Non-Alcoholic Fatty Liver Disease, the HEpatic Response to Oral Glucose, and the Effect of Semaglutide (NAFLD HEROES)

Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic
steatohepatitis (NASH), are leading causes of liver injury and are tightly associated with
obesity, diabetes and the metabolic syndrome. Despite recent advances, there is still a
considerable knowledge gap regarding the fundamental pathogenic mechanisms, and especially
regarding the transition from benign steatosis to steatohepatitis. Although NAFLD reflects
disordered energy metabolism in the liver, little information exists on the response of the
human liver to the acute caloric load of a meal. We hypothesize, based on preliminary
non-invasive results, that in patients with NAFLD, an oral carbohydrate load results in
preferential de novo lipogenesis (due to selective insulin resistance) and generation of
fatty acids (FA). We further hypothesize a spillover effect, wherein NASH patients have an
impairement of the hepatic ability to esterify the load of FA to triglycerides (TG) compared
to patients with steatosis, resulting in accumulation of lipotoxic intermediary metabolites.

GLP-1 receptor agonists (GLP-1RA) have demonstrated significant benefit in the treatment of
diabetes and obesity. Liraglutide was shown in a prospective trial to improve NASH histology
and other GLP-1RA have shown benefit in secondary analyses, consistent with a class effect.
However, response rates to GLP-1RA, as well as to other pharmacological interventions in
NAFLD, have not exceeded 50%, and there are no adequate baseline predictors of response that
could allow for selection of subjects for personalized treatment. Given that GLP-1RAs exert
their main activity in the post-prandial state, it is plausible that post-prandial parameters
may be more effective in predicting treatment response and can shed light on its mechanism.

Our aims in this study are (1) to assess the hepatic response to an acute oral carbohydrate
load; (2) to identify which baseline parameters can predict the clinical response of NAFLD
patients to a course of semaglutide, a novel GLP-1RA.

We propose a non-randomized, single-center, pilot exploratory study in which up to 32
subjects with NAFLD (16) or NASH (16) will initially undergo two liver biopsies, one in the
fasting state and one performed 2 hours after an oral 75g glucose load (OGTT biopsy). Tissue
samples obtained will be subjected to a comparative, paired analysis of gene expression,
protein phosphorylation in key signaling pathways, composition of tissue lipid species and
oxidative stress. Subjects will be treated with semaglutide (escalated to 1 mg/week) for 30
weeks in all subjects, and their clinical response will be assessed by ALT and 1H-Magnetic
resonance spectroscopy and a final (3rd) liver biopsy. Clinical responders will be compared
to non-responders with regards to their baseline fasting and post-prandial parameters, to
identify predictors of response.

The human hepatic response to a meal has never been studied at the tissue level and the
findings of this study are likely to generate important data and clarify some of the
fundamental questions regarding mechanisms of injury and insulin resistance. Furthermore, our
study aims at identifying predictors of response to GLP-1RA and allow for appropriate
selection of subjects for this class of medications, as well as to shed light on mechanism of
response.

- INCLUSION CRITERIA:

1. Histological evidence of hepatic steatosis on a liver biopsy within 12 months OR
evidence of fatty liver disease, as documented by imaging (ultrasound, CT, MRI,
MRI-PDFF, MR spectroscopy, or Fibroscan CAP greater than or equal to 285
db/M(25)) within 12 months.

2. Estimated average alcohol consumption < 30 g/d for men or < 20 g/d for women in
the 6 months prior to enrollment and no binge-drinking behavior.

3. Age > 18 years at enrollment.

Additional Inclusion Criteria for Treatment Phase

1. Presence of NAFLD (steatosis grade greater than or equal to 1 on NASH-CRN scoring
scale) on initial admission liver biopsy.

AND

2. Liver fat content greater than or equal to 10% by 1H-MRS on initial admission.

EXCLUSION CRITERIA:

1. Chronic infection with hepatitis C virus (HCV) or hepatitis B virus (HBV). Patients
who were treated successfully for HCV and achieved sustained virological response can
be eligible for enrollment > 18 months after treatment cessation. Patients receiving
antiviral therapy are ineligible.

2. HIV infection.

3. Concomitant liver disease such as autoimmune hepatitis, primary biliary cholangitis,
primary sclerosing cholangitis, Wilson s disease, alpha-1 antitrypsin deficiency,
hereditary hemochromatosis.

4. Presence of definite or probable drug-induced liver injury. In the case of
lipid-lowering, anti-hypertensive or anti-diabetic medications that are suspected to
cause aminotransferase elevation, patients will be eligible if treatment is associated
with stable enzyme levels for at least 6 months.

5. Decompensated advanced liver disease, defined as direct bilirubin > 0.5 g/dL, PT > 18
, albumin < 3 g/dL, MELD score > 12 (applicable only in patients without Gilbert s
syndrome), or history of ascites, encephalopathy, variceal bleeding, spontaneous
bacterial peritonitis or liver transplant

6. Treatment with medications known to cause fatty liver disease such as atypical
neuroleptics, tetracycline, methotrexate or tamoxifen

7. Uncontrolled hypo- or hyperthyroidism.

8. Thyroid nodules with ultrasonographic features suggestive of an increased risk of
thyroid cancer per radiologist reporting (hypoechoic, microcalcifications, twinkling
on B flow imaging, central vascularity, irregular margins, incomplete halo, nodule
taller than wide and documented enlargement of a nodule), or nodules associated with
an abnormal TSH (0.4 to 5 mU/L).

9. Active coronary artery disease, defined as persistent angina pectoris, reversible
ischemia on cardiac stress test or imaging, or the presence of significant coronary
artery disease on imaging or catheterization. Patients with coronary artery disease
that was treated by angioplasty or bypass surgery may be eligible if they have no
evidence of active disease >= 1 year after intervention, can safely stop antiplatelet
and anticoagulant medications before the performance of invasive procedures, and have
adequate ventricular function as assessed by echocardiography or cardiology
consultation. These patients will require cardiology consultation and clearance prior
to enrollment.

10. Congestive heart failure.

11. Chronic kidney disease, with creatinine clearance < 60 ml/min or eGFR <
60/ml/min/m(2).

12. Uncontrolled diabetes mellitus with HbA1c > 9% will exclude subjects. Patients with
diabetes may be enrolled only if they have HbA1c <=9%, have been on stable therapy
with lifestyle and/or metformin for at least 3 months prior to enrollment, and are not
foreseen to require change of antidiabetic medication or dose during the trial.

13. Use of insulin, sulfonylurea agents, thiazolidendiones, SGLT2 inhibitors, GLP-1
receptor agonists or DPP-4 inhibitors unless discontinued greater than or equal to 3
months before enrollment.

14. Contraindication or inability to perform a liver biopsy.

1. Patients with coagulopathy (PT/PTT values that are prolonged greater than or
equal to 3 seconds from the upper limit of the normal, including treatment with
oral and parenteral anticoagulants), thrombocytopenia (< 70,000), abnormal
bleeding time or platelet dysfunction. Antiplatelet agents taken for
cardiovascular prevention will not exclude patients, unless they cannot be
stopped safely for the performance of a liver biopsy.

2. Hemoglobin level < 11 g/dL

15. Contraindications to MRI (heart pacemakers, unless MRI safe, insulin pumps, implanted
hearing aids, neurostimulators, intracranial metal clips, metallic bodies in the eye,
metal hip replacements, sutures, extreme anxiety or fear of small spaces.)

16. History of gastric bypass or other bariatric surgery, partial or complete gastrectomy
and known maldigestion or malabsorption.

17. Treatment with orlistat.

18. Patients with uncontrolled eating disorders including anorexia and bulimia nervosa.

19. Patients with proliferative diabetic retinopathy.

20. Use of medications or supplements to treat NAFLD (approved or unapproved) unless
withdrawn greater than or equal to 3 months prior to enrollment or taken at a stable
dose for greater than or equal to 6 months.

21. Patients who had a liver biopsy performed less than or equal to 2 years before
enrollment, unless they are willing to undergo all of the trial biopsies, knowing that
these biopsies are purely for research and are not clinically indicated. This will be
clearly documented in the patients charts prior to enrollment.

22. Inability or unwillingness to receive subcutaneous injections.

23. Known or suspected allergy to trial medication(s), excipients, or related products.

24. Alcohol or substance abuse within the past 12 months.

25. For women of childbearing age, breast-feeding, pregnancy or inability (or
unwillingness) to practice contraception for the duration of the study or breast
feeding.

26. Personal or first-degree family member with history of medullary thyroid carcinoma or
subjects with known multiple endocrine neoplasia syndrome type 2 (MEN-2).

27. Actively pursuing an intensive weight loss regiment, aimed at losing > 10% of current
body weight, by following a different diet or exercise regimen over the study time
period or recent (<3 months) significant weight loss (>10%) .

28. The receipt of any investigational drug within 3 months prior to enrollment in this
trial.

29. Assessment by the principal investigator that the subject will be unlikely to complete
the study procedures, or that enrollment puts the subject at a significant risk
unspecified by the criteria above.

Enrollment Criteria for Healthy Volunteers (arm C)

1. No history of known liver disease

2. Not on any regular systemic medications (with the exception of oral contraceptives)

3. BMI less than or equal to 25 kg/m2

4. Non-diabetic

5. Normal transaminases (ALT less than or equal to 31 U/L for men or less than or equal
to 19 U/L for women, and AST less than or equal to 30 U/L)

6. Fasting glucose less than or equal to 95 mg/dL