Conditions: Progressive Multiple Sclerosis
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 59
Healthy volunteers: no

Brief summary:
This study aims to identify the safety and tolerability of bile acid supplementation in patients with progressive Multiple Sclerosis (MS). Participants will also be assessed for an impact of the bile acid on their immune system and gut microbiome. Half of the participants will receive the bile acid tauroursodeoxycholic acid (TUDCA) and half will receive placebo. The investigators believe participants who take TUDCA will have normalization of blood bile acid levels, a normalization of abnormal immune response and a normalization of the gut microbiome.

Detailed description:

Arms:
arm 1: Tauroursodeoxycholic acid (Taurolite) 250 mg four capsules by mouth, twice daily for 16 weeks. arm 2: Placebo oral capsule four capsules by mouth, twice daily for 16 weeks.

Interventions: intervention 1: Tauroursodeoxycholic Acid intervention 2: Placebo oral capsule
Intervention descriptions: intervention 1: Participants will be given 1 gram of Tauroursodeoxycholic acid twice daily in the form of four 250mg capsules. intervention 2: Participants will be given four capsules of the placebo twice daily.

Protocol document:
Prot_SAP_000.pdf: 
 
Page 1 of 21 
 
A phase 1/2 trial of Tauroursodeoxycholic acid 
supplementation in progressive MS patients 
 
NCT03423121 
 
1/11/2021 


Date: __01/11/2021__________________    
Principal Investigator: __Pavan Bhargava, MD_______________ 
Application Number: __IRB00144766_________ 
 
 
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Version 3 Dated:   06/2007 
 
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JHM IRB - eForm A – Protocol 
 
A phase 1/2 trial of Tauroursodeoxycholic acid supplementation in progressive MS patients 
 
 
*************************************************************************************************** 
 
 
1. 
Abstract 
 
People with multiple sclerosis have abnormalities in multiple metabolic pathways that can be 
identified using metabolomics. The aberrant pathways identified in people with MS, in our 
preliminary studies using untargeted metabolomics, include primary and secondary bile 
acid metabolism. Bile acids have several biological functions beyond aiding in lipid absorption 
in the gut, and can modulate the function of cells in the peripheral immune system and the CNS. 
Bile acid metabolism is also closely linked to the gut microbiota. Bile acid supplementation 
reduced neuroinflammation and was neuroprotective in models of various neurodegenerative 
and neuroinflammatory disorders. Bile acid supplementation has been tested in early trials in 
neurodegenerative disease and has been well tolerated. Whether bile acid supplementation is 
beneficial in MS is not known. 
 
The objective of this study is to test the safety and tolerability of supplementation with a bile 
acid – tauroursodeoxycholic acid (TUDCA) in people with progressive MS. We will also 
assess whether this intervention normalizes the abnormal bile acid levels in circulation and 
impacts the composition of the gut microbiota and peripheral immune cell function. 
 
Aims: 
 
1. Establish the safety of bile acid supplementation and determine whether this intervention 
leads to normalization of the bile acid profile in people with MS with abnormal bile acid 
metabolism  
 
2. Assess the effects of bile acid supplementation on the gut microbiota and the peripheral 
immune system in people with MS 
There is a need for adjunctive treatments that carry low risk and address the underlying 
pathophysiology of MS. This project aims to show that a bile acid – TUDCA is safe and well-
tolerated in MS patients and could result in beneficial effects on bile acid metabolism, gut 
microbiome and peripheral immune cell function in people with MS. If successful, this project 
could yield a new treatment strategy with both immunomodulatory and neuroprotective effects.  
 
2. 
Objectives  
 
Primary Objectives 
Aim 1. Establish the safety and tolerability of bile acid supplementation and determine 
whether this intervention leads to normalization of the bile acid profile in people with 
progressive MS with abnormal bile acid metabolism  


Date: __01/11/2021__________________    
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Hypothesis: Bile acid supplementation is safe and tolerable and will help normalize the bile acid 
profile in people with MS. 
Method: In a randomized, double-blind, placebo-controlled, phase 1/2 study of bile acid 
supplementation in people with progressive MS with abnormal bile acid metabolism, we will 
establish the safety and tolerability of this intervention and evaluate its effects on bile acid 
profiles as determined by targeted metabolomics. 
Aim 2. Assess the effects of bile acid supplementation on the gut microbiota and the 
peripheral immune system in people with MS 
Hypothesis: Bile acid supplementation leads to alterations in the gut microbiota and peripheral 
immune system in people with MS.  
Method: We will determine the change in gut microbiota composition following bile acid 
supplementation and determine changes in peripheral immune cell function in participants 
enrolled in the phase 1/2 trial of bile acid supplementation mentioned in Aim 1.  
 
3. 
Background  
 
Multiple Sclerosis is associated with alterations in multiple metabolic pathways including bile acid 
metabolism. Multiple sclerosis (MS) is a chronic autoimmune disorder 
that leads to inflammation and neurodegeneration involving the central 
nervous system. Abnormalities in metabolic pathways have been 
identified in multiple sclerosis, both in plasma and cerebrospinal 
fluid[1]–[3]. The majority of reports identified alterations in energy 
metabolism or oxidative stress; however, technological limitations 
prevented a large-scale evaluation of the metabolome in MS until 
recently. Now, recent advances allow for the use of global untargeted 
metabolomics to identify hundreds of small molecules in biological 
matrices using gas/liquid chromatography coupled with mass 
spectrometry[4]. We utilized untargeted global metabolomics to 
understand alterations in the metabolome of people with MS and 
identified alterations in multiple metabolic pathways. Both primary and 
secondary bile acid metabolism were identified as metabolic pathways 
that were altered in the MS population (see preliminary data). 
Intriguingly, we noted dysregulation of these pathways in both relapsing 
remitting MS (RRMS) and progressive MS (PMS). While the precise 
mechanism of this alteration is unknown, it may be linked to alterations 
in the gut microbiota or intestinal motility.   
Bile acids have multiple biological functions, and their metabolism 
is closely related to the gut microbiota. Bile acids are primarily 
produced in the liver through the action of multiple enzymes on 
cholesterol[5]. Primary bile acids are further modified by conjugation in 
the liver (with glycine or taurine) and are then stored in the gall bladder. They are secreted into the 
intestine and the majority of bile acids are converted to secondary bile acids and reabsorbed in the ileum 
and colon and transported back to the liver by the portal circulation (enterohepatic circulation)[5],[6]. In 
the gut, bile acids can undergo deconjugation (removal of glycine or taurine) through the action of bile 
salt hydrolases (BSH) found in several bacteria. The process of deconjugation allows further conversion 
Figure 1. Bile acid synthesis and 
metabolism. The two pathways of bile acid 
production and associated enzymes are 
depicted. The role of gut microbiota in 
secondary bile acid production and bile acid 
metabolism is also highlighted (From 
Wahlstrom et al[38]). 


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of primary to secondary bile acids through 7-dehydroxylation. While BSH activity is found in all major 
bacterial and archaeal divisions, The 7-dehydroxylation ability is limited to Clostridium (Cluster XIVa 
& XI) and Eubacterium, both belonging to Firmicuites phylum[7]. A previous study found a significant 
reduction in species belonging to Clostridial clusters XIVa & XI in people with MS[8]. Such an 
alteration could lead to a decrease in the secondary bile acid pool.  
Bile salts have differing 
affinities for the various 
classes of bile acid 
receptors, and hence 
alterations in the 
composition of the bile 
acid pool may have a 
significant impact even 
when the overall bile 
acid pool is unchanged. 
Table 1 lists some of the 
receptors at which bile acids are active and the affinities of individual bile acids at these receptors. The 
action of bile acids at these receptors have been linked to control of various metabolic functions 
including – lipid metabolism, glucose homeostasis and development of obesity[6].  
Bile acid supplementation can alter the gut microbiota in humans and animal models of 
autoimmune disease. A recent study in patients with primary biliary cholangitis noted alterations in the 
gut microbiota in this population. Treatment with ursodeoxycholic acid (UDCA) led to a partial 
restoration of the gut microbial profile to a normal state, suggesting that bile acid supplementation can 
lead to alterations in the composition of the gut microbiome[9]. Another recent study utilizing an animal 
model of inflammatory bowel disease, demonstrated the presence of an altered gut microbiota in mice 
with experimental colitis. UDCA treatment normalized the increased ratio of Firmicutes to 
Bacteroidetes[10]. In this study bile acid supplementation also prevented a decrease in Clostridium 
cluster XIVa which has been noted to be decreased in people with MS[10]. 
Bile acids have immunological functions and can modulate both innate and adaptive immunity. 
The gut is the largest immune organ of the body and alterations in the gut microbiome have a profound 
effect on neuroinflammation[11]. Recent studies in MS have noted alterations in gut microbiota, 
however the metabolic consequences of these alterations have not been elucidated[12],[13]. Metabolites 
produced by gut microbes, including bile acids, can have significant effects on immune cells[14]. Bile 
acids activate receptors on immune cells including the nuclear Farnesoid X receptor (FXR) and cell-
surface G-protein coupled receptors (TGR5). FXR receptors are found predominantly on myeloid cells, 
but are also present on lymphocytes and NK cells[15]. Treatment of mice with experimental 
autoimmune encephalomyelitis (EAE) with a FXR agonist resulted in amelioration of disease, through 
increased production of interleukin-10 (IL-10) from myeloid cells[15]. The TGR5 receptor is also found 
on microglia and myeloid cells and in animal models of neuroinflammation a TGR5 agonist led to a 
reduction in severity and inhibition of myeloid cell activation[16],[17].  
Bile acids are bioactive in the CNS and have neuroprotective effects. Bile acids and their precursors 
have been detected in brain tissue and cerebrospinal fluid[18]. Besides microglia, TGR5 receptors have 
also been described on astrocytes and neurons[19]. The activation of this receptor is associated with 
increased adenylate cyclase activity, increased intracellular Ca2+ and production of reactive oxygen 
species. Bile acids have also been postulated to have effects on mitochondrial function, as well as the 
unfolded protein response. Several studies have demonstrated that secondary bile acids such as UDCA, 
glycoursodeoxycholic acid (GUDCA) and TUDCA may have neuroprotective properties. In a mouse 
Table 1. Classes of bile acid receptors with a listing of bioactive endogenous ligands 


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model of Parkinson’s disease (PD) utilizing MPTP toxicity – TUDCA supplementation mediated 
neuroprotection through modulation of c-Jun N-terminal kinase (JNK) phosphorylation and reactive 
oxygen species production[20]. UDCA supplementation reversed mitochondrial dysfunction noted in the 
fibroblasts from patients with LRRK2 mutations linked to familial PD[21]. Neuroprotection in the retina, 
in various models (retinal detachment, optic nerve injury), has also been demonstrated with bile salt 
treatment[22]–[24]. This effect is thought to be mediated through a reduction in apoptosis and 
endoplasmic reticulum (ER) stress.  
Trials of bile acid supplementation have been conducted in other neurodegenerative conditions 
and have shown good safety profiles and CNS penetration. A recent trial of TUDCA in 34 ALS 
patients randomized to either TUDCA 1gm twice daily or placebo for 54 weeks revealed a higher rate of 
response (defined as slowing in the slope of decline on a standardized rating scale) in the TUDCA 
arm[25]. TUDCA was well tolerated and there were no serious adverse effects in this trial. Two patients 
in the TUDCA arm of the trial had mild diarrhea and none of the patients had any changes in safety 
laboratory parameters that were monitored. A previous trial of UDCA in ALS employed a cross-over 
design and utilized 3.5 gm/day of UDCA for 3 months[26]. This trial found a reduction in the slope of 
decline in one of the three outcomes utilized. There was a high attrition rate related to GI adverse effects 
of UDCA. Another trial of UDCA supplementation in ALS revealed a dose-dependent increase in CSF 
UDCA concentrations, demonstrating good CNS penetration[27]. The European Medicines Agency 
(EMA) recently accorded orphan drug status to TUDCA for the treatment of ALS in Europe. Trials are 
also ongoing in Huntington’s disease and PD (NCT02967250, NCT00514774).  
PRELIMINARY DATA 
People with MS have alterations in their metabolome compared to 
healthy controls. We performed untargeted global metabolomic 
profiling of plasma from people with RRMS and healthy controls (n=27 
in each group) utilizing gas or liquid chromatography coupled with 
tandem mass spectrometry. The mass spectra obtained were compared to 
a reference library to identify individual compounds and the relative 
abundance was calculated using the area under the curve of the mass 
spectra. Using this method, we identified over 600 metabolites in the 
plasma of study participants and were able to differentiate the two 
groups based on their metabolic profiles using a statistical model 
optimized for high-dimensional datasets -  orthogonal partial least 
squares -discriminant analysis (OPLS-DA) (Figure 2). 
People with MS have alterations in both primary and secondary bile 
acid metabolism. In the first discovery cohort described above we noted 
reductions in the levels of multiple primary and secondary bile acids in 
people with MS. To follow-up on our findings, we performed untargeted 
metabolomics in a validation cohort of 50 RRMS, 50 progressive MS 
and 50 healthy controls. In this study we again noted a reduction in multiple bile acids in RRMS and 
PMS groups (Figure 3A). We also investigated alterations in individual metabolic pathways using 
pathway dysregulation scores (calculated as described in Drier et al)[28]. This method generates a score 
for a metabolic pathway for each individual based on their distance from a principal components curve 
constructed based on healthy control data. These scores range from 0 to 1 with higher scores 
representing greater abnormality in the pathway. We found significant alterations in both primary and 
secondary bile acid metabolism in RRMS and PMS groups (Figure 3B & 3C). In this cohort, we also 
noted a correlation of PDS scores for secondary bile acid metabolism with those for xenobiotic 
Figure 2. Metabolomics differentiates 
people with MS from healthy controls. 
The plot depicts the first two 
components from an orthogonal partial 
least squares -discriminant analysis 
(OPLS-DA) model. The two groups can 
be clearly distinguished in this plot. 


Date: __01/11/2021__________________    
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metabolism (Xanthine and Benzoate, p<0.01 for both) indicative of a link between the gut microbiota 
and bile acid metabolism. 
Since pathway analysis utilizes a priori specification of metabolic pathways, we also used an agnostic 
method - weighted correlation network analysis (WGCNA) that identifies modules of highly correlated 
metabolites. We identified a module that contained bile acids and the eigen-metabolite values for this 
module differed significantly between RRMS, PMS and control groups with a reduction noted in bile 
acids in both RRMS and PMS groups (p<0.001 for both).  
 
In animal-models of MS supplementation with bile acids leads to improvement of disease. We then 
tested the hypothesis that supplementation with a bile acid (TUDCA) would alter the course of disease 
in a mouse model of MS - EAE. Seven-week old C57/BL6 mice were immunized with MOG35-55 and 
CFA subcutaneously along with injection of pertussis toxin intraperitoneally (IP). Mice were then 
monitored for development of disease, and at disease onset they were randomized to either TUDCA 500 
mg/kg or placebo (PBS) IP for 3 weeks. At the end of the experiment we noted a significant reduction in 
the severity of disease in the TUDCA treated group (Figure 4A) and a reduction in spinal cord 
demyelination (based on Black-gold staining) and macrophage infiltration (Iba1+ cells) on histology 
(Figure 4B). Recent studies from other groups corroborate our findings; Ho & Steinman demonstrated 
that supplementation with a synthetic bile acid resulted in reduction in severity of EAE[29]. 
Additionally, another independent group demonstrated the ability of a FXR agonist to reduce CNS 
inflammation through an IL-10 mediated pathway[15].  
Figure 3. Bile acid metabolism is 
altered in MS. Panel A shows a heat-
map of ratios of concentrations of 
various bile acids in RRMS and PMS 
groups compared to healthy control. 
The green color denotes levels of 
statistical significance with dark green 
(p<0.05) and light green (p<0.10). 
Panel B and C are dot plots of 
pathway deregulation scores for 
Primary (panel B) and Secondary 
(panel C) bile acid metabolism. In 
both these plots there is a similar 
trend for higher scores (greater 
dysfunction) in both MS groups 
compared to healthy controls.  


Date: __01/11/2021__________________    
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Metabolomics can be used to monitor the effects of therapeutic interventions on the metabolome. 
We utilized metabolomics to assess the effect of vitamin D supplementation on the metabolome in 
people with MS and healthy 
controls. In this study, 24 people 
with MS and 27 controls received 
5000 IU of vitamin D daily for 3 
months. We obtained plasma at 
beginning and end of the study and 
performed metabolomic profiling. 
We utilized WGCNA to identify 
metabolite modules and then 
utilized generalized estimating 
equations (GEE) to determine the 
change in metabolite modules 
following vitamin D treatment. 
We found a reduction in markers 
of oxidative stress and alterations 
in lipid metabolism in healthy 
controls following vitamin D 
supplementation but noted a lack 
of a similar effect in people with MS (Figure 5). This study demonstrated the ability of metabolomics to 
track changes in metabolic pathways following a therapeutic intervention.  
These preliminary data clearly demonstrate that people with MS have altered bile acid metabolism and 
that supplementation of a bile acid (TUDCA) had a beneficial effect in an animal model of MS. 
Additionally, we demonstrate that metabolomics can be used to monitor the effects of a therapeutic 
intervention in MS.  
We hypothesize that bile acid supplementation in people with MS will be safe, well tolerated and lead to 
normalization of the bile acid profile and favorable immune and neuroprotective effects 
 
4. 
Study Procedures 
 
a. Study design, including the sequence and timing of study procedures   
 
This will be a randomized, 
double-blind, placebo 
controlled trial of TUDCA 
1gm twice daily versus placebo 
in patients with progressive 
MS who satisfy the inclusion 
and exclusion criteria.  
A pictorial representation of 
the trial design is shown here. 
 
 
 
 
Figure 5. Metabolomics identifies the effects of vitamin D supplementation of oxidative 
stress and lipid metabolism. Panel A is a circos plot displaying contents of two metabolite 
modules that were altered by vitamin D supplementation – red module containing lipids and 
green module containing oxidative stress related metabolites. Panel B shows change in the 
green module (redox homeostasis) derived from a GEE model with a change noted in healthy 
control group but not in the MS group (p=0.006 for interaction).  


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Study personnel:  
 
Treating physician will assess eligibility criteria, obtain informed consent, obtain and update 
medical history and concomitant medication record, monitor patient safety, conduct physical 
examination, evaluate and report adverse events.  
 
Examining physician will perform EDSS evaluations. 
 
Study coordinator will be responsible for the study’s administrative duties, obtaining vital 
signs, conducting the MSFC, administering the suicidality questionnaire, performing the 
phone check-in, drawing and processing blood samples, obtaining and storing stool samples 
and performing urine pregnancy testing when indicated.  
 
Research technologist will isolate PBMCs from blood samples and will perform the 
immunophenotyping, prepare stool samples for metagenomic sequencing and prepare plasma 
samples for targeted metabolomics analyses.  
 
Study statistician from the Johns Hopkins School of Public Health will create the 
randomization schedule, monitor randomization assignments, generate the data dictionary 
and perform final data analysis. 
 
All personnel except the study statistician will be blinded to treatment assignment.  
 
 
Study visits and procedures: 
 
Study procedures that can be done remotely will be conducted via video visit to minimize 
patient contact and provide scheduling flexibility to participants.  
 
1. Screening visit (-2 weeks)**: At the screening visit participants will undergo the 
following procedures.  
a. Informed consent 
b. Collection of demographic and disease characteristics 
c. Collection of concomitant medication information 
d. Vital signs, physical and neurological examination* 
e. Verification of eligibility based on inclusion/ exclusion criteria 
f. Venipuncture for baseline safety laboratory testing* 
g. MSFC* and MSQOL-54 assessments 
h. Urine pregnancy test* 
i. Suicidality evaluation (C-SSRS) 
 
If the participant is eligible for the trial, they will then return for the baseline visit at which 
time they will be randomized to a treatment group and begin the trial. 
 
2. Baseline visit (0 weeks)**: 
Following procedures will occur at this visit: 
a. Screening laboratory tests will be reviewed to assess whether there is any 
contraindication for continuation in trial 
b. Update concomitant medication information 


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c. Update medical and disease history 
d. Vital signs, physical and neurological information* 
e. Venipuncture for PBMCs and safety laboratory tests* 
f. Stool specimen collection 
g. MSFC*, MSQOL-54 and Food frequency questionnaire assessments 
h. EDSS evaluation* 
i. Urine pregnancy test* 
j. Suicidality evaluation 
 
3. Mid-study visit (8 weeks) 
The following procedures will occur at this study visit. 
a. Assessment of adverse effects 
b. Review safety labs 
c. Update medical and disease history 
d. Update concomitant medication information 
e. Vital signs, physical and neurological examination* 
f. Venipuncture for PBMCs and safety labs* 
g. Stool specimen collection 
h. MSFC*, MSQOL-54 and Food frequency questionnaire assessments 
i. EDSS evaluation* 
j. Urine pregnancy test* 
k. Suicidality evaluation 
 
4. End-of-study visit (16 weeks) 
The following procedures will occur at this study visit. 
a. Assessment of adverse effects 
b. Review of safety labs 
c. Update medical and disease history 
d. Update concomitant medication information 
e. Vital signs, physical and neurological examination* 
f. Venipuncture for PBMCs and safety labs* 
g. Stool specimen collection 
h. MSFC*, MSQOL-54 and Food frequency questionnaire assessments 
i. EDSS evaluation* 
j. Urine pregnancy test* 
k. Suicidality evaluation 
 
*Must be done at in-person portion of visit 
 
** Due to the COVID-19 pandemic and to make it more convenient for the patients to 
participate, we decided to offer the option of three visits instead of four as an alternative. In 
this case, we will perform the research blood draw and stool sample at the screening visit, 
along with providing patients with medicine. We will ask patients not to start taking 
medicine until we receive the screening lab results and let them know whether they are 
eligible and can start the medication. 
 


Date: __01/11/2021__________________    
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Study procedure 
Screening 
 
Baseline  
Week 0 
Week 8 
 
Week 16 
 Written consent 
X 
Verify eligibility 
X 
Medical history, relapse assessment 
X 
X 
X 
X 
Medication review (also antibiotic and probiotic use) 
X 
X 
X 
X 
Vital signs, physical, neurologic (EDSS) exams* 
X 
X 
X 
X 
MS Functional Composite* & MSQOL-54 
X 
X 
X 
X 
Adverse event assessment 
X 
X 
X 
Food frequency questionnaire 
 
X 
X 
X 
Laboratory testing (CBC, CMP, HbA1c, Lipid profile, pregnancy test)* 
X 
X 
X 
X 
Blood for PBMC isolation* & Stool specimen collection 
 
X 
X 
X 
 Suicidality evaluation (C-SSRS) 
X 
X 
X 
X 
 
*Must be done at in-person portion of visit 
 
b. Study duration and number of study visits required of research participants. 
 
Study duration: 16 weeks.  
 
Number of study visits: 4 
 
Study visits 
1. Screening 
2. Baseline 
3. Mid-study visit 
4. End-of-study visit 
5. Unscheduled visit will occur if participant develops adverse events or new symptoms 
 
 
c. Blinding, including justification for blinding or not blinding the trial, if applicable. 
 
In this trial all study personnel and patients will be blinded to reduce any biased assessment 
of study outcomes including adverse effects related to the treatment as well as the included 
efficacy end-points (EDSS, MSFC, MSQOL-54), some of which are subjective and could 
thus be influenced by prior knowledge of treatment assignment.  
 
 
d. Justification of why participants will not receive routine care or will have current 
therapy stopped. 
 
Participants will continue to receive routine care for their multiple sclerosis. 
 
 


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e. Justification for inclusion of a placebo or non-treatment group. 
 
While this trial is primarily focused on establishing the safety and tolerability of TUDCA in 
patients with progressive MS, the inclusion of a placebo group will enable us to determine 
whether changes observed in serum bile acid profiles, gut microbiota, immunophenotype and 
clinical/ quality of life measures are related to TUDCA treatment. This would provide 
information regarding the utility of pursuing larger trials focused on clinical outcomes in this 
patient population. 
 
 
f. Definition of treatment failure or participant removal criteria. 
 
Patients selected for the study will be removed if they:  
1) They develop abnormal safety labs as defined below 
2) Develop a serious adverse event 
3) Inform us that they became pregnant during the study 
4) Are unable to follow the requirement of the study 
5) Request to be removed 
 
 
g. Description of what happens to participants receiving therapy when study ends or if a 
participant’s participation in the study ends prematurely. 
 
The subject’s medical care will continue unaltered when the study ends or if a subject is 
removed from the study. 
 
5. 
Inclusion/Exclusion Criteria 
 
The inclusion and exclusion criteria for participants in this trial are listed in the table below. 
 
Eligibility criterion 
Rationale 
Diagnosis of Progressive MS based on Lublin 
criteria 
We are focusing on people with PMS since they are 
more likely to have low bile acid levels 
Age ≥ 18 years 
To include only adults in the trial 
On the same therapy for the past 6 months and not 
expected to switch therapy in the next 6 months 
To avoid confounding by changes in 
immunomodulatory therapy during the trial 
No relapse in the past 3 months 
To avoid any confounding introduced by relapse 
Exclusion criterion 
Rationale 
No previous history of liver disease 
Confounding due to decreased bile acid production or 
altered bile acid metabolism 
No stage IV/V chronic kidney disease or other 
severe metabolic derangements 
To reduce risk of adverse effects or confounding due to 
other systemic disorders 
BMI < 15 kg/m2 and BMI > 40 kg/m2 
Reduce confounding due to extremes of BMI 
Female patients who are pregnant or nursing, or not Prevent potential teratogenicity  


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willing to use contraception 
Chronic antibiotic use 
Prevent confounding of microbiome analyses 
Corticosteroid treatment within the past 30 days 
Prevent confounding of treatment effect 
Known history of other neuroinflammatory, 
neurodegenerative or systemic autoimmune disease 
These diseases could require other treatments or be 
affected by the trial medication. 
No current or prior history of the following –  
• 
Bariatric surgery 
• 
Small intestinal surgery resulting in loss of 
length of the jejunum or ileum 
• 
Chronic diseases of the small intestine 
(e.g., Crohn’s Disease, gluten sensitive 
enteropathy, or other disorders that could 
lead to malabsorption) 
• 
Abnormal bile duct anatomy 
• 
Sclerosing cholangitis 
• 
Anti-mitochondrial antibodies 
 
These conditions could alter bile acid handling in the 
gut or bile acid levels in circulation 
 
 
 
6. 
Drugs/ Substances/ Devices 
 
a. The rationale for choosing the drug and dose or for choosing the device to be used. 
Tauroursodeoxycholic acid (TUDCA) is a hydrophilic bile acid that is normally produced 
endogenously in humans in the liver, by conjugation of taurine to UDCA. It is used in Europe to 
treat cholestatic liver disease and gallstones. TUDCA has been used in trials for liver cirrhosis, 
primary biliary cirrhosis, transthyretin related amyloidosis, cholesterol gallstones, diabetes, and 
ALS[30]–[33], with a good safety profile. Diarrhea was the most common side effect noted in 
these studies, which utilized varied doses of TUDCA.  
As noted in the background section, we chose TUDCA as an intervention since it has been 
previously demonstrated to be safe and have several beneficial effects (on ER stress, 
mitochondrial dysfunction, immunological effects and neuroprotection) that could potentially 
lead to benefits in patients with MS. Additionally since TUDCA can change the circulating bile 
acid pool this could potentially help correct this metabolic abnormality that we have identified in 
our preliminary studies. 
We will obtain TUDCA from Bruschettini S.r.l (Genoa, Italy). This medication is manufactured 
as 250 mg capsules and hence participants will take 4 capsules two times a day of either TUDCA 
or placebo. The study medication will be provided through the Johns Hopkins investigational 
drug services and will appear identical to maintain blinding. TUDCA is approved in Italy for the 
treatment of cholestasis and gallstones. It was recently accorded orphan drug status by the 
European Medicines Agency for the treatment of Amyotrophic lateral sclerosis. 
Dose: We chose a dose of 1 gm BID based on the trial of TUDCA in ALS that was recently 
completed across three centers in Italy[25]. Participants in this trial had no significant adverse 


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events on TUDCA, and there appeared to be some evidence for a benefit in this early phase 
study. This dose is higher than the dose utilized in treatment of cholestatic disease and is close to 
the 30 mg/kg dose utilized in a trial of UDCA in ALS that was well tolerated and showed no 
significant difference in serum and CSF concentrations compared to a higher dose of 50 
mg/kg[27],[30]. 
Pharmacokinetics of TUDCA: Previous studies have demonstrated that about 65% of the oral 
dose of TUDCA is absorbed and then undergoes first pass metabolism in the liver followed by 
extensive enterohepatic circulation[34]. Administration of bile acids such as UDCA and TUDCA 
results in a change in the overall serum bile acid concentration – between 1.9 – 8 fold and alters 
the composition of the bile acid pool[35],[36]. A trial of UDCA in ALS also demonstrated dose-
dependent increased in CSF concentrations of the bile acid[27]. 
 
 
b. Justification and safety information if FDA approved drugs will be administered for 
non-FDA approved indications or if doses or routes of administration or participant 
populations are changed. 
 
N/A 
 
c. Justification and safety information if non-FDA approved drugs without an IND will be 
administered.  
 
We are using a non-FDA approved drug, however this will be utilized either with an IND or 
with an IND waiver from the FDA. 
 
 
7. 
Study Statistics 
 
a. Primary outcome variable. 
The primary outcome of this study is the safety and tolerability of TUDCA supplementation. 
This will be based on number of adverse events occurring in the two treatment groups and 
the rate of drop-out in each study arm. This data will be documented at each study visit. 
 
b. Secondary outcome variables. 
 
1. Metabolomics – We will store plasma before and after the study intervention to assess the 
effect of the study medication on fasting bile acid levels. This will enable us to determine 
whether the study drug raised the levels of bile acids that were deficient in people with MS. 
Targeted metabolomics analyses will be performed through Metabolon Inc.  
2. Gut microbiota –The composition of the gut microbiota is closely related to bile acid 
metabolism, and alterations in bile acid receptor (FXR) signaling can significantly affect 
composition of the gut microbiota[37],[38]. We will collect first morning stool specimens 
prior to and following study treatment to determine the effect of bile acid supplementation on 
the gut microbiota in people with MS. 
3. Immunophenotyping – We will store PBMCs from before and after the study intervention to 
assess the effect of TUDCA supplementation on the peripheral immune system. Our 
laboratory has expertise in immunophenotyping people with MS and assessing the effect of 


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therapeutic interventions on peripheral immune cell function. These assays will be performed 
at the end of the study. 
4. Other outcomes – We will also collect information regarding quality of life using the 
MSQOL-54 instrument[39] administered at the visits noted in the table above. We will also 
measure lipid profile and hemoglobin A1c at baseline and end-of-study, since bile acid 
metabolism is linked to obesity and metabolic syndrome. 
 
Assessment of important covariates: We will collect demographic information including age, 
gender, cigarette smoking, waist circumference, and BMI which can affect circulating bile 
acid levels. Another critical factor that can affect bile acid metabolism and the gut microbiota 
is diet. Since TUDCA could potentially lead to GI side effects, this may result in a change in 
diet. To monitor changes in diet we will perform the Block Food frequency questionnaire 
before and after the study intervention48. Since antibiotics and probiotics can lead to changes 
in the gut microbiome and could also affect bile acid metabolism, we will collect detailed 
information on the use of both these covariates so they can be adjusted for in our final 
analyses. 
 
c. Statistical plan including sample size justification and interim data analysis. 
 
Safety data will be analyzed as they are captured in real time. Assessments will focus on AEs 
(including study treatment tolerability assessments, laboratory evaluations, vital signs and 
physical examination). The incidence rate of AEs will be recorded by system organ class, 
severity and by relationship to study treatment. Tolerability analysis will be based on the 
number (%) of participants who failed to complete the study due to adverse events. Lab 
values for each parameter will be summarized by shift tables. For quantitative parameters, we 
will present summary statistics for actual values and change from baseline. We will compare 
changes over the 16-week treatment period between the groups using mixed effects 
regression analyses to account for repeated measures using random subject-specific 
intercepts and slopes. 
 
Metabolomics data will be analyzed at the end of the study following quantitation of the 15 
bile acids included in our panel. Summary statistics will be used to characterize these 
outcomes. We will then assess normality of these measures and perform appropriate 
univariate testing to determine which bile acids are significantly altered by TUDCA 
supplementation. A mixed effects longitudinal model will also be used to test which bile 
acids are altered between visits across the two groups. Models will also adjust for important 
covariates including BMI, age, gender and measures derived from the food frequency 
questionnaire.  
 
Gut microbiota data, a priori analyses will assess changes in relative abundances of different 
gut bacteria as well as microbial genes that encode bile acid metabolizing enzymes 
(identified from the literature and NCBI Gene database) which occur as a result of TUDCA 
treatment. Exploratory analysis will assess the effects of TUDCA on the metagenome using a 
combination of pathway and set-based approaches previously applied in metagenomics 
analysis of MS and other diseases to identify other biologically relevant changes[41]–[43].  
 


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Immunophenotyping data, we will summarize data using summary statistics (mean and 
standard deviation), before and after study treatment. We will utilize paired t tests to compare 
proportions of various immune cell sub-populations at different time points. For both sets of 
data we will utilize mixed-effects regression models to assess the change in bacterial taxa/ 
metagenomics pathways/ immune cell subsets over time, and determine their relationship to 
changes in bile acid concentrations with TUDCA supplementation.   
 
Sample size justification 
Since this is primarily a safety study we based our sample size on the ability to detect 
changes in our secondary outcomes, especially metabolomics. Previous studies of 
supplementation of UDCA and TUDCA have resulted in alterations in the total serum bile 
acid levels (2-8 fold change) and the composition of the bile acid pool. Based on our 
preliminary data, to detect a 50 - 70% change in levels of an individual bile acid, with a 
power of 85% and two-tailed alpha of 0.05 the required sample size would be between 14 – 
27 in each arm. Accounting for a 10% drop-out rate, we will aim to recruit 30 participants in 
each arm of the trial. We have previously noted significant alterations in metabolic profiles in 
MS patients treated with vitamin D and dimethyl fumarate in studies utilizing between 18-24 
patients (as shown in preliminary data). 
From a safety stand-point, a sample size of 30 would enable us to conclude with 80% 
confidence that the rate of occurrence of serious adverse events with TUDCA treatment was 
less than 5% if none of the patients experienced a serious adverse event during the study[40].   
 
 
d. Early stopping rules. 
 
We will discontinue treatment if a participant develops abnormal safety labs (serum AST or 
ALT > 3 x upper limit of normal, serum bilirubin > 3 x upper limit of normal, eGFR < 
60ml/min/1.73m2), becomes pregnant during the study, cannot tolerate the drug, develops an 
adverse event grade 3 or higher that is possibly related to study treatment or wants to 
discontinue treatment. Any death that is at least possibly related to the study will put the 
study on hold.  The Data and Safety Monitoring Board will determine if it is safe to resume 
the study.  Subjects who become suicidal during the study (codes 1-4, 
http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidance
s/UCM225130.pdf) will be referred to a psychiatrist or to a psychiatric emergency room for 
evaluation at the discretion of the site treating physician. 
 
 
Data and Safety Monitoring Board (DSMB): An independent DSMB will be utilized for 
subject monitoring. The DSMB will consist of a neurologist, a hepatologist and a statistician. 
The DSMB will meet every 12 weeks and any time an SAE occurs. All SAEs will be 
reported to the DSMB delegates within 24 hours of the study staff becoming aware of the 
SAE. The DSMB will be required to review SAEs and respond with recommendations within 
two business days. If more than 3 subjects experience serious adverse events, the study will 
be put on hold and the DSMB will decide on whether the adverse events were secondary to 
study treatment. They will make a judgement as to whether the study should continue and, if 
so, whether a reduction in TUDCA dosing or other protocol modifications are recommended.  
 
 


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8. 
Risks 
 
a. Medical risks, listing all procedures, their major and minor risks and expected 
frequency. 
 
TUDCA treatment: In previous studies of TUDCA and UDCA – adverse effects include – 
mainly GI related effects – diarrhea, constipation, gas, nausea, vomiting, abdominal pain and 
anorexia, other adverse effects which are less frequent (< 1% each) include – headache, rash 
and pruritus [25],[27],[30].  
While no serious adverse effects have been note in previous studies, several of the described 
AEs could lead to issues with tolerability in MS patients especially those with bowel function 
abnormalities.  
In a recent trial of TUDCA 1 gm twice daily in patients with Amyotrophic lateral sclerosis 
there were no serious adverse effects.  
 
Venipuncture: The potential risks include discomfort, bleeding or bruising at site of blood 
draw, and in rare cases fainting or infection. 
 
Clinical examination (including EDSS, MSFC): There are no significant risks of this 
procedure. 
 
Stool specimen collection: There are no significant risks of this procedure. 
 
b. Steps taken to minimize the risks. 
 
TUDCA treatment: We are utilizing a dose of TUDCA that has recently been shown to be 
safe in a trial in another chronic neurological disorder. We are also monitoring safety labs 
and conducting neurological evaluations every 8 weeks to monitor for any adverse effects 
that may be related to the treatment.  
 
Venipuncture: Blood will be drawn by a trained study team member. 
 
 
c. Plan for reporting unanticipated problems or study deviations. 
 
Unexpected Adverse Event: An adverse event is considered unexpected when its nature or 
severity is not consistent with the product information (e.g. package insert safety 
information, the investigational plan, the investigator’s brochure, the protocol, or the 
informed consent form).  


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Serious Adverse Event: A serious adverse event (any adverse event that suggests a 
significant hazard, contraindication, side effect, or precaution) must be reported. This 
includes, but may not be limited to, death, a life-threatening event, inpatient hospitalization 
or prolongation of existing hospitalization, persistent or significant disability/ incapacity, 
congenital anomaly/ birth defect, other protocol-specified conditions, or an event that 
requires intervention to prevent permanent impairment or damage.  Serious adverse events 
will be collected from informed consent signing until 30 days after study completion or until 
30 days after a participant withdraws from the study. 
 
Grading of Adverse Events 
We will use the National Cancer Institute’s Common Terminology Criteria for Adverse 
Events (CTCAE) version 4.0 to report and grade all adverse events, whether or not they are 
related to disease progression or treatment.  The relationship between an adverse event and 
the study drug will be determined by the site investigator and recorded on the appropriate 
form.  
   
Serious Adverse Event Reporting The following process for reporting a serious adverse event 
will ensure compliance with the International Conference on Harmonisation guidelines: 
1. 
The Institutional Review Board will be notified in one business day of a serious 
adverse event. 
2. 
Standard reporting will occur if the event is serious, expected and drug-related, 
serious, expected and not drug-related, or serious, unexpected and not drug-related. 
3. 
Expedited reporting is required if the event is serious, unexpected and drug-related. 
This type of event must be reported to the appropriate authorities within 15 days 
unless it is fatal or life-threatening; a fatal or life-threatening event must be reported 
within 7 days.  
   
Pregnancy Reporting Requirements: Any pregnancy that occurs during a clinical study with 
an investigational drug will be reported to the IRB and DSMB, and pregnancies will be 
followed to their conclusion.  Female participants should immediately inform the investigator 
of pregnancies and will stop taking the study medications. The investigator will report 
pregnancies to the Institutional Review Board and DSMB within one business day. The 
investigator will counsel the participant about the risks of continuing the pregnancy 
 
 
d. Legal risks such as the risks that would be associated with breach of confidentiality. 
 
Each participant will be assigned a study identification number, and the data key will be kept 
in a separate, secure location. Electronic records will be stored on a secure, password-
protected network. Any paper records will be kept in a locked file cabinet inside a locked 
office. These measures should prevent a breach of privacy.  The consequences should be 
minimal, as the subjects with MS will have already established care and carried the diagnosis 
thereof.  There should be no social stigmata for the participants. 
 
 
e. Financial risks to the participants. 
 
There are no significant financial risks to participants from this study. 


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9. 
Benefits 
a. 
Description of the probable benefits for the participant and for society. 
 
There are no individual medical benefits for participating in this study. As there is no 
curative treatment for MS, there is a glaring need for more knowledge and more effective 
treatment approaches for individuals with progressive MS. This study has the potential to 
benefit progressive MS patients in the future. 
 
 
 
10. 
Payment and Remuneration 
a. 
Detail compensation for participants including possible total compensation, 
proposed bonus, and any proposed reductions or penalties for not completing the 
protocol. 
 
No compensation will be provided to subjects for participating in this trial. 
 
 
11. 
Costs 
a. 
Detail costs of study procedure(s) or drug (s) or substance(s) to participants and 
identify who will pay for them. 
 
The subjects will not be charged for any of the procedures in the study. They will only be 
responsible for the cost of any travel to and from the Hospital as well as any meals they 
eat while participating. The costs of parking at the Hospital garage will be covered. 
 
 
 
 
 
 
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