Therapy of Nonalcoholic Steatohepatitis (NASH) – Antioxidants and Cytoprotective Agents

Tropical Gastroenterology 2011;Suppl:S33–37

Therapy of nonalcoholic steatohepatitis (NASH) – antioxidants and cytoprotective agents

Ajay Duseja

ABSTRACT

Department of Hepatology Pathogenesis of nonalcoholic fatty liver disease (NAFLD) and its progressive form Postgraduate Institute of Medical nonalcoholic steatohepatitis (NASH) is not completely understood. Because of the role of Education & Research (PGIMER) oxidative stress in the pathogenesis, antioxidants and cytoprotective agents have been used Chandigarh – 160012, India. in the treatment of patients with NAFLD/NASH. Various antioxidants and cytoprotective Correspondence: agents used in these patients include Vitamin E, Vitamin C, Betaine, N- acetyl cysteine, silybin, Dr. Ajay Duseja lecithin, beta carotene, metadoxine, selenium and ursodeoxycholic acid (UDCA). Most of the Email: [email protected] data is available with vitamin E and UDCA, either in isolation or in combination with various agents. The available data on the use of various agents are either experimental or have small number of patients and randomized controlled trials with histological efficacy are sparse. More data is required before any conclusions are drawn regarding the use of anti oxidants and cytoprotective agents in patients NAFLD/NASH.

KEYWORDS: Nonalcoholic fatty liver disease, NAFLD, oxidative stress, vitamin E, ursodeoxycholic acid, UDCA

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a recently coined and human studies.6,7 In the absence of alcohol intake, patients entity and includes patients with simple steatosis and who either have metabolic syndrome or any of its components nonalcoholic steatohepatitis (NASH). Under the broad umbrella with insulin resistance, develop hepatic steatosis due to of NAFLD, NASH is an intermediate stage of liver damage and increased lipolysis and increased delivery of fatty acids from has the maximum propensity of progressing into cirrhosis and adipose tissue to liver.8 The increased load of fatty acids in hepatocellular carcinoma (HCC).1 Ludwig et al introduced the the hepatocytes increases the mitochondrial β-oxidation and term NASH in 1980 to describe histological changes increase in cytochrome P450 4A and cytochrome P4502E1 indistinguishable from alcoholic hepatitis in patients with no levels, leading to increase in reactive oxygen species. The or insignificant alcohol intake of less than 20g/day.2 Even though increased mitochondrial oxidative stress leads to the second the clinicopathological profile may be different from that hit from steatosis to steatohepatitis and fibrosis by three main reported from the west, NAFLD/NASH is common in India and mechanisms, namely lipid peroxidation, cytokine induction, is responsible for significant liver disease.3-5 and Fas ligand induction. Lipid peroxidation, apoptosis and recruitment of various cytokines lead on to hepatic Oxidative stress in the pathogenesis of NAFLD/ inflammation and or fibrosis in addition to steatosis and NASH progression from a stage of simple steatosis to NASH, setting the stage for future complications like cirrhosis and HCC. 9, 10 Pathogenesis of the NAFLD/NASH is not completely Data from India has also confirmed the role of oxidative understood. Oxidative stress has been shown to play an stress in the pathogenesis NAFLD/NASH. In one of the important role in the pathogenesis of NAFLD/NASH in animal studies from New Delhi, oxidative stress and antioxidant status

© Tropical Gastroenterology 2011 S34 Tropical Gastroenterology 2011;Suppl:S33–37 was studied in 29 prospectively enrolled patients with NAFLD/ analysis on the use of various drugs reported extreme NASH, 25 diseased controls with chronic viral hepatitis, and heterogeneity of trials of antioxidants and cytoprotective 23 healthy controls.11 Apart from standard biochemical agents, which prevents any firm conclusions on the effect of parameters, lipid peroxidation products were measured as antioxidants in patients with NAFLD/NASH.13 thiobarbituric acid reactive substances (TBARS). As measures Various antioxidants and cytoprotective agents used in the of antioxidant capacity, superoxide dismutase, vitamin C levels treatment of NAFLD/NASH include vitamin E, vitamin C, and ferric reducing ability of plasma were measured. Products betaine, N- acetyl cysteine, silybon, lecithin, beta carotene, of lipid peroxidation were significantly increased among metadoxine, selenium and ursodeoxycholic acid (UDCA). patients with NAFLD/NASH in comparison to chronic viral hepatitis or healthy controls.11 In an attempt to explore the Vitamin E alone interrelationship of oxidative stress and insulin resistance in NAFLD/NASH subjects with and without type 2 diabetes, 200 Studies on usage of vitamin E are available either as pilot studies subjects were recruited from the Chennai Urban Rural or randomized controlled trials comparing results with placebo, Epidemiology Study group and tested for TBARS, protein UDCA or pioglitazone. Combinations of vitamin E with vitamin carbonyl (PCC) and glutathione levels.12 TBARS and PCC levels C, pioglitazone and UDCA have also been studied in patients were significantly elevated and reduced to oxidized glutathione with NAFLD/NASH. (GSH/GSSG) ratio was significantly decreased in diabetic One of the earlier pilot studies showed the efficacy of subjects with NAFLD/NASH compared to all other groups, vitamin E in children with NAFLD/NASH, used in the dosage suggesting that oxidative stress markers are significantly of oral vitamin E between 400 and 1200 IU per day.14 Serum associated with NAFLD/NASH even after adjusting for age, biochemistry values were monitored monthly during treatment gender, BMI and glycemic status.12 We recently evaluated the and treated patients were followed up for 4 to 10 months. Serum role of oxidative stress in 25 patients with NAFLD/NASH and alanine aminotransferase (ALT) decreased from 175 ± 106 IU/L compared results with age and BMI matched 25 chronic viral to 40 ± 26 IU/L (P <0.001), serum aspartate aminotransferase hepatitis patients and 25 healthy volunteers (HV) (AST) decreased from 104 ± 61 IU/L to 33 ± 11 IU/L (P <0.002), (unpublished). Patients with NAFLD/NASH had significantly and alkaline phosphatase decreased from 279 ± 42 IU/L to 202 higher malondialdehyde (MDA) in comparison to patients with ± 66 IU/L (P <0.003) during treatment but returned to abnormal chronic viral hepatitis and healthy volunteers. NAFLD/NASH in those electing to stop treatment and liver remained diffusely patients also had higher conjugated dienes levels in comparison echogenic during therapy.14 Another pilot study evaluated to HVs and reduced glutathione levels, higher glutathione the effects of a step 1 American Heart Association diet plus peroxidase levels and lower catalase activity in comparison to aerobic exercise with or without 800 IU of vitamin E daily on HVs, suggesting higher oxidative stress in patients with cytokine profiles and liver enzyme levels in 16 patients with NAFLD/NASH. biopsy-proven NASH.15 Lifestyle modifications (low-fat diet and exercise) were associated with improvement in liver Role of Antioxidants and cytoprotective agents enzymes, cholesterol, and plasma hyaluronic acid levels in patients with NASH, whereas the vitamin E supplementation Given the important role of oxidative stress in the pathogenesis used in this short-term pilot study provided no apparent added of NAFLD/NASH, various antioxidants and cytoprotective benefit.15 A small-randomized trial compared the efficacy of agents have been used in the treatment of NAFLD/NASH alpha-tocopherol (vitamin E) with ursodeoxycholic acid [3 patients with variable success. Treatment by antioxidants also patients received UDCA (250 mg TID) and six aTP (100 mg shares the similar drawbacks as observed by other modes of TID)] in patients with NAFLD/NASH. Ultrasound showed treatment in patients with NAFLD. Most of the studies have favorable changes in four patients (44%), two in each group. either experimental design or have small number of patients. ALT and AST average diminished by 40% and normalization Studies most often have pilot design rather than being the was obtained in five of six patients in treatment with aTP (83%) randomized controlled trials and the histological efficacy has and in one of the UDCA group (33%) with no statistically not been shown in majority of studies. Rather, a recent meta- significant difference between two groups.16 Therapy of nonalcoholic steatohepatitis S35

Vitamin E + Vitamin C in steatosis (mean grade, 2.2 vs. 1.4; p < 0.02). Compared with baseline, combination therapy produced a significant decrease The efficacy of combination of alpha-tocopherol (vitamin E) in steatosis (mean, 2.3 vs. 1; p < 0.002), cytologic ballooning and vitamin C in reducing histologic inflammation and fibrosis (1.3 vs. 0.2; p < 0.01), Mallory’s hyaline (0.7 vs. 0.2; p < 0.04), was evaluated in a prospective, double-blind, randomized, and pericellular fibrosis (1.2 vs. 0.6; p < 0.03). Although vitamin placebo-controlled trial.17 Forty five patients were randomized E had no significant effects, combination therapy produced a to receive either vitamins E and C (1000 IU and 1000 mg, significant increase in metabolic clearance of glucose and a respectively) or placebo daily for 6 months, based on their decrease in fasting free fatty acid and insulin.20 More data on initial histologic diagnosis of NASH. Additionally, all patients this combination is required to substantiate the results. were given standard weight-loss counseling and encouraged to follow a low fat diet (<30 fat g/day). The pre- and Ursodeoxycholic acid posttreatment liver biopsies were reviewed by a single pathologist, who was blinded to the patient’s medication. No Ursodeoxycholic acid (UDCA) exerts its beneficial effect in improvement in necroinflammatory activity or ALT was seen liver diseases through a diverse, probably, complementary array with this combination of drug therapy.17 Though authors of mechanisms. By decreasing hydrophobic acids, UDCA reported a statistically significant improvement in fibrosis score improves oxidative stress and hence has been used in patients (p=0.002) with vitamin treatment in comparison to placebo, with NAFLD/NASH. In animal models of NASH, UDCA has careful analysis reveled that the improvement difference been found to be effective in enhancing the therapeutic effects in fibrosis was seen only in 2 patients.18 Hence this of low calorie diet.21 In a human study comparing clofibrate combination may warrant further studies before any and UDCA, treatment of NAFLD/NASH patients with UDCA conclusions are drawn. for 12 months resulted in significant improvement in alkaline phosphatase, ALT, and gamma-glutamyl-transferase (GGT), and Vitamin E vs. or with pioglitazone hepatic steatosis. Despite the known lipid-lowering effects of clofibrate, it did not appear to be of clinical benefit in the Pioglitazone is an insulin sensitizer and has been found to be treatment of NASH in this 1-year pilot study. 22 In another study effective in the treatment of patients with NAFLD/NASH. comparing UDCA with atorvastatin, serum ALT, GGT levels Studies have compared the results of vitamin E usage against reduced significantly inNAFLD/NASH patients receiving pioglitazone and in combination with pioglitazone in patients UDCA.23 Around three fourth (74%) of our patients with with NAFLD/NASH. In a recent RCT, 247 adults with NASH NAFLD/NASH also had biochemical response to lifestyle and without diabetes either received pioglitazone at a dose of modifications and ursodeoxycholic acid (UDCA) given for 6 30 mg daily (80 subjects), vitamin E at a dose of 800 IU daily (84 months.24 Since we did not have a control group, improvement subjects), or placebo (83 subjects), for 96 weeks.19 The primary in our patients may or may not be related to UDCA. In a recent outcome was an improvement in histologic features of randomized placebo controlled trial, 2 years of therapy with nonalcoholic steatohepatitis, as assessed with the use of a UDCA was not found to be better than placebo for patients composite of standardized scores for steatosis, lobular with NASH.25 In this trial 166 patients with liver biopsy-proven inflammation, hepatocellular ballooning, and fibrosis. Vitamin NASH were randomized to receive between 13 and 15 mg/kg/d E therapy, as compared with placebo, was associated with a of UDCA or placebo for 2 years. End points included changes significantly higher rate of improvement in NASH (43% vs. in liver test results and liver histology at 2 years of therapy. 19%, p=0.001), but the difference in the rate of improvement Pre- and post-treatment liver biopsies were available in 107 with pioglitazone as compared with placebo was not significant patients for review at the end of the study. UDCA was well (34% and 19%, respectively; p=0.04).19 Another prospective tolerated and body weight was stable during the study randomized study from the same group earlier, compared the duration. Serum liver biochemistries were stable or improved efficacy and safety of vitamin E alone (400 IU/day) vs. vitamin in both the UDCA and placebo-treated groups. Changes in the E (400 IU/day) and pioglitazone (30 mg/day) in nondiabetic, degree of steatosis, necroinflammation, or fibrosis that occurred noncirrhotic subjects with NASH (10 patients in each arm).20 with therapy were not significantly different between the UDCA Treatment with vitamin E only produced a significant decrease and placebo groups.25 S36 Tropical Gastroenterology 2011;Suppl:S33–37

It is thought that the dosage of UDCA may be important in Other agents causing improvement in patients with NAFLD/NASH. Though liver histology was not available, Ratziu et al in their preliminary Data on the use of other antioxidants and cytoprotective agents report suggested that 12 months high dose UDCA (30 mg/kg in patients with NAFLD/NASH are sparse and require more per day) was effective in improving AST, ALT and GGT levels studies before any conclusions are drawn. Although, betaine, in patients with NAFLD/NASH.26 On the other hand in a recent a naturally occurring metabolite of choline, has been found to RCT comparing high dose UDCA (23-28 mg/kg/day for 18 be effective in animal models, results could not be translated in months) with placebo in 185 patients with histologically proven its favor, in a recent human study.31-33 N-acetylcyseine (NAC), NASH, significant differences in the overall histology could a precursor of glutathione has been found to be effective in not be detected between the two treatment groups, except for improving liver enzymes and hepatic steatosis and a study has the improvement in lobular inflammation. With the exception shown that silybin, the active principle of Silibum marinatum, of gamma-glutamyl transferase, UDCA did not improve in combination with phospholipids and vitamin E is effective laboratory data.27 in improving body weight, insulin resistance, plasma markers of liver fibrosis.34, 35 Ursodeoxycholic acid + vitamin E In conclusion, amongst various antioxidants and cytoprotective agents, role of vitamin E looks promising in Since UDCA and vitamin E have been considered separately patients with NAFLD/NASH. More data in different as therapeutic options in patients with NAFLD/NASH with populations is required for the use of UDCA and combinations variable results, studies have looked at the combinations of of various antioxidants and cytoprotective agents are worth both drugs. In a randomized placebo-controlled trial of exploring. More studies are required for other antioxidants and ursodeoxycholic acid with vitamin E, patients with biopsy- cytoprotective agents before any conclusions are drawn for proven NASH (with a second biopsy at 2 years) were randomly their effectiveness in patients with NAFLD/NASH. assigned to receive UDCA, 12-15 mg/kg/day with vitamin E, 400 IU twice a day (UDCA/Vit E), UDCA with placebo (UDCA/ References P), or placebo/placebo (P/P).28 Forty eight patients were included, 15 in the UDCA/Vit E group, 18 in the UDCA/P group, 1. Falck-Ytter Y, Younossi ZM, Marchesini G, McCullough AJ. and 15 in the P/P group. Serum AST and ALT levels diminished Clinical features and natural history of nonalcoholic steatosis significantly in the UDCA/Vit E group. Neither the AST nor the syndromes. Semin Liver Dis. 2001;21:17–26. ALT levels improved in the P/P group and only the ALT levels 2. Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed in the UDCA/P group. Histologically, the activity index was disease. Mayo Clin Proc. 1980;55:434–8. unchanged at the end of the study in the P/P and UDCA/P 3. Duseja A, Das A, Dhiman RK, Chawla YK, Das R, Bhadada S, et groups, but it was significantly better in the UDCA/Vit E group, al. Indian patients with nonalcoholic fatty liver disease presenting mostly as a result of regression of steatosis.28 Another recent with raised transaminases are different at presentation. World J Gastroenterol. 2007;13:649–50. study evaluated the effect of ursodeoxycholic acid in 4. Duseja A, Chawla Y. Nonalcoholic fatty liver disease in India— combination with vitamin E on adipokines and apoptosis in how much? How soon? Trop Gastroenterol. 2005;26:1–3. patients with NASH. Combination treatment improved not only 5. Duseja A, Sharma B, Kumar A, Kapil S, Das A, Dhiman RK, et aminotransferase levels and liver histology of patients with al. Nonalcoholic fatty liver in a developing country is responsible for significant liver disease. Hepatology. 2010;52:2248–9. NASH, but also decreased hepatocellular apoptosis and 6. de Almeida BB, Mathias MG, Portari GV, Jordao AA. Chronic restored circulating levels of adiponectin.29 A retrospective acetonemia alters liver oxidative balance and lipid content in rats. study from Delhi, also found vitamin E based treatment (life A model of NASH? Exp Clin Endocrinol Diabetes. style modifications + UDCA + vitamin E) to be superior to 2010;118:61–3. 7. Machado MV, Ravasco P, Jesus L, Marques-Vidal P, Oliveira either lifestyle modifications alone or a combination of lifestyle CR, Proenca T, et al. Blood oxidative stress markers in non- modifications and UDCA in normalizing ALT in patients with alcoholic steatohepatitis and how it correlates with diet. Scand J NASH.30 Gastroenterol. 2008;43:95–102. Therapy of nonalcoholic steatohepatitis S37

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