam therapy, as well as for bile acid equilibrium re-establishment, providing a safe margin to eliminate any carry-over effect from the first treatment. Participants are asked to maintain their usual diet throughout the study period.
Study endpoints
At each visit (4 visits in total), we will perform a consistent battery of evaluations to capture the trial endpoints. Figure 1 provides an overview of the primary and secondary endpoints, and the measures used to assess them at various time points.
Primary endpoints:
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1.
Change in Plasma Bile Acid Levels: The primary efficacy endpoint is the reduction in total plasma bile acid concentration with colesevelam relative to placebo. Fasting blood samples for bile acid profiles will be collected at baseline and at the end of each treatment period. Plasma concentrations of total bile acids and individual BA species will be measured using targeted mass spectrometry methods. The primary efficacy analysis will compare the within-subject change in total BA, and individual BA component, from pre to postcolesevelam versus the change pre to postplacebo. We hypothesize that colesevelam therapy will produce a significant decline in BA levels.
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2.
Safety and Tolerability: This includes the incidence of adverse events (AEs) and serious adverse events (SAEs) during colesevelam therapy compared to placebo, as well as gastrointestinal-specific tolerability (eg, reports of constipation, bloating, abdominal discomfort). Tolerability and compliance will also be gauged by study adherence: pill counts, and participant diaries will be used to estimate the percentage of prescribed doses taken. All AEs will be systematically recorded. We will compare the frequency of AEs between the colesevelam and placebo treatment periods to identify any notable imbalances or safety concerns.
Secondary endpoints: A range of secondary outcomes will be assessed to explore the broader impact of BA reduction on Fontan patients:
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1.
Gut microbiome composition and functions: To investigate the gut microbiome’s role, stool samples will be collected from Fontan participants at baseline and at the end of each treatment period (total 4 time points per Fontan participant). We will perform shotgun metagenomic sequencing on these samples to characterize the complete intestinal microbiota composition and functions. Primary analyses will include alpha and beta diversity measures along with microbiota composition and functions (Kraken2, MetaPhlAn4, HUMAnN3). We will compare baseline microbiome profiles of Fontan vs controls to uncover how the composition of the microbiota community and its relevant functions are altered in the Fontan gut. More importantly, within Fontan patients, we will examine microbiome changes in response to colesevelam: we hypothesize that colesevelam reduces bile acid recirculation that in-turn leads to shifts in microbiome.
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2.
Fecal metabolomics and bile acid excretion: Paired with microbiome analysis, stool samples will also be analyzed by TMIC, University of Alberta, using global metabolomics elevated analysis (chemical isotope labeling LC-MS). This includes high metabolome coverage and excellent relative quantification, with measurement of additional metabolite classes such as acids (eg, short-chain fatty acids, bile acids, etc.). By design, colesevelam should increase fecal excretion of bile acids and the impacts on gut microbiome community would influence SCFA and various other metabolites. We will correlate changes in fecal BAs with changes in plasma BAs to quantify the sequestration effect.
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3.
Blood and urine biochemical markers: Beyond bile acids, we will explore a panel of metabolic and biological markers in blood and urine. Fasting blood tests at each major visit will include a comprehensive metabolic panel, such as, but not limited to short chain fatty acids, cytokine– chemokines, oxylipipins, lipidomics, etc. We will also collect plasma and urine for targeted metabolomic analysis. These samples will be stored in −80°C freezer, for batch analysis at a later date.
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4.
Hemodynamic parameters: We will noninvasively measure resting and exercise-challenge hemodynamics using noninvasive cardiac system (NICaS) at baseline and end of each period. Key parameters include stroke index, cardiac index, and cardiac power index. Measurements will be taken in the supine position at rest.
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