Aliment Pharmacol Ther 2005; 21: 709–721.
Bioequivalence of a new liquid formulation of ursodeoxycholic acid
(Ursofalk suspension) and Ursofalk capsules measured by plasma
pharmacokinetics and biliary enrichment
K. D. R. SETCH ELL*, L. GALZIG NA , N . O ’CONNELL*, G. BRUN ETTIà & H .-D. TAUSCH EL§
*Department of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA;  Clinical Biochemistry
Department of Laboratory Medicine, University of Padova, Padova, Italy; àKnoll Farmaceutici, Milan, Italy; §Dr Falk
Pharma, Freiburg, Germany
Accepted for publication 20 December 2004
SUMMARY
Background: Ursodeoxycholic acid is an approved ther-
apy for hepatobiliary disorders but in infants and
children compliance is compromised because it is
formulated exclusively as capsules, or tablets.
Aim: To determine the pharmacokinetics and bioequiv-
alence of a new liquid formulation of ursodeoxycholic
acid (Ursofalk suspension) with a standard capsule
(Ursofalk) in a randomized, unblinded, crossover
designed study of 24 healthy adults.
Methods: Equivalence was based on single bolus
oral plasma pharmacokinetics and biliary ursode-
oxycholic acid enrichments after repeat doses. Bil-
iary bile acid composition and hydrophobicity index
were also compared. Ursodeoxycholic acid was
measured in duodenal bile by high-performance
INTRODUCTION
Ursodeoxycholic acid (UDCA) has been used for several
decades for the treatment of hepatobiliary disorders,1–3
including cholelithiasis,4 biliary dyspepsia, bile reflux
gastritis.5 It is now prescribed for the treatment of
primary biliary cirrhosis,6–12 primary sclerosing cho-
langitis13–15 and a range of other adult cholestatic
Correspondence to: Dr K. D. R. Setchell, Department of Pathology,
Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229,
USA.
E-mail: kenneth.setchell@cchmc.org

2005 Blackwell Publishing Ltd
doi: 10.1111/j.1365-2036.2005.02385.
liquid chromatography and in plasma by mass
spectrometry.
Results: The mean percentage biliary ursodeoxycholic
acid enrichment after administration of the suspension
was not significantly different from that obtained with
capsules (44.2 ± 11.7% vs. 46.9 ± 10.2%, respectively).
The equivalence ratio was 0.94 (95% CI: 0.8–1.1),
establishing bioequivalence between suspension and
capsules. Both formulations reduced the biliary hydro-
phobicity index and no differences in bile acid composi-
tion were observed between formulations. The plasma
pharmacokinetics of both formulations was similar and
the tolerability of the suspension was excellent.
Conclusions: A new liquid formulation of ursodeoxy-
cholic acid suitable for paediatric patients is pharmaco-
logically bioequivalent to capsules when given as single,
or repeated oral doses.
conditions.16–21 Very recently, UDCA has been under
evaluation as a potential anticancer drug for the
prevention of colon cancer in high-risk individuals.22–24
Beneficial effects on biochemical and histological
markers of liver diseases have been reported for infants
and children with a variety of cholestatic disorders,
including biliary atresia, Byler disease, Alagille synd-
rome, progressive familiar intrahepatic cholestasis,25–28
inborn errors of bile acid synthesis,29 total parenteral
nutrition-associated liver disease30, 31 and cystic
fibrosis-associated liver disease32–37 treated with oral
UDCA. However, chronic treatment of infants and
709
710 K. D. R. SETCHELL et al.
young children is especially difficult because the thera-
peutic forms of UDCA available have since their
introduction been exclusively of either capsule, or tablet
formulations.38–41 Parents of infants and very young
children with liver disease generally have to devise
ingenious means of administering UDCA after unpack-
ing the capsules or crushing the tablets to disguise the
extreme bitter taste of this bile acid. This is usually
achieved with variable outcomes by mixing the powder
with flavoured liquids or foods. In many instances it still
proves difficult to administer complete doses and
compliance is often compromised. In order to overcome
these limitations a new oral liquid formulation of UDCA
(Ursofalk suspension) with significant masking of the
bitterness of UDCA was recently developed as an
alternative to the presently prescribed solid formula-
tions. This new formulation is mainly targeted for
infants and young children but may also offer an
alternative for adult patients who have difficulty in
swallowing capsules or tablets. Regulatory approval of
any new drug formulation, however, requires demon-
stration of bioequivalence to existing approved formu-
lations. The object of this study was to compare the
pharmacokinetics and determine the extent of bioequiv-
alence of this new liquid formulation (Ursofalk suspen-
sion) against an approved reference solid formulation
(Ursofalk 250-mg capsules) that has been in clinical use
for more than two decades for the treatment of liver
disease.
SUBJECTS AND METHODS
Study subjects
Healthy adults in the age range 18–55 years and
with normal body weight (body mass index,
BMI < 25) were enrolled in the study. The study
group comprised 11 females and 13 males. The study
protocol was approved by the Human Investigations
Ethical Committee in Montegrotto, Terme, Italy, and
written informed consent was obtained from each
subject. For inclusion in the study each subject had to
be in good health and free of acute or chronic
diseases as indicated by medical history, physical
examination, electrocardiogram (ECG) and laboratory
screening tests. Subjects taking any over-the-counter
(OTC) medication within 1 week of the study, antibi-
otics in the prior 2 months, had a history of alcohol
or drug abuse, undergone a cholecystectomy, were

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
taking oral contraceptives, or were pregnant or
lactating, were excluded from enrolment.
Study design
The study was an open-label randomized crossover
design. Prescreening of subjects was performed
7–30 days prior to entry, and baseline evaluations
were performed on days )2 and )1, before treatment
with drug. Each subject was given orally 750 mg UDCA
daily (equivalent to 10–15 mg/kg body weight/day) in
a single bolus dose as either a suspension (Ursofalk
suspension, 15 mL of a 50 mg/mL concentration), or as
3 · 250-mg capsules with a small amount of water for
13 days according to a randomization schedule. After a
14-day washout period to ensure plasma and biliary
UDCA levels returned to baseline values, the procedure
was repeated in each adult with a reversal of the
formulations. Because of the fact that the physical
appearance of the two formulations was markedly
different it was impossible to blind the treatment
regimens to the patient. However, all of the analytical
determinations were performed blinded to the analyst.
Subjects were studied as in-patients during the period
that samples were collected. For determination of biliary
UDCA enrichment, duodenal bile was obtained in the
morning using a special string device (Enterotest) that
was swallowed, positioned in the duodenum, and then
intramuscular ceruleine was used to induce gall-bladder
contraction.42 The bile-stained string was then removed
and bile was extracted by soaking the string in 80%
methanol. This procedure was performed 1 day before
administration of UDCA (baseline sample), and again at
the end of the treatment period (day 14). For determin-
ation of UDCA pharmacokinetics, blood samples (12 mL
each) were collected into heparinized tubes through an
indwelling catheter placed in the anticubital vein. This
was performed on first day of the respective treatments
and blood draws were taken at baseline and at 0.5, 1,
1.5, 2, 4, 8, 12 and 24 h after oral administration of
UDCA. The blood was centrifuged at 3000 rpm for
15 min and the plasma removed and frozen at )20 C
until later analysis. On the days of admission the
subjects were given a standard diet consisting of a total
daily calorie intake of 2200 kcal with 30% of calories
derived from fat. The meals were served at approxi-
mately 12.30 pm and 7 pm each day.
In addition to detailed bile acid analysis, the following
routine serum biochemistries were performed; serum
 PHARMACOKINETICS OF URSODEOXYCHOLATE SUSPENSION
aspartate aminotransferase (AST), alanine aminotransf-
erase (ALT), gamma-glutamyltransferase (c-GT), alka-
line phosphatase (ALP), total bilirubin, total cholesterol,
creatinine, blood urea nitrogen (BUN), uric acid,
glucose, sodium, potassium, calcium, total and serum
protein electrophoresis. Haematological measurements
included haemoglobin, blood count and prothrombin
time.
Tolerability and compliance
Each subject completed a questionnaire regarding
adverse events or symptoms at the end of each
treatment and a complete physical examination was
performed on completion of the study. Compliance was
assessed from measuring the number of capsules and
volume of suspension consumed by the end of the study
and from the plasma and biliary UDCA levels.
Sample bile acid analysis
Analysis of plasma and duodenal bile for UDCA and other
major bile acids was carried out using established and
validated methodologies based on stable-isotope dilution
gas chromatography–mass spectrometry with selected
ion monitoring (GC-MS-SIM) and by reverse-phase
high-performance liquid chromatography (HPLC).43
Measurement of plasma bile acid concentrations by
GC-MS-SIM
Bile acids were quantitatively extracted from plasma by
solid-phase extraction on small Bond-Elut C18 car-
tridges44, 45 after addition of deuterated internal stand-
ards, [2,2,4,4-2H4]ursodeoxycholic, [2,2,4,4-2H4]cholic
acid, [2,2,4,4-2H4]deoxycholic, [11,12-2H2]chenode-
oxycholic and [11,12-2H2]lithocholic and as described
in detail previously.46 Extracted bile acid conjugates
were then subjected to solvolysis and enzymic hydroly-
sis. After hydrolysis, unconjugated bile acids were
extracted on a Bond-Elut C18 cartridge and recovered
by elution with methanol. Bile acids were then isolated
and separated from neutral steroids by lipophilic anion-
exchange chromatography47 and converted to volatile
methyl ester-trimethylsilyl (Me-TMS) ether derivatives.
The Me-TMS ethers were analysed by GC-MS with SIM of
the specific ions m/z 383, m/z 370, m/z 372 and m/z
374 corresponding to specific electron ionization
(70 eV) fragments derived from monohydroxy-, dihyd-

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
711
roxy- and trihydroxy-5b-cholanaoates and their respect-
ive deuterated analogues used as internal standards.48
GC-MS was performed on an Autospec Q mass spectro-
meter (Waters Inc., Franklin, MA) using electron
ionization (70 eV). Confirmation of the identity of each
bile acid was established from the GC retention index of
the specific ions corresponding to UDCA and other
primary and secondary bile acids when compared with
authentic standards. Bile acids were quantified by
comparing the peak area of each bile acid to that of
the corresponding internal standards and interpolating
this ratio relative to a set of calibration standards of
known concentrations of bile acids. The individual and
total plasma bile acid concentrations were expressed in
lm and the relative content of each bile acid was
expressed as percentage of the total bile acid content.
The intra-assay precision of the method for the individ-
ual bile acids as measured from repeat analysis of the
same sample was <6% (coefficient of variation, CV). The
intra-assay precision of the method measured as CV for
UDCA was <5%, while the inter-assay CV was 11.8%.
UDCA enrichment in duodenal bile measured by HPLC
The relative proportion of UDCA and the principal
primary and secondary bile acid conjugates in the
Enterotest samples of duodenal bile was determined by
reverse-phase HPLC essentially as described by Rossi
et al.49 The stained Enterotest strings were soaked and
sonicated in 10 mL of methanol to solubilize bile acids42
for subsequent analysis by HPLC. The methanolic extract
was reduced in volume by evaporation under a stream of
nitrogen and reconstituted in the HPLC mobile phase
(100–500 lL). In cases where there was inadequate bile
staining of the Enterotest strings (string failures), UDCA
was measured by the more sensitive GC-MS method
exactly as described above for the plasma samples.
Reverse-phase HPLC was performed using a Thermo-
separation Spectrasystem HPLC system equipped with a
25 · 0.46 cm Hypersil 5 lm ODS column. Chromato-
graphic separation of bile acid conjugates was achieved
using a mobile phase of methanol and 0.01 m KH2PO4
(75:25, v/v), adjusted to pH 5.3 and flow rate of
1.5 mL/min, and bile acids were detected by UV
absorption at 205 nm.
The UDCA content was expressed as percentage of
total bile acids by calculating the sum of chromato-
graphic area for UDCA conjugated as a proportion of the
total area of bile acids. All individual bile acid curves
712 K. D. R. SETCHELL et al.
had r-values of ‡0.99. The interassay CV was 5%
for tauroursdeoxycholic acid (TUDCA) and 6.2% for
glycoursodeoxycholic acid (GUDCA) for a mixture of
standard bile acids and 14.6% for TUDCA and 7% for
GUDCA when an Enterotest extract was used as quality
control. The detection limit of the method was 5 lg/mL
(0.01 mm).
Assessment of drug bioequivalence
Bioequivalence was based upon comparisons of the
plasma UDCA concentrations after single bolus oral
administration and on the relative percentage biliary
UDCA enrichment after daily administration of both
formulations for 14 days. The pharmacokinetics was
determined from the appearance/disappearance profiles
for plasma UDCA concentrations. The extent of bio-
equivalence of the two formulations was determined
from measures of the apparent bioavailability assessed
from the area under the plasma UDCA concentration–
time curve (AUC0)12h), and from the maximum plasma
concentration (Cmax), and time taken to attain maxi-
mum plasma concentration (Tmax). The AUC up to the
12 and 24 h measured time-points (AUC0)12h and
AUC0)24h) was calculated by conventional linear tra-
pezoidal rule.
Biliary enrichment of UDCA at the end of each 14-day
treatment period was expressed as the relative percentage
of UDCA (GUDCA + TUDCA) to the total biliary bile acid
concentration. This pharmacokinetic parameter is a
measure of how much UDCA reaches the target organ
under steady-state conditions. Secondary measures that
were compared, included the change in the biliary bile
acid composition [chenodeoxycholic acid (CDCA), cholic
acid (CA), deoxycholic acid (DCA), lithocholic acid (LCA)]
and change in hydrophobicity index (HI) of duodenal bile
between baseline and postadministration of each formu-
lation. Bile acid HI was calculated according to Heuman’s
formula50 as follows: HI ¼ E · HIx · Fx where, HI is the
individual HI for the glyco- or tauro-conjugate of each bile
acid and Fx is the proportion of the individual bile acids in
the duodenal bile.
Statistical analysis
Biliary enrichment in UDCA was taken as the primary
end-point for estimation of the appropriate sample size.
A maximum allowed difference of 8% in UDCA biliary
enrichment (i.e. ±20% difference from the reference

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
product) is deemed under regulatory standards to be
necessary to establish equivalence between any two
drugs.
Assuming a 90% power, a one-sided significance level
of 5%, and an analytical standard deviation of 8% for
measurement of UDCA enrichment in bile, 17 patients
were needed to assess bioequivalence between the test
and the reference formulation. Taking into account a
possible 30% failure rate of the Enterotest method used
to collect duodenal bile, a total of 24 patients were
subsequently enrolled in the study.
The primary variables and HI were analysed by anova.
The statistical model tested the effects of drug treatment
sequence, subjects and treatments. In addition, this
model tested the two one-sided hypotheses at a 0.05
level of significance, by constructing the 90% confid-
ence interval (CI) for the estimate of the ratio between
the geometric mean values of biliary enrichment of
UDCA, AUCt, Cmax assuming logarithmic distribution.
For Tmax, a normal distribution was assumed to
construct the 90% CI for the difference between the
expected arithmetic mean values. The test (Ursofalk
suspension) and the reference formulation were to be
considered as equivalent if the 95% CI of the ratio of
geometric mean values of biliary enrichment in UDCA
and the AUCt and Cmax, lay between a range of 0.80
and 1.25.
RESULTS
Compliance
All study subjects successfully completed the treatment
periods with no significant deviation from the protocol.
The tolerability of both formulations was found to be
excellent and there were no reports of adverse events
during the study. Based upon the amounts of study drug
returned after each treatment period, compliance was
confirmed. The expected number of capsules and
volumes of liquid were taken by each subject. Compli-
ance was further indicated from measurements of the
plasma UDCA concentration that in all subjects was
significantly elevated above normal during treatment.
Biliary enrichment
The extent of bile staining of the terminal portion of the
string was variable among subjects irrespective of the
drug treatment, but adequate amounts of duodenal bile
 PHARMACOKINETICS OF URSODEOXYCHOLATE SUSPENSION 713

were obtained for HPLC analysis in all but five subjects.
In these five string failures concentrations of biliary bile
acids were too low for quantification by HPLC and these
were subsequently measured by GC-MS. The mean
(±s.d.) biliary enrichment of UDCA before and after
administration of test and reference formulations is
shown in Figure 1.
There was no significant difference in the baseline
biliary UDCA level which was expectedly low and in the
physiological reported range when expressed as a
percentage of the total biliary bile acids measured.1, 2
Geometric mean values and the ratio for test/reference
formulation for UDCA enrichment are shown in
Table 1. The 95% CI for the ratio of geometric mean
values of Ursofalk suspension and Ursofalk capsules was
within the accepted standard bioequivalence range
(0.8–1.25). Thus, the two formulations were found to
be identical in bioequivalence with regard to biliary
UDCA enrichment achieved under steady-state condi-
tions (Table 1).
Pharmacokinetics
The mean UDCA plasma concentration–time curves for
both formulations are shown in Figure 2. The profiles of
the plasma appearance/disappearance curves were
similar and characterized by a rapid increase in UDCA
in plasma followed by a slower elimination phase. The
time to attain maximal plasma UDCA concentration
(Tmax) was 3.56 h for Ursofalk suspension when
compared with 2.56 h for Ursofalk 250-mg capsules,
and this difference was not statistically significant
(Table 1). The absorption and elimination rates followed
first-order kinetics, and pharmacokinetic measures were
computed based on this model. The mean peak plasma
concentration (Cmax) and bioavailability as measured
from the AUC0)12h of the suspension and the reference
capsules were comparable and within the range of
equivalence. These data are summarized in Table 1.

Pharmacodynamic effects of UDCA administration

Figure 1. Arithmetic mean (±s.d.) biliary ursodeoxycholic acid
(UDCA) enrichment after 14 days administration of 750 mg/day
dose of UDCA given as Ursofalk capsules (reference) or as Ursofalk
suspension (test formulation).
There was a significant decrease in the HI of duo-
denal bile following administration of the suspension
(Table 1). Mean (±S.E.M.) values decreased from
0.31 ± 0.08 in the pre-treatment bile to 0.014 ±
0.028 (P < 0.05) after administration of the UDCA
suspension, and correspondingly from 0.33 ± 0.06 at
baseline to )0.033 ± 0.018 (P < 0.05) for the UDCA
capsules (Figure 3). The HI was decreased by 95%
during administration of suspension and 106% during

Table 1. Plasma pharmacokinetics after single dose administration of 750 mg ursodeoxycholic acid (UDCA) in the form of an Ursofalk
suspension (T) or as Ursofalk 250-mg capsules (R) and biliary UDCA enrichment and hydrophobicity index (HI) after a 14 days
administration of 750 mg UDCA/day

Parameters Suspension (T) Capsules (R) Ratio (T/R) 95% CI for T/R

Biliary enrichment (%)* 45.1 ± 1.0 47.9 ± 1.0 0.94 0.85–1.04

AUC0)12h (lmol h/L)* 150.5 ± 1.0 134.5 ± 1.0 1.12 1.01–1.24
Cmax (lm)* 22.7 ± 1.0 22.7 ± 1.0 1.00 0.81–1.24

Difference (T ) R) 95% CI for T ) R

Tmax (h)  3.56 ± 0.51 2.56 ± 0.51 1 )0.49 to 2.49
HI  0.014 ± 0.028 )0.033 ± 0.018 )0.05 )0.034 to 0.127

Values expressed as means ± S.E.M.

* Geometric mean.
  Arithmetic mean.

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721

714 K. D. R. SETCHELL et al.
Figure 2. Plasma ursodeoxycholic acid (UDCA) concentration
(lm, mean ± s.d.) profiles following single bolus oral administra-
tion of 750 mg Ursofalk suspension, or 750 mg Ursofalk capsules
to the same healthy adults.
Figure 3. Change in hydrophobicity index (HI, mean ± S.E.M.) of
duodenal bile after oral administration for 14 days of ursodeoxy-
cholic acid (UDCA) (750 mg/day) given as Ursofalk capsules
(reference) or as Ursofalk suspension (test formulation).
administration of the reference UDCA capsules, and
there was no statistically significant difference between
the two different formulations of UDCA.
Following both formulations, there was a significant
change in the composition of the biliary bile acid pool.
There occurred a decrease in the proportion of biliary
CA, from 37 to 18% during administration of the
suspension and from 35 to 21% when the capsules were
given. The proportion of CDCA similarly declined from
39% at baseline to 24% for suspension, and from 38 to
21% for capsules. A decrease in the relative proportion
of DCA was observed after both formulations, but LCA,
which accounted for a relatively small proportion of the
total biliary bile acids (<1.5%), did not change after
administration of either bile acid formulation. The ratio
of glycine-/taurine-conjugated biliary bile acids was not

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
significantly different between the two formulations
(6.51 ± 2.1 vs. 3.77 ± 3.04, mean ± s.d. for suspen-
sion vs. capsule).
DISCUSSION
The present study was designed to evaluate the
bioequivalence of a new liquid formulation of UDCA
(Ursofalk suspension) with that of a conventional
approved solid capsule in healthy volunteers at the
usual therapeutic doses of 10–15 mg/kg body weight/
day currently recommended for solid forms of UDCA in
the treatment of hepatobiliary disease. The new liquid
formulation was developed with the aim of achieving
the same UDCA release profile to a currently prescribed
reference formulation, Ursofalk 250-mg capsules. UDCA
is poorly soluble in water1, 2 and therefore, to achieve a
liquid formulation, an excipient was used to produce
a suspension. To mask the natural bitter taste of UDCA,
a sweetener was added. The widespread availability of a
liquid formulation is needed to facilitate compliance to
UDCA therapy particularly in infants and children with
cholestatic liver disease that presently have to be given
by the parents the powdered contents of commercial
UDCA capsules or tablets disguised in some form of food
sweetener. From a practical perspective, presently it is
also difficult to titrate accurate doses of UDCA in the
young patients. Based on the therapeutic dose range
(10–15 mg/kg body weight/day) an infant of 5–10 kg
body weight should be prescribed 50–150 mg/day,
however, currently approved capsules are marketed as
250 mg or 300 mg doses. Commercial availability of a
liquid formulation of UDCA would make it possible to
deliver the exact prescribed dose according to the body
weight of the infant. Furthermore, this formulation
could also be useful for adults who find it difficult to
swallow capsules.
Changes in a drug formulation may alter in vitro
dissolution or gastrointestinal absorption,51 and hence
affect bioavailability, and therefore it is necessary to
demonstrate bioavailability and bioequivalence of any
new formulation for approval to be granted by Drug
Regulatory Agencies. In vitro comparative dissolution
studies have shown that Ursofalk suspension and
Ursofalk capsules have equivalent release profiles
(manufacturer’s proprietary data – unpublished), and
this is supported by the clinical data presented here.
Bioequivalence was assessed under two different
conditions. Classical single bolus oral pharmacokinetics
 PHARMACOKINETICS OF URSODEOXYCHOLATE SUSPENSION
of the two formulations was carried out to determine
bioavailability and to compute pharmacokinetic char-
acteristics of the drug from the plasma appearance/
disappearance profiles of UDCA. Then, under steady-
state conditions of chronic (repeated) dosing for a period
of 2 weeks, the concentration of the UDCA at the site of
action, in this case the liver was compared from the
UDCA enrichment in duodenal bile samples.
The results of this study established that this new
liquid formulation of UDCA is equivalent to standard
Ursofalk capsules currently in clinical use with respect
to plasma UDCA pharmacokinetics and biliary UDCA
enrichment. UDCA absorption and ‘apparent bioavail-
ability’ determined plasma UDCA pharmacokinetic
parameters were similar following single dose adminis-
tration of the suspension and the capsules (Table 1).
The plasma concentration appearance/disappearance
curves of both formulations were similar and charac-
terized by rapid appearance of UDCA in plasma with
peak concentrations occurring 2 h after oral adminis-
tration followed by a slower terminal elimination phase.
This is consistent with other pharmacokinetic studies of
UDCA,38–41, 52 where in most cases the peak UDCA
concentrations are observed between 2 and 4 h after
oral administration. Delayed absorption, with peak
plasma UDCA concentrations has been achieved with
sustained release formulations of UDCA,41, 53 although
it was shown such formulations made no significant
difference to the biliary UDCA enrichment.41 The
AUC0)12h was calculated from the plasma UDCA
concentrations for the time-points 0–12 h only in order
to minimize effects due to enterohepatic recycling of
UDCA where otherwise a second peak plasma concen-
tration is often observed.54, 55 However, similar results
were obtained when AUC0)24h was computed for
the two formulations. These plasma profiles are quali-
tatively consistent with the findings of other investi-
gators who have compared various preparations of
UDCA.38–41 While it is difficult to accurately compare
the different published studies on UDCA pharmacoki-
netics because of differences in study designs and
analytical methods, there are considerable differences
among studies in the ‘apparent bioavailability’ of UDCA
when the reported plasma AUC are compared on a dose-
adjusted basis. This is perhaps not surprising when
significant differences in the in vitro dissolution rates for
different UDCA preparations have been demonstrated.51
For most drugs bioequivalence is based upon demon-
strating that a new formulation exhibits the same

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
715
absorption and plasma characteristics to an existing
approved formulation. Plasma concentrations levels
after single administration of UDCA are especially useful
for the in vivo evaluation of new formulations with
modified release (delayed, multiple release, etc.) because
they predict the rate and extent of intestinal absorption.
However, they are less informative in determining how
much of the drug reaches the target tissue. In the case
of UDCA, the target organ is the liver and gall-bladder,
and therefore measures of plasma bile acid concentra-
tions are not so informative of whether the drug is
reaching its target. Circulating bile acid concentration
reflects the balance between intestinal input and hepatic
extraction and in health the latter is efficient leading to
relatively low plasma bile acid concentrations.56 Also,
intestinal absorption of UDCA can be affected by the
formulation and co-administration of other agents.57–59
When UDCA is administered, plasma UDCA concentra-
tions are usually very low and poorly correlate with the
amount the bile acid that reaches the site of action, the
liver.60 Biliary enrichment is crucial to achieving
efficacy. It is biliary UDCA enrichment and not plasma
UDCA concentration that best correlates with decrease
in total bilirubin and Mayo risk score in patients with
primary biliary cirrhosis.60 In addition, the increase in
biliary UDCA correlates significantly with decreases in
serum transaminases and alkaline phosphatase.33, 61, 62
In this bioequivalence study, we determined the biliary
UDCA enrichment under steady-state conditions and
this differs from previous pharmacokinetic studies of
UDCA where bioavailability has been assessed only after
a single dose oral administration using the plasma
kinetics.38–41, 53, 63 Our study was conducted in healthy
subjects using a dose of 750 mg given once daily. The
decision to perform these studies in healthy volunteers,
rather than patients was based on the fact that similar
biliary UDCA enrichments (approximately 45%) are
observed for healthy volunteers and gallstone patients
following chronic administration of doses of 8–12 mg/
kg body weight/day (see Table 2) and because this is a
standard approach in drug evaluation. Lower biliary
UDCA enrichments are often observed in patients with
chronic cholestatic liver disease as indicated in Table 2
which summarizes many published studies. This lower
enrichment in liver disease is mainly explained by
impaired hepatic bile acid uptake and/or reduced
canalicular bile acid secretion,2 although severe chol-
estasis can impair intestinal absorption of UDCA,64 as
does the use of bile acid-binding agents.57, 58
 Table 2. Published studies comparing the extent of biliary UDCA enrichment expressed as a percentage of the total biliary bile acids
716

Study reference Study subjects (N) UDCA preparation Dose Biliary UDCA (%)

Healthy subjects
Federowski et al.68 Healthy volunteers (n ¼ 7) UDCA, Tokyo Tanabe, Japan 1000 mg/day 56 ± 7
Batta et al.69 Healthy volunteers (n ¼ 4) Actigall 300 mg capsules, Ciba-Geigy, USA 900 mg/day 55 ± 7
Galzigna et al.70 Healthy volunteers (n ¼ 24) Ursofalk 250-mg capsules, Germany 750 mg/day 47 ± 10
Galzigna et al.71 Healthy volunteers (n ¼ 24) Ursofalk suspension 750 mg/day 44 ± 11
Galzigna et al.71 Healthy volunteers (n ¼ 24) Ursofalk 150/300 mg capsules, Italy 750 mg/day 43 ± 9
Biliary disease patients
Makino and Nakagawa72 Gallstone patients (n ¼ 23) UDCA, Tokyo Tanabe, Japan 150 mg/day 28 ± 8
600 mg/day 53 ± 8
K. D. R. SETCHELL et al.

Roda et al.73 Gallstone patients (n ¼ 7) UDCA 125 mg capsules, Guliani, Italy 12 mg/kg body weight/day Approximately
55
Salen et al.74 Gallstone patients (n ¼ 21) UDCA 250 mg capsules, Tokyo Tanabe, Japan 250 mg/day 29 ± 3
1000 mg/day 51 ± 6
Ponz de Leon et al.75 Gallstone patients (n ¼ 11) UDCA capsules, Giuliani, Italy 15 mg/kg body weight/day 42 ± 17
Carulli et al.76 Gallstone patients (n ¼ 25) UDCA, Gipharmex, Italy 15 mg/kg body weight/day 54 ± 5
Stiehl et al.77 Gallstone patients (n ¼ 10) Ursofalk 250-mg capsules, Germany 12–16 mg/kg body weight/day 64 ± 2
Bateson et al.78 Gallstone patients (n ¼ 24) UDCA, Tokyo Tanabe, Japan 250 mg/day 26 ± 2
500 mg/day 35 ± 3
750 mg/day 50 ± 3
1000 mg/day 50 ± 5
Thistle et al.66 Gallstone patients (n ¼ 6) UDCA, Tokyo Tanabe, Japan 5.8 mg/kg body weight/day 40
10.6 mg/kg body weight/day 49
Ward et al.5 Gallstone patients (n ¼ not stated) Various formulations 150 mg/day 19–29
250–300 mg/day 26
450–500 mg/day 29–35
600–750 mg/day 42–53
>750 mg/day 50–64
von Bergmann et al.79 Gallstone patients (n ¼ 10) Ursofalk 250-mg capsules, Germany 1000 mg/day 43
Stiehl et al.80 Gallstone patients (n ¼ 5) Ursofalk 250-mg capsules, Germany 250 mg/day 29 ± 2
500 mg/day 41 ± 3
750 mg/day 46 ± 3
1000 mg/day 53 ± 2
1250 mg/day 53 ± 2
Bazzoli et al.81 Gallstone patients (n ¼ 39) Not known 400 mg/day 31 ± 3
Frenkiel et al.82 Gallstone patients (n ¼ 55) UDCA, Gipharmex, Italy 750 mg/day 53 ± 1
Zuin et al.83 Gallstone patients (n ¼ 12) UDCA 250 mg capsules, Italy 15 mg/kg body weight/day 55 ± 6
Fischer et al.84 Gallstone patients (n ¼ 7) Ursofalk 250-mg capsules, Germany 750 mg/day 43 ± 17
Miettinen et al.85 Gallstone patients (n ¼ 7) Adursal, Leiras 23–25 mg/kg body weight/day 62 ± 4
Shiffman et al.86 Gallstone patients (n ¼ 32) Actigall 300 mg capsules, Ciba-Geigy, USA 300 mg/day 14 ± 11
600 mg/day 21 ± 14
1200 mg/day 34 ± 22

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721

 Liver diseases
Podda et al.87 Chronic active hepatitis (n ¼ 18) UDCA 250 mg, Italy? 250 mg/day 22
500 mg/day 32
750 mg/day 34
Podda et al.87 PBC (n ¼ 18) UDCA 250 mg, Italy? 250 mg/day 25
500 mg/day 31
750 mg/day 37
Roda et al.88 PBC (n ¼ 7) UDCA, Italy? 600 mg/day 34 ± 1
Podda et al.16 Chronic active hepatitis (n ¼ 24) UDCA, Italy? 600 mg/day 31
Colombo et al.32 CF-liver disease (n ¼ 9) UDCA, Italy? 10–15 mg/kg body weight/day 25 ± 7
Nakagawa et al.89 CF-liver disease (n ¼ 5) UDCA, Italy? 10–15 mg/kg body weight/day 12–33
Crossignani et al.90 BRIC (n ¼ 1) UDCA, Italy? 750 mg/kg body weight/day 35
Mazzella et al.91 PBC (stages I–III; n ¼ 9) UDCA 300 mg, Italy? 600 mg/day 34 ± 1
Crosignani et al.92 PBC (n ¼ 10) UDCA, Italy? 500 mg/day 33 ± 1
Crosignani et al.93 Chronic active hepatitis (n ¼ 18) UDCA 250 mg, Italy? 250 mg/day 22 ± 1
500 mg/day 32 ± 3
750 mg/day 34 ± 2
Batta et al.69 PBC (n ¼ 14) Actigall 300 mg capsules, Ciba-Geigy, USA 900 mg/day 31 ± 12
Lindor et al.8 PBC (n ¼ 89) Ursofalk 250-mg capsules, Germany 13–15 mg/kg body weight/day 40
Combes et al.94 PBC (stages I–II; n ¼ 28) Actigall 300 mg capsules, Ciba-Geigy, USA 10–12 mg/kg body weight/day 37 ± 12
PBC (stages III–IV; n ¼ 49) 42 ± 17
PBC (stages I–IV; n ¼ 77) 43 ± 13

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721

Stiehl et al.14 PSC (n ¼ 10) Ursofalk 250-mg capsules, Germany 750 mg/day 31 ± 3
van de Meeberg et al.61 PBC (n ¼ 19) and PSC (n ¼ 8) Ursofalk 250-mg capsules, Germany 10 mg/kg body weight/day 41 ± 3
Fracchia et al.95 PBC (n ¼ 6), PSC (n ¼ 1) Not stated 600 mg/day 54 ± 12
Invernizzi et al.96 PBC (n ¼ 12) UDCA, Pharmacia, Italy 750 mg/day 29 ± 6
Combes et al.11 PBC (n ¼ 54) UDCA, Actigall 300 mg capsules, Ciba-Geigy, USA 10–12 mg/kg body weight/day 40 ± 16
Other conditions
Nilsell et al.97 Hyperlipdaemia (n ¼ 12) UDCA 125 mg capsules, Tokyo Tanabe, Japan 15 mg/kg body weight/day 50 ± 4
Angelin et al.98 Hyperlipidaemia (n ¼ 5) UDCA, Tokyo Tanabe, Japan 7.5 mg/kg body weight/day 46 ± 9
15 mg/kg body weight/day 50 ± 4
Mazella et al.99 Obesity–weight management (n ¼ 10) UDCA 150 mg capsules, Gipharmex, Italy 10 mg/kg body weight/day 45 ± 1
Mazella et al.99 Obesity–weight reduction (n ¼ 10) UDCA 150 mg capsules, Gipharmex, Italy 10 mg/kg body weight/day 49 ± 1

PBC, primary biliary cirrhosis; BRIC, benign recurrent intrahepatic cholestasis; PSC, primary sclerosing cholangitis; CF, cystic fibrosis; UDCA, ursodeoxycholic acid.
PHARMACOKINETICS OF URSODEOXYCHOLATE SUSPENSION
717
718 K. D. R. SETCHELL et al.
Unlike most drugs, where the site of action or target
tissue may be inaccessible to sampling, relatively low
invasive procedures can be used to sample duodenal
bile. In order to reduce the discomfort of conventional
duodenal intubation, a proven alternative method of
collecting duodenal bile was used. This previously
validated procedure involved the placement of a special
string (Enterotest) to sample duodenal bile.42 In most
subjects successful sampling was obtained, however,
there were five string failures, which is not uncommon
with this sampling method. These failures were evident
from the fact that the strings had negligible bile staining
and measurement of biliary enrichment was therefore
made by GC-MS rather than HPLC. Mean (±s.d.) biliary
UDCA enrichment after 2 weeks of dosing 750 mg/day
with the suspension was 46.9 ± 10.2% and this was
similar to that achieved with the reference capsule
(44.2 ± 11.7%). The ratio of geometric mean values
(Table 1) after treatment with suspension and capsule
was close to 1.0 and the 95% CI for the ratio of
treatment was within the regulatory accepted range of
equivalence for UDCA enrichment and plasma AUC and
Cmax values. Very rarely does biliary UDCA enrichment
exceed 50% (Table 2), unlike oral CDCA therapy that
results in >80% biliary enrichment.65–67 Significant
changes in biliary bile composition were observed for
the major bile acids with both UDCA formulations; the
proportions of CA and the more hydrophobic CDCA
were reduced compared with baseline as the bile was
concomitantly enriched in UDCA.
UDCA is a naturally occurring bile acid present in small
amounts in human bile.1, 2 Orally administered UDCA
reduces intestinal absorption of cholesterol and biliary
cholesterol secretion thus producing a state of reduced
saturation of cholesterol in bile, which in patients with
cholesterol gallstones leads to gradual dissolution of the
stone.1 The suspension and capsules of UDCA were
equally effective in reducing the HI of bile.
In conclusion, this study confirms that a new liquid
formulation of UDCA at the usual therapeutic dosage of
750 mg/day has comparable bioavailability and phar-
macokinetics with that of a reference solid capsulated
formulation and based on measures of AUC of the
plasma UDCA concentration profiles and biliary UDCA
enrichment the two formulations were bioequivalent.
Equivalence in the physiological effects on biliary HI
was also observed and the suspension was well-
tolerated and generally accepted without side-effects.
The availability of a liquid UDCA formulation will

2005 Blackwell Publishing Ltd, Aliment Pharmacol Ther 21, 709–721
greatly facilitate the treatment of paediatric patients
where solid forms of the drug are only available.
ACKNOWLEDGEMENTS
This study was supported by a grant from Dr Falk
Pharma, Freiburg, Germany.
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