Comparative Clinical Study Between Empagliflozin Versus Pioglitazone in Non-diabetic Patients With Non-alcoholic Steatohepatitis

This study aims to evaluate the possible beneficial effect of empagliflozin versus pioglitazone on non-diabetic patients with non-alcoholic steatohepatitis (NASH).

This study will be a randomized, comparative parallel study. The study will be conducted according to the ethical standards of Helsinki declaration in 1964 and its later amendments.

The study duration will be 24 weeks. The patients will be randomized into two groups:

Group 1: (Pioglitazone group; n=28) which will receive 30mg/day pioglitazone for 24 weeks.

Group 2: (Empagliflozin group; n=28) which will receive 10mg/day empagliflozin for 24 weeks.

Nonalcoholic fatty liver disease (NAFLD) is a condition of fat accumulation in the liver in the absence of alcohol consumption. Dyslipidemia predominantly hypertriglyceridemia, oxidative stress and insulin resistance play crucial roles in the pathogenesis of NASH. Non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) are the two major subtypes of NAFLD. The patients diagnosed with NASH can subsequently progress to liver fibrosis, which increases the risks of cirrhosis and liver cancer. In Egypt, the prevalence of NAFLD is rising owing to rising prevalence of obesity whereas NAFLD was diagnosed in 57.65% of a cohort of obese Egyptian adolescents. Furthermore, around 1 in 3 had steatosis, and 1 in 20 had moderate-to-advanced fibrosis in Egypt.

NASH is also associated with production of various atherogenic factors including pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) which has been proposed to be the key link between obesity and insulin resistance during NASH. Current guidelines recommend changing lifestyle patterns as first-line therapy strategy for NAFLD. However, there is no defined treatment for NAFLD, previous studies aimed at discovering new treatments for NAFLD and several treatment approaches have been proposed, such as insulin-sensitizers, lipid-lowering drugs, antioxidants, L-carnitine, pentoxifylline, probiotics, ezetimibe, sitagliptin, empagliflozin, pentoxifylline, dapagliflozin, pioglitazone, and lobeglitazone which were reported to reduce liver fat and improve ALT levels in patients with NASH and NAFLD through their anti-inflammatory, antioxidant, and antifibrogenic effects.

Cytokeratin 18 (CK18) is an intermediate filament protein expressed by hepatocytes. Cytokeratin 18 is released into the blood by cell death and hepatocyte apoptosis. Transforming growth factor beta-1 (TGF-β1) cytokine mediates the transformation of quiescent hepatic stellate cells (HSCs) into myofibroblast-like cells with an increased production of extra cellular matrix proteins. TGF-β1 concentration is elevated in patients with NASH as compared to patients with hepatic steatosis, suggesting that this cytokine is involved in the fibrogenesis during NASH.

Although pioglitazone has been proven to enhance metabolism and liver histology among patients with NAFLD, it has limitations in clinical use due to significant adverse events including weight gain, lower extremity edema and risk for heart failure. Pioglitazone is a peroxisome proliferator activated receptor γ (PPARγ) agonist which can protect the liver by improving insulin resistance in patients with NAFLD and T2DM. It has been shown that pioglitazone could improve biochemical and histological parameters in non-diabetic patients with NASH. Previous trial revealed that, pioglitazone improved hepatic fibrosis in patients with NAFLD.

Sodium-glucose co-transporter type-2 inhibitors (SGLT2i) are glucose-lowering agents that improve glucose panel, promote weight loss and reduce serum uric acid level. There is increasing interest regarding the implication of SGLT2i in the treatment of NAFLD, regardless of the co-existence of T2DM. The beneficial effects of SGLT2i on NAFLD appear to be mediated directly through regulation of Endoplasmic Reticulum stress, oxidative stress, low-grade inflammation, autophagy and apoptosis. Empagliflozin (SGLT2i) was reported to decrease the expression of pro-fibrotic genes such as alpha smooth muscle actin (α-SMA), collagen, type I, alpha 1(collagen1α1), Matrix Metalloproteinase -2 (MMP2), and Transforming growth factor beta (TGF-β).

Furthermore, Empagliflozin successively induced the phosphorylation of MST1/2 and YAP, the two central members of the Hippo signaling pathway, in the CDAHFD-induced liver fibrosis leading to inhibition of HSCs activation and proliferation. In EFFECT-II trial, Dapagliflozin (SGLT2i) was found to reduce the levels of all measured hepatocyte injury biomarkers including cytokeratin 18-M30.

This study will be a randomized, comparative parallel study. The patients will be recruited from Outpatient Clinic of the Endocrine and Diabetes Unit, Tanta University Hospital, Tanta, Egypt. The diagnosis of NASH will be confirmed by imaging technique (increased liver echogenicity, stronger echoes in the hepatic parenchyma, vessel blurring, and narrowing of the lumen of the hepatic veins), mild to moderate elevation in aminotransferase activities (\>2 but \<5 times upper limit of normal), hepatic steatosis index (HSI) \>36, HAIR score (hypertension, alanine aminotransferase level, insulin resistance) of 2 or 3. The study will be conducted according to the ethical standards of Helsinki declaration in 1964 and its later amendments. The study duration will be 24 weeks. The patients will be randomized into two groups:

Group 1: (Pioglitazone group; n=28) which will receive 30mg/day pioglitazone for 24 weeks.

Group 2: (Empagliflozin group; n=28) which will receive 10mg/day empagliflozin for 24 weeks.

* All patients will be submitted to demography, physical examination, and measurement of waist circumferences, weight, height, and calculation of body mass index (BMI).
* Ultrasonography of the liver will be carried out at baseline and at the end of the study.
* All patients will be assessed for complete blood count, glycated hemoglobin (HbA1c %), fasting blood glucose, fasting insulin, serum creatinine, liver panel, fasting lipid profile, serum Cytokeratin-18, serum transforming growth factor-beta1(TGF-β1), Malondialdehyde (MDA) and Tumor necrosis factor- alpha (TNF-α).
* The Homeostasis Model Assessment-insulin resistance (HOMA-IR), Hepatic steatosis index (HSI), HAIR score and Fibrosis risk scores including Fibrosis index based on the 4 factors (FIB-4) and Aspartate transaminase-to-platelet ratio index (APRI) will be calculated.
* All patients will be followed up by weekly telephone calls and monthly direct meetings according to scheduled visits to assess their adherence and to report any drug related adverse effects.

The primary outcome is the change in fibrosis indices (FIB-4 and APRI) and the liver enzymes. The secondary outcome is the change in measured biological markers mainly cytokeratin-18, MDA, TNF-α, and TGF-β1.

* Sample size calculation:

The sample size was calculated depending up-on a previous Randomized Controlled Trial included Fifty patients with NAFLD who were randomly assigned to either the empagliflozin group 10 mg daily or the control group (standard treatment without empagliflozin) for 20 weeks. The two groups showed a significant difference (P = 0.005) for the change in serum ALT level. In this context, the initial sample size of 50 patients will be sufficient to provide a good power to detect the effect. Assuming that, the attrition rate is 10%, the initial sample size will be 56 patients in both groups.

* Ethical approval:

The study will be conducted according to the ethical standards of Helsinki declaration in 1964 and its later amendments. The study will be approved by the Research Ethical Committee of Tanta University. The study will be registered as a clinical trial on clinicaltrial.gov. All participants will be informed about the benefits and risks of the study. Any unexpected risks that will appear during the course of the research will be clarified to the participants and to the ethical committee on time. The data of the enrolled patients will be confidential. All enrolled patients will give their written informed consents.

* Statistical analysis:

  1. The collected data will be tabulated using Microsoft® Office Excel, 2019 (Microsoft Corporation).
  2. The statistical analysis will be carried out using SPSS statistical package version 26.0 (IBM corporation software group, USA).
  3. Data will be tested for normality using Shapiro-Wilk test or Kolmogorov-Smirnov test.
  4. Parametric data will be analyzed using Paired and un-Paired t-test to compare the means within the same group and to compare the means of the two groups respectively.
  5. Non-parametric data will be analyzed using Mann Whitney U test to compare the means within the same group and to compare the means between groups.
  6. Categorical data will be analyzed using Chi-Square test.
  7. Fisher's exact test will be used to analyses the reported adverse effects.
  8. Correlation between variables will be assessed using Pearson or Spearman correlation coefficient which appropriate.
  9. Data will be expressed as the mean ±SD, medians, range, number and percent as appropriate.
  10. The significance level will be set at p≤ 0.05.

Eligibility Criteria:
Inclusion Criteria:

* Non-diabetic patients.
* Both males and females.
* Age \>18 years old.
* Patient with body mass index (BMI) \> 30 kg/m2.
* Patients with established diagnosis of NASH based on liver ultrasonography, mild to moderate elevation in aminotransferase activities (\>2 but \<5 times upper limit of normal), hepatic steatosis index (HSI) \>36, and HAIR score of 2 or 3.

Exclusion Criteria:

* Patients with BMI \> 40 kg/m2.
* Patients with type 2 diabetes mellitus (T2DM) on the basis of a fasting plasma glucose (FPG) level ≥ 126 mg/dl (7mmol/L) or glycated hemoglobin (HbA1c) \> 6.5% (48 mmol/mol).
* Alcohol consumption greater than 20 g per day for women or greater than 30 g for men for at least three consecutive months over the past 5 years.
* History of viral hepatitis, hemochromatosis, Wilson's disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, biliary obstruction, alpha-1 antitrypsin deficiency.
* Patients on medications interfere with lipid and carbohydrate metabolisms.
* Patients with heart failure (New York Heart Association (NYHA) class 2-4).
* Patients with history of cardiovascular events within the past 3 months.
* Patients with renal impairment (eGFR\> 45 mL/min/ 1.73 m2).
* Patients with cancer or with a history of cancer treatment over the past 2 years.
* Patients with thyroid disorder.
* Patients on medications associated with steatosis such as Non-steroidal anti-inflammatory drugs (NSAIDs), amiodarone, tamoxifen, estrogen, sodium valproate, corticosteroids, and methotrexate.
* Patients with inflammatory diseases.
* Patients on supplements known to have antioxidant activity such as vitamin E, vitamin C, zinc, and selenium.
* Pregnant and breastfeeding women.
* Females on oral contraceptive pills will be also excluded.