Efficacy and Safety of the Farnesoid X Receptor Agonist Obeticholic Acid in Patients With Type 2 Diabetes and Nonalcoholic Fatty Liver Disease

GASTROENTEROLOGY 2013;145:574–582

CLINICAL—LIVER Efficacy and Safety of the Farnesoid X Receptor Agonist Obeticholic Acid in Patients With Type 2 Diabetes and Nonalcoholic Fatty Liver Disease SUNDER MUDALIAR,1 ROBERT R. HENRY,1 ARUN J. SANYAL,2 LINDA MORROW,3 HANNS–ULRICH MARSCHALL,4 MARK KIPNES,5 LUCIANO ADORINI,6 CATHI I. SCIACCA,7 PAUL CLOPTON,1 ERIN CASTELLOE,7 PAUL DILLON,8 MARK PRUZANSKI,6 and DAVID SHAPIRO7 1

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Center for Metabolic Research, VA San Diego Healthcare System and University of California, San Diego, San Diego, California; 2Virginia Commonwealth University, Richmond, Virginia; 3Profil Institute for Clinical Research, Chula Vista, California; 4Department of Molecular and Clinical Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 5Diabetes and Glandular Clinic, San Antonio, Texas; 6Intercept Pharmaceuticals Inc, New York, New York; 7 Intercept Pharmaceuticals, Inc, San Diego, California; and 8Siemens Healthcare Diagnostics, Inc, Tarrytown, New York

See editorial on page 508. BACKGROUND & AIMS: Obeticholic acid (OCA; INT-747, 6a-ethyl-chenodeoxycholic acid) is a semisynthetic derivative of the primary human bile acid chenodeoxycholic acid, the natural agonist of the farnesoid X receptor, which is a nuclear hormone receptor that regulates glucose and lipid metabolism. In animal models, OCA decreases insulin resistance and hepatic steatosis. METHODS: We performed a double-blind, placebocontrolled, proof-of-concept study to evaluate the effects of OCA on insulin sensitivity in patients with nonalcoholic fatty liver disease and type 2 diabetes mellitus. Patients were randomly assigned to groups given placebo (n ¼ 23), 25 mg OCA (n ¼ 20), or 50 mg OCA (n ¼ 21) once daily for 6 weeks. A 2-stage hyperinsulinemiceuglycemic insulin clamp was used to measure insulin sensitivity before and after the 6-week treatment period. We also measured levels of liver enzymes, lipid analytes, fibroblast growth factor 19, 7a-hydroxy-4-cholesten-3-one (a BA precursor), endogenous bile acids, and markers of liver fibrosis. RESULTS: When patients were given a low-dose insulin infusion, insulin sensitivity increased by 28.0% from baseline in the group treated with 25 mg OCA (P ¼ .019) and 20.1% from baseline in the group treated with 50 mg OCA (P ¼ .060). Insulin sensitivity increased by 24.5% (P ¼ .011) in combined OCA groups, whereas it decreased by 5.5% in the placebo group. A similar pattern was observed in patients given a high-dose insulin infusion. The OCA groups had significant reductions in levels of g-glutamyltransferase and alanine aminotransferase and dose-related weight loss. They also had increased serum levels of low-density lipoprotein cholesterol and fibroblast growth factor 19, associated with decreased levels of 7a-hydroxy-4-cholesten-3-one and endogenous bile acids, indicating activation of farnesoid X receptor. Markers of liver fibrosis decreased significantly in the group treated with 25 mg OCA. Adverse experiences were similar among groups. CONCLUSIONS: In this phase 2 trial, administration of 25 or 50 mg OCA for 6 weeks

was well tolerated, increased insulin sensitivity, and reduced markers of liver inflammation and fibrosis in patients with type 2 diabetes mellitus and nonalcoholic fatty liver disease. Longer and larger studies are warranted. ClinicalTrials.gov, Number: NCT00501592. Keywords: Clinical Trial; Metabolic Syndrome; Treatment; Obesity.

T

ype 2 diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are components of the metabolic syndrome, a cluster of interrelated clinical features including insulin resistance, dyslipidemia, hypertension, and visceral obesity.1 The prevalence of type 2 diabetes mellitus is increasing worldwide and is projected to affect approximately 8% of the population by 2030.2 NAFLD is currently the most prevalent chronic liver disease, affecting 20%–40% of the population, and approximately 30% of patients with NAFLD will progress to nonalcoholic steatohepatitis (NASH).3 Type 2 diabetes mellitus and NAFLD are major health issues associated with the worldwide epidemic of obesity.4 Insulin resistance plays a major role in the pathogenesis of type 2 diabetes mellitus and NAFLD and is considered a key factor in the initiation and perpetuation of NASH.5 Although several drugs are available to improve insulin resistance in diabetes, none are currently approved for NAFLD or NASH.6 Given the role of insulin resistance in the pathogenesis of NASH, insulin sensitizers such as the thiazolidinediones have been extensively tested, showing significantly reduced liver inflammation and

Abbreviations used in this paper: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BA, bile acid; C4, 7a-hydroxy-4-cholesten-3one; FGF, fibroblast growth factor; FXR, farnesoid X receptor; GIR, glucose infusion rate; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; OCA, obeticholic acid. © 2013 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2013.05.042

steatosis but modest efficacy in the control of liver fibrosis in patients with NASH.7 Moreover, these compounds are associated with a significant increase in weight and concerns have been raised about the cardiovascular safety of rosiglitazone, although pioglitazone has been shown to improve all-cause mortality, including cardiovascular outcomes.8 In addition, long-term (5 years) therapy with thiazolidinediones in patients with type 2 diabetes mellitus may be associated with an increased risk of bladder cancer.9 Bile acids (BAs), classically recognized as detergent-like compounds involved in lipid absorption and cholesterol homeostasis,10 have more recently been shown to modulate several metabolic pathways that regulate glucose, lipid, and energy homeostasis by targeting the farnesoid X receptor (FXR)11 and the G protein–coupled receptor TGR5.12 In addition, FXR and TGR5 mediate antiinflammatory and antifibrotic properties,13,14 making them promising targets for the treatment of a number of metabolic and liver diseases. FXR, a member of the nuclear receptor superfamily, is mainly expressed in liver, intestine, kidney, and, to a lesser extent, adipose tissue. It regulates a wide variety of target genes critically involved in the control of BA synthesis and transport, lipid metabolism, and glucose homeostasis.11 In particular, FXR controls glucose metabolism through regulation of gluconeogenesis and glycogenolysis in the liver, as well as regulation of peripheral insulin sensitivity in striated muscle and adipose tissue,15–18 suggesting potential beneficial effects of FXR agonists in patients with diabetes and NAFLD.19 BAs have also been shown to induce insulin secretion by perfused pancreatic islets via an FXR activation mechanism.20 Finally, treatment with the synthetic FXR agonist GW4064 reduced insulin resistance in ob/ob and db/db mice,15,17 indicating that activation of FXR promotes insulin sensitivity. A BA-induced mechanism promoting insulin sensitivity relies on FXR-mediated production of fibroblast growth factor (FGF) 19 (and its mouse orthologue FGF15), an enterokine released by the ileal enterocyte that activates the cognate receptor FGFR4 in the liver to suppress CYP7A1/CYP8B1 expression, thus leading to reduced production of BAs.21,22 Treatment with FGF19 improves indices of dyslipidemia, hepatic steatosis, hyperinsulinemia, hyperleptinemia, and insulin sensitivity while reducing body weight and adiposity in mice fed a high-fat diet and ob/ob mice.23 In addition, treatment with FGF19 decreases hepatic triglyceride and free fatty acid levels as well as serum alanine aminotransferase (ALT) levels in FXR-deficient mice, ameliorating dysregulated hepatic lipogenesis due to absent FXR signaling.24 Treatment with FGF19 also restores glycogen loss in insulin-deficient diabetic animals, activating an insulin-independent endocrine pathway.25 Obeticholic acid (OCA; INT-747), a 6a-ethyl derivative of chenodeoxycholic acid, is a first-in-class selective FXR agonist with anticholestatic and hepatoprotective properties.26 OCA shows 100-fold greater FXR agonistic activity

FXR AND OCA IN DIABETES AND NAFLD 575

than chenodeoxycholic acid, the natural FXR agonist in humans.27 OCA does not activate other nuclear receptors,28 and its activities appear to be mediated only by FXR.29–31 A range of preclinical studies have shown that OCA increases insulin sensitivity and regulates glucose homeostasis, modulates lipid metabolism, and exerts antiinflammatory and antifibrotic effects in the liver, kidney, and intestine, the principal FXR-expressing organs.32 In light of the increasing preclinical evidence for the therapeutic potential of FXR agonists in the regulation of glucose and lipid metabolism, OCA was tested in a proof-ofconcept study in patients with type 2 diabetes mellitus and NAFLD and is reported here. To our knowledge, OCA is the first specifically designed FXR agonist to enter phase 2 clinical trials. This placebo-controlled study evaluated 2 doses of OCA, 25 and 50 mg, administered once daily for 6 weeks. The hyperinsulinemic-euglycemic clamp technique, considered the gold standard method to assess insulin sensitivity, was used to determine glucose infusion rate (GIR), the primary end point of the study. Several secondary end points were also assessed, including liver enzyme levels (because aminotransferase levels are related to the risk of allcause mortality)33 and FXR agonistic effects on FGF19, 7ahydroxy-4-cholesten-3-one (C4), and endogenous BA levels.

Patients and Methods Patients Patients with type 2 diabetes mellitus and NAFLD were enrolled in the study. The diagnosis of type 2 diabetes mellitus was based on the standard American Diabetes Association criteria.34 After other causes of liver disease were ruled out (including history or presence of hepatitis B virus, hepatitis C virus, primary biliary cirrhosis, or primary sclerosing cholangitis), presumed NAFLD was defined by one or more of the following criteria: ALT level 47 U/L for female and 56 U/L for male patients, aspartate aminotransferase (AST) level 47 U/L for female and 60 U/L for male patients, enlarged liver (shown by ultrasonography or other imaging technique), and diagnostic histologic findings shown on prior biopsy (in the prior 5 years). Exclusion criteria were highly elevated plasma AST (>155 U/L in female and >200 U/L in male patients) or ALT levels (>155 U/L in female and >185 U/L in male patients), bilirubin level greater than 2 times the upper limit of normal range, use of antidiabetic medications except for metformin or sulfonylureas, history or presence of alcohol abuse (defined as consumption of more than 210 mL of alcohol per week) or other substance abuse within the prior 2 years, and significant heart or renal disease. Written informed consent was obtained from all patients, which was approved by local institutional review boards. All authors had access to the study data and had reviewed and approved the final manuscript.

Study Design This was a multicenter, double-blind, randomized, placebo-controlled, multiple-dose, parallel-group exploratory study to evaluate the safety and efficacy of OCA on glucose and lipid homeostasis as well as on serum BA levels and markers of hepatic inflammation and fibrosis. Patients who met all inclusion and exclusion criteria were randomly assigned to receive 25 mg OCA, 50 mg OCA, or a matching placebo orally once daily

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for 6 weeks. Placebo and OCA supplies were provided by Intercept Pharmaceuticals, Inc. Eligible patients were assigned a 3-digit patient randomization number. This number was preprinted on the patient drug kit according to the master randomization schedule. The drug kit was dispensed by the site pharmacists. Randomized patient kits were packaged in blocks of 6; each block of 6 kits included 2 patient kits for each dose group (placebo, 25 mg OCA, and 50 mg OCA). Patients were admitted for an overnight fast and underwent a 2-step hyperinsulinemic-euglycemic clamp procedure performed both before the first and after the last dose of study treatment. Patients were required to stop the study if they developed significantly elevated ALT or AST levels (3 times the average pretreatment values) or an elevated bilirubin level 2 times the average pretreatment value. CLINICAL LIVER

Primary Outcomes The patients received a primed constant intravenous infusion of regular human insulin (Humulin, U 100; Eli Lilly, Indianapolis, IN) during the 2-step euglycemic clamp procedure as previously described.35 In the first step of the clamp, a lowdose submaximal insulin infusion rate (60 mU  m2 body surface area/min) was used for 180 minutes; in the second step, a high-dose insulin infusion rate (120 mU  m2 body surface area/ min) was used for an additional 120 minutes to maximally suppress endogenous glucose production and maximally stimulate peripheral glucose uptake. Throughout the clamp periods, blood glucose measurements were taken every 5 to 10 minutes and the glucose infusion rate was adjusted to maintain target euglycemia (approximately 95 mg/dL). At one site, the Biostator device (Life Science Instruments, Elkhart, IN) was used.36

Secondary Outcomes Serum FGF19 concentrations were assayed using the solidphase Quantikine FGF19 Immunoassay (R&D Systems, Minneapolis, MN). Serum C4 levels were determined by high-performance liquid chromatography as described.37 Serum BAs were analyzed using high-performance liquid chromatography/tandem mass spectrometry as described.38 All of these analytes were determined in fasting patients. Markers of liver fibrosis were assayed by iQur Ltd. (London, England) using the Enhanced Liver Fibrosis (ELF) test (Siemens Healthcare Diagnostics Inc, Tarrytown, NY). The ELF test combines, in an algorithm, measurements from assays for hyaluronic acid, procollagen III amino terminal peptide, and tissue inhibitor of metalloproteinase 1 to generate an ELF score that correlates strongly to the extent of liver fibrosis and/or cirrhosis.39 Caspase-cleaved keratin-18 was measured using the M30Apoptosense enzyme-linked immunosorbent assay (Peviva AB, Stockholm, Sweden).

Statistical Analysis The primary efficacy end point, change in GIR from predose baseline to posttreatment, was compared between the placebo and OCA treatment groups separately for both low-dose and high-dose insulin infusion periods using t tests for independent samples. Given the small sample size of this proofof-concept study, statistical analysis was also performed for the 2 OCA doses combined. FGF19, C4, and endogenous BA levels were summarized using descriptive statistics. ELF markers (score and individual components) were analyzed using the t test for independent samples. Safety variables were summarized with descriptive statistics. The Kruskal–Wallis test, a

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nonparametric analysis of variance using Wilcoxon scores, was conducted to test overall differences among treatment groups, considering absolute changes for the key analytes. Pairwise comparisons were performed using a 2-sided Wilcoxon– Mann–Whitney test. A 2-tailed P value of <.05 was considered statistically significant.

Results Study Patients Sixty-four patients were enrolled at 4 centers in the United States: Veterans Administration San Diego Healthcare System in San Diego, California (n ¼ 22), Diabetes and Glandular Diseases Clinic in San Antonio, Texas (n ¼ 7), Virginia Commonwealth University School of Medicine in Richmond, Virginia (n ¼ 4), and Profil Institute for Clinical Research in Chula Vista, California (n ¼ 31). Twenty-three patients were randomized to placebo, 20 were randomized to treatment with 25 mg OCA, and 21 were randomized to treatment with 50 mg OCA. Demographics and baseline characteristics are shown in Table 1, and patient disposition is summarized in Supplementary Figure 1. The 3 groups were well matched with respect to demographics, clinical and laboratory parameters, and NAFLD diagnosis inclusion criteria (Table 1). Most enrolled patients completed the study (56/64; 88%). Of the 8 patients who discontinued, the most frequently reported reason was protocol violation (4/64; 6%). Two patients (one each in the placebo and 50 mg OCA groups) developed significantly elevated ALT or AST levels (3 times the average pretreatment values). Two additional patients discontinued, both in the 50 mg OCA group: one due to withdrawal of consent and the other due to loss of follow-up (Supplementary Figure 1). Only patients who had both satisfactory baseline and endof-treatment euglycemic clamps were included in the GIR analysis (44/64; 69%) ( Supplementary Figure 1). Fourteen patients were excluded from the GIR analysis because of an error in the calculation of the GIR at a single site, which resulted in a significantly reduced dose of insulin infused during the clamp procedure.

Primary Outcomes Evaluation of efficacy. The results of GIR for lowdose and high-dose insulin infusion rates are shown in Table 2 as mean absolute change and mean percent change at day 43 versus day 0. At the low-dose insulin infusion rate, the mean percent change in GIR showed an improvement of 28.0% (40.2%) and 20.1% (32.6%) for the 25 mg and 50 mg OCA groups compared with a decrease of 5.5% (35.9%) in the placebo group (P ¼ .019 and P ¼ .060, respectively). For both OCA groups combined, the GIR increased by 24.5% compared with placebo (P ¼ .011). A similar pattern was observed at the high-dose insulin infusion rate. At the low-dose insulin infusion rate, the mean absolute change in GIR increased by 0.69 mg ∙ kg1 ∙ min1 and 0.24 mg ∙ kg1 ∙ min1 for the 25 mg and

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Table 1. Demographic and Baseline Characteristics of the Study Patients Treatment group Sex, n (%) Male Female Ethnicity, n (%) White Black Asian Hispanic Age (y) Mean  SD Range Body weight (kg) Mean  SD Range Body mass index (kg/m2) Mean  SD Range NALFD diagnosis inclusion criteria, n (%)a Ultrasonography/imaging Increased liver transaminase levels (ALT/AST) Liver biopsy Glucose and glycosylated hemoglobin Glucose (mg/dL) Glycosylated hemoglobin (%) Concomitant medications, n (%)a Biguanides HMG-CoA reductase inhibitors Angiotensin-converting enzyme inhibitors Sulfonamides a

Placebo (n ¼ 23)

25 mg OCA (n ¼ 20)

50 mg OCA (n ¼ 21)

10 (43) 13 (57)

14 (70) 6 (30)

9 (43) 12 (57)

11 5 3 4

(48) (22) (13) (17)

8 6 0 6

(40) (30) (0) (30)

8 7 0 6

(38) (33) (0) (29)

53.1  12.1 23.0–70.0

52.7  8.7 40.0–69.0

50.5  10.8 29.0–72.0

104.2  25.6 72.9–178.5

108.6  23.0 68.1–153.1

106.4  25.1 75.1–167.9

36.1  7.4 27.9–54.4

36.5  6.2 26.9–49.7

36.5  7.9 26.7–56.8

19 (83) 5 (22) 0 (0)

17 (85) 3 (15) 0 (0)

18 (86) 5 (24) 2 (10)

159  43 7.6  1.2

149  34 7.4  1.3

132  37 7.0  1.4

19 9 6 3

(83) (39) (26) (13)

16 9 5 7

(80) (45) (25) (35)

18 8 9 6

(86) (38) (43) (29)

Patients could be counted in more than one category.

50 mg OCA groups compared with a decrease of 0.51 mg ∙ kg1 ∙ min1 in patients receiving placebo (P ¼ .040 and P ¼ .278, respectively). The OCA treatment groups combined showed a significant increase in GIR (P ¼ .048) compared with placebo. A similar pattern was observed at the high-dose insulin infusion rate. Adverse events. Fourteen patients (61%) who received placebo, 9 patients (45%) treated with 25 mg OCA, and 16 patients (76%) treated with 50 mg OCA experienced at least one adverse event (Table 3). The incidence of all adverse events occurring in >1 patient are reported in Supplementary Table 1. Four severe adverse events occurred in 2 patients, one in the placebo and the other in the 50 mg

OCA group, who experienced an increased ALT or AST level and met protocol-mandated discontinuation criteria. These adverse events were considered by the investigator to be possibly related to study treatment; one resolved at the end of the study (50 mg OCA), and the other (placebo) was lost to follow-up. One patient (5%) treated with 25 mg OCA and 8 patients (38%) treated with 50 mg OCA experienced at least one adverse event considered by the investigator to be possibly or probably related to treatment compared with 6 patients (26%) who received placebo. Treatmentrelated AEs occurring in >1 patient/group were diarrhea (2 patients in the placebo group) and constipation (4 patients in the 50 mg OCA group).

Table 2. GIR: Low-Dose and High-Dose Insulin Rate Absolute values, mean  SD Insulin dose group/treatment groupa 1

b

Day 43

Absolute change Mean  SD

P valueb

Percent change Mean  SD

P valueb

1

Low-dose insulin (mg ∙ kg ∙ min ) Placebo (n ¼ 17) OCA 25 mg (n ¼ 15) OCA 50 mg (n ¼ 12) 25 mg and 50 mg combined (n ¼ 27) High-dose insulin (mg ∙ kg1 ∙ min1) Placebo (n ¼ 17) OCA 25 mg (n ¼ 15) OCA 50 mg (n ¼ 12) 25 mg and 50 mg combined (n ¼ 27) a

Day 0 3.96 3.04 4.04 3.48

   

2.25 0.83 2.72 1.93

3.45 3.73 4.27 3.97

   

1.55 1.14 1.81 1.47

0.51 0.69 0.24 0.49

   

1.88 1.12 1.62 1.36

.040 .278 .048

5.5 28.0 20.1 24.5

   

35.9 40.2 32.6 36.6

.019 .060 .011

7.14 6.43 7.23 6.78

   

2.56 2.13 2.84 2.45

6.53 7.16 7.65 7.38

   

2.27 2.14 2.36 2.21

0.61 0.73 0.42 0.59

   

1.88 1.53 1.42 1.46

.036 .122 .022

5.4 18.3 10.8 15.0

   

24.3 36.3 21.8 30.4

.036 .076 .025

Only patients who had both baseline and end-of-treatment valid results were included in the analysis. P value; t test for equality of means.

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Table 3. Summary of Treatment-Emergent AEs by Treatment Group Treatment group Placebo (n ¼ 23) Patients with any AEs, n (%) Patients with treatment-related AE, n (%)a Patients with serious AEs, n (%) Patient deaths, n (%) Patients who withdrew due to an AE, n (%) AE reports (entries)

OCA 25 mg (n ¼ 20)

14 (61) 6 (26) 0 (0) 0 (0) 1 (4) 29

9 1 0 0 0

OCA 50 mg (n ¼ 21)

Total (N ¼ 64)

16 (76) 8 (38) 0 (0) 0 (0) 1 (5) 35

39 (61) 15 (23) 0 (0) 0 (0) 2 (3) 81

(45) (5) (0) (0) (0) 17

AE, adverse event. a Related adverse events include possible or probable relationship.

Secondary Outcomes Liver enzymes and lipid analytes. Significant deCLINICAL LIVER

creases in ALT values of approximately 25% were observed after administration of 25 mg OCA as well as decreases in g-glutamyltransferase levels of approximately 50% with both doses of OCA (Table 4). Alkaline phosphatase values increased slightly in both OCA treatment groups compared with placebo (P < .01) but were within normal limits; AST values remained clinically stable. Levels of triglycerides were decreased after treatment with both doses of OCA (Table 4), significantly so in the 50 mg OCA group (P ¼ .02). A modest, nonsignificant increase in total cholesterol level and a modest but significant increase in low-density lipoprotein level were seen after administration of both doses of OCA. High-density lipoprotein cholesterol level was unchanged in the 25 mg OCA group and marginally but significantly decreased in the 50 mg OCA group. FGF19, C4, endogenous bile acids, and weight. FGF19 plasma levels were dose-dependently

increased compared with baseline after treatment with 25 mg OCA (92  13 to 177  23 ng/L; P ¼ .006) and more markedly after treatment with 50 mg OCA (79  10 to 255  42 ng/L; P < .0001), whereas they were unchanged in the placebo group (84  13 to 91  11 ng/L) (Figure 1A). The increased FGF19 levels were paralleled by significant reductions in C4 (Figure 1B) and total endogenous serum BAs (Figure 1C) in both dose groups compared with placebo. The mean baseline body weight was similar across treatment groups (104–109 kg; Table 1). A dose-related

reduction in body weight was observed at the end of the 6-week treatment period, with the 50 mg OCA group losing approximately twice the amount of weight (1.9%  2.2%; P ¼ .008) compared with the 25 mg OCA group (1.0%  1.6%; P ¼ .096). Weight loss was significant when both doses were combined as compared with the placebo group (P ¼ .011) (Figure 1D). Liver fibrosis markers and keratin-18 serum levels.

Baseline ELF scores were similar across treatment groups and indicated that 81% of the patients had mild-to-moderate or moderate liver fibrosis, while none had evidence of cirrhosis (Table 5). Although there was considerable variability in the results, a small increase in the mean ELF score was observed in the placebo group and essentially no change was seen in the 50 mg OCA group. However, there was a modest but significant reduction in the mean ELF score in the 25 mg OCA group compared with placebo (P ¼ .004). In this group, all 3 ELF component parameters (hyaluronic acid, procollagen III amino terminal peptide, and tissue inhibitor of metalloproteinase 1) showed significant reductions compared with placebo (Table 5). Because levels of serum caspase-cleaved keratin-18 fragments predict histologic NASH and severity of disease in patients with biopsy-proven NAFLD,40 they were determined at baseline and at day 43 in a small number of patients (n ¼ 5–7). No significant differences were observed between baseline and end-of-treatment values in any group (data not shown). It is conceivable that the lack of significant effects could be due to the small number of patients tested or that 6 weeks may not be a long enough treatment to see an effect induced by OCA on caspasecleaved keratin-18 levels.

Table 4. Liver Enzymes and Lipid Analytes Laboratory analytes (mean  SD/time point) Liver enzymes (U/L) AST ALT Alkaline phosphatase g-glutamyltransferase Lipids (mg/dL) Cholesterol Low-density lipoprotein cholesterol High-density lipoprotein cholesterol Triglycerides a

Placebo (n ¼ 23) Baseline

25 mg OCA (n ¼ 20)

Day 43

Baseline

50 mg OCA (n ¼ 21)

Day 43

P valuea

Baseline

Day 43

P valuea

32 37 77 40

   

27 25 26 53

36 48 77 45

   

48 58 21 50

32 41 72 72

   

21 40 26 104

30 31 86 35

   

22 27 37 38

.12 .003 .003 <.001

28 36 74 44

   

14 22 25 59

33 46 103 22

   

20 42 36 12

.73 .84 <.001 <.001

166 98 40 178

   

32 26 11 83

174 107 40 178

   

34 34 10 90

163 98 37 193

   

26 28 6 96

181 120 35 170

   

26 31 6 81

.08 .01 .42 .09

170 104 43 156

   

32 27 8 62

183 129 37 121

   

35 35 7 50

.15 .008 .01 .02

Wilcoxon–Mann–Whitney test of placebo vs OCA group.

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Figure 1. Increased FGF19 and decreased C4 and endogenous bile acid plasma levels and weight in patients with diabetes and NAFLD after treatment with OCA. Data are presented as mean  SEM. *P < .05, **P < .01, ***P < .001. Statistical significance is based on comparisons of absolute change from baseline between the OCA treatment groups and placebo.

Discussion In this proof-of-concept phase 2 study in patients with diabetes and NAFLD, treatment with OCA for 6 weeks led to improved insulin sensitivity, a small but meaningful decrease in body weight, and enhanced FGF19 serum levels, coupled with decreased C4 and endogenous BA levels, supportive of FXR agonism. The primary end

point of the study, insulin sensitization as determined by posttreatment versus pretreatment change in GIR during low-dose and high-dose insulin infusion periods, was achieved with significant improvements at the 25-mg dose as well as for both doses of OCA combined and at both insulin infusion dose levels. The improved GIR during both phases of the 2-step hyperinsulinemic clamp was

Table 5. Enhanced Liver Fibrosis Markers Mean ( SD) ELF Component/Treatment Group

Day 0

ELF score Placebo (n ¼ 23) 8.2 OCA 25 mg (n ¼ 20) 8.4 OCA 50 mg (n ¼ 20) 8.0 Hyaluronic acid (ng/mL) Placebo (n ¼ 23) 47.5 OCA 25 mg (n ¼ 20) 33.6 OCA 50 mg (n ¼ 20) 31.0 Procollagen 3 amino-terminal peptide (ng/mL) Placebo (n ¼ 23) 5.5 OCA 25 mg (n ¼ 20) 6.6 OCA 50 mg (n ¼ 20) 5.6 Tissue inhibitor of metalloproteinase 1 (ng/mL) Placebo (n ¼ 23) 609.1 649.9 OCA 25 mg (n ¼ 20) OCA 50 mg (n ¼ 20) 591.5 a

Mean Change ( SD) Day 43

(Day 43 - Day 0)

 1.2  0.9  1.0

8.5  1.2 8.2  0.9 8.1  1.0

 94.2  40.8  38.0

54.1  92.9 30.8  35.9 25.2  23.7

6.7  15.4 -2.9  14.5 -5.8  31.4

.05 .12

 2.7  2.5  3.1

6.0  2.3 6.1  2.2 6.2  3.6

0.5  1.3 -0.5  1.2 0.6  3.5

.02 .93

 151.2  127.0  101.5

Mean differences were tested using the t test for independent samples.

655.4  156.6 638.5  101.2 627.3  187.1

0.3  0.5 -0.2  0.4 0.03  0.8

P valuea

46.3  105.8 -11.4  57.5 35.8  158.9

.004 .21

.03 .80

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confirmatory of beneficial effects of OCA on both hepatic and peripheral insulin sensitivity.35,41 Treatment with OCA also induced a significant decrease in g-glutamyltransferase levels, a marker of fatty liver disease that is associated with prediabetes and diabetes in the general population,42 and a known risk factor for development of diabetes in patients with NAFLD.43 Because this was the first phase 2 study of OCA, we believed it was appropriate to test more than one dose of the drug. Although 10-mg/kg or 30-mg/kg doses were needed in preclinical studies to show the pharmacological effects of OCA, the results for most of the parameters evaluated suggest that the 25-mg and 50-mg doses of OCA may lie on the flat portion of the dose-response curve for OCA, given that no additional improvement was seen in most clinical parameters at the 50-mg dose with the exception of FGF19 levels and weight loss. However, when taking fasting glucose levels, glycosylated hemoglobin, and GIR measurements at baseline into account, it would appear that patients treated with 50 mg OCA had less severe diabetes, and this may also help explain the more attenuated response in this group. Importantly, in this study, OCA induced a marked doserelated increase in plasma levels of FGF19, a primary FXRresponsive transcriptional product.11 It is noteworthy that FGF19 is not up-regulated by ursodeoxycholic acid,44 a BA with no FXR agonistic activity26 that is used to treat primary biliary cirrhosis and has also been tested in patients with NASH at dosages ranging up to 30 mg ∙ kg1 ∙ day1, with inconclusive results across several studies.45–47 Thus, the marked induction of FGF19 production by treatment with OCA, with consequently reduced levels of the BA precursor C4 and endogenous BAs, confirms the FXR agonist activity of OCA in patients and supports a novel mechanistic explanation for the observed OCA-related improvement in insulin sensitivity and weight loss. We believe this is the first time that any drug has been shown to increase FGF19 levels in patients with diabetes and/or NAFLD. Modulation of intestinal homeostasis by OCA may extend beyond induction of FGF19. Activation of FXR by OCA has been shown in preclinical models to preserve the integrity of the intestinal epithelial barrier in vivo, possibly through an anti-inflammatory effect enhancing tight junctions.48 Interestingly, OCA also enhances colonic expression of cathelicidin, a natural antibiotic with microbicidal properties, suggesting its capacity to modulate the gut microbiota.48 The gut microbiota has recently been shown to influence the size and composition of the bile acid pool throughout the enterohepatic system via FXR-dependent mechanisms.49 Intriguingly, OCA can increase ileal FGF15 and suppress hepatic CYP7A1 expression in germ-free mice, showing its capacity to reverse their reduced FXR signaling and further supporting a potential cross talk between OCA and the gut microbiota. Activation of FXR lowers plasma high-density lipoprotein cholesterol levels by increasing reverse cholesterol transport and reducing intestinal cholesterol

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absorption.50 Consistent with these observations, treatment of hyperlipidemic Ldlr-deficient or Apoe-deficient mice with FXR agonists results in decreased atherosclerotic lesions.51,52 Interestingly, chenodeoxycholic acid, the parent BA derivatized to generate OCA, administered to patients for dissolution of gallstones led to decreased triglyceride levels and increased low-density lipoprotein cholesterol levels.53 These findings are all reflected in our study, showing reduced triglyceride and high-density lipoprotein cholesterol levels after treatment with OCA, accompanied by increased low-density lipoprotein cholesterol values. A comprehensive evaluation of the lipid profile modulation by treatment with OCA is currently ongoing. The ELF test has been validated as a scoring algorithm derived from 3 individual assays measuring noninvasive biomarkers of liver fibrosis,39 although validation of ELF markers as an end point to evaluate therapeutic efficacy is limited. We did not expect fibrosis to resolve after 6 weeks of treatment with OCA, but we were interested in evaluating the degree of fibrosis at baseline in our proof-ofconcept study. Indeed, baseline ELF scores indicated that 81% of the patients had mild-to-moderate or moderate liver fibrosis, while none had evidence of cirrhosis. Surprisingly, the ELF score showed significant improvement in the group treated with 25 mg OCA compared with placebo. In addition, all 3 ELF components were significantly reduced, suggesting a biologically relevant result. These unexpected data are consistent with a potentially beneficial effect of OCA on liver fibrosis despite the short 6-week duration of the study and could be explained by the observation that FXR activation in stellate cells promotes resolution of liver fibrosis.54,55 The effect of OCA on hepatic fibrosis will need to be defined in longer and larger studies given the relatively slow and variable progression of fibrosis. OCA appeared to be well tolerated in this patient population. Only mild constipation was seen more frequently in the patients treated with 50 mg (but not 25 mg) OCA compared with placebo. Of note, pruritus in these noncholestatic patients was no more common in the OCA-treated patients than in patients receiving placebo. With the exception of weight loss, 25 mg appeared to be at least as effective as 50 mg for the majority of end points evaluated, suggesting that lower doses should be evaluated in future studies. The present study has several limitations, including the relatively low number of patients and the short duration of the study. In addition, a number of patients had to be excluded from efficacy analysis due to an insulin dosing error in the clamp studies, but these patients were replaced by others. As a consequence, the size of the safety database was larger than originally planned and the data obtained met the objectives of the study. In conclusion, activation of FXR by OCA leads to increased insulin sensitivity in patients with NAFLD who have type 2 diabetes mellitus. This is associated with an FXR-mediated, dose-dependent increase in production of

FGF19, providing a plausible explanation not only for improved insulin sensitivity but also for weight loss, as well as for reduced C4 level and endogenous BA production. Overall, this proof-of-concept study, showing improved insulin sensitivity based at least in part on an FXR-induced FGF19-dependent mechanism of action, demonstrates the clinical relevance of this pathway and supports the potential of OCA to treat liver and metabolic diseases. Based primarily on the efficacy and safety shown by these promising results, and on the emerging preclinical evidence for potential beneficial effects of OCA in patients with NAFLD/NASH,32 the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health has selected OCA for a phase IIb clinical study in patients with NASH (http://www.clinicaltrials .gov; NCT01265498). The FXR Ligand NASH Treatment (FLINT) study is a double-blind, placebo-controlled, multicenter study that will evaluate whether treatment with 25 mg OCA daily for 72 weeks compared with placebo improves the severity of NASH as determined by centrally scored liver histology. FLINT has enrolled 280 patients at the 8 US centers comprising the National Institute of Diabetes and Digestive and Kidney Diseases–sponsored NASH Clinical Research Network. The primary end point of the 72-week study will be determined by liver biopsy and is defined as a decrease in NAFLD Activity Score of at least 2 points with no worsening of liver fibrosis.

Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi.org/10.1053/j.gastro.2013.05.042. References 1. Farrell GC. The liver and the waistline: fifty years of growth. J Gastroenterol Hepatol 2009;24(Suppl 3):S105–S118. 2. Lam DW, Leroith D. The worldwide diabetes epidemic. Curr Opin Endocrinol Diabetes Obes 2012;19:93–96. 3. Sanyal AJ. NASH: a global health problem. Hepatol Res 2011; 41:670–674. 4. Feneberg A, Malfertheiner P. Epidemic trends of obesity with impact on metabolism and digestive diseases. Dig Dis 2012;30:143–147. 5. Larter CZ, Chitturi S, Heydet D, et al. A fresh look at NASH pathogenesis. Part 1: the metabolic movers. J Gastroenterol Hepatol 2010;25:672–690. 6. Dowman JK, Armstrong MJ, Tomlinson JW, et al. Current therapeutic strategies in non-alcoholic fatty liver disease. Diabetes Obes Metab 2011;13:692–702. 7. Mahady SE, Webster AC, Walker S, et al. The role of thiazolidinediones in non-alcoholic steatohepatitis—a systematic review and meta analysis. J Hepatol 2011;55:1383–1390. 8. Lincoff AM, Wolski K, Nicholls SJ, et al. Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a meta-analysis of randomized trials. JAMA 2007;298:1180–1188. 9. Mamtani R, Haynes K, Bilker WB, et al. Association between longer therapy with thiazolidinediones and risk of bladder cancer: a cohort study. J Natl Cancer Inst 2012;104:1411–1421.

FXR AND OCA IN DIABETES AND NAFLD 581 10. Hofmann AF. Bile acids: trying to understand their chemistry and biology with the hope of helping patients. Hepatology 2009; 49:1403–1418. 11. Lefebvre P, Cariou B, Lien F, et al. Role of bile acids and bile acid receptors in metabolic regulation. Physiol Rev 2009;89:147–191. 12. Thomas C, Pellicciari R, Pruzanski M, et al. Targeting bile-acid signalling for metabolic diseases. Nat Rev Drug Discov 2008; 7:678–693. 13. Hollman DA, Milona A, van Erpecum KJ, et al. Anti-inflammatory and metabolic actions of FXR: insights into molecular mechanisms. Biochim Biophys Acta 2012;1821:1443–1452. 14. Pols TW, Noriega LG, Nomura M, et al. The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation. J Hepatol 2011;54:1263–1272. 15. Cariou B, van Harmelen K, Duran-Sandoval D, et al. The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice. J Biol Chem 2006;281:11039–11049. 16. Ma K, Saha PK, Chan L, et al. Farnesoid X receptor is essential for normal glucose homeostasis. J Clin Invest 2006;116:1102–1109. 17. Zhang Y, Lee FY, Barrera G, et al. Activation of the nuclear receptor FXR improves hyperglycemia and hyperlipidemia in diabetic mice. Proc Natl Acad Sci U S A 2006;103:1006–1011. 18. Rizzo G, Disante M, Mencarelli A, et al. The farnesoid X receptor promotes adipocyte differentiation and regulates adipose cell function in vivo. Mol Pharmacol 2006;70:1164–1173. 19. Fuchs M. Non-alcoholic fatty liver disease: the bile acid-activated farnesoid X receptor as an emerging treatment target. J Lipids 2012;2012:934396. 20. Dufer M, Horth K, Wagner R, et al. Bile acids acutely stimulate insulin secretion of mouse beta-cells via farnesoid X receptor activation and K(ATP) channel inhibition. Diabetes 2012;61:1479–1489. 21. Inagaki T, Choi M, Moschetta A, et al. Fibroblast growth factor 15 functions as an enterohepatic signal to regulate bile acid homeostasis. Cell Metab 2005;2:217–225. 22. Modica S, Petruzzelli M, Bellafante E, et al. Selective activation of nuclear bile acid receptor FXR in the intestine protects mice against cholestasis. Gastroenterology 2012;142:355–365 e1–4. 23. Fu L, John LM, Adams SH, et al. Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes. Endocrinology 2004;145:2594–2603. 24. Miyata M, Sakaida Y, Matsuzawa H, et al. Fibroblast growth factor 19 treatment ameliorates disruption of hepatic lipid metabolism in farnesoid x receptor (fxr)-null mice. Biol Pharm Bull 2011; 34:1885–1889. 25. Kir S, Beddow SA, Samuel VT, et al. FGF19 as a postprandial, insulinindependent activator of hepatic protein and glycogen synthesis. Science 2011;331:1621–1624. 26. Pellicciari R, Fiorucci S, Camaioni E, et al. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J Med Chem 2002;45:3569–3572. 27. Pellicciari R, Costantino G, Camaioni E, et al. Bile acid derivatives as ligands of the farnesoid X receptor. Synthesis, evaluation, and structure-activity relationship of a series of body and side chain modified analogues of chenodeoxycholic acid. J Med Chem 2004; 47:4559–4569. 28. Rizzo G, Passeri D, De Franco F, et al. Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist. Mol Pharmacol 2010;78:617–630. 29. Wang YD, Chen WD, Wang M, et al. Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response. Hepatology 2008;48:1632–1643. 30. Wang XX, Jiang T, Shen Y, et al. The farnesoid X receptor modulates renal lipid metabolism and diet-induced renal inflammation, fibrosis, and proteinuria. Am J Physiol Renal Physiol 2009; 297:1587–1596. 31. Wang XX, Jiang T, Shen Y, et al. Diabetic nephropathy is accelerated by farnesoid X receptor deficiency and inhibited by farnesoid X receptor activation in a type 1 diabetes model. Diabetes 2010; 59:2916–2927.

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MUDALIAR ET AL

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32. Adorini L, Pruzanski M, Shapiro D. Farnesoid X receptor targeting to treat nonalcoholic steatohepatitis. Drug Discov Today 2012; 17–18:988–997. 33. Kim HC, Nam CM, Jee SH, et al. Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study. BMJ 2004;328:983. 34. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2010;33(Suppl 1):S62–S69. 35. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979;237:E214–E223. 36. Heinemann L, Ampudia-Blasco FJ. Glucose clamps with the Biostator: a critical reappraisal. Horm Metab Res 1994;26:579–583. 37. Gälman C, Arvidsson I, Angelin B, et al. Monitoring hepatic cholesterol 7alpha-hydroxylase activity by assay of the stable bile acid intermediate 7alpha-hydroxy-4-cholesten-3-one in peripheral blood. J Lipid Res 2003;44:859–866. 38. Tagliacozzi D, Mozzi AF, Casetta B, et al. Quantitative analysis of bile acids in human plasma by liquid chromatography-electrospray tandem mass spectrometry: a simple and rapid one-step method. Clin Chem Lab Med 2003;41:1633–1641. 39. Guha IN, Parkes J, Roderick P, et al. Noninvasive markers of fibrosis in nonalcoholic fatty liver disease: validating the European Liver Fibrosis Panel and exploring simple markers. Hepatology 2008;47:455–640. 40. Feldstein AE, Wieckowska A, Lopez AR, et al. Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: a multicenter validation study. Hepatology 2009;50:1072–1078. 41. Muniyappa R, Lee S, Chen H, et al. Current approaches for assessing insulin sensitivity and resistance in vivo: advantages, limitations, and appropriate usage. Am J Physiol Endocrinol Metab 2008; 294:E15–E26. 42. Ruckert IM, Heier M, Rathmann W, et al. Association between markers of fatty liver disease and impaired glucose regulation in men and women from the general population: the KORA-F4-study. PLoS One 2011;6:e22932. 43. Arase Y, Suzuki F, Ikeda K, et al. Multivariate analysis of risk factors for the development of type 2 diabetes in nonalcoholic fatty liver disease. J Gastroenterol 2009;44:1064–1070. 44. Marschall HU, Wagner M, Zollner G, et al. Combined rifampicin and ursodeoxycholic Acid treatment does not amplify rifampicin effects on hepatic detoxification and transport systems in humans. Digestion 2012;86:244–249. 45. Lindor KD, Kowdley KV, Heathcote EJ, et al. Ursodeoxycholic acid for treatment of nonalcoholic steatohepatitis: results of a randomized trial. Hepatology 2004;39:770–778. 46. Leuschner UF, Lindenthal B, Herrmann G, et al. High-dose ursodeoxycholic acid therapy for nonalcoholic steatohepatitis: a double-blind, randomized, placebo-controlled trial. Hepatology 2010;52:472–479. 47. Ratziu V, de Ledinghen V, Oberti F, et al. A randomized controlled trial of high-dose ursodesoxycholic acid for nonalcoholic steatohepatitis. J Hepatol 2011;54:1011–1019. 48. Gadaleta RM, van Erpecum KJ, Oldenburg B, et al. Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease. Gut 2011;60:463–472.

GASTROENTEROLOGY Vol. 145, No. 3 49. Sayin SI, Wahlstrom A, Felin J, et al. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist. Cell Metab 2013; 17:225–235. 50. Zhang Y, Yin L, Anderson J, et al. Identification of novel pathways that control farnesoid X receptor-mediated hypocholesterolemia. J Biol Chem 2010;285:3035–3043. 51. Flatt B, Martin R, Wang TL, et al. Discovery of XL335 (WAY-362450), a highly potent, selective, and orally active agonist of the farnesoid X receptor (FXR). J Med Chem 2009;52:904–907. 52. Hartman HB, Gardell SJ, Petucci CJ, et al. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR-/- and apoE-/- mice. J Lipid Res 2009;50:1090–1100. 53. Schoenfield LJ, Lachin JM. Chenodiol (chenodeoxycholic acid) for dissolution of gallstones: the National Cooperative Gallstone Study. A controlled trial of efficacy and safety. Ann Intern Med 1981; 95:257–282. 54. Fiorucci S, Antonelli E, Rizzo G, et al. The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis. Gastroenterology 2004;127: 1497–1512. 55. Fickert P, Fuchsbichler A, Moustafa T, et al. Farnesoid X receptor critically determines the fibrotic response in mice but is expressed to a low extent in human hepatic stellate cells and periductal myofibroblasts. Am J Pathol 2009;175:2392–2405. Author names in bold designate shared co-first authorship. Received February 12, 2013. Accepted May 22, 2013. Reprint requests Address requests for reprints to: Sunder Mudaliar, MD, FRCP (Edin), FACP, FACE, Center for Metabolic Research, VA San Diego Healthcare System and University of California San Diego, San Diego, California 92161. e-mail: [email protected]. Conflicts of interest The authors disclose the following: Sunder Mudaliar is a consultant for and has received research grant support from Intercept Pharmaceuticals, Inc. Arun J. Sanyal has received research grant support from Intercept Pharmaceuticals, Inc. Linda Morrow has received research grant support from Intercept Pharmaceuticals, Inc, and is an employee of and shareholder in Profil Institute for Clinical Research. Mark Kipnes has received research grant support from Intercept Pharmaceuticals, Inc. Luciano Adorini is an employee of Intercept Pharmaceuticals, Inc. Cathi I. Sciacca is an employee of Intercept Pharmaceuticals, Inc. Erin Castelloe is an employee of Intercept Pharmaceuticals, Inc. Paul Dillon is an employee of Siemens Healthcare Diagnostics, the manufacturer of the ELF liver fibrosis tests. Mark Pruzanski is an employee of Intercept Pharmaceuticals, Inc. David Shapiro is an employee of Intercept Pharmaceuticals, Inc. The remaining authors disclose no conflicts. Funding Supported by a research grant from Intercept Pharmaceuticals, Inc.

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FXR AND OCA IN DIABETES AND NAFLD 582.e1

Supplementary Figure 1. Disposition of patients with type 2 diabetes mellitus and NAFLD in the study.

Supplementary Table 1. Incidence of All Adverse Events Occurring in >1 Patient in Any Treatment Group Treatment group System organ class/preferred term Patients with any adverse events General disorders and administrative site conditions Pyrexia Vessel puncture site pain Gastrointestinal disorders Constipation Diarrhea Infections and infestations Nasopharyngitis Upper respiratory tract infection Nervous system disorders Headache Skin and subcutaneous tissue disorders Pruritus Cardiac disorders Palpitations NOTE. All values are expressed as n (%).

Placebo (n ¼ 23) 14 4 2 2 4 0 2 5 2 2 4 1 3 2 0 0

(61) (17) (9) (9) (17) (0) (9) (22) (9) (9) (17) (4) (13) (9) (0) (0)

25 mg OCA (n ¼ 20) 9 3 0 1 1 0 0 2 0 0 2 1 1 0 2 2

(45) (15) (0) (5) (5) (0) (0) (10) (0) (0) (10) (5) (5) (0) (10) (10)

50 mg OCA (n ¼ 21) 16 5 0 4 6 5 0 2 0 1 3 3 4 1 0 0

(76) (24) (0) (19) (29) (24) (0) (10) (0) (5) (14) (14) (19) (5) (0) (0)