A Randomized Controlled Trial of High-dose Ursodesoxycholic Acid

8/3/2019 A Randomized Controlled Trial of High-dose Ursodesoxycholic Acid

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A randomized controlled trial of high-dose ursodesoxycholic acidfor nonalcoholic steatohepatitis

Vlad Ratziu1,⇑, Victor de Ledinghen2,, Fréderic Oberti3,, Philippe Mathurin4,,Claire Wartelle-Bladou5,, Christophe Renou6, Philippe Sogni7, Marianne Maynard8,

Dominique Larrey9, Lawrence Serf aty10, Dominique Bonnefont-Rousselot11, Jean-Philippe Bastard12,Marc Rivière13, Jean Spénard13,14 on behalf of the FRESGUN

1Université Pierre et Marie Curie, Assistance Publique – Hôpitaux de Paris, Hôpital Pitié Salpêtrière, Inserm UMR_S 938, Paris, France; 2Hôpitaldu Haut Leveque, Pessac Bersol, France; 3Centre Hospitalier Universitaire d’Angers, Angers, France; 4Hôpital Claude Huriez, Lille, France; 5CHG du

Pays d’Aix, Aix en Provence, France; 6CHG d’Hyères, Hyères, France; 7Hôpital Cochin, Paris, France; 8Hôpital Hotel-Dieu, Lyon, France; 9CHU Montpellier, Montpellier, France; 10Hôpital Saint-Antoine, Paris, France; 11Service de Biochimie Métabolique Hôpital Pitié Salpêtrière, France, and

EA 4466, Faculté de pharmacie, Université Paris Descartes, France; 12Hôpital Tenon, Paris, France; 13 Axcan Pharma, Inc. Quebec, Canada;14Université de Montréal, Quebec, Canada

Background & Aims: Nonalcoholic steatohepatitis (NASH) is aprevalent liver disease associated with increased morbidity and

mortality. Ursodeoxycholic acid (UDCA) may have antioxidant,

anti-inflammatory, and antifibrotic properties and may reduce

liver injury in NASH. To date, no studies have assessed the effi-

cacy and safety of high-dose UDCA (HD-UDCA) in patients with

NASH.

Methods: We conducted a 12-month, randomized, double-blind,

placebo-controlled multicenter trial to evaluate the efficacy and

safety of HD-UDCA (28–35 mg/kg per day) in 126 patients with

biopsy-proven NASH and elevated alanine aminotransferase

(ALT) levels. The primary study end point was reduction in ALT

levels from baseline in patients treated with HD-UDCA compared

with placebo. Secondary study end points were the proportion of patients with ALT normalization, relative reduction in the scores

of serum markers of fibrosis and hepatic inflammation, and safetyand tolerability.

Results: HD-UDCA significantly reduced mean ALT levelsÀ28.3%

from baseline after 12 months compared with À1.6% with pla-

cebo ( p <0.001). At the end of the trial, ALT levels normalized

(635 IU/L) in 24.5% of patients treated with HD-UDCA and in

4.8% of patients who received placebo ( p = 0.003). Both results

were not accounted for by changes in weight during the trial.

HD-UDCA significantly reduced the FibroTestÒ serum fibrosis

marker ( p <0.001) compared with placebo. HD-UDCA also signif-icantly improved markers of glycemic control and insulin resis-

tance. There were no safety issues in this population.

Conclusions: Treatment with HD-UDCA was safe, improved

aminotransferase levels, serum fibrosis markers, and selected

metabolic parameters. Studies with histologic end points are

warranted.

Ó 2011 Published by Elsevier B.V. on behalf of the European

Association for the Study of the Liver.

Introduction

Nonalcoholic fatty liver disease is rapidly emerging as the most

prevalent hepatic disorder in the Western world [13], where ithas become the most frequent cause of newly diagnosed cases

of chronic liver disease [58]. This increase in prevalence is mostly

attributable to the worldwide epidemics of obesity and diabetes

mellitus. Nonalcoholic steatohepatitis (NASH), which is part of

the spectrum of nonalcoholic fatty liver disease, is a progressive

condition that, in a minority of patients, can progress to cirrhosis,

end-stage liver disease, and hepatocellular carcinoma [7,46]. Sev-

eral long-term observational studies have shown that NASH sig-

nificantly reduces overall survival in affected patients compared

with age- and gender-matched individuals in the general popula-

tion and increases liver-related mortality 10-fold [1,12,19,53].

Currently, there are no approved medications for the treatment

of NASH, and the effectiveness of nonpharmacologic measuresin this patient population is insufficient [20]. Thus, there is an

unmet medical need for pharmacologic agents that are able tohalt or reverse disease progression.

Ursodeoxycholic acid (UDCA) is a natural, hydrophilic bile

acid that normally constitutes $3% of the human biliary pool [37].

The pharmacologic attributes, immunomodulatory functions, and

direct antiapoptotic properties of UDCA may interfere with the

progression of nonalcoholic fatty liver disease/NASH [6,24,29,

51,52]. UDCA reduces the mitochondrial membrane permeability

Journal of Hepatology 2011 vol. xxx j xxx–xxx

Keywords: Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis; Ami-

notransferases; Steatosis; Fibrosis; Randomized clinical trials; Insulin resistance.⇑ Corresponding author. Fax: +33 142161049.

E-mail address: [email protected] (V. Ratziu). VDL and FO contributed equally in the study. PM and CWB contributed

equally in the study. Members of the FRESGUN (French Study Group for Urso in NASH) are listed in

the Acknowledgements section.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase;

BMI, body mass index; HBA1c, glycosylated hemoglobin; HCC, hepatocellular

carcinoma; ITT, intent-to-treat; NAFLD, nonalcoholic fatty liver disease; NASH,

nonalcoholic steatohepatitis; PP, per-protocol; HD-UDCA, high-dose ursodeoxy-

cholic acid.

Research Article

Please cite this article in press as: Ratziu V et al. A randomized controlled trial of high-dose ursodesoxycholic acid for nonalcoholic steatohepatitis. J

Hepatol (2011), doi:10.1016/j.jhep.2010.08.030

mailto:[email protected]

http://dx.doi.org/10.1016/j.jhep.2010.08.030


mailto:[email protected]



and the release of hydrolytic enzymes from damaged hepatocytes

[23] and improves the resistance to reactive oxygen species [47]

and the expression of antiapoptotic signaling pathways [42].

UDCA also downregulates the production of the pro-inflamma-

tory tumor necrosis factor-a in patients with primary biliary cir-

rhosis [32], reduces serum levels of transforming growth factor-

a, restores defective natural killer cell activity, and may inhibit

fibrosis in patients with NASH [33].Three clinical trials, evaluating the efficacy of UDCA at doses

ranging from 12 to 15 mg/kg per day in patients with NASH, have

yielded variable results. Data from a nonrandomized, 1-year pro-

spective study in 40 patients showed that treatment with UDCA

produced significant improvements in alanine aminotransferase

(ALT) and c-glutamyl transpeptidase (GGT) levels and reduced

hepatic steatosis compared with clofibrate [25]. A 2-year, ran-domized controlled trial in 166 patients showed no differences

in liver function tests or histology between UDCA and placeboat the end of the trial [27]. Data from another 2-year trial, which

randomized patients to treatment with UDCA plus vitamin E,

UDCA alone, or placebo, showed that the combination of UDCA

plus vitamin E significantly reduced ALT levels and steatosis com-

pared with the other two treatments [11].

Many drugs have a dose–response relationship, and treatment

with UDCA at higher doses (20 mg/kg per day) has shown bene-

ficial effects on liver biochemistry and fibrosis in patients with

primary sclerosing cholangitis [31]. Based on these findings, we

hypothesized that higher doses of UDCA (28–35 mg/kg per day)

may have a beneficial effect in NASH. Because of previous contro-

versial results regarding the efficacy of UDCA in NASH, we

designed an exploratory trial with improvement in liver enzymes

and surrogate markers of fibrosis as efficacy endpoints. This

choice of endpoints assumed that only a significant and clinically

relevant improvement in these parameters would provide a solid

rationale for performing a subsequent trial testing histological

improvement.

Patients and methods

Study design

This multicenter, randomized, double-blind, parallel arm, placebo-controlled

phase II study of HD-UDCA was conducted in 15 centers in France between

December 2005 and October 2008. Inclusion criteria were: (1) age P18 years,

(2) increased ALT levels (>50 IU/L) on at least three occasions in the 12 months

preceding the screening, (3) ALT level >50 IU/L measured at screening in the cen-

tralized study laboratory, and (4) liver biopsy, performed within 18 months of

screening, demonstrating histologic changes compatible with NASH (i.e., steato-

sis >20% associated with hepatocyte ballooning and/or hepatic intralobular necro-

sis) after central review by a single pathologist who was blinded to the treatment

assignment.

Exclusion criteria were: (1) more than 1 normal ALT value in the year prior to

screening, (2) presence of steatosis with nonspecific inflammation that was

deemed insufficient for the diagnosis of steatohepatitis by the central pathologi-

cal review, (3) Child–Pugh class B or C cirrhosis, (4) daily alcohol consumption

P20 g in women and P30 g in men, (5) other causes of chronic liver disease,

(6) secondary NASH, (7) treatment with UDCA within the past 12 months, vitamin

E within the past 6 months, or glitazones within the past 3 years prior to screen-

ing, (8) newly instituted antihyperglycemic therapy (i.e., metformin, sulfona-

mides, insulin) within 4 months of screening, (9) loss of P15% of body weight

since liver biopsy, and (10) presence of hepatocellular carcinoma. Pregnant or

breastfeeding women were also excluded from the study.

Patients who satisfied the inclusion and exclusion criteria upon local evalua-

tion were prescreened by having their liver biopsy specimens sent for review by a

central pathologist (FC). Patients in whom the central evaluation confirmed the

histologic diagnosis of NASH were subsequently screened for inclusion (Fig. 1).

Patients (N = 126) were randomized in a 1:1 ratio in blocks of four, to receive

HD-UDCA 28–35 mg/kg per day (500-mg, film-coated, Urso-DSÒ tablets, Axcan

Pharma Inc., Mont-Saint-Hilaire, Quebec, Canada) or placebo for 12 months.

Treatments were administered in divided doses with meals. Patients were

encouraged to lose weight if overweight, to exercise, and to consume a healthy

diet, but no other specific dietary measures were instituted. Patients were advised

to return any unused medications at each study visit to allow calculations of com-

pliance by tablet counts. The study was independently monitored by UMANIS SA

(Levallois-Perret, France) and was conducted in compliance with guidelines for

Good Clinical Practice and with the principles stated in the Declaration of Hel-

sinki. The study protocol was approved by the ethics committees at each of the

participating institutions.

Study end points

The primary end point was relative reduction in ALT at the end of the treatment

period (month 12 [M12]). Secondary end points were the proportion of patients

with ALT normalization (i.e., <35 IU/L), relative reduction in the scores of serum

markers of fibrosis and hepatic inflammation (FibroTestÒ, ActiTestÒ; BioPredic-

tive, Paris, France), and safety and tolerability.

Data collection

Follow-up visits for physical examination and safety assessments were scheduled

every 3 months during treatment (i.e., M3, M6, M9, and M12). Liver function tests

were performed, serum glucose, serum insulin, and glycosylated hemoglobin lev-

els were measured, and serum fibrosis markers (FibroTest and ActiTest) and hyal-

uronic acid studies were performed with the results analyzed at a centralized

laboratory. Normal values for ALT and aspartate aminotransferase were 35 and

32 IU/L, respectively, in men and 27 and 28 IU/L, respectively, in women. Histo-

logic features of NASH were graded and staged according to the method of Kleiner

et al. [22]. Serum levels of adiponectin were determined by enzyme-linked

immunosorbent assay (Quantikine™ adiponectin, R&D Systems, Oxford, UK).

The sensitivity of this assay was 0.25 ng/ml and the inter- and intra-assay varia-

tions were <10%.

Statistical analysis

Clinically significant expected mean differences from baseline ALT, in the HD-

UDCA and placebo arms at the end of treatment, were estimated to be 50% and

20%, respectively. Assuming a = 0.05 and power = 0.91, the estimated sample size

for each study arm, according to the Yates correction method, was 54 patients.

Assuming a 10% withdrawal rate by treatment arm, the inclusion of 60 patients

per treatment arm was planned. Descriptive statistics were provided for each

treatment arm and for the entire population, according to the nature of the vari-

ables (i.e., qualitative or quantitative). Qualitative variables were presented as

actual values and percentages, while quantitative variables were presented as

actual, mean, standard deviation (SD), median, first and third quartiles, maxi-

mum, and minimum. Observations at screening/study entry and demographic

data were provided for the intent-to-treat and safety populations. Differences

from baseline within each treatment arm were calculated and compared for

HD-UDCA versus placebo using Fisher’s exact test (qualitative parameters or

parametric observations that did not meet the assumptions for parametric test-

ing) or Student’s t -test (quantitative parameters).

Results

Study participants

Fig. 1 shows the flow chart of patient participation in the trial.Among the 192 prescreened patients, the central pathologist

did not confirm the histological diagnosis of steatohepatitis in

26. Among the 146 screened patients, 19 no longer satisfied the

increased ALT criteria (>50 IU/L) on centralized measurement,

126 patients were randomized (62 in the HD-UDCA group and

64 in the placebo group) and 116 completed the trial. Baseline

patient characteristics are shown in Table 1. There were no signif-

icant differences between groups except for the proportion of

Research Article



2 Journal of Hepatology 2011 vol. xxx j xxx–xxx





treated arterial hypertension, which was higher, although only

borderline significantly so in the HD-UDCA group. Most patients

were asymptomatic at inclusion and remained so throughout the

course of the study. Asthenia was reported by 30% of patients in

the placebo arm and 48% in the HD-UDCA arm at inclusion,abdominal pain in 9% vs. 15%, and upper right quadrant pain in

6% vs. 19%, respectively.

Throughout the study period, no significant lifestyle changes

were reported in either treatment arm. The proportion of patients

reporting any amount of physical activity was 42% at baseline

and 56% at the end of treatment in the placebo arm ( p = 0.15)

and 44% and 50%, respectively, in the HD-UDCA arm ( p = 0.58).

Alcohol consumption remained stable throughout the study per-

iod in both arms.

Efficacy

Primary outcome

At the end of treatment (M12), the mean ALT reduction (±SD) was

significantly greater in the HD-UDCA arm (À28.3 ± 55.0%) than inthe placebo arm (À1.6 ± 35.4%; p <0.001). The median ALT reduc-

tion was also significantly different at M12 in patients treated

with HD-UDCA compared with placebo (À43.5% vs. À0.4%;

p <0.001). These results were confirmed in the per-protocol pop-ulation at M12, as patients who were treated with HD-UDCA

reported significantly greater median ALT reductions from base-line than patients treated with placebo (À33.9% vs. +3.1%;

p <0.001). Fig. 2 shows the kinetics of the biochemical response

during therapy (i.e., early and sustained ALT reduction in the

HD-UDCA arm, which persisted throughout the treatment

period). The median absolute ALT values at baseline and M3,

M6, M9, and M12 were 85, 62, 59, 58, and 55 IU/L, respectively,

in the HD-UDCA arm, and 81, 79, 83, 88, and 78 IU/L, respectively,

in the placebo arm, confirming the sustained reduction of ALT

that occurred during treatment with HD-UDCA. These resultswere confirmed in the per-protocol population, with a median

relative ALT reduction from baseline of 33.86% vs. a median

increase of 3.06% in the placebo arm ( p <0.001), at M12.

Secondary outcomes

Fig. 3 shows the proportion of patients with ALT normalization at

M6 and M12. At M12, 24.5% of patients treated with HD-UDCA

had normal ALT levels compared with 4.8% of patients who

received placebo ( p <0.003). FibroTest values also changed signif-

icantly during the course of the study. Median decreases were

observed at M6 (À18.0%) and M12 (À10.5%) in patients treated

with HD-UDCA compared with median increases (+3.8% and

+9.6%, respectively) at these time points, in patients who received

placebo ( p <0.006 for both time points).

Fig. 4 shows the absolute mean and relative FibroTest valuesfor all patients, as well as for patients with and without advanced

(bridging) fibrosis at baseline. Patients without advanced fibrosis

seemed to benefit more from HD-UDCA. In these patients, median

FibroTest scores declined by 15% after treatment with HD-UDCA

and increased by 11.2% after they received placebo ( p = 0.001). In

patients with advanced fibrosis, the median FibroTest scoredeclined by 10% in the HD-UDCA arm and increased by 7.6% in

the placebo arm ( p <0.002). Eighteen patients treated with HD-

UDCA reported stable or increased FibroTest values at M12 com-

pared with baseline, while 35 patients in the HD-UDCA treatment

192

146

126

64 PLACEBO62 HD-UDCA

61 PLACEBO55 HD-UDCA

PRESCREENING

SCREENING

RANDOMIZED

(ITT POPULATION)

COMPLETED RX

(PP POPULATION)

26 No NASH on central reading

19 refused to participate

1 high alcohol intake

19 ALT below 50 IU/L

1 exclusion criteria

(corticosteroid intake)

1 withdrew consent at baseline

2 lost to follow-up

2 stopped for AE,5 for personal reasons

Fig. 1. Study participation flow-chart. AE, adverse event; ALT, alanine aminotransferase; HD-UDCA, high-dose ursodeoxycholic acid; ITT, intent-to-treat; NASH,

nonalcoholic steatohepatitis; PP, per-protocol; RX, treatment.

JOURNAL OF HEPATOLOGY



Journal of Hepatology 2011 vol. xxx j xxx–xxx 3





arm reduced their FibroTest values by À5% or more. In these 35

FibroTest responders, the median GGT reduction was 62%.

Despite the unchanged or increased FibroTest values in the 18

FibroTest nonresponders, the median GGT reduction was À37%,

suggesting that the GGT reduction associated with HD-UDCA

accounts for only a portion of the FibroTest decrease.

Other efficacy end points

Aspartate aminotransferase reduction followed a similar trend as

ALT reduction, albeit to a lesser degree. Median aspartate amino-

transferase changes relative to baseline were À19.7% at M3,

À19.2% at M6, À18.6% at M9, and À18.9% at M12 in the HD-UDCA

arm compared with 0%, +7.3%, À2.5%, and 0%, respectively, in the

placebo arm. Treatment with HD-UDCA was associated with a

significant ( p <0.001) reduction in GGT levels (median change,

À62% vs. +15% in the placebo arm; Fig. 5). Alkaline phosphatase,

total bilirubin, and serum hyaluronic acid levels were not signif-

icantly affected. Asthenia improved significantly ( p <0.004) in the

HD-UDCA arm but not the placebo arm (48% of patients in the

HD-UDCA arm reported being tired at baseline compared with

22% at the end of treatment). There was also a trend (baseline

vs. M12) toward improvement of upper right quadrant pain

(19.4% vs. 7.4%; p = 0.10) and bloating (29% vs. 17%; p = 0.13) in

patients treated with HD-UDCA. In the placebo arm, the only sig-

nificant clinical change from baseline to M12 was improvement

in bloating (20% vs. 7%; p = 0.035).

Metabolic response

Patients treated with HD-UDCA experienced significant reduc-

tions in serum glucose, glycosylated hemoglobin, and seruminsulin levels, as well as homeostatic model assessment scores

compared with patients who received placebo (Table 2). High-density lipoprotein cholesterol, low-density lipoprotein choles-

terol, total cholesterol and serum triglycerides were unchanged.

Table 1. Patient characteristics at baseline (ITT/safety population).

Age, yr (SD)

Gender, % males

BMI, kg/m2 (SD)

Waist circumference, cm (SD)Systolic BP, mm Hg (SD)

Diastolic BP, mm Hg (SD)

Type 2 diabetes mellitus, n (%)

Arterial hypertension, n (%)

Dyslipidemia, n (%)

Alcohol consumption

Teetotalers, n (%)

Daily consumption,* n (g)

ALT, IU/L (SD)

AST, IU/L (SD)

GGT, IU/L (SD)

Bilirubin, µmol/L (SD)

Glucose, mmol/L (SD)

Insulin, µUI/L†

HOMA†

HbA1c, % (SD)

HDL-cholesterol, mmol/L (SD)

Liver biopsy

Steatosis grade, n

20%–33%

33%–66%

>66%

NAS score, mean (SD)

3–4, %

5, %

>5, %

Fibrosis stage, %

0

1

2

3

4

49.8 (10.2)

76

30.9 (5.2)

106 (12)134 (12)

82 (8)

24 (39)

30 (48)

39 (85)

40 (65)

13 (10)

109 (70)

61 (31)

122 (148)

12 (6)

5.6 (1.8)

14.5 (15)3.74 (5.50)

6.4 (1.7)

1.26 (0.4)

19

36

45

5.15 (1.17)

31

29

40

7

29

16

40

8

49.6 (12.6)

75

30.9 (5.1)

106 (13)137 (17)

84 (11)

16 (25)

20 (31)

34 (76)

44 (69)

11 (10)

103 (69)

59 (31)

126 (118)

14 (8)

5.5 (1.5)

14.9 (20)3.47 (6.5)

5.9 (0.9)

1.26 (0.3)

12

52

36

4.88 (1.00)

39

33

28

11

23

30

28

8

0.93

0.92

0.92

0.760.22

0.52

0.10

0.05

0.30

0.71

0.67

0.60

0.67

0.86

0.32

0.63

0.930.98

0.51

0.54

0.17

0.29

HD-UDCA(n = 62)

Characteristic Placebo(n = 64)

p

⁄Among drinkers.Median values (interquartile range).

0.0

0.2

0.4

0.6

0.8

1.0

1.2

MO M3 M6 M9 M12

64 61 61 61 62

62 57 57 51 53

Placebo, n

HD-UDCA, n

Placebo

HD-UDCA

M e a n f o l d A L T e l e v a t i o

n f r o m M

0 , m e a n

Fig. 2. ALT levels during treatment. Results are expressed as means ± SD of fold

elevation over baseline values at each time point during treatment with HD-

UDCA (triangles) and placebo (circles). ALT, alanine aminotransferase; HD-UDCA,

high-dose ursodeoxycholic acid; M, month; SD, standard deviation.

Placebo

HD-UDCA

0

5

10

15

20

25

30

0 0

14.3

6.5

24.5

4.8

M0 M6 M12 M0 M6 M12

% o

f p a t i e n t s w i t h N A L T

Fig. 3. Proportion of patients with ALT normalization (<35 IU/L) at end of

treatment (M12). p <0.003 between groups. NALT, normalized alanine amino-

transferase; HD-UDCA, high-dose ursodeoxycholic acid; M, month.

Research Article








There was a moderate but not statistically significant ( p <0.09)

reduction in mean body weight (±SD) in patients treated with

HD-UDCA (À1.65 ± 3.82 kg) compared with placebo (À0.37 ±

4.14 kg), which amounted to a relative change from baseline

(±SD) of À1.75 ± 4.00% and À0.4 ± 4.0% in the HD-UDCA and pla-

cebo arms, respectively. In patients treated with HD-UDCA, the

change in weight did not correlate with changes in serum glucose

levels (r = 0.09; p = 0.54), serum insulin levels (r = 0.03; p = 0.83),

homeostatic model assessment scores (r = À0.08; p = 0.96), or

glycosylated hemoglobin levels (r = 0.11; p = 0.43), suggestingthat the metabolic effects associated with HD-UDCA were not

attributed to weight changes during treatment. Moreover, among

patients with normalized ALT levels at M12, weight loss tended

to be lower in the HD-UDCA arm (n = 13; mean relative change

from baseline ± SD, À3.02 ± 4.90%) than in the placebo arm

(n = 3; À9.26 ± 4.40%; p = 0.06). The same weight loss trend was

reported by patients without ALT normalization in the HD-UDCA

arm (n = 39; À1.34 ± 3.8%) compared with patients in the placebo

arm (n = 58; 0 ± 3.8%; p <0.08), suggesting that ALT normalization

was also not fully attributable to weight loss. There was a trend

toward a more profound reduction of serum adiponectin levelsfrom baseline to M12 in patients given placebo (À22%; 36

patients with paired M12 and M0 measurements; median

change, 0.78 [interquartile range, 0.58]; p = 0.12) than with HD-UDCA (À5%; 29 patients; median change, 0.95 [interquartile

range, 0.61]).

Predictors of response

Multivariate analyses showed that independent predictors of ALT

reduction were treatment with HD-UDCA ( p <0.0001), high base-

line ALT levels ( p = 0.0003), increased age ( p = 0.001), and male

gender ( p <0.02). When an alternate definition of treatment

response was used (i.e., >30% reduction in ALT levels at the end

of treatment compared with baseline), HD-UDCA was associated

with an odds-ratio of 6.2 (95% confidence interval, 2.6–14.8) for

meeting this end point. No histologic features (e.g., steatosis,

fibrosis, nonalcoholic fatty liver disease activity score) were asso-

ciated with ALT reduction.

Safety

There were no deaths, cirrhotic complications (including progres-

sion to liver failure), or reports of hepatocellular carcinoma in the

study. Two patients in the HD-UDCA arm discontinued the treat-

ment because of diarrhea and abdominal discomfort. There was

no difference in the incidence of severe adverse events (AEs) or

intensity of non-severe AEs between the two treatment arms.

Most AEs were emergent but mild. Three patients in the HD-

UDCA arm required dosage changes compared with none in the

placebo arm. The most commonly reported AEs were gastrointes-

tinal (Table 3). Diarrhea and abdominal discomfort occurred

more frequently in the HD-UDCA arm than in the placebo arm.

Discussion

HD-UDCA significantly reduced aminotransferase levels in

patients with histologically proven NASH in this randomized,double-blind placebo-controlled trial, and a quarter of the HD-

UDCA-treated patients normalized ALT during the course of ther-

apy. Although there was a modest, albeit not significant, decrease

in body weight in the HD-UDCA arm, this did not account for the

1.0

0.8

0.6

0.4

0.2

0.0

F

i b r o

T e s

t V a

l u e

% o f

f i b r o

T e s

t r e

d u c

t i o n

f

r o m

M 0

, m e a n

M0 M6 M12 M0 M6 M12

100

-100

75

-75

50

-50

25

-25

0

1.0

0.8

0.6

0.4

0.2

0.0

F i b r o

T e s

t V a

l u e

M0 M6 M12

1.0

0.8

0.6

0.4

0.2

0.0

F i b r o

T e s

t V a

l u e

M0 M6 M12

A B

C D

Fig. 4. Changes in FibroTest values during treatment. (A) Absolute mean

change. (B) Relative mean change. (C) Absolute mean change in patients with

baseline fibrosis. (D) Absolute mean change in patients without baseline fibrosis.

M, month.

PlaceboHD-UDCA

-60

-50

-40

-30

-20

-10

0

10

20

30

-28 (55)

-2 (35)-8 (59)

9 (37)

-51 (28)

19 (48)

ALTM12 vs M0

ASTM12 vs M0

GGTM12 vs M0

-44% vs. 0%Medianreduction

-19% vs. 0%

p <0.001 %

c h a

n g e ,

m e a n

( S D )

-62% vs. 15%

Fig. 5. Biochemical response (LFTs). Mean percent change at end of treatment

(M12) compared with baseline (M0) for ALT, AST, and GGT in the HD-UDCA arm

(black boxes) and placebo arm (white boxes). Negative numbers indicate a

reduction during the trial. ALT, alanine aminotransferase; AST, aspartate amino-

transferase; GGT, c-glutamyl transpeptidase; LFTs, liver function tests; M, month;

SD, standard deviation.

Table 2. Relative change in glycemic parameters and insulin resistance

surrogate markers at M12⁄.

-2.2

-2.3

-19

-20

Serum glucose level

HbA1c level

Insulin level

HOMA score

+3.9

+5.2

-0.2

+6

0.002

<0.001

0.038

<0.009

HD-UDCAParameter Placebo p

⁄Percent relative change between baseline median values and M12.









ALT reduction. HD-UDCA-treated patients who normalized ALTlevels lost only 3% of their body weight, an amount insufficient

to effect ALT normalization. In contrast, the few patients in the

placebo arm who normalized ALT had a weight loss of 9.3%,

which is comparable to currently reported data. ALT reduction

was documented after the first 3 months of treatment and was

sustained throughout the treatment period in patients treated

with HD-UDCA. This is in contrast to the stability of ALT values

over 1 year in patients who received placebo, and furtherstrengthens the assertion that ALT reduction was due to treat-

ment with HD-UDCA. Additional confirmation was provided by

the multivariate analysis, which identified treatment with HD-

UDCA as an independent predictor of ALT reduction.

It is tempting to speculate that reduction in ALT levels reflects

improvement in hepatic inflammation. Data have shown that ALT

(but not GGT or other biologic parameters) is a valid proxy for

evaluating improvement in hepatic necroinflammation. In one

study that compared 2 years of treatment with UDCA to placebo

in 102 patients with NASH, Suzuki et al. showed that, after

adjustment for baseline ALT, rates of ALT change during treat-

ment were significantly correlated with changes in inflammationbut not steatosis or fibrosis [54]. ALT changes alone predicted his-

tologic improvement or worsening, as shown by area under the

receiver operating characteristic curves (0.72–0.77) [54]. There-fore, data from the current trial, showing a strong and sustained

ALT reduction, suggest that HD-UDCA may improve hepatic necr-

oinflammation. This appears biologically plausible as several

in vivo and in vitro actions of UDCA are consistent with hepato-

protective actions. UDCA displays antiapoptotic effects [47,48]

by eliciting survival signals through the stimulation of the MAPK

pathway [42], and pro-apoptotic diseases such as NASH [14]

could benefit from such antiapoptotic activity. A small pilot study

showed that UDCA reduces the number of apoptotic hepatocytes

by almost half [4]. UDCA could also improve hepatic inflamma-

tion by altering the metabolism of arachidonic acid [18]. Further,

effects such as membrane stabilization, changes in hepatic lipid

content [5], and immunomodulation may also result in cytopro-

tection from oxidative stress and cell death. Unfortunately, these

experimental data and the results from this study are not direct

evidence of a histologic benefit. While only well-conducted trialswith histologic end points can provide a formal demonstration of

the benefits of HD-UDCA, the data presented herein provide the

proof of principle on which future trials may be based.

Biochemical markers of liver fibrosis have been extensively

validated in patients with viral hepatitis including longitudinal

validation in therapeutic trials [38,40,41] where changes in Fibro-

Test scores correlated with changes in histologic stages of fibrosis

[39]. Specific validations in patients with NASH are available for

some of these markers [3,8,15,45]. Data from the current study

showed that patients treated with HD-UDCA (but not placebo)experienced a significant reduction in FibroTest scores. Arguably,

at least part of the FibroTest decrease could have been due to

reductions in GGT (one of the components of the FibroTest score),

although HD-UDCA reduced GGT levels by a median of 32% even

in patients with stable or increased FibroTest values. While this

suggests that the reduction in GGT did not fully account for thereduction in FibroTest scores, additional demonstration of the

potential antifibrotic effects of HD-UDCA is needed, using differ-

ent markers that are not based on GGT values. Serum hyaluronic

acid levels did not change during this trial. However, this marker

has insufficient diagnostic performance, in particular in the pre-

cirrhotic stage.

Data on the efficacy of UDCA in NASH are limited and contra-

dictory. In the largest randomized trial conducted to date, Lindor

et al. showed that treatment with UDCA over 2 years reduced ALTlevels to the same extent as placebo [27]. There was no histologic

benefit of treatment except for improvement in steatosis in 46%of UDCA-treated patients and 37% of patients receiving placebo.

Remarkably, the magnitude of the ALT reduction in patients trea-

ted with UDCA in that study was similar to data from the present

trial, although the high rate of ALT and steatotic improvement

reported in the placebo arm in the study by Lindor et al. stands

in sharp contrast to the results of the current and previous trials

[11,44], where these changes were minimal. It is unclear whether

methodologic issues or study conduct might explain the results

observed in the Lindor et al. trial. There was a high dropout rate

in the UDCA arm (only 56 of 80 patients had available end-of-

treatment data) and a quarter of randomized patients did not

complete the trial (half due to AEs, a percentage that is much

higher than in the present study). In a second randomized trial,

Dufour et al. did not show any significant benefit of UDCA over

placebo on ALT reduction, although the combination of UDCA

and vitamin E was beneficial [11]. While that study was under-

powered (17 or less patients per arm), patients in the UDCA

arm experienced a mean reduction of 35 IU/L even though base-line ALT levels were higher in the UDCA arm than in the placebo

arm. Finally, in an older, controlled, but not randomized study,Laurin et al. first suggested a beneficial effect of UDCA on ALT lev-

els and steatosis [25]. While these trials provide conflicting

results, they were all conducted with a standard dose of UDCA

(13 to 15 mg/kg). We hypothesized that a higher dose might

result in a more marked biologic effect based on results from tri-

als in diseases other than NASH. In patients with primary scleros-

ing cholangitis, bile enrichment with UDCA increases from 43%,

at doses of 10–13 mg/kg, to 58%, at doses of 26–32 mg/kg [49].

It remains to be demonstrated in vivo whether the other cytopro-

tective effects of UDCA suspected to be beneficial in NASH are

also dose dependent. Currently available data from trials of HD-

UDCA are conflicting. A benefit has been reported in some trials

in patients with primary sclerosing cholangitis [10,16,31] butnot in others [28]. Further, positive results have been reported

in patients with cystic fibrosis [9,56] but not with primary biliarycirrhosis [2]. Recently, Leuschner et al. presented the results of an

18-month randomized trial of higher doses of UDCA (23–28 mg/kg) without a beneficial histological effect compared with pla-

cebo [26]. However, the inclusion criteria were complex, mixing

both histological and clinical and biological features. The validity

of the histological features, as a case definition of NASH, was also

questionable because a non-validated composite score was used

Table 3. Emergent AEs occurring in >10% of patients.

24

17

16

15

10

5

3

Diarrhea, n

Abdominal cramps, n

Abdominal discomfort, n

Asthenia, n

Flatulence, n

Back pain, n

Pruritus, n

11

12

7

25

15

7

7

<0.01

0.29

<0.04

0.09

0.37

0.76

0.32

HD-UDCA(n = 62)

Parameter Placebo(n = 64)

p

Research Article








and steatosis was not a compulsory feature. Importantly, akin to

the Lindor trial, but in sharp contrast to the current and other tri-

als, UDCA was unable to reduce ALT levels significantly more

than placebo [43,44]. Therefore, besides non-standardized inclu-

sion criteria and an unusually long inclusion period (6 years), the

understanding of the differences in the course of the placebo arm

might be key to solving the discrepancies between different trial

results. The current trial adds to the data showing an efficacy of HD-UDCA, at least on biochemical parameters in NASH patients.

An unexpected finding from the current trial is the significant

improvement in some metabolic parameters, including glycemic

control, fasting insulin levels, and surrogate markers of insulin

resistance. The magnitude of this benefit could not be explained

by the minimal, non-significant weight loss experienced by

patients in the HD-UDCA arm. If this effect is confirmed, it mayoffer an explanation for the improvements in steatosis that were

reported in some UDCA trials. Interestingly, a much smaller trialdid not find any effect of UDCA alone on serum glucose, serum

insulin, or adiponectin [4], which directly challenges the results

obtained here. More data are necessary but the results from the

present trial open the field of the metabolic actions of UDCA to

further investigation. A biologic explanation for our results is

not readily available. UDCA reduces serum levels of tumor necro-

sis factor-a [32] a pro-oxidant cytokine that promotes insulin

resistance [17,55]. Standard-dose UDCA is not a potent modulator

of TGR5 [21,50], farnesoid X receptor-a [30,35], or the small hete-

rodimeric partner [57], which are all important regulators of lipid

and glucose metabolism. It seems improbable, although it has not

yet been disproved, that higher doses of UDCA may result in a sig-

nificant modulation of these nuclear/cellular receptors. On the

other hand, taurine-conjugated UDCA, acting as a chemical chap-

erone, alleviates the endoplasmic reticulum stress [59] that occurs

in obesity, improves insulin action in the liver, restores systemic

insulin sensitivity, and clears fatty liver in rodents [34]. Whether

HD-UDCA results in similar actions remains to be proven. Data

presented here certainly provide a rationale for a further explora-

tion of beneficial metabolic effects of HD-UDCA.An important finding of this study is the excellent safety pro-

file of HD-UDCA in patients with NASH. Only two patients had tostop taking the drug (due to digestive AEs) and no worsening of

liver function was reported. Previous studies testing high doses

of UDCA (20–32 mg/kg) did not show any new or severe AEs,

and the overall tolerance to HD-UDCA was similar to that of reg-

ular doses [2,10,16]. However, a recent trial of high-dose UDCA

did show an unanticipated and high rate of progression of liver

disease in patients with primary sclerosing cholangitis [28].

Moreover, in alcoholic cirrhosis with jaundice, the standard-dose

UDCA treatment increased the rate of cirrhosis complications

over placebo, although part of this deleterious effect may not

have been independent of the baseline severity of liver disease

[36]. These adverse outcomes were not observed in the current

study, where participants did not have jaundice, hepatic insuffi-ciency, decompensated cirrhosis, or obstructive biliary disease.

HD-UDCA appears safe for use in patients with NASH without

jaundice or decompensated cirrhosis.

In conclusion, this study demonstrates that HD-UDCA, 28–35 mg/kg per day, is safe and well-tolerated in patients with

NASH. Treatment with HD-UDCA resulted in a strong and sus-

tained reduction of ALT levels, suggesting that the numerous

hepatoprotective actions of this molecule, which were previously

documented in experimental/preclinical models, may result in an

improvement of hepatic cell injury and inflammation in people

with NASH. The potency of HD-UDCA in reducing biochemical

surrogates of liver fibrosis in NASH awaits confirmatory data with

different noninvasive markers. Unexpectedly, HD-UDCA resulted

in beneficial effects on glycemic control and insulin sensitivity

contingent to putative, although yet uncharacterized metabolic

actions and not mere weight loss. The benefits reported with

HD-UDCA in this trial support the initiation of larger studies withhistologic end points in patients with NASH.

Conflict of interest

This trial was sponsored and funded by Axcan Pharma S.A. V. Rat-

ziu is a consultant to Astellas Pharma, Axcan Pharma, Gilead Sci-

ences, Genentech, Intercept Pharmaceuticals, and sanofi-aventis.

Acknowledgments

The authors wish to thank the following practitioner and other

staff involved in the FRESGUN (French Study Group for Urso in

NASH):Si Nafa Si Ahmed (CHR La Source, Orléans), S. Ben Ali (Hôpital

Saint-Joseph, Marseille), R. Anty (Hôpital de l’Archet II, Nice), F.

Bailly (Hôpital HOTEL DIEU, Lyon), C. Bernhardt (Hôpital Pitié-

Salpêtrière, Paris), M. Bourlière (Hôpital Saint-Joseph, Mareseille),

J. Boursier (CHU d’Angers, Angers), J.P. Bronowicki (Hôpital Bra-

bois, Nancy), C. Bureau (Hôpital PURPAN, Toulouse), V. Canva

(Hôpital Huriez, Lille), X. Causse (CHR La Source, Orléans), F.

Charlotte (Hôpital Pitié-Salpêtrière, Paris), O. Chazouillères

(Hôpital Saint-Antoine, Paris), S. Darhancy (Hôpital Huriez, Lille),

Y. Deugnier (Hôpital Pontchaillou, Rennes), N. Dib (CHU d’Angers,

Angers), M. Doffoel (Hôpital Civil-Strasbourg), J. Foucher (Hôpital

du Haut-Levêque, Bordeaux), F. Habersetzer (Hôpital Civil, Stras-

bourg), F. Lainé (Hôpital Pontchaillou, Rennes), P. Lebray (Hôpital

Pitié-Salpêtrière, Paris), S. Métivier (Hôpital PURPAN, Toulouse),

J.B. Nousbaum (CHU La Cavale Blanche, Brest), V. Oulès (HôpitalSaint-Joseph, Marseille), J.M. Péron (Hôpital PURPAN, Toulouse),M. Picon-Coste (CHG du Pays d’Aix, Aix en Provence), P. Podevin

(Hôpital COCHIN, Paris), F. Tanné (CHU La Cavale Blanche, Brest),

A. Tran (Hôpital de l’Archet II; Nice).

The authors also wish to thank P. Colin and G. Chauvière for their

input in the design of this trial, M. Mouret for statistical expertise,

and J. Wert for editorial assistance in the production of this man-

uscript, which were sponsored by Axcan Pharma Inc.

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A Randomized Controlled Trial of High-dose Ursodesoxycholic Acid