Pilot Randomized Controlled Trial of Spironolactone in Young Women With Nonalcoholic Steatohepatitis (NASH)
Phase: PHASE1
Conditions: NASH - Nonalcoholic Steatohepatitis
Sponsor: University of California, San Francisco

Clinical Research Protocol
Pilot Randomized Controlled Trial of Spironolactone in Women With Nonalcoholic 
Steatohepatitis (NASH)
PRINCIPAL INVESTIGATOR:
MONIKA SARKAR, MD
ASSOCIATE CLINIC PROFESSOR OF 
GASTROENTEROLOGY AND HEPATOLOGY
INSTITUTIONS:
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO

Clinical Research Protocol
Pilot Randomized Controlled Trial of Spironolactone in Women With Nonalcoholic 
Steatohepatitis (NASH)
Version Date:
June 9, 2020
Investigational Product:
Spironolactone
IND Number:
IND is not indicated as discussed with Melanie Hassel 
(communication attached)
Funding Organization:
National Institutes of Health
National Institute of Diabetes and Digestive and Kidney 
Diseases
Principal Investigator:
Name:
Monika Sarkar, MD 
Telephone:
(415) 502-2656
Fax:
(415) 476-0659
E-mail:
monika.sarkar@ucsf.edu
Medical Monitor:
Name:
Marcelle Cedars, 
MD Telephone: 415-353-3032
Fax:
(415) 476-0659
E-mail:
marcelle.cedars@ucsf.edu
Approval:
PI or Sponsor Signature (Name and Title)
Date
This confidential information about an investigational product is provided for the 
exclusive use of investigators of this product and is subject to recall at any time. 
The information in this document may not be disclosed unless federal or state law 
or regulations require such disclosure. Subject to the foregoing, this information 
may be disclosed only to those persons involved in the study who have a need to 
know, with the obligation not to further disseminate this information.

PROTOCOL AGREEMENT
I have read the protocol specified below. In my formal capacity as Investigator, my duties 
include ensuring the safety of the study subjects enrolled under my supervision and 
providing the University of California, San Francisco Institutional Review Board (IRB) with 
complete and timely information, as outlined in the protocol. It is understood that all 
information pertaining to the study will be held strictly confidential and that this 
confidentiality requirement applies to all study staff at this site. Furthermore, on behalf of 
the study staff and myself, I agree to maintain the procedures required to carry out the 
study in accordance with accepted GCP principles and to abide by the terms of this 
protocol.
Protocol Title: Pilot Randomized Controlled Trial of Spironolactone in Women With 
Nonalcoholic Steatohepatitis (NASH)
Protocol Date: June 9, 2020
Investigator Signature
Date
Print Name and Title 
Site #
1
Site Name
University of California San Francisco 
Address
Liver Disease & Liver Transplant Clinic 
350 Parnassus Ave.
Suite 300
San Francisco, CA 94143
Phone Number
415-353-2318

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TABLE OF CONTENTS
Table of Contents..............................................................................................................4
List of Abbreviations..........................................................................................................6
Protocol Synopsis. ............................................................................................................8
1 BACKGROUND................................................................................................................12
1.1
Overview of Clinical Studies.......................................................................12
2 STUDY RATIONALE.................................................................................................13
2.1
Risk / Benefit Assessment........................................................................13
3 STUDY OBJECTIVES...............................................................................................15
3.1
Primary Objective .....................................................................................15
3.2
Secondary Objectives................................................................................15
4 STUDY DESIGN........................................................................................................15
4.1
Study Overview..........................................................................................15
5 CRITERIA FOR EVALUATION ...............................................................................16
5.1
Primary Endpoints......................................................................................16
5.2
Secondary Endpoints.................................................................................16
5.3
Safety Evaluations .....................................................................................17
6 SUBJECT SELECTION.............................................................................................17
6.1
Study Population........................................................................................17
6.2
Inclusion Criteria ........................................................................................17
6.3
Exclusion Criteria.......................................................................................17
7 CONCURRENT MEDICATIONS .............................................................................18
7.1
Allowed...........................................................................................................18
7.2
Prohibited .......................................................................................................18
8 STUDY TREATMENTS .............................................................................................19
8.1
Method of Assigning Subjects to Treatment Groups………………………19
8.2
Blinding.....................................................................................................19
8.3
Test and Control Formulation ....................................................................19
8.4
Supply of Study Drug at the Site ..............................................................20
8.5
Dosage Regimen .......................................................................................20
8.6
Administration Instructions .......................................................................20
8.7
Study Drug Accountability……………………………………………………..21
8.8.  Measures of Treatment Compliance…………………………………………21
9 STUDY PROCEDURES AND GUIDELINES ...........................................................21
9.1
Clinical Assessments................................................................................21
9.2
Clinical Laboratory Measurements ...........................................................22
9.3
Liver Biopsy..............................................................................................23
9.4
Magnetic Resonance Imaging..................................................................23
10 EVALUATIONS BY VISIT......................................................................................23
10.1 Visit 1 (Screening) ....................................................................................23
10.2 Visit 2 (Day 1/Baseline)..............................................................................23

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10.3 Visit 3 – 4 (Months 1, 3)....................................................................................24
10.4 Visit 5 (Month 6 End of Study/Treatment).........................................................24
10.5 Visit 6 (Follow-up) .............................................................................................24
10.6 Optional Visits (Months 9, 12)……………………………………………………...24
11 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION ........................25
11.1 Adverse Events...............................................................................................25
11.2 Serious Adverse Experiences (SAE)...............................................................26
11.3 Investigational Product Adjustment & Interruption Criteria.................................26
11.4 Medical Monitoring............................................................................................27
12 DISCONTINUATION AND REPLACEMENT OF SUBJECTS .................................27
12.1 Early Discontinuation of Study Drug..................................................................27
12.2 Withdrawal of Subjects .....................................................................................27
12.3 Replacement of Subjects..................................................................................27
13 PROTOCOL VIOLATIONS........................................................................................27
14 DATA SAFETY MONITORING................................................................................28
15 STATISTICAL METHODS AND CONSIDERATIONS..............................................28
15.1 Data Sets Analyzed ..........................................................................................28
15.2 Demographic and Baseline Characteristics......................................................28
15.3 Analysis of Primary Endpoint............................................................................28
15.4 Analysis of Secondary Endpoints.....................................................................28
15.5 Interim Analysis...............................................................................................29
15.6 Sample Size and Randomization .....................................................................30
16 DATA COLLECTION, RETENTION AND MONITORING.........................................29
16.1 Data Collection Instruments..............................................................................29
16.2 Data Management Procedures .......................................................................29
16.3 Data Quality Control and Reporting..................................................................30
16.4 Archival of Data.................................................................................................30
16.5 Availability and Retention of Investigational Records.......................................30
16.6 Monitoring .......................................................................................................30
16.7 Subject Confidentiality ......................................................................................30
17 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS......................30
17.1 Protocol Amendments ......................................................................................31
17.2 Institutional Review Boards and Independent Ethics Committees...................31
17.3 Informed Consent Form...................................................................................31
17.4 Publications.......................................................................................................32
17.5 Investigator Responsibilities .............................................................................32
Appendix 1 Schedule of Events. .......................................................................................34
References...............................................................................................................................35

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List of Abbreviations
AE
adverse event
ALT
alanine aminotransferase
AST
aspartate aminotransferase
AUDIT
Alcohol Use and Disorders Identification Test 
CARDIA
Coronary Artery Risk Development in Young Adults 
CFR
Code of Federal Regulations
CBC
Complete blood count
CFR
Code of Federal Regulations
CRF
case report form
CTCAE
Common Terminology Criteria for Adverse Events
DHEAS
Dehydroepiandrosterone
DMC
Data Monitoring Committee
DSMB
Data Safety Monitoring Board
eCRF
electronic case report form
EDC
Electronic Data Capture
EU
European Union
FDA
Food and Drug Administration
GCP
Good Clinical Practice
HbA1c
Hemoglobin A1c
HCV
Hepatitis C virus
HCV Ab
Hepatitis C virus antibody
HIPAA
Health Insurance Portability and Accountability Act of 1996
HIV
Human Immunodeficiency Virus
HOMA-IR
Homeostatic model assessment for insulin resistance
HRPP
Human Research Protection Program
ICF
informed consent form
ICH
International Council on Harmonisation
IEC
Independent Ethics Committee
IRB
Institutional Review Board
IND
Investigational New Drug
INR
International normalized ratio
LDL
low-density lipoprotein
LDL-R
low-density lipoprotein receptors
MeDRA
Medical Dictionary for Regulatory Activities
MRE
Magnetic Resonance Elastography
MRI
Magnetic Resonance Imaging
MRI-PDFF
Magnetic Resonance Imaging Proton Density Fat Fraction

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NAFLD
Nonalcoholic Fatty Liver Disease
NAS
NAFLD Activity Score
NASH
Nonalcoholic Steatohepatitis
NF
National Formulary and Drug Standards Laboratory
PI
Principal Investigator
PCOS
Polycystic Ovary Syndrome
NIDDK
National Institute of Diabetes and Digestive and Kidney 
Diseases
SAE
serious adverse experience
SAP
Statistical Analysis Plan
SGOT
serum glutamic oxaloacetic transaminase
SGPT
serum glutamate pyruvate transaminase
UCSF
University of California San Francisco
U.S.
United States
USP
United States Pharmacopeial Convention, Incorporated

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PROTOCOL SYNOPSIS
TITLE
Pilot Randomized Controlled Trial of Spironolactone in Women With 
Nonalcoholic Steatohepatitis (NASH)
SPONSOR
Monika Sarkar
FUNDING 
ORGANIZATION
NIDDK
NUMBER OF SITES
1
RATIONALE
Nonalcoholic steatohepatitis (NASH), or fat-related liver inflammation 
and scarring is projected to be the leading cause of cirrhosis in the 
United States (U.S.) within the next few years. Women are at 
disproportionate risk for NASH, with approximately 15 million U.S. 
women affected. There is an urgent need to understand risk factors for 
NASH and its progression in women, and sex hormones may provide 
a missing link.
Our preliminary data support a detrimental role of androgens, or “male 
sex hormones” on fatty liver in women but no studies have evaluated 
whether androgens are associated with liver inflammation and/or 
scarring from fatty liver (aka NASH). To better understand the 
mechanism by which androgens might promote NASH and/or 
metabolic co-factors that contribute to NASH, we are conducting a 
pilot clinical trial to primarily assess the feasibility of using an 
androgen blocking medication, spironolactone, in women with NASH. 
Spironolactone was selected because it is has been commonly 
prescribed for decades with good safety profile and tolerability to treat 
symptoms of high androgens, like acne and hirsutism in women.
Though primarily a feasibility-focused study, we also aim to explore 
the pathways by which blocking testosterone receptors might alter 
the biologic processes that promote NASH and its associated 
metabolic co-morbidities in women.
STUDY DESIGN
This is a single center, double-blind, placebo-controlled, randomized, 
(2:1) parallel group pilot clinical trial of spironolactone in women with 
biopsy-proven NASH receiving 6 to 12 months of spironolactone or 
placebo.
PRIMARY 
OBJECTIVES
To establish the feasibility and tolerability of the intervention and study 
outcomes measures.
SECONDARY 
OBJECTIVES

To examine the effect of spironolactone on features of NASH on 
MRE and MRI-PDFF

To examine the effect of spironolactone on features of NASH on 
liver biopsy

To examine the effect of spironolactone on metabolic 
parameters that contribute to NASH progression

To examine the effect of spironolactone on sex hormone levels 
and androgen receptor expression in liver tissue

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NUMBER 
OF 
SUBJECTS
30 total women:
20 randomized to spironolactone and 10 randomized to placebo
SUBJECT 
SELECTION
CRITERIA
Key Inclusion Criteria:
- Women with NASH identified on standard of care liver biopsy 
performed for clinical purposes.
Key Exclusion Criteria:
- Uncontrolled diabetes
- Alcohol consumption >2 drinks per day for at least 3 consecutive 
months over the previous 5 years
- Other chronic liver disease (i.e. hepatitis B virus, hepatitis C virus, 
autoimmune hepatitis) or cirrhosis from any cause
- Recent or planned upcoming weight reduction surgery within five years 
of diagnosis of biopsy-confirmed NASH
- HIV infection
- Drugs associated with fatty liver (i.e. amiodarone, methotrexate, 
systemic glucocorticoids, tamoxifen, anabolic steroids, valproic acid) for 
more than 4 weeks prior to baseline or during study
- Recent, current, or planned upcoming pregnancy or current 
perimenopausal status
- Renal impairment (glomerular filtration rate <45 ml/min/1.73m or 
potassium levels > 5.0 mmol/L)
- Androgen receptor antagonist use (i.e. flutamine, spironolactone or 
flutamide) for more than 3 months within one year prior to baseline 
bi
TEST PRODUCT, 
DOSE, AND ROUTE 
OF ADMINISTRATION
Spironolactone 100mg. Product will be administered orally once daily 
for a length of 6 to 12 months.
CONTROL PRODUCT, 
DOSE AND ROUTE 
OF ADMINISTRATION
Placebo. Product will be administered orally once daily for a length of 
6 months.
DURATION OF 
SUBJECT 
PARTICIPATION AND 
DURATION OF 
STUDY
Subjects will be on study for up to 10 or 16 months
Screening: up to one month
Treatment: 6 to 12 months
Follow-up: up to 3 months from end of treatment
The total duration of the study is expected to be 4 years. Women will be 
enrolled and treated on a rolling basis, with a 30 month total recruitment 
period, 6 to 12 months of treatment per participant, and up to 3 months 
for the final subject follow-up post treatment.

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CONCOMMITANT 
MEDICATIONS- 
PROHIBITED
- Androgen receptor antagonists (i.e. flutamine or flutamide) including 
current use of spironolactone
- Epleronone as also blocks aldosterone receptor
- Drugs associated with fatty liver (i.e. amiodarone, methotrexate, 
systemic glucocorticoids, tamoxifen, anabolic steroids, valproic acid)
EFFICACY 
EVALUATIONS
As a pilot trial this study is not powered to detect an effect of 
spironolactone on NASH progression in women. The primary goal of 
the study is rather to evaluate the feasibility and tolerability of the study 
intervention and outcome measures in women with NASH while also 
exploring the mechanisms by which blocking testosterone receptors 
may alter NASH severity or associated metabolic comorbidities leading 
to NASH. If the intervention is deemed to be feasible, and we detect a 
signal that spironolactone may alter these biologic processes, then 
these data would be used to support a larger trial aimed at assessing 
the efficacy of spironolactone or other related androgen modulating 
medication on the treatment of NASH in women.
PRIMARY 
ENDPOINTS
Feasibility endpoints:
●
Adherence and acceptability of spironolactone in women with 
NASH
●
Adherence and acceptability of study outcome measures (MRI 
and optional EOT biopsy)
Determine the range of NASH on baseline biopsy to inform inclusion 
criteria for larger trials
SECONDARY 
ENDPOINTS
●
Biochemical endpoints: serum lipids, HOMA-IR, liver enzymes
●
Radiologic endpoints: liver fat quantity, visceral fat volume, liver 
inflammation, liver scarring
●
Histologic endpoints: Change in NAFLD Activity Score (NAS)
●
Expression of androgen receptors in the liver and circulating 
hormone levels.
SAFETY 
EVALUATIONS
Spironolactone is an old, commonly used medication to treat 
symptoms of high androgens in women therefore has a longstanding 
favorable side effect profile. Change in clinical safety labs and in 
person review of potential side effects will be assessed at each study 
visit. Incidence of adverse events will be collected.

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PLANNED INTERIM 
ANALYSES
The Data Safety Monitoring Board (DSMB) will review the progress of 
the study and perform an interim review of the safety data after the first 
10 participants have completed 3 months of spironolactone, or at any 
time that a comprehensive review of safety is felt to be necessary. The 
committee will provide recommendations to Dr. Sarkar whether the 
nature, frequency, and severity of adverse effects associated with 
study treatment warrant the early termination of the study in the best 
interests of the participants, whether the study should continue as 
planned, or the study should continue with modifications. The DSMB 
may also provide recommendations as needed regarding study design, 
or changes to study conduct.
STATISTICS
Primary Analysis 
Plan
The focus of the pilot trial will be feasibility testing and obtaining 
preliminary assessments of the effects of spironolactone. Exact 95% 
confidence intervals for rates of agreement to participate, meeting 
eligibility criteria, completing follow-up will be calculated using the 
binomial distribution. Between-group analyses will be performed 
according to the intention-to-treat principle. We will attempt to collect 
complete follow-up data on every randomized participant, regardless of 
adherence to treatment. As randomization in this small sample may not 
result in the perfect balance of relevant biological variables (e.g., age or 
weight between groups) we will perform analyses adjusting for any of 
these that are substantially imbalanced. A planned interim analysis will 
be conducted after the first 10 participants (25%) have completed 3 
months of spironolactone. Interim results will provide useful preliminary 
safety and feasibility data.
Rationale for Number 
of Subjects
The sample size of 30 was determined based on budget and feasibility 
within a 4-year grant timeline. The sample size is also reasonable for 
pilot trial, and will allow us to estimate effects and feasibility with 
reasonable precision.

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1    BACKGROUND
Nonalcoholic steatohepatitis (NASH), or fat-related liver inflammation and scarring is 
projected to become the leading cause of cirrhosis, primary liver cancer, and the leading 
indication for liver transplantation in the United States (U.S.) within the next few years.(1,
2) Women are at disproportionate risk for NASH, with approximately 15 million U.S. 
women affected, and therapeutic options in NASH are limited. There is an urgent need to 
understand risk factors for NASH and its progression in women, and our preliminary data 
support a detrimental role of androgens, or “male sex hormones” on this process in 
women.(3)
To better understand the mechanism by which androgens might promote NASH, we are 
conducting a pilot clinical trial using a competitive inhibitor of the testosterone receptor 
known as spironolactone. Spironolactone was chosen because it is well tolerated and 
has a favorable safety profile without the associated liver side effects of other androgen 
blocking medications. Moreover, it has been commonly prescribed for decades to treat 
symptoms of hyperandrogenism in women, such as hirsutism and acne. While this trial 
is a feasibility-focused study, we do aim to obtain preliminary data on pathways by 
which blocking testosterone receptors might alter the biologic processes that promote 
NASH in women, including measures of NASH severity on biopsy and comprehensive 
serologic and imaging measures of metabolic co-morbidities.
1.1
Overview of Clinical Studies
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver 
disease, affecting one third of the United States (U.S.) population.(4) NAFLD 
encompasses a spectrum of pathology including isolated steatosis in approximately 
75% of individuals, with the remaining 25% having more serious fat-related liver 
inflammation and scarring, known as non-alcoholic steatohepatitis (NASH).(4) While 
isolated steatosis is more common in men, women have higher rates of NASH, with an 
estimated 15 million women in the U.S. affected.(5, 6) To prevent liver-related deaths 
due to NASH- associated cirrhosis and liver cancer- there is an urgent need to 
understand key risk factors for NASH in women and sex hormones may provide an 
important link.
Androgens, traditionally called “male sex hormones”, are produced in men and women. 
Data on androgens in women with NAFLD have largely focused on hyperandrogenic 
women with Polycystic Ovary Syndrome (PCOS).(7) Indeed half of these women have 
imaging-confirmed steatosis and liver enzyme elevation.(8, 9) In our prior work in the 
prospective Coronary Artery Risk Development in Young Adults (CARDIA) cohort we 
showed that higher testosterone levels in pre-menopausal women were associated with 
subsequent risk of NAFLD in midlife. Importantly this association was present even 
among women without hyperandrogenism suggesting that androgens may play an 
important role in the pathogenesis of NAFLD in a broader spectrum of women that 
extends beyond the well-recognized subgroup of women with PCOS.(3) Although the
●•••••

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mechanism linking androgens to NAFLD in women is not well-defined, we found that 
visceral adiposity was a significant mediator of this association, therefore may play an 
important role along the causal pathway from testosterone to NAFLD in women.
Consistent with our preliminary data, clinical studies have demonstrated increased 
visceral adiposity in post-menopausal women treated with synthetic testosterone.(10) 
Testosterone has been further shown to directly regulate lipid metabolism within both 
visceral and subcutaneous adipocytes in women.(11) Androgens have been shown to 
regulate cholesterol clearance in the liver.(12) In mouse models of NASH, spironolactone 
has also been shown to improve HOMA-IR and serum lipids, key metabolic factors that 
contribute to NASH in humans.(13) Taken together, these data suggest a complex 
interplay of sex hormones within the liver and need for investigation into their role in 
NAFLD-related injury in women.
2
STUDY RATIONALE
Women have disproportionate risk for NASH and androgens might promote NASH, or 
contribute to metabolic derangements that contribute to NASH. Spironolactone is a 
commonly used medication in women to treat high testosterone levels, and data in animal 
models and humans support a role of testosterone in promoting metabolic disease 
including fatty liver. This study will assess whether measures of NASH and associated 
metabolic co-morbidities that promote NASH may be altered from blocking testosterone 
receptors with spironolactone. The goal of this study is to assess the feasibility of the 
intervention and the selected study outcomes, as well as to determine whether biologic 
processes that lead to NASH are affected by testosterone blockade. If we detect a signal 
of these biologic effects, our data could be used to support a future, larger clinical trial 
aimed at evaluating the efficacy of an androgen-modulating therapy to treat NASH in 
women that incorporates the feasibility results identified in this pilot trial.
2.1
Risk / Benefit Assessment
The risks involved in this pilot clinical trial are reasonable in relation to the anticipated 
benefits to the participant and to society, including understanding the role of androgens in 
the pathway of NASH and associated metabolic conditions, which may provide 
preliminary feasibility and biologic data to support larger trials to develop hormone 
targeted therapy to treat NASH in women.
The specific risks and methods for mitigating these are as follows:
1) Privacy breach: Eligible participants will be initially contacted in person during 
clinic visits by the principal investigator or research assistant to ascertain interest in 
study participation. If participants are interested and come for a screening visit, they 
will be sent the informed consent form prior to the visit to allow ample time to review. 
At the study visit, the PI or study coordinator will read aloud the consent form with the

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participant, describing the information we are seeking, procedures and indications, the 
voluntary nature of the study, the risks and benefits, and the ability to refuse or 
withdraw at any time without adverse consequences. The participant’s understanding 
of the study and its requirements will be confirmed, and any questions they have will 
be addressed prior to signing consent. A copy of the consent will be retained for the 
participant and the participant’s file, which is kept in a locked cabinet in a locked 
office.
There is a risk of loss of confidentiality for subjects in the study, despite maximal 
measures to minimize this risk. Maximal safeguards to maintain participants’ confidentiality 
will be employed, including: 1) unique study identification numbers on all forms and 
specimens 2) maintaining identifiers in a password-protected database, and 3) restricting 
access to the password protected database and locked study documents to essential 
study personnel.
2) Risks of frequent phlebotomy: The risks of routine blood drawing are rare but 
can include pain, bleeding, bruising at the site, anemia, dizziness, and very rarely 
an infection at the site. Only trained phlebotomists will perform blood draws. 
Phlebotomy risks will be minimized by the use of trained personnel and application 
of pressure after the blood draw. Should any adverse event occur, medical 
attention by a licensed physician will be provided immediately.
3) Risks of liver biopsy: About 20% of persons having a liver biopsy have some degree 
of pain over the liver that may last a few minutes to several hours. This rarely requires 
pain medication and usually resolves within 1-2 days, with most people not needing to 
alter their daily activities or work schedule. A rare complication of liver biopsy is severe 
bleeding such that a blood transfusion or an operation is needed to stop the bleeding. 
These complications occur in less than one in 1,000 times. Very rarely (in less than one in 
10,000 reported cases), death has occurred from bleeding after a liver biopsy. Other rare 
complications of liver biopsy include infection, and puncture of the gallbladder, lung or 
kidney. These complications occur in less than one in 1,000 times.
On the day of the biopsy, women will receive baseline CBC and coagulation tests to 
ensure a platelet count > 50,000 and INR < 1.5. Women with values that do not reach 
these parameters will not undergo biopsy due to increased bleeding risks, although this is 
not anticipated to be the case as women with cirrhosis that might have such hematologic 
abnormalities are excluded at baseline. Women with renal failure will also have already 
been excluded. To reduce bleeding risk, patients are instructed to hold aspirin and non- 
steroidal anti- inflammatory medications for 7 days prior to the procedure. Participants are 
observed in our procedural care unit for 2 hours after their liver biopsy, where vital signs 
are obtained every 15 minutes for one hour and then again 1 hour later for a total 2 hour 
observation period post biopsy. A complete blood count is performed at 1.5 hours after the 
biopsy and must be reviewed by the hepatologist prior to discharge home to ensure < 10% 
decline in hematocrit. If greater hematocrit decline, the patient will undergo additional 
evaluation including ultrasound to ensure no significant post procedural bleeding. Patients 
are instructed to limit physical activity, including straining or lifting over 10 pounds, for 48 
hours and are provided with emergency contact information for the on-call hepatologist. If 
any injury occurs, medical attention by a licensed physician will be provided immediately.
4) Risks of Spironolactone: Spironolactone is an old, commonly prescribed blood

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pressure medication and diuretic, and also commonly used for treating symptoms of 
high androgens including acne and hirsutism at a starting dose of 100mg daily. This 
medication has a longstanding history of favorable side effects profiles. The most 
likely side effect is increased urination. Other side effects with a dose of 100mg 
daily in the absence of renal failure (an exclusion criterion for the current study) are < 
1%.
Potential side effects include breast tenderness, tiredness, headache, dizziness, 
lightheadedness, diarrhea, cramping, nausea, vomiting, fever, or confusion. There is 
a low risk of renal insufficiency, electrolyte disturbances, dizziness and/or 
dehydration. To minimize any harm, all participants will have baseline renal and 
electrolytes obtained, an in-person study visit within one month of treatment initiation 
and then every 3 months thereafter to obtain subjective information regarding 
medication tolerability, to obtain vital signs, as well as laboratory assessment of 
sodium, potassium, and creatinine levels. All values will be reviewed by the principal 
investigator and Data Safety Monitoring Board to determine whether the current dose 
may be maintained, decreased, or discontinued.
Spironolactone is categorized as Pregnancy Class C due to potential risk of 
feminization seen in animal studies of male fetuses, though no studies have 
implicated spironolactone in these birth defects in humans. Urine pregnancy testing 
will be performed every 4 weeks in all pre- menopausal women. Urine pregnancy 
testing is not required for women who are not sexually active with men. As sex 
organ development begins between 6-8 weeks of gestational age, spironolactone 
would be discontinued no later than week 4 of conception to avoid fetal risk if a 
woman were to become pregnant.
3
STUDY OBJECTIVES
3.1
Primary Objective
To establish the feasibility and tolerability of the intervention and study outcomes 
measures.
3.2
Secondary Objectives

To examine the effect of spironolactone on features of NASH on MRE and MRI-
PDFF

To examine the effect of spironolactone on features of NASH on liver biopsy

To examine the effect of spironolactone on metabolic parameters that contribute 
to NASH progression

To examine the effect of spironolactone on sex hormone levels and androgen 
receptor expression in liver tissue
4
STUDY DESIGN
4.1
Study Overview
This is a single center, double-blind, placebo-controlled, randomized (2:1), parallel group 
pilot clinical trial. 30 women are targeted for enrollment. Each participant will be 
administered a single dose of spironolactone or placebo once daily for a total of 6 
months. In person evaluations will take place at Month 1, 3, 6, and at the follow up visit 
within 3 months of end of treatment (up to Month 9). Participants may opt to extend the 

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study treatment period to 12 months. Participants who opt to extend will complete 
additional in person evaluations at Month 9 and Month 12. Screening data will be 
reviewed to determine subject eligibility.  Subjects who meet all inclusion criteria and 
none of the exclusion criteria will be entered into the study.
The following treatment regimens will be used:
●
Experimental treatment – spironolactone, 100 mg once daily
●
Placebo or Comparator – one capsule, once daily
Total duration of study treatment will be 6 months or 12 months. Total duration of the 
study is expected to be ≤10 or ≤16 months (including up to 1 month for screening).
5
CRITERIA FOR EVALUATION
5.1
Primary Endpoints
This is a pilot clinical trial that is largely feasibility focused. The primary goal is to 
establish if spironolactone is feasible in women with NASH, and if our study outcome 
measures are reasonable. The feasibility endpoints will include:

Rates (and reasons) for the following: a) % women that decline/women 
contacted for study inclusion (i.e. need for a second liver biopsy, concern 
regarding randomization to placebo) b) % women enrolled/women screened 
(i.e. exclusion criteria too narrow), c) study dropout (i.e. medication side 
effects, too frequent study visits, and/or phlebotomy)

Describe the range of NASH histology seen on biopsy in consideration of 
designing a future clinical trial.

Radiologic endpoints: liver fat quantity, visceral fat volume, liver 
inflammation, liver scarring. These endpoints will be examined because 
they offer a non invasive way of assessing visceral fat, another metabolic 
factor that may be altered by testosterone blockade, as well as other non 
invasive measures of assessing liver fat and inflammation that could 
potentially allow for avoidance of liver biopsy in future studies.
5.2
Secondary Endpoints
●
Biochemical endpoints: serum lipids, HOMA-IR, liver enzymes. These endpoints will 
be examined because existing data outside of NASH suggest that androgen blockade 
may alter these metabolic pathways. These metabolic pathways are also keys players 
in the development and progression of NASH, therefore understanding the biologic 
mechanism of androgen blockade in women with NASH could be used to support the 
need for a larger clinical trial aimed at evaluating such hormone modulation to treat 
NASH in women.
●
Histologic endpoints- Change in the NAFLD activity score (NAS), which measures 
different components of NASH on liver biopsy. If we detect a signal that 
spironolactone may affect NASH histology, these data could also help support need 
for larger trial evaluating efficacy of androgen modulating therapy for treatment of 
NASH in women.
●
Expression of androgen receptors in the liver and circulating hormone levels. It is not 
known whether spironolactone changes expression of androgen receptors in the liver, 
and this research question can help us to understand the mechanism of 
spironolactone effects on the liver. Likewise, data are conflicting on whether 
spironolactone changes circulating hormone levels, and this pathway will be studied 
by measuring hormone levels pre and post treatment.

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5.3
Safety Evaluations
●
In-person visits will be conducted at baseline, at one month (given highest risk of 
adverse events in first month of drug exposure) and every 3 months thereafter. 
Additionally women will be seen within 3 months after end of treatment to ensure 
resolution of any drug related side effects. In-person study visits will include a 
physical exam with vital signs, anthropomorphic measurements, assessment of 
adherence with pill counts, assessment of side effects, and performance of safety 
monitoring labs including electrolytes, renal function and urine pregnancy tests. Each 
visit will also include contraceptive counseling for pre-menopausal women because 
spironolactone has potential risk of feminization seen in animal studies of male 
fetuses, though no studies have implicated spironolactone in these birth defects in 
humans. Pregnancy testing will performed every month to ensure that if a participant 
were to become pregnant, spironolactone is discontinued within 4 weeks of 
conception. This will ensure no fetal risks as human sex organ development begins 
between 6-8 gestational weeks.
●
There will be continuous safety surveillance of participants through the reporting of 
incidence of adverse events (AEs). AEs will be recorded in a systematic manner at 
every study visit and when otherwise volunteered by the participant.
6
SUBJECT SELECTION
6.1
Study Population
Subjects with a diagnosis of NASH who meet the inclusion and exclusion criteria will be 
eligible for participation in this study.
6.2
Inclusion Criteria
1.
Women 18 to 45 years of age at Baseline Visit.
2.
Documentation of NASH diagnosis confirmed on baseline liver biopsy (performed as 
clinical care) prior to study enrollment.
3.
Written informed consent (and assent when applicable) obtained from subject and 
ability for subject to comply with the requirements of the study.
6.3
Exclusion Criteria
1.
Pregnant, breastfeeding, or unwilling to practice birth control during participation in 
the study
2.
Presence of a condition or abnormality that in the opinion of the Investigator would 
compromise the safety of the patient or the quality of the data
3.
Uncontrolled diabetes (HbA1c 9.5% or higher within 60 days prior to enrollment)
4.
Routine alcohol consumption >7 drinks per week during the preceding 3 months prior 
to baseline liver biopsy.
5.
Other forms of chronic liver disease including hepatitis B virus infection (hepatitis B 
surface antigen positive), chronic hepatitis C virus (HCV) infection (HCV Ab and HCV 
ribonucleic acid positive), autoimmune disorders (e.g., primary biliary cholangitis, 
primary sclerosing cholangitis, and autoimmune hepatitis), drug-induced 
hepatotoxicity, Wilson disease, iron overload, and alpha-1-antitryspin deficiency, 
based on medical history and/or centralized review of liver histology

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6.
Any upcoming weight reduction surgery (e.g., Roux-en-Y or gastric bypass) or weight 
reduction surgery within five years prior to diagnosis of biopsy-confirmed NASH.
7.
HIV infection
8.
Receipt of drugs associated with NAFLD (i.e. amiodarone, methotrexate, systemic 
glucocorticoids, tamoxifen, anabolic steroids, valproic acid) for more than 4 weeks 
prior to baseline biopsy
9.
Perimenopausal status (defined as within 3 years of self-reported menopause) due to 
unstable hormonal levels during that time
10. Renal impairment defined as glomerular filtration rate <45 ml/min/1.73m or 
potassium levels > 5.0 mmol/L due to the diuretic effect of spironolactone
11. Participation in another clinical trial of an investigational drug or device
12. History of medication non adherence as noted upon chart review or patient report of 
difficulty with medication adherence
13. Androgen receptor antagonist use (i.e. flutamine, spironolactone or flutamide) for 
more than 3 months within one year prior to baseline biopsy.
14. Epleronone use as this is a diuretic that also blocks the aldosterone receptor and 
could compound side effects
15. Cirrhosis on baseline biopsy as this condition leads to altered sex hormone 
metabolism
16. Unstable dosing (i.e. dose increase, intermittent use, or initiation) of Vitamin E 
anytime during the 3 months prior to baseline biopsy
17. Significant weight loss (at least 10% decrease in body weight) over preceding 3 
months prior to baseline biopsy
18. Contraindication to MRI scanning (e.g. presence of permanent pacemakers, 
implanted cardiac devices, etc.)
19. Unstable dosing (i.e. dose increase, intermittent use, or initiation) of diabetes 
medications or lipid lowering medications anytime during the 3 months prior 
to baseline biopsy 
7
CONCURRENT MEDICATIONS
All subjects should be maintained on the same medications throughout the entire study 
period, as medically feasible, with no introduction of new chronic therapies.
7.1
Allowed Medications and Treatments
Standard therapy for management of metabolic co-morbidities is allowed except for 
treatments noted in the exclusion criteria described above and as noted in the prohibited 
medications section below.
7.2
Prohibited Medications and Treatments
Flutamide, spironolactone, or flutamine (because also blocks androgen receptors) and 
eplerenone (because same diuretic effect as spironolactone and could increase renal 
side effects) are prohibited during the study and administration will be considered a 
protocol violation.

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Drugs associated with NAFLD (i.e. amiodarone, methotrexate, systemic glucocorticoids, 
tamoxifen, anabolic steroids, valproic acid) taken for more than 4 weeks are not allowed 
between baseline and follow-up biopsies.
Any investigational drug throughout the study is prohibited.
8
STUDY TREATMENTS
8.1
Method of Assigning Subjects to Treatment Groups
The UCSF pharmacy will generate a randomization table using random number 
generating software, assigning patients on a 2:1 basis to spironolactone or placebo in a 
blocked randomization scheme. Patients will be assigned to a treatment arm in the order 
to which they present for the first day of dosing. Patients assigned to a treatment arm 
who later drop out of the study will not be replaced in the randomization scheme.
8.2
Blinding
Due to the objectives of the study, the identity of test and control treatments will not be 
known to investigators, research staff, or patients. The following study procedures will be 
in place to ensure double-blind administration of study treatments
Access to the randomization code will be strictly controlled.
A color and size-matched placebo capsule that looks identical to the spironolactone 
capsule will be used.
Packaging and labeling of test and control treatments will be identical to maintain the 
blind.
No active drug measurements will be obtained.
The study blind will be broken on completion of the clinical study, after all study endpoints 
have been ascertained by blinded study coordinators and after the study database has 
been locked. At that time, the investigators, research staff, and participants will be 
informed about their treatment group assignments.
During the study, the blind may be broken only in emergencies when knowledge of the 
patient’s treatment group is necessary for further patient management. Spironolactone 
has a longstanding favorable side effect profile. Therefore the only condition that will 
definitively lead to participant unblinding is pregnancy. The UCSF investigational 
pharmacy would then be notified and responsible for unblinding.
8.3
Test and Control Formulation
Spironolactone and matching placebo will be prepared by a California state-licensed 
compounding pharmacy.
Spironolactone capsules will be prepared from USP grade powder at a dose of 100 mg.
Matching placebo capsules of the same color, mass, and appearance to the 
spironolactone capsules will be filled using microcrystalline cellulose powder.

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8.4
Supply of Study Drug at the Site
The UCSF Investigational Drug Service (IDS) will receive the bulk supplies of 
spironolactone and matching placebo from the compounding pharmacy. Capsules will be 
stored, accounted for, assigned per the randomization schedule and packaged and 
labeled for dispensing to the subject by the IDS pharmacy staff once ordered by the 
investigator on a study order/prescription form.
Drug will be provided by the manufacturer to the UCSF Investigational Pharmacy every 
three months. Subjects who were randomized and not treated will be immediately 
replaced when an “earlier” slot becomes available due to the dropped subject.
8.4.1
Packaging and Labeling
The UCSF Investigational Drug Service (IDS) will receive the bulk supplies of 
spironolactone and matching placebo from the compounding pharmacy. Capsules will be 
stored, accounted for, assigned per the randomization schedule and packaged and 
labeled for dispensing to the subject by the IDS pharmacy staff once ordered by the 
investigator on a study order/prescription form.
The IDS generates labels in compliance with California State pharmacy regulations, e.g., 
bottles will bear the name and address of pharmacy, name of subject, date of dispensing, 
name of the prescriber, instructions for use, expiry date and name, strength and quantity 
of the material dispensed. Capsules will be labeled as “spironolactone or placebo” to 
maintain the blind.
8.4.2
Storage
Study drug will be stored by the study site at controlled room temperature, 15 to 30ºC (59 
to 86ºF). If the temperature of the study drug storage exceeds or falls below this range, 
this will be reported to the Sponsor or designee and captured as a deviation. Subjects will 
be instructed to store the medication in original packaging (plastic bottles and protected 
from light) at room temperature according to the instructions outlined on the Drug 
Administration Instructions.
8.4.3
Dispensing
An order form/prescription will be sent to the IDS pharmacy. Drug will be selected, 
counted and labeled by the IDS staff based on the randomization schedule. Labeled 
bottles will be picked up from the pharmacy by the investigator’s staff and dispensed to 
the subjects by the research staff during a study visit. Participants will receive 3 month 
supplies.
8.5
Dosage Regimen
Participants will take either spironolactone or placebo by mouth once daily in the 
morning. Morning is preferred due to the diuretic effect of spironolactone, which may be 
less convenient at nighttime. Absorption is not affected by fasting state therefore 
participants may take this with or without food.
8.6
Administration Instructions
At the participant’s baseline visit she will receive the pill bottle and be shown the pills with 
instructions on how to take this.

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8.7
Study Drug Accountability
An accurate and current accounting of the dispensing and return of study drug for each 
subject will be maintained on an ongoing basis by a member of the study site staff. The 
number of study drug dispensed and returned by the subject will be recorded on the 
Investigational Drug Accountability Record.
8.8
Measures of Treatment Compliance
Subjects should be instructed to retain all bottles of investigational product, even if empty, 
and to return them to the Investigator at the indicated visits. The Investigator or designee 
should perform investigational product accountability (i.e., count of returned capsules) 
and, if applicable, follow-up with the subject to retrieve any investigational product bottles 
that have not been returned. If the Investigator has concerns about a subject’s dosing 
compliance, she should discuss this with the subject, assess the reasons for 
noncompliance and the likelihood that the subject will remain noncompliant, and notify 
the Sponsor accordingly.
9
STUDY PROCEDURES AND GUIDELINES
A Schedule of Events representing the required testing procedures to be performed for 
the duration of the study is diagrammed in Appendix 1.
Prior to conducting any study-related activities, written informed consent and the Health 
Insurance Portability and Accountability Act (HIPAA) authorization must be signed and 
dated by the subject. If appropriate, assent must also be obtained prior to conducting 
any study-related activities.
9.1
Clinical Assessments
9.1.1
Concomitant Medications
All concomitant medication and concurrent therapies will be documented at Screening 
and at all subsequent in person study visits and at early termination when applicable. 
Dose, route, unit, frequency of administration, indication for administration, and dates of 
medication will be captured.
9.1.2
Demographics
Demographic information (i.e. date of birth, race, ethnicity) will be recorded at Screening.
9.1.3
Medical History
Relevant medical history, including history of current disease, alcohol use, and other 
pertinent information regarding underlying diseases will be recorded at Screening.
9.1.4
Physical Examination
A complete physical examination will be performed by either the investigator or a 
subinvestigator who is a physician or other licensed medical professional at Screening. 
An abbreviated physical examination tailored to the symptoms the subject reports will be 
done at all subsequent in person visits. New abnormal physical exam findings must be 
documented and will be followed by a physician or other qualified staff at the next 
scheduled visit.

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9.1.5
Vital Signs
Body temperature, blood pressure, pulse and respirations will be performed after resting 
for 5 minutes at all in person study visits.
9.1.6
Anthropomorphic measures
Height will be measured at Screening. Weight will be measured at all in person study 
visits. Waist circumference will be measured at all in person study visits.
9.1.7
Adverse Events
Information regarding occurrence of adverse events will be captured throughout the 
study. Duration (start and stop dates), severity/grade, outcome, treatment and relation to 
study drug will be recorded on the case report form (CRF).
9.2
Clinical Laboratory Measurements
9.2.1
Blood Chemistry Profile
Blood will be obtained and sent to the site’s clinical chemistry lab for determination of 
serum sodium, potassium, creatinine at in person study visits. Liver tests (aspartate 
aminotransferase (AST/SGOT), alanine aminotransferase (ALT/SGPT), alkaline 
phosphatase, total bilirubin, and direct bilirubin), hemoglobin A1c, glucose, insulin, and 
lipid panel will be performed at each visit.
9.2.2
Pregnancy Test
Women of childbearing potential are required to perform a pregnancy test every 4 weeks. 
A urine pregnancy test will be obtained from participants of childbearing age prior to their 
participation in the study and at every in person study visit. Women who are not sexually 
active with men may decline the urine pregnancy test.                                                                                      
Home pregnancy test kits will be supplied at each visit to women of childbearing potential 
and these are to be performed as per the kit instructions every 4 weeks between study 
visits from Day 1/Baseline to End of Study/End of Treatment.
The site will call the participant every 4 weeks in between visits to record the result of the 
home pregnancy test. In the event of a positive result the patient must discontinue study 
drug immediately and report the result to the site as soon as possible.
9.2.3
Sex Hormone Measurements
A comprehensive panel of sex hormones (testosterone, androstendione, DHEAS, 
estrone, estradiol, follicle stimulating hormone, and sex hormone binding globulin) will be 
performed at baseline, Month 6, and Month 12 (if subject opts to extend the study 
period). These will be sent to the research laboratory of Dr.Toni Zeigler at the University 
of Wisconsin National Primate Laboratory.
9.2.4.  Constitution of biobank
Samples of blood and liver tissue will be kept from patients who provide their written 
informed consent for the purpose of conducting future studies investigating the link 
between reproductive health in women and liver disease. An additional amount of blood 
(2 Tbsp) will be collected along with the main study lab samples at each visit. 
Approximately 1 cm of the 3 cm core of tissue obtained in the Month 6 (or Month 12) 

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End of Study/ End of Treatment Visit liver biopsy (optional procedure) will be stored in 
the biobank.
Specimens will be stored at the UCSF Clinical Research Services Moffitt site in a 
specimen bank. Specimens and certain non-identifying medical information may be 
shared with other scientists or companies not at UCSF.
9.3
Liver Biopsy
A core liver biopsy will be performed prior to Screening and at the End of Study/ End of 
Treatment Visit (either Month 6 or Month 12). The liver biopsy at the End of Study/End of 
Treatment is optional for the subject. A historical biopsy prior to the Day 1/Baseline Visit 
will be used as the screening biopsy. A full pathology report confirming diagnosis of 
NASH must be available.
The liver biopsy specimens will be collected in a sterile specimen cup containing 0.1% 
ethanol. The cup will be labeled and sent to UCSF Pathology. Pathology will mount tissue 
onto a formalin block and send to UCSF Histology for slide preparation and reading.
9.4
Magnetic Resonance Imaging
Liver stiffness assessments will be performed by MRE (shear wave 60 Hz) and MRI- 
PDFF at the Baseline/Day 1 Visit, Month 6, and Month 12 (if subject opts to extend to 12 
months). The MRE and MRI-PDFF assessments will occur sequentially. The scans will 
be read by a radiologist in the UCSF Department of Radiology & Biomedical Imaging.
10
EVALUATIONS BY VISIT
10.1
Visit 1 (Screening)

Review the study with the subject and obtain written informed consent and HIPAA 
authorization.

Assign the subject a unique screening number.

Record demographics data.

Record medical history, in relation to inclusion/exclusion criteria.

Record concomitant medications.

Perform a complete physical examination.

Perform and record vital signs (blood pressure, pulse), height, and weight.

Measure waist circumference.

Collect blood for clinical laboratory tests 

Collect urine sample and perform pregnancy test for women of childbearing 
potential.
10.2
Visit 2 (Day 1/Baseline)

Record any Adverse Experiences.

Concomitant medications review.

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
Assess liver stiffness by MRE and MRI-PDFF

Randomize to treatment group, dispense study drug, and provide instructions on 
how to take it.
10.3
Visits 3 – 4 (Months 1 and 3)

Record any Adverse Experiences.

Record changes to concomitant medications.

Perform abbreviated physical examination.

Perform and record vital signs (blood pressure, pulse) and weight.

Measure waist circumference.

Collect blood for clinical laboratory tests.

Collect urine sample and perform pregnancy test for women of childbearing 
potential.

Perform pill count for compliance assessment.

Collect all unused study drug.

Dispense study drug.
10.4
Visit 5 (Month 6 End of Study/Treatment)

Record any Adverse Experiences.

Record changes to concomitant medications.

Perform abbreviated physical examination.

Perform and record vital signs (blood pressure, pulse) and weight.

Measure waist circumference

Collect blood for clinical laboratory tests 

Collect urine sample and perform pregnancy test for women of childbearing 
potential.

Obtain liver biopsy (optional for subject).

Assess liver stiffness by MRE and MRI-PDFF

Perform pill count for compliance assessment.

Collect all unused study drug.
10.5
Visit 6 (Follow-Up)

Record any Adverse Experiences.

Record changes to concomitant medications.
10.6
Optional Visits (if subject opts to extend study period)

Month 9 visit will include the same evaluations as listed for Visits 3 and 4

Month 12 Visit will serve as the end of study/treatment visit (liver biopsy will be 
completed at Month 12 instead of Month 6 if subject wishes to extend study period).

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11
ADVERSE EXPERIENCE REPORTING AND 
DOCUMENTATION
11.1
Adverse Events
An adverse event (AE) is any untoward medical occurrence in a clinical investigation of a 
patient-administered pharmaceutical product and that does not necessarily have a causal 
relationship with the treatment.  An AE is therefore any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom or disease temporally associated 
with the administration of an investigational product, whether or not related to that 
investigational product. An unexpected AE is one of a type not identified in nature, 
severity, or frequency in the current Investigator’s Brochure or of greater severity or 
frequency than expected based on the information in the Investigator’s Brochure.
The Investigator or research assistant will probe, via discussion with the subject, for the 
occurrence of AEs during each subject visit and record the information in the site’s 
source documents. Adverse events will be recorded in the patient CRF. Adverse events 
will be described by duration (start and stop dates and times), severity, outcome, 
treatment and relation to study drug, or if unrelated, the cause.
11.1.1 AE Severity
The National Cancer Institute’s Common Terminology Criteria for Adverse Events 
(CTCAE) Version 3.0 should be used to assess and grade AE severity, including 
laboratory abnormalities judged to be clinically significant. The modified criteria can be 
found in the study manual.  If the experience is not covered in the modified criteria, the 
guidelines shown in Table 1 below should be used to grade severity. It should be pointed 
out that the term “severe” is a measure of intensity and that a severe AE is not 
necessarily serious.
Table 1. AE Severity Grading
Severity (Toxicity Grade)
Description
Mild (1)
Transient or mild discomfort; no limitation in activity; no medical 
intervention or therapy required. The subject may be aware of 
the sign or symptom but tolerates it reasonably well.
Moderate (2)
Mild to moderate limitation in activity, no or minimal medical 
intervention/therapy required.
Severe (3)
Marked limitation in activity, medical intervention/therapy 
required, hospitalizations possible.
Life-threatening (4)
The subject is at risk of death due to the adverse experience as 
it occurred. This does not refer to an experience that 
hypothetically might have caused death if it were more severe.
11.1.2 AE Relationship to Study Drug
The relationship of an AE to the study drug should be assessed using the following the 
guidelines in Table 2.
Table 2. AE Relationship to Study Drug

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Relationship 
to Drug
Comment
Definitely
Previously known toxicity of agent; or an event that follows a reasonable 
temporal sequence from administration of the drug; that follows a known or 
expected response pattern to the suspected drug; that is confirmed by 
stopping or reducing the dosage of the drug; and that is not explained by any 
other reasonable hypothesis.
Probably
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to the suspected 
drug; that is confirmed by stopping or reducing the dosage of the drug; and 
that is unlikely to be explained by the known characteristics of the subject’s 
clinical state or by other interventions.
Possibly
An event that follows a reasonable temporal sequence from administration of 
the drug; that follows a known or expected response pattern to that 
suspected drug; but that could readily have been produced by a number of 
other factors.
Unrelated
An event that can be determined with certainty to have no relationship to the 
study drug.
11.2
Serious Adverse Experiences (SAE)
An SAE is defined as any AE occurring at any dose that results in any of the following 
outcomes:
●
Death
●
a life-threatening adverse experience
●
inpatient hospitalization or prolongation of existing hospitalization
●
a persistent or significant disability/incapacity
●a congenital anomaly/birth defect
Other important medical events may also be considered an SAE when, based on 
appropriate medical judgment, they jeopardize the subject or require intervention to 
prevent one of the outcomes listed.
11.2.1 Serious Adverse Experience Reporting
Study sites will document all SAEs that occur (whether or not related to study drug) per  
UCSF HRPP Guidelines. The collection period for all SAEs will begin after informed 
consent is obtained and end after procedures for the final study visit have been 
completed.
In accordance with the standard operating procedures and policies of the local 
Institutional Review Board (IRB)/Independent Ethics Committee (IEC), the site 
investigator will report SAEs to the IRB/IEC.
11.3
Investigational Product Adjustment & Interruption Criteria
Dosage for investigational product should be held constant throughout the course of the 
study. In the event that the subject experiences adverse experiences that the Investigator 
suspects are related to the investigational product, a dosage adjustment or interruption of 
investigational product may be implemented. Subjects may also be permanently 
discontinued from investigational product by the Investigator at any time for clinical safety 

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concerns (see section 12.3).
Subjects who are temporarily discontinued from investigational product are expected 
to continue in the study and should follow the regular visit schedule for the duration 
of the study. Rechallenge with investigational product may be initiated at the 
Investigator’s discretion upon discussion with the medical monitor. Additional safety 
monitoring may be initiated.
11.4
Medical Monitoring
Dr. Marcelle Cedars should be contacted directly at these numbers to report medical 
concerns or questions regarding safety.
Phone: 415-353-3032 
   Email: marcelle.cedars@ucsf.edu
12
DISCONTINUATION AND REPLACEMENT OF SUBJECTS
12.1
Early Discontinuation of Study Drug
A subject may be discontinued from study treatment at any time if the subject, the 
investigator, or the Sponsor feels that it is not in the subject’s best interest to continue. 
The following is a list of possible reasons for study treatment discontinuation:

Subject withdrawal of consent (or assent)

Subject is not compliant with study procedures

Protocol violation requiring discontinuation of study treatment

Adverse event that in the opinion of the investigator would be in the best interest 
of the subject to discontinue study treatment

Lost to follow-up

Positive pregnancy test
If a subject is withdrawn from treatment due to an adverse event, the subject will be 
followed and treated by the Investigator until the abnormal parameter or symptom has 
resolved or stabilized.
All subjects who discontinue study treatment should come in for End of Treatment visit as 
soon as possible and be encouraged to complete all visit procedures.
12.2
Withdrawal of Subjects
A subject may be withdrawn from the study at any time if the subject, the investigator, or 
the Sponsor feels that it is not in the subject’s best interest to continue.
All subjects are free to withdraw from participation at any time, for any reason, specified 
or unspecified, and without prejudice.
Reasonable attempts will be made by the investigator to provide a reason for subject 
withdrawals. The reason for the subject’s withdrawal from the study will be specified in 
the subject’s source documents. Refer to Section 10 for End of Treatment procedures.
12.3
Replacement of Subjects
Subjects who withdraw from the study treatment will be replaced.

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13
PROTOCOL VIOLATIONS
A protocol violation occurs when the subject, investigator, or Sponsor fails to adhere to 
significant protocol requirements affecting the inclusion, exclusion, subject safety and 
primary endpoint criteria. Protocol violations for this study include, but are not limited to, 
the following:

Failure to meet inclusion/exclusion criteria

Use of a prohibited concomitant medication

Failure to comply with Good Clinical Practice (GCP) guidelines.
The Sponsor will determine if a protocol violation will result in withdrawal of a subject.
When a protocol violation occurs, it will be discussed with the investigator and a Protocol 
Violation Form detailing the violation will be generated. This form will be signed by a 
Sponsor representative and the Investigator. A copy of the form will be filed in the site’s 
regulatory binder and in the Sponsor’s files.
14
DATA SAFETY MONITORING
The Data Safety Monitoring Board (DSMB) will include one reproductive endocrinologist 
(Heather Huddleston, MD) and one hepatologist (Jennifer C.Lai, MD) who are UCSF 
faculty who are not directly involved in the study itself, but have expertise in use of 
spironolactone to treat elevated androgens in women, and expertise in liver disease 
respectively. This DSMB will review data relating to safety and efficacy, conduct and 
review interim analyses, and ensure the continued scientific validity and merit of the 
study. Further details regarding the timing and content of the interim reviews is included 
in the statistical section below.
15
STATISTICAL METHODS AND CONSIDERATIONS
Prior to the analysis of the final study data, a detailed Statistical Analysis Plan (SAP) will 
be written describing all analyses that will be performed. The SAP will contain any 
modifications to the analysis plan described below.
15.1
Data Sets Analyzed
All eligible patients will be included in the feasibility analyses because a key feature of 
this study is to determine the proportion of eligible patients that agree to participate. Only 
those randomized participants will be included in the analyses of secondary outcomes, 
though analyses will be conducted and presented separately as intention to treat, per 
protocol, and as treated, as the information gleaned from these different analytic 
approaches provides different, clinically relevant information.
All eligible patients who are randomized into the study and receive at least one dose of 
the study drug (the Safety Population) will be included in the safety analysis.
15.2
Demographic and Baseline Characteristics
The following demographic variables at screening will be summarized by treatment 
group: race, ethnicity, age, body mass index, HOMA-IR, lipid levels, androgen levels, 
PCOS diagnosis, and menopausal status.

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15.3
Analysis of Primary Endpoint
Exact 95% confidence intervals for rates of agreement to participate, meeting eligibility 
criteria, and completing follow-up will be calculated using the binomial distribution.
15.4
Analysis of Secondary Endpoints
Logistic regression will be used to assess the association of spironolactone with each 
dichotomous outcome, and ordinal logistic or linear regression used to evaluate the 
association of spironolactone with change in either ordered categories or continuous 
measures, where appropriate.
Safety and tolerability data will be summarized by treatment group.
Adverse event rates will be coded by body system and MedDRA classification term. 
Adverse events will be tabulated by treatment group and will include the number of 
patients for whom the event occurred, the rate of occurrence, and the severity and 
relationship to study drug.
15.5
Interim Analysis
Planned interim analyses will be conducted after 10 women (~33%) have completed 
three months of treatment. Interim results will provide useful preliminary safety and 
feasibility data.
15.6
Sample Size and Randomization
The sample size of 30 with 2:1 randomization was determined based on budget and 
feasibility within a 4-year grant timeline. The sample size is also reasonable for pilot trial, 
and will allow us to estimate effects and feasibility with reasonable precision. The 2:1 
randomization was selected to increase the chance that women would receive the 
treatment, and therefore potentially increase study enrollment, while still allowing the 
investigator to obtain the necessary training in conducting a placebo-controlled trial in 
preparation for a larger trial in the future, and allowing for preliminary endpoint 
assessments in the treatment as compared to placebo groups.
16
DATA COLLECTION, RETENTION AND MONITORING
16.1
Data Collection Instruments
The Investigator will prepare and maintain adequate and accurate source documents 
designed to record all observations and other pertinent data for each subject treated with 
the study drug.
Study personnel at each site will enter data from source documents corresponding to a 
subject’s visit into the protocol-specific electronic Case Report Form (eCRF) when the 
information corresponding to that visit is available. Subjects will not be identified by 
name in the study database or on any study documents to be collected by the Sponsor 
(or designee), but will be identified by a subject number and initials.
If a correction is required for an eCRF, the time and date stamps track the person 
entering or updating eCRF data and creates an electronic audit trail.
The Investigator is responsible for all information collected on subjects enrolled in this 
study. All data collected during the course of this study must be reviewed and verified for 
completeness and accuracy by the Investigator. A copy of the CRF will remain at the 
Investigator’s site at the completion of the study.

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16.2
Data Management Procedures
The data will be entered into a validated database. The Data Management group will be 
responsible for data processing, in accordance with procedural documentation.
Database lock will occur once quality assurance procedures have been completed.
All procedures for the handling and analysis of data will be conducted using good 
computing practices meeting FDA guidelines for the handling and analysis of data for 
clinical trials.
16.3
Data Quality Control and Reporting
After data have been entered into the study database, a system of computerized data 
validation checks will be implemented and applied to the database on a regular basis. 
Queries are entered, tracked, and resolved through the EDC system directly. The study 
database will be updated in accordance with the resolved queries.  All changes to the 
study database will be documented.
16.4
Archival of Data
The database is safeguarded against unauthorized access by established security 
procedures; appropriate backup copies of the database and related software files will be 
maintained.  Databases are backed up by the database administrator in conjunction with 
any updates or changes to the database.
At critical junctures of the protocol (e.g., production of interim reports and final reports), 
data for analysis is locked and cleaned per established procedures.
16.5
Availability and Retention of Investigational Records
The Investigator must make study data accessible to the monitor, other authorized 
representatives of the Sponsor (or designee), IRB/IEC, and Regulatory Agency (e.g., 
FDA) inspectors upon request. A file for each subject must be maintained that includes 
the signed Informed Consent, HIPAA Authorization and Assent Form and copies of all 
source documentation related to that subject. The Investigator must ensure the reliability 
and availability of source documents from which the information on the CRF was derived.
All study documents (patient files, signed informed consent forms, copies of CRFs, Study 
File Notebook, etc.) must be kept secured for a period of two years following marketing of 
the investigational product or for two years after centers have been notified that the IND 
has been discontinued. There may be other circumstances for which the Sponsor is 
required to maintain study records and, therefore, the Sponsor should be contacted prior 
to removing study records for any reason.
16.6
Monitoring
Monitoring visits will be conducted by representatives of the Sponsor according to the
U.S. CFR Title 21 Parts 50, 56, and 312 and ICH Guidelines for GCP (E6).
By signing 
this protocol, the Investigator grants permission to the Sponsor (or designee), and 
appropriate regulatory authorities to conduct on-site monitoring and/or auditing of all 
appropriate study documentation.
16.7
Subject Confidentiality
In order to maintain subject confidentiality, only a subject number and subject initials will 
identify all study subjects on CRFs and other documentation submitted to the Sponsor. 

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Additional subject confidentiality issues (if applicable) are covered in the Clinical Study 
Agreement.
17
ADMINISTRATIVE, ETHICAL, REGULATORY 
CONSIDERATIONS
The study will be conducted according to the Declaration of Helsinki, Protection of 
Human Volunteers (21 CFR 50), Institutional Review Boards (21 CFR 56), and 
Obligations of Clinical Investigators (21 CFR 312).
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other 
records will be identified by a coded number and initials only. All study records will be 
kept in a locked file cabinet and code sheets linking a patient’s name to a patient 
identification number will be stored separately in another locked file cabinet.  Clinical 
information will not be released without written permission of the subject, except as 
necessary for monitoring by the FDA. The Investigator must also comply with all 
applicable privacy regulations (e.g., Health Insurance Portability and Accountability Act of 
1996, EU Data Protection Directive 95/46/EC).
17.1
Protocol Amendments
Any amendment to the protocol will be written by the Sponsor. Protocol amendments 
cannot be implemented without prior written IRB/IEC approval except as necessary to 
eliminate immediate safety hazards to patients. A protocol amendment intended to 
eliminate an apparent immediate hazard to patients may be implemented immediately, 
provided the IRB is notified within five working days.
17.2
Institutional Review Boards and Independent Ethics Committees
The protocol and consent form will be reviewed and approved by the IRB/IEC of each 
participating center prior to study initiation. Serious adverse experiences regardless of 
causality will be reported to the IRB/IEC in accordance with the standard operating 
procedures and policies of the IRB/IEC, and the Investigator will keep the IRB/IEC 
informed as to the progress of the study. The Investigator will obtain assurance of 
IRB/IEC compliance with regulations.
Any documents that the IRB/IEC may need to fulfill its responsibilities (such as protocol, 
protocol amendments, Investigator’s Brochure, consent forms, information concerning 
patient recruitment, payment or compensation procedures, or other pertinent information) 
will be submitted to the IRB/IEC. The IRB/IECs written unconditional approval of the 
study protocol and the informed consent form will be in the possession of the Investigator 
before the study is initiated. The IRB/IECs unconditional approval statement will be 
transmitted by the Investigator to the Sponsor or designee prior to the shipment of study 
supplies to the site. This approval must refer to the study by exact protocol title and 
number and should identify the documents reviewed and the date of review.
Protocol and/or informed consent modifications or changes may not be initiated without 
prior written IRB/IEC approval except when necessary to eliminate immediate hazards to 
the patients or when the change(s) involves only logistical or administrative aspects of 
the study. Such modifications will be submitted to the IRB/IEC and written verification 
that the modification was submitted and subsequently approved should be obtained.
The IRB/IEC must be informed of revisions to other documents originally submitted for 
review; serious and/or unexpected adverse experiences occurring during the study in 
accordance with the standard operating procedures and policies of the IRB; new 

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information that may affect adversely the safety of the patients of the conduct of the 
study; an annual update and/or request for re-approval; and when the study has been 
completed.
17.3
Informed Consent Form
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH 
GCP, US Code of Federal Regulations for Protection of Human Subjects (21 CFR 
50.25[a,b], CFR 50.27, and CFR Part 56, Subpart A), the Health Insurance Portability 
and Accountability Act (HIPAA, if applicable), and local regulations.
The Investigator will prepare the informed consent form, assent and HIPAA authorization 
and provide the documents to the Sponsor or designee for approval prior to submission 
to the IRB/IEC. The consent form generated by the Investigator must be acceptable to 
the Sponsor and be approved by the IRB/IEC. The written consent document will 
embody the elements of informed consent as described in the International Conference 
on Harmonisation and will also comply with local regulations. The Investigator will send 
an IRB/IEC-approved copy of the Informed Consent Form to the Sponsor (or designee) 
for the study file.
A properly executed, written, informed consent will be obtained from each subject prior to 
entering the subject into the trial. Information should be given in both oral and written 
form and subjects must be given ample opportunity to inquire about details of the study. If 
appropriate and required by the local IRB/IEC, assent from the subject will also be 
obtained. If a subject is unable to sign the informed consent form (ICF) and the HIPAA 
authorization, a legal representative may sign for the subject.  A copy of the signed 
consent form (and assent) will be given to the subject and the original will be maintained 
with the subject’s records.
17.4
Publications
The preparation and submittal for publication of manuscripts containing the study results 
shall be in accordance with a process determined by mutual written agreement among  
the study Sponsor and participating institutions. The publication or presentation of any 
study results shall comply with all applicable privacy laws, including, but not limited to, the 
Health Insurance Portability and Accountability Act of 1996.
17.5
Investigator Responsibilities
By signing the Agreement of Investigator form, the Investigator agrees to:
1.
Conduct the study in accordance with the protocol and only make changes after 
notifying the Sponsor (or designee), except when to protect the safety, rights or 
welfare of subjects.
2.
Personally conduct or supervise the study (or investigation).
3.
Ensure that the requirements relating to obtaining informed consent and IRB review 
and approval meet federal guidelines, as stated in § 21 CFR, parts 50 and 56.
4.
Report to the Sponsor or designee any AEs that occur in the course of the study, in 
accordance with §21 CFR 312.64.
5.
Ensure that all associates, colleagues and employees assisting in the conduct of the 
study are informed about their obligations in meeting the above commitments.
6.
Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to 
make those records available for inspection with the Sponsor (or designee).

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7.
Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be 
responsible for initial and continuing review and approval of the clinical study.
8.
Promptly report to the IRB and the Sponsor (or designee) all changes in the research 
activity and all unanticipated problems involving risks to subjects or others (to include 
amendments and IND safety reports).
9.
Seek IRB approval before any changes are made in the research study, except when 
necessary to eliminate hazards to the patients/subjects.
10. Comply with all other requirements regarding the obligations of clinical investigators 
and all other pertinent requirements listed in § 21 CFR part 312.

APPENDIX 1. Schedule of Events
Screening
Baseline/ 
Day 1
Month 1
Month 3
Month 6
End of Study/ 
Treatment
Month 9*
Month 12*
End of Study/ 
Treatment
Follow-Up within 3 
months of 
EOS/EOT
Visit 1
Visit 2
Visit 3
Visit 4
Visit 5
Visit 6
Visit 7
Obtain informed consent
X
Review your medical history
X
Review medications you are taking
X
X
X
X
X
X
X
X
Perform complete physical exam
X
Perform physical exam depending 
on your symptoms
X
X
X
X
X
Measure your vital signs (blood 
pressure, heart rate)
X
X
X
X
X
X
Measure your weight and height
Xa
X
X
X
X
X
Measure your waist circumference
X
X
X
Review your general health 
(side effects and illnesses)
X
X
X
X
X
X
X
Draw samples of your blood for 
to check your general health, 
liver health, cholesterol, 
glucose, and insulin levels
X
X
X
X
X
X
Draw samples of your blood to 
check your sex hormone levels
X
X
X
If you are a female who can 
have children, collect a sample 
of your urine for pregnancy 
testing
Xb
X
X
X
X
X
Collect a small liver sample (biopsy)
Xc
X*
X*
Scan your liver: MRI-PDFF and MRE
X
X
X
Give you study drug and 
instructions on how to take it
X
X
X
X
X
X
Review the amount of study drug 
you have taken and collect unused 
t d d
X
X
X
X
X
a Height only measured at Screening
  b In addition, home pregnancy tests are to be performed by women who can have children every 4 weeks from Day 1 until End of Study/Treatment 
 c Performed prior to screening as part of your standard of care for NASH
  *Optional visits/procedures
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