Clinical trial NCT03364335
Conditions: Obesity
Sponsor: NuSirt Biopharma
Start date: 2018-01-08; Completion date: 2018-08-17
Enrollment: 267 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a 24-week study to evaluate the effects of two fixed-dose combinations of leucine and sildenafil or two fixed-dose combinations of leucine, sildenafil and metformin compared to placebo. The primary objective of this study is to evaluate the percentage change in body weight in subjects from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24).

Detailed description:
This is a randomized, 24-week, placebo-controlled, double-blind study to evaluate the effects of two fixed-dose combinations of leucine and sildenafil or two fixed-dose combinations of leucine, sildenafil and metformin compared to placebo. Subjects meeting all the inclusion criteria and no exclusion criteria will be randomized to one of five study arms.

The primary objective of this study is to evaluate the percentage change in body weight in subjects from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) receiving two fixed-dose combinations of leucine and sildenafil or two fixed-dose combinations of leucine, sildenafil and metformin compared to placebo. Secondary objectives will also assess changes in absolute body weight, percentage of patients with ≥5% body weight loss, change in waist circumference, change in HbA1c, change in fasting glucose, change in blood lipids such as cholesterol, LDL, HDL, triglycerides, change in blood pressure and changes in in C-reactive protein. In addition this study will evaluate the safety and tolerability of NS-0200 and NS-0300.

Patients will have one screening visits which will determine their eligibility based on lab tests. Once qualified, patients will be randomly assigned to either one of the treatment groups or the placebo control group and monitored for a total of 24 weeks. Patients will return to the clinic each month for lab tests, and routine examinations.

Arms: arm 1: Each dose of placebo will consist of three tablets, identical in appearance to those used in the two active treatment arms, containing 93.5% Microcrystalline cellulose PH 102, 5.0% Crospovidone XL 10, 1.0% Silica gel (Syloid 244), 0.5% Magnesium stearate I MF3V for the leucine matched placebo and 99.5% Avicel PH200 , 0.5% magnesium stearate (w/w) and Opadry II White coating 3% weight gain for the sildenafil matched placebo. arm 2: 3 capsules BID consisting of 2 capsules containing 550 mg L-leucine each and 1 capsule containing 1.0 mg of sildenafil arm 3: 3 capsules BID consisting of 2 capsules containing 550 mg L-leucine each and 1 capsule containing 4.0 mg of sildenafil arm 4: 3 capsules BID consisting of 2 capsules containing 550 mg L-leucine and 250 mg metformin and 1 capsule containing 1.0 mg of sildenafil arm 5: 3 capsules BID consisting of 2 capsules containing 550 mg L-leucine and 250 mg metformin and 1 capsule containing 4.0 mg of sildenafil
Interventions: intervention 1: Leu Sil 1.0mg intervention 2: Leu Sil 4.0mg intervention 3: Leu Met Sil 1.0mg intervention 4: Leu Met Sil 4.0mg intervention 5: Placebo
Intervention descriptions: intervention 1: Leu 1100 mg + 1mg Sil BID intervention 2: Leu 1100 mg + 4mg Sil BID intervention 3: Leu 1100 mg + Met 500mg + 1mg Sil BID intervention 4: Leu 1100 mg + Met 500mg + 4mg Sil BID intervention 5: Placebo

Protocol documents:
Prot_000.pdf: 
 
Clinical Study Protocol NS-WM-01 
 
A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY TO 
EVALUATE THE EFFECT OF TWO FIXED-DOSE LEUCINE AND 
SILDENAFIL COMBINATIONS (NS-0300) OR TWO FIXED-DOSE LEUCINE, 
SILDENAFIL AND METFORMIN COMBINATIONS (NS-0200) VERSUS 
PLACEBO ON BODY WEIGHT IN OBESITY 
 
Phase 2 
 
Original Protocol: August 29, 2017 
Amendment #1: 
October 26, 2017 
Amendment #2:        November 16, 2017 
Amendment #3: 
December 4, 2017 
 
 
IND Number: 
135732 
 
 
 
 
Sponsor: 
NuSirt Sciences, Inc. 
D.B.A NuSirt Biopharma 
3835 Cleghorn Avenue, Suite 200 
Nashville, TN 37215 
 
Medical Monitor-: 
Orville G. Kolterman, MD 
Chief Medical Officer 
Cell Phone: (858) 342-7057 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Confidentiality Statement 
 
This protocol is a confidential communication and is the property of NuSirt Biopharma. Acceptance implies an 
agreement not to disclose the information contained herein that is not otherwise publicly available, except for 
use by an institutional review board (IRB) for the purpose of obtaining approval to conduct the study. The IRB 
is requested and expected to maintain confidentiality. This document may not be used or published in whole or 
in part without the consent of NuSirt Biopharma. 
 
 




NuSirt Biopharma 
Clinical Study Protocol NS-WM-01 
Page 3 
 
 
 
PROTOCOL SUMMARY 
 
SPONSOR: NuSirt Sciences, Inc. (D.B.A NuSirt Biopharma) 
STUDY TITLE: A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED STUDY TO 
EVALUATE THE EFFECT OF TWO FIXED-DOSE LEUCINE AND SILDENAFIL COMBINATIONS 
(NS-0300) OR TWO FIXED-DOSE LEUCINE, SILDENAFIL AND METFORMIN COMBINATIONS 
(NS-0200) VERSUS PLACEBO ON BODY WEIGHT IN OBESITY 
STUDY PHASE: 2 
PRIMARY OBJECTIVE 
• To evaluate the percentage body weight change in subjects from Baseline/Visit 2 (Day1) to Study 
Termination/Visit 8 (Day 168/Week 24) receiving two fixed-dose combinations of leucine and sildenafil 
or two fixed-dose combinations of leucine, sildenafil and metformin compared to placebo 
SECONDARY OBJECTIVES 
To evaluate the effects of receiving two fixed-dose combinations of leucine and sildenafil or two fixed-dose 
combinations of leucine, sildenafil and metformin compared to placebo in obese patients on the following 
parameters: 
• Change in absolute body weight from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 
168/Week 24) 
• Change in percentage of patients with ≥5% body weight loss from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in waist circumference from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 
24) 
• Change in blood lipids (total, HDL- and LDL-cholesterol; triglycerides) from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
• Change in glycemic control (fasting glucose and HbA1c) from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in blood pressure (systolic and diastolic) from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
• Change in inflammatory markers (hsCRP)from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 
168/Week 24) 
• Exploratory endpoint of percentage of patients with ≥10% body weight loss from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
STUDY DESIGN 
This is a 24-week, randomized placebo-controlled, double-blind study designed to evaluate the effect of two 
fixed-dose combinations (FDC) of leucine and sildenafil or two FDC of leucine, sildenafil and metformin 
compared to placebo on body weight.   
Subjects meeting all inclusion criteria with no exclusion criteria will be randomized to one of five treatment 
arms: 
Treatment Arms (Protocol NS-WM-01) 
Treatment 
Arm 
Subjects (N) 
Randomized 
Dosing 
Regimen 
Study Medication BID* 
 Leucine 
 Sildenafil 
Metformin 
A 
B 
C  
D 
E 
50 
50 
50 
50 
50 
Placebo 
FDC 
FDC 
FDC 
FDC 
 N/A 
  1100 mg 
  1100 mg 
  1100 mg 
  1100 mg 
 N/A 
 1.0mg 
 4.0 mg 
 1.0 mg 
 4.0 mg 
 N/A 
  N/A 
  N/A 
  500 mg 
  500 mg 
FDC = Fixed Dose Combination 
*Note: Total daily dose is twice the doses listed in table 


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Clinical Study Protocol NS-WM-01 
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The study will include 2 periods: Screening (to determine if inclusion/exclusion criteria are met), and a 24-
week, randomized treatment period (Visit 2 to Visit 8) with Visit 2 as the first dose of study medication.  
Study Design (Protocol NS-WM-01) 
 
 
s 
 
Screening 
Period
24 Week Treatment Period
Visit 1
(Day -7)
(Week -1)
Visit 2
(Day 1)
Phone 
Contact
(Day 14)
(Week 2)
Visit 3
(Day 28)
(Week 4)
Visit 4
(Day 56)
(Week 8)
Visit 5
(Day 84)
(Week 12)
Phone 
Contact
(Day 70)
(Week 10)
Visit 6
(Day 112)
(Week 16)
First Dose of 
Study Medication
Study
Termination
Phone 
Contact
(Day 98)
(Week 14)
Phone 
Contact
(Day 42)
(Week 6)
Phone 
Contact
(Day 126)
(Week 18)
Visit 7
(Day 140)
(Week 20)
Phone 
Contact
(Day 154)
(Week 22)
Visit 8
(Day 168)
(Week 24)


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Clinical Study Protocol NS-WM-01 
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VISIT STRUCTURE AND STUDY DURATION 
This study will include a total of 8 visits to the clinical study site: Screening /Visit 1 (Day-7/Week-1), 
Baseline/Visit 2 (Day 1), Visit 3 (Day 28/Week 4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), 
Visit 6 (Day 112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 (Day 168/Week 24). 
In addition, subjects will be contacted on Day 14/Week 2, Day 42/Week 6, Day 70/Week 10, Day 98/Week 
14, Day 126/Week 18 and Day 154/Week 22 to maintain engagement and enhance subject retention. 
All study visits should occur within a visit window of ±3 days. Total study duration should not exceed a 
maximum of 28 weeks. 
STUDY POPULATION 
A sufficient number of subjects will be exposed to study screening procedures to enroll approximately 250 
subjects, a maximum of 70% female, with a goal of having 200 subjects completing all assessments over 24 
weeks.  
STUDY MEDICATIONS 
Subjects will be assigned to one of five treatment groups based on a prospectively defined randomization 
schedule (see Study Design). Each treatment group will be asked to ingest 3 tablets of blinded study 
medication 30 minutes before the morning and evening meals. 
• Fixed Dose Combination Arms: Each dose of study medication will consist of three tablets, two tablets 
containing either 550mg L-leucine or 550 mg L-leucine/250 mg metformin  and one tablet containing either 
1.0 mg or 4.0 mg of sildenafil. 
• Placebo Arm:  Each dose of placebo will consist of three tablets, identical in appearance to those used in the 
two active treatment arms, containing 93.5% Microcrystalline cellulose PH 102, 5.0% Crospovidone XL 10, 
1.0% Silica gel (Syloid 244), 0.5% Magnesium stearate I MF3V for the leucine matched placebo and 99.5% 
Avicel PH200 , 0.5% magnesium stearate (w/w) and Opadry II White coating 3% weight gain for the sildenafil 
matched placebo. 
Study medication will be self-administered by the patient orally 30 minutes before the morning, and 
evening meals. 
EFFICACY ASSESSMENTS 
• Percentage change in body weight 
• Change in absolute body weight 
• Percentage of patients with ≥5% body weight loss 
• Change in waist circumference 
• Change in lipids: 
• Cholesterol 
• LDL 
• HDL 
• Triglycerides 
• Change in glycemic control (fasting glucose and HbA1c) 
• Change in blood pressure (systolic and diastolic) 
• Change in inflammatory markers (hsCRP) 
• Exploratory endpoint of percentage of patients with ≥10% body weight loss  
SAFETY ASSESSMENTS 
• Adverse events (AE), treatment emergent AEs (TEAEs), and Serious  Adverse Event (SAEs) reports 
• Vital signs 
• Physical examination 
• 12-lead ECGs at Screening and Study Termination of study participation 
• Clinical chemistry panel, hematology panel, and urinalysis 
• Screening β-hCG for females to insure that the subject is not pregnant 
• Change in concomitant medications 


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Clinical Study Protocol NS-WM-01 
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STATISTICAL CONSIDERATIONS 
Analysis Populations: 
Enrolled: The Enrolled Population will consist of all subjects who signed the informed consent to 
participate in the study, and had Screening visit electronic case report forms (eCRFs) collected. 
Randomized: The Randomized Population will consist of all Enrolled subjects who were randomized at 
Baseline/Visit 2 (Day 1) 
Intent-To-Treat (ITT): The ITT Population will include all randomized subjects who receive at least one 
dose of study medication. 
Per-Protocol (PP): The PP Population will include all ITT subjects who had adequate exposure to the 
randomized study medication (>85%) during the 24-week period as defined in the final statistical analysis 
plan, have completed all study visits and have adequately complied with the protocol as assessed by the 
investigator and sponsor prior to database lock.  The final definition of the PP Population will be 
documented in an addendum to the prospectively developed statistical analysis plan (SAP) prior to 
unblinding any data. 
 
Analysis Endpoints: 
Primary Efficacy Endpoint:  The primary efficacy endpoint is percentage change from Baseline/Visit 2 
(Day1) to Study Termination/Visit 8 (Day 168/Week 24) in body weight 
Primary Safety and Tolerability Endpoint:  The primary safety and tolerability endpoint is the incidence 
of all treatment-emergent adverse events. 
 
Statistical Analyses: 
Descriptive statistics (n, mean, SD, SE, median, min, and max) will be used to summarize values of the 
primary efficacy endpoint, change in percent body weight from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) and intermediate study visits as applicable by treatment group.  
These summaries will be presented for the PP Population with observed data and the ITT Population with 
Last Observation Carried Forward (LOCF) approach.  A mixed-model of analysis of variance (ANCOVA) 
will be used to analyze change in body weight over the study duration in PP and ITT Populations.  The 
ANCOVA will include treatment group and time as the main effects with baseline weight as the covariate.  
The LS means, SEs, and the corresponding 95% confidence intervals (CIs) for the changes from baseline to 
Week 24 will be derived from the model for each treatment.  Each of the fixed-dose combinations of 
leucine, sildenafil, and/or metformin treatment groups (Treatment B, C, D and E) will be compared to 
placebo group (Treatment A), and the LS means for the treatment difference (Treatment B, C D or E minus 
Treatment A), the SEs, the associated 95% CIs, and the p-values will be computed. 
 
SAMPLE SIZE 
 
A sufficient number of individuals will be screened to enroll at least 250 subjects (~50/treatment arm).  
With an assumption of 20% dropout rate, approximately 200 subjects (~40/treatment arm) are expected to 
complete treatment through Study Termination/Visit 8 (Day 168/Week 24).   
Using a two-sided, two-sample comparison (t-test) of means at the alpha=0.05 level of significance, sample 
sizes of 23 subjects per treatment will provide approximately 90% power to detect a mean treatment 
difference of 3 kg body weight (~3.0-3.3% weight loss over 24 weeks), assuming a common standard 
deviation of 25%.  However, detection of significant changes in obesity co-morbidities require a higher 
sample size; detection of a 6 mm Hg change in blood pressure will require a sample size of 40 subjects per 
treatment group at 80% power; accordingly, the sample size has been increased to 40/treatment arm to 
enable assessment of this secondary endpoint. These assumptions of effect size and standard deviation are 
based on data derived from the previous clinical trial of NS-0200 (NS-0200-01) and represent the 
anticipated treatment effect of effective fixed-dose combinations of leucine and sildenafil, with or without 
metformin, compared to placebo. 


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Clinical Study Protocol NS-WM-01 
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TABLE OF CONTENTS 
LIST OF ABBREVIATIONS 
10 
1 INTRODUCTION .................................................................................................................... 11 
2 STUDY OBJECTIVES ............................................................................................................... 11 
2.1 
Primary Objective .................................................................................................................. 11 
2.2 
Secondary Objectives ............................................................................................................ 12 
3 STUDY DESIGN ..................................................................................................................... 12 
3.1 
Design Description ................................................................................................................ 12 
3.2 
Visit Structure and Study Duration ........................................................................................ 13 
4 STUDY POPULATION ............................................................................................................. 14 
4.1 
Population to Be Studied ....................................................................................................... 14 
4.2 
Inclusion Criteria.................................................................................................................... 14 
4.3 
Exclusion Criteria ................................................................................................................... 15 
4.4 
Removal of Subjects from Study Participation ...................................................................... 16 
4.5 
Subject Restrictions ............................................................................................................... 17 
4.6 
Concomitant Medications ..................................................................................................... 18 
5 STUDY MEDICATIONS ........................................................................................................... 18 
5.1 
Formulation, Packaging, and Storage .................................................................................... 18 
5.2 
Dispensing of Study Medication ............................................................................................ 18 
5.3 
Dose Administration, Route and Schedule ............................................................................ 19 
5.4 
Randomization Schedule and Blinding Procedures ............................................................... 19 
5.5 
Drug Accountability ............................................................................................................... 19 
6 STUDY METHODS ................................................................................................................. 20 
6.1 
Screening (Visit 1/ Day-7/ Week-1) ...................................................................................... 20 
6.2 
Baseline/Visit2 (Day 1) .......................................................................................................... 21 
6.3 
Telephone Contact (Day14/Week 2) ..................................................................................... 22 
6.4 
Visit 3 (Day28/ Week 4) ........................................................................................................ 22 
6.5 
Telephone Contact (Day42/Week 6) ..................................................................................... 22 
6.6 
Visit 4 (Day 56/Week 8) ........................................................................................................ 22 
6.7 
Telephone Contact (Day 70/Week 10) .................................................................................. 23 
6.8 
Visit 5 (Day 84/Week 12) ...................................................................................................... 23 
6.9 
Telephone Contact (Day 98/Week 14) .................................................................................. 23 
6.10 Visit 6 (Day 112/Week 16) .................................................................................................... 24 
6.11 Telephone Contact (Day 126/Week 18) ................................................................................ 24 
6.12 Visit 7 (Day 140/Week 20) .................................................................................................... 24 
6.13 Telephone Contact (Day 154/Week 22) ............................................................................... 24 
6.14 Study Termination (Visit 8/Day 168/Week 24) or Early Termination .................................... 25 
6.15 Ethical Safety Considerations ................................................................................................ 25 
7 EFFICACY ASSESSMENTS ....................................................................................................... 26 
7.1 
Percentage Change in Body Weight ...................................................................................... 26 
7.2 
Absolute Body Weight ........................................................................................................... 26 
7.3 
Percentage of patients with ≥5% and ≥10% body weight loss .............................................. 26 
7.4 
Waist Circumference ............................................................................................................. 27 
7.5 
Fasting Lipids ......................................................................................................................... 27 
7.6 
Fasting Glucose...................................................................................................................... 27 
7.7 
HbA1c .................................................................................................................................... 27 
7.8 
Blood Pressure ...................................................................................................................... 27 
7.9 
hsC-Reactive Protein ............................................................................................................. 28 


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Clinical Study Protocol NS-WM-01 
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7.10 Biomarkers ............................................................................................................................ 28 
8 SAFETY ASSESSMENTS .......................................................................................................... 28 
8.1 
Targeted Safety Surveillance Items ....................................................................................... 28 
8.1.1 
Metformin Associated Lactate Acidosis ................................................................................. 28 
8.1.2 
Nephrotoxic Effects of Radiocontrast Agents in Patients Taking Metformin ......................... 29 
8.1.3 
Risk of Hypoglycemia in Patients Taking Metformin ............................................................. 29 
8.1.4 
Hypotension ........................................................................................................................... 29 
8.1.5 
Changes in Vision .................................................................................................................. 29 
8.1.6 
Leucine Toxicity ...................................................................................................................... 30 
8.2 
Reporting of Adverse Events ................................................................................................. 30 
8.3 
Adverse Events and Subject Withdrawals ............................................................................. 30 
8.4 
Clinical Laboratory Evaluations.............................................................................................. 33 
8.4.1 
Chemistry ............................................................................................................................... 33 
8.4.2 
Hematology ............................................................................................................................ 33 
8.4.3 
Lipids ...................................................................................................................................... 34 
8.4.4 
Urinalysis ................................................................................................................................ 34 
8.5 
Other Clinical Laboratory Evaluations ................................................................................... 34 
8.5.1 
Pregnancy Testing .................................................................................................................. 34 
8.5.2 
Urine Drug Screen .................................................................................................................. 34 
8.6 
Vital Signs, Physical Examination Findings, and ECGs ............................................................ 34 
8.6.1 
Vital Signs ............................................................................................................................... 35 
8.6.2 
Physical Examinations ............................................................................................................ 35 
8.6.3 
Electrocardiograms ................................................................................................................ 35 
9 BLOOD VOLUME ................................................................................................................... 35 
10 DISCONTINUING AND UNBLINDING OF SUBJECTS ............................................................... 35 
10.1 Unblinding of Subjects .......................................................................................................... 36 
11 DATA MANAGEMENT ........................................................................................................ 37 
12 STATISTICAL CONSIDERATIONS .......................................................................................... 37 
12.1 Analysis Populations ........................................................................................................... 37 
12.2 Study Subjects .................................................................................................................... 38 
12.3 Primary Endpoint Analysis .................................................................................................. 38 
12.4 Secondary Endpoint Analysis .............................................................................................. 38 
12.5 Safety Analysis .................................................................................................................... 39 
13 SAMPLE SIZE AND POWER CALCULATION ........................................................................... 39 
14 INVESTIGATOR AND SPONSOR OBLIGATIONS ..................................................................... 40 
14.1 Medical Supervision ........................................................................................................... 40 
14.2 Study Initiation and Discontinuation .................................................................................. 40 
14.3 Laboratory Accreditation .................................................................................................... 41 
14.4 Data Reporting .................................................................................................................... 41 
14.5 Study Monitoring ................................................................................................................ 41 
14.6 Record Retention ................................................................................................................ 42 
14.7 Deviation from the Protocol ............................................................................................... 42 
14.8 Quality Assurance ............................................................................................................... 43 
15 DISCLOSURE OF DATA AND PUBLICATIONS ......................................................................... 43 
16 ETHICAL CONSIDERATIONS ................................................................................................. 44 
17 INFORMED CONSENT ......................................................................................................... 44 
18 FINAL REPORT .................................................................................................................... 44 
19 REFERENCE LIST ................................................................................................................. 45 
20 INVESTIGATOR’S ACCEPTANCE ........................................................................................... 47 
 


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Clinical Study Protocol NS-WM-01 
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LIST OF IN-TEXT TABLES 
Table 1: Treatment Arms (Protocol NS-WM-01) ................................................................................................. 13 
 
LIST OF IN-TEXT FIGURES 
Figure 1: Study Design (Protocol NS-WM-01) .................................................................................................... 13 
 
LIST OF APPENDICES 
Appendix 1: Study Plan (Protocol NS-WM-01)  ...................................................................................................... 48 


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Clinical Study Protocol NS-WM-01 
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LIST OF ABBREVIATIONS 
AE 
adverse event 
ALT 
alanine transaminase 
AMPK 
5’ adenosine monophosphate-activated protein kinase 
AST 
aspartate transaminase 
β−hCG 
beta subunit of human chorionic gonadotropin 
BID 
twice daily 
BMI 
body mass index 
CI 
confidence interval 
CFR 
Code of Federal Regulations 
CPK 
creatine phosphokinase 
CK-MB 
creatine kinase-MB 
CRF 
case report form 
CRO 
contract research organization 
ECG 
electrocardiogram 
eCRF 
electronic case report form 
EDC 
electronic data capture 
FDA 
Food and Drug Administration 
FDC 
fixed dose combination 
mg 
milligram 
GCP 
Good Clinical Practices 
hr 
hour(s) 
HbA1c 
glycosylated hemoglobin 
HDL-C 
high density lipoprotein-cholesterol 
HIPAA 
Health Insurance Portability and Accountability Act 
hs-CRP 
High sensitivty C-reactive protein 
ICF 
Informed Consent Form 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
LDL-C 
low density lipoprotein-cholesterol 
LOCF 
last observation carried forward 
MedDRA 
Medical Dictionary for Regulatory Activities 
min 
minute(s) 
NDA 
New Drug Application 
SAE 
serious adverse event 
SD 
standard deviation 
Sirt 1 
NAD-dependent deacetylase sirtuin 1 
TEAE 
treatment-emergent adverse event 


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Clinical Study Protocol NS-WM-01 
Page 11 
 
 
1 
INTRODUCTION AND BACKGROUND 
 
Global obesity prevalence has doubled over the past three decades, and the obesity epidemic 
now represents a significant public health risk in the US and other developed countries as well 
as in the developing world.  In the US, >35% of the adult population and 17% of the pediatric 
and adolescent populations are obese, and ~70% of the population is overweight or obese [1,2]. 
Obesity is associated with significant mortality risk, as shown by a 9-13 year reduction in 
lifespan and >300,000 obesity-related deaths annually in the US [3–7].  Further, obesity 
increases the risk of co-morbidities, including cardiovascular disease, type 2 diabetes, 
hypertension, dyslipidemia, obstructive sleep apnea, and some cancers [4–8].  The risk of 
obesity-related co-morbidities increases with increasing body mass index (BMI); conversely, 
even modest reduction in BMI resulting from a 5-10% weight loss reduces the risk and severity 
of these co-morbidities [9,10]. 
 
Lifestyle intervention focused on reducing caloric intake and increasing caloric expenditure 
are the first-line approach to obesity management; however, patient adherence is generally 
poor and obesity control through lifestyle intervention alone rarely exhibits sustained success.  
Accordingly, pharmacotherapy is an important adjunct to diet and exercise to achieve 
sustained weight reduction.  The proposed fixed dose combination of the amino acid leucine 
and two well characterized agents with established good safety profiles is expected to 
modulate energy metabolism and facilitate sustained weight loss.  
 
AMPK and Sirt1 are well known regulators of lipid and energy metabolism. Activation of 
Sirt1 and AMPK stimulates mitochondrial biogenesis and fat oxidation with concomitant 
suppression of lipid synthesis and storage [11–16]. NuSirt Biopharma has demonstrated that 
L-leucine has a unique role as an activator of the Sirt1/AMPK pathway, serving as a partial 
mimetic of caloric restriction, and thereby modulates Sirt1-dependent lipid and energy 
metabolism [17–19]. NuSirt has shown that addition of L-leucine to metformin (fixed dose 
combination, NS-0100) results in a novel synergistic interaction that enables significant dose 
reduction of metformin with no loss of anti-diabetic efficacy (IND# 121112) and that a triple 
combination of L-leucine with low-dose metformin and sildenafil (NS-0200) regresses 
steatohepatitis in preclinical studies [19,20] and reduces hepatic fat in non-alcoholic fatty liver 
disease patients with elevated liver enzymes (IND #126214). Further, data from the Phase 2A 
trial conducted under this IND also provided a robust weight loss signal accompanied by 
decreases in obesity associated co-morbidities.  This finding is supported by both mechanism 
of action and preclinical studies demonstrating reductions in adiposity in a rodent obesity 
model. 
 
NuSirt also has preclinical data which suggests that metformin may not be a necessary 
component to achieve weight loss; we therefore will evaluate a related fixed dose combination 
of leucine and sildenafil in the absence of metformin (NS-0300) in this study.  The superior 
combination will then be utilized for further development.   
 
2 
STUDY OBJECTIVES 
 
2.1 
Primary Objective 
The primary objective of this study is: 
• To evaluate the percentage change in weight in subjects from : Baseline/Visit 2 
(Day 1) to Study Termination/Visit 8 (Day 168/Week 24) receiving two fixed-dose 


NuSirt Biopharma 
Clinical Study Protocol NS-WM-01 
Page 12 
 
combinations of leucine and sildenafil or two fixed-dose combinations of leucine, 
sildenafil and metformin compared to placebo 
2.2 
Secondary Objectives 
The secondary objectives of this study are to evaluate the effects of two fixed-dose 
combinations of leucine and sildenafil or two fixed-dose combinations of leucine, 
sildenafil and metformin compared to placebo in obese on the following parameters: 
• Absolute body weight from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
• Percentage of patients with ≥5% body weight loss from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
• Change in waist circumference from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in blood lipids (cholesterol, LDL, HDL, triglycerides) from Baseline/Visit 
2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
• Change in fasting glucose from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in HbA1cfrom Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 
168/Week 24) 
• Change in Blood pressure (diastolic and systolic) from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
• Change in hsCRP from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 
168/Week 24) 
• Safety and tolerability 
• Exploratory endpoint of percentage of patients with ≥10% body weight loss from 
Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
 
3 
STUDY DESIGN 
 
3.1 
Design Description 
This is a randomized, 24-week, placebo-controlled, double-blind study to evaluate the effects 
of two fixed-dose combinations of leucine and sildenafil or two fixed-dose combinations of 
leucine, sildenafil and metformin compared to placebo. 
Subjects meeting all inclusion criteria with no exclusion criteria will be randomized to one of 
five treatment arms (Table 1). 
 
 


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Table 1: Treatment Arms (Protocol NS-WM-01) 
Treatment 
Arm 
Subjects (N) 
Randomized 
Dosing 
Regimen 
Study Medication Dosed Twice/Day1 
Leucine 
Sildenafil 
Metformin 
A 
50 
Placebo 
N/A 
N/A 
N/A 
B 
50 
FDC 
1100 mg 
1 mg 
N/A 
C 
50 
FDC 
1100 mg 
4 mg 
N/A 
D 
50 
FDC 
1100 mg 
1 mg 
500mg 
E 
50 
FDC 
1100 mg 
4 mg 
500mg 
 
FDC = Fixed Dose Combination 
*Note: The total daily dose is twice that listed for each compound 
 
 
The study design is depicted in Figure 1. The study will include a total of 2 periods: 
Screening (including a 7 day period for lab results) and a 24-week treatment period (Day 1 
to Day 168) with the first dose of study medication being administered on Day 1.  
 
Figure 1: Study Design (Protocol NS-WM-01) 
 
 
 
 
 
 
3.2 
Visit Structure and Study Duration 
This study will include a total of 8 visits to the clinical study site (Figure 1) as follows: 
Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 (Day 1), Visit 3 (Day 28/ Week 4), Visit 
4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 112/Week 16), Visit 7 (Day 
140/Week 20) and Study Termination/Visit 8 (Day 168/Week 24). In addition, subjects will 
receive six telephone contacts on Day 14/Week 2, Day 42/Week 6, Day 70/Week 10, 
Day98/Week 14, Day126/ Week 18 and Day 154/Week 22. 
For all subjects, Visit 1 should be Day -7 ± 3 days relative to Baseline/Visit 2 (Day 1). 
During the 24-week treatment period, the visit window will not exceed ± 3 days for Visit 3 
(Day 28/ Week 4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 
112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 (Day 168/Week 
24). Total study duration should not exceed a maximum of 28 weeks. 
 
 
Screening 
Period
24 Week Treatment Period
Visit 1
(Day -7)
(Week -1)
Visit 2
(Day 1)
Phone 
Contact
(Day 14)
(Week 2)
Visit 3
(Day 28)
(Week 4)
Visit 4
(Day 56)
(Week 8)
Visit 5
(Day 84)
(Week 12)
Phone 
Contact
(Day 70)
(Week 10)
Visit 6
(Day 112)
(Week 16)
First Dose of 
Study Medication
Study
Termination
Phone 
Contact
(Day 98)
(Week 14)
Phone 
Contact
(Day 42)
(Week 6)
Phone 
Contact
(Day 126)
(Week 18)
Visit 7
(Day 140)
(Week 20)
Phone 
Contact
(Day 154)
(Week 22)
Visit 8
(Day 168)
(Week 24)


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4 
STUDY POPULATION 
 
4.1 
Population to Be Studied 
A sufficient number of obese subjects will be exposed to study screening procedures to 
enroll approximately 250 subjects, with a maximum of 70% female, with a goal of having 
at least 200 subjects completing all assessments over the 24-week assessment period. The 
definition of obesity is a BMI ≥30 kg/m2.  For purposes of this study we will not include 
individuals with a BMI >45 kg/m2.   
 
4.2 
Inclusion Criteria 
Individuals meeting all of the following criteria will be considered for admission to the 
study: 
1. Age ≥18 and ≤65 at study entry. 
2. Is male, or female and, if female, meets all of the following criteria: 
a. Not breastfeeding 
b. Post-menopausal or negative serum pregnancy test result (human chorionic 
gonadotropin, beta subunit [β-hCG]) at Screening/Visit 1 (Day-7/Week-1) (not 
required for hysterectomized females) 
c. If of childbearing potential (including peri-menopausal women who have had a 
menstrual period within one year) must practice and be willing to continue to practice 
appropriate birth control (defined as a method which results in a low failure rate, i.e., 
less than 1% per year, when used consistently and correctly, such as double barrier 
methods [male condom with spermicide, with or without cervical cap or diaphragm], 
implants, injectables, oral contraceptives [must have been using for at least the last 3 
months], some intrauterine contraceptive devices, tubal ligation, or in an established 
relationship with a vasectomized or same sex partner) during the entire duration of the 
study 
3. Stable body weight (±5%) and health over the last 3 months. 
4. Has a BMI between 30 kg/m2 and 45 kg/m2 
5. Stable diet within the last three months 
6. Subjects on antidepressants, excluding those listed in the exclusion criteria, must have 
been on a stable dose regiment for at least 3 months prior to study enrollment and 
maintain stable dose during the study 
7. Clinical laboratory tests (hematology, clinical chemistry, and urinalysis) either normal, 
not clinically significant or with abnormalities consistent with obesity 
8. Is able to read, understand, and sign the informed consent forms (ICF) and, when 
applicable, an authorization to use and disclose protected health information form 
(consistent with Health Insurance Portability and Accountability Act of 1996 [HIPAA] 
legislation), communicate with the investigator, and understand and comply with 
protocol requirements.  


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4.3 
Exclusion Criteria 
Subjects who meet any of the following criteria will be excluded from the study. 
1. Diagnosis of diabetes or on a diabetes medication 
2. HbA1c ≥6.5% at Screening/Visit 1 (Day-7/Week-1) 
3. Severe renal impairment (eGFR < 45 mg/mL/1.73 m2) and/or patients with acute or 
chronic metabolic acidosis.  Acidosis is defined as >25mmol/L computed without K.  
Normal is 8-16, but acidosis is >25 
4. Use of any of the following medications in the eight weeks prior to entering screening 
for study participation and during the study: 
a. Use of any anti-diabetes medication including metformin and any combination drug 
that contains metformin 
b. Sildenafil 
c. Tadalafil 
d. Vardenafil 
e. OCT2/MATE inhibitors (e.g. cimetidine, quinidine, and pyrimethamine) 
f. Riociguat (guanylate cyclase stimulant) 
g. Alpha blockers 
h. Nitrates (e.g. oral nitrates, sublingual nitroglycerine and nitroglycerine patches) 
i. Potent CYP3A4 inhibitors (e.g. clarithromycin, telithromycin, nefazodone, 
itraconazole, ketoconazole, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, 
ritonavir, saquinavir, tipranavir) 
j. All antihypertensive medications 
k. Medications associated with weight changes 
• 
Drugs approved for the treatment of obesity 
• 
Cypropheptadine or medroxyprogesterone 
• 
Atypical anti-psychotic drugs 
• 
Tricyclic antidepressants 
• 
Lithium, MAO’s, glucocorticoids 
• 
SSRI’s or SNRI’s 
• 
Antiepileptic drugs 
• 
Systemic corticosteroids 
• 
Stimulants e.g. amphetamines 
l. Any dietary supplement that is labeled for weight management or maintenance of 
healthy weight 
5. Diagnosis or evidence of eating disorders 
6. ≥ 5% weight change in the last 3 months 


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7. Subjects who have had bariatric surgery 
8. An infection requiring antibiotic treatment within the last 30 days 
9. Any chronic inflammatory disease, e.g. inflammatory bowel disease rheumatoid arthritis, 
vasculitis, systemic lupus erythematosus 
10. Diseases interfering with metabolism (including metabolism of branched chain amino 
acids) and/or ingestive behavior (myxedema, Cushing’s disease, diabetes, schizophrenia, 
major psychoses, maple syrup urine disease, unmanaged depression etc.) 
11. History of alcohol abuse (defined ≥ 21 drinks per week for males and > 14 drinks per 
week for females), within the past 3 months or failure on urinary drug screen 
12. History of substance abuse (including alcohol abuse as defined above) in the past 12 
months or a positive screen for drugs (opioids without a prescription), including 
marijuana, of abuse or alcohol at screening. 
13. Has received any investigational drug within 3 months of Screening. 
14. Has donated blood within 3 months before Screening or is planning to donate blood 
during the study (due to HbA1c reading at screening) 
15. Other medical conditions that may diminish life expectancy to <2 years, including 
known cancers 
16. Have been diagnosed with metastatic carcinomas in the last 5 years 
17. Has known allergies or hypersensitivity to metformin, sildenafil or leucine 
18. Have suffered myocardial infarction, stroke, arrhythmia in the last 6 months 
19. Resting hypotension (BP <90/50 mmHg) or severe hypertension (BP >170/110 mmHg) If 
both or one of the parameters for diastolic or systolic BP is meet 
20. Cardiac failure or coronary artery disease causing unstable angina 
21. History or evidence at screening of left ventricular outflow obstruction (e.g., aortic 
stenosis, idiopathic hypertrophic subaortic stenosis) and those with severely impaired 
autonomic control of blood pressure 
22. At risk for priapism due to anatomical deformation of the penis (e.g., angulation, 
cavernosal fibrosis, Peyronie’s disease) or other conditions that predispose to priapism 
(e.g., sickle cell anemia, multiple myeloma, or leukemia) 
23. Clinical evidence of hepatic impairment and/or ALT/AST >5X ULN 
24. Is an immediate family member (spouse, parent, child, or sibling; biological or legally 
adopted) of personnel directly affiliated with the study at the clinical study site, or NuSirt 
Biopharma. 
25. Is employed by NuSirt Biopharma (defined as an employee, temporary contract 
worker, or designee responsible for the conduct of the study). 
 
4.4 
Removal of Subjects from Study Participation 
Participants are to be removed from the study if any of the following conditions are met after 
study entry: 


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• Withdrawal of Consent - Subject wishes to withdraw from the study as outlined in the 
informed consent. All subjects reserved the right to withdraw from the study without 
prejudice to themselves or their future medical care. This implies a direct request to 
withdraw from the study and a refusal of further contact. 
• Adverse Event - Subject experiences an adverse event, which in the investigator’s 
opinion necessitates withdrawal from the study.   
• Investigator Decision - Investigator assessment that it is in the subject’s best interest 
to terminate participation. 
• Protocol Violation – Includes subject non-compliance, documented pregnancy, study 
entry violation, and/or start of an unacceptable concomitant medication (listed in 
section 4.3). 
• Lost To Follow-Up - Subject fails to return for study visits and cannot be reached 
with reasonable attempts.  At the close of the study one final attempt to contact 
the subject will be made to attempt to determine the subjects well-being. 
• Administrative Reason – Includes discontinuation of the study protocol by the 
sponsor, the Food and Drug Administration (FDA), or other regulatory 
authority, and/or discontinuation of the study site’s participation in the study 
protocol. 
• Pregnancy  
Any withdrawal from the study will be fully documented on the subject’s eCRF and in the 
subject’s study record at the site. The documentation will include reasons for the withdrawal 
and details of any sequelae (followed through until the symptoms resolved or returned to 
baseline levels) if the reason was an adverse event. 
Every effort will be made to conduct all protocol specified procedures and examinations 
required to complete the study. Optimally, all procedures should be performed within 24 
to 48 hours of termination and, if possible, prior to initiating new therapies.  Diligent 
attempts will be made to follow through endpoint assessment those patients who do not 
withdraw consent and/or are not lost to follow up. 
Subjects who discontinue from the study after randomization and prior to completion of 
Study Termination /Visit 8 will be considered early withdrawals and, if enrollment is still 
open, enrollment may be expanded to ensure a full complement of evaluable subjects. 
 
4.5 
Subject Restrictions 
Once screened and qualified for entry, subjects will be instructed as follows: 
• Take no new prescription or over-the-counter medications without prior 
notification of the investigator. For restrictions on concomitant medications, refer 
to Section 4.3  
• Remain on their usual diet and exercise regimen throughout the study  
 
 


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• Fast overnight for at least 10 hours (no food or beverage except water) prior to each 
visit 
• Do not donate blood for the duration of the study 
The sponsor should be contacted if the investigator is informed of any restriction violations. 
The sponsor will decide whether a subject with restriction violations will be granted a 
deviation and allowed to continue study participation. 
 
4.6 
 Concomitant Medications 
Subjects must follow the medication restrictions outlined in the inclusion and exclusion 
criteria (Section 4.3) during the study. Dosages for certain concomitant medications should 
be maintained constant during the study (Section 4.2 and 4.3) unless instructed otherwise by 
the investigator or a treating physician. Any change in regimen for any concomitant 
medication, including restricted concomitant medications, must be reported to the sponsor 
and recorded in the eCRF. 
The sponsor should be contacted if the investigator is informed of consistent concomitant 
medication restriction violations. The sponsor will decide whether the subject will be allowed 
to continue study participation. 
 
5 
STUDY MEDICATIONS 
Study medications administered in this protocol include either placebo or one of four 
combinations of leucine, sildenafil and/or metformin (See Section 5.1).  All drugs will be 
provided as tablets with identical appearance to double-blind the medications to subjects 
and study personnel.  Study medication is to be kept at ambient temperature at all times.  
Any deviations will require the drug to be temporarily quarantined and the deviation 
reported to NuSirt.  NuSirt will then make an assessment as to how to proceed. 
 
5.1 
Formulation, Packaging, and Storage 
Fixed Dose Combination Arms: Each dose of study medication will consist of three tablets, 
two tablets containing either 550 mg L-leucine alone or 550mg L-leucine in combination with 
250 mg of Metformin and one tablet either 1.0 mg or 4.0 mg of sildenafil. 
Placebo Arm: Each dose of placebo will consist of three tablets, identical in appearance 
to those used in the two active treatment arms, containing 93.5% Microcrystalline 
cellulose PH 102, 5.0% Crospovidone XL 10, 1.0% Silica gel (Syloid 244), 0.5% 
Magnesium stearate I MF3V for the leucine matched placebo and 99.5% Avicel PH200 , 
0.5% magnesium stearate (w/w) and Opadry II White coating 3% weight gain for the 
sildenafil matched placebo. 
 
5.2 
Dispensing of Study Medication 
Study materials will be provided to subjects by the investigator, medically qualified sub- 
investigator named on form FDA 1572, or other qualified study-site personnel. Under no 
circumstance will the investigator or sub-investigator(s) allow the study medication to be 
used other than as directed by the protocol or to be administered to any persons other than 


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subjects who have signed an informed consent and are participating in the study. 
Study-site personnel will be responsible for instructing subjects to administer the correct 
doses of study medication as shown in Appendix 1. 
Study medication will be provided in blinded, randomized kits. Each kit dispensed will be 
labeled with a unique package number. 
 
5.3 
Dose Administration, Route and Schedule 
Study medication will be self-administered by the patient as 3 tablets taken orally BID within 
30 minutes prior to the morning and evening meals. Each dose of study medication will 
consist of 3 tablets, which will be identical in appearance across all treatment groups. 
 
5.4 
Randomization Schedule and Blinding Procedures 
A randomization schedule will be prepared and not shared with anyone directly involved in 
study conduct. Subjects will be assigned to one of five treatment groups (Table 1) based 
on the randomization schedule. 
Subjects will be assigned a randomization number on Baseline/Visit 2 (Day 1). The 
randomization numbers will be allocated in strict chronological order. The number will 
uniquely identify the subject and dictate the assigned, blinded treatment group. 
The sponsor personnel involved in study conduct, study staff and subject will be blinded to 
the identity of study medication during the study. The study medication will be provided to 
the study centers in a blinded fashion. The study center pharmacist will use the subject’s 
assigned randomization number to ensure that the appropriate blinded study medication is 
allocated at each visit. The study centers will be provided with sufficient study medication to 
supply all randomized subjects. 
 
5.5 
Drug Accountability 
The investigator listed on form FDA 1572 will be responsible for all clinical supplies 
(study medication) accountability. Upon receipt of all study medication, the contents of 
each shipment will be verified against the enclosed shipping form. After verification, the 
form will be signed and dated, a copy will be retained for the investigator’s file, and the 
original will be returned to the sponsor. The sponsor will be notified immediately if any 
irregularities, discrepancies, or damages are identified. 
The investigational materials will be prescribed only by the investigator or sub-
investigators. Under no circumstances will the investigator or sub-investigators allow the 
investigational drug to be used other than as specified in the protocol. 
Drug dispensing and returns will be recorded on a sponsor-provided or sponsor-approved 
drug disposition form, to provide a complete accounting of the receipt, disposition, and 
return of study medication. At the completion of the study, all remaining used and unused 
study medication, empty containers, and copies of the completed drug disposition forms 
will be returned to the sponsor or designee. 
 


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6 
STUDY METHODS 
A detailed schedule of study procedures is summarized in Appendix 1. 
Prior to all screening and study visits, subjects will be asked to fast overnight for at least 10 
hours (i.e., no food or beverage except water). Subjects should report to the clinical study 
site in the morning of each scheduled visit (ideally between 0700 and 0900 hours). For all 
study visits after randomization, subjects should refrain from administering the morning dose 
of study medication prior to the visit, and bring the study medication along to the study-site.  
The morning dose of study medication will be taken while at the clinic after all study 
procedures have been completed from one of the two newly dispensed study medication kits. 
 
6.1 
Screening (Visit 1/ Day-7/ Week-1) 
The following procedures will be performed in order when possible, except for the ICF which 
must be done before any other items, at Screening: 
• The informed consent and HIPAA authorization forms will be signed prior to 
performing any procedures required by the protocol 
• Review of inclusion/exclusion criteria to evaluate subject eligibility 
• Complete medical history 
• Adverse event review 
• Review of concomitant medications 
• Vital signs; blood pressure to be done first and using the equipment provided 
by NuSirt as instructed in section 7.8 
• Complete height and weight assessment for BMI calculation 
• Complete physical examination  
• 12-lead ECG 
• Blood sample collection (fasting samples) for the following assessments (please follow 
the laboratory manual for the procedures and order of collection): 
− 
serum β-hCG test for all female subjects, regardless of menopausal status (not 
required if subject has had a hysterectomy) 
− 
Chemistry panel includes glucose, urea nitrogen, creatinine, total protein, albumin, 
uric acid, total and fractionated bilirubin, alkaline phosphatase, alanine 
aminotransferase (ALT), aspartate aminotransferase (AST), gamma 
glutamyltranspeptidase (GGT), creatine phosphokinase (CPK), creatine kinase-MB 
(CK-MB, If CPK is elevated), sodium, potassium, chloride, bicarbonate, phosphate, 
calcium, magnesium 
− 
Lipid Panel includes total cholesterol, HDL-C, LDL-C and triglycerides  
− 
Hematology panel (includes red cell count, hemoglobin, hematocrit, white cell 
count, platelets, differential count, mean cell volume, mean corpuscular 
hemoglobin, and mean corpuscular hemoglobin concentration) 
− 
HbA1c 


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Clinical Study Protocol NS-WM-01 
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• Urine sample collection for the following assessments: 
− 
urinalysis 
− 
drug and alcohol screen 
Abnormal results of laboratory tests, not addressed in the Inclusion/Exclusion criteria, 
consistent with and associated with obesity will be evaluated on a case by case basis with 
input from investigator in consultation with the medical monitor. With the exception of 
abnormal laboratory tests consistent with obesity as mentioned above, individuals will be 
disqualified if results of any laboratory tests are more than twice the upper limit of normal 
and clinically significant as judged by the investigator in consultation with the medical 
monitor.  
 
6.2 
Baseline/Visit 2 (Day 1) 
 
Note: All individuals will be counselled at Baseline/Visit 2 (Day 1) that they are not to 
change eating or exercise routines during the trial. 
The following procedures will be completed at Visit 2: 
• Review of concomitant medicines 
• Adverse event review 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the 
equipment provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Randomization to study medication 
• Blood sample collection (fasting samples) for the following assessments (please follow 
the laboratory manual for the procedures and order of collection): 
− 
Chemistry panel 
− 
Hematology panel 
− 
Lipid panel 
− 
hsC-reactive protein (hsCRP) 
− 
HbA1c 
− 
Biomarkers (Note:  These samples will be archived for future exploratory analyses.  
See Section 7.10)  
• Urine sample collection for urinalysis assessment  
• First dose of study medication taken in clinic 


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Clinical Study Protocol NS-WM-01 
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• Dispense study medication for 4 weeks (each kit has sufficient study medication for 2 
weeks + 1 day) 
 
6.3 
Telephone Contact (Day 14/Week 2) 
The investigator and/or qualified study-site personnel will contact subjects by telephone on 
Day 14 to review adverse events and study medication compliance. 
 
6.4 
Visit 3 (Day 28/Week 4) 
The following procedures will be completed at Visit 3: 
• Review of concomitant medicines 
• Review of Adverse Events 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Return of unused study medication 
• Review of study medication compliance 
• Dispense study medication for next 4 weeks 
 
6.5 
Telephone Contact (Day 42/Week 6) 
The investigator and/or qualified study-site personnel will contact subjects by telephone on 
Day 56 to review adverse events and study medication compliance. 
 
6.6 
Visit 4 (Day 56/Week 8) 
The following procedures will be completed at Visit 4: 
• Review of concomitant medicines 
• Review of Adverse Events 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 


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Clinical Study Protocol NS-WM-01 
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• Return of unused study medication 
• Review of study medication compliance 
• Dispense study medication for next 4 weeks 
 
6.7 
Telephone Contact (Day 70/Week 10) 
• The investigator and/or qualified study-site personnel will contact subjects by telephone 
on Day 70 to review adverse events and study medication compliance. 
 
6.8 
Visit 5 (Day 84/Week 12) 
The following procedures will be completed at Visit 5: 
• Review of concomitant medicines 
• Review of Adverse Events 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Blood sample collection (fasting samples) for the following assessments (please follow 
the laboratory manual for the procedures and order of collection): 
− 
Chemistry panel 
− 
Hematology panel 
− 
Lipid panel 
− 
hsC-reactive protein 
− 
HbA1c 
− 
Biomarkers (Note:  These samples will be archived for future exploratory analyses.  
See Section 7.10)  
• Return of unused study medication 
• Review of study medication compliance 
• Dispense study medication for next 4 weeks 
 
6.9 
Telephone Contact (Day 98/Week 14) 
• The investigator and/or qualified study-site personnel will contact subjects by 
telephone on Day 98 to review adverse events and study medication compliance. 
 
 


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6.10 
Visit 6 (Day 112/Week 16) 
The following procedures will be completed at Visit 6: 
• Review of concomitant medicines 
• Review of Adverse Events 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Return of unused study medication 
• Review of study medication compliance 
• Dispense study medication for next 4 weeks 
 
6.11 
Telephone Contact (Day 126/Week 18) 
• The investigator and/or qualified study-site personnel will contact subjects by 
telephone on Day 126 to review adverse events and study medication compliance. 
 
6.12 
Visit 7 (Day 140/Week 20) 
The following procedures will be completed at Visit 7: 
• Review of concomitant medicines 
• Review of Adverse Events 
• Measurement of vital signs:  
− 
Blood pressure (sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Return of unused study medication 
• Review of study medication compliance 
• Dispense study medication for next 4 weeks 
 
6.13 
Telephone Contact (Day 154/Week 22) 
• The investigator and/or qualified study-site personnel will contact subjects by 


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Clinical Study Protocol NS-WM-01 
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telephone on Day 154 to review adverse events and study medication compliance. 
 
6.14 
Study Termination/Visit 8 (Day 168/Week 24) or Early Termination 
The following procedures will be completed at Visit 8 or the termination visit for subjects who 
leave the study prior to study completion: 
• Complete medical history 
• Review of concomitant medicines 
• Adverse event review 
• Measurement of vital signs:  
− 
Blood pressure(sitting systolic and diastolic blood pressure using the equipment 
provided by NuSirt) 
− 
Heart rate 
• Body weight assessment 
• Waist Circumference measurement (using the tape measure provided by NuSirt) 
• Complete physical examination 
• 12-lead ECG 
• Blood sample collection (fasting samples) for the following assessments (please follow 
the laboratory manual for the procedures and order of collection): 
− 
Chemistry panel 
− 
Hematology panel 
− 
Lipid panel 
− 
hsC-reactive protein 
− 
HbA1c 
− 
Biomarkers (Note:  These samples will be archived for future exploratory analyses.  
See Section 7.10)  
• Urine sample collection for urinalysis assessment 
• Return of unused study medication 
• Review of study medication compliance 
 
6.15 
Ethical Safety Considerations 
 
Metformin, on rare occasions, has been associated with lactic acidosis in patients on higher 
doses than allowed in this study.  In addition, these events have usually occurred in patients 
encountering major medical illnesses superimposed on their diabetes.  These issues are 
addressed in Section 8.1 with an emphasis on early detection. 
 
Treatment with sildenafil at the approved doses of 50 to 100 mg (≥20 times the doses employed 


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Page 26 
 
in this study) has been associated with the following rare adverse events: 
 
1. Sudden loss of vision 
2. Ringing in the ears 
3. Loss of hearing 
4. Ischemic cardiovascular events 
 
Should any study participant experience one of these events, study drug is to be held, 
appropriate medical evaluation arranged and the medical monitor contacted.  It will then be the 
determination of the medical monitor, after discussions with the PI, whether the subject will be 
removed from the study.  
 
7 
EFFICACY ASSESSMENTS 
 
7.1 
Percent Change in Body Weight 
Body weight will be assessed at Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 
(Day1), Visit 3 (Day 28/Week4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), 
Visit 6 (Day 112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 
(Day 168/Week 24) or Early Termination.  Percentage change is defined as Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination weight minus Baseline/Visit 
2 (Day1) weight divided by Baseline/Visit 2 (Day1) weight multiplied by 100.  Weight 
measurements will be done without shoes or street clothing; subjects will be weighed in 
undergarments with gowns (front and back). All scales will have certified calibration and 
sites will maintain their current calibaration schedule.  When possible the same scale that is 
used for a subject at Screening/Visit 1 (Day-7/Week-1) will be used for all remaining visits 
for that subject.  All weights are to be made and reported in kilograms to the first decimal 
place. 
 
7.2 
Absolute Body Weight 
Body weight will be assessed at Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 
(Day1), Visit 3 (Day 28/Week4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), 
Visit 6 (Day 112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 
(Day 168/Week 24) or Early Termination.  All weights are to be made and reported in 
kilograms to the first decimal place. 
 
7.3 
Percentage of patients with ≥5% and ≥10% body weight loss 
Body weight will be assessed at Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 
(Day1), Visit 3 (Day 28/Week4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), 
Visit 6 (Day 112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 
(Day 168/Week 24) or Early Termination.  Percentage change is defined as Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination weight minus Baseline/Visit 
2 (Day1) weight divided by Baseline/Visit 2 (Day1) weight.  All weights are to be made 
and reported in kilograms to the first decimal place. 
 
 


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7.4 
Waist Circumference 
Waist circumference will be measured at Baseline/Visit 2 (Day1), Visit 3 (Day 28/Week4), 
Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 112/Week 16), Visit 7 
(Day 140/Week 20) and Study Termination/Visit 8 (Day 168/Week 24) or Early 
Termination.  Waist circumference is measured at the end of several consecutive natural 
breaths, at a level parallel to the floor, midpoint between the top of the iliac crest and the 
lower margin of the last palpable rib in the mid axillary line.  Measurements are made with 
a stretch‐resistant tape (which will be provided by NuSirt) that is wrapped snugly around 
the subject, but not to the point that the tape is constricting.  The tape is kept level and 
parallel to the floor at the point of measurement.  Subjects will be standing upright during 
the measurement, with arms relaxed at the side, feet evenly spread apart and body weight 
evenly distributed.  All measurements are to be made and reported in centimeters to the 
first decimal place.  
 
7.5 
Fasting Lipids 
Fasting lipid concentrations (total cholesterol, LDL, HDL, triglycerides) will be 
measured in all subjects from blood samples collected at Screening/Visit 1 (Day-
7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) and Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
 
7.6 
Fasting Glucose 
Fasting glucose will be measured in all subjects from blood samples collected at 
Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) and 
Study Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
 
7.7 
HbA1c 
HbA1c will be measured in all subjects from blood samples collected at Screening/Visit 1 
(Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) and Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
 
7.8 
Blood Pressure 
Blood Pressure will be measured in all subjects at Screening/Visit 1 (Day-7/Week-1), 
Baseline/Visit 2 (Day1), Visit 3 (Day 28/Week4), Visit 4 (Day 56/Week 8), Visit 5 (Day 
84/Week 12), Visit 6 (Day 112/Week 16), Visit 7 (Day 140/Week 20) and Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination. Blood pressure will be taken 
using the Omron HEM-907XL provided by NuSirt. Qualified site personnel will program 
the machine to capture a series of 3 measurements starting with a 5 minute delay followed 
by a delay of 2 minutes between the first and second reading and the second and third 
reading. The patient is to be in a seated position with their back resting on the back of the 
chair, feet flat on the floor and arm resting comfortably approximately at heart level.  The 
appropriate cuff size, cuff placement and wrapping of the cuff on the subjects arm will be 


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applied following the instruction manual provided with the equipment.  Once the patient and 
equipment is set, site personnel will leave the room for approximately 10 minutes.  After the 
measurements are completed, qualified site personnel will then document the 3 
measurements, as well as the average provided by the machine (refer to the Omron HEM-
907XL instruction manual for detailed instructions on all the directions listed above). 
 
7.9 
hsC-Reactive Protein 
hsC-Reactive Protein will be measured in all subjects from blood samples collected at 
Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) and Study Termination/Visit 8 (Day 
168/Week 24) or Early Termination. 
 
7.10 
Biomarkers 
  Biomarkers will be measured in all subjects from blood samples collected at Baseline/Visit 2 
(Day1), Visit 5 (Day 84/Week 12) and Study Termination/Visit 8 (Day 168/Week 24) or 
Early Termination. Biomarker analysis will not include genetic testing. Biomarker analysis 
may include but is not limited to assessments of adiponectin, TNFα, IL-6, PAI-1, and a 
lipidomic panel. Additional non-genetic biomarkers identified subsequent to trial initiation 
may also be used. 
 
8 
SAFETY ASSESSMENTS 
Safety will be assessed throughout the study by examination of adverse events, concomitant 
medications, clinical laboratory evaluations, vital signs, physical examinations, and 12-lead 
ECGs. Safety assessments by visit are listed in Appendix 1. 
8.1 
Targeted Safety Surveillance Items 
This study represents the second evaluation of the concomitant administration of leucine, 
sildenafil and metformin in combination and the initial evaluation of the concomitant 
administration of leucine and sildenafil in combination.  Given that leucine is a naturally 
occurring amino acid that is regularly ingested as part of normal dietary practices, the well-
established safety profiles of sildenafil and metformin when administered at higher doses and 
the observed safety of the drug combination in preclinical studies to date, the assessed risk is 
low.  However, the fact that this represents the second systematic administration of the 
leucine, sildenafil and metformin combination and the initial systemic administration of the 
leucine and sildenafil combination to humans, constant attention to safety monitoring is 
warranted.  The following issues need careful attention throughout the course of the study. 
 
8.1.1 Metformin Associated Lactic Acidosis 
Although its occurrence is extremely rare, lactic acidosis has been reported in 
patients using full dose metformin (1500 to 2500 mg/day).  Given that lactic 
acidosis is a serious condition and the possibility that the leucine-metformin-
sildenafil combination might potentiate this effect, study staff need to have a 
heightened awareness of this possibility.   


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The initial signs and symptoms associated with lactic acidosis are subtle, vague 
and non-specific including anorexia, nausea, vomiting, abdominal pain, 
lethargy and, with advancing severity, hyperventilation and hypotension.  Since 
plasma lactate levels are erratic and of little help until the severity of the 
disorder has advanced, it is important to be attentive to the electrolyte 
measurements on the chemistry panel and investigate further any anion gap that 
is noted.  Also, study staff should be attentive to patient reports and implement 
appropriate clinical evaluation of persistent complaints of lethargy and fatigue.  
If in doubt, contact the medical monitor. Study medication should be stopped 
immediately if any evidence of lactic acidiosis is seen. 
Also, since intravascular volume depletion can increase the risk for lactic 
acidosis, the study medication should be discontinued prior to any procedures, 
surgical or otherwise, that require restricted fluid intake. 
8.1.2 Nephrotoxic Effects of Radiocontrast Agents in Patients Taking 
Metformin 
The administration of iodinated radiocontrast agents has been associated with 
acute nephrotoxic effects when administered to patients taking metformin.  
Thus, it is recommended that study medication be discontinued for 24 hours 
prior to the patient receiving any iodinated radiocontrast material.  Study 
medication should then be resumed once the patient has returned to their usual 
diet and fluid intake. 
8.1.3 Risk of Hypoglycemia in Patients Taking Metformin 
While metformin is a glucose lowering agent, it is unusual for patients taking 
only metformin to experience hypoglycemia.  (Hypoglycemia associated with 
metformin use is seen almost exclusively when used in combination with a 
sulfonylurea or insulin.)  However, since the experience is limited with leucine, 
metformin and sildenafil administered together, it is possible that the risk of 
hypoglycemia could be observed.  Therefore, patients will be instructed 
regarding the signs and symptoms of hypoglycemia and advised to ingest 
carbohydrate should they encounter the symptoms of hypoglycemia as well as 
to notify the study site. Study medication should be stopped immediately if any 
evidence of hypoglycemia is seen. 
8.1.4 Hypotension 
Sildenafil, at doses as low as 5mg TID, has been shown to cause hypotension. 
Although rare, if any signs of hypotension are present , in addition to reporting 
as an adverse event, please notify the medical monitor within 24 hours. Study 
medication should be stopped immediately if any evidence of hypotension is 
seen. 
8.1.5 Changes in Vision 
Sildenafil, at doses of 50 to 100 mg, has been associated with sudden loss of 
vision.  In addition, patients with diabetes mellitus are at increased risk for 
developing macular edema.  While it is unlikely that macular edema will be an 


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issue in this study of only 24-weeks duration.  Should a patient report blurry 
vision and/or a change in color vision, they should be referred for evaluation to 
an ophthalmologist. 
Sildenafil, at doses of 50 to 100mg, has been associated with non-arteritic 
anterior ischemic optic neuropathy. Although rare, if any signs of non-arteritic 
anterior ischemic optic neuropathy are present , in addition to reporting as an 
adverse event, please notify the medical monitor within 24 hours. Study 
medication should be stopped immediately if any evidence of non-arteritic 
anterior ischemic optic neuropathy is seen. 
8.1.6 Leucine Toxicity 
Leucine, when consumed as a dietary supplement at high doses (>500 
mg/kg/day), has been associated with an increase in blood ammonia levels.  
While it’s unlikely that the doses employed in this trial are adequate to elevate 
blood ammonia levels, the potential for synergism between leucine, metformin 
and sildenafil exists.  Also, the potential exists for the combination to magnify 
the hypoglycemic effects of metformin. 
Therefore, any study participant presenting with neurological symptoms should 
be evaluated for low blood glucose values and elevated ammonia levels.  If 
either are found, in addition to reporting as an adverse event, please notify the 
medical monitor within 24 hours. Study medication should be stopped 
immediately if any evidence of leucine toxicity is seen. 
8.2 
Reporting of Adverse Events 
Adverse events will be reported in accordance with the National Cancer Institute- Common 
Terminology Criteria for Adverse Events (CTCAE), version 4.03.  (Appendix 2).  The 
CTCAE grades the severity of the AE based upon Grades 1 through 5 and lists unique 
clinical descriptions of severity for each AE. 
Grade 1:  Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated. 
Grade 2:  Moderate; minimal, local or noninvasive intervention indicated or limiting 
age-appropriate instrumental ADL. 
Grade 3:  Severe or medically significant but not immediately life-threatening 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-
care ADL 
Grade 4:  Life threatening consequences; urgent intervention indicated 
Grade 5:  Death related to AE 
In the event of the occurrence of a same category of three or more grade III CTCAE or 2-
grade IV CTCAE or one grade V CTCAE, the trial will be paused, evaluated for safety by 
the Medical Monitor and allowed to proceed only after the Medical Monitor determines the 
causality is not associated with the investigational fixed-drug combination.  


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8.3 
Adverse Events and Subject Withdrawals  
Adverse Event 
Any untoward medical occurrence associated with the use of a therapy in humans, whether 
or not considered treatment related. 
 
Life-Threatening 
Any untoward medical occurrence that places the patient or subject at immediate risk of 
death. It does not include an adverse event or suspected adverse reaction that, had it occurred 
in a more severe form, might have caused death. 
 
Serious Adverse Event (SAE) 
ANY SAE THAT OCCURS AFTER THE SIGNING OF THE ICF THROUGH 30 
DAYS AFTER ADMINISTRATION OF THE LAST DOSE OF STUDY 
MEDICATION MUST BE REPORTED IMMEDIATELY (WITHIN 24 HOURS OF 
KNOWLEDGE) TO THE SPONSOR’S MEDICAL MONITOR (858-342-7057). FAX 
THE SAE REPORT FORM TO THE NUMBER LISTED ON THE FORM 
An adverse event is considered "Serious" if, in the view of either the investigator or sponsor, 
it results in any of the following outcomes: 
• Death 
• A life-threatening adverse event 
• Inpatient hospitalization or prolongation of existing hospitalization 
• A persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• A congenital anomaly/birth defect. 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered serious when, based upon appropriate medical judgment, 
they may jeopardize the patient or subject and may require medical or surgical intervention to 
prevent one of the outcomes listed in this definition. 
Any SAE, unexpected events or death encountered during the course of the investigation and 
for a reasonable time thereafter will be reported to the principal and/or co-investigators by 
the subject (immediately by telephone and subsequently in writing within five (5) days). The 
investigator will immediately report the SAE/unexpected event or death within 24 hours of 
being notified to the sponsor for review and assessment/causality confirmation, and to the 
IRB (per FDA requirements). 
 
Unexpected Adverse Event:  
An adverse event is considered “unexpected” if it is not listed in the investigator brochure or 
is not listed at the specificity or severity that has been observed. 
FDA guidelines recognize that: 
1. “Individual adverse event reports generally require an evaluation of their relevance and 


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significance to the study, including an evaluation of other adverse events, before they can 
be considered to be an unanticipated problem,” and 
2. “All reports to the IRB of unanticipated problems should explain clearly why the event 
described represents a ‘problem’ for the study and why it is ‘unanticipated.’’ 
Investigators are required to report unexpected adverse events that fit the following criteria 
within 10 working days of the time the investigator has become informed of the event(s) to 
the IRB and sponsor. 
The following guidelines provide further definition to unexpected adverse events: 
• Event is unexpected (in terms of nature, severity, or frequency) given (a) the research 
procedures that are described in the protocol-related documents, such as the IRB- 
approved research protocol and informed consent document, or the investigator 
brochure; and (b) the characteristics of the subject population being studied, 
• Related or possibly related to participation in the research (possibly related means 
there is a reasonable possibility that the incident, experience, or outcome may have 
been caused by the procedures involved in the research); and 
• Suggests that the research places subjects or others at a greater risk of harm 
(including physical, psychological, economic, or social harm) than was previously 
known or recognized. 
If the adverse event is clearly not related to the study drug, device, procedures, or washout 
process, it would not represent a risk to other subjects in the research or a “problem” for the 
study and, therefore, does not have to be reported to the IRB. 
 
Intensity: 
The intensity of each adverse event will be characterized as mild, moderate, or severe, as 
follows: 
• MILD: Usually transient, requires no special treatment, and does not interfere with 
the subject’s daily activities. 
• MODERATE: Usually causes a low level of inconvenience or concern to the subject 
and may interfere with daily activities, but is usually ameliorated by simple 
therapeutic measures. 
• SEVERE: Interrupts a subject’s usual daily activities and generally requires 
systemic drug therapy or other treatment. 
Causality: 
The investigator will grade the association of the adverse event as UNRELATED or 
RELATED to study medication. The following criteria should be considered for determining 
relatedness: 
• UNRELATED: The adverse event is judged to be produced by the subject's clinical 
state or by other therapies administered to the subject. 
• RELATED: The adverse event is judged to be related to the administration of study 
medication. 


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Pregnancy: 
If a patient becomes pregnant during the study or within 30 days of discontinuing study drug, 
the Investigator should report the pregnancy to the NuSirt Medical Monitor within 24 hours 
of being notified. The NuSirt Medical Monitor will then forward the Exposure In Utero form 
to the Investigator for completion. A patient becoming pregnant while on study drug will 
immediately be withdrawn from the study and Early Termination study procedures will be 
performed. A patient who becomes pregnant should be followed by the Investigator until 
completion of the pregnancy. If the pregnancy ends for any reason before the anticipated 
date, the Investigator should notify NuSirt Medical Monitor. At the completion of the 
pregnancy, the Investigator should document the outcome of the pregnancy. If the outcome 
of the pregnancy meets the criteria for immediate classification as an SAE (ie, postpartum 
complication, spontaneous abortion, stillbirth, neonatal death, or congenital anomaly) the 
Investigator should follow the procedures for reporting an SAE.  If a pregnancy occurs 
during the study, the data is to be gathered regardless of the conclusion of the study and 
provided to NuSirt immediately after collection. 
 
8.4 
Clinical Laboratory Evaluations 
The investigator or medically qualified physician listed on the form FDA 1572 will evaluate 
all screening and safety laboratory reports, and will sign and date the review. Any out of 
range laboratory results should be assessed for clinical significance. The investigator should 
follow all clinically significant laboratory abnormalities occurring during the study that were 
not present at baseline. These abnormalities should be evaluated with additional tests, if 
necessary, until the underlying cause is diagnosed or resolution occurs. The diagnosis and 
resolution date must be reported to the sponsor. Samples will be collected according to the 
schedules presented in Appendix 1 and Section 6. 
The centralized laboratory will provide specific instructions for collection, processing, 
packaging, and shipping of all samples. 
8.4.1 Chemistry 
Chemistry assessments will be performed from blood samples to be collected at 
Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) and 
Study Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
Chemistry assessments will include the following: glucose, urea nitrogen, creatinine, total 
protein, albumin, uric acid, total and fractionated bilirubin, alkaline phosphatase, alanine 
aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltranspeptidase 
(GGT), creatine phosphokinase (CPK), creatine kinase-MB (CK-MB, If CPK is elevated), 
sodium, potassium, chloride, bicarbonate, phosphate, calcium, magnesium (or other routine 
chemistry panels as approved by the sponsor). 
 
8.4.2 Hematology 
Hematology assessments will be performed from blood samples to be collected at 
Screening/Visit 1 (Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) 


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and Study Termination/Visit 8 (Day 168/Week 24) or Early Termination  
Hematology assessments will include the following: red cell count, hemoglobin, 
hematocrit, white cell count, platelets, differential count, mean cell volume, mean 
corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (or other 
routine hematology assessments as approved by the sponsor). 
 
8.4.3 Lipids 
Lipid assessments will be performed from blood samples to be collected at 
Screening/Visit (Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 5 (Day 84/Week 12) 
and Study Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
Lipid assessments will include the following: Total cholesterol, HDL, LDL and 
triglycerides 
 
8.4.4 Urinalysis 
Urinalysis assessments will be performed from samples to be collected at Screening/Visit 
1 (Day-7/Week-1), Baseline/Visit 2 (Day 1), and Study Termination/Visit 8 (Day 168/Week 
24) or Early Termination. 
Urinalysis assessments will include the following: pH, specific gravity, glucose, blood, 
ketones, protein, and microscopic analysis (or other routine urinalysis as approved by the 
sponsor). 
8.5 
Other Clinical Laboratory Evaluations 
 
8.5.1 Pregnancy Testing 
For all female subjects, regardless of childbearing status (unless subject has had a 
hysterectomy), pregnancy tests will be performed from blood samples collected at 
Screening/Visit 1 (Day-7/Week-1). 
 
8.5.2 Urine Drug Screen 
A urine screen for drugs of abuse and alcohol will be performed from a urine sample 
collected at Screening/Visit 1 (Day-7/Week-1). Positive results due to prescription 
medications (e.g., opioid-containing pain medications) are not necessarily 
exclusionary as long as it can be documented that they have a prescription.  
 
8.6 Vital Signs, Physical Examination Findings, and ECGs 
Clinically significant abnormalities in vital signs, physical examination findings, and ECGs 
at Study Termination must be followed up by the investigator and evaluated with additional 
tests if necessary, until the underlying cause is diagnosed or resolution occurs. The 
diagnosis and resolution date must be reported to the sponsor. 
Measurements will be performed as discussed in this section and according to the 


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schedules presented in Appendix 1 and Section 6. 
 
8.6.1 Vital Signs 
Vital sign measurements (heart rate and blood pressure) will be performed for each subject 
at Screening/Visit (Day-7/Week-1), Baseline/Visit 2 (Day1), Visit 3 (Day 28/Week4), Visit 4 
(Day 56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 112/Week 16), Visit 7 (Day 
140/Week 20) and Study Termination/Visit 8 (Day 168/Week 24) or Early Termination, 
according to the schedule presented in Appendix 1.   
 
8.6.2 Physical Examinations 
A complete physical examination will be performed at Screening/Visit 1 (Day-7/Week-1), 
Visit 5 (Day 84/Week 12) and Study Termination/Visit 8 (Day 168/Week 24) or Early 
Termination. Complete physical examinations will include evaluation of all body systems 
listed in the eCRFs. 
 
8.6.3 Electrocardiograms 
Standard 12-Lead ECGs will be performed at Screening/Visit 1 (Day-7/Week-1) and 
Study Termination/Visit 8 (Day 168/Week 24) or Early Termination. 
Standard 12-lead ECGs will be performed after at minimum 5 minutes of quiet rest with 
the subject in a supine position. If the ECG must be performed with the subject in another 
position (sitting, standing, etc.), the investigator should record the alternate position on the 
ECG eCRF page. This data also needs to be in the medical record. The investigator should 
date and sign the ECG tracing and record the clinical significance of any abnormal result 
on the tracing. ECGs will be interpreted by a qualified physician (the investigator or 
qualified designee) at the clinical study site. 
 
9 
BLOOD VOLUME 
During this study, blood will be drawn for various analytes and panels, including chemistry, 
lipids, hematology, and efficacy assessments. The total amount of blood to be drawn 
(during the entire study) is expected to be <90mL or ~3 ounces for each subject. 
 
10 DISCONTINUING AND UNBLINDING OF SUBJECTS 
Every effort should be made to conduct all protocol-required procedures to complete the 
study. Subjects may be removed from the study for the following reasons: 
• Withdrawal of Consent - Subject wishes to withdraw from the study as outlined in the 
informed consent. All subjects reserved the right to withdraw from the study without 
prejudice to themselves or their future medical care. 
• Adverse Event - Subject experiences an adverse event which, in the investigator’s 
opinion, necessitates withdrawal from the study.   
• Investigator Decision - Investigator assessment that it is in the subject’s best interest 
is to terminate participation.  


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• Protocol Violation – Includes subject non-compliance, documented pregnancy, study 
entry violation, and/or start of an unacceptable concomitant medication. 
• Lost To Follow-Up - Subject fails to return for study visits and cannot be reached 
with reasonable attempts. 
• Administrative Reason – Includes discontinuation of the study protocol by the 
sponsor, the Food and Drug Administration (FDA), or other regulatory 
authority, and/or discontinuation of the study site’ participation in the study 
protocol. 
• Pregnancy 
 
Any withdrawal from the study will be fully documented on the subject’s eCRF and in 
the subject’s study record at the site. The documentation will include reasons for the 
withdrawal and details of any sequelae (followed through until the symptoms resolved or 
returned to baseline levels) if the reason was an adverse event. 
Every effort will be made to conduct all protocol specified procedures and examinations 
required to complete the study. Optimally, all procedures should be performed within 24 
to 48 hours of termination and, if possible, prior to initiating new therapies.  Diligent 
attempts will be made to follow through endpoint assessment those patients who do not 
withdraw consent and/or are not lost to follow up. 
Only withdrawls for withdrawal of consent or lost to follow-up will not continue to be 
followed. For all other patients – regardless of whether or not study drug is discontinued – 
every effort should be made to continue all visits in the study for safety and should be 
encouraged to complete all study procedures.Subjects who discontinue from the study 
after randomization and prior to completion of Study Termination /Visit 8 will be 
considered early withdrawals and, if enrollment is still open, enrollment may be expanded 
to ensure a full complement of evaluable subjects. 
 
10.1 
Unblinding of Subjects 
The subject, investigator (including all study-site personnel), and sponsor will be blinded to 
the identity of the assigned study medication during the 24-week treatment period (Visit 2 
to Visit 8). Every effort should be made to ensure that subjects remain blinded to their 
treatment. The investigator is to notify the sponsor’s medical monitor if an event occurs 
that requires a subject’s treatment assignment to be unblinded. The code may be unblinded 
only after an irrevocable decision has been made to withdraw a subject from the study and 
if immediate knowledge of the study medication is needed to optimize the clinical 
management of the subject. 
 
 


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11 DATA MANAGEMENT 
A designated contract research organization (CRO) will perform all data management 
activities, including the writing of a data management plan, outlining the data management 
system and procedures to be used. The data management plan will detail the procedure for 
handling and documenting protocol deviations. 
Clinical study data will be recorded (captured) electronically by study site personnel on 
eCRFs. The eCRF will be a validated clinical data management system according to 
procedures that comply with the Code of Federal Regulations 21 Part 11 and the 
International Conference on Harmonization (ICH) Guidelines for Good Clinical Practice 
(Topic E6, April 2000) Section 5.5.3. The eCRF data will be entered by study-site 
personnel and then reviewed and electronically signed by the investigator listed on form 
FDA 1572. All study-site personnel must use an electronic signature access method to 
enter, review, or correct study data. Electronic signature procedures shall comply with the 
CFR title 21 part II and the ICH guidelines for good clinical practice (GCP) (Topic E6, 
April 2000) section 5.5.3. Passwords and electronic signatures will be strictly confidential. 
All eCRF data will be downloaded from the electronic data capture (EDC) system and 
reformatted into SAS data sets. The sponsor’s data management department will receive 
electronic transfers of laboratory data from a central laboratory as well as other data from 
third-party vendors as appropriate. The electronic data format of all transfers will be agreed 
upon with the sponsor. 
The clinical monitoring staff will verify data recorded in the EDC system with source 
documents at the clinical study sites according to the data management plan and clinical 
monitoring plan. The data will be subjected to consistency and validation checks within the 
EDC system with supplemental validation following download to a SAS data set. 
Adverse events will be coded using a current version of medical dictionary for regulatory 
activities (MedDRA), and concomitant medications using a current version of the WHO drug 
dictionary. The sponsor will perform a medical safety review of the coding. Completed 
eCRF images with a date- and time-stamped electronic audit trail indicating the user, the data 
entered, and any reason for change (if applicable) will be archived at the investigator’s site 
and archived with backup at the sponsor’s site. 
 
12 STATISTICAL CONSIDERATIONS 
A detailed statistical analysis plan (SAP) describing each of the planned analyses in detail will 
be prepared and signed before any of the data are unblinded. 
12.1 
Analysis Populations: 
Enrolled: The Enrolled Population will consist of all subjects who signed the informed consent 
to participate in the study, and had Screening visit electronic case report form (eCRF) 
collected. 
Randomized: The Randomized Populations will consist of all Enrolled subjects who were 
randomized at Baseline/Visit 2 (Day 1) 


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Intent-To-Treat (ITT): The ITT Population will include all randomized subjects who receive at 
least one dose of study medication. 
Per-Protocol (PP): The PP Population will include all ITT subjects who had adequate exposure 
to the randomized study medication (>85%) during the 24-week period, completed Visit 8 
(Day 112/Week16) and have adequately complied with the protocol as assessed by the 
investigator and sponsor prior to database lock.  The final definition of the PP Population will 
be documented in the prospectively developed SAP prior to undertaking any data analysis. 
 
12.2 
Study Subjects 
Demographic and baseline characteristics will be summarized descriptively by treatment 
group.  Demographic and baseline characteristics include, but are not limited to:  gender, age, 
race, ethnicity, body weight, height, body mass index (BMI), HbA1c, and fasting glucose. 
Baseline is defined as the last non-missing observation obtained prior to the administration of 
the first dose of randomized study medication at Baseline/Visit 2 (Day 1). 
 
12.3 
Primary Endpoint Analysis 
The primary efficacy endpoint is percentage body weight change from Baseline/Visit 2 (Day 
1) to Study Termination/Visit 8 (Day 168/Week 24). 
Descriptive statistics (n, mean, SD, SE, median, min, and max) will be used to summarize 
values of the primary efficacy endpoint, percentage body weight change from Baseline/Visit 2 
(Day 1) to Study Termination/Visit 8 (Day 168/Week 24) and intermediate study visits as 
applicable by treatment group.  These summaries will be presented for the PP Population using 
only observed data and the ITT Population employing the LOCF approach. 
A mixed-model of analysis of variance (ANCOVA) will be used to analyze change in body 
weight over the study duration in PP and ITT Populations.  The ANCOVA will include 
treatment group and time as the main effects with baseline weight as the covariate.  The LS 
means, SEs, and the corresponding 95% confidence intervals (CIs) for the changes from 
baseline to Week 24 will be derived from the model for each treatment.  Each of the fixed-dose 
combinations of leucine, sildenafil, and/or metformin treatment groups (Treatment B, C, D and 
E) will be compared to placebo group (Treatment A), and the LS means for the treatment 
difference (Treatment B, C D or E minus Treatment A), the SEs, the associated 95% CIs, and 
the p-values will be computed. 
 
12.4 
Secondary Endpoint Analysis 
All secondary efficacy endpoints will be analyzed with mixed-effects models similar to the 
primary endpoint analyses.  For endpoints that require logarithm-transformation to ensure data 
normality, the change from baseline will be calculated as the log-transformed post-baseline 
data minus the log-transformed baseline data.  These changes from baseline data will be 
examined as the dependent variable and the log of the baseline covariate will be included as a 
covariate.  The LS means, SEs, and the corresponding 95% CIs for the changes from 


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Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) will be derived from 
the ANCOVA model for each treatment.  Each of the fixed-dose combinations of leucine and 
sildenafil and leucine, sildenafil and metformin treatment groups (Treatment B, C, D and E) 
will be compared to placebo group (Treatment A), and the LS means for the treatment 
difference (Treatment B, C, D and E minus Treatment A), the SEs, and the associated 95% CIs 
and p-values will be computed.  The resulting estimates will be exponentially transformed 
back to the original scale to yield geometric LS mean ratios, SEs of the geometric mean ratios 
(calculated as geometric LS mean ratio multiplied by the SE of the LS mean of the log-
transformed data), and the 95% CIs of the geometric LS mean ratios. 
 
12.5 
Safety Analyses 
All reported terms (investigator descriptions) for AEs will be coded using the most recent 
version of the Medical Dictionary for Regulatory Activities (MedDRA).  TEAEs are defined as 
adverse events with an onset or worsening after the first randomized dose.  Number and 
percentage of subjects with TEAEs will be summarized by treatment group, system organ 
classification, and preferred term.  AEs occurring prior to receiving the first randomized dose 
(e.g., during the Screening and Pre-Treatment Periods) will not be summarized but will be 
provided in listings.   
TEAEs leading to discontinuation from the study, TEAEs possibly or probably related to study 
treatment, serious TEAEs, death, TEAEs by severity (or intensity), and TEAEs with maximum 
severity (or intensity) and causality, will be summarized.   
Additionally, TEAEs of special interest, e.g., specific gastrointestinal symptoms, will be 
summarized separately if deemed necessary. 
All hematology, clinical chemistry, and urinalysis results will be listed by treatment group, 
subject, and visit, including scheduled and unscheduled/repeat measurements.  Laboratory 
assessments that are outside of normal ranges and/or with potential clinical importance will be 
flagged.  Baseline values, the values at each visit, and changes from baseline values will be 
summarized descriptively for each of the quantitative laboratory assessments by treatment 
group.  Additionally, shift tables of hematology, clinical chemistry, and urinalysis results will 
be generated to summarize the normal and abnormal (abnormal high and abnormal low) status 
changes from baseline to each post-baseline visit. 
For vital signs, baseline values, the values at each visit, and changes from baseline values will 
be summarized by treatment group. 
ECG and physical examination data will be provided in listing. 
 
13. 
SAMPLE SIZE AND POWER CALCULATION 
A sufficient number of individuals will be screened to enroll at least 250 subjects (50/per 
treatment).  With an assumption of 20% dropout rate, approximately 200 subjects (40/per 
treatment) are expected to complete treatment through Study Termination/Visit 8 (Day 
168/Week 24).   


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Using a two-sided, two-sample comparison (t-test) of means at the alpha=0.05 level of 
significance, sample sizes of 23 subjects per treatment will provide approximately 90% 
power to detect a mean treatment difference of 3 kg body weight (~3.0-3.3% weight loss 
over 24 weeks), assuming a common standard deviation of 25%.  However, detection of 
significant changes in obesity co-morbidities require a higher sample size; detection of a 6 
mm Hg change in blood pressure will require a sample size of 40 subjects per treatment 
group at 80% power; accordingly, the sample size has been increased to 40/treatment arm to 
enable assessment of this secondary endpoint. These assumptions of effect size and standard 
deviation are based on data derived from the previous clinical trial of NS-0200 (NS-0200-01) 
and represents the anticipated treatment effect of effective fixed-dose combinations of 
leucine and sildenafil, with or without metformin, compared to placebo 
14 INVESTIGATOR AND SPONSOR OBLIGATIONS 
 
14.1 
Medical Supervision 
Medical supervision is the responsibility of the investigator named on form FDA 1572. The 
investigator may delegate day-to-day activities to a sub-investigator listed on form FDA 
1572, but retains overall responsibility for ensuring that the study is conducted properly and 
in accordance with the study protocol. A list will be maintained that includes all qualified 
persons to whom the investigator may delegate significant study related duties. The 
investigator is responsible for ensuring that drugs and devices are available for treating 
possible medical emergencies, or that emergency medical facilities are available and 
accessible. The investigator is responsible for ensuring that the study is conducted according 
to GCP, other applicable regulatory guidelines, and sound medical practices. 
 
14.2 
Study Initiation and Discontinuation 
Prior to initiation of the study, the investigator must provide the sponsor with the following 
documents (copies of which must be retained by the investigator): 
• Signed original copy of the protocol acceptance statement (Section 19) that commits 
the investigator to follow the protocol exactly and to conduct the study according to 
GCP. 
• Original copy of the investigator’s brochure acceptance page signed by the 
investigator. 
• Completed and signed original form FDA 1572. 
• Current curriculum vitae, as indicated by version date in the footer along with the 
investigator’s signature and date. Also required is a state license for the investigator 
and for other medically qualified sub-investigators. 
• Signed financial disclosure forms for the investigator and all sub-investigators listed 
on form FDA 1572. 
• Signed copy of the IRB approval letter that lists the approved items. 
• List of the IRB members who voted on the approval, including their specialty and 
affiliation, or the IRB assurance numbers if the roster cannot be obtained. Copy of 


NuSirt Biopharma 
Clinical Study Protocol NS-WM-01 
Page 41 
 
the IRB approved ICF. 
• Copy of the IRB approved authorization to use and disclose protected health 
information form, consistent with HIPAA legislation, if applicable. 
 
Upon receipt of all necessary paperwork, the sponsor will arrange for all study materials to 
be delivered to the clinical study site. The sponsor or designee will train all personnel 
expected to be involved in the study. This training may include a review of the study 
protocol, instructions for eCRF completion, and a review of overall responsibilities, 
including drug accountability and study file maintenance. 
The sponsor has the right to terminate the study at any time for any of the following reasons: 
• Non-adherence of study-site personnel to the protocol or GCP 
• Unavailability of the investigator or study-site personnel to the sponsor’s monitoring 
personnel or designee(s) 
• Other administrative reasons 
Additionally, individual subjects may be excluded from the study if a medical records review 
indicates protocol violations or if other factors appear to jeopardize the validity of the study. 
Throughout the course of the study, the investigator is to make a reasonable effort to 
maintain the enrollment rate that was agreed upon with the sponsor. The investigator will 
also make a reasonable effort to enroll appropriate subjects. The sponsor may elect to 
terminate the study at a given site if the enrollment rate lags or if significant numbers of non- 
evaluable subjects are enrolled. 
 
14.3 
Laboratory Accreditation 
A central laboratory will be  used for all samples in this study, NuSirt Biopharma will 
provide copies of the central lab accreditation, licensure and related reference ranges.  
 
14.4 
Data Reporting 
Data for each subject will be recorded on source documents and the eCRF. A source 
document and an eCRF must be completed at each study visit for every subject enrolled in 
the study. When data are complete, the investigator or medically qualified sub-investigator 
listed on form FDA 1572 will apply his/her electronic signature on the eCRF indicating that 
he/she has reviewed and approves of the data collected on eCRF.  
 
14.5 
Study Monitoring 
The investigator will allow qualified sponsor representatives or designee(s) to conduct 
periodic audits and review of all eCRFs, office, clinical, and laboratory records for each 
subject at each clinical study site. Reviews of study data will be performed during routine 
monitoring visits, both during the study and following study completion. These visits are to 
provide the sponsor with the opportunity to evaluate study progress, verify the accuracy and 
completeness of eCRF, and ensure that all protocol requirements, applicable US FDA or 


NuSirt Biopharma 
Clinical Study Protocol NS-WM-01 
Page 42 
 
country-specific regulations, and investigator obligations are being fulfilled. Finally, any 
inconsistencies in the study records should be resolved during these visits. 
The sponsor may terminate study participation by a clinical study site if study-site personnel 
do not follow the protocol or GCP. Additionally, individual subjects may be excluded if a 
medical records review indicates protocol violations or if other factors appear to jeopardize 
the validity of the study. 
 
14.6 
Record Retention 
Source documents may include a subject’s medical record, hospital charts, clinic charts, 
procedural records, the principal investigator study files, as well as the results of diagnostic 
and safety tests. 
The PI has the responsibility of maintaining a comprehensive and centralized filing system 
containing all study-related documentation in accordance with the FDA or local regulations, 
whichever are more stringent. These files must be available for inspection by the sponsor and 
regulatory authorities (i.e., FDA) at any time and must consist of at least the following 
elements: subject files, containing the completed electronic case report forms (eCRFs), 
supporting source documentation from the medical record including laboratory data and the 
ICF; regulatory files, containing the protocol with all amendments and investigator signature 
pages, copies of all other regulatory documentation, and all correspondence between the site 
and IRB and sponsor; and drug accountability files, including a complete account of the 
receipt and disposition of the test article. 
The investigator must maintain study documents: (a) for a minimum of two years following 
the date the marketing application (NDA) is approved for the indication for which the drug 
was investigated, or (b) for a minimum of two years following the release date of the final 
report, if no marketing application is to be filed by the sponsor, or if the marketing 
application is not approved for the indication of which the drug was investigated or is 
discontinued and the FDA has been notified, or (c) for a minimum of 15 years after the 
completion or discontinuation of the study to be filed in support of registration in the 
European Union. If the investigator relocates, retires for any reason or withdraws from the 
study, the study records may be transferred to an acceptable designee, such as another 
investigator, another institution, or to the sponsor. The investigator must obtain written 
permission from the sponsor before disposing any study records. 
 
14.7 
Deviation from the Protocol 
The investigator is not to deviate from the protocol. In medical emergencies, the investigator 
will use medical judgment and will remove the subject from immediate hazard. The 
investigator will immediately notify the sponsor and IRB regarding the nature of the 
emergency and the course of action taken. The investigator is to notify the sponsor of any 
inadvertent protocol deviations upon discovery, and is to document the deviations 
appropriately in the study files or on the eCRFs. The sponsor assumes no responsibility or 
liability for any unapproved deviations. 
Major changes in the protocol initiated by the sponsor will be provided as an amendment and 


NuSirt Biopharma 
Clinical Study Protocol NS-WM-01 
Page 43 
 
will be approved by the IRB prior to implementation. 
 
14.8 
Quality Assurance 
NuSirt Biopharma may conduct a discretionary quality assurance audit of this study. If such 
an audit occurs, the investigator agrees to allow the auditor direct access to all relevant 
documents and to allocate his or her time and that of the study-site personnel to the auditor to 
discuss findings and any relevant issues. In addition, regulatory agencies may conduct a 
regulatory inspection of this study. If such an inspection occurs, the investigator agrees to 
notify NuSirt Biopharma upon notification by the regulatory agency. The investigator agrees 
to allow the inspector direct access to all relevant documents and to allocate his or her time 
and that of the study-site personnel to the inspector to discuss findings and any relevant 
issues. The investigator will allow NuSirt Biopharma personnel to be present as an observer 
during a regulatory inspection, if requested. 
 
15 DISCLOSURE OF DATA AND PUBLICATIONS 
Subjects’ medical information obtained as a result of this study is considered confidential, 
and disclosure to third parties other than those noted below is prohibited. Subject to any 
applicable authorization(s), all reports and communications relating to subjects in this study 
will identify subjects only by initials and number. Medical information resulting from a 
subject’s participation in this study may be given to the subject’s personal physician, other 
authorized parties, or to the appropriate medical personnel responsible for the subject’s 
welfare. 
Data generated in this study will be available for inspection on request by the FDA or other 
government regulatory agency auditors, the sponsor, the sponsor’s medical monitor (or 
designee) and its corporate partners for the study drug, if any, and their designated 
representatives, the IRB, and other authorized parties. 
If requested, the investigator agrees to furnish the sponsor with complete subject 
identification in a confidential disclosure for long term follow-up if needed. This disclosure 
will be treated in accordance with applicable law, with strict adherence to professional 
standards of confidentiality and will be filed by the sponsor with adequate security and 
restricted accessibility. 
All information concerning the study medication and the sponsor’s operations (such as patent 
applications, formulas, manufacturing processes, basic scientific data, or other information 
supplied by the sponsor and not previously published) are considered confidential and shall 
remain the sole property of the sponsor. The investigator agrees to use this information only 
in conducting this study and to not use it for other purposes without the sponsor’s prior 
written consent. 
The information developed in this clinical study will be used by the sponsor in the clinical 
development of this program and therefore, may be disclosed by the sponsor as required, to 
authorized parties (including its corporate partners for the study drug, if any, and their 
designated representatives), other clinical investigators, pharmaceutical companies, the FDA, 
and other government agencies. 


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Clinical Study Protocol NS-WM-01 
Page 44 
 
Any information, inventions, discoveries (whether patentable or not), innovations, 
suggestions, ideas, and reports made or developed by the investigator(s) as a result of 
conducting this study shall be promptly disclosed to the sponsor and shall be the sole 
property of the sponsor. The investigator agrees, upon the sponsor’s request and at the 
sponsor’s expense, to execute such documents and to take such other actions as the sponsor 
deems necessary or appropriate to obtain patents in the sponsor’s name covering any of the 
foregoing. 
The results of this study may be published under the direction of the sponsor. Results will 
not be published without the sponsor’s prior review and approval. 
 
16 ETHICAL CONSIDERATIONS 
The study will be conducted in accordance with the Declaration of Helsinki (1964), including 
the current Seoul revision (2008), and consistent with good clinical practice and applicable 
laws and regulations. 
The protocol and ICF will be reviewed and approved by a duly constituted IRB before 
individuals are screened for study entry. The investigator will ensure that all aspects of the 
IRB review are conducted in accordance with current institutional, local, and national 
regulations. A letter documenting the IRB approval will be provided to the sponsor prior to 
initiation of the study. Amendments to the protocol will be subject to the same requirements 
as the original protocol. The investigator will submit all periodic reports and updates that the 
IRB may require, including any final closeout reports. The investigator will inform the IRB 
of any reportable adverse events. 
 
17 INFORMED CONSENT 
Each individual will be provided with oral and written information describing the nature, 
purpose and duration of the study, participation/termination conditions, and risks and 
benefits. Prior to initiation of any study-related procedures, subjects (and their parent or 
legal guardian for subjects under 18 years of age) will sign and date the ICF to participate in 
the study. Subjects will also sign and date an authorization form required under HIPAA, if 
applicable, that authorizes the use and disclosure of the subject’s protected health 
information. The signed original ICF (or child assent form as applicable) and HIPAA 
authorization forms will be retained with the study center’s records and each subject will 
receive a copy of each form they have signed. 
 
18 FINAL REPORT 
All data, including subject characteristics, methodology, and clinical findings, will be 
presented in a final clinical/statistical report to be prepared by the sponsor or designee. 


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Clinical Study Protocol NS-WM-01 
Page 45 
 
 
19 REFERENCES 
 
[1] 
Flegal KM, Kruszon-Moran D, Carroll MD, Fryar CD, Ogden CL. Trends in obesity 
among adults in the united states, 2005 to 2014. Jama 2016;315:2284–91. 
doi:10.1001/jama.2016.6458. 
[2] 
Ogden C, Carroll M, Lawman H, Fryar C, Kruszon-Moran D, Kit B, et al. Trends in 
obesity prevalence among children and adolescents in the united states, 1988-1994 
through 2013-2014. Jama 2016;315:2292–9. doi:10.1001/jama.2016.6361. 
[3] 
Fontaine KR, Redden DT, Wang C, Westfall AO, Allison DB. Years of life lost due to 
obesity. J Am Med Assoc 2003;289:187–93. doi:10.1016/S0140-6736(09)60318-4. 
[4] 
MacMahon S, Baigent C, Duffy S, Rodgers A, Tominaga S, Chambless L, et al. Body-
mass index and cause-specific mortality in 900 000 adults: Collaborative analyses of 57 
prospective studies. Lancet 2009;373:1083–96. doi:10.1016/S0140-6736(09)60318-4. 
[5] 
Masters RK, Powers DA, Link BG. Obesity and US mortality risk over the adult life 
course. Am J Epidemiol 2013;177:431–42. doi:10.1093/aje/kws325. 
[6] 
Ding M, Hu Y, Schwartz J, Koh WP, Yuan JM, Sesso HD, et al. Delineation of body 
mass index trajectory predicting lowest risk of??mortality in U.S. men using generalized 
additive mixed model. Ann Epidemiol 2016;26:698–703. 
doi:10.1016/j.annepidem.2016.08.006. 
[7] 
The Global Mortality Collaboration. Body-mass index and all-cause mortality: 
individual-participant-data meta-analysis of 239 prospective studies in four continents. 
Lancet 2016;388:776–86. doi:10.1016/S0140-6736(16)30175-1. 
[8] 
Pi-Sunyer X. The Medical Risks of Obesity. Postgrad Med 2009;121:21–33. 
doi:10.3810/pgm.2009.11.2074.The. 
[9] 
Klein S, Burke LE, Bray G a, Blair S, Allison DB, Pi-Sunyer X, et al. Clinical 
implications of obesity with specific focus on cardiovascular disease: a statement for 
professionals from the American Heart Association Council on Nutrition, Physical 
Activity, and Metabolism: endorsed by the American College of Cardiology Found. 
Circulation 2004;110:2952–67. doi:10.1161/01.CIR.0000145546.97738.1E. 
[10] Wing R, Lang W, Wadden T, Safford M, Knowler W, Bertoni A, et al. Benefits of 
Modest Weight Loss in Improving Cardiovascular Risk Factors in Overweight and Obese 
Individuals With Type 2 Diabetes. Diabetes Care 2011;34:1481–6. doi:10.2337/dc10-
2415. 
[11] Chang H-C, Guarente L. SIRT1 and other sirtuins in metabolism. Trends Endocrinol 
Metab 2014;25:138–45. doi:10.1016/j.tem.2013.12.001. 
[12] Ruderman NB, Xu XJ, Nelson L, Cacicedo JM, Saha AK, Lan F, et al. AMPK and 
SIRT1: a long-standing partnership? Am J Physiol Endocrinol Metab 2010;298:E751-60. 
doi:10.1152/ajpendo.00745.2009. 
[13] Pfluger PT, Herranz D, Velasco-miguel S, Serrano M, Tscho MH. Sirt1 protects against 
high-fat diet-induced. PNAS 2008;105:9793–8. 
[14] Zhang W, Zhang X, Wang H, Guo X, Li H, Wang Y, et al. AMP-Activated Protein 
Kinase  1 Protects Against Diet-Induced Insulin Resistance and Obesity. Diabetes 
2012;61:3114–25. doi:10.2337/db11-1373. 
[15] Liu Y, Wan Q, Guan Q, Gao L, Zhao J. High-fat diet feeding impairs both the expression 
and activity of AMPKa in rats’ skeletal muscle. Biochem Biophys Res Commun 
2006;339:701–7. doi:10.1016/j.bbrc.2005.11.068. 
 
 


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[16] Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin resistance, and the 
metabolic syndrome. J Clin Invest 2013;123:2764–72. doi:10.1172/JCI67227. 
[17] Banerjee J, Bruckbauer A, Zemel MB. Activation of the AMPK/Sirt1 pathway by a 
leucine–metformin combination increases insulin sensitivity in skeletal muscle, and 
stimulates glucose and lipid metabolism and increases life span in Caenorhabditis 
elegans. Metabolism 2016:1–13. doi:10.1016/j.metabol.2016.06.011. 
[18] Bruckbauer A, Zemel MB. Synergistic effects of polyphenols and methylxanthines with 
Leucine on AMPK/Sirtuin-mediated metabolism in muscle cells and adipocytes. PLoS 
One 2014;9:e89166. doi:10.1371/journal.pone.0089166. 
[19] Bruckbauer A, Banerjee J, Fu L, Li F, Cao Q, Cui X, et al. A Combination of Leucine , 
Metformin , and Sildenafil Treats Nonalcoholic Fatty Liver Disease and Steatohepatitis in 
Mice. Int J Hepatol 2016;2016:16 pages. doi:10.1155/2016/9185987. 
[20] Fu L, Bruckbauer A, Li F, Cao Q, Cui X, Wu R, et al. Interaction between Metformin and 
Leucine in Reducing Hyperlipidemia and Hepatic Lipid accumulation in Diet-Induced 
Obese Mice. Metabolism 2015. doi:10.1016/j.metabol.2015.07.006. 
 
 
 


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20 INVESTIGATOR’S ACCEPTANCE 
 
 
Study Title: 
A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED 
STUDY TO EVALUATE THE EFFECT OF TWO FIXED-DOSE 
LEUCINE AND SILDENAFIL COMBINATIONS (NS-0300) OR TWO 
FIXED-DOSE LEUCINE, SILDENAFIL AND METFORMIN 
COMBINATIONS (NS-0200) VERSUS PLACEBO ON BODY 
WEIGHT IN OBESITY 
 
Final Date: 
December 4, 2017 
 
 
I have read the protocol described above. I agree to comply with all applicable regulations 
and to conduct the study as described in the protocol. I agree to its terms and will conduct 
the study according to Good Clinical Practices. 
 
 
 
 
 
 
Investigator Name (Printed) 
 
 
 
 
 
 
 
Investigator Signature  
 
 
 
Date


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Clinical Study Protocol NS-WM-01 
Page 48 
 
 
Appendix 1: Study Plan (Protocol NS-WM-01) 
 
 
 
 
 
Screening 
24-Week Treatment Period 
STUDY 
PROCEDURES 
Visit 1 
(Screen) 
Visit 2 
(Baseline) 
Phone 
Contact 
Visit 3 
Phone 
Contact 
Visit 4 
Phone 
Contact 
Visit 5 
Phone 
Contact 
Visit 
6 
Phone 
Contact 
Visit 7 
Phone 
Contact 
Visit 8 
(Study 
Termination) 
Day-7 
(± 3 day) 
Day 1 
 
Day 14 
(± 3 day) 
Day 28 
(± 3 day) 
Day 42 
(± 3 day) 
Day 56 
(± 3 day) 
Day 70 
(± 3 day) 
Day 84 
(± 3 day) 
Day 98 
(± 3 day) 
   Day 112 
(± 3 day) 
Day 126 
(± 3 day) 
Day 140 
(± 3 day) 
Day 154 
(± 3 day) 
Day 168 
(± 3 day) 
Informed Consent 
And HIPAA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
Adverse Event 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medication Review 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Vital Signs 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Waist Circumference 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
EKG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
β-hCG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chemistry and 
Hematology 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Blood Lipids 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
hsC-reactive protein 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
HbA1c 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Biomarkers 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Drug and Alcohol 
Screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
First Dose Of Study 
Medication 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Return Unused Study 
Medication 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Assess Medication 
Compliance 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Dispense Study 
Medication 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
Maximum study duration is 28 weeks 
All visits must be fasting 

SAP_001.pdf: 




                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
3 
 
TABLE OF CONTENTS 
1
INTRODUCTION ......................................................................................................................... 5
2
STUDY OBJECTIVES ................................................................................................................. 5
2.1
Primary Objectives ............................................................................................................... 5
2.2
Secondary Objectives ............................................................................................................ 5
3
INVESTIGATIONAL PLAN ....................................................................................................... 6
3.1
Study Design ........................................................................................................................ 6
3.2
Study Procedures and Visit Structure..................................................................................... 7
3.3
Randomization Schedule and Blinding Procedures .............................................................. 10
3.4
Dose Administration ........................................................................................................... 10
3.5
Study Population................................................................................................................. 11
4
STUDY ENDPOINTS ................................................................................................................. 11
4.1
Primary Study Endpoints .................................................................................................... 11
4.2
Secondary Study Endpoints ................................................................................................. 11
4.3
Safety and Tolerability Endpoints........................................................................................ 12
4.4
Exploratory Endpoint .......................................................................................................... 12
5
ANALYSIS POPULATIONS ..................................................................................................... 13
5.1
Enrolled Population ............................................................................................................ 13
5.2
Randomized Population ...................................................................................................... 13
5.3
Intent-to-Treat (ITT) Population .......................................................................................... 13
5.4
Per-Protocol (PP) Population............................................................................................... 13
6
INTERIM ANALYSIS ................................................................................................................ 14
7
STUDY SUBJECTS .................................................................................................................... 14
7.1
Subject Disposition ............................................................................................................. 14
7.2
Demographic and Baseline Characteristics .......................................................................... 14
7.3
Medical History .................................................................................................................. 15
7.4
Inclusion/Exclusion Criteria ................................................................................................ 15
7.5
Treatment Compliance ........................................................................................................ 15
7.6
Duration of Exposure .......................................................................................................... 16
7.7
Protocol Deviations............................................................................................................. 16
7.8
Concomitant Medications ................................................................................................... 17
8
STATISTICAL METHODS OF ANALYSIS ............................................................................. 17
8.1
General Considerations ....................................................................................................... 17
8.1.1
Statistical Notation and Presentation ...................................................................... 18
8.1.2
Hypothesis Testing Significance Level .................................................................. 18
8.1.3
Software ............................................................................................................... 19
8.1.4
Definition of Study Baseline .................................................................................. 19
8.1.5
Handling of Multiple Observations ........................................................................ 19
8.1.6
Handling of Missing Data ...................................................................................... 19
8.2
Derivation of Change in Endpoint Parameters ..................................................................... 20
8.2.1
Calculation of Change from Baseline ..................................................................... 20
8.2.2
Calculation of the Percent Change ......................................................................... 21
8.3
Analysis of Baseline Differences ......................................................................................... 21
8.4
Efficacy Analyses ............................................................................................................... 21
8.4.1
Primary Endpoint Analyses ................................................................................... 21
8.4.2
Secondary Endpoint Analyses ............................................................................... 22
8.4.3
Exploratory Analysis ............................................................................................. 23
8.5
Safety Analyses .................................................................................................................. 24
8.5.1
Adverse Events ..................................................................................................... 24
8.5.2
Clinical Laboratory Evaluations............................................................................. 25


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
4 
 
8.5.3
Vital Signs ............................................................................................................ 26
8.5.4
Electrocardiograms ............................................................................................... 26
8.5.5
Physical Examination ............................................................................................ 26
9
SAMPLE SIZE AND POWER ................................................................................................... 26
10
REFERENCES ........................................................................................................................... 27
11
PRELIMINARY TABLE OF CONTENTS................................................................................ 29
12
ADDENDUM .............................................................................................................................. 34
12.1 Rationale ............................................................................................................................ 34
12.2 Incorrect Weight Measurement ........................................................................................... 34
12.3 Antibiotic Use..................................................................................................................... 35
 
LIST OF TABLES 
Table 1:   Treatment Arms (Protocol NS-WM-01)...................................................................................... 6
Table 2:   Study Plan for Protocol NS-WM-01 ........................................................................................... 8
 
LIST OF FIGURES 
Figure 1:   Study Design for Protocol NS-WM-01 ...................................................................................... 7
 
 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
5 
 
1
INTRODUCTION 
The purpose of this Statistical Analysis Plan (SAP) is to describe the statistical 
methodology to be used in analyzing data in this Phase II, Randomized, Double-Blind, 
Placebo-Controlled Study to evaluate the effect of two fixed-dose leucine and sildenafil 
combinations (NS-0300) or two fixed-dose leucine, sildenafil and metformin 
combinations (NS-0200) versus placebo on body weight in obesity. The SAP outlines the 
data derivations and statistical programming specifications for the tables, figures and 
listings.  
The analyses described in this SAP are based on the Clinical Study Protocol (CSP) NS-
WM-01 Amendment #3 dated 04 December 2017, and describes the variables and 
populations, anticipated data transformations and other details of the analyses not 
provided in the CSP. 
The SAP will be finalized prior to database lock and describes the statistical analysis as 
anticipated. If circumstances should arise during the study rendering this analysis 
inappropriate, or if in the meantime improved methods of analysis should come to light, 
different analyses may be made. Any protocol amendment or significant change between 
the planned analysis and this SAP that occurs prior to database lock will be addressed in a 
SAP amendment. If any significant deviation occurs between the planned analysis and 
this SAP after database lock, the reasons for such deviations and all alternative or 
additional statistical analyses to be performed will be described in a SAP Addendum. 
2
STUDY OBJECTIVES 
2.1
Primary Objectives 
The primary objective of the study is to evaluate the percentage body weight change in 
subjects from Baseline/Visit 2 (Day1) to Study Termination/Visit 8 (Day 168/Week 24) 
receiving two fixed-dose combinations of leucine and sildenafil or two fixed-dose 
combinations of leucine, sildenafil and metformin compared to placebo. 
2.2
Secondary Objectives 
The secondary objectives of the study are to evaluate the effects of receiving two fixed-
dose combinations of leucine and sildenafil or two fixed-dose combinations of leucine, 
sildenafil and metformin compared to placebo in obese patients on the following 
parameters: 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
6 
 
• Absolute body weight from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
• Percentage of patients with ≥5% body weight loss from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
• Change in waist circumference from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in blood lipids (cholesterol, calculated LDL, HDL, triglycerides) from 
Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
• Change in fasting glucose from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• Change in HbA1c from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
• Change in blood pressure (diastolic and systolic) from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) 
• Change in hsCRP from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 
168/Week 24) 
• Safety and tolerability 
• Exploratory endpoint of percentage of patients with ≥10% body weight loss from 
Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
3
INVESTIGATIONAL PLAN 
3.1
Study Design 
This is a randomized, 24-week, placebo-controlled, double-blind study to evaluate the 
effects of two fixed-dose combinations of leucine and sildenafil or two fixed-dose 
combinations of leucine, sildenafil and metformin compared to placebo.  
Subjects meeting all inclusion criteria with no exclusion criteria will be randomized to 
one of the five treatment arms in 1:1:1:1:1 ratio (Table 1). 
Table 1:   Treatment Arms (Protocol NS-WM-01) 
Treatment 
Arm 
Subjects (N) 
Randomized 
Dosing 
Regimen 
Study Medication Dosed Twice/Day1 
Leucine 
Sildenafil 
Metformin 
A 
50 
Placebo 
N/A 
N/A 
N/A 
B 
50 
FDC 
1100 mg 
1 mg 
N/A 
C 
50 
FDC 
1100 mg 
4 mg 
N/A 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
7 
 
D 
50 
FDC 
1100 mg 
1 mg 
500mg 
E 
50 
FDC 
1100 mg 
4 mg 
500mg 
FDC = Fixed Dose Combination 
*Note: The total daily dose is twice that listed for each compound 
The study design is depicted in Figure 1.  The study includes a total of 2 periods: 
Screening (including a 7-day period for lab results) and a 24-week treatment period (Day 
1 to Day 168) with the first dose of study medication being administered on Day 1. 
Figure 1:   Study Design for Protocol NS-WM-01 

3.2
Study Procedures and Visit Structure 
This study includes a total of 8 visits to the clinical study site as follows: Screening/Visit 
1 (Day -7/Week -1), Baseline/Visit 2 (Day 1), Visit 3 (Day 28/ Week 4), Visit 4 (Day 
56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 112/Week 16), Visit 7 (Day 
140/Week 20) and Study Termination/Visit 8 (Day 168/Week 24). In addition, subjects 
will receive six telephone contacts on Day 14/Week 2, Day 42/Week 6, Day 70/Week 10, 
Day 98/Week 14, Day 126/ Week 18 and Day 154/Week 22. 
For all subjects, Visit 1 should be Day -7 ± 3 days relative to Baseline/Visit 2 (Day 1). 
During the 24-week treatment period, the visit window will not exceed ± 3 days for Visit 3 
(Day 28/ Week 4), Visit 4 (Day 56/Week 8), Visit 5 (Day 84/Week 12), Visit 6 (Day 
112/Week 16), Visit 7 (Day 140/Week 20) and Study Termination/Visit 8 (Day 
168/Week 24). All study visits should occur within a visit window of ±3 days. Total 
study duration should not exceed a maximum of 28 weeks (or 196 days). 
The following study plan (Table 2) provides an overview of the clinical schedule of 
assessments and events for the study.
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NS-WM-01 Statistical Analysis Plan 
 
8 
 
Table 2:   Study Plan for Protocol NS-WM-01 
 
 
 
 
Screening 
24-Week Treatment Period 
STUDY 
PROCEDURES 
Visit 1 
(Screen) 
Visit 2 
(Baseline) 
Phone 
Contact 
Visit 3 
Phone 
Contact 
Visit 4 
Phone 
Contact 
Visit 5 
Phone 
Contact 
Visit 
6 
Phone 
Contact 
Visit 7 
Phone 
Contact 
Visit 8 
(Study 
Day-7 
( 3 d
)
Day 1 
Day 14 
( 3 d
)
Day 28 
( 3 d
)
Day 42 
( 3 d
)
Day 56 
( 3 d
)
Day 70 
( 3 d
)
Day 84 
( 3 d
)
Day 98 
( 3 d
)
    Day 112 
( 3 d
)
Day 126 
( 3 d
)
Day 140 
( 3 d
)
Day 154 
( 3 d
)
Day 168 
( 3 d
)
Informed Consent 
and HIPAA 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Adverse Event 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant 
Medication Review 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Vital Signs 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Height 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Weight 
X 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Waist Circumference 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
EKG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X 
-hCG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Chemistry and 
Hematology 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Blood Lipids 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
hsC-reactive protein 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
HbA1c 
X 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 
Biomarkers 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
X 


                                                                                                                                                                           
 
NS-WM-01 Statistical Analysis Plan 
 
9 
 
Drug and Alcohol 
Screen 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
First Dose Of Study 
Medication 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Return Unused Study 
Medication 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
Assess Medication 
Compliance 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Dispense Study 
Medication 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
 
 
 
    Maximum study duration is 28 weeks 
    All visits must be fasting 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
10 

3.3
Randomization Schedule and Blinding Procedures 
A randomization schedule was provided by Bioclinica’s Biostatistician. The 
randomization schedule was generated utilizing the Bioclinica’s Trident® software and is 
not to be shared with anyone directly involved in the study conduct. Subjects who meet all 
study requirements based on inclusion and exclusion criteria are to be assigned to one of the 
five treatment groups on Baseline/Visit 2 (Day 1) (Table 1) based on the randomization 
schedule. The randomization numbers are allocated in strict chronological order. The 
number uniquely identifies the subject and dictate the assigned, blinded treatment group. 
Each randomized subject received a 4-digit randomization number (i.e., 1001, 1002, etc.). 
Randomized subjects who withdraw or are withdrawn from clinical study participation 
for any reason, regardless of whether any treatment was administered or not, retain their 
randomization number and the subject is not allowed to re-enter the study. Replacement 
subjects are assigned a randomization number as the subject they are replacing plus 1000, 
i.e., a subject replacing Subject 1001, will be assigned the subject number 2001. The 
replacement is assigned to the same treatment sequence as the subject being replaced  
regardless of where the previous subject withdrew. 
No stratification factors are planned in this study for randomization or analysis purposes. 
No cohorts or subgroups are planned in this study for randomization or analysis purposes. 
The sponsor personnel involved in study conduct, study staff and subject are to be blinded 
to the identity of study medication during the study. The study medication is provided to 
the study centers in a blinded fashion. The study center pharmacist uses the subject’s 
assigned randomization number to ensure that the appropriate blinded study medication is 
allocated at each visit. The study centers are provided with sufficient study medication to 
supply all randomized subjects.    
3.4
Dose Administration 
Study medication will be self-administered by the patient as three tablets taken orally 
BID within 30 minutes prior to the morning and evening meals. Each dose of study 
medication will consist of three tablets, which will be identical in appearance across all 
treatment groups.  
Fixed Dose Combination Arms: Each dose of study medication consists of three 
tablets - two tablets containing either 550 mg L-leucine alone or 550 mg L-leucine in 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
11 

combination with 250 mg of Metformin and one tablet either 1.0 mg or 4.0 mg of 
sildenafil. 
Placebo Arm: Each dose of placebo consists of three tablets, identical in appearance 
to those used in the active treatment arms – two tablets of leucine alone or leucine + 
metformin matched placebo and one tablet of sildenafil matched placebo. 
3.5
Study Population 
A sufficient number of obese subjects will be exposed to study screening 7 days (± 3 
days) before the planned start of study dosing. A complete list of all eligibility 
criteria is available in section 4.2 and 4.3 of the study protocol. The study plans to 
enroll approximately 250 subjects with obesity, with a maximum of 70% female, 
with a goal of having at least 200 subjects completing all assessments over the 24-
week (up to 28-week) assessment period from multiple study sites in the US. The 
definition of obesity used for this study is a Body Mass Index (BMI) ≥ 30 kg/m2. 
Individuals with a BMI > 45 kg/m2 will not be included in this study.  
4
STUDY ENDPOINTS 
4.1
Primary Study Endpoints 
To evaluate the percentage body weight change in subjects from Baseline/Visit 2 (Day1) 
to Study Termination/Visit 8 (Day 168/Week 24) receiving two fixed-dose combinations 
of leucine and sildenafil or two fixed-dose combinations of leucine, sildenafil and 
metformin compared to placebo. 
4.2
Secondary Study Endpoints 
The secondary endpoints of the study are to evaluate the effects of receiving two fixed-
dose combinations of leucine and sildenafil or two fixed-dose combinations of leucine, 
sildenafil and metformin compared to placebo in obese patients on the following 
parameters: 
• The change in absolute body weight from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24)  
• The percentage of patients with ≥5% body weight loss from Baseline/Visit 
2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
• The change in waist circumference from Baseline/Visit 2 (Day 1) to Study 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
12 

Termination/Visit 8 (Day 168/Week 24) 
• The change in blood lipids (cholesterol, calculated LDL, HDL, triglycerides) from 
Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
• The change in fasting glucose from Baseline/Visit 2 (Day 1) to Study 
Termination/Visit 8 (Day 168/Week 24) 
• The change in HbA1c from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
• The change in blood pressure (diastolic and systolic) from Baseline/Visit 2 (Day 1) 
to Study Termination/Visit 8 (Day 168/Week 24) 
• The change in hsCRP from Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 
(Day 168/Week 24) 
4.3
Safety and Tolerability Endpoints 
Safety and tolerability will be assessed by evaluating the following endpoints: 
• Extent of exposure 
• Incidence of adverse events 
• Adverse events reported by ≥ 5% of patients 
• Incidence of deaths and serious adverse events 
• Laboratory safety tests (hematology, biochemistry, and urinalysis) 
• Vital signs 
• ECG 
• Physical examination, including weight 
• Concomitant medication 
4.4
Exploratory Endpoints 
• The percentage of patients with ≥10% body weight loss from Baseline/Visit 2 
(Day 1) to Study Termination/Visit 8 (Day 168/Week 24) 
In addition, exploratory analysis of comorbidities independent of weight loss i.e. to 
determine if the comorbidities change occurs independent of the weight loss is also 
planned.  
The comorbidities that will be analyzed are: 
• Hypertension i.e. Systolic Blood Pressure (SBP) and Diastolic Blood Pressure 
(DBP) 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
13 

• Hyperlipidemia i.e. total cholesterol, HDL cholesterol, LDL cholesterol, and 
triglycerides 
• Diabetes Mellitus i.e. fasting blood sugar and HbA1c 
• Cardiovascular Diseases i.e. hsCRP  

5
ANALYSIS POPULATIONS 
All safety analyses will be performed by the actual treatment received.  
 
5.1
Enrolled Population 
The Enrolled Population will consist of all subjects who signed the informed consent to 
participate in the study, and had Screening visit electronic case report form (eCRF) 
collected.  Screen failure subjects will be excluded from the Enrolled Population. 
5.2
Randomized Population 
The Randomized Population will consist of all Enrolled subjects who were randomized at 
Baseline/Visit 2 (Day 1) to any of the five study treatments according to the study 
randomization schedule regardless of whether they receive study drug.  
5.3
Intent-to-Treat (ITT) Population 
The ITT Population will include all randomized subjects who receive at least one dose of 
study medication and have at least one follow-up primary efficacy assessment done. It 
will be assumed all patients who took one dose of study drug will have been assessed 
for one or more components of efficacy during follow-up, which begins immediately 
at administration of the first dose. To be included in the ITT population, the date of 
first dose of study drug must be entered in the eCRF data. 
5.4
Per-Protocol (PP) Population 
The PP Population will include all ITT subjects who had adequate exposure to the 
randomized study medication during the 24-week period, completed Visit 8 (Day 168/Week 
24) and have adequately complied with the protocol as assessed by the investigator and 
sponsor prior to database lock. Specifically, subjects should have completed the Visit 8 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
14 

protocol procedure with valid measurements at Visit 8 (i.e. Week 24 up to Week 28) 
without major protocol deviations, and have received more than 80% of planned study 
medication during the treatment period.   
PP Population will be the primary analysis population. A memo-to-file will be created if 
it is necessary to change this selection based on blinded data review on treatment 
compliance, availability of Visit 8 measurements, and protocol deviation data prior to 
database lock. 
The primary and secondary efficacy endpoints will also be analyzed using both the ITT 
as well as the PP populations. The safety endpoints will be analyzed using the ITT 
population. 
6
INTERIM ANALYSIS 
There is no formal interim analysis planned for this study. 
7
STUDY SUBJECTS 
All data from the Enrolled Population will be provided in listings. Summary tables will 
be provided for treatment arms A to E. 
7.1
Subject Disposition 
The completion status, date of completion or discontinuation, the reason for 
discontinuation will be listed for the Enrolled population. Also, the record of informed 
consent and subject status at end of screening will be listed for the Enrolled population.  
The subject disposition summary table of analysis populations for all enrolled patients 
will be summarized by treatment group and overall.  
The number of subjects randomized and the frequency and percentage of subjects 
completing the study, subjects withdrawing early, and primary reason for withdrawal will 
be summarized by treatment group and overall.  
7.2
Demographic and Baseline Characteristics 
The demographics and baseline characteristics summary will be presented in tables by 
treatment group and overall. Descriptive summary statistics will be calculated for 
continuous variables including age (years) at entry, height (cm), baseline weight (kg) and 
body mass index (BMI) (kg/m2), HbA1c, and fasting plasma glucose at screening. This 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
15 

summarization will be presented by treatment group and overall for ITT Population and 
the PP Population.  
Frequency counts will be tabulated for categorical variables including gender, race, 
ethnicity and childbearing potential. This summarization will be presented by treatment 
group and overall for the ITT Population and the PP Population.   
All ages will be calculated in Express from date of birth (DOB) in years. Baseline BMI 
will be derived as follows using baseline weight and height: 
BMI (Kg/m2) = weight (kg) at baseline / (height (m))2 
All patient demographic and baseline data will be presented in a listing for the Enrolled 
Population. 
7.3
Medical History 
Medical history will be captured at the screening visit and coded using version 19.0 of the 
Medical Dictionary for Regulatory Activities (MedDRA®). Medical history will be 
summarized by system organ class and preferred term. The information of onset and end 
date and time, or ongoing, will also be collected. All Medical History data will be 
presented in a listing for Enrolled Population. 
Medical history will be summarized in a table with descriptive statistics (frequencies and 
percentages) by medical history code and treatment group for the ITT Population.  
7.4
Inclusion/Exclusion Criteria 
The inclusion and exclusion criteria are defined in sections 4.2 and 4.3 of the CSP. All 
enrolled subjects who did not meet an inclusion criterion and all enrolled subjects who 
met an exclusion criterion will be presented in a listing.  
Inclusion and exclusion criteria failures in each study will be summarized separately 
(frequencies and percentages) for the ITT Population in a table. 
7.5
Treatment Compliance 
The compliance ratio for each study medication will be calculated as the total number of 
received doses of that medication within the 24-week treatment period divided by the 
total number of required doses of the same medication in the same period. 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
16 

Treatment compliance percentage for a subject will be defined as: 
Treatment Compliance Percentage (%) = Treatment Compliance Ratio x 100 
Treatment compliance will be summarized by treatment group for the ITT Population. 
7.6
Duration of Exposure 
Duration of exposure is defined as the total number of days a patient is exposed to study 
medication. Duration of exposure is calculated as the total number of days from the first 
dose date (Day 1) to the last dose date as recorded on the eCRF (last dose date minus first 
dose date, plus 1). Duration of exposure is therefore the duration of the interval during 
which the patient was exposed, but not necessarily the number of days on which a patient 
was exposed if the patient missed dosing on at least one day. If the last day of treatment 
is missing but Visit 8 occurred, Visit 8 will be used for the last day of treatment for 
purposes of calculating duration of exposure.  
Duration of exposurewill be summarized by treatment group for the ITT Population. 
7.7
Protocol Deviations 
A protocol violation is a deviation from the study protocol that may affect the patient’s 
rights, safety, or well-being and/or the completeness, accuracy and reliability of the study 
data. Protocol deviations will be collected and/or computed throughout the study. The 
deviations include, but are not limited to, patients who: 
• Do not meet important inclusion/exclusion criteria 
• Incorrect drug dispensed (dispensed different study medication than randomized) 
and administered 
• Have substantial use of any disallowed medication 
• Have study treatment compliance < 80% for the assigned treatment  
• Have early (before -3 days) or late (after +3 days) visits, inadequate/missed 
efficacy or safety assessment 
All protocol deviations will be recorded by the Investigator and will be listed by subject 
for the Enrolled Population. 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
17 

Protocol deviations based on the Inclusion/Exclusion criteria at time of enrollment will be 
summarized by treatment group for the ITT Population. Other protocol deviations 
occurring during the trial conduct will also be summarized by treatment group for the ITT 
Population.   
The protocol deviations will be reviewed by clinical and statistical personnel prior to 
database lock and unblinding, and will be used to determine the subject’s eligibility for 
inclusion in the PP Population. 
7.8
Concomitant Medications 
The WHO DRUG Dictionary will be used to categorize verbatim descriptions of 
medications into the Anatomic Therapeutic Chemistry (ATC) classification system. Each 
verbatim name will be classified by anatomical main group (ATC level 1), therapeutic 
subgroup (ATC level 2), pharmacological subgroup (ATC level 3), chemical subgroup 
(ATC level 4), and trade name. 
The number and percentage of subjects receiving concomitant and new concomitant 
medications will be summarized by treatment group and ATC classification (ATC level 2 
and level 4) for the ITT Population. Concomitant medications include prior concomitant 
and new concomitant medications, in which the prior concomitant medications refer to 
the medications that have a start date prior to receiving the first randomized dose and 
continue to be taken on or after that, and the new concomitant medications refer to the 
medications that have been used on or after receiving the first randomized dose.  
Dosages for certain concomitant medications should be maintained constant during the 
study (as mentioned in Section 4.2 and 4.3 of the CSP) unless instructed otherwise by the 
investigator or a treating physician. Any change in regimen for any concomitant 
medication, including restricted concomitant medications, must be reported to the 
sponsor and recorded in the eCRF and will be captured in prior and concomitant 
medication listings. 
8
STATISTICAL METHODS OF ANALYSIS 
8.1
General Considerations 
In general, efficacy analyses will be performed with the PP and ITT Populations. Safety 
analyses will be performed with the ITT Population. Safety analyses will be performed 
by the actual treatment received.  


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
18 

All data collected during the study for Enrolled Population will be included in data 
listings.  
Only those evaluations that were measured after randomization and events that occur or 
have onset after randomization will be used in analyses of all efficacy and safety 
endpoints. 
8.1.1
Statistical Notation and Presentation 
For descriptive statistical summaries, the mean, sample size (n), standard deviation (SD), 
standard error (SE), median, minimum (min), and maximum (max) will be calculated for 
continuous variables. For logarithm-transformed data, the geometric mean and standard 
error of the geometric mean will also be provided.  
For categorical variables, frequency and percentage in each category will be provided. 
When counts data are presented, the percent will be suppressed when the count is zero to 
draw attention to the non-zero counts. The denominator for all percentages will be the 
number of patients in each treatment group with data unless otherwise specified. Data 
will be summarized for baseline, endpoint and by visit (where applicable). 
Min and max values will be rounded to the precision of the original value. Means, least 
squares (LS) means, and medians will be rounded to 1 decimal place greater than the 
precision of the original value. SDs, SEs, and 95% confidence intervals (CIs) will be 
rounded to 2 decimal places greater than the precision of the original value. Percentages 
for summarizing categorical data will be rounded to one decimal place. 
P-values will be presented with 4 decimal places and values less than 0.0001 will be 
presented as <0.0001. 
All dates will be displayed in DDMMMYYYY format. Visits will be referred to as 
shown in the protocol: “Screening (Visit 1)”, “Baseline (Visit 2)”, “Visit 3”, “Visit 4”, 
“Visit 5”, “Visit 6”, “Visit 7” and “Visit 8”. 
8.1.2
Hypothesis Testing Significance Level 
All inferential statistical testing will be two-sided and conducted at the 0.05 significance 
level. For confidence intervals (CIs) a level of confidence of 95% will be used. No 
adjustment of type I error for multiple comparisons is planned for this early phase study. 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
19 

8.1.3
Software 
All statistical analyses will be performed using Statistical Analysis Software (SAS®) 
(SAS® Institute Inc., Cary, North Carolina, United States of America [USA]) Version 9.2 
or higher. Graphics may be generated using other software packages. 
8.1.4
Definition of Study Baseline 
Baseline is defined as the last assessment with non-missing values prior to the first dose 
of study medication. All baseline measurements must have been collected prior to 
administration of the first dose of study medication. Measurements that are obtained after 
the first dose of study medication will be considered as post-baseline values. If a given 
variable is not measured for a given patient prior to the first dose of study medication, 
then that patient will be considered not to have a baseline value for that variable. If no 
baseline values exist for lab measurements, then abnormalities that occur post-baseline 
will be considered as new abnormalities.  
8.1.5
Handling of Multiple Observations 
A patient may have multiple scheduled or unscheduled visits that are associated with a 
protocol defined visit (nominal visit). Each unscheduled visit is assigned a nominal 
scheduled visit number in the clinical database; therefore, no additional computer 
programming will be done to attribute the unscheduled visit following specific visit 
windows. Similarly, there will be no additional computer programming to derive/modify 
the scheduled visit number even if such a visit falls outside of the protocol-defined visit 
window. 
All values, scheduled or unscheduled, will be presented in data listings. However, 
multiple observations will be pre-processed before summarizing or analyzing the data. If 
an unscheduled visit occurs in a visit window with an existing scheduled visit, the 
assessment at the scheduled visit will be used for data summary and analysis. If no 
scheduled visit assessment exists for a visit window but at least one unscheduled visit 
assessment is available within that visit window, then the latest unscheduled visit within 
the visit window will be used for data summary and analysis.     
8.1.6
Handling of Missing Data 
Subjects who discontinue from the study prior to completing all study procedures through 
Study Termination (Day 168/Visit 8), but have data collected for at least one post-
baseline visit, will have their missing values imputed using the values at the last 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
20 

scheduled or unscheduled visit in accordance with the Last Observation Carry Forward 
(LOCF) approach. This LOCF value will be presented as the value of Endpoint Visit in 
descriptive statistical summary table. Values at baseline will not be carried forward.    
Missing or partial start dates for adverse events (AEs) will be imputed with the following 
algorithms. Note that for AE start date, the imputation rule is to conservatively capture 
AEs with missing start dates as treatment-emergent adverse events (TEAEs). 
• If “day” is the only missing field, impute the “day” as the later one between the 
first day of the month and the first dose date if their “month” are the same.  
• If “day” and “month” are the only missing fields, impute the “day” and “month” 
as the later one between January 1 of the year and the first dose date if their 
“year” are the same. 
• If “day”, “month”, and “year” are all missing, to be conservative, the event will be 
assumed to occur on the same day as the first dose was administered.   
Missing or partial start dates for concomitant medications data will also be imputed using 
the above algorithms. Missing or partial stop dates for concomitant medications data will 
be imputed using the following algorithms. 
• If “day” is the only missing field, impute the “day” as the last day of the month. 
• If “day” and “month” are the only missing fields, impute the “day” and “month” 
as the last day of the year. 
• If “day”, “month”, and “year” are all missing, leave as missing. 
Date imputation will only be used for computational purposes; e.g., treatment-emergent 
status.  Actual data values as they appear in the original CRFs will be shown in the data 
listings. 
8.2
Derivation of Change in Endpoint Parameters 
8.2.1
Calculation of Change from Baseline 
The change from baseline is defined as post-baseline assessment minus baseline 
assessment. For example, the absolute change in body weight from Baseline/Visit 2 (Day 
1) to Study Termination/Visit 8 (Day 168/Week 24) is defined as the body weight at 
Study Termination/Visit 8 (Day 168/Week 24) minus the body weight at Baseline/Visit 2 
(Day 1). 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
21 

8.2.2
Calculation of the Percent Change 
The percentage change from baseline is defined as post-baseline assessment minus 
baseline assessment, divided by the baseline assessment, and multiplied by 100: 
		 7 8
$%&%"!#%"!#
%"!#
9  . 
The primary endpoint of the percent change from Baseline/Visit 2 (Day 1) in body weight 
is defined as the body weight at Study Termination/Visit 8 (Day 168/Week 24) minus the 
body weight at Baseline/Visit 2 (Day 1), divided by the Baseline/Visit 2 (Day 1) body 
weight value, and multiplied by 100: 
		
 7 8
! &(45+6),2/-0*4-10! &'*3,.-0,
! &'*3,.-0,
9  . 
8.3
Analysis of Baseline Differences 
All primary and secondary endpoints will be analyzed for any baseline differences among 
treatment groups for PP and ITT Populations. All continuous outcomes will be analyzed 
using an analysis of variance (ANOVA) model and Dunnett’s Test will be used to 
compare each of the active Treatments B, C, D and E with Treatment A (placebo). For all 
Categorical outcomes, Fisher’s Exact Test will be used to compare Treatments B, C, D 
and E with Treatment A. 
8.4
Efficacy Analyses 
8.4.1
Primary Endpoint Analyses 
The primary efficacy endpoint is the percent change from Baseline/Visit 2 (Day 1) to 
Study Termination/Visit 8 (Day 168/Week 24) in body weight. 
Descriptive statistics (n, mean, SD, SE, median, min, and max) will be used to summarize 
values of the primary efficacy endpoint, change in body weight from Baseline/Visit 2 
(Day 1) to Study Termination/Visit 8 (Day 168/Week 24) and intermediate study visits as 
applicable by treatment group. 
A mixed-model of analysis of covariance (ANCOVA) will be used to analyze percentage 
change in body weight from Baseline to Week 24 in PP and ITT Populations. The 
ANCOVA will include treatment group and time as the main effects with baseline weight 
as the covariate. The LS means, SEs, and the corresponding 95% CIs for the changes 
from baseline to Week 24 will be derived from the model for each treatment. Each of the 
fixed-dose combinations of leucine, sildenafil, and/or metformin treatment groups 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
22 

(Treatment B, C, D and E) will be compared to placebo group (Treatment A), and the LS 
means for the treatment difference (Treatment B, C D or E minus Treatment A), the SEs, 
the associated 95% CIs, and the p-values will be computed. 
To make the treatment comparison the following SAS code may be used as reference:  
 
PROC MIXED data = dataset ; 
CLASS treatment time ; 
MODEL change = treatment time baseline ; 
LSMEAN treatment / pdiff ALPHA=0.05 CL ; 
ESTIMATE 'B vs. A' treatment -1 1 0 0 0  / CL; 
ESTIMATE 'C vs. A' treatment -1 0 1 0 0  / CL; 
ESTIMATE 'D vs. A' treatment -1 0 0 1 0  / CL; 
ESTIMATE 'E vs. A' treatment -1 0 0 0 1  / CL; 
RUN; 
 
Results of these statistical analyses will be summarized in tables. 
8.4.2
Secondary Endpoint Analyses 
All secondary efficacy endpoints will be analyzed with mixed-effects models similar to the 
primary endpoint analyses. For endpoints that require logarithm-transformation to ensure 
data normality, the change from baseline will be calculated as the log-transformed post-
baseline data minus the log-transformed baseline data. These changes from baseline data will 
be examined as the dependent variable and the log of the baseline covariate will be included 
as a covariate. The LS means, SEs, and the corresponding 95% CIs for the changes from 
Baseline/Visit 2 (Day 1) to Study Termination/Visit 8 (Day 168/Week 24) will be derived 
from the ANCOVA model for each treatment. Each of the fixed-dose combinations of 
leucine and sildenafil and leucine, sildenafil and metformin treatment groups (Treatment B, 
C, D and E) will be compared to placebo group (Treatment A), and the LS means for the 
treatment difference (Treatment B, C, D and E minus Treatment A), the SEs, and the 
associated 95% CIs and p-values will be computed.  The resulting estimates will be 
exponentially transformed back to the original scale to yield geometric LS mean ratios, SEs 
of the geometric mean ratios (calculated as geometric LS mean ratio multiplied by the SE of 
the LS mean of the log- transformed data), and the 95% CIs of the geometric LS mean 
ratios. 
Proportion of subjects with ≥ 5% body weight loss from baseline will be calculated and 
summarized for PP and ITT Populations. Pearson’s Chi Square Test will be used for 
analysis of proportions. Fisher Exact Test will be used when any expected cell count will 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
23 

be less than 5. Each of the fixed-dose combinations of leucine, sildenafil, and/or 
metformin treatment groups (Treatment B, C, D and E) will be compared to placebo 
group (Treatment A), and the following statistics will be computed: proportion difference 
(proportion of subjects for Treatment B, C D or E minus Treatment A), the SEs, the 
associated 95% CIs, and the p-values. 
8.4.3
Exploratory Analysis 
Proportion of subjects with ≥10% body weight loss from baseline will be calculated and 
summarized for PP and ITT Populations. Pearson’s Chi Square Test will be used for 
analysis of proportions. Fisher Exact Test will be used when any expected cell count will 
be less than 5. Each of the fixed-dose combinations of leucine, sildenafil, and/or 
metformin treatment groups (Treatment B, C, D and E) will be compared to placebo 
group (Treatment A), and the following statistics will be computed: proportion difference 
(proportion of subjects for Treatment B, C D or E minus Treatment A), the SEs, the 
associated 95% CIs, and the p-values. 
NuSirt is interested in analysis of comorbidities independent of weight loss i.e. to 
determine if the comorbidities change occurs independent of the weight loss. Although 
this analysis is exploratory in nature and may not provide a true test of treatment effect, it 
is considered important to examine, and thus will be analyzed with all relevant caveats 
presented.  
The comorbidities that will be analyzed (which are also secondary endpoints) are: 
• Hypertension i.e. Systolic Blood Pressure (SBP) and Diastolic Blood Pressure 
(DBP) 
• Hyperlipidemia i.e. total cholesterol, HDL cholesterol, LDL cholesterol, and 
triglycerides 
• Diabetes Mellitus i.e. fasting blood sugar and HbA1c 
• Cardiovascular Diseases i.e. hsCRP  
Analysis methods will follow those of the primary efficacy endpoint. Mixed-model of 
analysis of covariance (ANCOVA) will be used to analyze change in each comorbidity 
from Baseline to Week 24 in PP and ITT Populations. The ANCOVA will include 
treatment group and time as the main effects with percentage of weight loss as the 
covariate. The LS means, SEs, and the corresponding 95% CIs for the changes from 
baseline to Week 24 will be derived from the model for each treatment. Each of the 
fixed-dose combinations of leucine, sildenafil, and/or metformin treatment groups 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
24 

(Treatment B, C, D and E) will be compared to placebo group (Treatment A), and the LS 
means for the treatment difference (Treatment B, C D or E minus Treatment A), the SEs, 
the associated 95% CIs, and the p-values will be computed. 
Subgroup analysis for group of patients that are above certain threshold limits at baseline 
for each of the comorbidities will also be performed. Below are the baseline value cutoffs 
that will be used for the subgroup analysis: 
• Hypertension: SPB >130 or DBP>80 mm Hg.  Note: Analysis for the change in 
this comorbidity will be conducted using both parameters for all subjects who 
meet one or both of these criteria. 
• Triglycerides: >150 mg/dL 
• Cholesterol: >200 mg/dL 
• LDL-C: >130 mg/dL 
• HDL-C: <50 (men)/<40 (women). Note: Cutoffs are divided by gender, but 
analysis will be for all subjects with low HDL. 
• FBG: >100 
• HbA1c: > 5.7%. 
• Hs-CRP: > 3 mg/L 
NOTE: Table for the exploratory analysis will have the same specifications as the table 
for the analysis of secondary endpoints. Therefore, the same shells will be used as the 
reference. 
8.5
Safety Analyses 
8.5.1
Adverse Events 
The safety and tolerability endpoint is defined as the incidence of treatment-emergent 
adverse events (TEAEs). 
All reported terms (investigator descriptions) for AEs will be coded using the most recent 
version of the Medical Dictionary for Regulatory Activities (MedDRA). TEAEs are 
defined as adverse events with an onset or worsening after the first randomized dose.  
Number and percentage of subjects with TEAEs will be summarized by treatment group, 
system organ classification, and preferred term. AEs occurring prior to receiving the first 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
25 

randomized dose (e.g., during the Screening and Pre-Treatment Periods) will not be 
summarized but will be provided in listings.   
TEAEs leading to discontinuation from the study, TEAEs possibly or probably related to 
study treatment, serious TEAEs, death, TEAEs by severity (or intensity), and TEAEs 
with maximum severity (or intensity) and causality, will be summarized.   
Additionally, TEAEs of special interest, e.g., specific gastrointestinal symptoms, will be 
summarized separately if deemed necessary. 
Occurrence of adverse events in each of the active treatment groups (B, C, D, E, B and C 
pooled, D and E pooled) will be compared to the placebo group (Treatment A) using 
Pearson’s Chi Square Test. Fisher Exact Test will be used when any expected cell count 
will be less than 5. Statistical tests will be performed for all TRAEs; TRAEs by system 
organ class, intensity and treatment; TRAEs by system organ class, relationship of study 
medication and treatment; all serious TRAEs; serious TRAEs by system organ class and 
treatment; all TRAEs leading to study discontinuation; TRAEs leading to study 
discontinuation by system organ class and treatment; all drug-related TRAEs; all drug-
related serious TRAEs; and frequent (≥ 5%) TRAEs by preferred term and treatment. For 
each statistical test, Relative Risk Ratio (RRR) will be reported and is defined as the ratio 
of probability of occurrence of an event in Treatment B (or C, D, E, B and C pooled, D 
and E pooled) to the probability of occurrence of an event in Treatment A. RRR will be 
reported along with the associated 95% CI, and the p-value. If any need arises for ad hoc 
analysis (for example, to explore any trend in occurrence of adverse events with the 
increase of dose, or exposure-adjusted incidence rate to consider potential time effects), 
the reasons for such additions and method used for additional statistical analyses to be 
performed will be described in a SAP Addendum. 
8.5.2
Clinical Laboratory Evaluations 
All hematology, clinical chemistry, and urinalysis results will be listed by treatment 
group, subject, and visit, including scheduled and unscheduled/repeat measurements.  
Laboratory assessments that are outside of normal ranges and/or with potential clinical 
importance will be flagged.  Baseline values, the values at each visit, and changes from 
baseline values will be summarized descriptively for each of the quantitative laboratory 
assessments by treatment group.  Additionally, shift tables of hematology, clinical 
chemistry, and urinalysis results will be generated to summarize the normal and abnormal 
(abnormal high and abnormal low) status changes from baseline to each post-baseline 
visit. 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
26 

8.5.3
Vital Signs 
The observed data for blood pressure (systolic and diastolic), pulse, and body temperature 
will be listed by subject, treatment, and time point. Change from baseline for blood 
pressure (systolic and diastolic) and pulse rate will be derived and listed by subject, 
treatment, and time point.  
For systolic blood pressure, diastolic blood pressure and pulse rate the observed and 
change from baseline data will be summarized by treatment and time point. Summaries 
will include tables of descriptive statistics showing the number of observations (n), mean, 
SD, median, minimum, and maximum value. 
8.5.4
Electrocardiograms 
The 12-lead ECG parameters of normal, abnormal without clinical significance, and 
abnormal with clinical significance on Screening/Visit 2 (Day 1) and Study 
Termination/Visit 8 (Day 168/Week 24) or Early Termination visit, and changes from 
baseline values will be summarized by treatment group. Summaries will include tables of 
descriptive statistics showing the number of observations (n), mean, SD, median, 
minimum, and maximum value. 
8.5.5
Physical Examination 
Physical examination data will be listed. 
9
SAMPLE SIZE AND POWER 
A sufficient number of individuals will be screened to enroll at least 250 subjects (50/per 
treatment). With an assumption of 20% dropout rate, approximately 200 subjects (40/per 
treatment) are expected to complete treatment through Study Termination/Visit 8 (Day 
168/Week 24). 
Using a two-sided, two-sample comparison (t-test) of means at the alpha=0.05 level of 
significance, sample sizes of 23 subjects per treatment will provide approximately 90% 
power to detect a mean treatment difference of 3 kg body weight (~3.0-3.3% weight 
loss over 24 weeks), assuming a common standard deviation of 25%. However, 
detection of significant changes in obesity co-morbidities require a higher sample size; 
detection of a 6 mm Hg change in blood pressure will require a sample size of 40 
subjects per treatment group at 80% power; accordingly, the sample size has been 
increased to 40/treatment arm to enable assessment of this secondary endpoint. These 
assumptions of effect size and standard deviation are based on data derived from the 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
27 

previous clinical trial of NS-0200 (NS-0200-01) and represents the anticipated 
treatment effect of effective fixed-dose combinations of leucine and sildenafil, with or 
without metformin, compared to placebo. 
10 REFERENCES 
[1]
Flegal KM, Kruszon-Moran D, Carroll MD, Fryar CD, Ogden CL. 
Trends in obesity among adults in the united states, 2005 to 2014. Jama 
2016;315:2284–91. doi:10.1001/jama.2016.6458. 
[2]
Ogden C, Carroll M, Lawman H, Fryar C, Kruszon-Moran D, Kit B, et 
al. Trends in obesity prevalence among children and adolescents in the 
united states, 1988-1994 through 2013-2014. Jama 2016;315:2292–9. 
doi:10.1001/jama.2016.6361. 
[3]
Fontaine KR, Redden DT, Wang C, Westfall AO, Allison DB. Years of life 
lost due to obesity. J Am Med Assoc 2003;289:187–93. 
doi:10.1016/S0140-6736(09)60318-4. 
[4]
MacMahon S, Baigent C, Duffy S, Rodgers A, Tominaga S, Chambless L, 
et al. Body- mass index and cause-specific mortality in 900 000 adults: 
Collaborative analyses of 57 prospective studies. Lancet 2009;373:1083–96. 
doi:10.1016/S0140-6736(09)60318-4. 
[5]
Masters RK, Powers DA, Link BG. Obesity and US mortality risk over 
the adult life course. Am J Epidemiol 2013;177:431–42. 
doi:10.1093/aje/kws325. 
[6]
Ding M, Hu Y, Schwartz J, Koh WP, Yuan JM, Sesso HD, et al. Delineation 
of body mass index trajectory predicting lowest risk of??mortality in U.S. 
men using generalized additive mixed model. Ann Epidemiol 2016;26:698–
703. doi:10.1016/j.annepidem.2016.08.006. 
[7]
The Global Mortality Collaboration. Body-mass index and all-cause 
mortality: individual-participant-data meta-analysis of 239 prospective 
studies in four continents. Lancet 2016;388:776–86. doi:10.1016/S0140-
6736(16)30175-1. 
[8]
Pi-Sunyer X. The Medical Risks of Obesity. Postgrad Med 
2009;121:21–33. doi:10.3810/pgm.2009.11.2074.The. 
[9]
Klein S, Burke LE, Bray G a, Blair S, Allison DB, Pi-Sunyer X, et al. 
Clinical implications of obesity with specific focus on cardiovascular 
disease: a statement for professionals from the American Heart 
Association Council on Nutrition, Physical Activity, and Metabolism: 
endorsed by the American College of Cardiology Found. Circulation 
2004;110:2952–67. doi:10.1161/01.CIR.0000145546.97738.1E. 
[10]
Wing R, Lang W, Wadden T, Safford M, Knowler W, Bertoni A, et al. 


                                                                                     
 
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Benefits of Modest Weight Loss in Improving Cardiovascular Risk Factors in 
Overweight and Obese Individuals With Type 2 Diabetes. Diabetes Care 
2011;34:1481–6. doi:10.2337/dc10- 2415. 
[11]
Chang H-C, Guarente L. SIRT1 and other sirtuins in metabolism. Trends 
Endocrinol Metab 2014;25:138–45. doi:10.1016/j.tem.2013.12.001. 
[12]
Ruderman NB, Xu XJ, Nelson L, Cacicedo JM, Saha AK, Lan F, et al. AMPK 
and SIRT1: a long-standing partnership? Am J Physiol Endocrinol Metab 
2010;298:E751-60. doi:10.1152/ajpendo.00745.2009. 
[13]
Pfluger PT, Herranz D, Velasco-miguel S, Serrano M, Tscho MH. Sirt1 
protects against high-fat diet-induced. PNAS 2008;105:9793–8. 
[14]
Zhang W, Zhang X, Wang H, Guo X, Li H, Wang Y, et al. AMP-
Activated Protein Kinase 1 Protects Against Diet-Induced Insulin 
Resistance and Obesity. Diabetes 2012;61:3114–25. doi:10.2337/db11-
1373. 
[15]
Liu Y, Wan Q, Guan Q, Gao L, Zhao J. High-fat diet feeding impairs both the 
expression and activity of AMPKa in rats’ skeletal muscle. Biochem Biophys 
Res Commun 2006;339:701–7. doi:10.1016/j.bbrc.2005.11.068. 
[16]
Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin 
resistance, and the metabolic syndrome. J Clin Invest 2013;123:2764–72. 
doi:10.1172/JCI67227. 
[17]
Banerjee J, Bruckbauer A, Zemel MB. Activation of the AMPK/Sirt1 
pathway by a leucine–metformin combination increases insulin 
sensitivity in skeletal muscle, and stimulates glucose and lipid 
metabolism and increases life span in Caenorhabditis elegans. 
Metabolism 2016:1–13. doi:10.1016/j.metabol.2016.06.011. 
[18]
Bruckbauer A, Zemel MB. Synergistic effects of polyphenols and 
methylxanthines with Leucine on AMPK/Sirtuin-mediated metabolism in 
muscle cells and adipocytes. PLoS One 2014;9:e89166. 
doi:10.1371/journal.pone.0089166. 
[19]
Bruckbauer A, Banerjee J, Fu L, Li F, Cao Q, Cui X, et al. A Combination of 
Leucine , Metformin , and Sildenafil Treats Nonalcoholic Fatty Liver Disease 
and Steatohepatitis in Mice. Int J Hepatol 2016;2016:16 pages. 
doi:10.1155/2016/9185987. 
[20]
Fu L, Bruckbauer A, Li F, Cao Q, Cui X, Wu R, et al. Interaction between 
Metformin and Leucine in Reducing Hyperlipidemia and Hepatic Lipid 
accumulation in Diet-Induced Obese Mice. Metabolism 2015. 
doi:10.1016/j.metabol.2015.07.006. 



                                                                                     
 
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29 

11 PRELIMINARY TABLE OF CONTENTS* 
Number 
Description 
Population 
Tables 
14.1.1 
Analysis Populations by Treatment 
Enrolled 
14.1.2.1 
Subject Disposition by Treatment 
Randomized 
14.1.2.2 
Subject Enrollment by Treatment and Site 
Randomized 
14.1.3.1.1 
Demographics and Baseline Characteristics by Treatment 
– Continuous Variables 
PP 
14.1.3.1.2 
Demographics and Baseline Characteristics by Treatment 
– Continuous Variables 
ITT 
14.1.3.2.1 
Demographics and Baseline Characteristics by Treatment 
– Categorical Variables 
PP 
14.1.3.2.2 
Demographics and Baseline Characteristics by Treatment 
– Categorical Variables 
ITT 
14.1.3.3.1a Analysis of Baseline Differences: Body Weight, BMI and 
Waist Circumference 
PP 
14.1.3.3.1b Analysis of Baseline Differences: Body Weight, BMI and 
Waist Circumference 
ITT 
14.1.3.3.2a Analysis of Baseline Differences: Fasting Plasma Glucose, 
HbA1c and hsC-Reactive Protein 
PP 
14.1.3.3.2b Analysis of Baseline Differences: Fasting Plasma Glucose, 
HbA1c and hsC-Reactive Protein 
ITT 
14.1.3.3.3a Analysis of Baseline Differences: Fasting Plasma Lipids 
PP 
14.1.3.3.3b Analysis of Baseline Differences: Fasting Plasma Lipids 
ITT 
14.1.3.3.4a Analysis of Baseline Differences: Hematology 
PP 
14.1.3.3.4b Analysis of Baseline Differences: Hematology 
ITT 
14.1.3.3.5a Analysis of Baseline Differences:Clinical Chemistry and 
Lipid Values 
PP 
14.1.3.3.5b Analysis of Baseline Differences:Clinical Chemistry and 
Lipid Values 
ITT 
14.1.3.3.6a Analysis of Baseline Differences: Urinalysis Values 
PP 
14.1.3.3.6b Analysis of Baseline Differences: Urinalysis Values 
ITT 
14.1.3.3.7a Analysis of Baseline Differences:Other Laboratory Values PP 
14.1.3.3.7b Analysis of Baseline Differences:Other Laboratory Values ITT 
14.1.3.3.8a Analysis of Baseline Differences: Blood Pressure 
PP 
14.1.3.3.8b Analysis of Baseline Differences: Blood Pressure 
ITT 
14.1.4 
Medical History 
ITT 

* This section is intended as a guideline only and is not to constrain in any way the final summary tables, 
figures, or listings that will accompany the case study report. Additional analyses or modifications will be 
as needed, and the final table of contents will reflect all analyses as they are performed. 


                                                                                     
 
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14.1.5 
Deviations from Inclusion/Exclusion Criteria at Screening 
by Treatment 
ITT 
14.1.6 
Treatment Compliance 
ITT 
14.1.7 
Duration of Exposure by Treatment 
ITT 
14.1.8.1 
All Concomitant Medications by Anatomic Therapeutic 
Chemical Classification and Treatment 
ITT 
14.1.8.2 
New Concomitant Medications by Anatomic Therapeutic 
Chemical Classification and Treatment 
ITT 
14.1.8.3 
Prior Concomitant Medications by Anatomic Therapeutic 
Chemical Classification and Treatment 
ITT 
14.2.1.1 
ANCOVA Analysis for Primary Endpoint –  Percent 
Change in Body Weight from Baseline 
PP 
14.2.1.2 
Descriptive Statistics for Primary Endpoint – Body Weight 
Summary and Percent Change from Baseline by Treatment 
and Visit 
PP 
14.2.1.3 
ANCOVA Analysis for Primary Endpoint –  Percent 
Change in Body Weight from Baseline 
ITT 
14.2.1.4 
Descriptive Statistics for Primary Endpoint – Body Weight 
Summary and Percent Change from Baseline by Treatment 
and Visit 
ITT 
14.2.2.1 
ANCOVA Analysis for Secondary Endpoint – Change in 
Absolute Body Weight from Baseline 
PP 
14.2.2.2 
Descriptive Statistics for Secondary Endpoint – Absolute 
Body Weight Summary and Change from Baseline by 
Treatment and Visit 
PP 
14.2.2.3 
ANCOVA Analysis for Secondary Endpoint – Change in 
Absolute Body Weight from Baseline 
ITT 
14.2.2.4 
Descriptive Statistics for Secondary Endpoint – Absolute 
Body Weight Summary and Change from Baseline by 
Treatment and Visit 
ITT 
14.2.3.1 
Analysis for Secondary Endpoint - Percentage of Subjects 
with ≥5% and ≥10% Relative Percentage Reduction from 
Baseline in Body Weight by Treatment 
PP 
14.2.3.2 
Percentage of Subjects with ≥5% and ≥10% Relative 
Percentage Reduction from Baseline in Body Weight by 
Treatment 
PP 
14.2.3.3 
Analysis for Secondary Endpoint - Percentage of Subjects 
with ≥5% and ≥10% Relative Percentage Reduction from 
Baseline in Body Weight by Treatment 
ITT 
14.2.3.4 
Percentage of Subjects with ≥5% and ≥10% Relative 
Percentage Reduction from Baseline in Body Weight by 
Treatment 
ITT 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
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14.2.4.1 
ANCOVA Analysis for Secondary Endpoint – Change in 
Waist Circumference from Baseline 
PP 
14.2.4.2 
Descriptive Statistics for Secondary Endpoint – Waist 
Circumference Summary and Change from Baseline by 
Treatment and Visit 
PP 
14.2.4.3 
ANCOVA Analysis for Secondary Endpoint – Change in 
Waist Circumference from Baseline 
ITT 
14.2.4.4 
Descriptive Statistics for Secondary Endpoint – Waist 
Circumference Summary and Change from Baseline by 
Treatment and Visit 
ITT 
14.2.5.1 
ANCOVA Analysis for Secondary Endpoints – Change in 
Fasting Plasma Lipids from Baseline 
PP 
14.2.5.2 
Descriptive Statistics for Secondary Endpoints – Fasting 
Plasma Lipids Summary and Change from Baseline by 
Treatment and Visit 
PP 
14.2.5.3 
ANCOVA Analysis for Secondary Endpoints – Change in 
Fasting Plasma Lipids from Baseline 
ITT 
14.2.5.4 
Descriptive Statistics for Secondary Endpoints – Fasting 
Plasma Lipids Summary and Change from Baseline by 
Treatment and Visit 
ITT 
14.2.6.1 
ANCOVA Analysis for Secondary Endpoints – Change in 
Fasting Glucose from Baseline 
PP 
14.2.6.2 
Descriptive Statistics for Secondary Endpoints –  Change 
in Fasting Glucose Summary and Change from Baseline 
by Treatment and Visit 
PP 
14.2.6.3 
ANCOVA Analysis for Secondary Endpoints – Change in 
Fasting Glucose from Baseline 
ITT 
14.2.6.4 
Descriptive Statistics for Secondary Endpoints –  Change 
in Fasting Glucose Summary and Change from Baseline 
by Treatment and Visit 
ITT 
14.2.7.1 
ANCOVA Analysis for Secondary Endpoint – Change in 
HbA1c from Baseline 
PP 
14.2.7.2 
Descriptive Statistics for Secondary Endpoint – HbA1c 
Summary and Change from Baseline by Treatment and 
Visit 
PP 
14.2.7.3 
ANCOVA Analysis for Secondary Endpoint – Change in 
HbA1c from Baseline 
ITT 
14.2.7.4 
Descriptive Statistics for Secondary Endpoint – HbA1c 
Summary and Change from Baseline by Treatment and 
Visit 
ITT 
14.2.8.1 
ANCOVA Analysis for Secondary Endpoint – Change in 
Blood Pressure from Baseline 
PP 


                                                                                     
 
NS-WM-01 Statistical Analysis Plan 
 
 
 
 
 
 
 
 
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14.2.8.2 
Descriptive Statistics for Secondary Endpoint – Blood 
Pressure Summary and Change from Baseline by 
Treatment and Visit 
PP 
14.2.8.3 
ANCOVA Analysis for Secondary Endpoint – Change in 
Blood Pressure from Baseline 
ITT 
14.2.8.4 
Descriptive Statistics for Secondary Endpoint – Blood 
Pressure Summary and Change from Baseline by 
Treatment and Visit 
ITT 
14.2.9.1 
ANCOVA Analysis for Secondary Endpoints – Change in 
hsCRP from Baseline 
PP 
14.2.9.2 
Descriptive Statistics for Secondary Endpoints – hsCRP 
Summary and Change from Baseline by Treatment and 
Visit 
PP 
14.2.9.3 
ANCOVA Analysis for Secondary Endpoints – Change in 
hsCRP from Baseline 
ITT 
14.2.9.4 
Descriptive Statistics for Secondary Endpoints – hsCRP 
Summary and Change from Baseline by Treatment and 
Visit 
ITT 
14.3.1.1.1 
Summary of Treatment-Emergent Adverse Events by 
Treatment 
ITT 
14.3.1.1.2 
Analysis of Treatment-Emergent Adverse Events 
ITT 
14.3.1.2.1 
Treatment-Emergent Adverse Events by System Organ 
Class, Preferred Term, Intensity, and Treatment 
ITT 
14.3.1.2.2 
Analysis of Treatment-Emergent Adverse Events by 
System Organ Class, Intensity and Treatment 
ITT 
14.3.1.3.1 
Serious Treatment-Emergent Adverse Events by System 
Organ Class, Preferred Term, and Treatment 
ITT 
14.3.1.3.2 
Analysisof Serious Treatment-Emergent Adverse Events 
by System Organ Class and Treatment 
ITT 
14.3.1.4.1 
Treatment-Emergent Adverse Events by System Organ 
Class, Preferred Term, Relationship to Study Medication, 
and Treatment 
ITT 
14.3.1.4.2 
Analysis of Treatment-Emergent Adverse Events by 
System Organ Class, Relationship to Study Medication 
and Treatment 
ITT 
14.3.1.5.1 
Treatment-Emergent Adverse Events Leading to 
Discontinuation by System Organ Class, Preferred Term, 
and Treatment 
ITT 
14.3.1.5.2 
Analysis of Treatment-Emergent Adverse Events Leading 
to Discontinuation by System Organ Class and Treatment 
ITT 
14.3.1.6.1 
Frequent (≥5%) Treatment-Emergent Adverse Events by 
Preferred Term and Treatment 
ITT 


                                                                                     
 
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14.3.1.6.2 
Analysis of Frequent (≥ 5%) Treatment-Emergent Adverse 
Events by Preferred Term and Treatment 
ITT 
14.3.2.1 
Hematology Values and Change from Baseline by Visit 
and Treatment 
ITT 
14.3.2.2 
Clinical Chemistry Values and Change from Baseline by 
Visit and Treatment 
ITT 
14.3.2.3 
Urinalysis Values and Change from Baseline by Visit and 
Treatment 
ITT 
14.3.2.4 
Other Laboratory Values and Change from Baseline by 
Visit and Treatment 
ITT 
14.3.3.1 
Hematology Shift Table by Visit and Treatment 
ITT 
14.3.3.2 
Clinical Chemistry Shift Table by Visit and Treatment 
ITT 
14.3.3.3 
Urinalysis Shift Table by Visit and Treatment 
ITT 
14.3.3.4 
Other Laboratory Shift Table by Visit and Treatment 
ITT 
14.3.4.1 
Number of Hematology Values Outside of Normal Range 
ITT 
14.3.4.2 
Number of Clinical Chemistry Values Outside of Normal 
Range 
ITT 
14.3.4.3 
Number of Urinalysis Values Outside of Normal Range 
ITT 
14.3.4.4 
Number of Other Laboratory Values Outside of Normal 
Range 
ITT 
14.3.5 
12-Lead Electrocardiograms Change from Baseline by 
Treatment and Visit 
ITT 
14.3.6 
Vital Signs and Change from Baseline by Visit and 
Treatment 
ITT 
Listings 
16.2.1.1 
Subject Disposition and Reason for Exiting the Study 
Enrolled 
16.2.1.2 
Record of Informed Consent and Subject Eligibility at the 
End of Screening 
Enrolled 
16.2.1.3 
Subject Randomization 
Enrolled 
16.2.1.4 
Analysis Populations 
Enrolled 
16.2.1.5 
Demographic and Baseline Characteristics 
Enrolled 
16.2.1.6 
Medical History 
Enrolled 
16.2.1.7 
Concomitant Medications 
Enrolled 
16.2.1.8 
Protocol Deviations 
Enrolled 
16.2.1.9 
Study Medications and Treatment Compliance 
Enrolled 
16.2.2.1 
Body Weight, Height, BMI and Waist Circumference 
Enrolled 
16.2.2.2 
Fasting Plasma Glucose, HbA1c and hsC-Reactive Protein 
Enrolled 
16.2.2.3 
Fasting Lipid Values 
Enrolled 
16.2.3.1.1 
Adverse Events 
Enrolled 
16.2.3.1.2 
Serious Adverse Events 
Enrolled 
16.2.3.1.3 
Adverse Events Resulting in Study Discontinuation 
Enrolled 


                                                                                     
 
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16.2.3.1.4 
Adverse Events Resulting in Death 
Enrolled 
16.2.3.2.1 
Central Laboratory Summary 
Enrolled 
16.2.3.2.2 
Hematology Values 
Enrolled 
16.2.3.2.3 
Clinical Chemistry and Lipids Values 
Enrolled 
16.2.3.2.4 
Urinalysis Values 
Enrolled 
16.2.3.2.5 
Other Laboratory Values 
Enrolled 
16.2.3.3 
Vital Signs 
Enrolled 
16.2.3.4 
Physical Examinations 
Enrolled 
16.2.3.5 
12-Lead Electrocardiograms 
Enrolled 
16.2.4.1 
Phone Contacts 
Enrolled 
16.2.4.2 
Unscheduled Visits 
Enrolled 

12 ADDENDUM 
12.1
Rationale 
This addendum is to document the deviations from the study protocol that were observed 
after the initiation of the study NS-WM-001. This section clearly explains each deviation 
and describe proposed actions needed to be taken for the remaining duration of the study, 
and any effect on the statistical analysis.  
12.2
Incorrect Weight Measurement 
During the IMV's for the study NS-WM-01, it has come to NuSirt’s attention that two 
sites, Site 09/Young and Site 11/Studdard, have done the weight measurement for all 
their subjects incorrectly. These two sites took the weight measurement in light street 
cloths, no shoes and empty pockets instead of no street clothes, no shoes and two gowns 
as instructed by the protocol. This was done for both Screening/Visit 1 and Baseline/Visit 
2 by the time it was discovered by the clinical monitors. As NuSirt is interested in the 
change in weight during the trial for all participants, the two sites were instructed to 
continue to weigh all subjects enrolled at their sites on light street clothes, no shoes and 
empty pockets. This was done to limit skewing of the data by switching to the per 
protocol method after baseline measurements were already done. Due to this fact all 
subjects enrolled at these two sites with incorrect weight measurement may be excluded 
from the statistical analysis of this study. The decision to exclude these subjects will be 
made prior database lock. 


                                                                                     
 
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12.3
Antibiotic Use 
NuSirt has decided to flag subjects that take antibiotics during the last 30 days of the 
study. These subjects may have changes in weight that are due to infection or other 
causes for the antibiotic use. Due to this reason all such subjects will be excluded from 
the statistical analysis of this study.