Conjugated Cholic Acid for the Treatment of Inborn Errors in Bile Acid Synthesis Involving Side-Chain Conjugation
Phase: PHASE3
Conditions: Bile Acid Synthesis Defect, Inborn Error of Bile Acid Metabolism, Inborn Error of Bile Acid Conjugation
Sponsor: Children's Hospital Medical Center, Cincinnati

 
 
 
 
     Protocol Updated: 31Jan2018   
 
 
 
 
 
 
CONJUGATED CHOLIC ACID FOR THE  
TREATMENT OF INBORN ERRORS 
 IN BILE ACID SYNTHESIS INVOLVING  
SIDE-CHAIN CONJUGATION 
 
 
NCT01589523 
 
 
Date:  31Jan2018 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
     Protocol Updated: 31Jan2018   
 
 
 
 
 
CONJUGATED CHOLIC ACID FOR THE TREATMENT OF INBORN ERRORS IN 
BILE ACID SYNTHESIS INVOLVING SIDE-CHAIN CONJUGATION 
 
 
Date:  31Jan2018  
 
To:  
Institutional Review Board on Studies in Human Beings and the Scientific Advisory 
Committee, GCRC 
 
Cincinnati Children’s Hospital Medical Center 
 
From: Kenneth D.R. Setchell, Ph.D., Division of Pediatric Pathology and James E. Heubi, 
M.D./ Division of Gastroenterology, Hepatology and Nutrition, Co Principal 
Investigators 
Kevin Bove.M.D., Division of Pediatric Pathology, Coinvestigator 
 
Nadine Brown, Ph.D., Division of Pediatric Pathology 
 
Abstract 
Inborn errors of bile acid metabolism have been established as a well recognized cause 
of neonatal cholestasis and fat-soluble vitamin malabsorption.  Although there is 
extensive experience with metabolic defects in the biosynthetic pathway, few patients 
have identified with defects in conjugation with taurine or glycine that allows bile acids to 
become effective detergents.  This proposal is designed to study the effect of defects of 
conjugation of bile acids on growth and fat-soluble vitamin malabsorption.  Study subjects 
will have liver function studies performed, serum and urinary bile acid measurements, 
vitamin levels, growth measurements, bile acid pool size measurements made by stable 
isotope dilution mass-spectrometry, and measurements of absorption of two fat-soluble 
vitamins, tocopherol and vitamin D. Subjects will be treated orally with conjugates of 
cholic acid with follow-up laboratories performed as an outpatient and then subjects will 
have all of the initial studies repeated during an inpatient stay 3-12 months after starting 
treatment.    
 
I. Purpose/Specific Aims 
 
The purpose of this proposal is to define the pathophysiology of these newly 
described genetic defects of bile acid biosynthesis related to impaired conjugation of bile 
acids, and to examine the effect of oral bile acid therapy with conjugated bile acids on the 
clinical course of these disorders.  Specifically, we propose to address the following 
interrelated hypotheses: 
 
1. 
Unique alterations in bile acid metabolism, specifically affecting the 
conjugation of primary bile acids during early life, lead to reduced intraluminal bile 
acids concentrations and associated reductions in absorption of fats and fat-
soluble vitamins.   

 
 
 
 
     Protocol Updated: 31Jan2018   
 
2.  
Treatment of affected infants/children with conjugated bile acids will lead to 
resolution of liver disease, if present, and improvements in fat and fat-soluble 
vitamin absorption  
 
In order to address the above interrelated hypotheses we will perform the following: 
1.  We will determine the extent of alteration in biliary bile acid composition and 
measure the primary bile acid pool sizes by the administration of stable-isotope 
labeled primary bile acids, and correlate these with the concentration of 
unconjugated bile acids and with markers of liver dysfunction. 
2.  We will administer the conjugate of the primary bile acid, glycocholic acid, to 
patients with disorders in bile acid conjugation, monitor its metabolic fate and 
determine the effect of bile acid therapy on biliary bile acid composition and it ability 
to correct the clinical manifestation of fat and fat-soluble vitamin malabsorption.  
3.  Where liver dysfunction is present at diagnosis and liver biopsies have been 
obtained during the evaluation of the patient, a single follow-up liver biopsy will be 
performed after treatment to assess the effect of therapy on liver histology and 
ultrastructural pathology.  
 
 
II. Significance of the study in relation to human health 
Liver disease in infancy has many definable causes, including known infections, 
anatomic defects, well delineated metabolic errors, and inherited or acquired defects in 
hepatobiliary structure and function.  However, "idiopathic" neonatal hepatitis syndromes 
make up by far the largest proportion (35-40%) of cases of liver disease in neonates and 
children [1].  The frequent pattern of intrafamilial recurrence for many of these cases 
suggested to us a genetic basis, and this was subsequently substantiated by our 
description some years ago of two inborn errors in bile acid synthesis presenting as 
progressive cholestatic conditions.  More recently we have described three additional 
unique defects in bile acid synthesis. 
 
 
Because of the essential and multiple roles that bile acids play in gastrointestinal 
physiology, it was our contention that specific defects in the conversion of cholesterol into 
the primary bile acids, cholic and chenodeoxycholic acids, contribute to or are causal in 
the pathogenesis of idiopathic cholestasis.  Such defects would lead to an overproduction 
of potentially hepatotoxic "atypical" bile acids that would be synthesized from 
intermediates accumulating proximal to the enzyme defect, exacerbated by inadequate 
production of the primary bile acids that are essential for promoting bile-flow, and 
consequently would be expected to present as progressive cholestatic conditions. 
 
The two "primary" bile acids synthesized by the human liver, cholic and 
chenodeoxycholic acids, serve a number of important physiological functions.  In addition 
to facilitating fat absorption from the gastrointestinal tract, primary bile acids provide the 
major driving force for the promotion and secretion of bile [2] and are therefore essential 
to the maintenance of a normal enterohepatic circulation.  The pathways for bile acid 
synthesis from cholesterol are complex [3].  At least 15 enzymes, located within various 
subcellular fractions of the hepatocyte, participate in bile acid synthesis [4, 5].  Prior to 
initiating this research program, only one primary enzyme defect in bile acid synthesis 
was known, and this was the rare lipid-storage disease of Cerebrotendinous 
Xanthomatosis, shown to be due to point mutations in the gene encoding the cholesterol 

 
 
 
 
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C-27 hydroxylase enzyme, a condition that until recently was not associated with liver 
disease.   Seven genetic defects involving enzymes in the pathway for bile acid synthesis 
from cholesterol are now known, six of which were discovered by the Principal 
Investigator and coworkers [6,7,8 and unpublished data].  These disorders are manifest 
as progressive cholestatic conditions and indicate that inborn errors in bile acid synthesis 
should now be classified as a new category of metabolic liver disease.  As a 
consequence, our laboratory has become an important international resource for 
screening for these defects and referral of affected patients. 
 
These conditions were identified as a result of our application of the technique of 
liquid secondary ionization mass spectrometry (LSIMS, formerly FAB-MS), which affords 
definitive identification of derangements in bile acid synthesis, and consequently, 
identification of genetic defects in bile acid synthesis.   By this approach the first two new 
defects in bile acid synthesis, manifest as neonatal and late-onset chronic cholestasis, 
were discovered in infants and children and later inborn errors in bile acid synthesis 
became associated with unexplained fat-soluble vitamin malabsorption syndromes in 
some patients  
 
(a) 
3-Hydroxy-C27-steroid dehydrogenase/isomerase deficiency  [6]  
(b) 
4-3-Oxosteroid 5-reductase deficiency [7]  
(c) 
2-Methylacyl CoA racemase deficiency (8), accounting for chronic liver disease 
in a 3 week old infant, initially presenting as fat-soluble vitamin malabsorption.  
(d) 
Oxysterol-7-hydroxylase presenting as severe and fatal liver disease in infancy 
(e) 
Sterol 27-hydroxylase deficiency, accounting for transient cholestatic liver 
disease in early life and later presenting as the rare lipid-storage disease of 
cerebrotendinous xanthomatosis 
(f)  
Defects of bile acid conjugation (see Preliminary Results) 
(g)  
Cholesterol 7-hydroxylase deficiency described by Pugelli et al and presenting 
in adults as a condition of dyslipidemia that is unresponsive to statin treatment 
 
 
In addition to the above defects, bile acid synthesis is severely compromised in 
conditions in which there are abnormalities in peroxisomal assembly, structure, or 
function, because the peroxisome packages key enzymes responsible for -oxidation of 
the side-chain of cholestanoic acid intermediates in the pathway for primary bile acid 
synthesis.  These peroxisomopathies, particularly when severe, as in Zellweger’s 
syndrome, present with progressive cholestatic liver disease. Peroxisomal disorders can 
be subdivided into several groups [9, 10].  In disorders of peroxisome biogenesis, the 
organelle is not formed normally and multiple peroxisomal functions are defective.  
Included in this category are the Zellweger syndrome, neonatal adrenoleukodystrophy, 
infantile Refsum syndrome, and hyperpipecolic acidemia.  A second group is 
characterized by a single enzyme defect, without disturbance of peroxisomal structure or 
other peroxisomal functions. In all of the above described conditions, primary bile acid 
synthesis is markedly reduced or absent.  Instead, the liver of these patients synthesizes 
increased amounts of the intermediates in the pathway proximal to the enzyme defect, 
and these are subsequently metabolized to bile acids retaining the nuclear structure of 
the substrates for the missing enzyme.  We have speculated that the cholestasis and liver 
disease result from the lack of primary bile acids that are essential for promoting bile-flow 

 
 
 
 
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and/or the accumulation of atypical bile acids that are potentially cholestatic and 
hepatotoxic.  
 
III. 
Previous Work done in the Area By our Group 
 
3-Hydroxy-C27-steroid dehydrogenase/isomerase deficiency 
 
We first described this defect in 1987 [6], but since then it has been recognized in 
>25 patients. Since 1986, 2 subjects have been studied on the GCRC at Children’s 
Hospital and an additional 6 subjects have been investigated by KDRS/JEH in Paris. The 
condition is fatal if not diagnosed at an early enough stage to permit therapeutic 
intervention with primary bile acids. As a result of our efforts at least three children have 
avoided the need for liver transplantation (all were on transplant waiting lists) and have 
seen remarkable resolution of liver histology and normalization of serum liver enzymes.  
This enzyme defect involves the second step in primary bile acid synthesis from 
cholesterol. Age at onset and preliminary diagnosis have been variable, ranging from 3 
mos - 15 yrs, confirming that cases of late-onset chronic cholestasis can be explained by 
inborn errors in bile acid synthesis [11]. Interestingly, this inborn error is frequently but not 
exclusively associated with an elevated serum bilirubin and transaminases, or a normal 
serum gamma-GT concentration, and this biochemical picture is  often a useful clinical 
marker for the defect [12, 13].  Furthermore, serum bile acid concentrations are normal 
when measure by conventional laboratory methods and seemingly incompatible with the 
extent of cholestasis.  In many of the patients, the early presentation of the disease was 
unremarkable with normal liver function tests, save degree of fat malabsorption. We 
recently have published a mutational analysis on a cohort of subjects with this defect (14) 
 
4-3-Oxosteroid 5 beta-reductase deficiency 
 
Soon 
after 
the 
identification 
of 
the 
3-hydroxy-C27-steroid 
dehydrogenase/isomerase deficiency, we described a second inborn error in bile acid 
synthesis involving nuclear modification, also manifest as progressive neonatal liver 
disease [7].  This defect involved a deficiency of the cytosolic 4-3-oxosteroid 5-
reductase enzyme responsible for the catalytic conversion of 7-hydroxy- and 7,12-
dihydroxy-4-cholesten-3-one into the corresponding 3-oxo-5(H) analogues [15]. We 
first reported the defect in monochorionic twin boys born from our GCRC with a marked 
and progressive cholestasis [7].  A previous sibling born with neonatal hepatitis had died 
of liver failure following a similar clinical course and the parents subsequently gave birth 
to a fourth affected boy and later a healthy girl. Thus far, 20 patients have been identified 
with this disorder, including four infants in which a prior diagnosis of neonatal 
hemochromatosis was made [16].  In all of these cases, liver function tests revealed an 
elevation in serum transaminases, a marked hyperbilirubinemia and coagulopathy.  
Unlike the 3-hydroxy-C27-steroid dehydrogenase/isomerase deficiency, this defect is 
not commonly associated with a normal serum GGT concentration. 
 
Liver biopsies from these patients showed marked lobular disarray as a result of 
giant-cell and pseudoacinar transformation of hepatocytes, hepatocellular and canalicular 
bile stasis and extrahepatic medullary hematopoiesis.  Electron micrographs showed 
small, slit-like bile canaliculi, lacking the usual microvilli and containing variable amounts 

 
 
 
 
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of electron-dense material [17,18, 19].  Since primary bile acids are involved in the 
canalicular transport of iron [20,21] it is uncertain whether the iron storage defect is 
secondary to the bile acid inborn error, or vice-versa.  In contrast to the previously 
described defect, the4-3-oxosteroid 5-reductase deficiency presents early in life and in 
most of the cases identified, the diagnosis has been made too late to permit initiation of 
bile acid therapy.  
 
 
 
2- Methylacyl-CoA Racemase Deficiency 
 
We described a new defect in bile acid synthesis in a 3-week old infant who 
presented with fat-soluble vitamin deficiency and a mild elevation in serum 
transaminases. The patient, who appeared normal, was investigated because of the 
death of a sibling a year earlier from an intracranial hemorrhage due to massive vitamin 
K deficiency.  Analysis of the urine by mass spectrometry revealed a virtual lack of primary 
bile acids, and in their place the liver was synthesizing large quantities of cholestanoic 
acids.  These unusual C27 bile acids are typical of bile acids found in the alligator. While 
the urine and bile of this patient showed striking similarities in bile acid synthesis to that 
found in patients with the cerebrohepatorenal syndrome of Zellweger, however, clinically 
this patient had none of the features or symptoms of peroxisomopathies, and liver biopsy 
indicated the presence of normal peroxisomes.  
 
Remarkable in this case was the fact that the liver from the earlier deceased 
sibling, presumed to have the same defect, was transplanted and the recipient 
experienced problems absorbing cyclosporin; this was corrected with bile acid therapy. 
Analysis of urine collected from the recipient confirmed the same genetic defect in the 
transplanted liver and this was consequently a unique example of transplantation of a 
metabolic defect.  In collaboration with investigators at Kennedy Kreiger Institute we 
characterized this child and his sibling’s defect to be a deficiency of 2-methylacyl-CoA 
racemase. Because of reports of adults with this defect with neurologic disease, we are 
watching her closely and treating her with cholic acid and a phytate reduced diet (22). 
 
 
Bile Acid Synthesis in Peroxisomal Disorders 
 
Elevated levels of DHCA, THCA and a C29-dicarboxylic bile acid in biological fluids 
are a consistent feature of Zellweger syndrome, neonatal adrenoleukodystrophy, infantile 
Refsum's disease and pseudo-Zellweger syndrome [23-26].  Although bile acid synthetic 
rates are low in these patients [27,28], normal or increased serum concentrations of 
primary bile acids are frequently found which are a consequence of impaired hepatic 
function.  Additional metabolism of THCA by microsomal hydroxylation in the side-chain 
(to produce C-24 hydroxylated varanic acid isomers) and the nucleus (to produce C-1 
and C-6 tetrahydroxy-cholestanoic acids) gives rise to many tetrahydroxylated 
cholestanoic acids that are excreted in urine and these metabolites are of diagnostic value 
[29-31].  Differential diagnosis of these peroxisomopathies is challenging and requires a 
battery of tests to examine all steps in the -oxidation of bile acids and fatty acids, 
complemented by immunoblotting techniques to identify the enzymes.   
 
Severe liver disease is a common manifestation of patients with peroxisomal 
dysfunction and is almost universal in the Zellweger patient and mild in neonatal 
adrenoleukodystrophy and infantile Refsum's disease [9].  Infantile Refsum's disease is 

 
 
 
 
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a very mild form of the Zellweger syndrome, and the severity of the symptoms and 
biochemical abnormalities can be used to differentially diagnose these syndromes.  The 
progressive liver disease that develops in Zellweger syndrome may be a result of the 
increased production and accumulation of cholestanoic (C27) bile acids combined with 
reduced concentrations of the primary bile acids, cholic and chenodeoxycholic acids. 
Publication of the results of the studies in these patients is planned for 2005.  
 
Bile acid Conjugation Defect 
 
Under another protocol approved by the IRB (CHMC #90-10-10) we studied a boy 
from Saudi Arabia who appears to be only the second child to be identified with a defect 
of bile acid conjugation.  As a neonate, he had cholestasis that led to surgery for 
presumed biliary atresia and the performance of a portoenterostomy.  At age 5, he had 
mild liver disease with pruritus. In addition, this Saudi boy has a 9 year-old sibling with a 
history or rickets in infancy. After FAB-MS analysis of the urine and mutation analysis of 
the genes encoding bile acid conjugation both siblings we found to be have the same 
defect in bile acid conjugation.  A previous child had been identified in collaboration with 
an investigator at UCSD.  Unfortunately, both the Saudi and the UCSD patients have 
subsequently been lost to follow-up.  A publication regarding the mutational analysis and 
biochemical description of these patients is now in preparation.  Three further infants have 
recently been found with this amidation defect and are candidates for bile acid therapy 
with conjugated cholic acid. 
 
Treatment of inborn errors in bile acid synthesis 
 
Untreated, the progressive cholestasis characteristic of these genetic defects 
eventually leads to death due to cirrhosis and liver failure. The choice of therapeutic dose 
(ranging 50-750 mg/day) has been based upon the estimated normal bile acid pool-size 
for age and titration against the disappearance of the atypical endogenous bile acids as 
measured by mass spectrometry.  Bile acid therapy has proven effective in suppressing 
the urinary excretion of the atypical bile acids although our experience thus far suggests 
that it is not possible to completely shut down endogenous bile acid synthesis, but 
significant down-regulation occurs rapidly after initiating primary bile acid therapy. 
Concomitant with this response is a marked clinical improvement with concentrations of 
serum liver enzymes and bilirubin consistently showing a gradual return to normal values. 
The clinical and biochemical response to therapy is rapid with early therapeutic 
intervention.  
Liver biopsies performed before and 6 months after oral bile acid therapy in 
several of the patients have revealed a significant reduction in the extent of 
inflammation and cholestasis, and EM studies have shown a normalization of bile 
canalicular structure in several patients.  We speculate that oral bile acids may have led 
to maturation in canalicular structure and secretory function.  The value of oral bile acid 
therapy for patients with these inborn errors in bile acid synthesis is apparent from the 
fact that only those patients who have been treated have survived and have gone on to 
lead essentially normal lives, while three patients have avoided unnecessary liver 
transplantation.  
Although 
the 
cerebrohepatorenal 
syndrome 
(Zellweger) 
and 
neonatal 
adrenoleukodystrophy are strictly defects in peroxisome dysfunction, bile acid synthesis 

 
 
 
 
     Protocol Updated: 31Jan2018   
 
is significantly affected.  The biochemical defect involves failure to adequately synthesize 
the primary bile acids, cholic and chenodeoxycholic acid, although synthesis is usually 
not completely impaired.  Because of enzyme blocks related to peroxisomal dysfunction, 
levels of dihydroxycholestanoic and trihyroxycholestanoic acids and a C29- dicarboxylic 
bile acid are increased in biologic fluids including urine, bile and serum.  Based upon our 
hypothesis that atypical, hydrophobic bile acids may be hepatotoxic particularly when 
present in contracted primary bile acid pools, we developed an orphan drug grant 
application (FD-R-000995, Treatment of Peroxisomal Disorders with Bile Acids) which 
was funded beginning 9/1/94.  To date, 18 subjects have been evaluated with a protocol 
which includes, identification and enrollment of subjects, collection of bile to assess pool-
sizes of the primary bile acids utilizing stable isotope dilution techniques, percutaneous 
liver biopsy, collection of urine and serum for assessment bile acid profiles and treatment 
with 15 mg/kg/day of cholic acid.  Enrollment is proceeding with variable results to date.  
At least in 10 subjects, bile acid therapy appears to have stabilized the liver disease both 
by histologic and biochemical measures.  In the remaining 10 subjects, liver and 
neurologic disease has progressed despite therapy with 9-10 dying during the course of 
these studies and 2 lost to follow-up.   
 
 
IV. Experimental Design and Methods 
 
Study Subjects:  
 
These will consist of infants, children and adolescents of any race or gender who 
have had samples sent to the Mass Spectrometry Laboratory for assessment for defects 
in bile acid metabolism because of the presence of cholestasis, or unexplained fat and 
fat-soluble vitamin malabsorption. They will have been identified by urine bile acid 
analysis by fast atom bombardment ionization mass spectrometry as having defects in 
bile acid conjugation based upon characteristic mass spectra.  Prior to our planned 
studies, patients will have undergone a thorough evaluation to define the etiology of their 
cholestasis or fat-soluble vitamin deficiency. Conventional screening studies (urine 
culture and clinitest, 1-antitrypsin phenotype, STORCH titers, thyroid function tests, liver 
ultrasound, liver "function" tests and percutaneous liver biopsy) will have been performed. 
Patients with other organ dysfunction will not be excluded.  Only patients identified with 
inborn errors of bile acid synthesis caused by defective bile acid conjugation will be 
candidates for entry to this study. The possible effect of radiation exposure during this 
study on an unborn baby is unknown. Because of this possibility, all females that may be 
able to become pregnant will have a urine pregnancy test done before starting the study, 
and before any x-ray exposure. It is anticipated that such defects are rare; however, there 
are 3 candidates for study at this time and we anticipate identification of one or more new 
patients each year that would be candidates for study on this protocol. 
 
Study Protocol:   
 
Once a patient is identified by KDRS as having a potential defect in bile acid 
conjugation, the attending physician will be contacted by JEH to discuss enrollment of the 
patient in the study.  He will explain the study to the parents/patients, and if they are 
willing, the subject will be brought to the Children's Hospital Clinical Research Center.  

 
 
 
 
     Protocol Updated: 31Jan2018   
 
On admission, the patient will have studies to assess liver function (bilirubin, ALT, AST, 
GGT, 
alkaline 
phosphatase, 
cholesterol, 
albumin, 
prothrombin 
time), 
partial 
thromboplastin time, serum tocopherol, serum retinol, serum 25-OH vitamin D and 
PIVKA-II.  A full nutritional evaluation will be performed including anthropometrics (height, 
weight. weight/height and OFC).  Serum will be obtained to perform a complete bile acid 
analysis by gas-liquid chromatography and urine will be obtained for LSIMS and GC-MS 
analysis (total blood for study purposes <10 cc).  
 
Primary bile acid pool-sizes will be measured to determine whether the 
hypothesized contraction of pools might play a role in the pathogenesis of cholestasis in 
these subjects.  [2,2,4,4-2H4]Cholic acid (15 mg) and [11,12-2H2]chenodeoxycholic acid 
(15 mg) will be given by mouth at or soon after 2100 h on the evening of Day 1 on the 
GCRC.  Nothing will be taken by mouth after midnight but a glass of water may be taken 
up to 6 hours before the procedure to collect bile on day 2. After an overnight fast, a 
nasoduodenal tube will be passed with the aid of fluoroscopy.  Bile will be collected after 
gallbladder contraction with the octapeptide CCK.  After bile collection, the tube will be 
removed. A blood sample will be taken for DNA sequencing and detection of mutations 
in the two genes encoding the bile acid conjugation enzymes. On Day 3, after four hour 
fast 100 IU/kg liquid emulsified dl-alpha-tocopherol will be given orally with collection of 
blood (1.0 cc/sample) at 0, +4, +8, +12, +18, +24, +48 hours after ingestion (32,33). On 
Day 4, after a 4 hour fast 1,000 IU/kg Vitamin D3 will be ingested in 3-5 oz of formula and 
blood (1.0 cc/sample) collected at 0, 6, 12, and 24 hours as previously described (34,35).   
Formula may be resumed 1 hour after the test dose of vitamin D.  After completion of the 
last serum sample, the patient will begin therapy with 15 mg/kg glycocholic acid (IND 
#50,046) by mouth and discharged from the GCRC the morning of Day 5 after receiving 
at least 2 doses of the bile acid treatment.  Liver function tests (as described above), and 
serum and urine for bile acid analysis by GC-MS and LSIMS. Three to twelve months 
after initiation of therapy, the patient will return to the GCRC for evaluation and 
measurement of liver function tests, nutritional assessment, measurement of the fasting 
primary bile acid pool-sizes, measurement of vitamin E and 25-OH vitamin D absorption.  
If bile acid therapy is considered to be successful based upon clinical findings, bile acids 
will be continued indefinitely with periodic follow-up by our group, or by the referring 
physician.  Samples of urine will be collected every 3-12 months to assess compliance 
and response to therapy.  Liver function tests will be performed at the discretion of the 
attending physician as the standard of care. It is anticipated that they will be collected at 
least once/year. This follow-up would be considered standard care for patients affected 
with cholestatic disorders of this nature.    
 
Study Drug: 
 
The study drug, glycocholic acid is administered under an IND approved by the 
FDA, and is not considered a drug used as standard care for this condition.  Glycocholic 
acid powder is stored in the CCHMC Investigational Pharmacy Department and will be 
formulated and dispensed by the Investigational Pharmacy personnel and then shipped 
by overnight courier directly to the patient.  We will dispense 3 months supply of the drug 
at a time, for as long as the drug is available. 
 
Analytical Methods:   

 
 
 
 
     Protocol Updated: 31Jan2018   
 
 
Preliminary analysis of urine obtained prior to enrollment and follow up samples 
will be performed using LSIMS.  Samples identified with abnormal patterns of bile acid 
excretion will be subjected to GC-MS analysis to afford characterization of a specific 
metabolic defect.  Serum bile acids will be measured by GC-MS.  All methods are well 
validated and currently being performed in the Mass Spectrometry Center of the 
Children's Hospital [36, 37].  Bile acid pools will be measured by the technique of Duane 
et al. [38]. Serum 25-OH vitamin D and tocopherol levels will be performed using 
established methods in the GCRC Core Laboratory. Both tests are performed for research 
as well as clinical testing and are CLIA certified tests.  Serum vitamin D levels will be 
measured by HPLC in the laboratories of Bruce Hollis at the Medical University of South 
Carolina (39).  DNA sequencing will be performed by routine techniques in the Laboratory 
of Dr. David Russell, Dallas, TX. This sequence analysis is for research purposes only, 
and will not be used in any clinical decision making. 
 
Data Analysis: 
 
There is no sample-size for this study as the sample-size is based upon the 
number of subjects identified with this rare metabolic defect. Pool sizes for the conjugates 
of the primary bile acids (chenodeoxycholic and cholic acids) will be compared before 
and after treatment using the paired Student's T-test.  Assessment of the relative 
contribution of unconjugated bile acids to the total pool size will be made before and after 
therapy by the paired Student's T-test.  Serial changes in liver function tests (total and 
direct bilirubin, AST, ALT, GGT, alkaline phosphatase, cholesterol and serum bile acids) 
will be evaluated by ANOVA using methods for repeated measures.  Comparisons of the 
serum vitamin D and tocopherol absorption tests will be compared by paired T-tests with 
comparisons of the maximal rise of 25-OHD before and after therapy and the area under 
the curve (ng/ml*hr) for vitamin D or area under the curve (mg/ml*hr) for tocopherol.  
Comparisons will be made to previously published results from our laboratories 
(32,34,35).  It is not anticipated that the results of the present study will provide enough 
information to prove our hypothesis; however, the results will provide a good working 
model to test in a rodent model of this disorder which is being developed currently.  
 
Special Considerations 
 
1.  Radiation Exposure:  Fluoroscopy will be performed on 2 occasions.  
Fluoroscopy time for will be kept to the least possible to complete the intubations. It is 
anticipated that about 90 seconds of fluoroscopy time will be required for each 
intubation that will lead to an average exposure of 225 mrems with a total exposure of 
450 mrem/year for the entire study.  Each exposure is approximately equivalent to that 
received from 2 abdominal x-rays and is approximately the same as one year’s natural 
radiation exposure in the Cincinnati, Ohio region, estimated at 320 mrem/year. Stable 
isotopes produce no radiation exposure and are considered safe.  
   
 
 
2.  Participation in Other Approved Protocols  
None 
 
 
 
3.  Investigational Drugs.  
 
 
Glycocholic acid  (IND 50,046) 

 
 
 
 
     Protocol Updated: 31Jan2018   
 
 
4.  CCHMC facilities to be utilized 
 
All studies will be performed on the Clinical Research Center and in 
outpatient  
 
 
laboratories of choice of the study subjects for follow-up 
samples. 
 
V/VI.   Potential Risks and Discomforts 
Venipuncture may be accompanied by pain and bruising at the site.  Cumulative 
blood losses at each of the two hospitalizations on the GCRC will be 21 ccs which is 
approximately 5% of the blood volume of even the smallest enrolled subject (6 kg x 70 x 
0.05 = 21 cc).   The passage of a nasoduodenal tube may result in mild-to-moderate 
discomfort.  The nasopharynx will be anesthetized with a topical anesthetic to minimize 
discomfort.  Fluoroscopy required for passage of the tube results in modest radiation 
exposure.  Fluoroscopy will be performed on 2 occasions.  This will produce an average 
exposure of 225 mrem for each intubation and 450 mrem for the two exposures.  The 
scientific value of the information obtained from this study as it relates to our basic 
understanding of cholestasis and specifically as it related to the pathogenesis of disease 
in the study subjects makes this radiation exposure warranted. 
There are no recognized hazards associated with the use of small quantities of 
stable isotopes as proposed in the current protocol. There may be some nausea, 
abdominal cramping with the use of CCK-octapeptide for collection of bile.  Although 
diarrhea might be a potential complication of bile acid therapy, no significant problem to 
date has been found in the infants and children treated with UDCA or cholic acid.  
Treatment of more than 100 cholestatic children with ursodeoxycholic acid has not led to 
any measurable deterioration in hepatic function.  In fact, administration of 
ursodeoxycholic acid uniformly leads to reductions in serum concentrations of bilirubin, 
transaminases, and cholesterol in most cholestatic conditions, while primary bile acid 
therapy has led to normalization of liver function in all patients studied with inborn errors 
in bile acid synthesis.  Similarly, although diarrhea might be a potential complication of 
bile acid therapy, no significant problem has been found in the infants and children treated 
with cholic or ursodeoxycholic acids. It is not anticipated that there will be a different safety 
profile when conjugates of cholic acid are used for treatment.  
 
Risk/Benefit Assessment.   The above risks are considered to be more than minimal 
to these patients; however, the subjects may directly benefit from the proposed therapy 
with conjugated bile acids.  In addition, it is possible that patients with these conditions 
as a class may benefit by our improved understanding of the pathogenesis of this 
disease process and the optimization of the therapeutic approach. 
 
Data and Safety Monitoring Plan 
This study represents more than minimal risk to subjects with potential benefit.  This is a 
treatment intervention similar to one that has not been associated with any significant 
adverse events associated with the administration of the drug since the initiation of 
treatment studies in 1994.  Currently, we review any adverse events for causality and 
they are kept in tabular form in the investigator’s password protected database. For any 
serious adverse events or unanticipated adverse events, the investigator would report 

 
 
 
 
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deaths to the IRB, GCRC and FDA within 2 days and other SAEs and unanticipated AEs 
would be reported to the IRB, GCRC and FDA within 10 working days of the investigator’s 
knowledge of their occurrence. Any SAEs that are considered related or probably related 
to the use of glycocholic acid will result in halting of enrollment of subjects and review by 
an Independent Medical Monitor.  John Bucuvalas, M.D. will serve as Lead Independent 
Medical Monitor.  He will be responsible for the timely evaluation of any AE’s, SAE’s and 
unanticipated AE’s for causality and relationship to the study drug at least every six 
months.   
.     
VII.   Estimated Time to Complete Study. 
More than 5-10 years.  
 
VIII.  Funding 
Funding is provided from the NIH supported Cholestatic Liver Consortium (CLiC) Grant 
# 5U54DK078377-04 
 
 
IX.   
Payment for Studies 
 All costs related to visit 2 will be assigned to the grant from CLiC.  There will be 
no incentives provided for participation in this study. There are no funds available to 
cover transportation costs to the study site for study related visits. 
 
X.  
Methods to be used in Procuring Informed Consent 
 
A verbal explanation will be given to the parent/guardian by phone prior to 
coming to CCHMC for Visit 1.  Written consent will be obtained at screening before 
enrollment in the study at CCHMC.  The informed consent form is attached. 
 
XI.   
Attending Physician Approval 
Not applicable 
 
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