Clinical trial NCT03182686
Conditions: Osteoarthritis, Knee Knee Osteoarthritis Knee Arthritis Knee Pain Chronic
Keywords: osteoarthritis osteoarthritis of the knee OA OAK Kellgren Lawrence (KL)
Sponsor: Ampio Pharmaceuticals. Inc.
Start date: 2017-06-19; Completion date: 2017-12-07
Enrollment: 168 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a phase 3 randomized study to confirm the efficacy of an intra-articular injection of Ampion™ in adults with pain due to severe osteoarthritis of the knee.

Detailed description:
A Randomized Study to Confirm the Efficacy of an Intra-Articular Injection of Ampion™ in Adults With Pain Due to Severe Osteoarthritis of the Knee

There will be a 7-day screening period for each subject followed by a 12-week participation period.

The primary trial objective is to evaluate the clinical efficacy of Ampion using the Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) (using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) 3.1 Index and Patient's Global Assessment as assessments).

The secondary trial objectives are to evaluate the safety of a single intra-articular injection (4 mL) of Ampion.

Arms: arm 1: 4 mL injection of Ampion arm 2: 4 mL injection of Placebo
Interventions: intervention 1: 4 mL injection of Ampion intervention 2: 4 mL Injection of Placebo
Intervention descriptions: intervention 1: 4 mL injection of Ampion intervention 2: 4 mL Injection of Placebo

Protocol documents:
Prot_000.pdf: 
 
 
 
 
A PHASE 3 RANDOMIZED STUDY TO 
CONFIRM THE EFFICACY OF AN INTRA- 
ARTICULAR INJECTION OF AMPION™ IN 
ADULTS WITH PAIN DUE TO SEVERE 
OSTEOARTHRITIS OF THE KNEE 
 
STUDY NUMBER: AP-003-C 
 
NCT 03182686 
 
9 November 2017 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
1 
 
 
 
 
CLINICAL STUDY PROTOCOL 
 
A PHASE 3 RANDOMIZED STUDY TO CONFIRM THE 
EFFICACY OF AN INTRA- ARTICULAR INJECTION OF 
AMPION™ IN ADULTS WITH PAIN DUE TO SEVERE 
OSTEOARTHRITIS OF THE KNEE 
 
 
 
STUDY NUMBER: AP-003-C 
 
 
Drug Development Phase: 
Phase 3 
Investigational Product: 
Ampion™ 
Indication: 
Severe Osteoarthritis of the knee 
Sponsor: 
Ampio Pharmaceuticals, Inc. 
373 Inverness Parkway, Suite 200 
Englewood, CO 80112 
Date: 
Version 1.3 09 November 2017 
Conduct: In accordance with the ethical principles that originate from the Declaration of 
Helsinki and that are consistent with International Conference on Harmonisation (ICH) 
guidelines on Good Clinical Practice (GCP) and regulatory requirements as applicable. 
 
 
 
 
 
 
Confidential Information 
This document is the sole property of Ampio Pharmaceuticals, Inc.  This document and 
any and all information contained herein has to be considered and treated as strictly 
confidential.  This document shall be used only for the purpose of the disclosure herein 
provided.  No disclosure or publication shall be made without the prior written consent of 
Ampio Pharmaceuticals, Inc. 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
2 
 
 
 
 
 
PROTOCOL SIGNATURE PAGE 
 
I have read, understood, and agree to comply with the contents of this clinical protocol, 
Study No. AP-003-C version 1.3 dated 09 November 2017, and agree to meet all 
obligations as detailed in all applicable regulations and guidelines. 
 
 
 
 
 
 
 
Signed by: 
 
 
 
 
 
 
 
 
 
<Study personnel signature> 
<Enter date> 
 
 
 
 
 
 
 
 
 
 
 
<Printed name> 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
3 
 
 
PROTOCOL SYNOPSIS 
 
Sponsor: 
Ampio Pharmaceuticals, Inc. 
Investigational Product: 
Ampion™ 
Developmental Phase: 
Phase 3 
Title of Study: 
A Phase 3 Randomized Study to Confirm the Efficacy of an Intra-Articular Injection of 
Ampion in Adults with Pain Due to Severe Osteoarthritis of the Knee 
Protocol Number: 
AP-003-C 
Study Center(s): 
Approximately 14 sites 
Indication: 
Severe osteoarthritis (OA) of the knee (Kellgren Lawrence Grade 4) 
Number of subjects: 
171 subjects total 
Objectives: 
The primary trial objective is to evaluate the efficacy of a single 4 mL Ampion™ intra-
articular injection. 
The secondary trial objective is to evaluate the safety of an intra-articular injection of Ampion 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
4 
 
 
Sponsor: 
Ampio Pharmaceuticals, Inc. 
Investigational Product: 
Ampion™ 
Developmental Phase: 
Phase 3 
Methods: 
The study will have a 7-day screening period for each patient followed by a 12-week participation 
period. A total of 171 subjects with knee pain and decreased function due to severe osteoarthritis 
of the knee (KL IV) will be enrolled.  In order to reduce the potential for bias in reporting 
outcomes, subjects will be randomized 6:1 (Ampion intra-articular injection or saline intra-
articular injection).  Study visits will include: Screening visit, Baseline, a 24-hour, post-injection 
follow up call, Week 2 phone visit, Week 6, Week 10 phone visit, and Week 12 for a total of 7 
visits.  Screening, Baseline, Week 6 and Week 12 are in-office visits. 
The clinical effects of Ampion treatment on OA pain, function and PGA will be evaluated during 
in-clinic visits at 6 and 12 weeks, and telephone contacts at 2 and 10 weeks, using the Western 
Ontario and McMaster Universities Arthritis Index (WOMAC®), and the Patient’s Global 
Assessment of disease severity (PGA). 
The WOMAC® is a validated osteoarthritis scoring system and sets the standard for the 
subject response. In order not to bias the collection of data, only questions from the 
validated WOMAC scale and PGA will be asked of subjects. 
Clinical benefit will be established by evaluation of OMERACT-OARSI using the WOMAC 5-
point Likert scale and PGA as assessments during the in-office visits and phone visits. Safety will 
be assessed by recording adverse events (post-injection and at all follow up contacts), physical 
examination and vitals (Baseline, Weeks 6 and 12), and laboratory tests (Screening and Week 12). 
Diagnosis and Main Criteria for Inclusion: 
1. Able to provide written informed consent to participate in the study; 
2. Willing and able to comply with all study requirements and instructions of the site study 
staff; 
3. Male or female, 40 years to 85 years old (inclusive); 
4. Must be ambulatory; 
5. Study knee must have a clinical diagnosis of OA supported by radiological evidence 
(Kellgren Lawrence Grade 4) which is assessed locally (x-rays within the past 6 months 
of screening are acceptable); 
6. Moderate to moderately-severe OA pain in the study knee (rating of at least 1.5 on the 
WOMAC A, 5-point Likert pain subscale); 
7. Moderate to moderately-severe OA function in the study knee (rating of at least 1.5 on 
the WOMAC C, 5-point Likert Function subscale); 
8. WOMAC A, 5-point Likert pain subscale <1.5 in the contralateral knee; 
9. Ability to discontinue NSAID use at Screening visit and/or 72 hours prior to the 
Baseline visit and for the duration of the clinical study (low-dose Aspirin (81 mg) is 
allowed during the study); 
10. No analgesia (including acetaminophen) taken 24 hours prior to an efficacy measure; 
11. No known clinically significant liver abnormality (e.g. cirrhosis, transplant, etc.).  
Main Criteria for Exclusion: 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
5 
 
 
Sponsor: 
Ampio Pharmaceuticals, Inc. 
Investigational Product: 
Ampion™ 
Developmental Phase: 
Phase 3 
1. As a result of medical review and screening investigation, the Principal Investigator 
considers the subject unfit for the study 
2. A history of allergic reactions to human albumin (reaction to non-human albumin such as 
egg albumin is not an exclusion criterion) 
3. A history of allergic reactions to excipients in 5% human albumin (N-acetyltryptophan, 
sodium caprylate) 
4. Presence of tense effusions 
5. Inflammatory or crystal arthropathies, acute fractures, history of aseptic necrosis or joint 
replacement in the affected knee, as assessed locally by the Principal Investigator 
6. Isolated patella femoral syndrome, also known as chondromalacia 
7. Any other disease or condition interfering with the free use and evaluation of the study 
knee for the duration of the trial (e.g. cancer, congenital defects, spine osteoarthritis) 
8. Major injury to the study knee within the 12 months prior to screening 
9. Severe hip osteoarthritis ipsilateral to the study knee 
10. Any pain that could interfere with the assessment of study knee pain (e.g. pain in any 
other part of the lower extremities, pain radiating to the knee) 
11. Any pharmacological or non-pharmacological treatment targeting OA started or changed 
during the 4 weeks prior to randomization or likely to be changed during the duration of 
the study 
12. Pregnancy or planning to become pregnant during the study. 
13. Use of the following medications: 
a. No IA injected medications in the study knee during the study. No Hylauronic Acid 
(HA) injections in the study knee at least 12 weeks prior to Baseline.  No steroid 
injections in the study knee at least 4 weeks prior to Baseline. 
b. No analgesics containing opioids. 
c. NSAIDs are not permitted during the study; acetaminophen is available as a rescue 
medication during the study from the provided supply. 
d. No topical treatment on the study knee during the study 
e. No significant anticoagulant therapy (e.g. Heparin or Lovenox) during the study 
(treatment such as low-dose Aspirin (81 mg) and Plavix are allowed) 
f. No systemic treatments that may interfere with safety or efficacy assessments during 
the study 
g. No immunosuppressants 
h. No use of systemic or intra-articular corticosteroids 
14. No human albumin treatment in the 3 months before randomization or throughout the 
duration of the study 
Test Product, Dose and Mode of Administration: 
Ampion, 4 mL, single intra-articular injection in the knee 
 Reference Therapy, Dose and Mode of Administration: 
Saline 4 mL; single intra-articular injection in the knee  


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
6 
 
 
Sponsor: 
Ampio Pharmaceuticals, Inc. 
Investigational Product: 
Ampion™ 
Developmental Phase: 
Phase 3 
Study Duration: 
 
12 weeks 
Criteria for Evaluation: 
Efficacy: 
WOMAC Osteoarthritis Index 3.1, 5-point Likert scale Patient 
Global Assessment 
Safety: 
Safety will be assessed by recording adverse events (post-injection and at all follow-up 
contacts), change in laboratory analysis (Screening to Week 12), physical examination and 
vitals (Baseline, Weeks 6 and 12). 
Clinical Endpoints: 
Primary Efficacy Endpoint: 
- 
Evaluate improvement of OMERACT-OARSI response of a single 4 mL Ampion intra-
articular injection from Baseline to Week 12 
Secondary Efficacy Endpoints: 
- 
Evaluate improvement in the composite endpoint of pain and function (WOMAC A and 
C) from Baseline to Week 12  
- 
Evaluate improvement in PGA from Baseline to Week 12  
- 
Evaluate improvement in the composite endpoint of pain and function (WOMAC A and 
C), compared to saline from all single-injection Ampion studies 
Statistical Methods:   
For the primary endpoint evaluating the proportion of patients who are OMERACT-OARSI 
responders, the following hypothesis will be tested. 
 
 H0:π ≤ π0 versus HA:π > π0      
 
 Where π0 is the hypothesized value for the proportion of responders.  The value will be 30% 
in this study.  This test will be tested using an exact binomial test.  That is, given the sample 
size of n, the number of responders X, and the value of π0 =0.30, then probability that X or 
more events would be observed will be calculated as the p-value.  Since this is a one-sided 
test, the alpha level will be 0.025. 
 
Blinding and Randomization 
 
Subjects will be assigned in a 6:1 ratio to treatment by a randomization schedule developed 
and maintained by an independent statistician. Ampion and saline will be provided in blinded 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
7 
 
study vials labeled with the appropriate information and packed into patient kits.  Each patient 
kit will contain labeled vials, blinded for drug content, syringes, needles and rescue 
medication.   
The Sponsor, the investigator, and all study staff having a role in the day-to-day conduct of the 
study will remain blinded to treatment. 
A comprehensive presentation of the data management and statistical analysis plan will be 
approved by Ampio Pharmaceuticals, Inc. prior to unblinding of study data. 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
8 
 
 
 
TABLE OF CONTENTS 
PROTOCOL SYNOPSIS. ......................................................................................................... 3 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS  .......................................... 12 
1 
INTRODUCTION .......................................................................................................... 14 
1.1 
Study Drug  ........................................................................................................... 14 
1.2 
Background to the Disease.  .................................................................................. 15 
1.3 
Previous Human Experience  ................................................................................ 17 
1.4 
Preclinical Data  .................................................................................................... 17 
1.4.1 
Pharmacology Studies  ................................................................................ 17 
1.4.2 
Toxicity and Safety Studies  ....................................................................... 18 
1.5 
Clinical Experience  .............................................................................................. 18 
2 
RATIONALE FOR THE STUDY .................................................................................. 23 
2.1 
Rationale for the Doses and the Dosing Regimen  ................................................ 23 
3 
STUDY DESIGN. ........................................................................................................... 24 
3.1 
Study Design Overview  ....................................................................................... 24 
3.2 
STUDY OBJECTIVES  ........................................................................................ 24 
3.2.1 
Primary Objective  ...................................................................................... 24 
3.2.2 
Secondary Objective  .................................................................................. 24 
3.3 
Safety Monitoring committee  .............................................................................. 24 
3.4 
Stopping Rules  ..................................................................................................... 25 
3.5 
Study Endpoints  ................................................................................................... 25 
3.5.1 
Primary Endpoint  ....................................................................................... 25 
3.5.2 
Secondary Endpoint  ................................................................................... 25 
3.5.3 
Exploratory Endpoint  ................................................................................. 25 
3.6 
Blinding and Randomization  ............................................................................... 25 
4 
SELECTION OF SUBJECTS. ........................................................................................ 26 
4.1 
Number of Subjects.  ............................................................................................. 26 
4.2 
Recruitment Methods  ........................................................................................... 26 
4.3 
Inclusion Criteria  .................................................................................................. 26 
4.4 
Exclusion Criteria  ................................................................................................ 27 
4.5 
Inclusion of Subjects Incapable of Giving Informed Consent  ............................. 27 
5 
STUDY PLAN AND PROCEDURES  .......................................................................... 28 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
9 
 
5.1 
Description of Study visits  ................................................................................... 28 
5.1.1 
Visit 1 (Day -7 through Day 0, in-clinic); Screening.  ................................ 28 
5.1.2 
Visit 2 (Day 0, in-clinic) Baseline/Randomization/Treatment  .................. 29 
5.1.3 
Visit 3 (Day 1, telephone contact)  ............................................................. 29 
5.1.4 
Visit 4 (Week 2 ± 7 days, telephone contact)  ............................................ 29 
5.1.5 
Visit 5 (Week 6 ± 7 days, in-clinic)  ........................................................... 30 
5.1.6 
Visit 6 (Week 10 ± 7 days, telephone contact)  .......................................... 30 
5.1.7 
Visit 7 (Week 12 ± 7 days, in-clinic)  ......................................................... 30 
5.1.8 
Early Termination Visit  ............................................................................. 31 
5.1.9 
Unscheduled Visits  .................................................................................... 31 
5.1.10 
Missed Visits  .............................................................................................. 31 
5.1.11 
CONCOMITANT MEDICATIONS AND RESCUE MEDICATION  .......32 
6 
METHODS OF ASSESSMENT  .................................................................................... 33 
6.1 
Efficacy Assessments  ........................................................................................... 34 
6.1.1 
WOMAC® Osteoarthritis Index (Bellamy 1988)  ...................................... 34 
6.1.2 
Patient’s Global Assessment of Disease Severity (PGA)  .......................... 34 
6.2 
Safety Parameters  ................................................................................................. 34 
6.2.1 
Vital Signs  .................................................................................................. 35 
6.2.2 
Clinical laboratory tests  ............................................................................. 35 
7 
DISCONTINUATION CRITERIA  ................................................................................ 37 
7.1 
Early Discontinuation of the Study  ...................................................................... 37 
7.2 
Early Discontinuation of Individual Subjects  ...................................................... 37 
8 
TREATMENT  ............................................................................................................... 38 
8.1 
Dosing and administration of study medication  .................................................. 38 
8.2 
Drug Storage and Accountability  ......................................................................... 38 
8.3 
Treatment Compliance  ......................................................................................... 39 
9 
ADVERSE EVENTS  ..................................................................................................... 40 
9.1 
Definition of an adverse event  .............................................................................. 40 
9.2 
Definition of a Serious Adverse Event  ................................................................. 40 
9.3 
Recording of adverse events and serious adverse events  ..................................... 41 
9.3.1 
AE Follow up  ............................................................................................. 41 
9.3.2 
Overdose  .................................................................................................... 42 
9.4 
Serious Adverse Event Reporting  ........................................................................ 42 
9.4.1 
Reporting Requirements  ............................................................................ 42 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
10 
 
9.4.2 
SAE Information  ........................................................................................ 42 
10 STATISTICAL METHODS  .......................................................................................... 43 
10.1 
GENERAL CONSIDERATIONS  ........................................................................ 43 
10.1.1 
Statistical and Analytical Plan:  .................................................................. 43 
10.2 
STATISTICAL OBJECTIVES  ............................................................................ 43 
10.3 
ANALYSIS POPULATIONS  .............................................................................. 44 
10.3.1 
Safety Analysis Population:  ....................................................................... 44 
10.3.2 
Intent-to-treat Population:  .......................................................................... 44 
10.3.3 
Per Protocol Population:  ............................................................................ 44 
10.3.4 
Hypotheses Tested  ..................................................................................... 44 
10.3.5 
Exploratory Endpoints  ............................................................................... 46 
10.3.6 
Definition of Study Visits  .......................................................................... 46 
10.3.7 
Number of subjects to receive study drug  .................................................. 46 
10.3.8 
Disposition of subjects  ............................................................................... 47 
10.3.9 
Interim analysis  .......................................................................................... 47 
10.3.10 Blinding and randomization  ....................................................................... 47 
10.3.11 Data presentation  ........................................................................................ 47 
10.3.11.1 
Demographic and Baseline Characteristics:  .................................... 47 
10.3.11.2 
Medical History and Physical Examination:  .................................... 47 
10.3.11.3 
Concomitant Medications or Treatments:  ........................................ 47 
10.3.11.4 
Safety data:  ....................................................................................... 48 
10.4 
Missing Data  ........................................................................................................ 48 
11 REGULATORY, ETHICAL AND LEGAL OBLIGATIONS  ...................................... 49 
11.1 
Declaration of Helsinki  ........................................................................................ 49 
11.2 
Good Clinical Practice  ......................................................................................... 49 
11.3 
Institutional Review Boards/Ethics Committees  ................................................. 49 
11.4 
Regulatory Authority Approval  ........................................................................... 49 
11.5 
Informed Consent.  ................................................................................................ 49 
11.6 
Subject Confidentiality and Disclosure ................................................................ 49 
11.7 
Collection, Monitoring and Auditing Study Documentation, and Data 
Storage  ................................................................................................................. 50 
11.7.1 
Collection of Data and Monitoring Procedures  ......................................... 50 
11.7.2 
Auditing Procedure  .................................................................................... 51 
11.7.3 
Retention of Documents ............................................................................. 51 
11.8 
Disclosure of Information  .................................................................................... 51 
11.9 
Discontinuation of the Study  ............................................................................... 51 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
11 
 
11.10 Study Report, Publication Policy and Archiving of Study Documentation  .......... 51 
11.10.1 Study Report and Publication Policy  .......................................................... 51 
11.10.2 Study Documents  ....................................................................................... 52 
11.10.3 Archiving of Documents  ............................................................................ 52 
12 REFERENCES  ............................................................................................................... 53 
13 APPENDICES  ............................................................................................................... 56 
13.1 
Contact List  .......................................................................................................... 56 
 
 
 
LIST OF IN-TEXT TABLES 
 
 
 
Table 1.5.1 
Summary of the Studies Conducted with Ampion Administered as an 
Intra-Articular Injection to the Knee ............................................................... 18 
Table 1.5.2 
Descriptive Statistics and Analysis Results for KL 4 Subjects for Each 
Study and All Studies Combined .................................................................... 20 
Table 1.5.3 
Responder Analysis Across All Single Injection Studies and KL Grades using 
OMERACT-OARSI Criteria………………………………………………….21 
Table 1.5.4 
Summary of Treatment-Emergent Adverse Events ........................................ 21 
Table 6.1 
Schedule of Assessments and Procedures ....................................................... 36 
Table 9.1 
Definitions of AE Severity. ............................................................................. 40 
Table 9.2 
Guidelines for Determining the Relationship (if any) Between Adverse 
Event and the Study Drug ............................................................................... 40 
 
 
LIST OF IN-TEXT FIGURES 
 
 
Figure 1 
LS Mean Change for Ampion-Treated Patients Over Time and by 
Study………………………………………………………………………….20 
 
Figure 2 
Kellgren Lawrence Grading System………………………………………….34 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
12 
 
 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
 
Abbreviation 
Definition 
C 
degrees Celsius 
F 
degrees Fahrenheit 
g 
Microgram 
ACR 
American College of Rheumatology 
AE 
adverse event 
ALP 
alkaline phosphatase 
ALT 
alanine transaminase (SGPT) 
ARDS 
adult respiratory distress syndrome 
AST 
aspartate transaminase (SGOT) 
BP 
Blood pressure 
CFR 
Code of Federal Regulations 
CI 
confidence interval 
Cm 
Centimeter 
CRF 
case report form 
CRO 
Clinical Research Organization 
Da 
Dalton 
DA-DKP 
Asp-Ala diketopiperazine 
dL 
Deciliter 
eDC 
electronic data capture 
FDA 
Food and Drug Administration 
G 
11.26 x (RPM/1000)2 x Radius (cm) 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
GI 
Gastrointestinal 
HSA 
human serum albumin 
HCG 
Human chorionic gonadotropin 
IA 
intra-articular 
ICF 
informed consent form 
ICH 
International Conference on Harmonisation 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
ITT 
intent-to-treat 
kDa 
Kilodalton 
KL 
Kellgren Lawrence 
LDH 
lactic dehydrogenase 
LK 
Likert 
MCH 
mean cell hemoglobin 
MCHC 
mean cell hemoglobin concentration 
MCID 
Minimal Clinically Important Difference 
MCV 
mean cell volume 
Mg 
Milligram 
mL 
Milliliter 
NA 
not applicable 
Ng 
Nanogram 
NSAID 
non-steroidal anti-inflammatory drug 
OA 
Osteoarthritis 
OAK 
osteoarthritis of the knee 
OMERACT-OARSI 
outcome measures in rheumatology clinical trials and osteoarthritis research 
PGA 
patient’s global assessment of disease severity 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
13 
 
 
Abbreviation 
Definition 
PP 
per protocol population 
Radius 
distance (in centimeters) from the center of rotation to the bottom of tube in 
the rotor 
Rap-1 
Regulatory protein  
RBC 
red blood cell 
REB 
Research Ethics Board 
RPM 
Rounds Per Minute 
SAE 
serious adverse event 
SD 
standard deviation 
SEM 
standard error of the mean 
SMC 
Safety Monitoring Committee 
SOP 
standard operation procedure 
TEAE 
treatment-emergent adverse event 
TEER 
trans endothelial electrical resistance 
VAS 
Visual Analog Scale 
WBC 
white blood cell 
WCC 
white cell count 
WO 
Washout 
WOMAC 
Western Ontario and McMaster Universities Arthritis Index 


Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
  
 
       
14 
 
 
1 
INTRODUCTION 
 
HSA has a long history of clinical use as a colloid replacement therapy, dating back over 
60 years.  Currently, HSA is approved for the indications of hypovolemia, hypoalbuminemia, 
prevention of central volume depletion after paracentesis due to cirrhotic ascites, ovarian 
hyperstimulation, adult respiratory distress syndrome (ARDS), acute nephrosis, hemolytic 
disease of the newborn and burns.  In addition to its effects on oncotic pressure, HSA has 
pharmacological effects including decreased inflammation (Quinlan 2005), decreased 
vascular permeability (Evans 2002) and no adverse effect on cardiac safety (Vincent JL, 
2004). 
 
Ampion™, the < 5 kilodalton (kDa) ultrafiltrate of 5% human serum albumin (HSA), is 
being developed to provide relief for the pain of severe osteoarthritis of the knee. There are 
no currently FDA approved drugs for the indication of pain from severe (KL 4) osteoarthritis 
of the knee for which Ampion is indicated. Ampion is a heterogeneous solution that contains 
several known small molecules and unknown components that may act synergistically. The 
individual contribution(s) of the unknown components to the biologic activity of Ampion 
may not be quantifiable since over 1200 peptides and proteins have been identified in 
commercial HSA preparations [Gay, 2010]. 
 
Bar-Or et al found that commercial preparations of HSA are heterogeneous in terms of 
posttranslational modifications, including C- and N-terminal truncation, cysteinylation, 
glycation and nitrosylation (Bar-Or D, 2005). An important modification is the cleavage of 
the two N-terminal amino acids Aspartate (D) and Alanine (A). This occurs by the action of a 
naturally occurring dipeptidase (Bar-Or 2013) present in plasma and also bound to or 
associated with leukocytes and endothelial cells (Ohnuma 2006), and activated during the 
FDA required heat-treatment of commercial preparations of HSA. After cleavage, the 
dipeptide undergoes cyclisation to form a diketopiperazine (aspartylalanyl diketopiperazine 
[DA-DKP]). DA-DKP is found in the low molecular weight fraction (< 5000 Da) of 
commercial lots of HSA at concentrations ranging from approximately 100 μM.in vitro 
studies and its action in human subjects suggest that it is an active ingredient in the 
pharmacological effects of HSA and of Ampion™. Other components, such as N-acetyl 
tryptophan and its metabolites and sodium caprylate present in HSA and Ampion, act 
synergistically with DA-DKP on numerous molecular pathways involved in inflammation, 
nociceptive and neuropathic pain, vascular permeability and the resolution of inflammation 
in various cell types including synoviocytes, chondrocytes, PBMC, macrophages, memory T 
cells, endothelial cells and mesenchymal stem cells (Thomas, GW 2016) (Fredrick ED 2016) 
(Bar-Or 2015) ( Shimonkevitz 2008) (Bar-Or 2006) (Bar-Or 2005) 
 
1.1 
STUDY DRUG 
 
Ampion™ is the < 5 kDa ultrafiltrate of 5% HSA. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
15 
 
 
1.2 
BACKGROUND TO THE DISEASE 
 
Osteoarthritis (OA) remains a leading contributor to global disability, with both hip and knee 
OA accounting for over 17 million years lived with disability (Cross 2014). OA is the most 
common form of arthritis and affects up to 38 million adults in the US alone. OA is caused 
by inflammation of the soft tissue and bony structures of the joint which worsens over time 
and leads to progressive thinning of articular cartilage, narrowing of the joint space, synovial 
membrane thickening, osteophyte formation and increased density of subchondral bone. 
These changes eventually result in chronic pain and disability and deterioration of the joint 
despite drug therapy may require eventual surgery for total joint replacement. OA of the 
knee is defined in stages as noted by Kellgren Lawrence grading: Grade 0 (normal knee; no 
osteophytes or joint space narrowing), Grade 1 (possible osteophytic lipping and doubtful 
narrowing of joint space), Grade 2 (definite osteophytes and possible narrowing of joint 
space), Grade 3 (moderate multiple osteophytes, definite narrowing of joint space and some 
sclerosis, and possible deformity of the bone ends) and Grade 4 (large osteophytes, marked 
narrowing of joint space, severe sclerosis, and definite deformity of bone ends). 
 
Patients with severe OAK, defined radiologically as KL grade 4, have few treatment options 
for pain management. Even with reported difficulties in managing symptoms, including pain, 
a large number of KL 4 patients do not discuss pain or OA during consultations. Many 
patients perceived that they just had to put up with pain until their joint replacement because 
there was nothing else that could be done (McHugh 2007). The net result is that the patients 
control their pain by limiting their activity, and this can progress to the point of losing the 
ability to ambulate in the community.  There is a consequent loss of the benefits of activity 
and the conditioning that this provides, leading to a deterioration of health in general, 
including both physical and psychological effects (Cisternas 2016). Difficult-to-treat pain 
conditions, such as severe OAK, are a challenge for both medical and surgical doctors and 
patients. There is no doubt that patients with chronic pain in general suffer from an unmet 
need, as FDA agreed stating that there is an unmet medical need among patients with severe 
osteoarthritis. 
 
The pain associated with osteoarthritis is now thought to be multifactorial. The pathogenesis of 
pain from OA is comprised of both nociceptive and neuropathic pain pathways resulting from 
severely damaged cartilage, exposed subchondral bone, joint innervation, and less defined 
pathways including the synovium. A recent publication in January 2016 (Akinci 2016) noted 
that the balance between nociceptive and neuropathic pain shifts towards neuropathic pain as 
the severity of the condition increases. The multifactorial nature of the pain of osteoarthritis 
may make pain control more difficult, particularly in patients with severe OA (KL4)  
 
The presence of synovitis in KL 4 patients indicates active ‘inflammatory’ disease process. 
These findings were supported with a linear observation of an increasing trend correlated with 
the severity of synovitis related to advancing KL grades. Additionally, KL 4 patients have 
been shown to have lesions that fluctuate with pain, (i.e., fluctuating bone marrow lesions 
and synovitis) related to a high grade of cartilage loss in KL 4 patients (Guermazi 2015). 
These findings were further supported using a unique in vivo imaging technique that detected 
activated and not resting macrophages (Kraus 2016). In a patient cohort that included patients 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
16 
 
 
with severe (KL 4) OAK, the quantity of knee-related activated macrophages was 
statistically associated with more severe knee pain and radiographic severity of knee OAK. 
 
These data demonstrated that greater macrophage mediated inflammation is a cause of more 
severe pain in KL 4 OAK and provides a rationale whereby Ampion, demonstrated in vitro 
with anti-inflammatory and anti-macrophage activity, may mediate symptomatic relief in this 
patient group. These data further demonstrate the need to examine patients by disease 
severity and supply treatment options for severely diseased patients (Guermazi 2015). 
Ampion has shown anti-inflammatory activity in vitro against macrophages, monocytes, and 
immune cells. 
 
When the clinical trials of Ampion were designed, it was assumed that the vehicle for the 
product, saline, would have little to no effect in reducing pain, stiffness, or improving physical 
function. This control was determined not to be a true placebo, but rather turned out to be 
active treatment.  
The Osteoarthritis Research Society International (OARSI) and Standing Committee for 
Clinical Trials Response Criteria Initiative Outcome Measures in Rheumatology (OMERACT) 
committee, in concert with the international rheumatology community, worked to develop a 
uniform core set of outcome measures for osteoarthritis. While the committee initially 
developed two sets of responder criteria to present the results of changes after treatment in three 
symptomatic domains (pain, function, and patient’s global assessment) as a single variable for 
clinical trials, the committee ultimately worked to create a simplified set of responder criteria, 
Scenario D, which captured the criteria for response.  
In the original plan, for each domain, a response was defined by both a relative and an absolute 
change, with different cut-offs with regard to the drug, the route of administration and the OA 
localization. The formal OMERACT-OARSI scenarios defined a responder as  
a. having achieved a high degree of improvement in pain or a moderate degree of 
improvement in 2 of the 3 response domains (pain, function, global assessment) 
(scenario A) OR  
b. as having achieved a high degree of improvement in either pain or function, or a 
moderate degree of improvement in 2 of the 3 response domains (scenario B) 
The composite index permits presentation of results of symptom modifying clinical trials in OA 
based on individual patient responses (responder yes/no). The use of composite indices was still 
somewhat controversial in that the original OARSI criteria did not account for multiplicity of 
comparisons, however OMERACT-OARSI worked to rectify this and their conclusions were 
presented at the OMERACT 6 conference.  
OMERACT and OARSI established a task force aimed at evaluating: (1) the variability of 
observed placebo and active treatment effects using the OARSI responder criteria; and (2) the 
possibility of proposing a simplified set of criteria. Because individual study endpoints in 
studies of NSAIDs in patients with hip or knee osteoarthritis were highly correlated, 
OMERACT-OARSI evaluated six different scenarios to determine whether a simplified set of 
criteria could be developed. The conclusions of the task force were presented and discussed 
during the OMERACT 6 conference, where a simplified set of responder criteria (OMERACT-
OARSI set of criteria) was proposed. Scenario D was chosen by the members, indicating the 
importance of having both a relative change and an absolute change as part of the criteria for 
response.  


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
17 
 
The criteria for a responder using the Likert Scale, where the 20-point change on the VAS scale 
is the same as a 1-point change on the Likert Scale and, similarly, the 10-point change in the 
VAS scale is the same as a 0.5-point change on the Likert Scale, are defined as: 
 
1. High improvement in pain or in function ≥ 50% and absolute change in Likert pain scale 
of ≥1 OR 
2. Improvement in at least two of the 3 following: 
a. pain ≥ 20% and absolute change in Likert score ≥0.5 
b. function ≥ 20% and absolute change in Likert score ≥0.5 
c. patient’s global assessment ≥ 20% and absolute change in Likert score ≥0.5 
 
1.3 
PREVIOUS HUMAN EXPERIENCE 
 
In addition to the extensive clinical experience with HSA, Ampio Pharmaceuticals, Inc. has 
completed multiple clinical studies to evaluate the treatment effect of Ampion in subjects 
with OAK. These studies are summarized in Section 1.5. 
 
1.4 
PRECLINICAL DATA 
 
1.4.1 
Pharmacology Studies 
 
In vitro pharmacology studies demonstrated that a low molecular weight fraction 
(< 3000 daltons [Da]) of more than five commercial preparations of HSA, and a cyclized 
dipeptide contained in that fraction, Asp-Ala diketopiperazine (DA-DKP) have a range of 
anti-inflammatory properties, which may be expected to ameliorate the symptoms of 
inflammation, including pain, in man (Bar-Or et al 2006). These studies showed reduced 
inflammatory cytokine production from human T lymphocytes in vitro via modulation of 
signal transduction through increased expression of Rap-1, and inhibition of macrophage 
activation. In addition, DA-DKP enhanced integrity of human endothelial cell junctions in 
the transendothelial electrical resistance (TEER) assay, which would result in decreased 
vascular permeability to the influx of inflammatory cells in vivo. No pharmacokinetics 
studies were performed as the plasma levels of the constituents of the < 5000 Da preparation, 
including DA-DKP, after intra-articular injection of a therapeutic dose in man or in 
laboratory animals are anticipated to be below the limits of detection. For more information, 
consult the Investigator Brochure. 
 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
18 
 
 
1.4.2 
Toxicity and Safety Studies 
 
HSA and other components of serum albumin preparations are species specific and may be 
expected to be immunogenic if repeatedly injected into non-human species, even at very low 
concentrations.  For this reason, it was not possible to perform formal toxicological studies in 
animals. 
 
1.5 
CLINICAL EXPERIENCE 
 
 
Ampio has conducted multiple clinical studies to evaluate the treatment effect and safety of 
Ampion in subjects with OAK. Ampion has been administered across 6 clinical studies as a 
single intra articular (IA) injection or as three IA injections administered every two weeks. 3 
single-dose pivotal Phase 3 studies, 2 multi-dose studies, and 1 multiphase single-dose phase 
1b study have been completed.  An overview of the efficacy studies is provided in the Table 
1.5.1 
 
For the clinical development program, all studies were conducted in subjects with moderate 
to moderately severe pain due to OA of the knee. Patients were required to have x-ray 
findings demonstrating KL grade 2, 3, or 4. All studies enrolled patients with KL 4. In 
addition, eligible subjects had at least moderate pain at Baseline (defined as a score of at least 
1.5 on the WOMAC Index 3.1, 5-point Likert pain subscale at screening) in the study knee, 
which was to have been symptomatic for greater than 6 months with a clinical diagnosis of 
OA confirmed by radiological evidence. The clinical effects of treatment on OA pain were 
evaluated during clinic visits, with phone call follow-ups at intermittent times. 
 
 
Table 1.5.1 
Summary of the Studies Conducted with Ampion™ Administered as an Intra Articular Injection to the Knee 
 
Study 
Phase 
N 
N, 
KL 2, 3, 4 
Route of 
Administratio 
n 
Primary 
outcome 
Primary 
endpoint 
Additional 
outcomes 
Test 
product 
Single-Injection Phase 3 Studies in Support of Efficacy & Safety 
AP-003- 
A 
3 
329 
115 / 139 / 75 
Single IA 
injection 
WOMA 
C A 
pain, 5- 
point 
Likert 
scale 
Week 12 
WOMAC B 
WOMAC C 
PGA 
OMERACT 
-OARSI 
Rescue 
analgesia 
Ampion, 4 
mL or 
10ml* 
AP-004 
3 
538 
133 / 195/ 
210 
Single IA 
injection 
WOMA 
C A 
pain, 5- 
point 
Likert 
scale 
Week 12 
WOMAC C 
PGA 
WOMAC A 
pain over 12 
weeks 
Ampion, 4 
mL 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
19 
 
 
Study 
Phase 
N 
N, 
KL 2, 3, 4 
Route of 
Administratio 
n 
Primary 
outcome 
Primary 
endpoint 
Additional 
outcomes 
Test 
product 
AP-003- 
B 
3 
480 
101 / 247 / 
132 
Single IA 
injection 
WOMA 
C A 
pain, 5- 
point 
Likert 
scale 
Week 12 
WOMAC B 
WOMAC C 
PGA 
Rescue 
analgesia 
WOMAC A 
pain with 
movement, 
pain at rest 
Ampion, 4 
mL 
Additional Clinical Studies in Support of Safety 
AP-007 
1 / 2 
47 
2 / 23/ 15 
Multiple IA 
injection; 
3 injections, 
every 2 weeks 
WOMA 
C A 
pain, 5- 
point 
Likert 
scale 
Week 20 
WOMAC B 
WOMAC C 
PGA 
Physical 
activity 
Radiologic 
changes 
Ampion, 4 
mL 
AP-008 
3 
342 
0 / 114 / 228 
Multiple IA 
injection; 
3 injections, 
every 2 weeks 
WOMA 
C A 
pain, 5- 
point 
Likert 
scale 
Week 20 
WOMAC B 
WOMAC C 
PGA 
Physical 
activity 
WOMAC A 
pain with 
movement 
Ampion, 4 
mL 
AIK 
1 / 2 
103 
33 / 69 / 1 
Single IA 
injection 
Pain, 10- 
point 
NRS 
Day 3, 8, 
30 and 
84 
WOMAC 
total, parts 
A, B, C 
Range in 
motion 
Rescue 
analgesia 
Ampion 10 
ml, alone or 
in solution 
with 
betamethaso 
ne and/or 
lidocaine 
and/or 
saline 
Total 
1839 
*Both doses of Ampion, 4 mL and 10 mL, are safe, efficacious and well tolerated. In the absence of a difference in efficacy 
for the 4 mL and 10 mL Ampion doses, the lower dose of 4 mL will be used for subsequent phase 3 studies. 
 
In all three well-controlled, single-injection studies, Ampion demonstrated a numerically 
greater effect in the KL 4 patients compared to saline, indicating a reproducible and 
consistent response. Analysis of the combined data demonstrate a statistically significant 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
20 
 
 
result in this underserved patient population. Table 1.5.2 presents the data for KL 4 patients 
from all single-injection studies, analyzed according to the SAP for the respective study. 
 
 
Table 1.5.2 
Descriptive Statistics and Analysis Results for KL 4 Subjects for Each Study and All Studies Combined 
 
 
 
 
 
Figure 1 visually represents the consistent reduction in pain for Ampion-treated patients over 
a 12-week period. 
Figure 1. 
LS Mean Change for Ampion-Treated Patients Over Time and by Study 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
21 
 
 
 
In accordance with FDA guidance on osteoarthritis, responder analysis according to the OMERACT-
OARSI criteria was applied to previously conducted clinical trials, Table 1.5.3. 
 
Table 1.5.3 
Responder analysis in KL 4 patients across single injection studies using OMERACT-OARSI criteria 
 
Study ID 
Ampion Responder (%) 
AP-003-A 
59.4% 
AP-003-B 
59.7% 
AP-004 
57.7% 
Combined 
58.6% 
 
 
In addition to the efficacy analysis described in this section, Ampion has also demonstrated a 
robust safety profile across the three well-controlled, single-injection studies, as well as the 
multiple-injection and early phase trials (Table 1.5.4). No drug-related Serious Adverse Events 
(SAEs) have been reported in 1,839 patients treated during the clinical development program. 
The Adverse Event (AE) profile is similar for Ampion and saline, with a majority of the AEs 
of minor or moderate severity and unrelated to treatment. 
 
Table 1.5.4 
Summary of Treatment-Emergent Adverse Events 
 
Treatment emergent AE 
Saline 
Ampion 
Overall 
N=907 
N=932 
One or more TEAE 
425 (47%) 
421 (45%) 
One or more related TEAE 
63 (7%) 
73 (8%) 
KL grade 4 
N=338 
N=326 
One or more TEAE 
150 (44%) 
145 (44%) 
One or more related TEAE 
20 (6%) 
24 (7%) 
 
 
 
 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
22 
 
 
CONCLUSIONS 
 
Clinical efficacy observed in previous single-injection studies suggest that Ampion safely 
relieves pain due to severe, KL 4, osteoarthritis of the knee, the most severe form of OAK, 
for which there is no FDA approved treatment alternative. While opioids can treat pain, and 
total knee replacement can excise the affected tissues, the ability to treat OAK pain and 
inflammation locally is an unmet medical need.  Currently, when KL 4 patients are treated 
for OAK, they are receiving treatment using intra-articular therapy (such as 
viscosupplementation) that were not studied, nor required in their device approvals to 
demonstrate efficacy in the KL 4 population (Rutjes 2012). Without surgery, with only a 
single intra-articular injection of Ampion, KL 4 patients are able to achieve clinically 
meaningful pain relief. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
23 
 
 
2 
RATIONALE FOR THE STUDY 
 
This is a phase 3 randomized study is to confirm the efficacy of an intra-articular injection of 
Ampion™ and is designed to follow on from the completed single-injection Ampion studies. 
This study explores the clinical impact of Ampion-treatment in patients with severe OA 
using criteria developed by OMERACT-OARSI (Pham 2004).  
 
The appropriateness of a within-patient multi-component endpoint is generally determined by 
clinical considerations, and as such, the OMERACT-OARSI responder criteria, which evaluates 
pain, function and patient global assessment, and has been deemed clinically meaningful for OA 
trials, remains an endpoint in this clinical investigation. 
 
In addition to evaluating response to treatment (Y/N), the study will evaluate a composite 
endpoint of pain and function, as well as improvement in PGA, using OMERACT-OARSI 
criteria. Analysis of Ampion response to saline from previously conducted single-injection 
Ampion studies will also be evaluated.   
 
Ampio plans to evaluate patients who complete study AP-003-C in an open label extension 
study. The protocol for the open-label extension study will be submitted under separate cover.  
 
2.1 
RATIONALE FOR THE DOSES AND THE DOSING REGIMEN 
 
This trial will use the 4 mL volume of Ampion used in the single-injection studies (AP- 003-A, 
AP-004, and AP-003-B). 


Ampion™ 
Version 1.3       09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
24 
 
 
3 
STUDY DESIGN 
 
3.1 
STUDY DESIGN OVERVIEW 
 
A confirmatory, randomized, phase 3 study with a 7-day screening period for each subject 
followed by a 12-week participation period. A total of 171 subjects with knee pain and 
decreased function due to severe osteoarthritis (KL 4) will be enrolled in a 6:1 randomization 
schema: 4 mL Ampion intra-articular injection: 4 mL saline intra-articular injection.  Study 
visits will include: Screening visit, Baseline, a 24-hour, a post- injection follow-up phone call, 
Week 2 phone visit, Week 6, Week 10 phone visit, and Week 12 for a total of 7 visits.  
Screening, Baseline, Week 6 and Week 12 are in-office visits. 
 
The clinical effects of Ampion treatment on OA pain will be evaluated during in-clinic visits 
at 6 and 12 weeks, and telephone contacts at 2 and 10 weeks, using the Western Ontario and 
McMaster Universities Arthritis Index (WOMAC®) and the Patient’s Global Assessment of 
disease severity (PGA). 
 
The WOMAC® is a validated Osteoarthritis scoring system and sets the standard for the 
subject response. In order not to bias the collection of data, only questions from the 
validated WOMAC scale and PGA will be asked of subjects. 
 
Clinical benefit will be established by evaluation of the OMERACT-OARSI response using 
the WOMAC and PGA scales as assessed during the in-office visits and phone visits. Safety 
will be assessed by recording adverse events (post-injection and at all follow-up contacts), 
physical examinations, vitals (Baseline, Weeks 6 and 12), and laboratory analysis (Screening 
and Week 12). 
 
3.2 
STUDY OBJECTIVES 
 
3.2.1 
Primary Objective 
 
The primary trial objective is to evaluate the clinical efficacy of a single intra-articular 
injection (4 mL) of Ampion. 
3.2.2 
Secondary Objective 
 
The secondary trial objectives are to evaluate the safety of a single intra-articular injection (4 
mL) of Ampion. 
3.3 
SAFETY MONITORING COMTTEE 
 
A Safety Monitoring Committee (SMC) will be established to review the safety of Ampion 
as the study progresses. The SMC will consist of independent clinicians not involved in the 
clinical trial.  The SMC will be primarily responsible for reviewing any serious Adverse 
Event (SAE) and other clinically important safety findings (e.g., discontinuations due to 
 
AEs) that may occur during the study.  A charter will be developed to detail the SMC review 
responsibilities, the frequency of meetings, and data evaluation plans. 
 
3.4 
STOPPING RULES 
 


Ampion™ 
Version 1.3       09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
25 
 
The entire study may be stopped under defined circumstances as outlined in Section 7. 
 
3.5 
STUDY ENDPOINTS 
 
3.5.1 
Primary Endpoint 
 
- 
Evaluate improvement of OMERACT-OARSI response of a single 4 mL Ampion intra-
articular injection from Baseline to Week 12 
3.5.2 
Secondary Endpoint 
 
- 
Evaluate improvement in the composite endpoint of pain and function (WOMAC A and 
C) from Baseline to Week 12  
- 
Evaluate improvement in PGA from Baseline to Week 12  
- 
Evaluate improvement in the composite endpoint of pain and function (WOMAC A and 
C) compared to saline from all single-injection Ampion studies 
 
3.5.3 
Exploratory Endpoint 
 
 N/A 
 
3.6 
BLINDING AND RANDOMIZATION 
 
All subjects will be randomized 6:1 to receive Ampion 4 mL intra-articular injection or 4 mL 
saline intra-articular injection in the study knee. 
 
A comprehensive presentation of the data management and statistical analysis plan will be 
approved by Ampio Pharmaceuticals, Inc. prior to final analysis of study data. 


Ampion™ 
Version 1.3          09  November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
26 
 
 
4 
SELECTION OF SUBJECTS 
 
4.1 
NUMBER OF SUBJECTS 
 
A total of approximately 171 subjects (146 Ampion; 25 Saline) will be enrolled in the study 
to a single (1) intra-articular injection of 4 mL Ampion or 4 mL saline (6:1). 
 
4.2 
RECRUITMENT METHODS 
 
Subjects will be recruited from the population being seen by Investigators at the clinical sites 
participating in the study.  In addition, notifications about the opportunity for subjects to 
participate in a clinical trial will be sent to referring physicians.  A description of the clinical 
trial will also be posted at ClinicalTrials.gov, and advertisements and/or other notices may be 
produced to advise potential study subjects on how they may obtain information about study 
participation. All such materials will be reviewed and approved by the Institutional Review 
Board (IRB) prior to their publication or dissemination. 
 
4.3 
INCLUSION CRITERIA 
 
Subjects should fulfill all of the following inclusion criteria: 
 
1. Able to provide written informed consent to participate in the study; 
2. Willing and able to comply with all study requirements and instructions of the site study 
staff; 
3. Male or female, 40 years to 85 years old (inclusive); 
4. Must be ambulatory; 
5. Study knee must have a clinical diagnosis of OA supported by radiological evidence 
(Kellgren Lawrence Grade 4) which is assessed locally (x-rays within the past 6 
months of screening are acceptable); 
6. Moderate to moderately-severe OA pain in the study knee (rating of at least 1.5 on the 
WOMAC A, 5-point Likert Pain Subscale); 
7. Moderate to moderately-severe OA function in the study knee (rating of at least 1.5 
on the WOMAC C, 5-point Likert Function Subscale); 
8. WOMAC A, 5-point Likert pain subscale <1.5 in the contralateral knee,; 
9. Ability to discontinue NSAID use at Screening visit and/or 72 hours prior to the 
Baseline visit and for the duration of the clinical study (low-dose Aspirin (81 mg) is 
allowed during the study); 
10. No analgesia (including acetaminophen) taken 24 hours prior to an efficacy measure; 
11. No known clinically significant liver abnormality (e.g. cirrhosis, transplant, etc.). 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
27 
 
 
4.4 
EXCLUSION CRITERIA 
 
Subjects fulfilling one or more of the following criteria may not be enrolled in the study: 
 
1. As a result of medical review and screening investigation, the Principal Investigator 
considers the subject unfit for the study 
2. A history of allergic reactions to human albumin (reaction to non-human albumin such as 
egg albumin is not an exclusion criterion) 
3. A history of allergic reactions to excipients in 5% human albumin (N-acetyltryptophan, 
sodium caprylate) 
4. Presence of tense effusions 
5. Inflammatory or crystal arthropathies, acute fractures, history of aseptic necrosis or joint 
replacement in the affected knee, as assessed locally by the Principal Investigator 
6. Isolated patella femoral syndrome, also known as chondromalacia 
7. Any other disease or condition interfering with the free use and evaluation of the study 
knee for the duration of the trial (e.g. cancer, congenital defects, spine osteoarthritis) 
8. Major injury to the study knee within the 12 months prior to screening 
9. Severe hip osteoarthritis ipsilateral to the study knee 
10. Any pain that could interfere with the assessment of study knee pain (e.g. pain in any 
other part of the lower extremities, pain radiating to the knee) 
11. Any pharmacological or non-pharmacological treatment targeting OA started or changed 
during the 4 weeks prior to randomization or likely to be changed during the duration of 
the study 
12. Pregnancy or planning to become pregnant during the study 
13. Use of the following medications: 
a. No IA injected medications in the study knee during the study (or 12 weeks prior to 
Baseline). 
b. No analgesics containing opioids. 
c. NSAIDs are not permitted during the study; acetaminophen is available as a rescue 
medication during the study from the provided supply. 
d. No topical treatment on the study knee during the study 
e. No significant anticoagulant therapy (e.g. Heparin or Lovenox) during the study 
(treatment such as low-dose Aspirin (81 mg) and Plavix are allowed) 
f. No systemic treatments that may interfere with safety or efficacy assessments during 
the study 
g. No immunosuppressants 
h. No use of systemic or intra-articular corticosteroids 
14. No human albumin treatment in the 3 months before randomization or throughout the 
duration of the study 
 
4.5 
INCLUSION OF SUBJECTS INCAPABLE OF GIVING INFORMED 
CONSENT 
 
No subject incapable of giving informed consent may be enrolled in the study. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
28 
 
 
5 
STUDY PLAN AND PROCEDURES 
 
Subjects will be randomized into the study 6:1 to receive an IA injection of 4 mL Ampion™ 
or 4 mL saline on Visit 2, Day 0 (Baseline). Telephone contact will be made with the subject 
24 hours after the IA injection. The subject will be followed for 12 weeks and the clinical 
effects of treatment on OA pain will be evaluated during clinic visits at 6 and 12 weeks, and 
during telephone contacts at 2 and 10 weeks, using the WOMAC osteoarthritis Index 3.1 
(pain subscore, stiffness subscore and function subscore) and an overall global severity 
assessment (Patient’s Global Assessment [PGA]). Safety will be assessed by recording 
adverse events (through 24 hours post-dose and at all follow up in-clinic visits and telephone 
contacts), vital signs (Baseline, and Weeks 6 and 12), recording prior and concomitant 
medications including start/stop dates, indication, dose and frequency (through 24 hours 
post-dose and at all follow up in-clinic visits and telephone contacts), physical examination 
(Baseline and Weeks 6 and 12), and laboratory tests (Screening and Week 12). The 
assessments and procedures performed at each subject visit or contact are described in 
Section 5.1 and in Table 6.1. 
 
 
5.1 
DESCRIPTION OF STUDY VISITS 
 
5.1.1 
Visit 1 (Day -7 through Day 0, in-clinic); Screening 
 
The following procedures will be performed at Visit 1 (Screening): 
 
• Obtain written informed consent before the start of any study specific procedure. 
• Review medical history including all previous treatments for OA. 
• Record prior and concomitant medications including start/stop dates, indication, dose 
and frequency. 
• Record demographic data including date of birth, gender and race. 
• Measure and record height and weight. 
• Perform and record physical examination. 
• Record WOMAC Index 3.1, 5-point Likert scale (sections A-C) for the study knee 
• Record WOMAC Index 3.1, 5-point Likert scale (section A only) for the contralateral 
knee 
• Record PGA 
• Record vital signs. (Measure body temperature, systolic / diastolic blood pressure [BP] 
and pulse rate). 
• Evaluate all inclusion and exclusion criteria to ensure that subjects meet all inclusion 
criteria and none of the exclusion criteria. If subject meets all inclusion/exclusion 
criteria up to this point, move forward with x- ray and laboratory assessments. 
• Obtain x-ray of study knee and evaluate for KL grade (x-rays within 6 months of the 
screening date are acceptable) 
• Collect blood sample for local laboratory assessment.  
• Collect urine hCG in women of child-bearing potential. Evaluate for pregnancy.  
 
 
 
 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
29 
 
 
5.1.2 
Visit 2 (Day 0, in-clinic) Baseline/Randomization/Treatment 
 
The following procedures will be performed at Visit 2 (Baseline/Randomization/Treatment): 
 
• Confirm eligibility by assessing all inclusion/exclusion criteria from the screening visit 
again at Baseline. Confirm KL grade from Principal Investigator. 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for the evaluation of 
the study knee. Confirm Baseline WOMAC Pain (WOMAC A) and WOMAC 
Function (WOMAC C) meet Inclusion #6 and #7 prior to enrollment. 
• Perform PGA evaluation of the study knee. 
• Record concomitant medications. 
• Perform and record physical examination. 
• Record vital signs. (Measure body temperature, systolic / diastolic blood pressure [BP] 
and pulse rate) pre- and post-injection. 
• If subject meets all inclusion/exclusion, subject can be enrolled into study 
• Perform intra-articular injection of study drug. 
• Record post-injection AEs if observed. 
• Issue rescue medication (acetaminophen). 
 
5.1.3 
Visit 3 (Day 1, telephone contact) 
 
The following procedures will be performed at Visit 3: 
 
• Record AEs. 
• Record concomitant medications. 
• Review rescue medication use. 
 
5.1.4 
Visit 4 (Week 2 ± 7 days, telephone contact) 
 
The following procedures will be performed at Visit 4: 
 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for evaluation of the 
study knee. 
• Perform PGA evaluation of the study knee. 
• Record AEs. 
• Record concomitant medications. 
• Review rescue medication use. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
30 
 
 
5.1.5 
Visit 5 (Week 6 ± 7 days, in-clinic) 
 
The following procedures will be performed at Visit 5: 
 
• Perform and record physical examination. 
• Record vital signs. (Measure body temperature, systolic / diastolic blood pressure [BP] 
and pulse rate). 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for the evaluation of 
the study knee. 
• Perform PGA evaluation of the study knee. 
• Record AEs. 
• Record concomitant medications. 
• Review rescue medication use. 
 
5.1.6 
Visit 6 (Week 10 ± 7 days, telephone contact) 
 
The following procedures will be performed at Visit 6: 
 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for the evaluation of 
the study knee. 
• Perform PGA evaluation of the study knee. 
• Record AEs. 
• Record concomitant medications. 
• Review rescue medication use. 
 
5.1.7 
Visit 7 (Week 12 ± 7 days, in-clinic) 
 
The following procedures will be performed at Visit 7: 
 
• Perform and record physical examination (including weight). 
• Record vital signs. (Measure body temperature, systolic / diastolic blood pressure [BP] 
and pulse rate). 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for the evaluation of 
the study knee. 
• Perform PGA evaluation of the study knee. 
• Collect blood sample for local laboratory assessment.  
• Record AEs. 
• Obtain information from subject about medical care for knee OA prior to participation in 
the current study (medical history). 
• Record concomitant medications. 
• Review, count, and collect rescue medication. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
31 
 
 
 
5.1.8 
Early Termination Visit 
 
The following procedures will be performed at Early Termination Visit: 
 
• Perform and record physical examination, including weight. 
• Record vital signs. (Measure body temperature, systolic / diastolic blood pressure [BP] 
and pulse rate). 
• Perform WOMAC (sections A-C), using the 5-point Likert scale for the evaluation of 
the study knee. 
• Perform PGA evaluations of the study knee. 
• Collect blood sample for laboratory assessment. 
• Record AEs. 
• Obtain information from subject about medical care for knee OA prior to participation in 
the current study (medical history). 
• Record concomitant medications. 
• Review, count, and collect rescue medication. 
 
5.1.9 
Unscheduled Visits 
 
Additional visits may be scheduled at the discretion of the Investigator, for example as part 
of follow up of AEs. Unscheduled Visits, if patient is terminated from the study, should 
follow the visit structure described for the Early Termination visit. All unscheduled visits 
should include review of potential AEs. 
 
5.1.10 
Missed Visits 
 
Subjects unable to complete a study visit as scheduled should be re-scheduled for a 
replacement visit as soon as possible. If a subject misses any scheduled follow up visit and 
cannot be seen prior to the start of the visit range for the next visit, the visit is considered 
missed. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
32 
 
 
5.1.11 
CONCOMITANT MEDICATIONS AND RESCUE MEDICATION 
 
The following medications/therapies are NOT allowed during this clinical study: 
 
1. No IA injected medications in the study knee during the study. 
2. No analgesics containing opioids. 
3. NSAIDs may not be used during the study (low-dose Aspirin (81 mg) is allowed). 
Acetaminophen is available as a rescue medication during the study from the 
provided supply. 
4. No non-pharmacological treatment targeting OA started or changed during the study. 
5. No topical treatment on the study knee during the study. 
6. No significant anticoagulant therapy (e.g. Heparin or Lovenox) during the study 
(treatment such as low-dose Aspirin (81 mg) or Plavix is allowed) 
7. No systemic treatments that may interfere with safety or efficacy assessments during 
the study. 
8. No immunosuppressants. 
9. No use of systemic or intra-articular corticosteroids 
10. No human albumin treatment during the study. 
 
Any medication used during the study should be recorded. All concomitant medication start 
and stop dates, total daily dose, route and indication should to be recorded. 
 
The only allowed analgesia medication during the 12-week study is 500 mg of 
acetaminophen, 1 tablet every 4 hours as required by the subject, not to exceed 3,000 mg of 
acetaminophen in a 24-hour period. Dose must be reduced if using other medications that 
contain acetaminophen (cold medicines, etc.). 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
33 
 
 
6 
METHODS OF ASSESSMENT 
 
Demographic Data 
 
At Visit 1 (Screening), subject demographic data will be collected.  These include: date of 
birth, gender and race. 
 
Medical History 
 
At Visit 1 (Screening) a complete medical history, including prior interventions to the study 
knee, will be obtained from each subject. 
 
Concomitant Medications 
 
Detailed history of medications will be documented for each subject at Visit 1 (Screening) 
and Visit 2 (Baseline).  Concomitant medications (especially any changes in medication) will 
be documented for each subject at each scheduled visit. 
 
Kellgren-Lawrence Grading Scale 
 
Radiographic images should be evaluated according to the Kellgren-Lawrence Grading 
Scale as noted below in Figure 2. 
 
  FIGURE 2: Kellgren-Lawrence Grading System 
Grade 
Description 
0 
Normal knee without osteophytes or joint space narrowing 
1 
Possible osteophytic lipping and doubtful narrowing of joint space 
2 
Definite osteophytes and possible narrowing of joint space 
3 
Moderate multiple osteophytes, definite narrowing of joint space and some 
sclerosis and possible deformity of bone ends 
4 
Large osteophytes, marked narrowing of joint space, severe sclerosis and 
definite deformity of bone ends 
 
Physical Examination and Vital Signs 
 
Height in feet and inches will be measured at Visit 1 (Screening). 
 
Body weight in pounds (lb) will be measured at Visit 1 (Screening) and at Visit 7 (Week 12 
or Early Termination Visit).  
 
Body temperature (deg F) will be measured at each visit. 
Systolic and diastolic BP and pulse rate will be measured with the subject in a seated 
position. 
 
The full physical examination will consist of examining the following body systems: 
cardiovascular, respiratory, abdominal, skin and musculoskeletal other than the knee. The 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
34 
 
 
physical examination of the target knee will consist of evaluating the knee joint for effusion 
and tenderness on palpation.  
 
Laboratory Assessments 
Blood will be collected at Screening and Week 12 and analyzed by a local lab. Reference 
ranges must be available to evaluate lab values during the course of the study. 
 
See Table 6.1 for full schedule of assessments and procedures 
 
 
 
6.1 
EFFICACY ASSESSMENTS 
 
Note: Efficacy questionnaire questions will be asked “with reference to study knee” i.e. to 
obtain scores specific for the treated knee. 
 
6.1.1 
WOMAC® Osteoarthritis Index (Bellamy 1988) 
 
WOMAC Index 3.1 (sections A-C), using the 5-point Likert scale, should be completed by 
subjects at Screening, pre-dose and pre-aspiration as applicable on Day 0 and at Weeks 2, 6, 
10 and 12. All subjects are required to take at least 5 minutes to complete the questionnaire. 
Subjects are asked about their pain, stiffness, and function in the knee (study joint) due to 
arthritis during the last 24 hours. 
 
Subjects respond to each subscale by using a 5-point Likert score (0 = none, 1 = mild, 2 = 
moderate, 3 = severe, 4 = extreme). 
 
6.1.2 
Patient’s Global Assessment of Disease Severity (PGA) 
 
The PGA should be completed by subjects at Screening, pre-dose and pre-aspiration as 
applicable on Day 0 (prior to injection), and at Weeks 2, 6, 10 and 12. 
 
Subjects are asked the following question: “Considering all the ways in which your arthritis 
affects you, please indicate how you are doing.” 
 
Subjects respond by using a 5-point adjectival Likert score (0 = very well, 1 = well, 2 = fair, 
3 = poor, 4 = very poor). 
 
6.2 
SAFETY PARAMETERS 
 
Based on results of previous studies of a single intra-articular injection of Ampion™ < 5 kDa 
ultrafiltrate of 5% HSA in adults with OA of the knee in which no clinically significant 
differences between active and saline were found, safety will be assessed by recording 
adverse events, vital signs, results of physical examination, recording prior and concomitant 
medications and safety labs. 
 
 
 
 
 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
35 
 
 
 
6.2.1 
Vital Signs 
 
Vital signs (radial pulse rate, blood pressure, and body temperature) should be recorded at 
Screening, pre- and post–treatment on Day 0, and then on visits at Weeks 6 and 12. 
 
Vital signs should be taken after the subject has rested in a seated position for at least 
5 minutes. 
 
6.2.2 
Clinical laboratory tests 
 
Subjects in the will undergo a blood draw at Screening and Week 12. Blood draws should be 
done after subject meets all other inclusion/exclusion criteria at Screening; Blood draws 
should be done at Week 12 after all WOMAC and PGA assessments are completed.  All 
clinically significant values will be followed up at the Investigator’s discretion. Blood 
laboratory results must be reviewed and compared to reference ranges throughout the study to 
evaluate for potential adverse events. Blood draws will be performed in compliance with 
standard laboratory procedures.  
 
The following analysis will be conducted by the local laboratory: 
 
Serum biochemistry: Sodium, potassium, chloride, bicarbonate, glucose, urea, creatinine, 
creatine kinase, urate, phosphate, total calcium, cholesterol, albumin, globulins, protein, total 
bilirubin, conjugated bilirubin, gamma glutamyltransferase (GGT), alkaline phosphatase 
(ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate 
dehydrogenase (LD). 
 
Hematology: Hemoglobin, red blood cell count, hematocrit, mean cell volume (MCV), mean 
cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelets, white cell 
count, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. 
 
In addition to the tests stated above, all female subjects with child-bearing potential will be 
tested for serum hCG (for exclusion #12/pregnancy). 


 
36 
 
Ampion™ 
Version 1.3    09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
 
 
TABLE 6.1 SCHEDULE OF ASSESSMENTS AND PROCEDURES 
 
 
Visits are in clinic except for Day 1 and Weeks 2 and 10 when subjects will be contacted by telephone 
1 
X-ray must be acquired at Screening to satisfy inclusion criteria #5.  X-rays obtained within 6 months of screening are acceptable. 
2 
At Week 12 and Early Termination visits, ask subject if they have previously sought out treatment for OAK prior to this study  
*Includes height and weight at Screening (Visit 1) and Week 12 (Visit 7) 
 
Screening 
VISIT 1 
Baseline 
Randomization 
Treatment 
VISIT 2 
Post-treatment 
check 
(telephone 
contact) 
VISIT 3 
Week 2 
(telephone 
contact) 
VISIT 4 
Week 6 
VISIT 5 
Week 10 
(telephone 
contact) 
VISIT 6 
Week 12 
Final Visit 
VISIT 7 
Early Termination 
Visit 
# Day 
1 
Day-7 to 0 
2 
Day 0 
3 
Day 1 
4 
Day 14 ± 7 
5 
Day 42 ± 7 
6 
Day 70 ±7 
7 
Day 84 ± 7 
Informed Consent 
X 
Inclusion/exclusion criteria 
X 
X 
Medical history/prior medications2 
X 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination* 
X* 
X 
X 
X* 
X* 
Vital Signs 
X 
X
=
X 
X 
X 
Randomization 
X 
WOMAC A-C, 5-point Likert Scale 
X 
X 
X 
X 
X 
X 
X 
Patient’s global assessment (PGA) 
X 
X 
X 
X 
X 
X 
X 
X-ray1 
X 
Clinical laboratory tests 
X 
 
X 
X 
Treatment with study drug 
X 
Rescue medication dispensed/collected 
X 
 
X 
X 
Review Rescue medication 
X 
X 
X 
X 
X 
X 
Adverse Events 
X 
X 
X 
X 
X 
X 
X 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
37 
 
 
 
7 
DISCONTINUATION CRITERIA 
 
7.1 
EARLY DISCONTINUATION OF THE STUDY 
 
It is agreed that for reasonable cause, either the investigator or the Sponsor may terminate 
this study, provided a written notice is submitted at a reasonable time in advance of intended 
termination; if by the investigator notice is to be submitted to Ampio Pharmaceuticals, Inc., 
and if by the Sponsor, notice will be provided to each investigator. 
 
If a severe local reaction or drug-related SAE occurs at any time during the study, the Safety 
Monitoring Committee will review the case immediately. 
 
The study will be immediately suspended and no additional Ampion treatments administered 
pending review and discussion of all appropriate study data by the SMC if 1 or more subjects 
develop any of the following adverse events deemed to be possibly, probably, or definitely 
related to Ampion by the Investigator and/or Medical Monitor, based upon close temporal 
relationship or other factors: 
 
• Death 
• Anaphylaxis (angioedema, hypotension, shock, bronchospasm, hypoxia, or 
respiratory distress) 
• Induction of autoimmune arthritis 
• Hepatic failure 
• Aplastic anemia. 
 
The study will not be restarted until all parties have agreed to the course of action to be taken 
and the IRB/EC has been notified. 
 
7.2 
EARLY DISCONTINUATION OF INDIVIDUAL SUBJECTS 
 
Subjects are to be withdrawn from the study for any of the following reasons: 
 
• Withdrawal of informed consent 
• Subject is lost to follow up. 
 
Subjects will also be withdrawn at any time if the investigator concludes that it would be in 
the subject’s best interest for any reason.  Protocol violations do not lead to subject 
withdrawal unless they constitute a significant risk to the subject’s safety. 
 
Subjects can voluntarily withdraw from the trial for any reason at any time. They are to be 
considered withdrawn if they state an intention to withdraw, fail to return for visits, or 
become lost to follow up for any reason. Subjects withdrawing from the study because of an 
AE should be followed for at least 30 days, resolution of the AE or until no further 
improvement is expected, whichever comes first. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
38 
 
 
8 
TREATMENT 
 
Eligible subjects will be randomized (6:1) to a single 4 mL Ampion intra-articular injection 
or a single 4 mL saline injection into the study knee. 
 
Subjects will be randomized to an assigned subject number after confirmation of 
eligibility, just prior to treatment. 
 
8.1 
DOSING AND ADMINISTRATION OF STUDY MEDICATION 
 
Appropriately trained site personnel should administer the study treatment. 
 
If both knees are osteoarthritic, then at Screening the Investigator should select the knee that 
best satisfies the requirements for the study (see inclusion criteria 6 - 7) as the study knee.  At 
the time of dose administration, the study knee should be treated with received 
investigational product in accordance with the randomization schedule.  The other knee 
should receive standard of care. 
 
Aspiration of the study knee should only occur if an effusion is present. Aspiration and 
volume of aspiration should be noted on the study source notes/eCRF. The study treatment 
should be administered as an injection into the knee joint space under sterile prep conditions, 
(i.e. prior to injection, the knee should be cleaned with an antiseptic).   
 
The recommended procedures is to administer the injection into the knee joint space with the 
subject in the seated position with the treatment knee flexed at 90°. The area of injection is 
inferior lateral to the patella; lateral level of the joint line.  
 
The Principal Investigator may determine whether anesthesia of the treatment area with a 
topical anesthesia or nothing is appropriate. Injection of lidocaine prior to the Ampion 
injection is not allowed.  
 
The needle should be passed through the fat pad to the firm surface of the intercondylar 
notch. Following the withdrawal of the needle, it is recommended that fingertip pressure be 
applied to the injection site, and then a sterile dressing (BandAid) is used to cover the 
injection site. Injection should proceed easily.  
 
The recommended needle for this injection is a 25-gauge needle that is 1.5 inches long. 
 
Failure to easily inject should be documented as a potential non-inter-articular injection. The 
injector and date of administration should be recorded in the study source notes/eCRF. 
 
Subjects should be advised to abstain from submersing their knee in water for at least 24 
hours (swimming pool, baths, lakes, ect.).  Showering is acceptable. 
 
8.2 
DRUG STORAGE AND ACCOUNTABILITY 
 
Study drug should be stored at room temperature (59º – 77ºF or 15º – 25ºC) in a secure area 
with restricted access and temperature monitoring.  A temperature monitor calibration must 
be provided to the CRO prior to receiving study drug. 
 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
39 
 
 
 
The Investigator, the clinical site staff, or other personnel authorized to store and dispense 
investigational product is responsible for ensuring that the investigational product used in the 
clinical study is securely maintained as specified by the Sponsor and in accordance with the 
applicable regulatory requirements. 
 
All investigational product is to be dispensed in accordance with the Investigator’s 
prescription and it is the Investigator’s responsibility to ensure that an accurate record is 
maintained of investigational product issued and returned. 
 
If any quality issue is noticed upon the receipt or use of an investigational product (i.e. 
deficiencies in condition, packaging, appearance, associated documentation, labeling, expiry 
date, temperature, etc.), Ampio Pharmaceuticals, Inc. must be promptly notified.  
Temperature monitors, and instructions to download the temperature data to Ampio 
Pharmaceuticals, Inc., will be included with each site shipment of study drug. 
 
Under no circumstances may the Investigator supply investigational product to a third party, 
allow the investigational product to be used other than as directed by this clinical study 
protocol, or dispose of investigational product in any other manner. 
 
All investigational product, used or unused during the study, must be retained in the study kit 
until notification from Ampio or designated representative. Ampio, or designated 
representative will instruct sites on study drug returns and/or destruction of study drug. 
 
8.3 
TREATMENT COMPLIANCE 
 
The injection of study drug will be performed by the Investigator or a designated member of 
the site clinical staff.  A 5mL syringe, a 25-gauge needle, and a bottle of rescue medication 
will be included in every subject kit.   
 
During monitoring visits, monitors will visually inspect each vial to ensure full injection 
volume was used.  Compliance with treatment is thus assured. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
40 
 
 
9 
ADVERSE EVENTS 
 
9.1 
DEFINITION OF AN ADVERSE EVENT 
 
An adverse event (AE) is defined as any undesired medical occurrence in a subject or clinical 
investigation subject receiving a pharmaceutical product and which does not necessarily have 
a causal relationship with this treatment. An AE can therefore be any unfavorable sign and 
unintended sign (including an abnormal laboratory finding), symptom, or disease temporarily 
associated with the use of a study drug, whether or not related to the study drug. 
 
Assessment of severity of an AE will be rated according to categories listed in TABLE 9.1. 
 
TABLE 9.1 
DEFINITIONS OF AE SEVERITY 
 
Grade 1 (MILD): 
The symptom is barely noticeable to the study subject and does not influence performance or functioning. 
Concomitant medication is not ordinarily indicated for relief of mild AEs. 
Grade 2 (MODERATE): 
The symptom is of sufficient severity to make the study subject uncomfortable and to influence performance 
of daily activities. Concomitant medication may be indicated for relief of moderate AEs. 
Grade 3 (SEVERE): 
The symptom causes severe discomfort, sometimes of such severity that the study subject cannot continue in 
the study. Daily activities are significantly impaired or prevented by the symptom. Concomitant medication 
may be indicated for relief of severe AEs. 
 
Determination of the relationship between the AE and the study drug will be made using the 
guidelines presented in TABLE 9.2. 
 
TABLE 9.2 GUIDELINES FOR DETERMINING THE RELATIONSHIP (IF ANY) 
BETWEEN ADVERSE EVENT AND THE STUDY DRUG 
 
Unrelated 
The adverse event is unlikely to have been caused by study drug. 
Possibly related 
It is unclear whether the adverse event may have been caused by study drug. 
Related 
The adverse event is likely to have been caused by study drug. 
 
 
9.2 
DEFINITION OF A SERIOUS ADVERSE EVENT 
 
A Serious Adverse Event (SAE) is any untoward medical occurrence that occurs at any dose 
that: 
 
• Results in death 
• Is life-threatening (subject is at immediate risk of death from the event as it occurred) 
• Requires in-patient hospitalization (formal admission to a hospital for medical reasons) or 
prolongation of existing hospitalization 
• Results in persistent or significant disability/incapacity 
• Results in a congenital anomaly/birth defect 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
41 
 
 
Important medical events that may not result in death, are not life-threatening, or do not 
require hospitalization may be considered SAEs when, based on appropriate medical 
judgment, they may jeopardize the subject and may require medical or surgical intervention 
to prevent one of the outcomes listed in this definition.  Examples of such events include 
allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood 
dyscrasias or convulsions that do not result in in-patient hospitalization, or the development 
of drug dependency or drug abuse. 
 
Hospitalizations for elective surgery or other medical procedures that are not related to a 
treatment-emergent AE are not considered SAEs. 
 
9.3 
RECORDING OF ADVERSE EVENTS AND SERIOUS ADVERSE 
EVENTS 
 
Recording and reporting of adverse events should be in accordance with the FDA final 
“Guidance for Industry and Investigators Safety Reporting Requirements for INDs and 
BA/BE Studies” of December 2012. 
 
Any AE is to be recorded in the eCRF. In order to avoid vague, ambiguous, or colloquial 
expressions, the AE should be recorded in standard medical terminology rather than the 
subject’s own words.  Whenever possible, the investigator should combine signs and 
symptoms that constitute a single diagnosis. 
 
The existence of an AE may be concluded from a spontaneous report of the subject; from the 
physical examination; or from special tests e.g., laboratory assessments, where applicable, or 
other study-specified tests (source of AE). 
 
The reporting period begins from the time that the subject receives IA injection at the 
Baseline visit through subject’s Final Visit at 12 weeks. Any events continuing at study exit 
will be followed for 30 days or to resolution, or until no improvement is expected, whichever 
comes first. Any SAE occurring after the reporting period must be promptly reported if a 
causal relationship to the investigational drug is suspected. If the subject begins a new 
therapy, the safety reporting period ends at the time the new treatment is started, however, 
death must always be reported when it occurs during the 12-week study period, irrespective 
of intervening treatment. 
 
Each AE is to be evaluated for duration, severity, seriousness, and causal relationship to the 
investigational drug.  The action taken and the outcome must also be recorded. 
 
9.3.1 
AE Follow up 
 
All AEs occurring during the study are to be followed up in accordance with good medical 
practice until they are resolved, stabilized or judged no longer clinically significant or, if a 
chronic condition, until fully characterized.  Any AEs that are considered drug-related 
(possibly related, definitely related) must be followed for 30 days, or to resolution, or until no 
improvement is expected, whichever occurs first. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
42 
 
 
9.3.2 
Overdose 
 
No information on treatment of overdose of Ampion is currently available. However, it is 
common medical practice to administer 5% HSA, the sole starting material of Ampion, in 
multiple infusions until hemorrhage is controlled thereby resulting in higher systemic 
concentrations of the significant components of Ampion.  
 
In the case of overdose, the subject should be followed as for an AE and appropriate 
supportive medical treatment instigated. 
 
9.4 
SERIOUS ADVERSE EVENT REPORTING 
 
9.4.1 
Reporting Requirements 
 
Unexpected serious suspected adverse reactions are subject to expedited reporting to FDA. 
ALL SAEs must be entered into the eCRF within 24 hours of first knowledge of the event by 
study personnel. It is important that the investigator provide his/her assessment of 
relationship to study drug at the time of the initial report. Entry of an SAE into the eCRF 
triggers an automatic alert to the CRO safety team. The following information must be 
reported: 
 
• Protocol number 
• Site and/or Investigator number 
• Subject number 
• Demographic data 
• Brief description of the event 
• Onset date and time 
• Resolution date and time, if the event resolved 
• Current status, if event not yet resolved 
• Any concomitant treatment and medication 
• Investigator’s assessment of whether the SAE was related to Investigative product or 
not. 
 
The CRO Safety Associate will contact the site for clarification of data entered onto the 
eCRF, or to obtain missing information.  In the event of questions regarding SAE reporting, 
the site may contact the appropriate individual as in Section 9.4.2. 
 
9.4.2 
SAE Information 
 
Ampio Pharmaceuticals Inc., or their designee CRO, is responsible for submitting reports of 
AEs associated with the use of the drug that are both serious and unexpected to FDA 
according to 21 CFR 312.32 and the final guidance (2012). All investigators participating in 
ongoing clinical studies with the study medication will receive copies of these reports for 
prompt submission to their Institutional Review Board (IRB) or Ethics committee (EC). 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
43 
 
 
10 STATISTICAL METHODS 
 
10.1 GENERAL CONSIDERATIONS 
 
This section describes the rules and conventions to be used in the presentation and analysis of 
the data.  A comprehensive presentation of the data management and statistical analysis plan 
will be approved by Ampio Pharmaceuticals, Inc., prior to study completion. 
 
10.1.1 
Statistical and Analytical Plan: 
 
This is a randomized, confirmatory study in subjects with severe OA (KL 4) of the knee. 
Subjects will be followed over a 12-week treatment period to assess the efficacy of 
Ampion™ on changes in pain, stiffness and function, and patient satisfaction (PGA) and to 
assess the safety of Ampion™ by monitoring adverse events. 
 
A within-patient, multi-component endpoint, OMERACT-OARSI responder analysis, 
evaluating pain, function, and PGA, will be used as primary analysis for this study. 
The saline group in this study is designed to maintain a blind the study is not powered to 
make inference about patients receiving saline; therefore, the results from patients receiving 
saline will be summarized but no comparisons or inferences will be made. 
 
10.2 STATISTICAL OBJECTIVES 
 
The primary statistical objective is to evaluate the efficacy of a single intra-articular injection 
of Ampion. Efficacy will be evaluated by the percentage of patients who are considered an 
OMERACT-OARSI responder at 12 weeks after a single 4 mL Ampion™ intra-articular 
injection at Baseline. 
OMERACT-OARSI response is evaluated by: 
A patient in this study will be considered a responder for the purpose of efficacy analysis if the 
following criteria is met. 
1) The patient has:  
a. a percent improvement in pain (WOMAC pain) from baseline of ≥50% and an 
absolute change in pain from baseline of ≥ 1 point; or  
b. a percent improvement in function (WOMAC function) from baseline of ≥50% and 
an absolute change in function from baseline of ≥1 point.   
Meeting one of the above criterion designates the patient as a responder.  If the patient does 
not meet this criterion, then the patient must meet the criterion below to be considered a 
responder. 
2) The patient demonstrates improvement in at least 2 of the following: 
a. Improvement in pain (WOMAC pain) over baseline of ≥20% and a 0.5-point 
absolute change in pain 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
44 
 
b. Improvement in function (WOMAC function) over baseline of ≥20% and a 0.5-
point absolute change in function 
c. Improvement in patient global assessment (PGA) over baseline of ≥20% and a 0.5-
point absolute change in PGA 
The second statistical objectives are to: 
 
˗ Evaluate the improvement in the composite endpoint of pain and function (WOMAC A 
and WOMAC C) from Baseline to Week 12  
- 
Evaluate the improvement in patient global assessment (PGA) from Baseline to Week 
12  
- 
Evaluate improvement in the composite endpoint of pain and function (WOMAC A and 
C) compared to saline from all single-injection Ampion studies. 
 
The composite endpoint of pain and function (WOMAC A and WOMAC C) will be evaluated 
using a “controlled” OMERACT-OARSI responder criteria, defined as: 
 
 Improvement in pain and function (WOMAC A and WOMAC C) over baseline of 
≥20% and a 0.5 point absolute change 
Improvement in patient global assessment will also follow OARSI’s recommendation for a 
clinically meaningful improvement, defined as: 
 
 Improvement in patient global assessment (PGA) over baseline of ≥20% and a 0.5 
point absolute change in PGA 
 
10.3 ANALYSIS POPULATIONS 
 
10.3.1 
Safety Analysis Population: 
 
The safety analysis population is defined as all subjects who receive study medication 
(Ampion or saline). Subjects will be analyzed as treated. 
 
10.3.2 
Intent-to-treat Population: 
 
The intent-to-treat (ITT) analysis population is defined as all subjects who are randomized, 
regardless of whether or not they have received the investigational product, or have a post- 
baseline visit. All efficacy analyses will be performed in the ITT population. Subjects will 
be analyzed as treated. 
 
10.3.3 
Per Protocol Population: 
 
The per protocol analysis population is defined as all subjects included in the ITT analysis 
who met all entry criteria, completed the Week 12 visit, and had no major protocol 
violations. All efficacy analyses will be repeated in the per-protocol population. These 
analyses will be supportive of the ITT analysis. Subjects will be analyzed as treated. 
 
10.3.4 
Hypotheses Tested 
 
For the primary endpoint of the proportion of patients who are responders according to the 
OMERACT-OARSI criteria, the following hypothesis will be tested. 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
45 
 
 
H0:π ≤ π0 versus HA:π > π0      
 
Where π0 is the hypothesized value for the proportion of responders.  The value will be 30% 
in this study.  This test will be tested using an exact binomial test.  That is, given the sample 
size of n, the number of responders X, and the value of π0 =0.30, then probability that X or 
more events would be observed will be calculated as the p-value.  Since this is a one sided 
test, the alpha level will be 0.025. 
 
The primary hypothesis and these secondary hypotheses will be tested in a hierarchical 
manner with the primary first and the secondary hypotheses in the order listed.  Using this 
approach, there is no adjustment necessary in the overall significance level.  All testing will 
be conducted using an overall significance level of 5%.  If the tests are one sided, the 
significance level will be 2.5%. 
 
The secondary endpoints are represented by the proportion of patients who are responders as 
identified by the following response criteria: 
 
- 
Improvement in pain and function (WOMAC A and WOMAC C) over baseline of 
≥20% and a 0.5 point absolute change 
 
- 
Improvement in patient global assessment (PGA) over baseline of ≥20% and a 0.5 point 
absolute change in PGA 
 
- 
Improvement in pain and function (WOMAC A and WOMAC C) over baseline of 
≥20% and a 0.5 point absolute change compared to saline  
The hypotheses for the first two secondary endpoints are identical and are: 
 
H0:π ≤ 30% versus HA:π > 30%      
 
These will be tested using the exact binomial test. 
 
The hypothesis for the third secondary endpoint will be tested as: 
 
H0:πA ≤ πHS versus HA: πA > πHS 
 
Where πA is the response rate from Ampion in this study and πHS is the response rate of the 
saline control from previous single-injection Ampion studies.  This will be tested using 
Fisher’s exact test. 
 
10.3.5 
Exploratory Endpoints 
 
None 
 
10.3.6 
Definition of Study Visits 
 
This clinical trial has a total of 7 study visits, including telephone contacts, during the 12-
week study (see Table 6.2).  The time on study for each subject observation will be defined 
relative to Day 0/Baseline, the day of the initial dose. For analysis, the Baseline measure is 
the latest measure prior to initiation of treatment. 
 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
46 
 
10.3.7 
Number of subjects to receive study drug 
 
A total of approximately 171 subjects (146 Ampion; 25 Saline) will be enrolled in the study 
to a single (1) intra-articular injection of 4 mL Ampion or 4 mL saline (6:1). 
 
Since the sample size for adequate power of the primary and second secondary hypotheses 
is almost identical given the anticipated results of previous studies, the sample size for this 
study is based on the following hypothesis.  
 
H0:π ≤ 30% versus HA:π > 30%     
 
A sample size of 146 patients yields a greater than 90% power of rejecting the null 
hypothesis when the anticipated proportion under the alternative hypothesis is 45%.  
 
10.3.8 
Disposition of subjects 
 
Disposition of subjects, including study completion status and response to therapy as 
measured by WOMAC A, WOMAC C and PGA subscores, will be summarized by age 
group, race, and gender for each of the analysis populations. 
 
10.3.9 
Interim analysis 
 
There will be no interim analysis. 
 
10.3.10 Blinding and randomization 
 
All subjects will be randomized 6:1 to receive Ampion 4 mL or saline 4 mL as an intra-
articular injection in the study knee. 
 
A comprehensive presentation of the data management and statistical analysis plan will be 
approved by Ampio Pharmaceuticals, Inc. prior to final analysis to adjust for any changes to 
the protocol or unexpected issues in the study conduct and data that affect the planned 
analysis. 
 
10.3.11 Data presentation 
 
10.3.11.1 Demographic and Baseline Characteristics: 
 
Demographic (e.g., age, sex, race, and ethnicity) and Baseline characteristics (e.g., weight, 
height, prior injection of another intra-arterial therapeutic for OA of the knee) summarized 
using descriptive statistics, overall and by treatment group for the ITT analysis population. 
 
10.3.11.2 Medical History and Physical Examination: 
 
The number and percent of subjects with past and current medical disorders at the time of 
randomization will be presented overall for the ITT analysis population. Results of any 
abnormalities documented from the abbreviated physical examination at Baseline and Week 
12, will be summarized overall for the safety and ITT analysis populations. 
 
10.3.11.3 Concomitant Medications or Treatments: 
 
The number and percent of subjects receiving concomitant medications or treatments prior to 
and during the study and at the final visit will be tabulated and presented overall for the ITT 


Ampion™ 
Version 1.3 09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
47 
 
analysis population.  Concomitant medications/treatments will be summarized using 
descriptive statistics and will be presented by type of drug (WHO DRUG classification) 
overall for the safety and ITT analysis populations. 
 
10.3.11.4 Safety data: 
 
Safety data will be evaluated by changes in vital sign measurements, physical exam, 
laboratory analysis, and the frequency and severity of AEs. Concomitant medication will 
be recorded for safety. 
 
Adverse events: 
 
The Investigator is responsible for monitoring the safety of subjects who have enrolled in the 
study. All AEs considered related, or possibly related to Ampion™, will be followed until 
the event resolves or stabilized without further change. Subjects will be followed for the 
occurrence of AEs until 12 weeks after the first dose of study medication. 
 
Investigators are required to document all AEs occurring during the clinical trial, 
commencing with the first day of treatment through the end of study. AEs should be 
properly documented on the appropriate CRF pages. 
 
The severity of AEs (mild, moderate, severe), relatedness (related, possibly related, 
unrelated) along with the duration, action taken, and outcome (e.g., study withdrawal) will 
also be recorded.  In addition, events meeting the criteria of a Serious Adverse Event (SAE) 
must be reported to the Sponsor within 24 hours on the SAE reporting forms. 
 
10.4 MISSING DATA 
 
All data collected under this study protocol will be included in the assessment of subject 
safety.  Missing or incomplete AE data will assume greatest relationship to study drug and/or 
severity. 
 
For the primary effectiveness endpoint (WOMAC A, WOMAC C, and PGA), missing Week 
12 values will be imputed when the ITT analysis population is used. Worst Observation 
Carried Forward (WOCF) will be selected as the primary method of imputing missing 12 
Week data. Alternate imputation methods employed will include multiple imputations (SAS 
PROC MI and SAS PROC MIANALYZE) and Last Observation Carried Forward (LOCF). 
These sensitivity analyses will be conducted for the primary effectiveness endpoint, to ensure 
that the primary method of imputation chosen is robust with respect to imputation method 
used. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
48 
 
 
11 REGULATORY, ETHICAL AND LEGAL OBLIGATIONS 
 
11.1 DECLARATION OF HELSINKI 
 
The Principal Investigator will ensure that this Study is conducted in accordance with the 
most recent revision of the Declaration of Helsinki. 
 
11.2 GOOD CLINICAL PRACTICE 
 
The Study will be conducted according to the study protocol and to Standard Operating 
Procedures (SOPs) that meet the guidelines provided by the International Conference on 
Harmonisation (ICH) for Good Clinical Practice in clinical studies. 
 
11.3 INSTITUTIONAL REVIEW BOARDS/ETHICS COMMITTEES 
 
Before implementing this study, the protocol, the proposed subject informed consent forms 
and other information for the subjects, must be reviewed by a properly constituted committee 
or committees responsible for approving clinical studies. The IRB/IEC written, signed 
approval letter/form must contain approval of the designated investigator, the protocol 
(identifying protocol title, date and version number), and of the subject informed consent 
form (date, version). 
 
 
Any change or addition to the protocol can only be made in a written protocol amendment, 
which must be approved by the Sponsor, the IRB/IEC and the Health Authorities. 
 
11.4 REGULATORY AUTHORITY APPROVAL 
 
Before this study is implemented, the protocol must be approved by the relevant regulatory 
authority. 
 
11.5 INFORMED CONSENT 
 
The investigator must fully inform the subject of all pertinent aspects of the trial including 
the written information approved/favorably assessed by the IRB/IEC. 
 
Prior to the start of the pre-study examination, the written informed consent form must be 
signed and personally dated by the subject and by the physician who conducted the informed 
consent discussion. One copy of the written information and signed consent form must be 
given to each subject and 1 copy must be retained in the investigator's study records. 
 
11.6 SUBJECT CONFIDENTIALITY AND DISCLOSURE 
 
Data on subjects collected on eCRFs during the trial will be documented in an anonymous 
fashion and the subject will only be identified by the subject number, and by his/her initials 
If, as an exception, it is necessary for safety or regulatory reasons to identify the subject, all 
parties are bound to keep this information confidential. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
49 
 
 
The investigator will guarantee that all persons involved will respect the confidentiality of 
any information concerning the trial subjects. All parties involved in the study will maintain 
strict confidentiality to assure that neither the person nor the family privacy of a subject 
participating in the trial is violated.  Likewise, the appropriate measures shall be taken to 
prevent access of non-authorized persons to the trial data. 
 
11.7 COLLECTION, MONITORING AND AUDITING STUDY 
DOCUMENTATION, AND DATA STORAGE 
 
11.7.1 
Collection of Data and Monitoring Procedures 
 
This study will use a 21 CFR Part 11 compliant electronic data capture system (eDC). An 
electronic case report form (eCRF) is used for data recording. All data requested on the 
eCRF must be entered and all missing data must be accounted for. 
 
The data will be checked for completeness and correctness as it is entered by the real-time 
online checks applied by the eDC system. Off-line checks will also be run to perform any 
additional data review required.  Discrepancy reports will be generated accordingly and 
transferred to the study center for resolution by the investigator or his/her designee. 
 
Accurate and reliable data collection will be assured by verification and cross–check of the 
eCRF against the investigator’s records by the study monitor (source document verification), 
and the maintenance of a study drug–dispensing log by the investigator. 
 
Before study initiation, at a site initiation visit or at an investigator’s meeting, a Sponsor 
representative will review the protocol and case report forms with the investigators and their 
staff.  During the study a monitor will visit the site regularly to check the completeness of 
subject records, the accuracy of entries on the case report forms, the adherence to the 
protocol and to Good Clinical Practice and the progress of enrollment. The monitor will 
ensure during on-site visits that study medication is being stored, dispensed and accounted 
for according to specifications. Key trial personnel must be available to assist the monitors 
during these visits. 
 
The investigator must give the monitor access to relevant hospital or clinical records, to 
confirm their consistency with the case report form entries.  No information in these records 
about the identity of the subjects will leave the study center. Monitoring standards require 
full verification for the presence of informed consent, adherence to the inclusion/exclusion 
criteria, documentation of SAEs and the recording of primary efficacy and safety variables. 
Additional checks of the consistency of the source data with the eCRFs are performed 
according to the study-specific monitoring plan. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
50 
 
 
11.7.2 
Auditing Procedure 
 
In addition to the routine monitoring procedures the Sponsor or the regulatory authority can 
conduct an audit or an inspection (during the study or after its completion) to evaluate 
compliance with the protocol and the principles of Good Clinical Practice. 
 
The investigator agrees that representatives of the Sponsor and Regulatory Authorities will 
have direct access, both during and after the course of this study, to audit and review all 
study-relevant medical records. 
 
11.7.3 
Retention of Documents 
 
The investigator must maintain source documents for each subject in the study, consisting of 
all demographic and medical information, including laboratory data, etc., and keep a copy of 
the signed informed consent form. All information on case report forms must be traceable to 
these source documents in the subject's file. Data without a written or electronic record will 
be defined before trial start and will be recorded directly on the case report forms, which will 
be documented as being the source data. 
 
11.8 DISCLOSURE OF INFORMATION 
 
All information provided to the investigator by Ampio Pharmaceuticals, Inc. or their 
designee, will be kept strictly confidential. No disclosure shall be made except in accordance 
with a right of publication granted to the investigator. 
 
No information about this study or its progress will be provided to anyone not involved in the 
study other than to Ampio Pharmaceuticals, Inc. or its authorized representatives, or in 
confidence to the IRB, or similar committee, except if required by law. 
 
11.9 DISCONTINUATION OF THE STUDY 
 
It is agreed that, for reasonable cause, either the investigator or Ampio Pharmaceuticals, Inc., 
may terminate the investigator’s participation in this study after submission of a written 
notice.  Ampio Pharmaceuticals, Inc., may terminate the study at any time upon immediate 
notice for any reason, including the Sponsor’s belief that discontinuation of the study is 
necessary for the safety of subjects. 
 
11.10 STUDY REPORT, PUBLICATION POLICY AND ARCHIVING OF 
STUDY DOCUMENTATION 
 
11.10.1 Study Report and Publication Policy 
 
An ICH-compliant integrated clinical and statistical report will be prepared upon completion 
of the study and data analysis. The results of the study will be published in a relevant peer- 
reviewed journal, with authorship status and ranking designated according to the 
acknowledged contributions of participating investigators, institutions and the Sponsor. 


Ampion™ 
Version 1.3         09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
51 
 
 
11.10.2 Study Documents 
 
The investigator must maintain source documents for each subject in the study, consisting of 
all demographic and medical information, questionnaires, including laboratory data, 
radiology data (as applicable), etc., and keep a copy of the signed informed consent form.  
All information on the e-case report forms must be traceable to these source documents in 
the subject's file. 
 
Data without a written or electronic record will be defined before trial start and will be 
recorded directly on the e-case report forms, which will be documented as being the source 
data. 
 
11.10.3 Archiving of Documents 
 
Essential documents, as listed below, must be retained by the investigator for as long as 
needed to comply with national and international regulations. The Sponsor will notify the 
investigator(s)/institution(s) when the study-related records are no longer required. The 
investigator agrees to adhere to the document retention procedures by signing the protocol. 
Essential documents include: 
 
• IRB/IEC/REB approvals for the study protocol and all amendments 
• All source documents and laboratory records 
• CRF copies (electronic copies on a CDROM) 
• Subjects' informed consent forms (with study number and title of trial) 
• FDA form 1572 
• Any other pertinent study documents. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
52 
 
 
12 REFERENCES 
 
1. Akinci A. Predictive factors and clinical biomarkers for treatment in patients with 
chronic pain caused by osteoarthrosis with a central sensitization component. 
International Journal of Clinical Practice. 2016; 70; 31-44. 
 
2. Bar-Or D. The filamentous actin cytoskeleton organization and the endothelial cell 
barrier. Crit Care Med. 2013 Feb;41(2):686-7. 
 
3. Bar-Or D, Bar-Or R, Rael LT, et al. Heterogeneity and oxidation status of commercial 
human albumin preparations in clinical use. Crit Care Med. 2005;33:1638-41. 
 
4. Bar-Or D, Thomas GW, Bar-Or R, et al. Commercial human albumin preparations for 
clinical use are immunosuppressive in vitro. Crit Care Med. 2006;34:1707-12. 
 
5. Bar-Or D, Rael LT, Thomas GW, Brody EN. Inflammatory pathways in knee 
osteoarthritis: potential targets for treatment. Curr Rheumatol Rev. 2015. 
 
6. Bar-Or D, Thomas GW, Rael LT, Gersch ED, Rubinstein P, Brody E. Low Molecular 
Weight Fraction of Commercial Human Serum Albumin Induces Morphologic and 
Transcriptional Changes of Bone Marrow-Derived Mesenchymal Stem Cells. Stem Cells 
Translational Medicine. 2015; 4: 1-11. 
 
7. Bar-Or D, Slone DS, Mains CW, Rael LT. Dipeptidyl peptidase IV activity in 
commercial solutions of human serum albumin. Anal Biochem. 2013 Oct 1;441(1):13-7. 
 
8. Bellamy N, Buchanan WW, Goldsmith CH et al.  Validation study of WOMAC: a health 
status instrument for measuring clinically important patient relevant outcomes to 
antirheumatic drug therapy in subjects with osteoarthritis of the hip or knee. J Rheumatol. 
1988 Dec;15(12):1833-40. 
 
9. Cisternas 2016, Royal College of Physicians of London, (1993). // Center for Disease 
Control and Prevention, Arthritis // Litwic, A., Dennison, E., Cooper, C. 2013, 
Epidemiology and Burden of Osteoarthritis. Br Med Bull. (2013); 105: 185-199. 
 
10. Dillon CF, Rasch EK, Gu Q, Hirsch R. Prevalence of knee osteoarthritis in the United 
States: arthritis data from the Third National Health and Nutrition Examination Survey 
1991-94. J Rheumatol. 2006 Nov;33(11):2271-9. 
 
11. Dworkin et al. The Journal of Pain, Vol. 9, No 2, 2008 
 
12. Evans TW. Review article: albumin as a drug – biological effects of albumin unrelated to 
oncotic pressure. Aliment Pharmacol Ther. 2002;16 (Suppl. 5); 6-11. 
 
13. Felson DT, Anderson JJ, Naimark A, et al.  Obesity and knee osteoarthritis: The 
Framingham study. Ann Int Med. 1988;109:18-24 
 
14. Frederick ED, Hausburg MA, Thomas GW, Rael LT, Brody E, Bar-Or D. The low 
molecular weight fraction of human serum albumin upregulates COX2, prostaglandin E2, 
and prostaglandin D2 under inflammatory conditions in osteoarthritic knee synoviocytes. 
Biochemistry and Biophysics Reports. 2016; (8), pp. 68-74. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
53 
 
 
15. Greco et al. The American Journal of Sports Medicine, Vol. 38, No. 5. 2010 
 
16. Guccione AA. Arthritis and the process of disablement. Phys Ther. 1994 May;74(5):408- 
14. 
 
17. Kraus V. Direct in vivo evidence of activated macrophages in human osteoarthritis. 
Osteoarthritis and Cartilage. 2016; 24(9):1613-1621. 
 
18. Mallinckrodt CH, Clark WS, David SR. Accounting for dropout bias using mixed-effects 
models. J Biopharm Stat. 2001 Feb-May;11(1-2):9-21. 
 
19. Navarro-Sarabia F, Coronel P, Collantes E, Navarro FJ, de la Serna AR, Naranjo A, 
Gimeno M, Herrero-Beaumont G; AMELIA study group.  A 40-month multicentre, 
randomized placebo-controlled study to assess the efficacy and carry-over effect of 
repeated intra-articular injections of hyaluronic acid in knee osteoarthritis: the AMELIA 
project. Ann Rheum Dis. 2011;70:1957-62. 
 
20. Ohnuma K, Inoue H, Uchiyama M, Yamochi T, Hosono O, Dang NH, Morimoto C. T- 
cell activation via CD26 and caveolin-1 in rheumatoid synovium. Mod Rheumatol. 
2006;16(1):3-13. 
 
21. Peterfy C.G., M.D. Ph.D., Guermazi A M.D., S. Zaim M.D., P. F. J. Tirman M.D., Y. 
Miaux M.D., D. White Ph.D., M. Kothari Ph.D., Y. Lu Ph.D., K. Fye M.D., S. Zhao 
Ph.D. H. K. Genant M.D.  Whole-Organ Magnetic Resonance Imaging Score (WORMS) 
of the knee. OsteoArthritis and Cartilage (2004) 12, 177–190. 
 
22. Quinlan GJ, Martin GS, Evans TW. Albumin: biochemical properties and therapeutic 
potential. Hepatology. 2005;41:1211-1219. 
 
23. Rutjes A, Juni P, et al. Viscosupplementation for Osteoarthritis of the Knee. Annals of 
Internal Medicine. 2012; 157:180-191. 
 
24. Shimonkevitz R, Thomas GW, Slone DS, et al. A diketopiperazine fragment of human 
serum albumin modulates T-lymphocyte cytokine production through Rap1. J Trauma. 
2008;64:35-41. 
 
25. Thomas GW, Rael LT, Hausburg M, Frederick ED, Brody E, Bar-Or D. The Low 
Molecular Weight Fraction of Commercial Human Serum Albumin Induces Acetylation 
of α-Tubulin and Reduces Transcytosis in Retinal Endothelial Cells. Biochemical and 
Biophysical Research Communications. 2016 Sep 30;478(4):1780-5. 
 
26. Thomas GW, Rael LT, Hausburg M, Frederick ED, Mains CW, Slone D, Carrick MM, 
Bar-Or D. The low molecular weight fraction of human serum albumin upregulates 
production of 15d-PGJ(2) in Peripheral Blood Mononuclear Cells. Biochem Biophys Res 
Commun. 2016 May 13;473(4):1328-33. Epub 2016 Apr 16. 
 
27. Thomas GW, Rael LT, Mains CW, Slone D, Carrick MM, Bar-Or R, Bar-Or D. Anti- 
Inflammatory Activity in the Low Molecular Weight Fraction of Commercial Human 
Serum Albumin (LMWF5A). J Immunoassay Immunochem. 2016;37(1):55-67. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
54 
 
 
28. Vincent JL, Navickis RJ, Wilkes MM. Morbidity in hospitalized subjects receiving 
human albumin: A meta-analysis of randomized, controlled trials* Crit Care Med. 
2004;32:2029–38. 
 
29. Zhang W, Nuki G, Moskowitz R.W. et al.  OARSI recommendations for the management 
of hip and knee osteoarthritis Part III: changes in evidence following systematic 
cumulative update of research published through January 2009. Osteoarthritis and 
Cartilage. 2010;18:476-499. 


Ampion™ 
Version 1.3          09 November 2017 
Clinical Study Protocol: AP-003-C 
Confidential 
 
 
55 
 
 
13 APPENDICES 
 
13.1 CONTACT LIST 
 
SPONSOR: 
Ampio Pharmaceuticals, Inc. 
373 Inverness Parkway, Suite 200 
Englewood, CO 80112 
720-437-6500 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

SAP_001.pdf: 
 
 
 
 
A PHASE 3 RANDOMIZED STUDY TO 
CONFIRM THE EFFICACY OF AN INTRA- 
ARTICULAR INJECTION OF AMPION™ IN 
ADULTS WITH PAIN DUE TO SEVERE 
OSTEOARTHRITIS OF THE KNEE 
 
STATISTICAL ANALYSIS PLAN 
 
STUDY NUMBER: AP-003-C 
 
NCT 03182686 
 
14 November 2017 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 1 of 18 
 
  
CONFIDENTIAL 
  
 
 
 
STATISTICAL ANALYSIS PLAN APPROVAL SHEET 
Protocol Number:  AP-003-C 
Product: Ampion™ 
Protocol Title: A Phase 3 Randomized Study to Confirm the Efficacy of an 
Intra-Articular Injection of Ampion™ in Adults with Pain Due to Severe 
Osteoarthritis of the Knee 
  
 
Author:  Gary Stevens, Ph.D 
Version:  2.00 
Version date: November 14, 2017 
  
Approved by: 
 
 
 
Name                                                                             Date 
 
 
 
Name                                                                             Date 
 
 
 
Name                                                                             Date 
 
 
 
 
 
  
 
 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 2 of 18 
 
  
CONFIDENTIAL 
  
 
STATISTICAL ANALYSIS PLAN 
A Phase 3 Randomized Study to Confirm the Efficacy of an Intra-Articular 
Injection of Ampion™ in Adults with Pain Due to Severe Osteoarthritis 
of the Knee Protocol Number AP-003-C 
Version: 
 
2.00 
 
Date: 
14 November 2017 
 
Biostatistician: 
Gary Stevens, Ph.D. 
  
 
 
 
 
 
 
 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 3 of 18 
 
  
CONFIDENTIAL 
  
 
Table of Contents 
 
1. 
INTRODUCTION .................................................................................................... 5 
2. 
OBJECTIVES ......................................................................................................... 5 
2.1 Primary Objective ........................................................................................... 5 
2.2 Secondary Objective ...................................................................................... 5 
2.3 Study Design .................................................................................................. 5 
2.4 Sample Size ................................................................................................... 6 
3. 
PRIMARY STUDY ENDPOINTS ............................................................................. 6 
3.1 Primary Endpoint ............................................................................................ 6 
3.2 Secondary Endpoints ..................................................................................... 7 
4. 
HYPOTHESES AND METHODS ............................................................................ 8 
4.1 Primary Hypothesis ........................................................................................ 8 
4.2 Secondary Hypotheses .................................................................................. 8 
4.3 Exploratory Analysis ....................................................................................... 9 
4.4 Definition of Study Visits ................................................................................. 9 
4.5 Number of subjects to receive study drug....................................................... 9 
4.6 Disposition of subjects .................................................................................... 9 
4.7 Interim analysis .............................................................................................. 9 
4.8 Blinding and randomization ............................................................................ 9 
4.9 Data presentation ......................................................................................... 10 
4.9.1 
Demographic and Baseline Characteristics: .................................... 10 
4.9.2 
Medical History and Physical Examination: ..................................... 10 
4.9.3 
Concomitant Medications or Treatments: ........................................ 10 
4.9.4 
Safety data: ..................................................................................... 10 
4.10 Missing Data ................................................................................................ 11 
4.11 Detection of Bias .......................................................................................... 11 
4.12 Outliers ........................................................................................................ 11 
4.13 Testing/Validation Plan................................................................................. 11 
5. 
DEFINITIONS ....................................................................................................... 11 
5.1 Abbreviations ............................................................................................... 11 
5.2 Definitions .................................................................................................... 12 
6. 
CHANGES FROM PROTOCOL-SPECIFIED ANALYSES .................................... 14 
7. 
LIST OF PLANNED TABLES, FIGURES, AND LISTINGS .................................... 14 
8. 
LITERATURE CITATIONS  REFERENCES ........................................................ 16 
9. 
HANDLING OF MISSING OR INCOMPLETE DATES FOR ADVERSE 
EVENTS AND CONCOMITANT MEDICATIONS .................................................. 16 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 4 of 18 
 
  
CONFIDENTIAL 
  
 
9.1 Imputation Rules for Partial or Missing Stop Dates ....................................... 16 
10. Schedule of Assessments and Procedures ........................................................... 18 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 5 of 18 
 
  
CONFIDENTIAL 
  
 
1. 
INTRODUCTION 
This statistical analysis plan (SAP) outlines the proposed statistical methods to be 
implemented during the review and analysis of the data to ensure confirmation with 
categories determined by the CRF or the anticipated ranges for continuous variables and 
analysis of data collected within the scope of Ampion Protocol AP-003-C, “A Phase 3 
Randomized Study to Confirm the Efficacy of an Intra-Articular Injection of Ampion™ 
in Adults with Pain Due to Severe Osteoarthritis of the Knee,” protocol version 1.3 dated 
09 November 2017. 
It is not intended that each and every table, listing, or graph will be included in the 
clinical study report (CSR). It is also possible that additional analyses will be conducted 
after review of the data.  Any analyses or summaries not specified in the SAP, but 
performed after review of the data, will be identified in the CSR as post hoc. 
2. 
OBJECTIVES 
 
2.1 Primary Objective 
 
The primary trial objective is to evaluate the clinical efficacy of a single intra-articular 
injection (4 mL) of Ampion. 
2.2 Secondary Objective 
 
The secondary trial objectives is to evaluate the safety of a single intra-articular injection 
(4 mL) of Ampion. 
2.3 Study Design 
This is a randomized, double-blind study with a 7-day screening period for each patient 
followed by a 12-week participation period.  A total of 171 subjects with osteoarthritis of 
the knee (OAK) will be randomized 6:1 across 2 study arms, 4 mL Ampion™ and 4 mL 
saline. Deferred treatment of pain in the most severe form of the disease is unacceptable 
to both patients and physicians, and a larger saline arm is not possible or necessary for 
true comparator purposes in severe OAK subjects. The saline arm is for control of 
unintended bias and is not intended for analysis; only the active Ampion arm will be 
analyzed for efficacy.  
Severe OAK is a painful, incurable, and progressive disease with a highly predictable 
outcome. Methodological challenges exist in this debilitated patient population where an 
unmet medical need exists and there is no valid, licensed or approved control or therapy 
for severe OAK patients. Therefore, in addition to the 6:1 randomization that is in place 
to eliminate any unintended bias, the study plan will assess a historic control using all the 
severe OAK saline-treated patients from all previously completed, randomized, saline-
controlled, single-injection Ampion studies. The historic control for this clinical trial has 
a similar demographic and concomitant treatment profile to the current study, which 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 6 of 18 
 
  
CONFIDENTIAL 
  
 
allows an effective comparison. Analysis of Ampion and its comparison to this historic 
saline-control group will ensure a meaningful comparison and quantitative assessment of 
Ampion effect, in addition to the demonstration of an effect of at least some minimum 
size, which is considered essential for acceptance of efficacy, specifically in cases where 
the active control is not a licensed product (e.g. some vaccine trials, IGIV trials, etc.).  
   
The clinical effects of treatment on OAK pain will be evaluated during clinic visits at 6 
and 12 weeks, and telephone contacts at 2, and 10 weeks, using the Western Ontario and 
McMaster Universities Arthritis Index (WOMAC®) osteoarthritis Index 3.1 and the 
Patient’s Global Assessment of disease severity (PGA). 
The WOMAC® is a validated scoring system and sets the standard for the patient 
response. In order not to bias the collection of data, only questions from the validated 
WOMAC scale will be asked of subjects.  
Clinical benefit will be established by significant improvement on the WOMAC Index 
3.1 and the PGA as assessed at Weeks 2, 6, 10, and 12. Safety will be assessed by 
recording adverse events (through 24 hours post-dose and at all follow up contacts) and 
physical examination and vitals (Baseline, Weeks 6, and 12), and clinical laboratory tests 
(Screening and Week 12). 
2.4 
Sample Size  
A total of approximately 171 subjects (146 Ampion; 25 Saline) will be enrolled in the 
study to a single (1) intra-articular injection of 4 mL Ampion or 4 mL saline (6:1). 
 
The sample size for this study is based on the primary endpoint and is based on the 
following hypotheses.   
 
H0:π ≤ 30% versus HA:π > 30%     
 
A sample size of 146 patients in the active Ampion arm yields a greater than 90% power 
of rejecting the null hypothesis when the anticipated proportion under the alternative 
hypothesis is 45%. This power is applicable for the demonstration of an effect of at least 
some minimum size, defined in the current trial as greater than a 30% responder rate.  
 
3. 
PRIMARY STUDY ENDPOINTS  
3.1 Primary Endpoint  
 
The primary trial endpoint is to evaluate improvement of OMERACT-OARSI response 
of a single 4 mL Ampion™ intra-articular injection from Baseline to Week 12.  
A patient in this study will be considered an OMERACT-OARSI responder for the 
purpose of efficacy analysis if the following criteria is met: 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 7 of 18 
 
  
CONFIDENTIAL 
  
 
1) The patient has a percent improvement in pain (WOMAC pain) from baseline of 
≥50% and an absolute change in pain from baseline of ≥ 1 point or the patient has a 
percent improvement in function (WOMAC function) from baseline of ≥50% and an 
absolute change in function from baseline of ≥1 point.  Meeting this criterion 
designates the patient as a responder.  If the patient does not meet this criterion, then 
2) The patient demonstrates improvement in at least 2 of the following: 
 Improvement in pain (WOMAC A) over baseline of ≥20% and a 0.5 point 
absolute change in pain 
 Improvement in function (WOMAC function) over baseline of ≥20% and a 
0.5 point absolute change in function 
 
 Improvement in patient global assessment (PGA) over baseline of ≥20% and 
a 0.5 point absolute change in PGA 
 
3.2 Secondary Endpoints 
The secondary trial endpoints are to evaluate improvement in a composite endpoint of 
pain and function, defined as OMERACT-OARSI “controlled” responder, supported by 
patient global assessment following a single 4 mL Ampion intra-articular injection from 
Baseline to Week 12.  
Secondary endpoints are to evaluate improvement of: 
- 
OMERACT-OARSI “controlled” response from Baseline to Week 12 
- 
PGA response from Baseline to Week 12 
- 
OMERACT-OARSI “controlled” responder from Baseline to Week 12 in 
comparison to historic saline 
A patient in this study will be considered an OMERACT-OARSI “controlled” responder 
for the purpose of efficacy analysis if the following criteria is met: 
1) The patient demonstrates improvement in both: 
 Improvement in pain (WOMAC A) over baseline of ≥20% and a 0.5 point 
absolute change in pain 
 Improvement in function (WOMAC function) over baseline of ≥20% and a 
0.5 point absolute change in function 
 
A patient in this study will be considered to have a PGA response if the following 
criteria is met: 
 Improvement in patient global assessment (PGA) over baseline of  ≥20% and 
a 0.5 point absolute change in PGA 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 8 of 18 
 
  
CONFIDENTIAL 
  
 
4. 
HYPOTHESES AND METHODS 
4.1 Primary Hypothesis 
 
Demonstration of an effect of at least some minimum size is considered essential for 
acceptance of efficacy, and this is defined in the current trial as a greater than 30% 
responder rate based on review of KOL opinion, literature, and clinical benefit in 
severely diseased OAK patients.  For the primary endpoint of the proportion of patients 
who are responders to the OMERACT-OARSI criteria from above, the following 
hypothesis will be tested. 
 
H0:π ≤ π0 versus HA:π > π0      
 
Where π0 is the hypothesized clinically significant value for the proportion of responders.  
The value will be 30% in this study.  This test will be tested using an exact binomial test.  
That is, given the sample size of n, the number of responders X, and the value of π0 
=0.30, then probability that X or more events would be observed will be calculated as the 
p-value.  Since this is a one-sided test, the alpha level will be 0.025. 
 
4.2 Secondary Hypotheses 
 
The primary hypothesis and these secondary hypotheses will be tested in a hierarchical 
manner with the primary first and the secondary hypotheses in the order listed.  Using 
this approach, there is no adjustment necessary in the overall significance level.  All 
testing will be conducted using an overall significance level of 5%.  If the tests are one 
sided, the significance level will be 2.5%. 
 
For the secondary endpoint of the proportion of patients who are “controlled responders” 
to the OMERACT-OARSI criteria as defined in section 3.2 above, the following 
hypothesis will be tested. 
 
H0:π ≤ π0 versus HA:π > π0      
 
Where π0 is the hypothesized clinically significant value for the proportion of responders.  
The value will be 30% in this study.  This test will be tested using an exact binomial test.  
That is, given the sample size of n, the number of responders X, and the value of π0 
=0.30, then probability that X or more events would be observed will be calculated as the 
p-value.  Since this is a one sided test, the alpha level will be 0.025. 
 
This composite analysis of OMERACT-OARSI “controlled responder” will be supported 
by Patient Global Assessment.  Patient Global Assessment will be tested against the same 
hypothesis:   
H0:π ≤ π0 versus HA:π > π0      
 
The 3rd secondary trial endpoint, evaluating the improvement in a composite endpoint of 
pain and function (WOMAC A and C), defined as OMERACT-OARSI “controlled” 
response, will demonstrate that the active arm from this study is superior to analysis of all 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 9 of 18 
 
  
CONFIDENTIAL 
  
 
historic saline-control arms from previous single-injection Ampion studies, will be 
analyzed using the following hypothesis. 
 
 
H0:πA ≤ πHS versus HA: πA > πHS 
 
Where πA is the response rate from Ampion in this study and πHS is the response rate of 
the saline control from previous single-injection Ampion studies.  This will be tested 
using Fisher’s exact test. 
 
4.3 Exploratory Analysis 
 
If it is deemed to be clinically relevant, the analyses will be conducted using the 
appropriate adjustment methodologies. 
 
 
4.4 Definition of Study Visits 
 
This clinical trial has a total of 7 study visits during the 12-week study (see Table 6.2).  
The time on study for each subject observation will be defined relative to Day 
0/Baseline, the day of the initial dose. For analysis, the Baseline measure is the latest 
measure prior to initiation of treatment. 
 
4.5 Number of subjects to receive study drug 
 
A total of approximately 171 subjects (146 Ampion; 25 Saline) will be enrolled in the 
study to a single (1) intra-articular injection of 4 mL Ampion or 4 mL saline (6:1). 
 
4.6 Disposition of subjects 
 
Disposition of subjects, including study completion status and response to therapy as 
measured by WOMAC subscores and PGA, will be summarized by age group, race, and 
gender for each of the analysis populations. 
 
4.7 Interim analysis 
 
There will be no interim analysis. 
 
4.8 Blinding and randomization 
 
All subjects will be randomized 6:1 to receive Ampion 4 mL or saline 4 mL as an intra-
articular injection in the study knee. 
 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 10 of 18 
 
  
CONFIDENTIAL 
  
 
4.9 Data presentation 
 
4.9.1 Demographic and Baseline Characteristics: 
 
Demographic (e.g., age, sex, race, and ethnicity) and Baseline characteristics (e.g., 
weight, height, prior injection of another intra-articular therapeutic for OA of the knee) 
will be summarized using descriptive statistics, overall and by treatment group for the 
ITT analysis population. Demographic data for the historic control will be provided 
and summarized.  
 
4.9.2 Medical History and Physical Examination: 
 
The number and percent of subjects with past and current medical disorders at the time of 
randomization will be presented overall for the ITT analysis population. Results of any 
abnormalities documented from the abbreviated physical examination at Baseline and 
Week 12, will be summarized overall for the safety and ITT analysis populations. 
 
4.9.3 Concomitant Medications or Treatments: 
 
The number and percent of subjects receiving concomitant medications or treatments 
prior to and during the study and at the final visit will be tabulated and presented overall 
for the ITT analysis population.  Concomitant medications/treatments will be 
summarized using descriptive statistics and will be presented by type of drug (WHO 
DRUG classification) overall for the safety and ITT analysis populations. Concomitant 
medication and treatment data for the historic control will be provided and summarized.  
 
4.9.4 Safety data: 
 
Safety data will be evaluated by changes in vital sign measurements, physical exam, 
laboratory analysis, and the frequency and severity of AEs. Concomitant medication 
will be recorded for safety. 
 
Adverse events: 
 
The Investigator is responsible for monitoring the safety of subjects who have enrolled 
in the study. All AEs considered related, or possibly related to Ampion™, will be 
followed until the event resolves or stabilizes without further change. Subjects will be 
followed for the occurrence of AEs until 12 weeks after the first dose of study 
medication. 
 
Investigators are required to document all AEs occurring during the clinical trial, 
commencing with the first day of treatment through the end of study. AEs should be 
properly documented on the appropriate CRF pages. 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 11 of 18 
 
  
CONFIDENTIAL 
  
 
The severity of AEs (mild, moderate, severe), relatedness (related, possibly related, 
unrelated) along with the duration, action taken, and outcome (e.g., study withdrawal) 
will also be recorded.  In addition, events meeting the criteria of a Serious Adverse 
Event (SAE) must be reported to the Sponsor within 24 hours on the SAE reporting 
forms. 
4.10 MISSING DATA 
All data collected under this study protocol will be included in the assessment of subject 
safety.  Missing or incomplete AE data will assume greatest relationship to study drug 
and/or severity. 
 
For the primary effectiveness endpoints (WOMAC A, WOMAC C, and PGA), missing 
Week 12 values will be imputed when the ITT analysis population is used. The Worst 
Observation Carried Forward (WOCF) will be selected as the primary method of 
imputing missing 12 Week data. Alternate imputation methods employed will include 
multiple imputations (SAS PROC MI and SAS PROC MIANALYZE) and Last 
Observation Carried Forward (LOCF).  These sensitivity analyses will be conducted for 
the primary effectiveness endpoint, to ensure that the primary method of imputation 
chosen is robust with respect to imputation method 
4.11  Detection of Bias 
Any breaking of the blind for individual subjects prior to formal unblinding will be 
documented in the clinical study report.  Reason for unblinding will also be documented.  
Data collected after unblinding will be noted.   
4.12  Outliers 
No formal outlier tests are planned. Values that are outside the pre-defined range, such as 
a WOMAC score not between zero and four, or a PGA score not between one and five, 
would be queried and excluded if necessary prior to database lock. 
4.13  Testing/Validation Plan 
All statistical analyses will be programmed using SAS® software version 9.3, or later.  
Testing and validation plans for all programs will be developed in accordance with 
contract statistical organization guidelines and will include independent programming of 
tables and analyses. 
 
5. 
DEFINITIONS 
5.1 Abbreviations 
 
Abbreviation 
 
Definition 
AE 
Adverse Event 
ANCOVA 
Analysis of Covariance 
ATC 
Anatomical Therapeutic Chemical Classification System 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 12 of 18 
 
  
CONFIDENTIAL 
  
 
CSR 
Clinical Summary Report 
ICH 
International Conference on Harmonisation  
MedDRA® 
Medical Dictionary for Regulatory Activities 
mL 
Milliliter 
NA 
Not applicable 
ng 
Nanogram 
NSAID 
Non-steroidal anti-inflammatory drug 
OA 
Osteoarthritis 
OAK 
Osteoarthritis of the knee 
OMERACT-OARSI 
Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis 
Research-Osteoarthritis Research Society International 
PGA 
Patient’s global assessment of disease severity 
PP 
Per protocol population 
SAE 
Serious adverse event 
SAS 
Statistical Software from SAS Institute 
SD 
Standard deviation 
SEM 
Standard error of the mean 
TEAE 
Treatment-emergent adverse event 
WO 
Washout 
WOMAC 
Western Ontario and McMaster Universities Arthritis Index 
 
5.2 Definitions 
Adverse Event (AE) 
An adverse event (AE) is defined as any undesired medical occurrence in a patient or 
clinical investigation patient receiving a pharmaceutical product and which does not 
necessarily have a causal relationship with this treatment.  An AE can therefore be any 
unfavorable sign and unintended sign (including an abnormal laboratory finding 
comparing pre-treatment to post-treatment), symptom, or disease temporarily associated 
with the use of a study drug, whether or not related to the study drug. 
AEs will be graded for severity using the following categories.  Missing grade will be 
assigned a grade of 3 (severe) in tabulations. 
 Grade 1 (MILD): The symptom is barely noticeable to the study patient and does not 
influence performance or functioning.  Concomitant medication is not ordinarily 
indicated for relief of mild AEs. 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 13 of 18 
 
  
CONFIDENTIAL 
  
 
Grade 2 (MODERATE): The symptom is of sufficient severity to make the study 
patient uncomfortable and to influence performance of daily activities.  Concomitant 
medication may be indicated for relief of moderate AEs. 
Grade 3 (SEVERE): The symptom causes severe discomfort, sometimes of such 
severity that the study patient cannot continue in the study.  Daily activities are 
significantly impaired or prevented by the symptom. Concomitant medication may be 
indicated for relief of severe AEs. 
 
Relationship to study drug will be coded using the following categories.  Missing 
relatedness will be assigned to “related” in tabulations. 
Unrelated:  The adverse event is unlikely to have been caused by study drug. 
Possibly related: It is unclear whether the adverse event may have been caused by study 
drug. 
Related: The adverse event is likely to have been caused by study drug.  
Serious Adverse Event: 
  A serious adverse event (SAE) is defined as an adverse event that 
 Results in death 
 Is life-threatening (patient is at immediate risk of death from the event as it occurred) 
 Requires in-patient hospitalization (formal admission to a hospital for medical 
reasons) or prolongation of existing hospitalization 
 Results in persistent or significant disability/incapacity 
 Results in a congenital anomaly/birth defect 
 
Treatment-Emergent AE: 
A Treatment-Emergent AE (TEAE) is any AE that begins or increases in severity after 
the initial dose of study drug. 
Age 
Subject’s age is defined as its integer value in years at enrollment.  
Baseline 
For any variable, unless otherwise defined, Baseline is the last assessment taken prior to 
the first study drug administration. 
Change from Baseline: 
The arithmetic difference between a post-Baseline value and the Baseline value: 
Change from Baseline = (Post-Baseline Value – Baseline Value) 
Percentage Change from Baseline = [(Post-baseline Value – Baseline Value) / 
Baseline Value] x 100 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 14 of 18 
 
  
CONFIDENTIAL 
  
 
 
End of Study 
End of study is at Visit 9 (day 84 ± 7 days), unless terminated early. 
Enrollment date 
Enrollment date is the same as the randomization date and is designated Day 0. 
Study drug 
Study drug in this study is Ampion or saline. 
Randomization date 
Randomization date is the day the subject is assigned a randomization number on study 
Day 0. 
Study Day 0 
Day 0 is defined as the first day that study drug is administered to the subject.   
Study Day 
Day of treatment: study day = (visit date - date of Study Day 0)  
6. 
CHANGES FROM PROTOCOL-SPECIFIED ANALYSES  
Modifications to the planned statistical analyses should be minimized. Nonetheless, the 
data obtained from the study may indicate that the planned analyses are inappropriate, 
that additional analyses need to be performed, or that the design of the study needs to be 
modified, due to factors such as the distribution of the data or imbalance in important 
covariates.  The study report will provide a detailed explanation for any deviations from 
the planned analyses. 
 
7. 
LIST OF PLANNED TABLES, FIGURES, AND LISTINGS 
Tables are categorized and numbered in accordance with ICH E3 guidelines. Each table, 
figure and listing is presented by treatment arm. All efficacy tables will be provided both 
for ITT and PP. Accountability tables will also include an overall column. Listings will 
be sorted by treatment, subject ID, and by visit, if multiple visits exist.  These will be 
provided in a separate document. 
 
Number 
Title 
14.1  Accountability 
 
14.1.1  
Accountability (Analysis Population: All Enrolled) 
14.1.2 
Enrollment by Site (Analysis Population: All Enrolled) 
14.1.3 
Analysis Populations (include ITT, safety, PP, and reasons for PP exclusion) 
14.1.4 
Subject Disposition (all screened) 
14.1.5 
Major Protocol Deviations (Analysis Population: All Enrolled) 
14.1.6.1 
Demographics and Baseline Characteristics (Analysis Population: ITT) 
14.1.6.2 
Demographics and Baseline Characteristics (Analysis Population: PP) 
14.2 Efficacy 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 15 of 18 
 
  
CONFIDENTIAL 
  
 
Number 
Title 
14.2.1.1 
Summary of OMERACT-OARSI Percent Responder Status (Analysis 
Population: ITT) 
14.2.1.2 
Summary of OMERACT-OARSI “Controlled” Responders (WOMAC A and 
C) 
14.2.1.3 
Summary of Responders Demonstrating a 20% Improvement and 0.5 Shift in 
PGA 
14.2.1.4 
Summary of OMERACT-OARSI “Controlled” Responders (WOMAC A and 
C) Compared to Historic Saline  
14.2.1.5 
Summary of OMERACT-OARSI Percent Responder Status for a 50% 
Threshold 
14.2.1.6 
Summary of Patients Rresponding to 50% Improvement in Pain and 1.0 
Absolute Change 
14.2.1.7 
Summary of Patients Responding to a 50% Improvement in Function and 1.0 
Absolute Change 
14.2.1.8 
Summary of Patients Responding to a 20% Improvement in Pain and 0.5 
Absolute Change 
14.2.1.9 
Summary of Patients responding to a 20% Improvement in Function and 0.5 
Absolute Change 
14.2.1.10 
Summary of Patients responding to a 20% Improvement in PGA and 0.5 
Absolute Change 
14.2.1.11 
Summary of WOMAC A mean change 
14.2.1.12 
Summary of WOMAC A percent change 
14.2.1.13 
Summary of WOMAC B mean change 
14.2.1.14 
Summary of WOMAC B percent change 
14.2.1.15 
Summary of WOMAC C mean change 
14.2.1.16 
Summary of WOMAC C percent change 
14.2.1.17 
Summary of WOMAC A and C mean change 
14.2.1.18 
Summary of WOMAC A and C percent change 
14.2.1.19 
Summary of PGA mean change 
14.2.1.20 
Summary of PGA percent change 
14.2.1.21 
Summary of WOMAC mean change 
14.2.1.22 
Summary of WOMAC percent change 
14.3 Safety 
 
14.3.1  
Overall Summary of TEAEs (Analysis Population: Safety) 
14.3.2  
Incidence of  TEAEs by System Organ Class and Preferred Term (Analysis 
Population: Safety) 
14.3.3 
Incidence of Treatment Emergent Related AEs by System Organ Class and 
Preferred Term 
14.3.4 
Incidence of Treatment Emergent Serious AEs by System Organ Class and 
Preferred Term 
14.3.5 
Incidence of TEAEs by Preferred Term in Descending Order of Frequency  
14.3.6 
Incidence of Treatment Emergent Related AEs by Preferred Term in 
Descending Order of Frequency  
14.3.7 
Summary of Lab Value and Change from Baseline 
14.3.8 
Shifts in Reference Range  
14.3.9.1 
Summary of Vital Signs and Change from Baseline: Pulse (bpm) 
14.3.9.2 
Summary of Vital Signs and Change from Baseline: Temperature (°F) 
14.3.9.3 
Summary of Vital Signs and Change from Baseline: Systolic Blood Pressure 
(mmHG) 
14.3.9.4 
Summary of Vital Signs and Change from Baseline: Diastolic Blood Pressure 
(mmHG) 
14.3.10.1 
Concomitant Medication Use by ATC Level 1 and WHO Preferred Term 
14.3.10.2 
Medication started on study by ATC Level 1 and WHO Preferred Term 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 16 of 18 
 
  
CONFIDENTIAL 
  
 
Number 
Title 
14.3.10.3 
Concomitant Medication Use by WHO Preferred Term in Descending Order of 
Use 
14.3.10.4 
Concomitant Medication Use by WHO Preferred Term in Descending Order of 
Use for Historic Control  
14.3.10.5 
Medication Started on Study by WHO Preferred Term in Descending Order of 
Use 
16- Listings 
 
16.1 
Randomization List 
16.2 
Inclusion/Exclusion Criteria 
16.3 
Protocol Deviations 
16.4 
Early Terminations (Include date of ET and reason for ET) 
16.5 
Exclusions from PP 
16.6 
Demographics and Baseline Characteristics  
16.7 
Demographics and Baseline Characteristics for Historic Control  
16.8 
Medical History 
16.9 
Concomitant Medications 
16.10 
WOMAC/PGA/OMERACT OARSI Responder Status 
16.11 
Analgesic Used within 24 hours of visit 
16.12 
Adverse Events 
16.13 
Serious Adverse Events 
16.14 
Laboratory Values 
16.15 
Physical Exam (weight (lbs), Height, any abnormality (if listed)) and Vital 
Signs (BP, Temp, Pulse) 
16.16 
X-ray Data (and abnormalities, if listed) 
16.17 
OMERACT-OARSI Responder Status (WOMAC A, C, and PGA) 
10.3 Figures 
 
10.3.1 
OMERACT-OARSI Responder Over 12 Weeks 
10.3.2 
WOMAC A Pain Scale Over 12 Weeks 
10.3.3 
WOMAC C Function Over 12 Weeks 
10.3.4 
PGA Over 12 Weeks 
10.3.5 
Rescue Med Use Over 12 Weeks 
 
 
 
8. 
LITERATURE CITATIONS  REFERENCES 
SAS Institute Inc. SAS Language: version 8 first edition. SAS Institute, Inc, Cary, NC, 
USA, 1990. 
9. 
HANDLING OF MISSING OR INCOMPLETE DATES FOR ADVERSE 
EVENTS AND CONCOMITANT MEDICATIONS 
9.1 Imputation Rules for Partial or Missing Stop Dates 
If the month and year are present, impute the last day of the month.  If only the year is 
present, impute December 31 of that year.  If the stop date is entirely missing, assume the 
event or medication is ongoing.  If a partial or complete stop date is present and the 
‘ongoing’ or ‘continuing’ box is checked, then it will be assumed that the AE or 
concomitant medication stopped and the stop date will be imputed, if partial.   
 
 


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 17 of 18 
 
  
CONFIDENTIAL 
  
 
 
Stop Date 
 
 
Complete: 
yyyymmdd 
Partial: yyyymm 
Partial: yyyy 
Missing 
Start Date 
 
<1st 
Dose 
1st 
Dose 
<1st Dose 
yyyymm 
1st Dose 
yyyymm 
<1st 
Dose 
yyyy 
1st 
Dose 
yyyy 
Partial: 
yyyymm 
=1st Dose 
yyyymm 
2 
1 
2 
1 
N/A 
1 
1 
 1st Dose 
yyyymm 
2 
2 
2 
2 
2 
Partial: 
yyyy 
=1st Dose 
yyyy 
3 
1 
3 
1 
N/A 
1 
1 
 1st Dose 
yyyy 
3 
3 
3 
3 
3 
Missing 
4 
1 
4 
1 
4 
1 
1 
 
1 = Impute the date of first dose 
2 = Impute the first of the month 
3 = Impute January 1 of the year 
4 = Impute January 1 of the stop year 
 
Note: For subjects who were never treated (first dose date is missing), partial start dates 
will be set to the first day of the partial month. 
 
Note: If the start date imputation leads to a start date that is after the stop date, then do 
not impute the start date.


Ampio AP-003-C 
Statistical Analysis Plan 
Version 2.0 Dated: 14 November 2017 
Page 18 of 18 
 
  
CONFIDENTIAL 
  
 
10. 
SCHEDULE OF ASSESSMENTS AND PROCEDURES 
 
Screening 
Baseline 
Randomization 
Treatment 
Post-
treatment 
check 
(telephone 
contact) 
Week 2 
(telephone 
contact) 
Week 6 
 
Week 10 
(telephone 
contact) 
Week 12 
Final Visit 
 
Early Termination 
Visit # 
Day # 
1 
Day-28 to 0 
2 
Day 0 
3 
Day 1 
4 
Day 14 ± 7 
6 
Day 42 ± 
7 
8 
Day 70 ±7 
9 
Day 84 ± 7 
 
Informed Consent 
X 
 
 
 
 
 
 
 
Inclusion/exclusion criteria 
X 
X 
 
 
 
 
 
 
Medical history/prior 
medications2 
X 
 
 
 
 
 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
X 
 
 
X 
 
X 
X 
Vital Signs 
X 
X 
 
 
X 
 
X 
X 
Randomization 
 
X 
 
 
 
 
 
 
WOMAC  
X 
X 
 
X 
X 
X 
X 
X 
Patient’s global assessment 
(PGA) 
X 
X 
 
X 
X 
X 
X 
X 
X-ray1 
X 
 
 
 
 
 
 
 
Clinical laboratory tests 
X 
 
 
 
 
 
X 
 
Treatment with study drug 
 
X 
 
 
 
 
 
 
Rescue medication dispensed 
 
X 
 
 
 
 
 
 
Review Rescue medication 
 
 
X 
X 
X 
X 
X 
X 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
 
Visits are in clinic except for Day 1 and Weeks 2 and 10 when subjects will be contacted by telephone 
1 X-ray must be acquired at Screening to satisfy inclusion criteria, “Index knee must be symptomatic for greater than 6 months with a clinical diagnosis of OA and supported by radiological evidence (Kellgren Lawrence Grade  IV)  
2   At Week 12 and Early Termination visits, ask subject if they have previously sought out treatment for OAK prior to this study.