A Randomized, Placebo-Controlled, Double-Blind Pilot Study to Evaluate the Effect of GRF6021 on Intracellular Signaling Cascades in Blood Leukocytes and Postoperative Recovery Following Primary Hip or Knee Arthroplasty
Phase: PHASE2
Conditions: Postoperative Recovery
Sponsor: Alkahest, Inc.

Official Title: A Randomized, Placebo-Controlled, Double-Blind Pilot Study to 
Evaluate the Effect of GRF6021 on Intracellular Signaling Cascades in 
Blood Leukocytes and Postoperative Recovery Following Primary Hip 
or Knee Arthroplasty 
NCT Number: NCT03981419  
Document Date: Protocol Version 3: 14-November-2019 

 
Protocol AKST6021-211 
 
 
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CONFIDENTIAL/PROPRIETARY                                                                                                   Page 2 of 69
 
 
 
TABLE OF CONTENTS 
TABLE OF CONTENTS ..................................................................................................................... 2 
LIST OF ABBREVIATIONS .............................................................................................................. 6 
PROTOCOL APPROVAL PAGE ...................................................................................................... 8 
STATEMENT OF COMPLIANCE .................................................................................................... 9 
PROTOCOL SUMMARY ................................................................................................................. 10 
SCHEMATIC OF STUDY DESIGN ................................................................................................ 12 
1 
KEY ROLES ........................................................................................................................... 12 
1.1 
AUTHORIZED REPRESENTATIVE (SIGNATORY) / RESPONSIBLE PARTY ........ 12 
1.2 
STUDY ORGANIZATION .................................................................................................... 12 
2 
INTRODUCTION .................................................................................................................. 12 
2.1 
BACKGROUND INFORMATION ...................................................................................... 12 
2.2 
RATIONALE .......................................................................................................................... 13 
2.3 
POTENTIAL RISKS AND BENEFITS ............................................................................... 15 
2.3.1 
KNOWN POTENTIAL RISKS ................................................................................................ 15 
2.3.2 
KNOWN POTENTIAL BENEFITS ......................................................................................... 16 
3 
OBJECTIVES AND PURPOSE ........................................................................................... 16 
4 
STUDY DESIGN AND ENDPOINTS .................................................................................. 16 
4.1 
DESCRIPTION OF THE STUDY DESIGN ........................................................................ 16 
4.2 
STUDY ENDPOINTS ............................................................................................................ 17 
4.2.1 
PRIMARY ENDPOINT ............................................................................................................ 17 
4.2.2 
SECONDARY ENDPOINTS ................................................................................................... 17 
5 
STUDY ENROLLMENT AND WITHDRAWAL .............................................................. 17 
5.1 
INCLUSION CRITERIA ....................................................................................................... 17 
5.2 
EXCLUSION CRITERIA ...................................................................................................... 18 
 

 
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5.3 
STRATEGIES FOR RECRUITMENT AND RETENTION ............................................. 19 
5.4 
SUBJECT WITHDRAWAL OR TERMINATION ............................................................ 19 
5.4.1 
REASONS FOR WITHDRAWAL OR TERMINATION ........................................................ 19 
5.4.2 
HANDLING OF PARTICIPANT WITHDRAWALS OR TERMINATION ........................... 20 
5.5 
PREMATURE TERMINATION OR SUSPENSION OF STUDY .................................... 20 
6 
STUDY AGENT ..................................................................................................................... 20 
6.1 
STUDY AGENT AND CONTROL DESCRIPTION .......................................................... 20 
6.1.1 
ACQUISITION ......................................................................................................................... 20 
6.1.2 
FORMULATION, APPEARANCE, PACKAGING, AND LABELING ................................. 20 
6.1.3 
PRODUCT STORAGE AND STABILITY .............................................................................. 21 
6.1.4 
PREPARATION ....................................................................................................................... 21 
6.1.5 
DOSING AND ADMINISTRATION ...................................................................................... 21 
6.1.6 
ROUTE OF ADMINISTRATION ............................................................................................ 21 
6.1.7 
DOSING SCHEDULE .............................................................................................................. 21 
6.1.8 
DURATION OF THERAPY .................................................................................................... 22 
6.2 
STUDY AGENT ACCOUNTABILITY ................................................................................ 22 
7 
STUDY PROCEDURES AND SCHEDULE ....................................................................... 22 
7.1 
STUDY PROCEDURES/EVALUATIONS .......................................................................... 22 
7.1.1 
STUDY SPECIFIC PROCEDURES ........................................................................................ 22 
7.2 
LABORATORY PROCEDURES/EVALUATIONS ........................................................... 27 
7.2.1 
CLINICAL LABORATORY EVALUATIONS ....................................................................... 27 
7.2.2 
SPECIMEN PREPARATION, HANDLING, STORAGE, AND SHIPPING .......................... 27 
7.3 
STUDY SCHEDULE .............................................................................................................. 28 
7.3.1 
SCREENING ............................................................................................................................ 28 
7.3.2 
BASELINE: REMOTE ............................................................................................................. 28 
7.3.3 
RANDOMIZATION ................................................................................................................. 28 
7.3.4 
BASELINE: ON-SITE/TREATMENT ..................................................................................... 28 
7.3.5 
FOLLOW-UP............................................................................................................................ 29 
7.3.6 
END OF STUDY VISIT ........................................................................................................... 29 
7.3.7 
EARLY TERMINATION ......................................................................................................... 30 
7.3.8 
SCHEDULE OF EVENTS TABLE .......................................................................................... 30 
7.4 
CONCOMITANT MEDICATIONS ..................................................................................... 30 
7.4.1 
ANESTHESIA GUIDELINES ................................................................................................. 30 
7.5 
PROHIBITED MEDICATIONS, TREATMENTS, AND PROCEDURES ...................... 30 
8 
ASSESSMENT OF SAFETY ................................................................................................ 30 
8.1 
SPECIFICATION OF SAFETY PARAMETERS .............................................................. 31 
8.1.1 
DEFINITION OF ADVERSE EVENTS .................................................................................. 31 
8.1.2 
DEFINITION OF SERIOUS ADVERSE EVENTS ................................................................. 32 
8.2 
CLASSIFICATION OF AN ADVERSE EVENT ................................................................ 32 
 

 
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8.2.1 
SEVERITY OF EVENT ........................................................................................................... 33 
8.2.2 
RELATIONSHIP TO STUDY AGENT ................................................................................... 33 
8.2.3 
EXPECTEDNESS .................................................................................................................... 33 
8.3 
TIME PERIOD/FREQUENCY FOR EVENT ASSESSMENT/FOLLOW-UP ................ 34 
8.3.1 
POST-STUDY AE AND SAE .................................................................................................. 34 
8.4 
REPORTING PROCEDURES .............................................................................................. 34 
8.4.1 
ADVERSE EVENT REPORTING ........................................................................................... 34 
8.4.2 
SERIOUS ADVERSE EVENT REPORTING ......................................................................... 35 
8.4.3 
ADVERSE EVENTS OF SPECIAL INTEREST ..................................................................... 36 
8.4.4 
REPORTING OF PREGNANCY ............................................................................................. 36 
8.5 
STUDY HALTING RULES ................................................................................................... 37 
8.6 
SAFETY OVERSIGHT ......................................................................................................... 37 
9 
CLINICAL MONITORING ................................................................................................. 37 
10 
STATISTICAL CONSIDERATIONS .................................................................................. 38 
10.1 
STATISTICAL DESIGN MODEL AND ANALYTICAL PLANS .................................... 38 
10.2 
STATISTICAL HYPOTHESES ........................................................................................... 38 
10.3 
ANALYSIS DATASETS ........................................................................................................ 38 
10.4 
DESCRIPTION OF STATISTICAL METHODS ............................................................... 39 
10.4.1 
GENERAL APPROACH .......................................................................................................... 39 
10.4.2 
ANALYSIS OF THE PRIMARY ENDPOINT ........................................................................ 39 
10.4.3 
ANALYSIS OF THE SECONDARY ENDPOINTS ................................................................ 40 
10.4.4 
ADHERENCE AND RETENTION ANALYSES .................................................................... 40 
10.4.5 
BASELINE DESCRIPTIVE STATISTICS .............................................................................. 40 
10.4.6 
PLANNED INTERIM ANALYSES ......................................................................................... 40 
10.4.7 
ADDITIONAL SUBGROUP ANALYSES .............................................................................. 40 
10.4.8 
MULTIPLE COMPARISON/MULTIPLICITY ....................................................................... 41 
10.4.9 
TABULATION OF INDIVIDUAL RESPONSE DATA ......................................................... 41 
10.4.10 
EXPLORATORY ANALYSES .............................................................................................. 41 
10.5 
SAMPLE SIZE ........................................................................................................................ 41 
10.6 
MEASURES TO MINIMIZE BIAS ...................................................................................... 41 
10.6.1 
ENROLLMENT/RANDOMIZATION ..................................................................................... 41 
10.6.2 
EVALUATION OF SUCCESS OF BLINDING ...................................................................... 41 
10.6.3 
BREAKING THE STUDY BLIND/SUBJECT CODE ............................................................ 41 
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS ............ 41 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS ........................................................... 42 
12.1 
ETHICAL STANDARD ......................................................................................................... 42 
 

 
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12.2 
INSTITUTIONAL REVIEW BOARD ................................................................................. 42 
12.3 
INFORMED CONSENT PROCESS .................................................................................... 42 
12.3.1 
CONSENT FORMS .................................................................................................................. 42 
12.3.2 
CONSENT PROCEDURES AND DOCUMENTATION ........................................................ 43 
12.4 
PARTICIPANT AND DATA CONFIDENTIALITY .......................................................... 43 
12.5 
FUTURE USE OF STORED SPECIMENS ......................................................................... 44 
13 
DATA HANDLING AND RECORD KEEPING ................................................................ 44 
13.1 
DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES ............................ 44 
13.1.1 
INVESTIGATOR RESPONSIBILITIES.................................................................................. 44 
13.1.2 
STUDY FILES .......................................................................................................................... 45 
13.2 
STUDY RECORDS RETENTION ....................................................................................... 45 
13.3 
PROTOCOL DEVIATIONS ................................................................................................. 45 
13.4 
PUBLICATION AND DATA SHARING POLICY ............................................................ 46 
14 
FINANCIAL DISCLOSURE AND CONFLICT OF INTEREST POLICY .................... 47 
15 
SCHEDULE OF EVENTS .................................................................................................... 48 
15.1 
SCHEDULE OF EVENTS TABLE ...................................................................................... 48 
16 
REFERENCES ....................................................................................................................... 51 
16.1 
PUBLISHED REFERENCES ............................................................................................... 51 
16.2 
UNPUBLISHED REFERENCES .......................................................................................... 53 
17 
APPENDICES ........................................................................................................................ 55 
18 
REVISION HISTORY ........................................................................................................... 62 
18.1 
SUMMARY OF CHANGES .................................................................................................. 62 
 
 
 

 
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LIST OF ABBREVIATIONS 
3D-CAM 
3-Minute Diagnostic Interview for Confusion 
AE 
Adverse event 
ALT 
Alanine aminotransferase 
ASA 
American Society of Anesthesiologists 
AST 
Aspartate aminotransferase 
BDI-II 
Beck Depression Index-II 
BP 
Blood pressure 
CBC 
Complete blood count 
CD68 
Cluster of differentiation 68 
CFR 
Code of Federal Regulations 
CLIA 
Clinical Laboratory Improvement Amendments 
CMP 
Clinical Monitoring Plan 
CREB 
Cyclic AMP-responsive element-binding protein  
CRF 
Case Report Form 
CRO 
Contract Research Organization 
csEN 
Cell signaling Elastic Net 
CyTOF 
Mass cytometry 
EBL 
Estimated blood loss 
ECG 
Electrocardiogram 
eGFR 
Estimated glomerular filtration rate 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GGT 
Gamma-glutamyl transferase 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human immunodeficiency virus 
HR 
Heart rate 
Iba1 
Ionized calcium binding adaptor molecule 1 
ICH 
International Conference on Harmonization 
ICH E6 R2 
International Conference on Harmonization Guidance for Industry, Good Clinical 
Practice: Consolidated Guidance, Revision 2 
IEC  
Independent Ethics Committee 
IgA 
Immunoglobulin A 
IRB 
Investigational Review Board 
ITT 
Intent-to-treat 
IV 
Intravenous 
LV 
Left ventricular 
MedDRA 
Medical Dictionary for Regulatory Activities 
MRI 
Magnetic resonance imaging 
NF-kB 
Nuclear factor kappa-light-chain-enhancer of activated B cells 
PACU 
Post-anesthesia care unit 
POMS 
Profile of Mood States 
PSS 
Perceived Stress Scale 
 

 
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PT 
Preferred Term 
PT/INR 
Prothrombin time/international normalized ratio 
PTT 
Partial thromboplastin time 
RBC 
Red blood cell 
RR 
Respiration rate 
SAE 
Serious adverse event 
SAP 
Statistical Analytical Plan 
SF-36 
Short Form-36 
SOC 
System Organ Class 
SRS 
Surgery Recovery Scale 
STAT3 
Signal transducer and activator of transcription 3 
SUSAR 
Suspected unexpected serious adverse reaction 
THA 
Total hip arthroplasty 
TKA 
Total knee arthroplasty 
UPCR 
Urine protein-to-creatinine ratio 
US 
United States 
WOCBP 
Women of childbearing potential 
WOMAC 
Western Ontario and McMaster Universities Osteoarthritis Index 
 
 
 
 

 
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STATEMENT OF COMPLIANCE 
 
Protocol Title: 
A Randomized, Placebo-Controlled, Double-Blind Pilot Study to Evaluate the 
Effect of GRF6021 on Intracellular Signaling Cascades in Blood Leukocytes 
and Postoperative Recovery Following Primary Hip or Knee Arthroplasty 
Protocol Number: 
AKST6021-211 
 
Version/Date: 
V3.0_14NOV2019 
 
 
By my signature, I: 
• 
Confirm that my staff and I have carefully read and understand this protocol or protocol amendment 
and are thoroughly familiar with the appropriate use of the investigational agent described herein. 
• 
Agree to comply with the conduct and terms of the study specified herein and with any other study 
conduct procedures provided by the Sponsor, Alkahest, Inc., or their designee 
• 
Agree to assume responsibility for the proper conduct of the study at this site, including complying 
with current relevant versions of the US Food and Drug Administration (FDA) regulations, the 
International Conference on Harmonization (ICH) GCP guidelines, the Declaration of Helsinki, and all 
applicable rules, regulations, and federal, state, and local laws relating to the conduct of clinical studies 
and the protection of human subjects.  
• 
Agree not to implement deviations from or changes to the protocol or protocol amendments without 
agreement from the Sponsor and prior submission to and written approval (where required) from the 
Institutional Review Board (IRB) or Independent Ethics Committee (IEC), except when necessary to 
eliminate an immediate hazard to the subjects, or for administrative aspects of the study (where 
permitted by all applicable regulatory requirements).  
• 
Agree to onsite monitoring of all source documents by Alkahest, Inc. or designee and to onsite 
inspection of source documents by appropriate regulatory authorities, including but not limited to the 
FDA, local governing regulatory bodies, and IRB inspectors. 
 
 
 
 
Investigator's Signature 
 
Date 
 
 
 
Print Name 
 
 
 

 
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PROTOCOL SUMMARY 
 
 
Title: 
A Randomized, Placebo-Controlled, Double-Blind Pilot Study to Evaluate the 
Effect of GRF6021 on Intracellular Signaling Cascades in Blood Leukocytes 
and Postoperative Recovery Following Primary Hip or Knee Arthroplasty  
 
Précis:  
This is a randomized, placebo-controlled, double-blind pilot study to 
investigate the effects of GRF6021, a 5% human plasma protein fraction 
administered by intravenous (IV) infusion, on intracellular signaling cascades 
in blood leukocytes in subjects undergoing primary hip or knee arthroplasty. 
 
The study will enroll approximately 45 subjects with the aim of having 40 
evaluable subjects randomized in a 1:1 ratio to active treatment or placebo. 
Each subject will receive 4 infusions of 250 mL GRF6021 or 4 infusions of 
250 mL placebo (normal saline): 
 
1) 1 infusion on the day before surgery 
2) 1 infusion on the day of surgery within 4 hours before surgery start (first 
incision)  
3) 1 infusion on the day of surgery upon arrival in the postoperative care unit 
(within 5 hours after the first incision)  
4) 1 infusion on the day after surgery 
 
Each infusion will be administered over approximately 30 minutes according 
to Stanford Hospital procedures for colloid infusions, but infusion rates and 
infusion time will be adjusted according to tolerability as determined by the 
anesthesiologist and infusion nurse. The infusion procedure of active and 
placebo agents will be identical to maintain the blind. The following measures 
will be taken to ensure adequate blinding during infusions: blinding of 
subjects, study coordinators, physicians, and raters to treatment allocation; 
use of unblinded infusion nurses; and measures to block view of the vial and 
drip chamber throughout the infusion. 
 
Mass cytometry (CyTOF), a multiplexed, high-content immune profiling 
technology, will be used to provide high-resolution surveillance of circulating 
immune cells and the response to GRF6021 infusions on surgical recovery. 
Additional functional and quality of life assessments will be measured at 
baseline and approximately twice weekly for 6 weeks after hospital discharge. 
 
Objectives: 
The primary objective of the study is to investigate the effect of GRF6021 on 
intracellular signaling cascades in blood leukocytes as determined by CyTOF. 
Secondary objectives include the effect of GRF6021 on clinical recovery 
parameters in patients undergoing primary hip or knee arthroplasty, safety and 
tolerability of GRF6021, and the effects of GRF6021 on plasma proteomics.  
 
Endpoints: 
Primary Endpoint:  
• 
Effect of GRF6021 on intracellular signaling cascades in blood 
leukocytes as determined by CyTOF.  
 

 
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Secondary Endpoints:  
• Change from baseline in 3-Minute Diagnostic Interview for Confusion 
Assessment Method (3D-CAM). 
• Time to 50% recovery of baseline value on the Surgery Recovery 
Scale (SRS). 
• End of study treatment comparison of the Western Ontario and 
McMaster Universities Osteoarthritis Index (WOMAC) 
• Time to a score of < 12/40 on a subset of questions from the WOMAC, 
Pain Subscale. 
• Time to a score of < 18/60 on a subset of questions from the WOMAC, 
Physical Function Subscale. 
• Change from baseline in the Short Form-36 (SF-36). 
• Change from baseline in the Beck Depression Inventory-II (BDI-II).  
• Opioid analgesic consumption during hospital stay and after discharge 
to end of study. 
• Time to discharge. 
• Perioperative outcomes (e.g., surgery duration, anesthesia duration, 
post-anesthesia care unit (PACU) stay duration, American Society of 
Anesthesiologists (ASA) status, estimated blood loss (EBL), type and 
amount of intraoperative fluids administered, type and amount of 
intraoperative blood products, dose and type of anesthesia (general, 
regional), and intraoperative opioid dose).  
• Change in functional status using the ActiGraph wearable device 
providing measurements for physical activity/function and sleep 
(starting at least 3 days prior to surgery and continuing until the end of 
the study). 
• Change from baseline in clinical laboratory parameters. 
• Change from baseline in vital sign measurements. 
• Change from baseline in electrocardiogram (ECG). 
• The effects of GRF6021 on plasma proteomics.  
 
 
Population: 
The study will enroll approximately 45 subjects with the aim of having 40 
evaluable subjects between 50 and 85 years of age undergoing primary hip or 
knee arthroplasty. 
 
Phase: 
2 
 
Number of Sites: 
Single site pilot study (Stanford University) 
Description of Study 
Agent: 
GRF6021: A 5% human plasma protein fraction for IV infusion 
Study Duration: 
Approximately 12 months  
 
 
 
 
 

 
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physical, psychological, and social factors, inclusive of the return to normal activities, are important 
considerations (Martinez-Cano 2017, Fragiadakis 2015).  
 
Convalescence after surgery is highly variable with inter-individual variability in fatigue, pain, and functional 
impairment (Gaudillière 2014, Kehlet 2003). Past studies have explored the association of preoperative 
psychological status with outcomes of total joint replacement surgery (Lingard 2007, Judge 2012, Faller 2003, 
Ayers 2004), and additional studies have demonstrated pain catastrophizing or negative preoperative emotional 
state is responsible for poor outcomes with resultant delays in recovery (Witvrouw 2009, Edwards 2008). In 
addition to psychological variables, physiological factors also play a vital role in determining duration of 
recovery. The physiologic stress response, characterized by endocrine and neuroimmune mechanisms, may also 
influence arthroplasty outcomes. Psychological stressors can elicit neuroimmune responses mimicking those 
initiated by physical trauma and/or inflammation (Deak 2017). Due to these factors, researchers have gained 
renewed interest in unravelling immune mechanisms in response to trauma that determine postoperative recovery 
(Gaudillière 2014, Fragiadakis 2015).  
 
Surgical trauma triggers an intricate programmed immune response (Stoecklein 2012). Characteristics of this 
injury-mediated series of intracellular signaling cascades are associated with recovery from surgery. Specifically, 
changes in cyclic AMP-responsive element-binding protein 1 (CREB), signal transducer and activator of 
transcription 3 (STAT3), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) signaling in 
monocytes were associated with postoperative functional hip recovery, fatigue, and pain, respectively (Gaudilliere 
2014). Therefore, measuring the effect of GRF6021 on intracellular phosphorylation events in precisely 
phenotyped immune-cell subsets may increase our understanding of the immunomodulatory effects of GRF6021 
and its potential to enhance postoperative recovery. 
 
In 2011, data in parabiotic animal models suggested that differences in systemic chemokines, immune signaling 
molecules, and rejuvenating factors between young and old animals may explain the benefits in old animals of 
heterochronic parabiosis (Villeda 2011). Following these findings, the investigators performed a study to explore 
the therapeutic effects of systemic exposure of aged mice to young mouse plasma by intravenous injection 
(Villeda 2014). Systemic administration of young mouse plasma into aged mice improved age-related 
impairments. 
 
Results from nonclinical studies conducted by Alkahest (see Section 2.2, Rationale) have repeatedly demonstrated 
beneficial effect of the plasma protein fraction GRF6021 in age-related decline and histopathological endpoints in 
mouse models, including a reduction in neuroinflammation. Specifically, treatment with GRF6021 – in aged mice 
and in mouse models characterized by neuroinflammation – reduces the number of microglia labeled with either 
ionized calcium binding adaptor molecule 1 (Iba1) or cluster of differentiation 68 (CD68). Since microglia derive 
from the same monocytic lineage as blood monocytes and tissue macrophages, these data demonstrate that 
GRF6021 has a specific effect on cells of the monocytic lineage. Given this, testing the effect of GRF6021 on 
intracellular signaling events in monocytes in the context of THA or TKA will provide important information 
about whether GRF6021 has the potential to improve postoperative recovery in patients undergoing hip and knee 
arthroplasty. 
 
2.2 
RATIONALE 
 
Protocol AKST6021-211 will evaluate GRF6021 administered via intravenous (IV) infusion. GRF6021 is a 
purified 5% plasma protein fraction depleted of coagulation factors and gamma globulins that is manufactured by 
fractionation from pooled human plasma by Grifols Therapeutics, Inc. Additional processing steps with high 
pathogen clearance capacity are undertaken to reduce the potential for pathogen transmission. GRF6021 is 
 

 
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indicated for the treatment of hypovolemic shock due to burns, crushing injuries, abdominal emergencies, and any 
other condition where there is a predominant loss of plasma fluids. GRF6021 is also used more generally as a 
colloid for volume expansion. Fluid management is an essential part of surgical care as both inadequate and 
excessive fluid replacement can increase the stress on the circulatory system with deleterious consequences for 
tissue healing and recovery after surgery. Intraoperative and postoperative fluid management can involve 
crystalloids, colloids, or a combination thereof. Crystalloids (e.g. normal saline) are associated with interstitial 
and pulmonary edema, because up to 80% of the fluid volume can distribute to the interstitial (extravascular) 
space. Colloids (e.g. 5% albumin or 5% plasma protein fraction) provide better volume expansion with less 
interstitial edema; however, they are associated with potential hypersensitivity reactions (Kayilioglu 2015). There 
is controversy in the field as to which type of fluid replacement is more appropriate, and clinical care often 
depends on the preferences of the individual anesthesiologist and surgeon. Therefore, the use of either crystalloids 
and/or colloids (like GRF6021) is part of the intraoperative and postoperative standard of care in US hospitals.  
 
Nonclinical studies conducted by Alkahest have demonstrated that GRF6021 confers beneficial outcomes in 
cognitive decline, motor function, neuroinflammation, pain, and histopathological endpoints in mouse models 
(ALK-2015-R002, VIV-2016-R003, VIV-2016-R009, VIV-2016-R014, VIV-2016-R020, VIV-2016-R021, VIV-
2016-R024, VIV-2017-R025, VIV-2017-R036, VIV-2017-R038, VIV-2018-R069, 2018-CET01, VIV-89).  
 
Surgical trauma triggers an intricate programmed immune response (Stoecklein 2012). Characterization of this 
injury-mediated series of intracellular signaling cascades can provide insights about time to, and ease of, recovery 
from surgery. Therefore, measuring immune function responses at the single-cell level, and functional and 
phenotypic characterization of signals specific to immune cell subsets, may point to diagnostic signatures and 
potential therapeutic targets that could postoperatively improve patient recovery (Gaudillière 2014, Fragiadakis 
2015).  
 
In this randomized, placebo-controlled pilot study, approximately 45 patients will be enrolled (with the goal of 
obtaining 40 evaluable subjects) who are scheduled to undergo primary hip or knee arthroplasty without major co-
morbidities. Subjects will be randomized 1:1 (active: placebo) to evaluate the effect of GRF6021 on intracellular 
signaling cascades in blood leukocytes and the relationship of these immunomodulatory effects with postoperative 
recovery. In addition, the effects of GRF6021 on plasma proteomics will be investigated. Four (4) doses of 250 
mL of GRF6021 or placebo will be administered over 3 days. Each dose will be administered over approximately 
30 minutes as per Stanford Hospital standard procedures, but infusion rates and infusion time may be adjusted 
according to tolerability or other factors as determined clinically by the anesthesiologist and infusion nurse. 
Assessments of surgical recovery will be measured at baseline, during the inpatient stay, and for 6 weeks after 
hospital discharge. 
 
Human dose levels were selected to be consistent with the standard clinical use of GRF6021 as a colloid. In 
previous studies of patients undergoing THA at Stanford, the median volume of crystalloids for fluid management 
was 1,500 mL (interquartile range 1,000 to 2,000 mL) (Gaudilliere 2014). Given a conversion factor of 1.5-2 
between colloids and crystalloids, 1,500 mL of crystalloid is the equivalent of 750-1,000 mL of colloid. The total 
volume of GRF6021 that will be administered in this study is 1,000 mL.  
 
In mice, no safety concerns were observed using repeated daily (5-7) doses of 150 µL, which is the dosing 
regimen that produced robust anti-neuroinflammatory effects. Because GRF6021 contains a complex mixture of 
proteins with a molecular weight predominantly <100 kDa, and because the beneficial effects of these proteins on 
inflammation are believed to occur in the circulation, the concentration of these proteins per blood volume 
appears to be the scaling method most likely to accurately estimate the human potential effective dose.  
 
 

 
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Using isometric scaling based on blood volume, the mouse dose of 150 µL is equivalent to a human dose of 
413 mL, as outlined in Table 1. The dose proposed in this study is 250 mL GRF6021 per infusion. 
 
Table 1 
Isometric Scaling Based on Blood Volume 
 
Mouse 
Human Equivalent 
Dose (70 kg person) 
Human 
Proposed Dose 
GRF6021 Protein Content 
5% 
5% 
5% 
Dose Volume 
150 µL 
413 mL 
250 mL 
Protein per Dose 
7.5 mg 
20.7 g 
12.5 g 
Blood Volume 
2 mL 
5,500 mL 
5,500 mL 
Protein per Dose per mL of Blood 
3.75 mg/mL 
3.75 mg/mL 
2.3 mg/mL 
 
Given the long-standing clinical experience with GRF6021 in which doses of 250 mL to 1.5 L have been well 
tolerated, and the standard-of-care use of GRF6021 and similar colloids for fluid management, the investigational 
regimen of 4 doses of 250 mL of GRF6021 over a period of 3 days represents a reasonable and appropriate dosing 
regimen for evaluation in this clinical context (PSUR, PI, Bertrand 1959, Gahart 2019). 
 
2.3 
POTENTIAL RISKS AND BENEFITS 
 
2.3.1 
KNOWN POTENTIAL RISKS 
 
GRF6021, a 5% human plasma protein fraction, is reduced in coagulation factors and gamma globulins and 
serves as a viable source of soluble, infusible plasma proteins from healthy male and female donors. 
GRF6021 has been marketed since 1958 in the US, is approved in other parts of the world, and has a well-
established safety profile with a very low frequency of reported infusion reactions (0.002%) (PSUR). Based 
on the cumulative review of post-marketing safety surveillance data (PSUR), the benefit-risk balance for 
GRF6021 as an investigative product in individuals undergoing THA or TKA is favorable. Because it has 
been depleted of the majority of immunogenic proteins, GRF6021 is not expected to alter the blood typing 
characteristics of the recipient and should not sensitize humans to its subsequent administration (Bertrand 
1959). ABO antigen typing is not required prior to administration. Finally, the additional steps taken during 
production to remove viral pathogens provide an increased margin of pathogen safety in comparison to fresh 
frozen plasma.  
 
GRF6021 is not expected to significantly alter safety laboratory parameters, including urinalysis, coagulation 
times, prothrombin times, prothrombin consumption, platelet counts, or fibrinogen levels when given in 
quantities of up to 1000 mL (Bertrand 1959). Hypotension may occur, particularly following rapid infusion. 
The blood pressure may normalize spontaneously after the slowing or discontinuation of the infusion. 
Vasopressors will also correct the hypotension (PI). GRF6021 is contraindicated for use in patients on 
cardiopulmonary bypass as severe hypotension has been reported (Bland 1973). GRF6021 is contraindicated 
in patients with severe anemia, congestive heart failure, or increased blood volume (PI). 
 
GRF6021 is made from human plasma and may carry a risk of transmitting infectious agents, e.g., viruses, the 
variant Creutzfeldt-Jakob disease (vCJD) agent, and, theoretically, the Creutzfeldt-Jakob disease (CJD) agent. 
The risk that GRF6021 can transmit an infectious agent has been reduced by screening plasma donors for 
prior exposure, testing donated plasma, and including manufacturing steps with the capacity to inactivate 
and/or remove pathogens.  
 
 
 

 
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2.3.2 
KNOWN POTENTIAL BENEFITS 
 
There is no known benefit of administering GRF6021 to patients undergoing THA or TKA.  
 
3 
OBJECTIVES AND PURPOSE 
 
The primary objective of the study is to investigate the effect of GRF6021 on intracellular signaling cascades in 
blood leukocytes as determined by CyTOF. Secondary objectives include the investigation of GRF6021 on clinical 
recovery parameters in patients undergoing primary hip or knee arthroplasty, safety and tolerability of GRF6021, 
and the effects of GRF6021 on plasma proteomics.  
 
4 
STUDY DESIGN AND ENDPOINTS 
4.1 
DESCRIPTION OF THE STUDY DESIGN 
 
This will be a randomized, placebo-controlled pilot study conducted at a single site, Stanford University 
(Stanford, California, US) to investigate the effects of GRF6021, a 5% human plasma protein fraction 
administered by intravenous (IV) infusion, on intracellular signaling cascades in blood leukocytes in subjects 
undergoing primary hip or knee arthroplasty. 
 
Approximately 45 subjects will be enrolled with the aim of having 40 evaluable subjects randomized in a 1:1 ratio 
to active treatment or placebo. Each subject will receive 4 infusions of 250 mL of active GRF6021 or 4 infusions 
of 250 mL of placebo: 
 
1) 
1 infusion on the day before surgery 
2) 
1 infusion on the day of surgery within 4 hours before surgery start (first incision)  
3) 
1 infusion on the day of surgery upon arrival in the postoperative care unit (within 5 hours after the first 
incision)  
4) 
1 infusion on the day after surgery 
 
Each infusion will be administered over approximately 30 minutes according to Stanford Hospital standard of 
care, but infusion rates and infusion time may be adjusted for tolerability or other clinical reasons, as determined 
by the anesthesiologist and infusion nurse.  
 
CyTOF, a multiplexed, high-content immune profiling technology, will be utilized to provide high-resolution 
surveillance of circulating immune cells and the response to GRF6021 infusions on surgical recovery. Additional 
functional and quality of life assessments will be measured at baseline and for 6 weeks after hospital discharge. 
For a detailed overview of the timing of study assessments and interventions, please review Section 15 Schedule 
of Events. 
 
The overall duration of the study/recruitment period is approximately 12 months from study initiation (i.e., 
following consent of first subject) to study completion (i.e., last subject, last visit). The subject participation 
period is approximately 7 to 12 weeks from screening through end of study, unless prematurely discontinued.  
 
 
 
 
 
 

 
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4.2 
STUDY ENDPOINTS 
 
4.2.1 
PRIMARY ENDPOINT 
 
Primary Endpoint:  
• 
Effect of GRF6021 on intracellular signaling cascades in blood leukocytes as determined by CyTOF. 
 
4.2.2 
SECONDARY ENDPOINTS 
 
This study is not powered to detect statistically significant differences in outcomes. Secondary endpoints will 
be summarized over the study period from baseline values using descriptive statistics. 
 
Secondary Endpoints:  
• 
Change from baseline in 3D-CAM (Inouye 2003)(Appendix 1). 
• 
Time to 50% recovery of the baseline value in the SRS (Paddison 2011)(Appendix 2). 
• 
End of study treatment comparison of the Western Ontario and McMaster Universities Osteoarthritis 
Index (WOMAC) (Bellamy 1986, Bellamy 2002)(Appendix 3). 
• 
Time to a score of < 12/40 on a subset of questions from the WOMAC, Pain Subscale (Bellamy 1986, 
Bellamy 2002)(Appendix 3). 
• 
Time to a score of < 18/60 on a subset of questions from the WOMAC, Physical Function Subscale 
(Bellamy 1986, Bellamy 2002)(Appendix 3). 
• 
Change from baseline in the SF-36 (Laucis 2015)(Appendix 4). 
• 
Change from baseline in the BDI-II (Beck 1996)(Appendix 5).  
• 
Opioid analgesic consumption during hospital stay and after discharge to end of study. 
• 
Time to discharge.  
• 
Perioperative outcomes (e.g., surgery duration, anesthesia duration, PACU stay duration, ASA status, 
EBL, type and amount of intraoperative fluids administered, type and amount of intraoperative blood 
products, dose and type of anesthesia (general, regional), and intraoperative opioid dose). 
• 
Change in functional status using the ActiGraph wearable device  (ActiGraph Corporation 2019) 
providing measurements for physical activity/function and sleep (starting at least 3 days prior to 
surgery and continuing until the end of the study). 
• 
Change from baseline in clinical laboratory parameters. 
• 
Change from baseline in vital sign measurements. 
• 
Change from baseline in ECG. 
• 
The effects of GRF6021 on plasma proteomics.  
 
5 
STUDY ENROLLMENT AND WITHDRAWAL 
5.1 
INCLUSION CRITERIA 
 
In order to be eligible for inclusion, all subjects must meet the following criteria: 
1. Men and women scheduled to undergo primary THA or TKA. 
2. Aged 50-85 years at time of enrollment, inclusive. 
3. Renal function as defined by estimated glomerular filtration rate (eGFR) ≥ 45 mL/min/1.73 m2 using 
the Modification of Diet in Renal Disease (MDRD) study equation. 
4. Female subjects must not be pregnant or breastfeeding. Women of childbearing potential (WOCBP) 
must have a negative pregnancy test at screening. WOCBP must agree to use acceptable forms of 
contraception (Clinical Trial Facilitation Group) prior to study entry. A woman is considered of 
 

 
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childbearing potential following menarche and until becoming postmenopausal (no menses for at 
least 2 years without an alternative cause). Should a woman become pregnant or suspect she is 
pregnant while she or her partner is participating in the study, she should inform her treating 
physician immediately. 
5. The subject must understand and speak English and be able to understand and follow the study 
procedures, receive the treatment in the established timeframe, and continue during the follow-up 
interval.  
6. Provide a signed and dated informed consent form in accordance with local regulations and/or 
IRB/IEC guidelines. 
 
5.2 
EXCLUSION CRITERIA 
 
An individual will not be eligible for inclusion if any of the following criteria apply: 
1. Blood coagulation disorders, including severe liver disease, vitamin K deficiency, von Willebrand 
disease, hemophilia A or B or other known deficiencies in clotting factors (fibrinogen, prothrombin, 
factor VII, X, XI, XII, or XIII) that in the opinion of the investigator could put the subject at risk.  
2. Subjects who started chronic anticoagulant therapy (warfarin, heparin, low-molecular weight 
heparin, or Factor Xa inhibitors) in the last 6 months. Use of pre-, intra- and post-operative 
anticoagulation according to standard of care is permitted. Use of antiplatelet drugs (e.g., aspirin or 
clopidogrel) is permitted. 
3. History of a known hypercoagulable state, including Factor V Leiden thrombophilia, prothrombin 
gene mutation, deficiencies in antithrombin, protein C or protein S, elevated levels of fibrinogen, or 
dysfibrinogenemia.  
4. History of deep vein thrombosis or pulmonary embolism in the last 6 months and/or if it is 
associated with a known hypercoagulability disorder.  
5. History of disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, or 
heparin-induced thrombocytopenia in the last 5 years. 
6. Prior hypersensitivity reaction to any human blood product including plasma or a plasma protein 
fraction. 
7. Treatment with any human blood product, including transfusions and IV immunoglobulin, during the 
6 months prior to screening. 
8. History of immunoglobulin A (IgA) or haptoglobin deficiency; at screening a test for IgA must be 
performed to confirm detectable levels of IgA. 
9. History of complications of IV immunoglobulins, including stroke, anaphylaxis, or thromboembolic 
events.  
10. History of major surgery, trauma, or injury in the last 3 months or minor surgery in the last 1 month. 
11. History of unstable coronary heart disease (e.g. myocardial infarction or severe or unstable angina in 
the 6 months prior to dosing).  
12. Moderate or severe congestive heart failure (New York Heart Association Class III or IV or 
metabolic equivalent < 4). 
13. Uncontrolled high blood pressure (systolic blood pressure of 170 mmHg or higher and/or diastolic 
blood pressure of 110 mmHg or higher) despite adequate treatment during the 3 months prior to 
dosing. 
14. Current treatment with 4 or more antihypertensive medications of different classes. 
15. Clinically significant abnormalities on screening electrocardiogram (ECG) that in the opinion of the 
investigator could put the subject at risk, including QTc intervals (using Fridericia’s correction 
formula) of ≥ 450 ms in men and ≥ 470 ms in women.  
16. Current, active liver disease: > 3-fold elevation of liver enzymes (alanine aminotransferase (ALT) 
 

 
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and aspartate aminotransferase (AST) over upper limit of normal.  
17. Clinically significant abnormalities in complete blood count (CBC), complete metabolic panel, 
serum albumin, or coagulation. 
18. History of a major infection (e.g. bacterial pneumonia) in the last 3 months, or recent minor infection 
(e.g. influenza, urinary tract infection) or fever (measured body temperature above 100.5 degrees F 
on 2 consecutive days) that in the assessment of the Principal Investigator could interfere with the 
primary endpoint. 
19. Severe anemia (hemoglobin < 8 g/dL). 
20. Urine protein-to-creatinine ratio (UPCR) of > 1.5 grams of protein per gram of creatinine. 
21. Inadequate venous access to allow IV drug delivery or multiple blood draws.  
22. Concurrent participation in any other therapeutic treatment trial. If there was prior clinical trial 
participation, subject must have discontinued investigational agents for at least 30 days for small 
molecules, and 1 year for active or passive immunotherapies prior to screening.  
23. Current diseases associated with significant alterations in immune function, including, but not 
limited to, autoimmune disease and cancer, except adequately-treated squamous or basal-cell 
carcinoma. 
24. Ongoing immunosuppressant treatment or within 5 half-lives of the treatment. 
25. History of substance abuse (alcohol, drugs of abuse) in the last 2 years. 
26. Functional impairment of major joint or lower extremity other than joint undergoing surgery. 
27. Any other condition and/or situation that the investigator believes may interfere with the safety of 
the subject, study conduct, or interpretation of study data. 
 
5.3 
STRATEGIES FOR RECRUITMENT AND RETENTION 
 
The Sponsor does not anticipate any specific challenges in enrolling 45 subjects with the goal of obtaining at least 
40 evaluable subjects in this study. Subjects will be recruited continuously until the planned sample size is 
achieved. Subjects who withdraw or are withdrawn during screening, as well as subjects who discontinue, may be 
replaced (see Section 5.4.2).  
 
The expected length of participation in the study of approximately 7 to 12 weeks is not expected to be challenging 
to subjects. A description of the study will be included in local clinical trial databases, as required.  
 
5.4 
SUBJECT WITHDRAWAL OR TERMINATION 
 
5.4.1 
REASONS FOR WITHDRAWAL OR TERMINATION 
 
 
 
 
A subject may be withdrawn from the study for the following medical or administrative reasons: 
• 
Occurrence of an adverse event (AE) that represents an unacceptable risk to the subject and when 
continued participation in the investigational study is not warranted, in the judgment of the 
investigator or Sponsor Medical Monitor. The investigator must follow the subject until the AE 
resolves or is stable, unless the subject is lost to follow up. 
• 
Treatment with a prohibited concomitant medication other than the use of appropriate medications 
for the treatment of AEs under direction of the investigator. 
• 
Subject noncompliance, defined as refusal or inability to adhere to the trial schedule or procedures. 
• 
At the request of the subject (e.g., subject withdraws consent), investigator, Sponsor, or regulatory 
authority. 
• 
Pregnancy. 
 

 
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5.4.2 
HANDLING OF PARTICIPANT WITHDRAWALS OR TERMINATION  
 
 
Subjects will be encouraged to complete the study and all assessments. Subjects may voluntarily withdraw at 
any time, and the investigator may discontinue individual subjects from the study at any time.  
 
Approximately 45 subjects (1:1 ratio of GRF6021 to placebo) will be enrolled in the study with the aim of 
having 40 evaluable subjects. Evaluable subjects are those who received all 4 doses and had all blood draws 
for assessment of the primary endpoint. Subjects who discontinue or are unblinded may be replaced. Subjects 
who withdraw or are withdrawn during screening will be replaced.  
 
5.5 
PREMATURE TERMINATION OR SUSPENSION OF STUDY 
 
The Sponsor reserves the right to terminate the study at any time. Should this be necessary, the Sponsor and/or 
their representatives will arrange discontinuation procedures and notify FDA and the IRB. In terminating the 
study, the Sponsor and the investigator will continue to protect the subjects’ privacy and identity as required by 
relevant statutes and regulations. 
 
Alkahest, Inc. has the right to terminate the study at any time. Reasons for study or site termination may include, 
but are not limited to: 
• 
(Immediate) risk to subject safety. 
• 
Unsatisfactory subject enrollment. 
• 
Unacceptable Protocol Deviations as assessed by the Medical Monitor. 
• 
Inaccurate or incomplete data entry and recording/fabricated data. 
• 
Investigational site non-compliance with ICH/GCP. 
• 
Unacceptable emergent safety profile. 
 
6 
STUDY AGENT 
6.1 
STUDY AGENT AND CONTROL DESCRIPTION 
 
 
6.1.1 
ACQUISITION 
 
GRF6021 is manufactured by Grifols Therapeutics, Inc. (Clayton, North Carolina, US). The placebo control 
agent will be 0.9% sodium chloride for injection (normal saline) manufactured by Laboratorios Grifols, S.A. 
(Parets del Vallès, Barcelona, Spain). Both GRF6021 and placebo will be supplied to the site directly from a 
depot.  
 
6.1.2 
FORMULATION, APPEARANCE, PACKAGING, AND LABELING 
 
GRF6021 is prepared from large pools of human source plasma that has been depleted of coagulation factors 
and gamma globulins (PI). GRF6021 is iso-oncotic with normal human plasma and isotonic. Each 100 mL of 
GRF6021 contains 5 g selected plasma proteins buffered with sodium carbonate and stabilized with 0.004 M 
sodium caprylate and 0.004 M acetyltryptophan. The plasma proteins consist of approximately 88% normal 
human albumin, 12% alpha and beta globulins, and not more than 1% gamma globulin as determined by 
electrophoresis. The approximate concentrations of the main electrolytes in GRF6021 are: sodium 145 
 

 
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mEq/L, potassium 0.25 mEq/L, and chloride 100 mEq/L. 
 
Both GRF6021 and placebo will be supplied in 250 mL glass vials labeled for investigational use according to 
the local regulatory requirements for clinical studies.  
 
6.1.3 
PRODUCT STORAGE AND STABILITY 
 
The study agent and placebo should be stored at room temperature not exceeding 30°C (86°F) and not used 
after the expiration date. Solution that has been frozen should not be used. Solutions that are turbid should not 
be used. Administration of the study agent or placebo must begin within 4 hours of the container being 
entered. Vials that are cracked or have been previously entered or damaged should not be used, as this may 
have allowed the entry of microorganisms. Neither the study agent nor the placebo contain preservatives. 
 
6.1.4 
PREPARATION 
 
GRF6021 vials should be inspected visually for particulate matter and discoloration prior to administration, 
whenever solution and container permit. The study agent and placebo should be prepared in accordance with 
the GRF6021 Infusion Administration Manual that will be provided to the site (see Section 6.1.5 Dosing and 
Administration).  
 
6.1.5 
DOSING AND ADMINISTRATION 
 
The study agent/placebo will be infused in accordance with the GRF6021 Infusion Administration Manual. 
The purpose of the GRF6021 Infusion Administration Manual is to promote safe administration of GRF6021 
and to maintain appropriate blinding of staff and study subjects. The manual will include provisions for 
masking study agent/placebo and maintaining the staff and study subjects’ blind during each infusion. 
 
The study agent/placebo to be administered will be dispensed by an unblinded pharmacist (or other qualified 
personnel responsible for drug accountability) to blinded study personnel for transport to an unblinded 
infusion nurse. The study agent/placebo may also be dispensed directly to the unblinded infusion nurse. 
Administration of the study agent/placebo will be performed by the unblinded infusion nurse. Infusion nurses 
will be qualified by training and experience to administer infusions under the direction of the Principal 
Investigator. Only authorized infusion nurses who complete the training on the Infusion Administration 
Manual may administer the study agent/placebo, and only subjects enrolled in the study may receive the study 
agent/placebo in accordance with applicable regulatory requirements. 
 
6.1.6 
ROUTE OF ADMINISTRATION 
 
The study agent and placebo will be administered by IV route only. 
 
6.1.7 
DOSING SCHEDULE 
 
Subjects will be randomized to GRF6021 (Group 1: active dosing) or placebo (Group 2: placebo dosing). 
Subjects will receive 4 infusions of 250 mL per the following schedule: 
 
1) On the day before surgery 
2) On the day of surgery within 4 hours before surgery start (first incision)  
3) On the day of surgery upon arrival in the postoperative care unit (within 5 hours after the first incision)  
 

 
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4) On the day after surgery 
   
6.1.8 
DURATION OF THERAPY 
 
From screening to exit, the duration of study involvement for each subject is approximately 7 to 12 weeks. All 
subjects will receive 4 infusions over 3 days. The duration of therapy for a subject to be considered evaluable 
in the intent-to-treat population is 4 doses. 
 
6.2 
STUDY AGENT ACCOUNTABILITY 
 
Under the supervision of the Principal Investigator, the study pharmacist or other qualified unblinded personnel 
are responsible for ensuring adequate accountability of all used and unused study agent and placebo control agent. 
This includes acknowledgment of receipt of each shipment of study agent and placebo (quantity and condition), 
subject dispensing records, and returned or destroyed study agent/placebo. Dispensing records will document 
quantities received and quantities dispensed to subjects including the date dispensed, intended subject’s study 
identifier, initials of the individual responsible for dispensing, and initials of the infusion nurse administering the 
study agent. Drug accountability will be monitored by an unblinded Clinical Research Associate. 
 
Accountability records must be maintained and readily available for inspection by representatives of Alkahest, 
Inc. or their designee and are open to inspection by regulatory authorities at any time. The accounts of any study 
agent/placebo accidentally wasted or intentionally disposed of must be maintained.  
 
The disposal of used, partially used, or wasted study agent/placebo must be performed in accordance with the 
institution’s drug disposal policy. At study initiation, the clinical study monitor will evaluate the site’s standard 
operating procedure for study drug disposal/destruction in order to ensure that it complies with study 
requirements. At the end of the study, following final drug reconciliation by the monitor, the study site will be 
instructed by the Sponsor to return or destroy all unused study agent/placebo. A copy of the institution’s drug 
disposal policy should be maintained or referenced in the investigator’s study file. 
 
7 
STUDY PROCEDURES AND SCHEDULE 
7.1 
STUDY PROCEDURES/EVALUATIONS 
 
7.1.1 
STUDY SPECIFIC PROCEDURES 
 
7.1.1.1 Screening Procedures 
During the screening period, the following information will be obtained: 
• 
Medical history and comorbidities 
• 
Demographics 
• 
Review of medications 
• 
Vital signs 
• 
Physical examination 
• 
Blood and urine collection for laboratory evaluations 
 
Detailed descriptions of each of these procedures are provided in the sections immediately following. Some 
portions of information used to determine eligibility can be obtained from the subject directly or from an 
electronic medical record. Information pertaining to all study activities performed during screening, and the 
sequence of events, is provided in Section 7.3.1 Screening. 
 

 
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7.1.1.1.1 Medical History and Comorbidities 
The investigator or designee will obtain pertinent medical history during screening that will include: 
• 
Current/past illnesses and conditions, including a comprehensive review of comorbidities prior to 
surgery  
• 
Current symptoms of any active medical condition 
• 
Surgeries and procedures 
• 
Allergies 
• 
Social history (e.g. exercise, smoking, substance use) and current living situation 
 
7.1.1.1.2 Demographics 
Demographic information such as the subject’s age, ethnicity, and race will be collected at screening. 
 
7.1.1.1.3 Review of Medications 
The investigator or designee should review the subject’s current medications, including over-the-counter 
drugs, herbal supplements, and/or vitamins, as well as those taken by the subject in the past 1 month and any 
dose changes. In addition, any potential subjects who have had human blood product transfusions or 
immunosuppressants in the last 6 months will be excluded from the study. Assessment of eligibility should 
include a review of permitted and prohibited medications. Any additions, discontinuation, or dosage changes 
in medication during the course of the study will be recorded (see Section 7.1.1.2.2).  
 
7.1.1.1.4 Vital Signs 
Vital signs will include supine or seated systolic and diastolic blood pressure (BP) (mmHg), heart rate (HR) 
(beats per minute [bpm]), respiration rate (RR) (breaths per minute), and body temperature.  
 
7.1.1.1.5 Physical Examination 
A physical examination will be performed to assess the following organ systems: lungs/chest, heart, abdomen, 
neuro/spine.  Height and weight will also be measured at screening. Should the subject have a physical exam, 
including height and weight measurements, as part of standard of care during the screening window, then the 
results may be obtained from the subject’s medical record for the purposes of assessing eligibility.  
 
7.1.1.1.6 Biological Specimen Collection  
Biological samples will be analyzed at screening. Blood will be drawn by a qualified medical provider, and 
urine specimens will also be collected. The timing and frequency for specimen collection and laboratory 
evaluations to be performed are further described in Section 15 Schedule of Events.  
 
The labs that will be tested at screening include:  
• 
Pregnancy test. 
• 
Alkaline phosphatase, ALT, AST, bicarbonate, bilirubin (total), ionized calcium, chloride, creatinine, 
gamma-glutamyl transferase (GGT), glucose (random), lactate dehydrogenase, magnesium, 
phosphate, potassium, protein total, sodium, blood urea nitrogen, albumin, partial thromboplastin 
time (PTT), and prothrombin time/international normalized ratio (PT/INR).  
• 
CBC with differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils).  
• 
Urinalysis: blood, glucose, ketone, protein, pH, specific gravity, nitrite, leukocytes, urine creatinine, 
urine protein/creatinine ratio, (and reflex urine microscopy if indicated).  
• 
Infectious serology (anti-human immunodeficiency virus (HIV) antibodies, anti-hepatitis B surface 
antigen, anti-hepatitis B core antigen, anti-hepatitis C antibodies), direct antiglobulin test, red blood 
 

 
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cell (RBC) antibody screen (including direct Coombs and anti-RBC antibody), serum IgA, and 
haptoglobin.  
• 
Urine drug screen: cannabinoids, benzodiazepine, barbiturates, opiates, cocaine, amphetamines, 
phencyclidine.  
 
7.1.1.2 Procedures to Assess Safety 
Subjects enrolled in the trial will be monitored closely to assess safety and tolerability of the study agent and 
intervention. Study-specific procedures that will be used for this purpose are summarized below. Information 
regarding the timing and frequency of these procedures is provided in Section 7.3 Study Schedule.  
• 
Review of AEs 
• 
Review of medications 
• 
Vital signs 
• 
12-Lead ECGs 
• 
Targeted physical exams 
• 
Blood draw and urine collection for laboratory evaluations 
 
7.1.1.2.1 Review of Adverse Events 
AEs will be reviewed, documented, and reported as required at each visit, beginning at Screening. For 
definitions, guidance, and additional information regarding AEs, refer to Section 8.  
 
7.1.1.2.2 Review of Medications 
The investigator or designee should review the subject’s current medications, including over-the-counter 
drugs, herbal supplements and/or vitamins, as well as those taken by the subject since the last visit. Changes 
to the subject’s list of medications should be reviewed and recorded. Review of medications should occur at 
every visit.  
 
7.1.1.2.3 Vital Signs 
Refer to Section 7.1.1.1.4 for a description of vital signs. Vital signs will be collected at every treatment visit 
within 1 hour prior to infusion start and after infusion start, at 15, 30, 45, and 60 minutes (for further details 
see Section 15 Schedule of Events). Vital signs may be collected ad hoc during or after the infusion.  
 
7.1.1.2.4 12-Lead ECG 
A 12-lead ECG will be performed after the subject has rested quietly for at least 5 minutes in a supine or 
sitting position. In some cases, it may be appropriate to repeat abnormal ECGs to rule out technical factors 
contributing to ECG artifacts or abnormality. It is important that leads are placed in the same positions each 
time for consistency. The overall conclusion with the interpretation of the ECGs will be recorded on the 
appropriate CRF. The interpretation of the ECGs will be recorded as normal, abnormal but not clinically 
significant, or abnormal and clinically significant. Corrected QTc intervals will be calculated using 
Fridericia’s correction formula.  
 
7.1.1.2.5 Targeted Physical Exams 
A targeted physical exam, including auscultation of the heart and lungs and an assessment of peripheral 
edema, will be performed per the Study Schedule in Section 7.3. If performed as part of standard of care, then 
the results may be obtained from the subject’s medical record for review and assessment.  
 
 

 
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7.1.1.2.6 Blood and Urine Collection for Laboratory Evaluations 
In addition to screening evaluations (see Section 7.1.1.1.6), subjects will have blood and urine specimens 
collected for clinical evaluation (see Section 7.2.1 Clinical Laboratory Evaluations). Refer to Section 15 
Schedule of Events for timing and frequency of blood and urine collection for laboratory evaluations.  
 
The following labs will be performed within ~48 hours after the end of the infusion on Day 3 (exit lab panel): 
• 
Infectious serology (anti-HIV antibodies, anti-hepatitis B surface antigen, anti-hepatitis B core 
antigen, anti-hepatitis C antibodies), direct antiglobulin test, RBC antibody screen (including direct 
Coombs and anti-RBC antibody).  
• 
ALT, AST, bilirubin (total), ionized calcium, chloride, creatinine, potassium, protein total, sodium, 
blood urea nitrogen, PTT, PT/INR. 
• 
CBC with differential (basophils, eosinophils, lymphocytes, monocytes, neutrophils).  
• 
Urinalysis: blood, glucose, ketone, protein, pH, specific gravity, nitrite, leukocytes, urine creatinine, 
urine protein, urine protein/creatinine ratio, and reflex urine microscopy if indicated. 
• 
Pregnancy test. 
 
7.1.1.3 Procedures to Assess Efficacy 
Procedures to assess efficacy include investigations of intracellular signaling cascades in blood leukocytes as 
determined by CyTOF following administration of GRF6021. In addition, the effects of GRF6021 on clinical 
recovery parameters in patients undergoing THA and TKA, and the effects of GRF6021 on plasma 
proteomics will be evaluated. 
  
1. CyTOF 
2. 3D-CAM 
3. SRS 
4. WOMAC 
5. SF-36 
6. BDI-II 
7. POMS (covariate assessment) 
8. PSS (covariate assessment) 
9. Perioperative outcomes 
10. ActiGraph assessments 
11. Proteomic assessments 
 
Descriptions of each assessment are provided below. 
 
7.1.1.3.1 Mass Cytometry (CyTOF) 
Surgical trauma triggers an intricate programmed immune response (Stoecklein 2012). This injury-mediated 
series of intracellular signaling cascades provides insights about time and ease of surgical recovery. 
Therefore, measuring immune function responses at the single-cell level, and functional and phenotypic 
characterization of signals specific to immune cell subsets, may point to diagnostic signatures and potential 
therapeutic targets that could postoperatively improve patient recovery (Gaudillière 2014, Fragiadakis 2015). 
CyTOF provides high-dimensional numerical and functional characterization of the immune response to 
surgical trauma and enables the detection of biological mechanisms critically associated with relevant health-
associated outcomes (Gaudillière 2014). 
 
 

 
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7.1.1.3.2 3-Minute Diagnostic Interview for Confusion Assessment Method 
The 3D-CAM (Inouye 2003)(Appendix 1) is a brief verbal assessment tool used to test patients for delirium. 
Each item in the instrument directly informs one of the 4 CAM features including acute onset of mental status 
change or fluctuating course of cognition, inattention, disorganized thinking, and altered level of 
consciousness. For all items on the assessment, the patient’s answer is “incorrect,” “correct,” “no,” or “yes.”   
 
7.1.1.3.3 Surgery Recovery Scale 
The SRS (Paddington 2011)(Appendix 2) is a sensitive and simple tool for the assessment of functional 
recovery following major surgery. The scale contains 13 items. Impacts on daily activities are scored from 1 
to 5 or 6 (1= not at all). It’s considered the most broadly validated measure of surgical recovery available. 
 
7.1.1.3.4 Western Ontario and McMaster Universities Osteoarthritis Index  
The WOMAC (Pain and Physical Function Subscales) (Bellamy 1986, Bellamy 2002)(Appendix 3) was 
developed to assess pain, stiffness, and physical function in patients with knee and/or hip osteoarthritis, but it 
has been assessed among patients with many different conditions. The WOMAC is one of the most widely 
utilized self-report measures of lower extremity symptoms and function. It has been studied over a period of 
almost 30 years in many different contexts and patient populations, and there are abundant data regarding its 
utility and measurement properties. The Likert Scale version uses an 11-point scale anchored by the wording 
“no pain” and “extreme pain” for pain, and “no difficulty” and “extreme difficulty” for physical function. 
Higher scores on the WOMAC indicate worse pain and functional limitations. 
 
7.1.1.3.5 Short Form-36 
The SF-36 (Laucis 2015)(Appendix 4) is the most widely used health-related quality-of-life measure in 
research to date. The SF-36 yields 8 sub-scale scores and 2 summary scores for mental and physical health.  
 
7.1.1.3.6 Beck Depression Inventory-II 
The BDI-II (Beck 1996)(Appendix 5) was developed in 1996 and derived from the original BDI. It is a 
proprietary, 21-item self-administered survey scored on a scale of 0–3 in a list of four statements arranged in 
increasing severity about a particular symptom of depression. It is easy to complete, and relatively short 
compared to interview-based assessments. It was designed to assess mood within the most recent 2-week 
period, so comparison across assessments should reflect change over time. 
 
7.1.1.3.7 Profile of Mood States  
The POMS (Heuchert 2012)(Appendix 6) is widely used to assess affective traits, mood, and emotion. It 
contains 65 POMS items. It can be completed online or in a paper format in 3 to 5 minutes. For the purposes 
of this study, only 9 items in the “tension-anxiety” scale will be used as anxiety is an important covariate of 
postoperative pain.  
 
7.1.1.3.8 Perceived Stress Scale  
The PSS (Cohen 1983), is a widely used instrument consisting of 10 questions for measuring the degree to 
which situations in one’s life are stressful during the last month. Items were also designed to tap how 
unpredictable, uncontrollable, and overloaded respondents find their lives. The scale includes a number of 
queries about current levels of experienced stress. Answers are given as 0=never, 1=almost never, 
2=sometimes, 3=fairly often, and 4=very often. PSS scores are obtained by reversing responses (e.g., 0=4, 
1=3, 2=2, 3=1, and 4=0) to the 4 positively stated items (items 4,5,7, and 8) and then summing across all scale 
items. 
 
 

 
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7.1.1.3.9 Perioperative Outcomes 
Captured variables may include surgery duration, anesthesia duration, PACU stay duration, ASA status, EBL, 
type and amount of intraoperative fluids administered, type and amount of intraoperative blood products, dose 
and type of anesthesia (general, regional), and intraoperative opioid dose. 
 
7.1.1.3.10 ActiGraph Wearable Device 
At screening, subjects will be provided with an ActiGraph wearable device (ActiGraph Corporation 2019). 
Approximately -7 to -3 days prior to surgery, subjects will be instructed to wear the device which will then 
begin monitoring of physical activity, mobility, and sleep measures. Data collection is ongoing, and subjects 
will wear the device until admission, except during Visits 4a and 4b when it is removed for surgery.  
 
7.1.1.3.11 Proteomic Assessments 
Blood and plasma will be collected from subjects at multiple timepoints throughout the trial for proteomic 
assessments. Fasting samples are preferred. For information regarding the timing and procedures for sample 
collection and related requirements, refer to the study’s laboratory manual and Section 15 Schedule of Events.  
 
Plasma will be analyzed by proteomics using targeted approaches to assess the specific signature of proteins 
in subjects at baseline and to assess the changes in the proteome with repeated GRF6021 infusions. These 
methodologies will provide a broad overview of the proteins that are present in the plasma sample by 
assessing 1000-5000 analytes and allow generation of a proteomic signature. From this signature, it is hoped 
that key proteins that are drivers of postoperative recovery can be identified. Blood samples will be collected 
for analysis of emergent genetic markers. By understanding the composition and function of plasma samples 
from the trial, the goal is to identify the biomarkers relevant to further optimizing postoperative recovery in 
THA and TKA. 
 
7.2 
LABORATORY PROCEDURES/EVALUATIONS 
 
7.2.1 
CLINICAL LABORATORY EVALUATIONS 
 
Biological samples (e.g. whole blood, serum/plasma, urine) will be collected for laboratory evaluations in 
accordance with the Schedule of Events. Clinical sample processing and laboratory evaluations will be 
conducted by the local lab at Stanford Hospital according to their standard processes. Refer to the study’s 
laboratory manual for complete information regarding all exploratory biomarker (proteomics) samples to be 
collected. 
 
The investigator is responsible for determining and documenting whether out of range laboratory values are 
clinically significant. All clinically significant values will be recorded as AEs in the CRF and followed until 
determined to be stable or resolved, unless the subject is lost to follow up. Once resolved, the appropriate 
CRF page(s) will be updated. 
 
 
7.2.2 
SPECIMEN PREPARATION, HANDLING, STORAGE, AND SHIPPING 
 
For the biomarker (proteomics) samples, refer to the study’s laboratory manual for specimen preparation, 
handling, storage, and shipping procedures. All other laboratory samples will be collected and processed 
according to standard policies at Stanford Hospital. 
 
 

 
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7.3 
STUDY SCHEDULE 
 
 
7.3.1 
SCREENING 
 
Visit 1, Screening Visit (Day -42 to -3) 
□ Informed consent of subject as documented by a signed and dated informed consent form that must be 
obtained prior to any study-related assessments. 
□ Collect demographic information. 
□ Review subject’s current and prior medications, including opioid consumption assessment. 
□ Collect vital signs, height, and weight. 
□ Physical exam. 
□ Collect blood and urine samples required for Screening (including pregnancy test for WOCBP).  
□ Provide subject with ActiGraph wearable device and any training required for use of the device (the 
ActiGraph can also be mailed to the subject 1 week before the scheduled surgery).  
 
7.3.2 
BASELINE: REMOTE 
 
Subjects will be contacted by phone to obtain the remote Baseline Visit assessments. 
 
Visit 2, Baseline: Remote (Days -7 to -3) 
□ Review AEs and concomitant medications. 
□ SRS 
□ WOMAC 
□ SF-36 
□ BDI-II 
□ POMS 
□ PSS 
□ Subject will be instructed to begin wearing the ActiGraph device. 
 
7.3.3 
RANDOMIZATION 
 
Subjects will be randomized after eligibility has been confirmed. 
 
7.3.4 
BASELINE: ON-SITE/TREATMENT 
 
The on-site Baseline assessments will be gathered prior to the first infusion on Day 1. After the first infusion 
on Day 1, the subject will be discharged from the facility and come back the following morning (Day 2) for 
the inpatient period. The inpatient treatment period is 2 days during which each subject will receive 3 
additional doses of 250 mL of their assigned treatment allocation (i.e., either GRF6021 or placebo).  
 
Visits 3 through 5, Treatment (Days 1-3): 
□ Perform the following procedures on each day prior to administering (the first) study infusion: 
o Review AEs and concomitant medications. 
o 12-lead ECG (Day 1 and 3 Only). 
o Collect sample for CyTOF assessment (Including second infusion on Day 2). 
 

 
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o Obtain samples for proteomic assessments; fasting samples preferred (Including second infusion 
on Day 2). 
o Collect vital signs (BP, HR, RR, and body temperature) (Including second infusion on Day 2). 
o Targeted physical exam (Day 1 and 3 Only).  
o Pregnancy testing for WOCBP (Day 1 Only). 
o Administer 3D-CAM, SRS, and WOMAC (Day 1 and 3 Only). 
o ActiGraph device data collection (Day 1 and 3 Only). 
□ Administer study treatment per the GRF6021 Infusion Administration Manual: 
o On the day before surgery (Day 1). 
o On the day of surgery within 4 hours before surgery start (first incision) (Day 2).  
o On the day of surgery upon arrival in the PACU (within 5 hours after the first incision) (Day 2).  
o On the day after surgery (Day 3). 
□ 
Perform the following assessments following each infusion start: 
o BP and HR at 15, 30, 45, and 60 min after infusion start. 
o Body temperature and RR at 60 minutes. 
o Collection of perioperative data (Day 2 Only, after second infusion). 
o Opioid consumption assessment (Days 2 and 3 Only). 
o 12-lead ECG (Day 3+1 Only, after end of last infusion). 
o Exit lab panel including pregnancy test (Day 3 Only, after end of last infusion). 
o Administer 3D-CAM (Day 2 Only, after second infusion). 
 
7.3.5 
FOLLOW-UP 
 
All Follow-up Visits will be conducted remotely by phone or electronically. 
 
Visits 6 through 14 (Day 4 through Day 37±2) 
□ Review AEs and concomitant medications. 
□ Perform the following assessments: 
o 
SRS 
o 
WOMAC 
o 
Opioid consumption assessment. 
□ 
Review AEs and concomitant medications. 
□ 
Ongoing collections of data from wearable device. 
 
7.3.6 
END OF STUDY VISIT 
 
The End of Study Visit will be conducted remotely by phone and/or electronically. 
 
Visit 15, End of Study (Day 44 ± 2)  
□ Review AEs and concomitant medications. 
□ Perform the following assessments: 
o SRS 
o WOMAC 
o SF-36 
o BDI-II 
o Opioid consumption assessment. 
□ Subject will be instructed to return the ActiGraph wearable device. 
 
 

 
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7.3.7 
EARLY TERMINATION 
 
If a subject has received at least one infusion but is terminated or terminates from the study early, the site 
should try to perform all assessments scheduled at the End of Study Visit (Visit 15) and complete the exit lab 
panel if the subject is still at the site and available and willing.  
 
7.3.8 
SCHEDULE OF EVENTS TABLE 
 
A tabular summary of all procedures/assessments at each study visit can be found in Section 15 Schedule of 
Events. 
 
7.4 
CONCOMITANT MEDICATIONS 
 
All concomitant prescription medications taken during study participation will be recorded on the CRFs. For this 
protocol, a prescription medication is defined as a medication that can be prescribed only by a properly 
authorized/licensed clinician. Medications to be reported in the CRF are concomitant prescription medications, 
over-the-counter medications, and non-prescription medications. Medications that are considered standard use 
during surgery will not be collected in the CRF. 
 
7.4.1 
ANESTHESIA GUIDELINES 
 
For the purposes of this trial, it is recommended to:  
• 
Avoid adjuvant analgesic techniques that have immune-modulatory potential (e.g. ketamine or IV 
lidocaine infusions or bolus administration), unless clinically indicated. 
• 
Avoid other medications with immune-modulatory potential such as steroids (e.g. decadron), unless 
clinically indicated. 
 
7.5 
PROHIBITED MEDICATIONS, TREATMENTS, AND PROCEDURES 
 
The following concomitant medications are prohibited: 
• 
Concurrent participation in any other therapeutic treatment trial. If there was prior clinical trial 
participation, subject must have discontinued investigational agents for at least 30 days for small 
molecules, and 1 year for active or passive immunotherapies prior to screening.  
• 
Chronic use of anticoagulant therapy (e.g., heparin, warfarin, thrombin inhibitors, Factor Xa inhibitors). 
Anticoagulation used during surgery is permitted. Use of antiplatelet drugs (e.g., aspirin or clopidogrel) is 
acceptable. 
• 
Any drugs of the interferon class. 
• 
Systemic corticosteroids (e.g., hydrocortisone, cortisone, betamethasone, prednisone, prednisolone, 
triamcinolone, dexamethasone, fludrocortisone) for longer than 5 consecutive days. Ophthalmic, topical, 
intra-articular, and inhaled steroids are allowed. 
 
8 
ASSESSMENT OF SAFETY 
 
Assessment of subject safety is the responsibility of the Investigator or medically qualified designee. In 
extraordinary circumstances where knowledge of whether GRF6021 or placebo was received by a subject is 
 

 
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essential, the Investigator may unblind. Any instances of unblinding will be managed as indicated in Section 10.6.3 
Breaking the Study Blind/Subject Code. 
 
8.1 
SPECIFICATION OF SAFETY PARAMETERS 
 
8.1.1 
DEFINITION OF ADVERSE EVENTS 
 
Per 21 CFR 312.32(a), an AE is any untoward (unfavorable, harmful, or pathologic) medical occurrence in a 
subject administered a pharmaceutical (investigational) product even if the event does not necessarily have a 
causal relationship with this treatment.  
 
An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding that 
is deemed clinically significant), symptom, or disease temporally associated with the use of a medicinal 
(investigational) product, whether or not related to the medicinal (investigational) product.  
 
An AE does include any: 
• 
Exacerbation of a pre-existing illness. 
• 
Subjective or objective symptoms spontaneously offered by the subject and/or observed by the 
investigator or study staff. 
• 
Increase in frequency or intensity of a pre-existing episodic event or condition. 
• 
Condition detected or diagnosed after study drug administration even though it may have been 
present prior to the start of the study (unless it can be demonstrated by medical record review that 
the onset of the event preceded the date/time of Informed Consent). 
• 
Continuous persistent disease or symptoms present at baseline that worsen following the start of the 
study. 
• 
Symptoms associated with disease not previously reported by the subject. 
• 
Untoward medical occurrences considered by the investigator to be related to study-mandated 
procedures. 
• 
Abnormal assessments (e.g., change on physical examination, ECG findings), if they represent a 
clinically significant finding, that were not present at Baseline or worsened during the course of the 
study. 
• 
Laboratory test abnormalities, if they represent a clinically significant finding, symptomatic or not, 
which were not present at Baseline or worsened during the course of the study. 
 
An AE DOES NOT include a/an: 
• 
Elective medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, transfusion).  
• 
Pre-existing diseases or conditions present or detected at the start of the study that do not worsen. 
• 
Situations where an untoward medical occurrence has not occurred (e.g., hospitalization for cosmetic 
elective surgery, social and/or convenience admissions). 
• 
The disease or disorder being studied or sign or symptom associated with the disease or disorder 
unless more severe than expected for the subject’s condition. 
• 
Overdose of either study drug or concurrent medication without any signs or symptoms. 
 

 
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• 
Pregnancy. 
 
8.1.2 
DEFINITION OF SERIOUS ADVERSE EVENTS 
 
Note: if either the investigator or the Sponsor believes that the event is serious, the event must be considered 
serious and evaluated for expedited reporting.  
Note: the terms “severe” and “serious” are not synonymous. Severity (or intensity) refers to the grade of an 
AE. “Serious” is a regulatory definition.  
A serious adverse event (experience) or reaction is an untoward medical occurrence that, at any dose, fulfills 
one or more of the following criteria: 
a. Results in death (i.e., the AE actually causes or leads to death). 
b. Is life-threatening. 
• 
An AE is considered “life-threatening” if, in the view of either the investigator or Sponsor, its 
occurrence places the patient or subject at immediate risk of death; it does not include AEs which, 
had it occurred in a more severe form, might have caused death. 
c. Results in inpatient hospitalization or prolongation of existing hospitalization. 
• 
Hospitalization for elective treatment of a pre-existing condition that did not worsen during the study 
is not considered an AE; hospitalization for participating in this study is not considered an AE. 
• 
Complications that occur during hospitalization are AEs; if a complication prolongs hospitalization, 
the event is an SAE. 
• 
“Inpatient” hospitalization means the subject has been formally admitted to a hospital for medical 
reasons that may or may not be overnight; it does not include presentation at a casualty or emergency 
room unless the event meets the definition of an Important Medical Event (in the opinion of the 
Investigator or Sponsor). 
d. Results in persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
• 
The term ‘disability’ means a substantial disruption of a person’s ability to conduct normal life 
functions; this definition is not intended to include experiences of relatively minor medical 
significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, accidental 
trauma (i.e., sprained ankle) that may interfere or prevent everyday life functions but do not constitute 
a substantial disruption. 
e. Results in a congenital anomaly in the offspring of a subject who received drug. 
f. Results in an Important Medical Event. Important Medical Events are events that may not result in death, 
be life-threatening, or require hospitalization but may be considered an SAE when, based upon 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 medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug 
abuse. 
• 
Medical and scientific judgment should be used in deciding whether prompt reporting is appropriate 
in this situation. 
 
8.2 
CLASSIFICATION OF AN ADVERSE EVENT 
 
 

 
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purposes (FDA 2012). 
 
This definition of “unexpected” relies entirely on the Reference Safety Information in the Investigator’s 
Brochure as the basis for determining if newly acquired information generated from clinical trials or reported 
from other sources is unexpected. The suspected adverse reactions listed in the Investigator’s Brochure (i.e., 
“expected”) are those observed with the investigational drug and for which a causal relationship between the 
event and the drug is suspected or confirmed. 
 
Sponsor assessment of expectedness and relationship to study drug/causality will determine the need for 
expedited reporting of AEs. 
 
8.3 
TIME PERIOD/FREQUENCY FOR EVENT ASSESSMENT/FOLLOW-UP 
 
At every clinic visit, subjects will be assessed for AEs and SAEs. After the subject has had an opportunity to 
spontaneously mention any problems, the investigator should inquire about AEs by asking a non-leading question 
such as the following: 
1. 
“How are you feeling?” 
2. 
“Have you had any changes since your last assessment/visit?” 
3. 
“Have you taken any new medicines since your last assessment/visit?” 
 
8.3.1 
POST-STUDY AE AND SAE 
 
The investigator is not obligated to actively seek SAE information in former study subjects, but the 
investigator is encouraged to notify Alkahest, Inc. or their designee of any AE or SAE occurring within 30 
days after a subject completes the study (or has their last visit) that the investigator judges may be reasonably 
related to study treatment or study participation. 
 
8.4 
REPORTING PROCEDURES 
 
8.4.1 
ADVERSE EVENT REPORTING 
 
All subjects who have given informed consent will be evaluated for AEs. All AEs that occur after the time of 
treatment with the study agent will be considered Treatment Emergent AEs. Subjects with Treatment-
Emergent AEs must be followed until the AE is resolved or is stable, unless the subject is lost to follow up. 
 
Each AE or suspected adverse reaction must be described as follows: the date of onset, date of resolution, 
severity (mild, moderate, severe), frequency of the event (single episode, intermittent, continuous), action 
taken with study treatment (no action taken, treatment held, treatment discontinued), outcome, causality* 
(unrelated, possibly related, definitely related), and seriousness criteria. Each AE or suspected adverse 
reaction must be recorded separately. 
 
*Note: Causality assessment will be made only when the AE occurs after the subject has initiated at least one 
infusion of the study agent. An AE occurring before the subject’s exposure to study agent will always be 
labeled as “unrelated”. 
 
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CRF. Any SAE occurring during the study must be documented in the subject’s medical records and as an 
SAE in the CRF. 
 
A separate set of SAE pages should be used for each SAE. However, if at the time of initial reporting, 
multiple SAEs are present that are temporally and/or clinically related, they may be reported on the same SAE 
page. 
 
The investigator should attempt to establish a diagnosis of the event (that meets the definition of an AE or 
SAE) based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be 
documented as the AE and/or SAE and not the individual signs or symptoms. The diagnosis will become the 
basis for the verbatim term as reported by the investigator. If no diagnosis is known and clinical signs and 
symptoms are not present, the abnormal finding should be recorded. 
 
In addition to the investigator’s own description of the AE, each AE will be encoded according to the 
MedDRA. 
 
The investigator will take all appropriate and necessary therapeutic measures required for resolution of the 
AE, whether or not the AE is considered related to the investigational product. Any medication necessary for 
the treatment of an AE must be recorded on the concomitant medication CRF.  
 
The SAE pages of the CRF should be completed as thoroughly as possible and signed by the investigator or 
his/her designee before transmittal to the study Contract Research Organization (CRO). It is very important 
that the investigator provide his/her assessment of causality to study drug as well as an applicable diagnosis at 
the time of the initial SAE report. 
 
8.4.2 
SERIOUS ADVERSE EVENT REPORTING 
 
8.4.2.1 
Timeframes for Reporting SAEs 
Under 21 CFR 312.32(c), the Sponsor is required to notify FDA and all participating investigators in a safety 
report of potentially serious risks from clinical trials [i.e., Suspected Unexpected Serious Adverse Reactions 
(SUSARS)], as soon as possible after the Sponsor receives the safety information and determines that the 
information qualifies for reporting: 
• 
No later than 7 calendar days for events that are life threatening (in the opinion of the 
investigator or the Sponsor) or that involve Death as an outcome. 
• 
No later than 15 calendar days for all other SUSARS. 
 
As such, prompt notification of the Sponsor, and/or the Sponsor’s representatives, and promptly providing 
requested follow-up information regarding SAEs is essential so that ethical and regulatory responsibilities and 
legal obligations can be satisfied. Investigators are responsible for reporting SAEs according to the following 
timeframes:  
 
• 
All SAEs occurring during the study should be reported immediately. 
• 
The SAE Report Form and relevant source documents, if applicable, must be completed and emailed 
to drugsafety@inclin.com within 24 hours of observation or learning of the event.  
• 
Follow-up information must be sent to the CRO within 24 hours of receipt of information by the 
investigational site. 
 
SAEs will be followed until resolution, the condition stabilizes, the event is otherwise explained or is judged 
 

 
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by the investigator to be no longer clinically significant, or until the subject is lost to follow up. 
 
8.4.2.2 
SAE Information to Report 
All information available regarding an SAE must be submitted in the timeframes indicated. At a minimum, 
SAE reports must contain the subject ID, the SAE verbatim term, onset date, relationship to study 
drug/causality, and a brief narrative of the event. Please note that relationship to study drug/causality as 
well as the reported verbatim term are very important and should be included in the initial report as it 
may impact expedited regulatory reporting requirements for the event. The date of SAE discovery by the site 
staff should be documented in the source documents. 
 
The investigator must record all relevant information regarding an AE/SAE in the applicable sections of the 
CRF. It is not acceptable for the investigator to send photocopies of the subject’s medical records in lieu of 
completion of the appropriate AE/SAE pages. However, there may be instances when copies of medical 
records for certain cases are requested by the CRO and/or the Sponsor. If medical records are submitted to the 
CRO then all subject personal identifiers must be completely and thoroughly redacted prior to submission. 
 
A blank SAE Report Form and instructions for SAE reporting will be provided to the site and will be 
maintained in the investigator’s study file. The SAE Report Form must be completed and emailed to 
drugsafety@inclin.com according to the timeframes specified in Section 8.4.2.1. The SAE Report Form 
should include copies of relevant source documents, if applicable. Reconciliation of any discrepancy noted 
during monitoring and amending the eCRF is required. 
 
If new information about an SAE is received or corrections to data are needed, the investigator should 
complete a new SAE Report Form and check the “follow-up” box on the form. This follow-up SAE Report 
Form should be submitted within 24 hours of learning of the information, especially if the new information 
concerns seriousness, relatedness, or the event term of an AE. 
 
The site, acting under their local IRB, should submit all applicable events, unanticipated problems, and safety 
reports to the site’s local IRB, if applicable. All safety reporting deviations should also be submitted to their 
local IRB, if applicable. 
 
8.4.3 
ADVERSE EVENTS OF SPECIAL INTEREST 
 
The following will be considered AEs of special interest (AESI):  
• 
Clinically-significant peripheral edema or pulmonary edema. 
• 
Reduced kidney function (eGFR < 45 mL/min/1.73 m2). 
• 
Suspected transmission of blood-borne infectious agents. 
 
AESI occurring during the study should be reported within 48 hours of observation or learning of the event, 
unless the event is serious, in which case the event must be reported according to the timeframes specified in 
Section 8.4.2.1. 
 
 
8.4.4 
REPORTING OF PREGNANCY 
 
While pregnancy itself is not considered an AE, pregnancy occurring in a clinical study must be followed to 
collect information regarding the experiences of gestation and pregnancy with study agent exposure. The 
investigator must report any pregnancy that occurs in a female study subject subsequent to first exposure to 
 

 
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the study agent until End of Study, or 3 months following a subject’s last dose in the event of early 
termination. All pregnancies will be reported to the IRB, Sponsor, and CRO. In the event of a pregnancy, the 
subject will undergo continued safety follow-up through pregnancy outcome. 
 
Any pregnancy must be followed by the investigator until delivery or to the end of pregnancy. Any 
anomalies, complications, abnormal outcomes, or birth defect(s) observed in the child must be reported as an 
SAE within 24 hours of the investigator or study personnel’s first knowledge. 
 
8.5 
STUDY HALTING RULES 
 
If any of the following safety events occur, a Safety Evaluation Meeting (defined below) will be triggered: 
• 
Three or more SAEs in the same system/organ/class (SOC) that are assessed as possibly or definitely 
related to the study agent by the investigator and confirmed as such by the Sponsor (see Section 8.2.2 
Relationship to Study Agent). 
• 
Within or between any of the dosing groups: an overall pattern of symptomatic, clinical, or laboratory 
events associated with the study agent that the Sponsor’s Program Physician or designee consider a 
serious potential safety concern (e.g., suspicious overall pattern). 
Events that are more likely related to the infusion procedure, such as infiltration or hematoma, will not be 
considered “drug related” and will not contribute to the count of definitely-related SAEs that would trigger a 
Safety Evaluation Meeting. 
 
Safety Evaluation Meeting 
 
If safety events of potential concern occur during the trial (i.e., 3 related events in the same SOC or a suspicious 
overall pattern, as defined above) a Safety Evaluation Meeting may be triggered, and dosing may be temporarily 
halted based on the observations. The Sponsor will inform the FDA in the event of any temporary halt in dosing at 
any time during the conduct of the study. The purpose of the meeting is for the investigator, the Sponsor, and the 
CRO to discuss and evaluate the safety of the subjects using available aggregated safety data and without 
compromising study blinding, unless the Sponsor deems unblinding necessary for safety evaluation.  
 
Attendants at the Safety Evaluation Meeting will include the Program Physician of Alkahest (or his/her designee), 
the CRO, and available active investigators participating in the trial. After sufficient data review the Sponsor will 
choose one of the following courses of action: 
1. Continue dosing with no change to protocol. 
2. Halt dosing in all groups and stop the study.  
3. Continue with a modified protocol design and amend the protocol as appropriate. 
 
8.6 
SAFETY OVERSIGHT 
 
Safety oversight will be provided by the Sponsor’s Program Physician or his or her designee in concert with the 
site investigators. There will be no formal Data Safety Monitoring Board (DSMB) established. As needed, Safety 
Evaluation Meetings will be convened as described in Section 8.5 to monitor the ongoing safety of the study. The 
Sponsor’s Program Physician or designee is the final authority for safety oversight in the study. 
 
9 
CLINICAL MONITORING 
 

 
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Clinical site monitoring is conducted to ensure that the rights and well-being of human subjects are protected, that 
the reported trial data are accurate, complete, and verifiable, and that the conduct of the trial is in compliance with 
the currently approved protocol/amendment(s), with GCP, and with applicable regulatory requirement(s). 
 
• 
Monitoring for this study will be performed by the study CRO in accordance with the Clinical Monitoring 
Plan (CMP). 
• 
A mix of on-site and centralized monitoring will be performed to ensure the safety of clinical subjects and 
the accuracy and completeness of study data.  
• 
The Sponsor will be provided with copies of monitoring reports per the timelines specified within the 
CMP. 
• 
Details of clinical site monitoring tasks and scope are documented in the study’s CMP. The CMP 
describes in detail who will conduct monitoring, at what frequency monitoring will be done, at what level 
of detail monitoring will be performed, and the distribution of monitoring reports. 
• 
Independent audits may be conducted by the Sponsor in accordance with a quality oversight plan or 
equivalent to ensure monitoring practices are performed consistently and that monitors are following the 
CMP. 
 
10 
STATISTICAL CONSIDERATIONS 
10.1 STATISTICAL DESIGN MODEL AND ANALYTICAL PLANS 
 
Approximately 45 subjects (1:1 ratio of GRF6021 to placebo) will be enrolled in the study with the aim of having 
40 evaluable subjects. Evaluable subjects are those who received all 4 doses and had all blood draws for 
assessment of the primary endpoint.  
 
A Statistical Analysis Plan (SAP) with analytical details and assumptions will be developed and finalized before 
database lock and unblinding of the study data. 
 
10.2 STATISTICAL HYPOTHESES 
 
While we hypothesize that active treatment with GRF6021 will modulate the immune response to surgery, 
because this is an exploratory study, it is not designed to detect statistically significant differences between active 
and placebo on the primary endpoint. We will use a cell-signaling Elastic Net (csEN) approach to detect immune 
features (functional cellular attributes as well as cell abundance) that separate the active and placebo groups, and 
we hypothesize that active treatment will be associated with immunomodulatory changes that have previously 
been associated with improved clinical recovery. While a classical power calculation has not been performed, 
previous interventional studies from the Stanford group have demonstrated that a sample of 40 subjects is a 
pragmatic sample that enables the detection of clinically-relevant immunomodulatory changes, such as changes in 
signaling downstream of immune receptors, including interleukin-1 and Toll-like receptor 4.  
 
For secondary endpoints, the statistical approach toward efficacy endpoints will be primarily descriptive; within-
subject changes from baseline for each dosing group and among-group differences will be evaluated. The most 
important clinical outcomes will be the SRS (time to 50% recovery of the baseline score), WOMAC Pain (time to 
mild pain as indicated by a score <12/40), and WOMAC Physical Function (time to mild functional impairment 
as indicated by a score <18/60). 
 
10.3 ANALYSIS DATASETS 
 

 
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Four analysis datasets are possible; however, analyses may not necessarily be conducted with all four: 
• 
Intention-to-Treat (ITT) Dataset: all randomized participants. 
• 
Safety Dataset: all subjects who received at least one dose of the study agent.  
• 
Evaluable Dataset: all subjects who receive all 4 doses. 
– 
Per Protocol Dataset:  a subset of the ITT Dataset. A detailed description of the 
reasons for exclusion from the PP population will be included in the SAP. 
 
The presentation of baseline characteristics will be conducted on the ITT dataset. All safety analyses will be 
performed for the Safety Dataset. Analyses of the secondary endpoints will focus on the Evaluable and/or Per 
Protocol Datasets. 
 
10.4 DESCRIPTION OF STATISTICAL METHODS  
 
10.4.1 
GENERAL APPROACH 
 
Using the Evaluable and/or Per Protocol Datasets, all endpoints will be summarized serially over time 
using descriptive statistics to assess the within-subject changes and between-group differences. 
Overall baseline and demographic data will be summarized using descriptive statistics; between-
groups testing will be used to evaluate the effectiveness of the randomization in producing 
homogeneous pre-treatment groups.  
 
For analysis of the primary and secondary endpoints, the following will be considered:  
• 
For endpoints that are continuous in nature: 
– 
Number of observations, mean, median, minimum, and maximum, and standard deviation (SD) 
values will be presented as descriptive summary 
– 
For inferential statistics: 
 
If the Normality assumption is met, Paired t-test, or Analysis of Covariance (ANCOVA) using 
the baseline value as a covariate will be used 
 
If the Normality assumption is not met, a rank –ANCOVA analysis i.e., an ANCOVA analysis on 
rank-transformed data or other non-parametric methods will be used 
• For endpoints that are categorical in nature: 
– 
Frequency counts and percentages will be presented as descriptive summary 
– 
Chi-square test or Logit model will be used for inferential statistics 
 
Subject disposition (e.g., the number of subjects randomized, completed, and discontinued) will be 
summarized, and medical history data will be listed. Prior and concomitant medications taken from 
screening and during the study will be categorized by World Health Organization classification for 
therapeutic class and drug name, listed and summarized by number and percentage of subjects.  
 
Final analyses are not limited to the summaries described herein. As noted above, analytical details 
and assumptions will be fully presented in the SAP. 
 
10.4.2 
ANALYSIS OF THE PRIMARY ENDPOINT 
 
 
The study is not powered to detect significant changes in the immune signatures; however, using available 
data from analysis of this endpoint, including changes from baseline, the primary interest is to detect 
 

 
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immune features (and proteomic features) that separate the two groups. Machine leaning approaches using 
cross-validation algorithms will be the primary approach for such analysis. The output of this analysis will 
produce a list of significant immune and/or proteomic features.  
 
 
10.4.3 
ANALYSIS OF THE SECONDARY ENDPOINTS 
 
Using available data from analysis of the secondary endpoints, descriptive summaries will be developed. Of 
particular interest will be the within-subject changes from baseline and their distribution around a null value 
of zero and a comparison between groups to evaluate any trends in differences between subjects randomized 
to active and placebo agents.  
 
For safety endpoints, actual values and changes from baseline in clinical laboratory measurements and vital 
signs will be assessed and summarized. Abnormal lab or vital sign values will be determined and flagged in 
the listings. Laboratory shift tables or graphics displaying the change (number of subjects) relative to the 
reference range from baseline to each study visit may also be presented for each test. The investigator should 
exercise his or her medical and scientific judgment in deciding and documenting whether an abnormal 
laboratory finding, or other abnormal assessment is clinically significant. 
 
For safety endpoints that are continuous in nature (e.g. clinical laboratory parameters, systolic and diastolic 
blood pressure, heart rate, respiratory rate, body temperature) the mean, median, minimum, maximum, and 
standard deviation will be plotted over time. 
 
For safety endpoints that are categorical in nature (e.g. targeted physical exam or ECG abnormalities), the 
frequency counts and percentages will be presented as a descriptive summary. 
 
 
10.4.4 
ADHERENCE AND RETENTION ANALYSES 
 
Subject adherence with the study visit schedule, visit procedures, infusions, and subject retention will be 
assessed. Reasons for study discontinuation will be compared across groups and across other subgroups of 
subjects, as appropriate. 
 
10.4.5 
BASELINE DESCRIPTIVE STATISTICS 
 
See Section 10.4.1. 
 
 
10.4.6 
PLANNED INTERIM ANALYSES 
 
Safety will be monitored on an ongoing basis. If a Safety Evaluation Meeting is triggered (see Section 8.5), an 
ad hoc interim safety analysis will be performed. If such an ad hoc safety interim analysis is conducted, the 
treatment assignment will remain masked, unless unblinding is deemed necessary by the Sponsor for safety 
evaluation.  
 
10.4.7 
ADDITIONAL SUBGROUP ANALYSES 
 
Not applicable. 
 

 
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10.4.8 
MULTIPLE COMPARISON/MULTIPLICITY 
 
No adjustments for multiplicity will be employed. 
 
10.4.9 
TABULATION OF INDIVIDUAL RESPONSE DATA 
 
This will be further defined in the SAP. 
 
10.4.10 EXPLORATORY ANALYSES 
 
Not applicable. 
 
10.5 SAMPLE SIZE 
 
A total of approximately 45 subjects will be enrolled with the aim of having 40 evaluable subjects randomized in 
a 1:1 ratio to active treatment or placebo. The primary endpoint of this study (differences in immunological and/or 
proteomic signatures among patient groups), the principal computational approach (machine learning) used, and 
the exploratory nature of the study prevent a traditional power analysis. However, a sample size of 2 x 20 subjects 
was sufficient to detect relevant differences in previous studies examining interventions expected to have a similar 
effect size (Aghaeepour 2017). 
 
10.6 MEASURES TO MINIMIZE BIAS 
 
10.6.1 
ENROLLMENT/RANDOMIZATION 
 
To minimize the potential bias at the time of randomization, the study will be randomized in a 1:1 ratio 
(active: placebo), stratified by sex and surgical joint (hip, knee) to assure a balanced distribution of evaluable 
male and female subjects in both treatment groups.  
 
10.6.2 
EVALUATION OF SUCCESS OF BLINDING 
 
Success of blinding will be assessed based on all occurrences (intentional or unintentional) of unblinding of 
blinded study subjects or study personnel (e.g. investigators, study coordinators and other blinded site staff, 
the Sponsor or their representatives). All intentional and unintentional unblinding will be documented and 
reported. 
 
10.6.3 
BREAKING THE STUDY BLIND/SUBJECT CODE 
 
The study blind can be broken for safety reasons if the information is required for the management of SAEs or 
severe AEs. Any noted intentional or unintentional breaking of the blind should be reported to the Sponsor’s 
Program Physician. If unintentional unblinding occurs during the study, root cause analysis will be evaluated, 
and corrective actions implemented. 
 
11 
SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS 
 
 

 
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The site will maintain appropriate medical and research records for this trial, in compliance with ICH E6 R2 and 
regulatory and institutional requirements for the protection of confidentiality of subjects. The site will permit 
authorized representatives of regulatory agencies, the IRB, the Sponsor, or the Sponsor’s representatives to 
examine (and when permitted by applicable law, to copy) clinical records for the purposes of quality assurance 
reviews, audits, and evaluation of the study safety, progress, and data validity.  
 
Source data are all information, original records of clinical findings, observations, or other activities in a clinical 
trial necessary for the reconstruction and evaluation of the trial. Examples of these original documents and data 
records include, but are not limited to, hospital records, clinical and office charts, laboratory notes, memoranda, 
subject’s memory aids or evaluation checklists, pharmacy dispensing records, recorded audio tapes of counseling 
sessions, recorded data from automated instruments, copies or transcriptions certified after verification as being 
accurate and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, and subject files 
and records kept at the pharmacy, at the laboratories, and medico-technical departments involved in the clinical 
trial.  
 
It is not acceptable for the CRF to be the only record of a subject’s participation in the study. This is to ensure that 
anyone who would access the subject’s medical record has adequate knowledge that the subject is participating in a 
clinical trial. Source document templates will be developed for this study. 
 
12 
ETHICS/PROTECTION OF HUMAN SUBJECTS 
12.1 ETHICAL STANDARD 
 
The investigator will ensure that this study is conducted in full conformity with Regulations for the Protection of 
Human Subjects of Research codified in 42 CFR Part 11, 21 CFR Part 50, 21 CFR Part 54, 21 CFR Part 56, 21 
CFR Part 312, ICH E6 R2, and the Declaration of Helsinki. 
 
12.2 INSTITUTIONAL REVIEW BOARD 
 
This protocol and any accompanying material to be provided to the subject (such as advertisements, subject 
information sheets, or descriptions of the study used to obtain informed consent) will be submitted by the 
investigator to an IRB. Approval from the IRB must be obtained before starting the study and should be 
documented in a letter to the investigator specifying the protocol number, protocol version, documents reviewed, 
and date on which the committee met and granted the approval.  
 
All changes to the consent form will be IRB approved; a determination will be made regarding whether 
previously consented subjects need to be re-consented. 
 
Any modifications or amendments to the protocol must also be submitted to the IRB for approval prior to 
implementation. 
 
12.3 INFORMED CONSENT PROCESS 
 
12.3.1 
CONSENT FORMS 
 
Consent forms describing in detail the study agent, study procedures, and risks are given to the subject, and 
written documentation of informed consent is required prior to any study-related procedures. 
 
 

 
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12.3.2 
CONSENT PROCEDURES AND DOCUMENTATION 
 
It is the responsibility of the investigator or designee to obtain written informed consent from each subject 
participating in this study after adequate explanation of the aims, methods, objectives, and potential hazards 
of the study and prior to undertaking any study-related procedures. 
 
Subjects should have the opportunity to discuss the study with their family members or other advisors and the 
time to consider participation in the trial carefully. The subjects may withdraw consent at any time throughout 
the course of the trial. The rights and welfare of the subjects will be protected by emphasizing to them that the 
quality of their medical care will not be adversely affected if they decline to participate in this study. 
 
The investigator or designee must utilize an IRB-approved consent form that contains the elements required 
by ICH GCP and applicable regulatory requirements for documenting written informed consent. Each 
informed consent will be appropriately signed and dated by the subject and the person obtaining consent. A 
copy of the signed consent form will be provided to the subject. By signing the informed consent form, all 
parties agree they will complete the evaluations required by the study, unless they withdraw voluntarily or are 
terminated from the study for any reason. 
 
Investigators will be expected to maintain a screening log of all potential study candidates that includes 
limited information about the potential candidate (e.g., date of screening and reason for screen failure if 
appropriate).  
 
All subjects who provide consent will be assigned a unique study number. This number will be used to 
identify the subject throughout the clinical study and must be used on all study documentation related to the 
study subject. Once a number is assigned to a subject, that number will remain with that study subject and 
will not be reused. 
 
If an individual’s medical chart or results of diagnostic tests performed as part of an individual’s regular 
medical care are going to be used for screening, written informed consent must be obtained prior to review 
of that information in accordance with HIPAA. 
 
12.4 PARTICIPANT AND DATA CONFIDENTIALITY  
 
Subject confidentiality is held in strict trust by the participating investigators, their staff, the Sponsor, and their 
agents. This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the 
clinical information relating to subjects. Therefore, the study protocol, documentation, data, and all other 
information generated will be held in strict confidence. No information concerning the study or data will be 
released to any unauthorized third party without prior written approval of the Sponsor. 
 
The study monitor, other authorized representatives of the Sponsor, representatives of the IRB, or government 
regulatory agencies may inspect documents and records required to be maintained by the investigator, including 
but not limited to, medical records (office, clinic, or hospital) and pharmacy records for the subjects in this study. 
The clinical study site will permit access to such records. 
 
The investigator must assure that subjects’ anonymity will be strictly maintained and that their identities are 
protected from unauthorized parties. Only subject initials and an identification code (i.e., not names) should be 
recorded on non-local lab samples, requisitions, and any documents submitted to the CRO, Sponsor, and/or IRB. 
The investigator must keep a subject log showing codes, names, and addresses for all subjects screened and for all 
 

 
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subjects enrolled in the trial. The study subject’s contact information will be securely stored at each clinical site 
for internal use during the study. At the end of the study, all records will continue to be kept in a secure location 
for as long a period as dictated by local IRB and Institutional regulations. 
 
12.5 FUTURE USE OF STORED SPECIMENS 
 
With the subject’s approval, and as approved by local IRB, de-identified biological samples may be stored at 
Alkahest, or designee, for future use. These samples could be used for research. Alkahest will also maintain a 
code link that will allow linking of the biological samples to specific data from each subject, while maintaining 
masking of the identity of the study subject. Subjects will be consented, and this consent documented, to allow the 
Sponsor to store and use samples for further research. An individual subject can choose to withdraw consent to 
have biological specimens stored and used for future research. 
 
When the study is completed, access to study data and/or samples will be managed by Alkahest. In the event 
Alkahest transfers ownership to another commercial Sponsor, ownership of the samples may be transferred as 
well. 
 
13 
DATA HANDLING AND RECORD KEEPING 
13.1 DATA COLLECTION AND MANAGEMENT RESPONSIBILITIES 
 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of the site 
investigator. The investigator is responsible for ensuring the accuracy, completeness, and timeliness of the data 
reported. 
 
Source documents provide evidence for the existence of the subject and substantiate the integrity of the data 
collected. Source documents are filed at the investigator’s site. Data entered in the eCRFs that are transcribed 
from source documents must be consistent with the source documents or the discrepancies must be explained. All 
source documents should be completed in a neat, legible manner to ensure accurate interpretation of data. Black 
or blue ink is required to ensure clarity of reproduced copies. When making changes or corrections, cross out the 
original entry with a single line, and initial and date the change. DO NOT ERASE, OVERWRITE, OR USE 
CORRECTION FLUID OR TAPE ON THE ORIGINAL. The investigator may need to request previous medical 
records or transfer records, depending on the trial; also, current medical records must be available. 
 
For each subject who receives the study agent, the eCRF must be completed in a timely manner. The investigator 
will review and approve the eCRF for each study subject after all data have been entered, the eCRFs have been 
source document verified, and all queries have been resolved. This also applies to records for those subjects who 
fail to complete the study. If a subject withdraws from the study, the reason must be noted on the eCRF. If a 
subject is withdrawn from the study because of an AE, thorough efforts should be made to clearly document the 
outcome. 
 
All data collection and recordkeeping procedures must be compliant with applicable ICH GCP. 
 
13.1.1 
INVESTIGATOR RESPONSIBILITIES 
 
The investigator will comply with the protocol (which has been approved/given favorable opinion by an IRB), 
ICH GCP, and applicable regulatory requirements. The investigator is ultimately responsible for the conduct 
of all aspects of the study at the study site and verifies by signature the integrity of all data transmitted to the 
 

 
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Sponsor. The term “investigator” as used in this protocol as well as in other study documents, refers to the 
investigator or authorized study personnel that the investigator has designated to perform certain duties. Sub-
investigators or other authorized study personnel are eligible to sign for the investigator, except where the 
investigator’s signature is specifically required. 
 
13.1.2 
STUDY FILES 
 
The investigator must maintain adequate and accurate records to enable the conduct of the study to be fully 
documented and the study data to be subsequently verified. These documents should be classified into two 
separate categories (although not limited to) the following: (1) investigator’s study file, and (2) subject 
clinical source documents. 
 
The investigator’s study file will contain the protocol/amendments, eCRF, IRB approval with 
correspondence, informed consents, drug records, staff curriculum vitae and authorization forms, and other 
appropriate documents and study-specific manuals (e.g., lab manual). 
 
Subject clinical source documents would include (although are not limited to) the following: subject 
hospital/clinic records, physician’s and nurse’s notes, appointment book, original laboratory reports, ECG, 
radiologic imaging, X-ray, pathology and special assessment reports, consultant letters, screening and 
enrollment log, etc. 
 
13.2 STUDY RECORDS RETENTION 
 
Before the investigator destroys any material related to the clinical study, he/she must obtain approval in writing 
from the Sponsor. According to ICH (E6), essential documents should be retained until at least 2 years after the 
last approval of a marketing application in an ICH region and until there are no pending or contemplated 
marketing applications in an ICH region or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product. The Investigator must retain protocols; amendments; IRB 
approvals; copies of the Form FDA 1572 or equivalent; completed, signed, dated consent forms; patient medical 
records and original source documents; CRFs; drug accountability records; all study-related correspondence; and 
any other documents pertaining to the conduct of the study. These documents should be retained for a longer 
period, however, if required by the applicable regulatory requirements or by an agreement with the Sponsor. It is 
the responsibility of the Sponsor to inform the Investigator/institution as to when these documents no longer need 
to be retained. 
 
The investigator should keep a file where the full name and address of each subject and all signed informed 
consents are included for at least 15 years after completion of the trial. Any original study-related information that 
permits verification of inclusion and exclusion criteria, including clinical history, a copy of all data collection 
logs, and documents on the use of the study agent, must be stored for as long a time period as permitted by the 
center.  
 
Should the investigator wish to move study records to another location, arrangements must be made to store these 
in sealed containers so that they can be returned sealed to the investigator in case of a regulatory audit. Where 
source documents are required for the continued care of the subject, appropriate copies should be made for storage 
outside of the site. 
 
13.3 PROTOCOL DEVIATIONS 
 

 
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A Protocol Deviation is any noncompliance with the clinical trial protocol or with GCP. The noncompliance may 
be either on the part of the subject, the investigator, or the study site staff. When deviations occur, corrective 
actions are to be developed by the site and implemented promptly. 
 
These practices are consistent with ICH E6: 
• 4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
• 5.1 Quality Assurance and Quality Control, section 5.1.1 
• 5.20 Noncompliance, sections 5.20.1, and 5.20.2 
 
Protocol Deviations will be categorized as either Major or Minor and will be defined in the study-specific 
Protocol Deviation Guidance Plan, or equivalent.  
 
Major Protocol Deviations are departures from the approved protocol relating to the conduct of the study 
which may significantly impact the completeness, accuracy, and/or reliability of the study data or that may 
significantly affect the rights, safety or wellbeing of study participants. Major Protocol Deviations may 
result in data that are not deemed evaluable for the per protocol analysis and/or may require that subjects 
are discontinued from the study.  
 
Minor Protocol Deviations are departures from the approved protocol relating to the conduct of a study that does 
not affect the rights, safety, and/or wellbeing of study participants or the study outcomes or data quality. Minor 
Protocol Deviations do not require review by the Medical Monitor. Minor Protocol Deviations would not 
generally preclude subject data from the per protocol analysis population.  
All deviations will be logged and tracked by the site and CRO. Periodic review of Protocol Deviations will serve 
an indicator of site performance. 
 
It is the responsibility of the site to use continuous vigilance to identify and report deviations promptly to the 
study CRO and/or Sponsor. All deviations must be addressed in study source documents. Notification of Protocol 
Deviations must be sent to the local IRB/IEC per their guidelines. The investigator/study staff is responsible for 
knowing and adhering to their IRB/IEC requirements. 
 
13.4 PUBLICATION AND DATA SHARING POLICY 
 
In compliance with The International Committee of Medical Journal Editors (ICMJE) clinical trials registration 
policy and Section 801 of the Food and Drug Administration Amendments Act of 2007, this study will be 
registered by the Sponsor in ClinicalTrials.gov, a public trials registry which is sponsored by the National Library 
of Medicine. 
 
Notwithstanding the Sponsor’s requirements for registration and data sharing in ClinicalTrials.gov, any formal 
presentation or publication of data collected as a direct or indirect result of this trial will be considered as a joint 
publication by the investigator(s) and the Sponsor. The resulting publication will name investigators according to 
the policy of the chosen journal. Where it is not permitted for all investigators to be included as authors, the 
publication will name all investigators within the publication. 
 
The investigator will provide the Sponsor with copies of collaborative written publications (including abstracts 
and posters) at least 60 days in advance of submission. This will permit the Sponsor to review the communication 
for accuracy (thus avoiding potential discrepancies with submissions to regulatory authorities), to verify that 
 

 
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confidential information is not inadvertently divulged (including patent protection), to allow adequate input or 
supplementary information that may not have been available to the investigator, and to allow establishment of co-
authorship. 
 
Data will be reviewed by all participating investigators prior to publication. The study Sponsor will have 90 days 
to review all definitive publications, such as manuscripts and book chapters, and a minimum of 30 days to review 
all abstracts. 
 
14 
FINANCIAL DISCLOSURE AND CONFLICT OF INTEREST POLICY 
 
A separate financial disclosure agreement will be made between each Principal Investigator and Alkahest, Inc. or 
its authorized representative before the study agent is shipped. Each investigator will notify Alkahest, Inc. or its 
authorized representative of any relevant changes during the conduct of the study and for 1 year after the study has 
been completed. Alkahest and the study CRO will evaluate any disclosed conflicts of interest and will establish a 
mechanism for their management. 
 
 

 
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16 
REFERENCES 
16.1 PUBLISHED REFERENCES 
 
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arginine-enriched nutritional intervention in patients undergoing surgery. J Immunol. 2017;l1700421:1-10. 
 
Ayers DC, Franklin PD, Trief PM, Ploutz-Snyder R, Freund D. Psychological attributes of preoperative total joint 
replacement patients: Implications for optimal physical outcome. J Arthroplasty. 2004;19:125-130.  
 
Beck AT, Steer RA, Brown GK. Manual for The Beck Depression Inventory, Second Edition (BDI-II). San 
Antonio: Psychological Corporation; 1996. 
 
Bellamy N. WOMAC: a 20-year experiential review of a patient-centered self-reported health. J Rheumatol. 
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Bellamy N, Buchanan WW. A preliminary evaluation of the dimensionality and clinical importance of pain and 
disability in osteoarthritis of the hip and knee. Clin Rheumatol. 1986;5:231-241. 
 
Bertrand JJ, Feichtmeir TV, Kolomeyer N, Beatty JO, Murphy PL, Waldschmidt WD, et al. Clinical 
investigations with a heat-treated plasma protein fraction—Plasmanate®. Vox Sang. 1959;4:385-402. 
 
Bland JHL, Laver MB, Lowenstein E. Vasodilator effect of commercial 5% plasma protein fraction 
solutions. JAMA. 1973;224:1721-1724. 
 
Burnouf T. Modern plasma fractionation. Transfus Med Rev. 2007;21(2):101-17. 
 
Clinical Trial Facilitation Group. Recommendations related to contraception and pregnancy testing in clinical 
trials. Version 2014-09-15:1-13. 
 
Cohen S, Kamarck T, Mermelstein R. A Global Measure of Perceived Stress. Journal of Health and Social 
Behavior, 1983;385-396 
 
 
Deak T. A multispecies approach for understanding neuroimmune mechanisms of stress. Dialogues Clin 
Neurosci. 2017;19:37-53. 
 
Edwards RR, Kronfli T, Haythornthwaite JA, Smith MT, Mcguire L, Page GG. Association of catastrophizing 
with interleukin-6 responses to acute pain. Pain. 2008;140:135-144.  
 
Faller H, Kirschner S, König A. Psychological distress predicts functional outcomes at three and twelve months 
after total knee arthroplasty. Gen Hosp Psychiatry. 2003;25:372-373.  
 
Food and Drug Administration (FDA), U.S. Department of Health and Human Services. Guidance for Industry 
and Investigators: Safety Reporting Requirements for INDs and BA/BE Studies. December, 2012. 
 

 
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Fragiadakis GK, Gaudillière B, Ganio EA, Aghaeepour N, Tingle M, Nolan G, et al. Patient-specific immune 
states before surgery are strong correlates of surgical recovery. Anesthesiology. 2015;123:1241-1255.  
 
Gahart BL, Nazareno AR, Ortega MQ. Plasma protein fraction. In: Gahart’s 2019 Intravenous Medications: A 
Handbook for Nurses and Health Professionals. Elsevier, St. Louis, Missouri, 2019:1053-1054. 
 
Gebhard C, Stahli B, Jenni R, Tanner FC. Effect of age on left ventricular ejection fraction assessed by 
echocardiography. Eur Heart J. 2012;33:1. 
 
Gaudillière B, Fragiadakis GK, Bruggner R V, Nicolau M, Finck R, Tingle M, et al. Clinical recovery from 
surgery correlates with single-cell immune signatures. Sci Transl Med. 2014;6:1-12.  
 
Heuchert JP, McNair DM. POMS-2 manual: A profile of mood states. 2012. 
 
Inouye SK. The Confusion Assessment Method (CAM): Training Manual and Coding Guide. 2003; Boston, 
Hospital Elder Life Program, LLC. Available at: http: www.hospitalelderlifeprogram.org. 
 
Judge A, Arden NK, Cooper C, Javaid MK, Carr AJ, Field RE, et al. Predictors of outcomes of total knee 
replacement surgery. Rheumatology. 2012;51:1804-1813.  
 
 
Kayilioglu SI, Dinc T, Sozen I, Bostanoglu A, Cete M, Coskum F. Postoperative fluid management. World J Crit 
Care Med. 2015;4(3):192-201. 
 
Kehlet H, Dahl JB. Anaesthesia, surgery, and challenges in postoperative recovery. Lancet. 2003;362:1921-1928.  
 
Kurtz S, Ong K, Lau E, Mowat F, Halpern M. Projections of primary and revision hip and knee arthroplasty in the 
United States from 2005 to 2030. J Bone Joint Surg Am. 2007;89:780-785.  
 
Laucis NC, Hays RD, Bhattacharyya T. Scoring the SF-36 in orthpaedics: a brief guide. J Bone Joint Surg Am. 
2015;97”1628-1634. 
 
Lingard EA, Riddle DL. Impact of psychological distress on pain and function following knee arthroplasty. J 
Bone Joint Surg Am. 2007;89(6):1161-1169.  
 
 
Martinez-Cano JP, Herrera-Escobar JP, Arango Gutierrez AS, Sanchez Vergel A, Martinez-Rondanelli A. 
Prospective quality of life assessment after hip and knee arthroplasty: short- and mid-term follow-up results. 
Arthroplast Today. 2017;3:125-130. 
 
Mayer M, Naylor J, Harris I, Badge H, Adie S, Mills K, et al. Evidence base and practice variation in acute 
care processes for knee and hip arthroplasty surgeries. PLoS ONE 2017;12(7):e0180090. 
 
Paddison JS, Sammour T, Kahokehr A, Zarger-Shoshtari K, Hill AG. Development and validation of the Surgical 
Recovery Scale (SRS). J Surg Res. 2011;167:e85-E91. 
 
Prescribing Information (PI): Plasma Protein Fraction (Human) 5%, USP. June, 2018. 
 
 

 
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Stoecklein VM, Osuka A, Lederer JA. Trauma equals danger—damage control by the immune system. J Leukoc 
Biol. 2012;92:539-551. 
 
Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, et al. The ageing systemic milieu negatively 
regulates neurogenesis and cognitive function. Nature. 2011;477:90-94. 
 
Villeda SA, Plambeck KE, Middeldorp J, Castellano JM, Mosher KI, Luo J, et al. Young blood reverses age-
related impairments in cognitive function and synaptic plasticity in mice. Nat Med. 2014 Jun;20(6):659-663. 
 
Witvrouw E, Pattyn E, Almqvist KF, Crombez G, Accoe C, Cambier D, et al. Catastrophic thinking about pain as 
a predictor of length of hospital stay after total knee arthroplasty: A prospective study. Knee Surg Sports 
Traumatol Arthrosc. 2009;17:1189-1194.  
 
16.2 UNPUBLISHED REFERENCES 
 
2018-CET-01. Actual effects of plasma protein fraction (human), 5% on neuronal activation in aged wild type 
mice. 
 
ALK-2015-R002. Effects of young and old human plasma on cognition in NODscid mice. 
 
Periodic Safety Update Report (PSUR) for Plasma Protein Fraction (Human). April, 2018. 
 
VIV-2016-R003. Effects of young human plasma and plasma protein fraction (human), 5% on neurogenesis, 
inflammation and cognition. 
 
VIV-2016-R009. Effects of plasma protein fraction (human), 5% on cognition, behavior and histopathological 
endpoints in aged NSG mice. 
 
VIV-2016-R014. Young plasma and plasma protein fraction (human), 5% effects on behavior and survival in 
aging NODScid mice. 
 
VIV-2016-R020. Effects of plasma protein fraction (human), 5% on cognition. 
 
VIV-2016-R021. Effects of young and aged human plasma and plasma protein fraction (human), 5% on 
histopathological endpoints in aged NSG mice. 
 
VIV-2016-R024. Effects of human plasma and plasma protein fraction (human), 5% on cognition, behavior and 
histopathologic endpoints in aged NSG mice. 
 
VIV-2017-R025. Effects of plasma protein fraction (human), 5% on cognition in aged NSG mice (2). 
 
VIV-2017-R036. Effects of plasma protein fraction (human), 5% on histopathological endpoints in aged NSG 
mice. 
 
VIV-2017-R038. Effects of plasma protein fraction (human), 5% on cognition, behavior and histopathological 
endpoints in aged C57BL/6J mice. 
 
 

 
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VIV-2018-R069. Effects of plasma protein fraction (human), 5% on cognition and neurogenesis in aged, 
C57BL/6J mice. 
 
 

 
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17 
APPENDICES 
 
The assessments in this section and associated information are provided as EXAMPLES ONLY. The actual 
assessments, related source documents, and instructions for administration and scoring are included in the 
associated guides if available.  
 
 
 

 
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Appendix 1.  
3-Minute Diagnostic Interview for Confusion Assessment Method 
 
The 3D-CAM (Inouye 2003) is a brief verbal assessment tool used to test patients for delirium. Each item in the 
instrument directly informs one of the 4 CAM features. For all items, the patient’s answer is “incorrect” or “yes.”   
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
  
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
  

 
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Appendix 2.  
Surgery Recovery Scale 
 
The SRS (Paddison 2011) is a sensitive and simple tool for the assessment of functional recovery following major 
surgery. The scale contains 13 items. Impacts on daily activities are scored from 1 to 5 (1= not at all). It’s 
considered the most broadly validated measure of surgical recovery available. 
 
Surgery Recovery Scale 
During the last two days: 
I have been feeling energetic 
I have been feeling worn out 
I have been feeling vigorous 
I have been forgetful 
I have achieved very little with the day 
I have been feeling fatigued 
Physically, I have felt tired 
I have had to restrict how much I try to do in a day 
I have been feeling lively 
 
During the last two days, I have had enough energy to: 
Read a newspaper/book or watch TV 
Dress 
Do household chores 
Engage in leisure or recreational activities 
Shop or do errands 
 
 
Appendix 3.  
Western Ontario and McMaster Universities Osteoarthritis Index 
 
The WOMAC (Bellamy 1986, Bellamy 2002) was developed to assess pain, stiffness, and physical function in 
patients with knee and/or hip osteoarthritis, but it has been used among patients with many different conditions. 
The WOMAC is one of the most widely utilized self-report measures of lower extremity symptoms and function. It 
has been studied over a period of almost 30 years in many different contexts and patient populations, and there are 
abundant data regarding its utility and measurement properties. The Likert Scale version uses an 11-point scale 
anchored by “no pain” and “extreme pain” for pain, and “no difficulty” and “extreme difficulty” for physical 
function. Higher scores on the WOMAC indicate worse pain and functional limitations. 
 
 
 
 

 
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Appendix 4.  
Short Form-36 
 
The SF-36 (Laucis 2015) is the most widely used health-related quality-of-life measure in research to date. The SF-
36 yields 8 scale scores and 2 summary scores. The physical component summary and mental component summary 
scores.  
 
 
 
 
 
 
 
 
 
 
  
 
 
  
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
  
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
 
 
  
 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
  
 
 
 
  
 
 
  
 
 
 
 
 
  
 
 
 
 
 
  
 
 
  
 
 
 
  
 
 
  
 
 
  
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Appendix 5.  
Beck Depression Inventory-II 
 
The BDI-II (Beck 1996) is a 21-item self-administered survey is scored on a scale of 0–3 in a list of four 
statements arranged in increasing severity about a particular symptom of depression.  
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
  
 
  
  
 
 
 
 
  
  
 
 
  
 
 
  
 
 
 
  
 
 
  
 
 
 
 
  
 
 
 
 
  
 
  
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
  
 
 
  
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
 
 
 
  

 
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Appendix 6.  
Profile of Mood States 
 
The POMS (Heuchert 2012) is widely used to assess affective traits, mood, and emotion. It contains 65 POMS 
items. It can be completed online or in a paper format in 3 to 5 minutes. The subscale measurements include anger-
hostility, confusion-bewilderment, depression-dejection, fatigue-inertia, tension-anxiety, and vigor-activity. For the 
purposes of this study, only the 9 items in the “tension-anxiety” scale will be used. 
 
Descriptions of POMS Scales 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Section 5.2  
 
Previously read: Exclusion Criteria #2 Subjects on 
chronic anticoagulant therapy (warfarin, heparin, low-
molecular weight heparin, or Factor Xa inhibitors). Use 
of pre-, intra- and post-operative anticoagulation 
according to standard of care is permitted. Chronic use of 
antiplatelet drugs (e.g., aspirin or clopidogrel) is 
permitted. 
 
Now reads: Exclusion Criteria #2 Subjects who started 
chronic anticoagulant therapy (warfarin, heparin, low-
molecular weight heparin, or Factor Xa inhibitors) in the 
last 6 months. Use of pre-, intra- and post-operative 
anticoagulation according to standard of care is 
permitted. Use of antiplatelet drugs (e.g., aspirin or 
clopidogrel) is permitted. 
GRF6021 does not contain 
coagulation factors, and the 
volume of GRF6021 given to 
subjects in this trial will not 
cause hemodilution and 
secondary coagulopathy. Thus, 
the sentence was modified to 
clarify that subjects who have 
been off anticoagulation 
therapy for 6 months or longer 
may be eligible for enrollment.  
Subjects who had a recent 
medical event that warranted 
anticoagulation therapy will be 
excluded. 
Section 5.2  
Split Exclusion Criteria #3 into Exclusion Criteria #3 and 
#4, and revised length of time for Exclusion Criteria #4. 
 
Previously read: Exclusion Criteria #3 History of a 
known hypercoagulable state, including Factor V Leiden 
thrombophilia, prothrombin gene mutation, deficiencies 
in antithrombin, protein C or protein S, elevated levels of 
fibrinogen, or dysfibrinogenemia. A history of deep vein 
thrombosis or pulmonary embolism is only exclusionary 
if it occurred in the last 24 months and/or if it is 
associated with a known hypercoagulability disorder. 
 
Now reads:  
Exclusion Criteria #3 History of a known 
hypercoagulable state, including Factor V Leiden 
thrombophilia, prothrombin gene mutation, deficiencies 
in antithrombin, protein C or protein S, elevated levels of 
fibrinogen, or dysfibrinogenemia. 
 
Exclusion Criteria #4 History of deep vein thrombosis or 
pulmonary embolism in the last 6 months and/or if it is 
associated with a known hypercoagulability disorder.  
Human immunoglobulin 
concentrates for IV infusion 
(IVIg) are known to be 
associated with a risk of 
thromboembolic events; 
however, because the 
concentration of IgG in 
GRF6021 is very low in 
comparison with IVIg, there is 
no known risk of 
thromboembolic events 
associated with GRF6021. 
Section 5.2 
Removed: Exclusion Criteria #14 History of Torsades de 
Pointes dysrhythmia. 
GRF6021 is not associated with 
any known effects on ECG or 
electrolytes or any cardiac risk. 
Thus, The PI should use his/her 
clinical judgement to determine 
whether an ECG abnormality 
would warrant the exclusion of 
the subject. 
Section 5.2 
Previously read: Exclusion Criteria #15 Clinically 
significant abnormalities on screening electrocardiogram 
(ECG) including QTc intervals (using Fridericia’s 
GRF6021 is not associated with 
any known effects on ECG or 
electrolytes or any cardiac risk. 
 

 
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correction formula) of ≥ 450 ms in men and ≥ 470 ms in 
women. 
 
Now reads: Exclusion Criteria #15 Clinically significant 
abnormalities on screening electrocardiogram (ECG) that 
in the opinion of the investigator could put the subject at 
risk, including QTc intervals (using Fridericia’s 
correction formula) of ≥ 450 ms in men and ≥ 470 ms in 
women.  
Thus, The PI should use his/her 
clinical judgement to determine 
whether an ECG abnormality 
would warrant the exclusion of 
the subject. 
Section 5.2   
Previously read: Exclusion Criteria #19 History of a 
major infection (e.g. bacterial pneumonia) in the last 3 
months, or minor infection (e.g. influenza, urinary tract 
infection) or fever (measured body temperature above 
100.5 degrees F on 2 consecutive days) in the last 1 
month. 
 
Now reads: Exclusion Criteria #18 History of a major 
infection (e.g. bacterial pneumonia) in the last 3 months, 
or recent minor infection (e.g. influenza, urinary tract 
infection) or fever (measured body temperature above 
100.5 degrees F on 2 consecutive days) that in the 
assessment of the Principal Investigator could interfere 
with the primary endpoint. 
 
The PI should use his/her 
clinical judgment to determine 
whether the infectious or 
inflammatory process was 
systemic, in which case there 
may be effects on the primary 
endpoints. 
Section 5.2   
Previously read: Exclusion Criteria #20 Hemoglobin < 9 
g/dL in women and < 10 g/dL in men. 
 
Now reads:  Exclusion Criteria #19 Severe anemia 
(hemoglobin < 8 g/dL). 
GRF6021 is contraindicated in 
severe anemia because of the 
risk of hemodilution; however, 
there is no known risk in mild 
or moderate anemia. 
Section 5.2   
Previously read: Exclusion Criteria #25 
Immunosuppressant treatment in the last 6 months. 
 
Now reads: Exclusion Criteria #24 Ongoing 
immunosuppressant treatment or within 5 half-lives of 
the treatment. 
Sentence was modified to allow 
for subjects who stopped taking 
an immunosuppressant but 
whose immune function has 
returned to normal. 
Section 6.1.5 and 
throughout  
Clarified study personnel and administration of the study 
agent/placebo.   
Updated language per the 
current Infusion Administration 
Manual.  
Section 7.1.1.1.1.  Removed: Cumulative Illness Rating Scale  
The scale was determined not to 
be required for medical history, 
as it is being obtained from the 
electronic medical record.  
Section 7.1.1.1.5 
Previously read: A full physical examination will be 
performed to assess the following organ systems: 
lungs/chest, heart, abdomen, neuro/spine.  Height and 
weight will also be measured at screening. 
 
The original intent was for the 
physical exam that is performed 
as part of presurgical clinical 
care to be used to assess 
eligibility. Added sentence to 
clarify original intent. 
 

 
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Now reads: A physical examination will be performed to 
assess the following organ systems: lungs/chest, heart, 
abdomen, neuro/spine.  Height and weight will also be 
measured at screening. Should the subject have a 
physical exam, including height and weight 
measurements, as part of standard of care during the 
screening window, then the results may be obtained from 
the subject’s medical record for the purposes of assessing 
eligibility.  
 
 
Section 7.1.1.2.6 
Added window of ~48 hours for completion of the exit 
lab panel. 
To aid trial feasibility. 
Section 7.1.1.3.4 
and 17 
Revised summary of the WOMAC.  
To align with the licensed scale 
and Section 15.1.  
Section 7.1.1.3.7 
and 17 
Removed Profile of Mood States (POMS) Short Form 
and replaced with full-length POMS.  
The 9 items in the “tension-
anxiety” scale are present in the 
full-length POMS but are not 
all present in the Short Form.  
Section 7.3.4 
Under the section for “Perform the following procedures 
on each day prior to administering (the first) study 
infusion” 
• 
Added: “Including second infusion on Day 2” to 
bullets 3, 4, and 5. 
• 
Removed: Bullets 8, 9, and 11.  
 
Under the section for “Perform the following assessments 
following each infusion start” 
• 
Added: Administer 3D-CAM (Day 2 Only, after 
second infusion). 
Corrected and aligned section 
with Section 15.1 (Schedule of 
Events). 
Section 7.3.7 
Added exit lab panel to End of Study/Early Termination 
visit for subjects who have received at least one infusion 
but terminate from the study prior to completing the last 
infusion. 
To ensure samples for the 
primary endpoint and safety 
endpoints are collected should a 
subject discontinue 
participation early.  
Section 7.4.1 
Added: For the purposes of this trial, it is recommended 
to:  
• 
Avoid adjuvant analgesic techniques that have 
immune-modulatory potential (e.g. ketamine or 
IV lidocaine infusions or bolus administration), 
unless clinically indicated. 
• 
Avoid other medications with immune-
modulatory potential such as steroids (e.g. 
decadron), unless clinically indicated. 
Recommended guidelines 
added to avoid anesthetics and 
other medications that may 
have effects on the primary 
endpoint, unless these are 
clinically indicated.  
 
Section 8.4.3 
Removed: Systolic blood pressure (BP) <80 or > 180 mm 
Hg and/or diastolic BP <50 or >110 mm Hg, or a change 
of >30% from baseline in systolic and/or diastolic BP.  
Based on the evaluation of 
aggregate safety data from 
ongoing trials with this and 
similar plasma fractions, only 
 

 
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Section 5.3 
Content removed: During the study, financial support for 
meal and miscellaneous expenses will be available, as 
appropriate, consistent with local regulations and 
guidelines. Use of visit transport services may also be 
incorporated into the trial to support the subject in 
maintaining study visit compliance. 
Subjects will receive a 
moderate stipend for successful 
completion of the study and for 
incurred hotel expenses only. 
Section 5.4.1  
Previously read: Occurrence of an adverse event (AE) 
that represents an unacceptable risk to the subject and 
when continued participation in the investigational study 
is not warranted, in the judgment of the investigator, 
Sponsor, or medical monitor. 
 
Now reads: Occurrence of an adverse event (AE) that 
represents an unacceptable risk to the subject and when 
continued participation in the investigational study is not 
warranted, in the judgment of the investigator or Sponsor 
Medical Monitor. 
The Sponsor Program 
Physician will be acting as 
Medical Monitor for the study; 
sentence updated to correctly 
reflect Sponsor’s responsibility.  
Section 7.1.1.1.5 
Previously read: A full physical examination will be 
performed to assess the following organ systems: skin, 
ENT (ears, nose, and throat), head, eyes, lungs/chest, 
heart, abdomen, musculoskeletal, extremities, neurologic 
and lymphatic systems.  Height and weight will also be 
measured at screening. 
 
Now reads: A full physical examination will be 
performed to assess the following organ systems: 
lungs/chest, heart, abdomen, neuro/spine.  Height and 
weight will also be measured at screening. 
Physical examination modified 
to reflect  requirements for this 
study. 
Section 7.1.1.1.6 
Content removed: Samples that remain after study 
screening is complete will be stored at the study site in 
the event additional testing (e.g., further evaluation of an 
AE or assessment of effect) is required. Samples will be 
stored in a deidentified coded form. 
There will be no samples 
remaining following screening; 
therefore, storage will not be 
required. 
Sections 7.1.1.1.6 
and 15.1 
Removed methadone from urine drug screen. 
Evaluation not required for this 
study. 
Section 
7.1.1.3.10 
Previously read: Approximately -7 to -3 days prior to 
surgery, subjects will be instructed to wear the device 
which will then begin remote monitoring of physical 
activity, mobility, and sleep measures. 
 
Now reads: Approximately -7 to -3 days prior to surgery, 
subjects will be instructed to wear the device which will 
then begin monitoring of physical activity, mobility, and 
sleep measures. 
Removed word “remote” as the 
device does not provide data 
transmitted remotely. 
Sections 7.3 and 
15.1 
The following assessments were revised to occur as 
follows: 
• 
CyTOF: V3, V4a, V4b, V5 
• 
3D-CAM: V3, V4b, V5 
Revision to assessment dates 
and frequency to conform with 
original study design. 
 

 
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• 
SRS/WOMAC: V2, V3, V5-V15 
Section 7.3.1 
Bullet 3 previously read: Review subject’s current and 
prior medications. 
 
Bullet 3 now reads: Review subject’s current and prior 
medications, including opioid consumption assessment. 
 
Bullet 6 previously read: Collect blood and urine 
samples required for Screening. 
 
Now reads: Collect blood and urine samples required for 
Screening (including pregnancy test for WOCBP). 
 
Opioid consumption assessment 
will now be captured at 
screening (Visit 1) during 
review of medications. 
 
Pregnancy testing required at 
screening for WOCBP. 
Section 7.3.2 
Removed “Opioid consumption assessment.” 
Assessment will now be 
captured at screening (Visit 1) 
during review of medications. 
Section 7.3.4 
Added bullet1/sub-bullet 7: “Pregnancy testing for 
WOCBP (Day 1 Only).” 
Pregnancy testing should occur 
in WOCBP prior to the first 
infusion on Day 1. 
Section 8.4.4 
Previously read: The investigator must report any 
pregnancy that occurs in a female study subject or female 
partner of a male subject subsequent to first exposure to 
the study agent until End of Study, or 3 months following 
a subject’s last dose in the event of early termination. All 
pregnancies will be reported to the IRB, Sponsor, and 
CRO. In the event of a pregnancy, treatment will be 
discontinued, and the subject will undergo continued 
safety follow-up through pregnancy outcome. 
 
Now reads: The investigator must report any pregnancy 
that occurs in a female study subject subsequent to first 
exposure to the study agent until End of Study, or 3 
months following a subject’s last dose in the event of 
early termination. All pregnancies will be reported to the 
IRB, Sponsor, and CRO.  In the event of a pregnancy, the 
subject will undergo continued safety follow-up through 
pregnancy outcome. 
 
Content updated to reflect 
appropriate precautions for this 
study design. 
Sections 8.5 and 
8.6 
Term “CRO medical monitor(s)” was changed to “CRO.”  The Sponsor Program 
Physician will be acting as 
Medical Monitor for the study; 
terms updated to correctly 
reflect CRO responsibility. 
Section 15.1 
• Added footnote: “X3: To be performed following final 
infusion on Day3.” 
 
• Notes did not contain “e” footnote (previously 
deleted); alpha listing revised.  
• Clarification of 12-lead ECG 
timing. 
 
• Notes were revised for 
continuity of alpha listing.