Conditions: Postoperative Delirium Postoperative Cognitive Dysfunction
Keywords: Delirium Aging Elderly Postoperative Cognitive Dysfunction
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 203
Healthy volunteers: yes

Brief summary:
This research study will evaluate the effectiveness and estimate the feasibility of administering an investigational drug called 'CN-105' (the study drug), to prevent postoperative cognitive decline, delirium (serious confusion) and underlying brain inflammatory and brain activity changes in adults 60 years and older undergoing surgery.

Detailed description:
This research study will evaluate the effectiveness and estimate the feasibility of administering an investigational drug called 'CN-105' (the study drug), to prevent postoperative cognitive decline, delirium (serious confusion) and underlying brain inflammatory and brain activity changes in adults 60 years and older undergoing surgery. The word "investigational" means the study drug is still being tested in research studies and is not approved by the U.S. Food and Drug Administration (FDA).

It is hoped that CN-105 will block signaling via a gene known as ApoE4, the most common gene implicated in late life Alzheimer's disease.

Depending on when patients enroll in this study, participants will receive either a placebo or a progressively higher dose of CN-105 until the safest and best tolerated dose is reached. The study drug is given via IV (intravenous, meaning through a vein) infusion in the hospital. Study drug infusions will be given up to 4 days after surgery.

Participants will also perform memory and thinking tests, as well as complete a survey and functional assessments, both prior to surgery and again 6 weeks after surgery. Each of those research visits will last about 1 hour.

Additionally, the investigators will collect a blood sample and a cerebrospinal fluid (CSF) sample prior to the participant's surgery, 24 hours after surgery, and again 6 weeks after surgery. To obtain the CSF (cerebrospinal fluid) sample, investigators will perform a lumbar (the lower part of the spinal column) puncture. During surgery, investigators will also record participant brain waves from the scalp using an EEG (electroencephalography) monitor. An electroencephalography monitor reads the electrical activity of the brain in different places using a cap with sensors that is worn on the head.

Although previous studies have not found any associations between the study drug and any serious medical problems, investigators will monitor its effect on wound healing and postoperative infections.

Benefits of this study include the possibility of fewer problems in thinking and memory after surgery if this study drug works as hoped.

Risks of participation in this study include headache, infection/discomfort from the lumbar puncture, discomfort from the blood draw, and minor skin irritation or redness from the EEG and heart rate monitor procedures.

Arms:
arm 1: Cohort 1: 50 Patients Dose of CN-105: 0.1 mg/kg

Cohort 2: 50 Patients Dose of CN-105: 0.5 mg/kg

Cohort 3: 50 Patients Dose of CN-105: 1 mg/kg arm 2: Cohort 1: 17 Patients receiving placebo

Cohort 2: 17 Patients receiving placebo

Cohort 3: 17 Patients receiving placebo

Interventions: intervention 1: CN-105 intervention 2: Placebo
Intervention descriptions: intervention 1: Three doses of CN-105 will be used in three successive cohorts of 50 patients each.

0.1 mg/kg (cohort 1), 0.5 mg/kg (cohort 2), 1 mg/kg (cohort 3)

The study drug will be administered by IV every 6 hours, beginning 1 hour prior to surgery, until postoperative day 3 or hospital discharge, whichever occurs first, up to a maximum of 13 doses. intervention 2: Patients will receive placebo intravenously every 6 hours, beginning 1 hour prior to surgery, until postoperative day 3 or hospital discharge, whichever occurs first, up to a maximum of 13 doses, identical to those receiving the study drug.

Protocol document:
Prot_SAP_000.pdf: 
 
 
 
 
 
Modulating ApoE signalling to Reduce Brain 
inflammation, deLirium and postopErative cognitive 
dysfunction (MARBLE): 
A Phase 2 Trial to Evaluate the Efficacy and 
Feasibility of the Apolipoprotein E Mimetic 
Peptide, CN-105, in Preventing Postoperative 
Cognitive Dysfunction and Delirium and Blocking 
Their Underlying Neural Mechanisms 
 
 
 
Protocol Identifying Number: 20183129 
 
Protocol version: 7 
 
Principal Investigator: Miles Berger, MD, PhD 
 
IND Sponsor: Miles Berger, MD, PhD 
 
Funded by: Alzheimer’s Drug Discovery Foundation 
 
Document Date: 25 – May - 2022 
 
 
NCT03802396


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
2 
 
 
 
 
TABLE OF CONTENTS 
TABLE OF CONTENTS .................................................................................................. 2 
LIST OF ABBREVIATIONS ........................................................................................... 5 
STATEMENT OF COMPLIANCE................................................................................. 7 
PROTOCOL SUMMARY ................................................................................................ 8 
SCHEMATIC OF TRIAL DESIGN .............................................................................. 11 
1 
KEY ROLES ............................................................................................................ 12 
2 
INTRODUCTION: BACKGROUND AND RATIONALE ................................. 12 
2.1 
Background Information .................................................................................... 12 
2.2 
Rationale .............................................................................................................. 13 
2.3 
Potential Risks and Benefits ............................................................................... 13 
2.3.1 
Known Potential Risks .................................................................................. 13 
2.3.2 
Known Potential Benefits ............................................................................. 14 
3 
OBJECTIVES AND PURPOSE ............................................................................. 15 
3.1 
Primary Objectives.............................................................................................. 15 
3.2 
Secondary Objectives .......................................................................................... 15 
4 
TRIAL DESIGN AND ENDPOINTS ..................................................................... 16 
4.1 
Description of Trial Design. ................................................................................ 16 
4.2 
Trial Endpoints .................................................................................................... 18 
4.2.1 
Primary Endpoint.......................................................................................... 18 
4.2.2 
Secondary Endpoints .................................................................................... 18 
5 
TRIAL ENROLLMENT AND WITHDRAWAL ................................................. 19 
5.1 
Participant Inclusion Criteria ............................................................................ 19 
5.2 
Participant Exclusion Criteria ........................................................................... 19 
5.3 
Strategies for Recruitment and Retention ........................................................ 19 
5.4 
Participant Withdrawal or Termination ........................................................... 22 
5.4.1 
Reasons for Withdrawal or Termination .................................................... 22 
5.4.2 
Handling of Participant Withdrawals or Terminations ............................ 22 
5.4.3 
Premature Termination or Suspension of Trial ......................................... 22 
6 
STUDY AGENT ....................................................................................................... 24 
6.1 
Study Agent and Control Description ............................................................... 24 
6.1.1 
Acquisition ..................................................................................................... 24 
6.1.2 
Formulation, Appearance, Packaging, and Labeling ................................. 24 
6.1.3 
Product Storage and Stability ...................................................................... 24 
6.1.4 
Preparation .................................................................................................... 25 
6.2 
Dosage and Administration ................................................................................ 25 
6.2.1 
Route of Administration ............................................................................... 25 
6.2.2 
Starting Dose and Escalation Schedule ....................................................... 25 
6.2.3 
Dose Adjustments/Modifications/Delays ..................................................... 25 
6.2.4 
Duration of Therapy ..................................................................................... 26 
6.2.5 
Tracking of Doses .......................................................................................... 26 
6.3 
Study Agent Accountability Procedures ........................................................... 26 
7 
TRIAL PROCEDURES AND SCHEDULE .......................................................... 27 
7.1 
Trial-Specific Procedures ................................................................................... 27 
7.1.1 
Initial Lumbar Puncture and Blood Draw .................................................. 27 
7.1.2 
Postoperative Lumbar Puncture and Blood Draws ................................... 27 


MARBLE Study – Protocol Version 7 
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7.1.3 
Neurocognitive Testing, Delirium Testing, and Other Assessments ......... 28 
7.1.4 
Quality of Life Assessment ........................................................................... 29 
7.1.5 
Electroencephalographic Recording............................................................ 30 
7.2 
Standard-of-Care Trial Procedures .................................................................. 30 
7.2.1 
Anesthetic Treatment .................................................................................... 30 
7.3 
Laboratory Procedures/Evaluations.................................................................. 31 
7.3.1 
Clinical Laboratory Evaluations. ................................................................. 31 
7.3.2 
Other Assays or Procedures ......................................................................... 31 
7.3.3 
Specimen Preparation, Handling, and Storage .......................................... 31 
7.3.4 
Specimen Shipment ....................................................................................... 32 
7.4 
Trial Schedule ...................................................................................................... 32 
7.4.1 
Initial Consent and Visit ............................................................................... 32 
7.4.2 
Day 0 (Day of Surgery) ................................................................................. 33 
7.4.3 
Day 1 (First Day After Surgery, Hospitalization)....................................... 34 
7.4.4 
Days 2-4 (Hospitalization)............................................................................. 34 
7.4.5 
6 weeks (+/- 21 Days) Follow-Up .................................................................. 34 
7.4.6 
Schedule of Events ......................................................................................... 36 
8 
ASSESSMENT OF SAFETY .................................................................................. 38 
8.1 
Specification of Safety Parameters .................................................................... 38 
8.1.1 
Definition of Adverse Events ........................................................................ 38 
8.1.2 
Definition of Serious Adverse Events .......................................................... 38 
8.1.3 
Definition of Unanticipated Problems ......................................................... 39 
8.2 
Classification of an Adverse Event .................................................................... 40 
8.2.1 
Severity of Event ............................................................................................ 40 
8.2.2 
Relationship of Study Agent ......................................................................... 40 
8.2.3 
Expectedness .................................................................................................. 42 
8.3 
Time Period and Frequency for Event Assessment and Follow-up ................ 42 
8.4 
Reporting Procedures ......................................................................................... 42 
8.4.1 
Serious Adverse Event Reporting ................................................................ 43 
8.5 
Trial Halting Rules .............................................................................................. 43 
8.6 
Safety Oversight .................................................................................................. 44 
9 
CLINICAL MONITORING ................................................................................... 44 
10 STATISTICAL CONSIDERATIONS ................................................................... 44 
10.1 
Description of Statistical Methods and Analysis Plans .................................... 44 
10.1.1 
General Approach ......................................................................................... 44 
10.1.2 
Safety and Feasibility Endpoints .................................................................. 44 
10.1.3 
Analysis of the Secondary Endpoints .......................................................... 45 
10.2 
Sample Size .......................................................................................................... 46 
10.3 
Measures to Minimize Bias. ................................................................................ 46 
10.3.1 
Enrollment/Randomization/Masking Procedures ...................................... 46 
10.3.2 
Procedures to Ensure the Success of Blinding ............................................ 47 
11 SOURCE DOCUMENTS AND ACCESS TO DATA/DOCUMENTS ................ 48 
12 QUALITY ASSURANCE AND QUALITY CONTROL ..................................... 48 
13 ETHICS/PROTECTION OF HUMAN SUBJECTS ............................................ 49 
13.1 
Ethical Standard .................................................................................................. 49 
13.2 
Institutional Review Board ................................................................................. 49 
13.3 
Informed Consent Process .................................................................................. 49 
13.3.1 
Consent and Other Informational Documents Provided to Participants 49 


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13.3.2 
Consent Procedures and Documentation .................................................... 49 
13.4 
Participant and Data Confidentiality ................................................................ 50 
13.5 
Research Use of Stored Human Samples, Specimens, or Data ....................... 50 
14 DATA HANDLING AND RECORDKEEPING ................................................... 51 
14.1 
Data Collection and Management Responsibilities .......................................... 51 
14.2 
Trial Records Retention ...................................................................................... 51 
14.3 
Protocol Deviations.............................................................................................. 51 
14.4 
Publication and Data Sharing Policy ................................................................. 52 
15 CONFLICT OF INTEREST POLICY .................................................................. 52 
16 LITERATURE REFERENCES ............................................................................. 52 


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LIST OF ABBREVIATIONS 
 
AD 
Alzheimer’s disease 
AE 
adverse event 
apoE 
apolipoprotein E 
BIAC 
Brain Imaging and Analysis Center (Duke University) 
BVMT-R 
Brief Visuospatial Memory Test, Revised 
CES-D 
Center for Epidemiological Studies-Depression 
CFR 
United States Code of Federal Regulations 
COWAT 
Controlled Oral Word Association Test 
CRF 
case report form 
CSF 
cerebrospinal fluid 
DCC 
data coordinating committee 
DSMB 
Data Safety Monitoring Board 
DUHS 
Duke University Health System 
DUMC 
Duke University Medical Center 
EC 
ethics committee 
EEG 
electroencephalogram 
FDA 
Food and Drug Administration (US) 
GCP 
Good Clinical Practice 
HSCL 
Hopkins Symptom Checklist 
HVLT-R 
Hopkins Verbal Learning Test, Revised 
ICF 
informed consent form 
ICMJE 
International Committee of Medical Journal Editors 
IRB 
institutional review board 
IV 
intravenous 
LAR 
legally authorized representative 
LP 
lumbar puncture 
MADCO-PC 
Markers of Alzheimer’s Disease and neuroCognitive Outcomes after Perioperative Care 
MoCA 
Montreal Cognitive Assessment 
NIH 
National Institutes of Health (US) 
PI 
principal investigator 
POCD 
postoperative cognitive decline 
QOL 
quality of life 
SAE 
serious adverse event 


MARBLE Study – Protocol Version 7 
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SC 
steering committee 
SF-36 
Short Form-36 
STAI 
State Trait Anxiety Inventory 
TMT 
Trail Making Test 
VAS 
visual analog scale 
WAIS-III 
Wechsler Adult Intelligence Scale, 3rd Revision 
WTAR 
Wechsler Test of Adult Reading 


MARBLE Study – Protocol Version 7 
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STATEMENT OF COMPLIANCE 
 
The trial will be carried out in accordance with Good Clinical Practice as required by the 
following: 
• United States Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312 and/or 21 CFR Part 
812) 
• International Council on Harmonisation E6 
 
 
I agree to ensure that all staff members involved in the conduct of this trial are informed about 
their obligations in meeting the above commitments. 
 
 
Principal Investigator: Miles Berger, MD, PhD 
 
 
Signed:  
Date:    
 


MARBLE Study – Protocol Version 7 
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PROTOCOL SUMMARY 
 
Title: 
Modulating ApoE signaling to Reduce Brain inflammation, deLirium and 
postopErative cognitive dysfunction (MARBLE): A Phase 2 Trial to Evaluate the 
Efficacy and Feasibility of the Apolipoprotein E Mimetic Peptide CN-105 in 
Preventing Postoperative Cognitive Dysfunction and Delirium and Blocking Their 
Underlying Neural Mechanisms 
Summary: 
Participants age 60 and over who are scheduled to undergo non-cardiac, non- 
neurologic surgical procedures that will take more than 2 hours, and who will be 
admitted to the hospital after surgery, will be prospectively approached for 
enrollment in this trial. Participants will undergo preoperative cognitive testing and 
will be randomized to receive either the apolipoprotein E (apoE) mimetic peptide 
CN-105 or placebo. Three sequential cohorts of 67 patients will be randomized to 
receive CN-105 vs placebo; the CN-105 patients in each successive cohort will 
receive a higher/escalated dose of CN-105. Thus, we will enroll at least 201 
participants to reach the target of 150 participants who complete the trial. 
Trial participants will undergo cognitive testing and complete quality-of-life (QOL) 
assessments before surgery and again 6 weeks +/- 3 weeks after surgery. These 
participants will receive either 1 of 3 dose levels of CN-105 or placebo, within one 
hour prior to case start time and continuing every 6 hours until postoperative day 3 
or hospital discharge (whichever occurs first). We will also record noninvasive 
scalp electroencephalogram (EEG) data from these participants immediately 
before, during and after surgery. Cerebrospinal fluid (CSF) (up to 20 ml) and blood 
samples (up to 10 mL) will be collected prior to surgery (between the time of 
consent and the time the surgery will start), 24 +/- 2 hr after the scheduled surgery 
start/anesthesia induction and 6 weeks +/- 3 weeks after the scheduled surgery 
start/anesthesia induction. Cerebrospinal fluid samples will be assayed for amyloid 
beta, tau, and other Alzheimer‘s disease (AD)-associated markers, as well as 
inflammatory cytokines and markers; blood samples will be assayed for serum 
inflammatory markers and used for APOE genotyping studies. CN-105 levels will 
also be measured in both CSF and peripheral blood samples. Trial participants will 
return to the hospital 6 weeks +/- 3 weeks later to undergo repeat cognitive testing 
and CSF/blood draws. 
Endpoints: 
Primary: 
To determine if there is significant difference in grade II or higher adverse 
events (AEs) between participants who receive CN-105 vs those who receive 
placebo.  
Secondary: 
1. Compare the increase in CSF inflammatory cytokines (i.e. IL-6, IL-8, MCP-1, 
G-CSF) from before to 24 hr and 6 weeks after anesthesia and surgery in 
patients who receive CN-105 vs placebo. 
2. Compare the change in cognition (i.e. continuous cognitive change index) from 
before surgery to 6 weeks after surgery in patients who receive CN-105 vs 
placebo. 
3. To determine whether it is feasible to administer 90% of study doses within the 
required time window (every 6 hours +/- 90 minutes). 
4. Compare post-operative delirium rates and severity in patients who receive CN-
105 vs placebo (using the CAM-ICU in intubated patients and the 3D-CAM in 
non- intubated patients). 


MARBLE Study – Protocol Version 7 
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MARBLE Study – Protocol Version 7 
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Exploratory: 
5. Compare the change in CSF tau, phospho-tau and amyloid beta from before to 24
hours after anesthesia and surgery in patients who receive CN-105 vs 
       placebo. 
6. Compare serum chemistries (Na, K, Cl-, HCO3-, BUN, Cr, Glucose) and blood 
counts (white blood cell count, hemoglobin and hematocrit, and platelet count) 
before, 24 hours and 6 weeks after anesthesia and surgery in patients who 
received CN-105 vs placebo. 
7. Compare changes in quality of life from before to 6 weeks after surgery in 
patients who receive CN-105 vs placebo (using the SF-36 instrument). 
8. Compare intraoperative EEG spectral patterns, including “anteriorization” of 32-
channel EEG alpha power and levels of burst suppression, in patients who 
receive CN-105 vs placebo. 
9. To determine if the primary and secondary outcomes differ among patients who 
carry the APOE4 allele or other genetic polymorphisms related to inflammation, 
neuroimmune function, metabolism, brain function, and/or AD. 
10. Describe the relationship between anesthetic exposure (duration, average 
hourly exposure, total exposure) and CSF inflammatory cytokine changes and 
demographics (age, sex, and race). 
11. Describe the relationship between CSF changes in AD and 
inflammatory biomarkers, anesthetic exposure, and serum markers of 
metabolic and/or inflammatory status. 
12. Describe the relationship between anesthetic exposure, CSF and serum 
biomarker changes, and intraoperative 32 channel EEG parameters. 
13. Compare physical function (DASI) and mental health (STAI, SCL-90, and 
CES-D) before and after surgery in patients who receive CN-105 vs placebo. 
Population: 
Up to 250 participants age 60 and over who are scheduled to undergo non-
cardiac, non- neurologic surgical procedures that will take more than 2 hours and 
who will be admitted to the hospital after surgery. 
Phase: 
2 
Sites Enrolling 
Participants 
Duke University Medical Center 
Duke Regional Hospital 
Duke Raleigh Hospital 
Description of 
Study Agent: 
CN-105 is an ApoE mimetic pentapeptide (AC-VSRRR-NH2). It will be supplied as 
a frozen liquid in 10 mL amber glass vials with a Flurotec™ stopper and a white 
aluminum flip off cap. Each vial contains 4 mL of CN-105 at a concentration of 12.5 
mg/mL (50 mg CN-105 drug product per vial). 
Trial Duration: 
Up to 50 months 
Participant 
Duration: 
6 weeks +/- 3 weeks 


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SCHEMATIC OF TRIAL DESIGN 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Patient scheduled for surgical procedures in the operation room. 
Consent, enrollment, randomization to CN-105 vs placebo, and preoperative cognitive 
testing. 
Collect blood and cerebrospinal fluid samples between the time of consent and 
anesthesia/surgery start (preoperative sample), and 24 +/- 2 hr after anesthesia/surgery 
start; conduct delirium screening on each perioperative day that the participant remains in 
the hospital. Participants will receive either CN-105 or placebo at 6 hr intervals (for up to 
13 consecutive doses) from within one hour prior to case start time until postoperative 
day 3 or hospital discharge, whichever comes first. 
Participants will repeat cognitive testing and return for repeat lumbar puncture and 
blood draw 6 weeks +/- 3 weeks after surgery. 
Intraoperative course variables from anesthesia record, EEG setup, and physiologic 
monitors will be obtained after the completion of the surgery. 
Run biomarker and genotyping assays after CSF and blood samples obtained, analyze 
electroencephalographic and other data. 
Study is complete after all assays run and all data analyzed 


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1 KEY ROLES 
Principal Investigator: 
Miles Berger, MD, PhD 
Assistant Professor 
Neuroanesthesiology Division 
Anesthesiology Department 
Duke University Medical Center 
Durham, NC 
 
Sub-Investigators: 
Jeffrey Browndyke, PhD 
Daniel Laskowitz, MD 
Roberto Cabeza, PhD 
Marty Woldorff, PhD 
Niccolo Terrando, PhD 
 
Biostatisticians: 
Primary Study Statistician- Mary Cooter, MS 
Independent (DSMB) Statistician- Mathew Fuller, MS 
Advisory Statistician- Frank Rockhold, PhD 
 
Clinical Laboratory: 
ADNI Biomarker Core Lab, Otherwise TBD 
 
Data Management: 
Per REDCap database, under the supervision of Mary Cooter, MS 
 
2 INTRODUCTION: BACKGROUND AND RATIONALE 
2.1 
Background Information 
 
Alzheimer’s disease (AD), the most common type of dementia, is an increasingly rising and 
alarming public health issue afflicting more than 5 million Americans (Corriveau RA et al, 2017). 
Epidemiologic studies have linked anesthetic and surgery exposure to the development of dementia 
and AD, as well as milder forms of cognitive decline (i.e., postoperative cognitive decline [POCD] 
and delirium) (reviewed in Berger M. et al, 2014; Berger M. et al, 2015). Furthermore, animal and 
human studies have shown that anesthesia and surgery increase cerebrospinal fluid (CSF) levels of 
AD biomarkers, such as tau, and induce neuroinflammation (see Supplemental Appendix, Figures 1 
& 2, respectively). However, to date, no studies have examined the effects of interventions to block 
increases in these CSF AD and inflammatory biomarkers induced by anesthesia and surgery or have 
determined whether blocking changes in these biomarkers can improve postoperative cognitive 
function. We hypothesize that blocking changes in AD and neuroinflammatory biomarkers could 
prevent and/or at least ameliorate the risk of POCD and delirium, thus improving postoperative 
recovery and quality of life (QOL). 


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We propose to conduct a phase 2 trial in older adults undergoing non-cardiac surgery to evaluate the 
safety, feasibility, and efficacy of administering the apolipoprotein E (apoE) mimetic peptide, CN- 
105, to prevent postoperative cognitive dysfunction, delirium, and underlying pathologic 
neuroinflammatory and brain activity changes. 
 
2.2 
Rationale 
 
Postoperative cognitive dysfunction (POCD) and delirium occur in up to 40% of the ≥16 million 
Americans over age 60 who undergo anesthesia and non-cardiac surgery each year. POCD and 
delirium are each associated with decreased quality of life, increased one-year postoperative 
mortality risk, long term cognitive decline, and a possible increased risk for dementia. There are 
currently no FDA-approved drugs to prevent POCD or delirium, largely because we do not 
understand their underlying mechanisms. Neuro-inflammation and an exacerbation of A and tau 
pathology (i.e. AD pathology) are two mechanisms that have been identified in mouse models of 
POCD and delirium. Further, preliminary data (see Supplemental Appendix) suggest that neuro- 
inflammation and AD pathogenesis increase post-operatively in humans, potentially leading to 
POCD and delirium. Thus, blocking both neuro-inflammation and the acceleration of AD 
pathogenesis may help prevent POCD and delirium, as well as their long term sequelae. Several 
lines of evidence suggest that postoperative increases in neuro-inflammation and AD 
pathogenesis/biomarkers occur in a synergistic fashion, and together, likely contribute to delirium 
and POCD. Thus, an ideal drug to prevent delirium and POCD would block postoperative increases 
in both neuro-inflammation and AD pathogenesis/biomarkers. 
 
The APOE4 allele, the most common genetic risk factor for late onset AD, promotes neuro- 
inflammation, as well as amyloid beta and tau pathology. The APOE4 allele has also been 
associated with long term cognitive dysfunction after anesthesia and surgery. Thus, AegisCN LLC 
developed the 5-amino acid peptide CN-105 to block both of these APOE4–mediated effects. 
AegisCN LLC’s phase I study found that CN-105 is safe to administer at 1 mg/kg IV every 6 h for 
14 days (Guptill J et al, 2017), which is 24-100-fold the therapeutic dose in mouse models. Here, we 
will conduct a phase II randomized controlled study in older surgical patients to evaluate the safety, 
feasibility, and efficacy of using CN-105 to prevent POCD and delirium, as well as their underlying 
brain function and biomarker changes. This work rests upon rigorous methods we developed in the 
Markers of Alzheimer’s Disease and neuroCognitive Outcomes after Perioperative Care (MADCO- 
PC) trial to evaluate neuro-inflammation, CSF AD biomarker changes, and functional brain 
connectivity alterations in POCD and delirium (Giattino et al, 2017; Berger et al, manuscript in 
preparation, 2018; see Supplemental Appendix). In MADCO-PC, 140 older non-cardiac surgery 
patients underwent pre- and post-operative CSF biomarker measurements, intraoperative EEG 
recordings, and cognitive testing/delirium screening. Here, we will apply these methods to evaluate 
CN-105's efficacy in blocking perioperative increases in neuro-inflammation and CSF AD 
biomarkers, intraoperative brain activity alterations, and in reducing the incidence of POCD and 
delirium. 
 
2.3 
Potential Risks and Benefits 
 
2.3.1 
Known Potential Risks 
 
There are currently no known potential risks associated with CN-105 administration. CN-105 was 
not associated with any significant adverse safety events during a completed phase I trial (Guptill J 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
14 
 
 
 
et al, 2017) or a currently ongoing multicenter Phase II study. Both studies are evaluating CN-105 
using a similar dosing regimen as that described in this protocol. 
 
Except for LPs (discussed below), all other study procedures (e.g. blood draws, EEG recordings, 
etc.) are standard practice and pose minimal risk to patients. 
 
Lumbar punctures (LPs) are associated with the potential risk of a postdural headache, although this 
risk is less than 1% in patients over age 60 who have an LP performed with a 25g pencil-point spinal 
needle (or other appropriate spinal needle per Good Clinical Practice) under strictly sterile 
conditions. Nonetheless, all participants will be given contact information for Dr. Miles Berger or 
the trial physician on call and encouraged to be in touch at any time if they develop a headache after 
the LP or any other concerning signs or symptoms. If the trial participant calls and reports that he or 
she has developed a headache related to the LP (i.e., a postdural puncture headache), the trial 
physician will assess whether this headache is likely related to the LP. If the headache is thought to 
be related to the LP, then per standard of care, the trial physician will then initiate conservative 
treatment (bed rest, increased fluid and caffeine intake, etc.) for several days and/or perform an 
epidural blood patch (free of charge to the patient and his or her insurance). 
 
There is also a theoretical risk of developing an infection after a lumbar puncture, although this risk 
is minimal when using a clean sterile kit, sterile gloves and sterile skin prep, having all individuals 
in the procedure area where hats and masks, and using strictly sterile technique. We have previously 
performed over 600 LPs with no LP-related infections in the MADCO-PC and Investigating 
NeuroinflammaTion UnderlyIng Postoperative Brain ConnectiviTy Changes, POCD, Delirium in 
Older Adults (INTUIT) studies, further demonstrating the extremely low risk of post-LP infections. 
 
For patients undergoing surgery who have a primary cancer, such as lung, kidney, breast or prostate, 
if the patient has spine imaging (presumably PET or MRI) available in his/her Duke medical record, 
we will review that spine imaging prior to enrolling the patient in the study to make sure that the 
patient does not have metastatic disease in their spinal column, which could then theoretically get 
seeded into the intra-thecal space if the LP needle went thru the metastasis before entering the 
intrathecal space. If a patient has one of these cancers (lung, kidney, breast or prostate) that tend to 
metastasize to bone, and the patient has imaging evidence of metastatic disease in their spinal 
column, we will then exclude the patient from the study to avoid even the theoretical risk of seeding 
the intrathecal space with cancerous cells. If the patient has cancer and has not had spine imaging, 
that presumably means that the patient has not had back pain or other symptoms suggestive of spine 
metastasis and/or that the clinical team had a low enough suspicion for spine metastasis that they 
didn’t feel that it was necessary to obtain spine imaging on the patient. In such cases, these patients 
will still remain eligible for enrollment, assuming they meet other inclusion/exclusion criteria. We 
will not obtain spine imaging as part of the study in these situations if the primary clinical team did 
not order it and did not think it was indicated.  
 
In cases in which a patient has prior spine imaging in his or her Duke medical record, a physician 
member of the study team will review the images (and any official interpretations) in the patient’s 
chart and make a determination as to whether there is severe spinal stenosis. Patients with severe 
spinal stenosis will be excluded from this study due to the theoretical risk of causing myelopathy or 
radicular symptoms by introducing a needle into the intrathecal space. 
 
2.3.2 
Known Potential Benefits 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
15 
 
 
 
There are currently no known benefits of CN-105 administration. Since CN-105 has been shown to 
block neuro-inflammation in animal models and since neuro-inflammation is thought to play an 
etiologic role in postoperative delirium and cognitive dysfunction, CN-105 has the potential to 
reduce postoperative delirium and/or cognitive dysfunction severity and/or incidence. 
 
3 OBJECTIVES AND PURPOSE 
For additional details describing the objectives, please also see the endpoints described in the 
Protocol Summary, as well as the Statistical Considerations in Section 10. 
 
3.1 
Primary Objectives 
 
The primary objective of this trial is to evaluate the safety of administering the apoE mimetic 
peptide, CN-105, to older adults undergoing major non-cardiac or non-neurologic surgery. 
 
3.2 
Secondary Objectives 
 
Secondary objectives include the following: 
To determine whether CN-105 blocks postoperative neuroinflammation as measured by changes in levels of 
the following cytokines in the CSF: IL-6, IL-8, MCP-1, and G-CSF. 
To determine whether CN-105 blocks or reduces the risk (or severity) of CCI index change 
between CN-105 and placebo groups. 
To determine whether participants who receive CN-105 have lower postoperative delirium rates 
and severity than those who receive placebo. 
To determine whether it is feasible to administer 90% of study doses within the required time 
window (every 6 hours +/- 90 minutes). 
 
Additional exploratory objectives include: 
To determine whether CN-105 has effects on serum chemistries or blood counts. 
To describe the relationship between anesthetic exposure (both duration, average hourly 
exposure, total exposure) and CSF inflammatory cytokine changes and demographics (age, sex, 
and race). 
To describe the relationship between CSF changes in AD and inflammatory biomarkers, 
anesthetic exposure, and serum markers of metabolic and/or inflammatory status. 
To describe the relationship between anesthetic exposure, CSF and serum biomarker changes, 
and intraoperative changes in 32-channel (whole head) EEG parameters. 
To determine whether CN-105 blocks the increase in CSF tau levels from before to 24 hours 
after anesthesia and surgery and whether it modulates changes in CSF phospho-tau and 
amyloid beta levels from before to 24 hours after anesthesia and surgery. 
To determine whether participants who receive CN-105 (vs placebo) have improved QOL scores 
6 weeks after surgery. 
To determine whether participants who receive CN-105 (vs placebo) have reduced abnormalities 
in intraoperative EEG spectral power patterns. 
To determine if the other primary and secondary outcomes differ among patients who carry the 
ApoE4 allele or other genetic polymorphisms related to inflammation, neuroimmune function, 
metabolism, brain function, and/or AD. 
Compare physical function (DASI) and mental health (STAI, SCL-90, and CES-D) before and 
after surgery in patients who receive CN-105 vs placebo. 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
16 
 
 
 
 
4 TRIAL DESIGN AND ENDPOINTS 
4.1 
Description of Trial Design 
 
Trial initiation will start with the enrollment of the first participant, which will occur only after the 
external institutional review board (IRB) as well as the Duke University Medical Center IRB has 
approved the protocol. 
 
Participants age 60 and over who are scheduled to undergo non-cardiac, non-neurologic surgical 
procedures that will take more than 2 hours and who are scheduled to be admitted to the hospital 
after surgery at Duke University Medical Center (DUMC) or one of its affiliate hospitals (Duke 
Regional Hospital, Duke Raleigh Hospital) will be prospectively enrolled in this trial. 
 
Trial participants will undergo cognitive testing and complete QOL assessments within 2 months 
before surgery and again 6 weeks +/- 3 weeks after surgery. These participants will receive either 
CN-105 or placebo, within one hour before the scheduled or actual start time of surgery and 
continuing every 6 hours through postoperative day 3 (up to 13 doses maximum) or hospital 
discharge, whichever occurs first. Each case start time will be defined by the study team as either 
the start of the surgery/procedure or the start of anesthesia. 
 
We planned to enroll up to 250 patients in this phase 2 escalating dose study, with escalating CN-
105 doses occurring in three successive groups of 67 randomized patients each. Due to a clerical 
error by the Investigational Drug Services that prepares our drug/placebo doses, 69 patients were 
randomized to the first patient cohort rather than 67. Additionally, because enrollment happens at 
consent, and not all patients that consent will be randomized, we will keep enrolling patients until 
we reach 67 consented and randomized patients in each of the last two cohorts. 
In each group of 67 patients, 50 will receive a given dose of CN-105 and 17 will receive placebo. In 
our recent MADCO-PC study (Duke IRB protocol #45180), ~80% of patients returned for the 6 
week assessment. Thus, enrolling 67 patients in each group ensures ≥52 patients (i.e. 39 CN-105-
treated; 13 placebo treated) will complete the 6 week +/- 3 weeks postop assessment.  
There will be no baseline preoperative cognitive-status inclusion/exclusion criteria. Our prior work 
suggests ~15% of Duke orthopedic surgery patients over age 60 have MCI, ~5% have dementia, 
and ~80% are cognitively normal. All primary analyses will be performed on patients grouped 
together independent of baseline cognitive status and dose level; exploratory analyses will examine 
effects stratified by baseline cognitive strata, dose level, and by APOE4 carrier status. In each 
successive 67-patient group, the 50 drug-treated patients will receive a higher CN-105 dose: 0.1 
mg/kg in the first group, then 0.5 mg/kg in the second group, and then 1 mg/kg in the third group. 
In each case, CN-105 (or placebo) will be given IV every 6 hours (i.e. q6 h) from the first dose, 
which will occur within one hour of the scheduled or actual surgery start time, until just prior to 
hospital discharge or 3 days after surgery (up to 13 doses maximum), whichever occurs first. Each 
case start time will be defined by the study team as either the start of the surgery/procedure or the 
start of anesthesia (or the timed notes in the EMR for induction, intubation, or the start of anesthetic 
drug administration), or such times as planned/scheduled. If the case gets delayed by more than 6 hr 
and we’ve already given 1st dose, we will give an additional dose prior to surgery, but under no 
conditions will the patient be given more than 13 doses total of CN-105 
 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
17 
 
 
We will also record noninvasive electroencephalogram (EEG) data from the scalps of these 
participants just before,  during, and right after anesthesia and surgery. In the event that sufficient 
personnel or equipment are unavailable, or in the event that two MARBLE patients are having 
surgery at the same time, or in the event that there is insufficient time to place the EEG sensor on 
that patient’s head before the start of surgery, the study team may not record EEG from all patients 
or at all points before, during, and just after surgery/anesthesia.  
Cerebrospinal fluid and blood samples will each be collected before surgery (between the time of 
consent and the time of scheduled anesthesia/surgery start), 24 hours +/- 2 hours later after the start 
of anesthesia/surgery, 6 weeks +/- 3 weeks later, and potentially 1 year after surgery for some 
patients, depending on funding and the PIs discretion. Cerebrospinal fluid (CSF) samples (up 20 
mL each—approximately 4 teaspoons) will be collected via a sterile LP using a standard spinal 
anesthesia kit with a 25-g needle (or other appropriate spinal needle per Good Clinical Practice) 
under strictly sterile conditions. The study may provide funds for patients to stay at a local hotel 
near the hospital overnight if the patient is discharged on the day of their surgery and if driving 
back to Duke on post-op day 1 would not be possible or reasonable for the patient. This service 
will be provided based on the PI’s discretion, in part based on whether sufficient funds are 
available to support this expense. Blood samples (up to 10 mL each—approximately 2 teaspoons) 
will be obtained at these time points as well. Blood samples will be drawn from an IV, intra-
arterial line, or from a sterile venipuncture. Cerebrospinal fluid samples will be assayed for 
amyloid beta, tau, and other AD- associated markers, as well as for inflammatory cytokines and 
markers; blood samples will be assayed for serum inflammatory markers and used for genotyping 
studies. Trial participants will complete repeated cognitive testing and return to the hospital to 
undergo repeat CSF and blood draws 6 weeks +/- 3 weeks later, or may complete the 6 week 
cognitive testing by remote video if appropriate (i.e. due to the pandemic or other issues). We 
created a post-LP instruction sheet for patients and caregivers advising them on activities for the 
24 hours following their lumbar puncture and with Dr. Berger’s phone number, should they have 
questions or experience any complications following the LP. 
 
Patients may complete a post-op 1 year study visit (+/-2 months) at which time we will collect 
blood, perform another LP, and administer the same surveys and cognitive testing that were 
performed at the baseline and 6 week visits, provided that sufficient funds are available per the PI's 
judgement at that time. If funding become available after some 1 year time point visits have 
already been missed, we will potentially perform 1 year visits only on the remaining patients who 
are still within the correct time window. Likewise, in light of uncertainties surrounding cost and 
funding due to COVID-19, if other financial changes occur that make it impossible to continue 1 
year study visits or any other aspect of this protocol, specific patients may be discontinued based 
on the PI's judgement and discretion. 
 
Trial duration will be up to 72 months total for participant enrollment and completion of all trial 
activities, with approximately 6-8 weeks duration for each individual participant (and up to 1 year 
duration for those who have a 1 year follow up performed), assuming an enrollment rate of ~2 
patients per week. If the actual enrollment rate is slower than ~2 patients per week, the trial 
duration will be longer until we reach full enrollment, though the study duration per patient (~6-8 
weeks or 1 year for those with 1 year follow up time) will be unchanged. 
 
Surgical patients may participate in the trial more than once if they desire and are having a second 
surgery after they have already completed all trial procedures (including the 6 week postoperative 
time point). There are no known added risks to participants from taking part in the trial additional 
times, beyond the risks discussed in the informed consent form (ICF). 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
18 
 
 
 
4.2 
Trial Endpoints 
 
4.2.1 
Primary Endpoint 
 
The primary endpoint is to determine whether there is a significant difference in grade II or higher 
adverse events (AEs) between participants in each dose cohort of CN-105 vs. those who receive 
placebo. 
 
4.2.2 
Secondary Endpoints 
 
1. Measure the increase in CSF inflammatory cytokines (i.e. IL-6, IL-8, MCP-1, G-CSF) from 
before to 24 hr after and 6 weeks +/- 3 weeks after anesthesia and surgery in patients who 
receive CN-105 vs placebo. 
2. Measure the change in cognition (i.e. continuous cognitive change index) from before, 
immediately after, and 6 weeks +/- 3 weeks after surgery in patients who receive CN-105 vs 
placebo. 
3. Measure postoperative delirium rates in patients who receive CN-105 vs placebo (using the 
CAM-ICU in intubated patients and the 3D-CAM in non-intubated patients). 
4. To determine whether it is feasible to administer 90% of study doses within the required 
time window (every 6 hours +/- 90 minutes). 
 
Exploratory Endpoints: 
1. To describe the relationship between anesthetic exposure (duration, average hourly 
exposure, total exposure) and CSF inflammatory cytokine changes and demographics (age, 
sex, and race). 
2. To describe the relationship between CSF changes in AD and inflammatory 
biomarkers, anesthetic exposure, and serum markers of metabolic and/or inflammatory 
status. 
3. 
To describe the relationship between anesthetic exposure, CSF and serum biomarker 
changes, and intraoperative 32 channel EEG patterns.  
4. 
Measure serum chemistries (Na, K, Cl-, HCO3-, BUN, Cr, Glucose) and blood counts (white 
blood cell count, hemoglobin and hematocrit, and platelet count) before, 24 hours after, and 6 
weeks after anesthesia and surgery in patients who received CN-105 vs placebo. 
5. 
Measure changes in quality of life from before to 6 weeks +/- 3 weeks after surgery in patients who receive 
CN-105 vs placebo (using the SF-36 instrument). 
6. 
Measure intraoperative EEG spectral patterns, including previously observed “anteriorization” of 32-
channel EEG alpha power (e.g., Giattino et al., 2017) and levels of burst suppression in patients who 
receive CN-105 vs placebo. If necessary due to COVID-19-related issues or other safety concerns or 
exigencies, 1-2 channel EEG will be performed using a BIS EEG monitor (or other appropriate frontal 
EEG device) rather than using our standard 32 channel EEG caps to minimize patient contact. 
7. 
To determine if the other primary and secondary outcomes differ among patients who carry the ApoE4 
allele or other genetic polymorphisms related to inflammation, neuroimmune function, metabolism, brain 
function, and/or AD. 
8. 
Measure the change in CSF tau, phospho-tau, and amyloid beta from before to 24 hours (and to 6 weeks +/- 
3 weeks) after anesthesia and surgery in patients who receive CN-105 vs placebo. 
 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
19 
 
 
5 TRIAL ENROLLMENT AND WITHDRAWAL 
5.1      Participant Inclusion Criteria 
In order to be eligible to participate in this trial, an individual must meet all of the 
following criteria: 
1. Age ≥ 60 
2. Undergoing non-cardiac, non-neurologic surgical procedures; surgery scheduled to last 
> 2 hours; due to be admitted to the hospital following surgery.  
Note: Informed consent will be obtained from each subject whenever possible. In situations where a subject is 
not competent to give informed consent, then the legally authorized representative (LAR) will provide 
consent on behalf of the subject. We will initially use consent of the LAR for subjects deemed not to have 
medical decision making capacity; however, should these subjects regain medical decision-making capacity 
after enrollment, they will be re-consented prior to continuation of participation in study. The PI will 
withdraw LAR consented subjects if they display distress, are uncooperative, or if the LAR consented subject 
indicates in any way that he or she does not wish to proceed with the LP and other procedures. The LAR 
should be present at study procedures to act as an advocate and to request withdrawal if/as needed. The 
subject’s capacity to consent will be assessed at each research visit. 
 
5.2      Participant Exclusion Criteria 
 
An individual who meets any of the following criteria will be excluded from participation in this 
trial: 
1. Inmate of a correctional facility 
2. Scheduled to receive systemic chemotherapy between the time of the two cognitive testing 
sessions 
3. Known inability to undergo LPs due to anticoagulant use, severe anxiety, 
or other clinical contraindication known ahead of time. 
4. Inappropriate for study inclusion based on the judgement of the principal investigator. 
5. If a patient undergoes major head trauma that occurs between the pre- and post-operative 
cognitive testing sessions, then they will be withdrawn from the study. 
6. If a patient has lung, kidney, breast or prostate cancer, which tend to metastasize to 
bone, and the patient has imaging evidence of metastatic disease in their spinal column, 
the patient is excluded from the study to avoid even the theoretical risk of seeding the 
intrathecal space with cancerous cells. 
7. Patients with existing imaging in the electronic medical record showing severe spinal 
stenosis will be excluded from this study due to the theoretical risk of causing 
myelopathy or radicular symptoms by introducing a needle into the intrathecal space. 
 
5.3      Strategies for Recruitment and Retention 
 
Surgical patients will initially be approached in their hospital room (if they are admitted before 
surgery) and asked whether they are interested in hearing about the study. If the patient is not 
admitted before surgery, he or she will be approached in the preoperative screening clinic, 
preoperative optimization of surgical health, one of the surgery clinics, or approached via a phone 
call by a member of the clinical care team and/or the study team using IRB approved phone 
script. In addition, potentially eligible patients will be identified in the electronic medical record 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
20 
 
 
by a report, and those patients will be sent an automated message through Duke’s MyChart 
application briefly describing the study and including a link to contact the study team if they are 
interested in participating. Per the template from the Duke MyChart recruitment team, the 
message will also contain a link to one of our MARBLE recruitment videos in which Dr. Berger 
explains the study.  Patients will be consented before study activities begin.  
 
We will enroll participants of both sexes and all ethnicities and racial groups. Based on the 
established literature regarding enrolling African-Americans and other historically underprivileged 
minority groups in research studies and to encourage trust and intrinsic human connections (Yancey 
et al, 2006), we will make every effort to have an African-American study coordinator or assistant 
contact African-American participants. 
 
To help encourage study patient retention, study patients will be compensated $50 at baseline and 
$50 at the 6 week +/- 3 weeks follow up study visit ($25 for the 6-week postoperative cognitive 
testing and questionnaire completion, and $25 for undergoing the 6 week postoperative blood draw 
and lumbar puncture). Study patients will also receive up to $12 at each study visit (consent, pre-op, 
24hr, and 6 weeks +/- 3 weeks post-op) for valet and/or parking costs. Additionally, for study 
patients who need transportation, the study will also pay for a wheelchair accessible van or other 
appropriate transportation to bring them to and from Duke for study visits. If the study pays for 
transportation for the patient, then the patient will not receive a parking pass or parking voucher. For 
patients who wish to drive themselves to Duke and who say that paying for gas would be an issue, 
the study will provide reimbursement for gas mileage at $0.50 per mile (up to 150 miles each way). 
Total possible direct subject reimbursement is thus approximately $148 for patients who live close 
by and do not request gas compensation, up to $100 for patients whose transportation is provided for 
by the study, or up to a theoretical maximum of $748 for a study subject who lives 150 miles away 
and requests gas compensation. 
 
Once patients are contacted via phone (submitted as separate phone script) and express interest in 
the study, we may email them a link to one of the following videos, which features Dr. Berger 
introducing the MARBLE study. 
 
https://www.dropbox.com/sh/blfaucno13w2u6e/AAAKcu0wjf1wdsuJsJow5aOHa?dl=0 
 
Because the idea of a lumbar puncture is scary for some patients, we have also created a series of videos 
explaining and showing an actual lumbar puncture procedure. If patients are concerned about this procedure 
from a safety and adverse event perspective, we can email them a link to one of the following videos that 
introduce the procedure.  
 
https://duke.app.box.com/folder/87650948771  
 
Furthermore, to promote continued patient engagement and interest in MARBLE, we have created a quarterly 
MARBLE newsletter with the intent of distributing them to all patients who have been consented and enrolled 
in MARBLE if possible. We will attempt to distribute this newsletter to patients via email or mailed through the 
postal service. In addition, we will attempt to give a physical copy of the current newsletter to all newly 
consented patients at their baseline visit, unless this is not possible due to the covid19 crisis or other exigencies. 
Each newsletter may contain the following: a brief synopsis of the study, study progress (recruitment goals and 
current recruitment attained), tips for a healthy brain, recent presentations/accomplishments from our research 
group, and staff member spotlight (a picture and job description of one of our research staff members and a 
quote from them on their perspective on the study).  


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
21 
 
 
 
We have also created an information tri-fold flier describing the MARBLE study that includes a brief synopsis 
of the study, eligibility criteria, what is involved in the study, and contact information for the PI, Dr. Miles 
Berger. Flyers will be distributed at the clinics of Duke surgeons who may have eligible patients, at the 
anesthesia preoperative screening clinic, and at other relevant locations where potentially eligible patients will 
see these flyers.  


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
22 
 
 
 
5.4      Participant Withdrawal or Termination 
 
5.4.1     Reasons for Withdrawal or Termination 
 
Participants may withdraw from the study at any time by contacting the study team via email or in 
writing. Participants who prematurely discontinue study agent treatment will still be encouraged to 
return for the 42-day (6 week) follow-up study visit. 
 
Participants may also be withdrawn from the study at any time per the discretion of the principal 
investigator (PI). 
 
The PI will withdraw legally authorized representative (LAR)-consented participants if they display 
significant distress, are uncooperative, or if the LAR-consented participant indicates in any way that 
he or she does not wish to proceed with the LP or other procedures, per the PI’s clinical judgement. 
The LAR should be present during trial procedures to act as an advocate and to request withdrawal 
if needed, unless this is impossible due to hospital/health system regulations during the covid19 or 
other exigent circumstances. 
 
5.4.2     Handling of Participant Withdrawals or Terminations 
 
A recent phase I trial did not identify any specific adverse events or safety concerns with CN-105 
(Guptill J et al, 2017). Nonetheless, if at any time during the dosing period, participant safety is 
considered at risk due to study agent-related adverse events per the judgement of the external Data 
Safety Monitoring Board (or DSMB+) and the PI, then the study agent will be immediately 
discontinued. Although the DSMB+ is only scheduled to convene and review reports (including 
grade II or higher AE, and SAE information) at the completion of each 67 patient cohort, the PI 
may also request that the independent statistician prepare additional interim analyses and provide 
these to the DSMB+ at any time. For example, this may be useful and/or appropriate if there is a 
severe SAE (such as a patient death) and the PI wishes to have the DSMB+ review grade II or 
higher AE rates and data up to an including this severe SAE, and make interim recommendations to 
the study team. 
 
Since CN-105 modulates the inflammatory response, it could theoretically impair the response to 
infection. Thus, in an abundance of caution, the study agent will be discontinued in any patient who 
develops any intracerebral or systemic infection. 
 
If a participant has clinically significant laboratory and/or electrocardiographic abnormalities that 
are considered to be related to the study agent in the judgement of the Principal Investigator or the 
DSMB, study agent treatment discontinuation will be evaluated. 
 
Primary study analyses will be conducted using intention to treat analysis (i.e. with patients included 
in the groups to which they were randomized even if they did not correctly receive all doses to 
which they were randomized); secondary analysis may be conducted using as treated analyses (i.e. 
with patients grouped by the drug doses actually received). 
 
5.4.3     Premature Termination or Suspension of Trial 
 
At the end of each 67 patient dose cohort, grade II or higher AEs and any SAEs will be provided to 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
23 
 
 
and reviewed by the DSMB+ and the PI, who will then consider whether the study can continue or 
whether it needs to be stopped 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
24 
 
 
 
and/or whether other corrective action needs to take place. The external DSMB+ or the PI may also 
request additional interim analyses. 
 
The study may be stopped if after any group of 67 patients the rate of grade III or higher SAEs (per 
CTCAE guidelines) in drug-treated patients is at least 10% and more than three times the rate of 
such events in the placebo-treated group. Considering the wide range of surgical procedures and 
patient comorbidities that could contribute to adverse events seen in this study, the DSMB+ should 
use both this quantitative cutoff and its clinical judgement in considering whether to stop the study. 
The study may be modified at any time by the sponsor, the FDA, or the external IRB as part of their 
duties to ensure that research subjects are protected. 
 
6 STUDY AGENT 
6.1 
Study Agent and Control Description 
 
6.1.1 
Acquisition 
 
CN-105 will be supplied by AegisCN and manufactured under contract by University of Iowa 
Pharmaceuticals, Iowa City IA. Study drug will be shipped directly from the manufacturer to the 
Duke pharmacy, whose investigational drug service will then dispense the drug at each participating 
study site (Duke Hospital, Duke Regional Hospital, Duke Raleigh Hospital). 
 
6.1.2 
Formulation, Appearance, Packaging, and Labeling 
 
CN-105 will be supplied in amber glass vials containing 4 mL of a concentrated, 12.5-mg/mL, clear- 
to-slightly-yellow solution. The vials are stoppered and capped with over-seals. Cloudiness, change 
in color, and/or the presence of any particulate matter may indicate that the product has deteriorated; 
if any of these or other changes are noted, the affected vials will not be used, and the study team will 
communicate with AegisCN and the University of Iowa Pharmaceuticals as soon as possible. 
 
Vials of CN-105 will have the following labeling information: 
CN-105 12.5 mg/mL (50 mg/vial) 
Store frozen at -70°C 
Caution: New Drug—Limited by Federal (U.S.) Law to Investigational Use 
Dosing labels will be attached to each dosing syringe/infusion bag. The individual participant 
syringe/infusion bag label will include both preprinted fields and handwritten fields as follows: 
Protocol number 
Personal identification number 
Participant initials 
Date 
Time 
Volume 
6.1.3 
Product Storage and Stability 
 
If dosing solutions are prepared more than 1 hour before the planned start of the study drug infusion, 
the infusion bags containing the dosing solutions are to be refrigerated at 2 to 8°C in a secure and 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
25 
 
 
 
locked refrigerator (and/or in a refrigerator in a secure and locked room). The drug can be 
refrigerated for up to 24 hours until approximately 60 minutes before the planned start of the 
infusion. 
 
6.1.4 
Preparation 
 
Study drug will be diluted in normal saline by the Investigational Drug Service (IDS); placebo will 
be normal saline. The infusion will be administered using a 1.2-micron or smaller filter. 
 
6.2 
Dosage and Administration 
 
We planned to enroll up to 250 patients total in this phase 2 study to obtain three successive dose 
groups of 67 patients each. In each group of 67 patients, 50 will receive a given dose of CN-105, 
and 17 will receive placebo. Due to a clerical error by the Investigational Drug Services that 
prepares our drug/placebo doses, 69 patients were randomized to the first patient cohort rather than 
67.Because enrollment happens at consent, and not all patients that consent will be randomized, we 
will keep enrolling patients until we reach 67 consented and randomized patients in each cohort.  
 
In each successive 67 patient group, participants will be randomized the week or day of surgery to 
receive either CN-105 (see Table 1 for dose levels) or placebo, within one hour before the 
scheduled or actual surgery start time and continuing every 6 hours until postoperative day 3 or 
hospital discharge (whichever occurs first), up to a maximum of 13 doses.  
 
6.2.1 
Route of Administration 
 
Study drug or placebo will be administered intravenously over 5-10 min, up to maximum of 30 minutes. 
 
6.2.2 
Starting Dose and Escalation Schedule 
 
Patients who receive CN-105 will receive one of three dose levels depending on their cohort: 0.1 
mg/kg, 0.5 mg/kg, or 1 mg/kg, depending on whether they are in the first, second or third group of 
67 patients, respectively. The 6 week +/- 3 week follow up visits for patients in the first two cohorts 
may be completed while the next cohort (higher dose level) is starting to be enrolled. 
 
Table 1: Dose levels of CN-105 
 
Cohort 
Dose of CN-105 
Patients receiving drug 
Patients receiving placebo 
Dose level 1 
0.1 mg/kg 
50 
17 
Dose level 2 
0.5 mg/kg 
50 
17 
Dose level 3 
1 mg/kg 
50 
17 
 
6.2.3 
Dose Adjustments/Modifications/Delays 
 
At the end of each 67-patient enrollment/ randomization group, grade II or higher AE rates and 
safety information will be provided to the DSMB. Upon evaluating this information, the DSMB plus 
will then consider whether the study should be stopped or whether other corrective action should 
take place. While grade II and/or higher AE and safety information from a given 67 patient 
randomized group is being analyzed and provided to the DSMB plus, and while the DSMB plus is 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
26 
 
 
evaluating this data (as described above), the trial will continue with enrollment of the next 67 
patient group (who will receive either placebo or the next higher dose of CN-105). Due to a clerical 
error by the Investigational Drug Services that prepares our drug/placebo doses, 69 patients were 
randomized to the first patient cohort rather than 67.  
There is no data from either preclinical studies or our recent phase I study suggesting any dose- 
dependent toxicity or side effects from CN-105. Thus, there is no reason to halt the enrollment of a 
subsequent 67 patient group (which will include patients receiving a higher dose of CN-105) while 
the DSMB is awaiting and then analyzing data from the prior 67 patient group. Nonetheless, the 
DSMB plus will be empowered to make recommendations about whether the trial should be stopped 
or modified based on their evaluation of data in the prior 67 patient group(s). 
 
6.2.4 
Duration of Therapy 
 
Study drug will be administered every 6 hours, with the first dose within one hour of planned or 
actual case start time, and subsequent drug doses will be given every 6 hours thereafter until 
postoperative day 3 or hospital discharge, whichever occurs first, up to a maximum of 13 doses. 
Each case start time will be defined by the study team as either the start of the 
surgery/procedure or the start of anesthesia (or the timed notes in the EMR for induction, 
intubation, or the start of anesthetic drug administration). If the case gets delayed by more than 
6 hr and we’ve already given the first dose, we will give an additional dose prior to surgery, but 
under no conditions will the patient be given more than 13 doses total of CN-105 
 
6.2.5 
Tracking of Doses 
 
Since the immediate pre- and post-operative periods are busy and nurses frequently have numerous 
tasks to perform, it is important to measure how many CN-105 (or placebo) doses are given as 
ordered to assess feasibility of q6 h dosing. We will track whether each CN-105 (or placebo) dose is 
given within +/-90 min of the scheduled administration time per nursing records. 
 
We will report the observed rate of doses administered within the required time window and the 
95% confidence interval. Each dose will be counted as an independent observation. The total 
number of doses correctly administered (i.e. within 90 min of the scheduled administration time) 
divided by the number of prescribed doses will define our dosing feasibility rate. Post-hoc feasibility 
analysis will be performed both among all study patients (including those receiving placebo), and 
specifically among CN-105 drug patients only, as well as by dose level to determine if drug dose 
level affects feasibility. 
 
6.3 
Study Agent Accountability Procedures 
 
For each participant dose, details of study agent preparation and administration, i.e., time, date, dose 
level, volume, infusion time, etc., will be recorded in the source documents. 
 
Upon receipt of CN-105 shipments, the pharmacist/designee will verify the condition of the vials 
and document per instructions in the Manual of Operational Procedures. 
 
All used study agent vials, syringes, infusion sets, etc., will be kept in a secure location. After 
accountability is complete, used supplies may be discarded according to the site’s normal 
procedures. All study vials (used/unused) remaining at the completion of the study will either be 
returned to the sponsor/designee or will be destroyed at the site. In case of the latter, the site will 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
27 
 
 
maintain destruction records. 
 
7 TRIAL PROCEDURES AND SCHEDULE 
7.1 
Trial-Specific Procedures 
7.1.1 
Initial Lumbar Puncture and Blood Draw 
 
An LP will be performed prior to surgery (between the time of consent and the time of scheduled 
anesthesia/surgery start), with a standard spinal anesthesia kit containing a 25 gauge pencil-point 
needle (or other appropriate spinal needle per Good Clinical Practice) under strictly sterile 
conditions. The patient’s back will be sprayed with benzocaine numbing spray and we will wait 
approximately 10 minutes for analgesic relief prior to performing the lumbar puncture. This 
preoperative LP will be performed by an anesthesiology attending physician or senior 
anesthesiology resident or fellow under attending supervision, or a nurse practitioner or 
physician assistant under appropriate supervision. 
 
The LP will be performed using a standard spinal anesthesia kit per the manufacturer’s directions 
under sterile conditions at the L4–5 spinal level or the next interspace above or below this level, 
depending on which interspace appears larger by palpation. A curvilinear ultrasound probe will be 
used when necessary to help visualize the interspace. An initial 1 ml of CSF will be discarded (to 
reduce blood contamination of the CSF) before collecting a study sample of up to 20 ml of CSF. 
Blood (up to 10 mL) will be collected via sterile venipuncture, from an arterial line, or by the IV 
line placed before surgery. To assess their level of pain following the lumbar puncture procedure, 
subjects will be asked to answer the following question: How much did the lumbar puncture hurt 
(on a scale of 0-10, where 0 is no pain at all and 10 is the worst pain imaginable?’ 
 
LP vital signs will be collected and recorded by the study team before and after the LP if performed 
on outpatient basis (between the time of consent and surgery day).  
If the LP is performed while inpatient (i.e. on the day of surgery, or at 24 hrs after surgery), the 
study team will not collect and record pre and post LP vital signs, as these will already be recorded 
in the patient’s hospital electronic medical record per standard of care. 
 
7.1.2 
Postoperative Lumbar Puncture and Blood Draws 
 
LPs at 24 hours and at 6 weeks +/- 3 weeks after the start of anesthesia/surgery will be performed by 
an anesthesiology attending physician or senior anesthesiology resident or fellow under attending 
supervision, or a nurse practitioner or physician assistant under appropriate supervision. To assess 
their level of pain following each lumbar puncture procedure, subjects will be asked to answer the 
following question: How much did the lumbar puncture hurt (on a scale of 0-10, where 0 is no pain 
at all and 10 is the worst pain imaginable?’ 
 
If the participant is hospitalized overnight after the surgery, the 24-hour postoperative LP may be 
performed in the participant’s hospital room, in the preoperative holding area, or in other 
appropriate space for performing a sterile neuraxial procedure per Good Clinical Practice (GCP) 
guidelines. If the participant is unexpectedly discharged after surgery on the same day and does not 
spend the first night in the hospital, the patient will be asked to return for these procedures, and then 
the 24-hour postoperative LP will be performed in the same location as the 6 week +/- 3 weeks 
postoperative LPs are performed or in another safe location for performing sterile neuraxial 
anesthetic procedures per GCP guidelines. As for the preoperative LP, an initial 1 ml of CSF will be 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
28 
 
 
discarded (to reduce blood contamination of the CSF) before collecting up to a 20ml CSF sample for 
the study. 
 
The 6 week +/- 3 week postoperative LPs will also be performed in the preoperative or 
postoperative holding area, or in other clinical research space of the Duke Anesthesiology 
department that is appropriately outfitted for human subject procedures (i.e. presence of medical 
gases, suction, resuscitation equipment. As with the preop and 24 hr postop LPs, an initial 1 ml of 
CSF will be discarded (to reduce blood contamination of the CSF) before collecting up to a 20ml 
CSF sample for the study. 
 
The 24 hr and 6 week +/3 week postoperative LP vital signs will be collected and recorded by the 
study team before and after the LP if performed on outpatient basis. If the LP is performed while the 
patient is an inpatient, the study team will not collect and record vital signs, as these will already be 
obtained by the patient’s clinical care team and recorded in the patient’s hospital record per standard 
of care.  
The LP will then be performed at the L4-5 interspace or the interspace above or below this level, depending 
on which space feels more open by palpation and/or appears more open by ultrasound visualization. The LP 
will be performed using a spinal anesthesia kit per the manufacturer’s directions under sterile conditions. The 
participant may be continuously monitored for 15 minutes or longer if deemed necessary. 
Blood will be drawn by sterile venipuncture, or from an arterial or an IV line if already in place, at 
the same time as the lumbar punctures. 
 
7.1.3 
Neurocognitive Testing, Delirium Testing, and Other Assessments 
 
Neurocognitive testing and assessments may be performed remotely via teleconference or may be 
performed in-person in one of the study testing offices located in Duke South or other appropriate 
location   preoperatively (baseline) and/or at 6 weeks +/- 3 weeks after surgery. They will also 
occur for any patients who have a 1 year post-operative study visit. Tests from the following 
standardized tests and assessments list will be utilized, which have equivalent alternate forms (to 
minimize test-retest practice effects) and have English, French and Spanish language versions: 
a. Hopkins Verbal Learning Test, Revised (HVLT-R) - an auditory-verbal word-list task used 
to assess learning, short-term recall, long-term recall and recognition memory that is co- 
normed with the BVMT-R (see immediately below). 
b. Brief Visuospatial Memory Test, Revised (BVMT-R) – a visual measure of constructional 
praxis, spatial/figure learning, short-term recall, long-term recall and recognition memory 
that is co-normed with the HVLT-R (see immediately above). 
c. Digit Span subtest from the Wechsler Adult Intelligence Scale, 3rd Revision (WAIS-III) – a 
test of immediate auditory-verbal recall and complex attention (i.e., working memory). 
d. Digit Symbol Coding subtest from the Wechsler Adult Intelligence Scale, 3rd Revision 
(WAIS-III) - a matching-to-sample visual scanning and visuomotor production task. 
e. Trail Making Test (TMT), Parts A & B – a test of simple visuomotor scanning and complex 
sequencing and set-switching executive demands. 
f. Controlled Oral Word Association Test (COWAT) – a test of lexical verbal fluency and 
information retrieval efficiency. 
g. Grooved Pegboard Test – a timed test of bilateral manual dexterity and motor speed. 
h. Wechsler Test of Adult Reading (WTAR) – a non-phonemic reading test used to assess 
crystalized pronunciation knowledge; a reliable estimate of premorbid intellectual 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
29 
 
 
functioning. 
i. MoCA: used to screen for cognitive impairment. 
j. STOP-Bang brief questionnaire: to assess sleep apnea risk during one of the participants’ 
trial visits. 
k. Visual analogue scale (VAS) pain assessment at each trial visit (preoperative, 6 weeks +/- 3 
weeks postop. 
 
Other assessments performed at both the preoperative and 6 week (+/- 3 week) postop visits 
may include: 
a. List of medications participants are currently taking at each trial visit. 
b. Timed up and Go task/test, Romberg assessment, and falls assessments. In particular, 
patients will undergo a brief falls risk assessment questionnaire, which contains questions 
from the Elderly Falls Screening test (Cwikel J et al, Disabil Rehab, 1998) and the Fall Risk 
Screening test (Tromp et al, J Clin Epidemiology, 2001), and questions about whether they 
have fallen recently. 
Relevant tests listed above may be recorded using a digital voice recorder for quality assurance 
purposes. Quality assurance activities will include reviewing of testing audio files, generating 
performance review documents, and providing feedback on accuracy and completeness. There is no 
risk to the trial participants since participant identifiers will not be provided. Trial participants will 
not be identified by name on the recording. Only the trial number, test date, and initials of the tester 
will be provided on the digital file label. The link between trial number and participant identifiers 
will be maintained. These files will be stored on locked computers within locked offices to which 
only authorized research personnel will have access. These files will be destroyed after the primary 
outcome analysis of this research trial is complete. 
 
Delirium Testing will be performed twice daily starting on the day of surgery until postoperative 
day 5 or hospital discharge (whichever occurs first), as well as at the baseline study visit and the 6 
week +/- 3 weeks postoperative study visit, as well as the 1 year visit that some patients may have, 
using the CAM-ICU instrument in intubated patients (Ely EW et al, 2001) and the 3D-CAM 
instrument in non-intubated patients (Marcantonio E et al, 2014), based on the enhanced sensitivity 
of these instruments in these respective patient populations (Kuczmarska A et al, 2016). On the day 
of surgery, delirium testing will be performed once while the patient is in the post-anesthesia care 
unit (PACU) after surgery and once later in the afternoon or early evening on the day of surgery, 
unless the PACU delirium assessment is performed after 4:00 PM. In that case, no additional 
afternoon/evening testing assessment will be performed on that day. If the surgery ends late and if 
the PACU delirium assessment cannot be performed before 7:00 PM, then no delirium assessments 
will be performed on the day of surgery. Inter-rater reliability for these assessments will be assessed 
as previously described (Marcantonio E et al, 2014). 
 
7.1.4 
Quality of Life Assessment 
 
Our approach to measuring QOL for this trial involves the use of a battery of well-tested and well- 
validated instruments that together cover the relevant domains of interest. For this trial, we propose 
to use an assessment instrument that the Randt group developed, the SF-36 generic QOL instrument, 
from their longer instruments used in the Health Insurance Experiment and in the Medical Outcomes 
Study, as well as functional status measures (the Duke Activity Status Index [DASI]); psychological 
measures, including depression (the Center for Epidemiological Studies- Depression [CES-D]), the 
State Trait Anxiety Inventory [STAI], and the Hopkins Symptom Checklist [HSCL] (SCL-90); and 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
30 
 
 
social/role functioning (including employment status). 
 
Participants will complete these questionnaires during the baseline trial visit after completing all 
consent paperwork and again at the 6 week +/- 3 weeks postoperative visit. Any participant who is 
unable to complete this questionnaire at either trial visit will be given an e-copy or a paper copy of 
the questionnaire to fill out at home and to mail back to our study team at his or her convenience 
(in the case of paper questionnaires). Mail in questionnaires will be accepted from patients as long 
as they are completed by the patient within +/- 3 weeks of the preoperative study visit and before 
the surgery, or if they are completed within +/- 3weeks of the 6 week +/- 3 week postoperative 
study visit. 
 
7.1.5 
Electroencephalographic Recording 
 
Scalp recordings of electrical activity produced by the brain (EEGs) will be obtained for 3-60 minutes before the start of 
anesthesia, as well as during and right after anesthesia and surgery. The EEG will be recorded from 32 electrodes embedded 
in an elastic scalp electrocap, using a state-of-the-art, battery-operated, portable EEG system (Live-amps, Brain Vision LLC, 
Morrisville, NC). If necessary due to time constraints or other considerations, the study team may also use a BIS electrode 
strip to monitor the patient’s brain waves instead of the full 32 channel EEG cap setup.  The electroencephalographic data 
will be analyzed to determine the effect of CN-105 on EEG correlates of postoperative cognitive function and post-operative 
delirium, as well as to identify possible novel pre- and intraoperative EEG correlates of POCD, delirium, and/or 
neuroinflammation (with the latter as measured by CSF inflammatory cytokine levels at 24 hours after anesthesia and 
surgery). In the event that sufficient personnel or equipment are unavailable, or in the event that two MARBLE patients are 
having surgery at the same time, or in the event that there is insufficient time to place the EEG sensor on that patient’s head 
before the start of surgery, the study team may not record EEG from all patients or at all points before, during, and just after 
surgery/anesthesia 
In addition to passive EEG recordings, patients may also undergo auditory event related potential 
testing during EEG recording in which brief sounds will be administered via earphones during the 
pre-operative and/or intra-operative EEG recordings, when the pre-op and surgical workflow allows 
and when study personnel are available. The volume of these brief intermittently played sounds 
would be well within the range of normal daily exposures, i.e., <= 80 dB, a level that would be safe 
for even a full day of continuous exposure (MRC Institute of Hearing Research, 1996), which is 
much more than is proposed here. Patients may further undergo brief audiometric screening in 
which sounds are delivered via headphones to ensure that they are able to hear the presented sounds 
at a volume of 80 dB or less without auditory assist devices (i.e., without hearing aids, cochlear 
implants, or BAHA devices). This would not require any additional visits.  EEG responses to 
auditory event-related potentials have been associated with neurocognitive reserve previously 
(Speer and Soldan, 2015). Collecting this data may increase the team's ability to identify EEG-
based biomarkers of perioperative neurocognitive resilience. 
 
7.2 
Standard-of-Care Trial Procedures 
 
7.2.1 
Anesthetic Treatment 
 
All participants will receive either general anesthesia or sedation (i.e. monitored anesthesia care); 
patients may also receive regional or neuraxial nerve blocks as adjuncts. 
 
The duration of anesthetic exposure shall be recorded for each participant, and intraoperative 
anesthetic medication administration and physiologic monitoring data will be extracted from the 
electronic anesthetic records for post-hoc analyses. 
 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
31 
 
 
7.3 
Laboratory Procedures/Evaluations 
 
7.3.1 
Clinical Laboratory Evaluations 
 
A basic metabolic panel (i.e. Na+, K+, Cl-, CO2, BUN, Cr and glucose) and a complete blood count 
(with differential) will be measured at three time points: within 2 weeks before anesthesia and 
surgery (and before the first dose of CN-105), 1 day after the start of anesthesia/surgery (i.e., after 
~4 doses of CN-105), and at the 6 week +/- 3 weeks postoperative study visit. 
 
7.3.2 
Other Assays or Procedures 
 
We will measure CN-105 levels in both the serum and CSF at the 24-hour postoperative time point. 
This will help us determine how the CN-105 doses administered to patients in this study translate 
into serum drug concentrations, and will help us determine the extent of CN-105 penetration in the 
human CNS (as assayed in the CSF). We note that we do not plan to perform these measurements at 
the preoperative time point (as there should be no CN-105 in the serum or blood before the first dose 
is administered), or at the 6 week +/- 3 weeks postoperative study appointment (as the last CN-105 
dose will be given no later than on postoperative day 3, and thus CN-105 levels would be expected 
to be undetectable ~6 weeks later). 
 
We will perform serum and CSF CN-105 measurements together with our colleagues at MPI 
research (Mattawan, MI; now part of Charles River Laboratories), with whom AegisCN has 
previously performed these measurements. In brief, CN-105 will be detected via a previously 
validated high-performance liquid chromatography-tandem mass spectrometry assay (LC-MS/MS). 
The liquid chromatography system uses an ACE 3 C8 column, 2.1 x 50 mm (3 µm particle size) 
with an isocratic flow consisting of water/acetonitrile/heptafluorobutyric acid (76/24/0.05, v/v/v) at 
a flow rate of 200 µL/minute (Agilent 1200 Binary Pump) and a post column infusion consisting of 
isopropanol/acetic acid (100/1, v/v) at a flow rate of 800 µL/minute (Agilent 1100 LC Quaternary 
Pump). The analyte and internal standard (acebutolol hydrochloride) are detected using a SCIEX 
API 5000 triple quadrupole LC-MS/MS system equipped with an ESI (TurboIonSpray®) ionization 
source operated in the positive ion mode. Multiple reaction monitoring transitions of the respective 
[M+H]+ ions are used to monitor CN-105 and acebutolol hydrochloride. 
7.3.3 
Specimen Preparation, Handling, and Storage 
 
Blood samples will be used for genotyping and metabolic/inflammatory marker assays. Whole- 
blood specimens (10 mL total volume) will be collected sometime between the patient being 
consented and scheduled anesthesia/surgery start time, 24 +/- 2 hours later, and 6 weeks +/- 3 
weeks later. 
Whole-blood specimens will be collected with EDTA as the anticoagulant, obtained either from an 
arterial or IV catheter or from sterile venipuncture. 
 
Cerebrospinal fluid specimens will be analyzed for amyloid beta, tau, and other related AD- 
associated neural markers and inflammatory cytokines. Cerebrospinal fluid specimens will be 
collected when the LP is performed before surgery (i.e. between the time of consent and 
anesthesia/surgery start), 24 hours after the start of anesthesia/surgery, and at the 6 week +/- 3 weeks 
follow up visit. 
 
Cerebrospinal fluid and blood samples will be collected by a trained care provider under sterile 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
32 
 
 
conditions. Blood and CSF samples will be stored at -80°C. All assay measurements will be 
performed at DUMC or in the accredited laboratory of a collaborator. All CSF and blood samples 
will be held in freezers in the Department of Anesthesiology and/or DUMC research facilities.  
 
7.3.4 
Specimen Shipment 
 
Relevant specimens will be shipped on dry ice by FedEx overnight express to relevant collaborating 
labs for measurements, such as to MPI/Charles River and to the ADNI biomarker core lab at the 
University of Pennsylvania or other appropriate laboratory for tau, phospho-tau, and A 
measurements. 
7.4 
Trial Schedule 
 
7.4.1 
Initial Consent and Visit 
The following procedures and assessments will be conducted at the time of initial study consent 
or between the Initial study consent and the day of surgery:  
Informed consent 
Enrollment 
Demographics (age, sex, and race) 
Medical history 
Neurocognitive testing will be done either in-person or remotely via teleconference 
(may include WTAR; HVLT-R; BVMT-R; WAIS-III —Digit Span, Digit Symbol 
Coding subtests, TrailTMT, Parts A & B, COWAT and Grooved Pegboard; MoCA) 
as well as the STOPBang assessment, VAS pain assessment, Falls assessments, 
Timed Up and Go Test, and Romberg Test 
DASI 
Psychological measures (including CES-D, STAI, SCL-90, SF-36) 
Record concomitant medications administered per standard of care  
Delirium Screening with the 3D-CAM instrument 
Collect CSF sample/ LP prior to surgery (i.e. between the time of consent and the time of 
scheduled anesthesia/surgery start, which means it could be done at the initial visit or 
anytime between the time of consent at the initial visit and the time of scheduled 
anesthesia/surgery start on the day of surgery) 
 
Collect research blood sample prior to surgery (i.e. between the 
time of consent and the time of scheduled anesthesia/surgery start, 
which means it could be done at the initial visit or anytime between 
the time of consent at the initial visit and the time of scheduled 
anesthesia/surgery start on the day of surgery) 
 
LP vital signs will be collected and recorded by the study team 
before and after the LP if performed on outpatient basis (between 


MARBLE Study – Protocol Version 7 
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33 
 
 
the time of consent and surgery day).  
If the LP is performed while the patient is an inpatient (i.e. on the 
day of surgery), the study team will not collect and record vital 
signs, as these will already be obtained by the patient’s clinical care 
team and recorded in the hospital electronic medical record for the 
patient, per standard of care.  
-Randomization assignments will be made within 1 week prior to the patient’s scheduled surgery.  
 
7.4.2 
Day 0 (Day of Surgery) 
The following procedures and assessments will be conducted at this visit: 
 
Collect CSF sample/ LP prior to surgery (if not done at the time of the Initial Consent and 
Visit) 
Concomitant medications administered per standard of care.  
Collect blood sample (if not done at the time of the Initial Consent and Visit) 
Laboratory assessments 
Study drug or placebo administration (within one hour prior to scheduled or actual surgery start 
time, and subsequent drug doses will be given every 6 hours thereafter until 3 days after surgery or 
hospital discharge, whichever comes first, up to a maximum of 13 doses) by the patient’s clinical 
care team. Each case start time will be defined by the study team as either the start of the 
surgery/procedure or the start of anesthesia. If the case gets delayed by more than 6 hr and we’ve 
already given the first dose, we will give an additional dose prior to surgery, but under no 
conditions will the patient be given more than 13 doses total of CN-105. 
EEG (within 2 hours before surgery as well as during and just aftersurgery). In the event that sufficient 
personnel or equipment are unavailable, or in the event that two MARBLE patients are having surgery 
at the same time, or in the event that there is insufficient time to place the EEG sensor on that patient’s 
head before the start of surgery, the study team may not record EEG from all patients or at all points 
before, during, and just after surgery/anesthesia. Auditory tones will be played and auditory event-
related potentials will be recorded if the pre-op and surgical workflow allows and if study personnel are 
available. 
Vital signs (including heart rate) will be obtained and entered into the anesthetic 
record during the surgery by the clinical care team as per GCP, standard of care, and  
the American Society of Anesthesiology guidelines . 
Continuous heart rate monitoring will be obtained for up to 15 minutes prior to surgery and 
during the surgery, to assess heart rate variability (R-R interval) as an index of vagal nerve 
function. 
Grade II or higher AEs (and all SAEs) will be monitored and recorded from just after the 
first study drug or placebo administration until the end of the 6 week +/- 3 weeks 
postoperative study follow up visit. 
Delirium Screening may be administered with 3D-CAM or CAM-ICU up to two times after 
surgery. 
 


MARBLE Study – Protocol Version 7 
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34 
 
 
7.4.3 
Day 1 (First Day after Surgery, Hospitalization) 
The following procedures and assessments will be conducted at this visit: 
Collect CSF sample/ LP 
LP vital signs will be collected and recorded by the study team 
before and after the LP only if the LP is performed on an 
outpatient basis.  
Concomitant medications will be administered according to standard of care 
Collect blood sample  
Laboratory assessments 
 
Study drug or placebo administration (every 6 hours until 3 days after surgery or 
hospital discharge, whichever comes first, up to 13 doses maximum) by the 
patient’s clinical care team 
Grade II or higher AEs recorded 
Delirium Screening will be administered with 3D-CAM or CAM-ICU   
7.4.4 
Days 2-4 (Hospitalization) 
The following procedures and assessments will be conducted at this visit: 
Concomitant medications will be administered according to standard of care 
Study drug or placebo administration (every 6 hours until the 3rd day after surgery or 
hospital discharge, whichever comes first, for a maximum of 13 doses) given by the 
patient’s clinical care team. 
Grade II or higher AEs recorded 
Delirium Screening will be administered with 3D-CAM or CAM-ICU until postoperative day 5 or 
hospital discharge (whichever occurs first). 
 
7.4.5 
6 weeks (+/- 3 weeks) Follow-Up 
The following procedures and assessments will be conducted at this visit: 
Neurocognitive testing will be done either in-person or remotely via teleconference 
(may include HVLT-R; BVMT-R; WAIS-III —Digit Span, Digit Symbol Coding 
subtests, TMT Parts A & B, COWAT, MoCA, and Grooved Pegboard; STOPBang 
(only if it wasn’t performed at the preoperative visit) VAS pain assessment, Falls 
assessments, Timed Up and Go Test, Romberg Test) 
Laboratory assessments 
DASI 
Psychological measures (including CES-D, STAI, Hopkins SCL-90) 
SF-36 
Collect CSF samples / LP 
Vital signs will be collected and recorded by the study team before and after the LP, only 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
35 
 
 
if the patient is not already hospitalized as an inpatient at this time. 
Collect blood samples  
Grade II or higher AEs recorded 
Delirium assessment (using either the 3D-CAM instrument, or the CAM-ICU instrument in 
the unlikely case that the patient is intubated at the time of this study visit). 
STOP-Bang sleep apnea screening assessment may be performed, if it was not already 
completed at the baseline study visit or at another point during the study. 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
36 
 
 
 
 
7.4.6 
Schedule of Events 
 
Table 2: Schedule of Study Events for Trial 
 
Screening/ 
Baseline 
Study 
Visit 
Within 1 
week 
before 
surgery 
Day 0 
Surgery 
Day 1 a 
Hospitalization 
Days 2-4 
Hospitalization 
6 weeks 
(+/- 21 
days) 
Follow- 
up 
Informed consent 
X 
Enrollment 
X 
Randomization 
X 
Demographics (age, sex, and race) 
X 
Vital signs 
X b, e 
X b 
X b 
Medical history 
X 
Neurocognitive testing c 
X 
X 
Continuous Heart Rate Recording 
X 
Laboratory assessments (additional 
routine clinical pre- + post-op labs will 
also be recorded) 
X 
X 
X 
Duke Activity Status Index 
X 
X 
Psychological/physical measures d 
X 
X 
Short Form-36 
X 
X 
Lumbar puncture, Collect CSF samples 
X e 
X 
X 
Concomitant medications 
X 
X 
X 
X 
X 
Collect blood sample 
X f 
X 
X 
Drug administration g 
X 
X 
X 
Electroencephalogram (EEG) 
X h 
Grade II or higher AEs/ all serious AEs 
X 
X 
X 
X 
3D-CAM or CAM-ICU for delirium 
screening 
X 
X 
X 
Xi 
X 
Biomarker assays 
X 
X 
X 
Genotyping assays 
 
X f 
Inflammatory protein analysis 
X 
X 
X 
CSF protein analysis 
X 
X 
X 


MARBLE Study – Protocol Version 7 
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37 
 
 
 
 
 
AE, adverse event; CSF, cerebrospinal fluid; 
QOL, quality of life 
a 24 (+/- 2) hours post-surgery. 
b LP vital signs will be collected and recorded by the study team before and after the LP only if it is performed on an 
outpatient basis.  
c May include Hopkins Verbal Learning Test-Revised; BVMT-R ; Wechsler Adult Intelligence Scale-III—Digit Span, Digit 
Symbol subtests; Wechsler Test of Adult Reading; Trail Making Test Parts A & B; Grooved Pegboard; Montreal Cognitive 
Assessment (MoCA); STOPBang Questionnaire; visual analog scale pain assessment; 
d May include Center for Epidemiological Studies-Depression, State Trait Anxiety Inventory, Hopkins Symptom Checklist, 
Falls assessments, Timed Up and Go, and Romberg Assessments. 
e Between the time of consent and the scheduled anesthesia/surgery start time on the day of surgery. 
f Between the time of consent and the scheduled anesthesia/surgery start time on the day of surgery. 
g w/in 1 hr before the scheduled or actual start time of surgery and continuing every 6 hours through postoperative 
day 3 (up to 13 doses maximum) or hospital discharge, whichever occurs first  
h Immediately before , during, and after surgery/anesthesia. In the event that sufficient personnel or equipment are 
unavailable, or in the event that two MARBLE patients are having surgery at the same time, or in the event that 
there is insufficient time to place the EEG sensor on that patient’s head before the start of surgery, the study team 
may not record EEG from all patients or at all points before, during, and just after surgery/anesthesia. Auditory 
tones will be played, and auditory event-related potentials will be recorded if the pre-op and surgical workflow 
allows and if study personnel are available. 
I This will be administered until postoperative day 5 or hospital discharge (whichever occurs first). 
 


MARBLE Study – Protocol Version 7 
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38 
 
 
 
 
8 ASSESSMENT OF SAFETY 
8.1 
Specification of Safety Parameters 
 
8.1.1 
Definition of Adverse Events (AEs) 
 
An AE is any untoward medical occurrence associated with the use of a drug in a participant, 
whether or not considered drug related. An AE can, therefore, be any unfavorable and unintended 
sign, including an abnormal laboratory finding, for example, symptom, or disease temporally 
associated with the use of a pharmaceutical product. Diseases, signs, symptoms, or laboratory 
abnormalities already existing at randomization are not considered AEs unless they worsen (i.e., 
increase in intensity or frequency). Surgical procedures planned before randomization and the 
conditions leading to the need for these surgical procedures are not AEs. 
 
8.1.2 
Definition of Serious Adverse Events 
 
An AE is considered serious if any of the following occur or the investigator believes any of the 
following outcomes may occur: 
1. Death. 
2. Life-threatening AE: places the participant, in the view of the investigator, at immediate risk 
of death at the time of the event as it occurred. It does not include an AE that, had it occurred 
in a more severe form, might have caused death. 
3. Persistent or significant disability or incapacity or substantial disruption of the ability 
to conduct normal life functions. 
4. Congenital abnormality or birth defect. 
5. Requires inpatient hospitalization or prolongation of existing hospitalization with the 
following exceptions: 
Preplanned (before the trial) hospital admissions, unless the hospitalization is 
prolonged 
Planned admissions (as part of a trial, e.g. routine biopsies) 
Re-hospitalizations (which occur less than 24 hours after discharge 
from index hospitalization) 
Elective procedures 
Emergency room visits 
6. Important medical events that may not result in death, be life threatening, or require 
inpatient hospitalization may be considered a serious AE (SAE) when, based on appropriate 
medical judgment, they may jeopardize the participant and may require medical or surgical 
intervention to prevent one of the serious outcomes listed above. This determination is based 
on the opinion of the investigator. 
7. SAEs that are thought to be related (i.e. more likely than not to be related) to the study drug 
will be reported to the FDA and all other appropriate regulatory bodies within 15 days of the 
study time documenting the SAE. SAEs thought not to related to the study drug will 
reported on a yearly basis.  
 
 


MARBLE Study – Protocol Version 7 
PI: Miles Berger, MD, PhD 
39 
 
 
8.1.3 
Definition of Unanticipated Problems 
 
Unanticipated problems involving risks to participants or others include, in general, any incident, 
experience, or outcome that meets all of the following criteria: 


MARBLE Study – Protocol Version 7 
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40 
 
 
 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures 
that are described in the protocol-related documents, such as the IRB-approved research 
protocol and informed consent document; and (b) the characteristics of the participant 
population being studied 
Related or possibly related to participation in the research (“possibly related” means 
there is a reasonable possibility that the incident, experience, or outcome may have been 
caused by the procedures involved in the research) 
Suggests that the research places participants or others at a greater risk of harm (including 
physical, psychological, economic, or social harm) than was previously known or 
recognized 
 
8.2 
Classification of an Adverse Event 
 
8.2.1 
Severity of Event 
 
Assessment of severity of an event will be based on the following: 
Mild: Is generally transient, does not interfere with usual activities, and is easily tolerated by 
the participant. 
Moderate: Causes sufficient discomfort and interferes with usual activities. 
Severe: Causes considerable interference with usual activities and may be incapacitating. 
Whenever appropriate and/or possible, we will define AE’s by the NIH Common Terminology 
Criteria for Adverse Events (available online at 
https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_Refe
rence_5x7.pdf 
 
Multiple grade I (i.e. mild) AEs per CTCAE criterion typically occur after major non-cardiac surgery in 
older adults (i.e. our study population). Examples of such grade I AEs that occur in virtually every older 
adult after major non-cardiac surgery include hemoglobin declines to between 10 mg/dL and the lower 
limit of the normal range, serum Ca++ drops to between 8.0 and the lower limit of the normal range, 
WBC count increases to the 10-20 range, etc. Thus, to avoid capturing a massive amount of such grade I 
AEs that are unlikely to be related to study drug administration, here we will capture and report just those 
AEs of grade II or higher, and all SAEs.  
8.2.2 
Relationship of Study Agent 
 
Assessment of the relationship of an event to the study agent will be based on the following: 
Unrelated = None; Remote 
o None (unrelated, not related, no relation) 
 Unreasonable temporal relationship between administration of the study 
agent and the onset of the AE (e.g., the event occurred either before, or 
too long after, administration of the study agent for it to be considered 
drug related) 
 Causal relationship between the study agent and the AE is biologically 
implausible (e.g., death as a passenger in an automobile accident) 
 Clearly more likely alternative explanation for the AE is present (e.g., 
typical adverse reaction to a concomitant drug and/or typical disease- 


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related event) 
o   Remote (unlikely, doubtful, improbable) 
Little evidence to suggest there is a causal relationship and there is 
another reasonable explanation for the event. 
 
Related = Possible, Probable 


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o Possible 
 There is some evidence to suggest a causal relationship (e.g., the 
event occurred within a reasonable time frame after administration 
of the study agent); however, the influence of other factors may 
have contributed to the event (e.g., the participant’s clinical 
condition, other concomitant medications, or concurrent illness) 
o Probable 
 There is evidence to suggest a causal relationship between the event 
and the study agent, and the influence of other factors is unlikely. 
 
The investigator should consider the following when assessing causality: 
Temporal associations between the study agent and the event 
Pre-existing risk factors or concurrent illness 
Plausible mechanism 
8.2.3 
Expectedness 
 
The investigator will be responsible for determining whether a grade II or higher AE is expected or 
unexpected. A grade II or higher AE will be considered unexpected if the nature, severity, or 
frequency of the event is not consistent with the risk information previously described for the study 
agent in the protocol. 
 
8.3 
Time Period and Frequency for Event Assessment and Follow-up 
 
The investigator will record all reportable events starting from the time just after the first 
administration of study drug (or placebo) until the last trial-related procedure/visit. At each trial 
visit, the investigator or study team will inquire about the occurrence of grade II or higher AE and 
any SAEs since the last visit. Events will be followed for outcome information until resolution or 
stabilization. Adverse events of grade II or higher for participants who discontinue trial 
participation will be collected/reported through at least the time of discontinuation. 
 
When additional relevant information (final diagnosis, outcome, results of specific investigations, 
etc.) becomes available, the investigator will submit this additional information to the DSMB at the 
time of the next DSMB report (or earlier, if deemed appropriate by the PI). 
 
8.4 
Reporting Procedures 
 
All grade II or higher AEs that occur between the time immediately following the first 
administration of study drug (or placebo) until the last trial-related procedure/visit will be 
recorded. Adverse events may be volunteered by subjects and/or solicited at each assessment by 
the investigator or designee. 
 
Recording will be performed in a concise manner using standard, acceptable medical terms. The 
research team (and the Principal Investigator) will make every effort to establish a diagnosis of the 
event based on the presenting signs, symptoms, and/or clinical information. In such cases, the 
diagnosis will be documented as an AE/SAE. 


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Diseases, signs, symptoms, or laboratory abnormalities already existing at enrollment will not 
considered AEs unless they worsen (i.e., increase in intensity or frequency). Surgical procedures 
planned before enrollment and the conditions leading to these measures are not AEs, and the 
normal post-operative recovery course of a surgical procedure planned before enrollment also 
will not constitute an AE. Thus, for example, if a study patient has a planned knee replacement 
surgery before he or she enrolls in the study, the patient has this surgery, and a postoperative 
knee x-ray shows the normal postoperative presence of the replacement knee, this will not be 
considered an AE. 
 
If, in the investigator’s judgment, a clinically significant worsening from baseline is observed in any 
laboratory or other test parameter (e.g., ECG), physical examination finding, or vital sign, a 
corresponding clinical AE should be recorded. If the cause is not known, the abnormal test or 
finding should be recorded as an AE, using appropriate medical terminology (e.g., 
thrombocytopenia, peripheral edema, QT prolongation). If this AE is of grade II or higher per 
CTCAE criteria, this event will be recorded and reported to the DSMB+. 
 
8.4.1 
Serious Adverse Event Reporting 
 
8.4.1.1 
Reporting to the DSMB and IRB 
 
All dru-related SAEs (i.e. if thought to be drug- related, or more likely than not to be related to the 
drug), occurring between between the time immediately following the first administration of study 
drug (or placebo) until the last trial-related procedure/visit will be reported by the investigator or 
qualified designee within 5 working day of first becoming aware of the event to the IRB and 
DSMB. For SAEs deemed “probably” drug-related, reporting to the IRB and/or FDA will occur 
within appropriate time windows, per institutional and FDA requirements. 
 
8.4.1.2 
Reporting to Regulatory Authorities 
 
All SAEs will be reviewed for expectedness by the Principal Investigator or another investigator 
designated by the Principal Investigator. SAEs determined to be “probably” related to the 
investigational drug and that are unexpected according to the protocol will be reported in an 
expedited manner to the FDA, in accordance with 21 CFR 312.32. For unexpected drug-related 
SAEs that result in death or are considered life threatening, they will be reported to the FDA as 
soon as possible but in no case later than 7 calendar days after the sponsor's initial receipt of the 
information. For all other unexpected, drug-related SAEs, reporting to the FDA will occur no later 
than 15 days after the event. Non drug-related SAEs will be reported to the FDA on a yearly basis.  
 
8.5 
Trial Halting Rules 
 
At the end of each 67 patient dose cohort, grade II or higher AEs (and all SAEs) will be provided 
to and reviewed by the DSMB, which will then consider whether the study can continue or 
whether it needs to be stopped and/or other corrective action needs to take place. Due to a 
clerical error by the Investigational Drug Services that prepares our drug/placebo doses, 69 
patients were randomized to the first patient cohort rather than 67. Additionally, because 
enrollment happens at consent, and not all patients that consent will be randomized, we will keep 
enrolling patients until we reach 67 consented and randomized patients in each of the last two 
cohorts. 


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8.6 
Safety Oversight 
 
A DSMB composed of clinical/translational neuroscience investigators from outside of Duke (i.e. a 
DSMB+) will be convened before trial initiation and will evaluate the incidence of AEs in the 
placebo vs CN-105 treatment groups at the completion of each 67 patient cohort, as enrollment for 
the next 67 patient study cohort (and the next successive higher dose of CN-105) is beginning. 
 
The DSMB+ will be empowered to halt the study during enrollment of the next 67 patient cohort if 
the following two conditions are met during the DSMB+ review of the prior 67 patient study cohort. 
First, that there is an apparent, consistent and persistent evidence of net harm (i.e. grade II or higher 
AE’s, or SAEs, that occur at significantly increased frequency in patients who receive CN-105 vs 
placebo and which are viewed as likely resulting from CN-105 treatment rather than other factors or 
chance), and second that this net harm is perceived to overwhelm any benefit from study drug 
treatment and the overall knowledge to be obtained from the study. In this way, participants can be 
prevented from receiving higher doses of CN-105 if we determine that lower doses are consistently 
associated with an increased incidence of grade II or higher AEs. 
 
9 CLINICAL MONITORING 
Clinical site monitoring will be conducted by the on-site study team 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/amendments, with GCP, and with applicable regulatory requirements. The study team will 
perform this monitoring at least on a quarterly basis. 
 
10 STATISTICAL CONSIDERATIONS 
10.1 
Description of Statistical Methods and Analysis Plans 
 
10.1.1 
General Approach 
 
Data will be analyzed using standard analysis programs, including SAS and SPSS. R may also be 
used if necessary. 
 
10.1.2 
Safety and Feasibility Endpoints 
 
Safety Analysis: Our DSMB will examine all grade II or higher AEs and all SAEs in CN-105 and placebo treated patients. 
The expected rate of moderate and severe adverse events in this patient population is 5% each, based on historical Duke 
orthopedic surgery data; thus we expect similar AE rates in this trial in placebo- treated patients. At the end of each 67 
patient randomized group, the DSMB will examine grade II or higher AE rates in drug-treated vs placebo-treated patients, 
and consider whether the study should be stopped or whether other corrective action should take place. An independent 
statistician will prepare summary reports (including all grade II or higher AE rates in drug vs placebo treated patients) for 
review by the DSMB+ at the end of each 67 patient randomized group. Thus, the study team (and the primary study 
statistician) will thus remain blinded to patient treatment assignment throughout the course of the study. Due to a clerical 
error by the Investigational Drug Services that prepares our drug/placebo doses, 69 patients were randomized to the first 
patient cohort rather than 67. Additionally, because enrollment happens at consent, and not all patients that consent will be 
randomized, we will keep enrolling patients until we reach 67 consented and randomized patients in each of the last two 
cohorts. 
 
 


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Assuming that we continue with all three dose groups, at study completion we will have ≥117 total 
CN-105 treated patients and ≥39 placebo-treated patients. Given the aforementioned expected grade 
II or higher AE rates in placebo-treated patients, at completion we will have >80% power (with 
a=0.05) in a two- sample two-sided unpooled variance chi-square test to detect an absolute 
difference of 14.8% between grade II or higher AE rates seen in placebo vs CN-105 treated patients. 
We will also track the rates of other grade II or higher AEs recorded by the patient’s surgical team 
in the medical record; at study completion we will use 2 sided t tests to compare the rates of any 
other grade II or higher AEs between placebo vs CN-105 treated patients. 
 
Feasibility Analysis: We will report the observed rate of doses administered within the required 
time window and the 95% confidence interval. Each dose will be counted as an independent 
observation. The total number of doses correctly administered (i.e. within 90 min of the scheduled 
administration time) divided by the number of prescribed doses will define our dosing feasibility 
rate. Post-hoc feasibility analysis will be performed among all patients, as well as specifically in 
patients receiving the study drug only, and by dose level to determine if the drug itself or drug dose 
level affects feasibility. 
 
10.1.3 
Analysis of the Secondary Endpoints 
 
CSF Inflammatory Cytokine Analysis: Non-parametric 2-sided tests will be used to detect 
differences in CSF inflammatory cytokine increases from before surgery to 24 h after surgery in CN- 
105 vs placebo treated patients. For our primary analysis, we will group all three CN-105 drug dose 
groups together into one larger group of ≥117 CN-105 treated patients. In unpublished data from the 
MADCO-PC study, we observed a pre- to 24 h post-op CSF IL-6 change of 21.55 pg/ml (SD=44.72; 
log normal distribution; see Supplement Appendix Figure 2). Markov-chain Monte Carlo simulations 
based on these data demonstrate that this study (with ≥ 117 CN-105 treated and ≥ 39 placebo treated 
patients) will have >80% power (with 〈=0.05) in a 2-sided Wilcoxon Rank Sum test to detect a 34% 
smaller increase in IL-6 levels from before to 24 h after surgery in CN-105 vs placebo treated 
patients. Based on our preliminary MADCO-PC data, this sample size will also provide >80% power 
to detect a 36% smaller increase in G-CSF, a 32% smaller decrease in IL-8, and a 44% smaller 
decrease in MCP-1 levels from before surgery to 24 h after surgery in CN-105 vs placebo-treated 
patients. A post-hoc Kruskal-Wallis analysis will be used to compare the change in each of these 
inflammatory cytokines from before surgery to 24 h after surgery by CN-105 dose level. We will 
also examine CSF inflammatory cytokine levels 6 weeks +/- 3 weeks after surgery in all study 
patients to evaluate whether CN-105 treatment results in a long lasting reduction of CSF 
inflammatory cytokine levels. 
 
Cognitive Data Analysis: We will compare continuous cognitive index change scores (Newman 
MF et al, 2001) between patients who receive CN-105 vs placebo using a 2-sided t-test. Based on the 
cognitive change variance seen in MADCO-PC patients (SD=0.21), this study (with ≥117 CN-105 
treated and ≥ 39 placebo treated patients) will provide 80% power with 〈=0.05 to detect a half SD 
difference (i.e. 0.1 units) in CCI score between CN-105 vs placebo treated patients. Post-hoc 
ANOVA will determine if there are differences in cognitive index values by CN-105 dose level. We 
will also perform a chi-squared analysis (or Fisher’s exact test if needed) to determine whether the 
rate of POCD as a dichotomous outcome (defined by a 1 SD drop in any of the 4 cognitive domains 
we have previously studied- 1- verbal memory, 2- visual memory, 3- attention/concentration, and 4- 
executive function) differs between CN-105 vs placebo treated patients. 
 
Delirium analysis: Patients will be divided into those with delirium (i.e., 1 or more positive delirium 


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46 
 
 
assessments) vs those without delirium (no positive delirium assessments). We will compare 
delirium rates between CN-105 treated and placebo treated patients using a two-sided chi-square test. 
With an expected delirium rate of 40% in placebo treated patients, this study (with >117 CN-105 
treated patients, and >39 placebo treated patients) will provide 80% power to detect a 25% lower 
absolute rate of delirium in CN-105 vs placebo treated patients. Post-hoc ANOVA testing will 
evaluate for differences in delirium rates between CN-105 drug dose level groups, with post-hoc 
pairwise comparison tests as appropriate. Additional post-hoc analyses will evaluate delirium 
duration and severity between CN-105 vs placebo treated patients. 
 
 
10.2 
Sample Size 
 
We planned to enroll up to 250 patients total in this phase 2 escalating dose study, to obtain three 
successive groups of 67 randomized patients each. Because enrollment happens at consent, and not 
all patients that consent will be randomized, we will keep enrolling patients until we reach 67 
consented and randomized patients in each cohort. In each group of 67 patients, 50 will receive a 
given dose of CN-105 and 17 will receive placebo. In our recent MADCO-PC study, ~80% of 
patients returned for the 6 week assessment. Thus, enrolling 67 patients in each group ensures ≥52 
patients (i.e. 39 CN-105-treated; 13 placebo-treated) will complete the 3 month postop 
assessment.Due to a clerical error by the Investigational Drug Services that prepares our 
drug/placebo doses, 69 patients were randomized to the first patient cohort rather than 67. . There 
will be no baseline preoperative cognitive status inclusion/exclusion criteria. Our prior work 
suggests ~15% of Duke orthopedic surgery patients over age 60 have MCI, ~5% have dementia, 
and ~80% are cognitively normal. All primary analyses here will be performed on patients grouped 
together (independent of baseline cognitive status); exploratory analyses will examine effects 
stratified by baseline cognitive strata, dose level, and by APOE4 carrier status. 
 
Given the aforementioned expected 5% AE rates in placebo-treated patients, at completion we will 
have >80% power (with a=0.05) in a two-sample two-sided unpooled variance chi-square test to 
detect an absolute difference of 14.8% between grade II or higher AE rates seen in placebo vs CN-
105 treated patients (pooling the three dose levels of CN-105 together). We will also track the rates 
of other grade II or higher AEs recorded by the patient’s surgical team in the medical record; at study 
completion we will use 2-sided t tests to compare the rates of any other grade II or higher AEs 
between placebo vs CN-105 treated patients. 
 
10.3 
Measures to Minimize Bias 
 
10.3.1 
Enrollment/Randomization/Masking Procedures 
 
Participants will be randomized to receive either CN-105 or placebo after they consent to participate in the trial, within 1 
week prior to their index surgery. Participants will be enrolled in 1 of 3 dose cohorts (see Table 1) of 67 subjects each. An 
independent statistician will prepare dose stratified mixed block size randomization lists in a 3 to 1 ratio to achieve the 
desired distribution of drug and placebo. In each block of 67 trial participants, 50 will receive a given dose of CN-105 and 17 
will receive placebo, in a double-blind fashion. CN-105 will be dissolved in normal saline, and either it or normal saline 
placebo will be administered as an IV drip over 15-30 minutes. Due to a clerical error by the Investigational Drug Services 
that prepares our drug/placebo doses, 69 patients were randomized to the first patient cohort rather than 67.  Additionally, 
because enrollment happens at consent, and not all patients that consent will be randomized, we will keep enrolling patients 
until we reach 67 consented and randomized patients in each of the last two cohorts. 
CN-105 and placebo will be prepared in identical bags labelled simply with the trial participant’s 
number (1–203) to keep all trial staff and clinicians blinded to randomization. Only the 


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47 
 
 
investigational pharmacist will have the randomization list that explains which drug (CN-105 or 
placebo) each participant is to receive and what dose of CN-105 or placebo will be administered 
every 6 hours (+/- 90 minutes) starting within 1 h before the scheduled or actual start of 
anesthesia/surgery and continuing until the end of postoperative day 3 (or when the participant is 
discharged home, whichever comes first), up to a maximum of 13 doses. Each case start time will 
be defined by the study team as either the start of the surgery/procedure or the start of anesthesia. 
In order to minimize bias, investigators conducting the laboratory assays on the CSF and blood 
samples will be blinded to trial group assignment during the course of the trial. 
 
 
10.3.2 
Procedures to Ensure the Success of Blinding 
 
All personnel directly involved with study subjects (e.g., the PI, nurses, study coordinator, laboratory 
staff) will be blinded to treatment assignment (i.e. CN-105 vs placebo). All assays will be performed 
by laboratory staff blinded to treatment group/randomization. 
 
The independent study statistician will prepare a randomization assignment log/schedule for the 
study, which only he and the investigational pharmacy staff will have access to, the PI, the primary 
study statistician, the co-investigator(s), nor any other members of the study team will have access to 
the randomization assignment log/schedule. The randomization assignment log/schedule will be 
saved in a password protected file, to which only the independent study statistician and the 
investigational pharmacy staff will have access. The randomization assignment log/schedule will not 
be pulled up on a computer screen anytime the PI, primary study statistician, co-investigator(s) or 
any other members of the study team are in the room. Pharmacy personnel who will prepare the 
study drug will be un-blinded (by necessity). Pharmacy personnel will prepare infusion bags of CN- 
105 and placebo and label each bag with the subject number for whom it was intended. 
 
For DSMB reports, the primary study statistician will pull data in a blinded fashion and give it to the 
independent study statistician, who will prepare a report containing analysis of the unblinded data for 
the DSMB plus. 
 
In the event of blinding failure, we will document the exact nature of the unblinding incident and 
take it into account during study analysis. 
 
The independent statistician will share the randomization list with the primary study statistician only 
upon the completion of all 6 week postoperative follow-up visits, and only after all data has been 
double-entered into the study redcap database and checked for accuracy by both the primary study 
statistician and/or the PI (or another individual blinded to randomization and selected/designated to 
complete this task by the PI). The primary study statistician (Ms. Mary Cooter) will remain blinded 
through the duration of the study, and will be unblinded (and receive a copy of the randomization 
assignment log/schedule) only once all assays have been completed and all data have been double 
entered into the study database (with any disagreements resolved), i.e. once the final study data set is 
completed and locked. Only at this point will the primary study statistician be unblinded, in order to 
complete the final study data analysis. 
 
In the event of a suspected safety issue regarding the study drug and to ensure patient safety, the 
DSMB-Plus has the authority to unblind the study PI and the project manager at any time during the 
study if necessary. If the study continues, the primary study statistician and the rest of the study 
team will remain blinded to help ensure the integrity of data collection and analysis.   


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11 SOURCE DOCUMENTS AND ACCESS TO DATA/DOCUMENTS 
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, CRFs, hospital records, clinical 
and office charts, laboratory notes, memoranda, participant 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 participant files and records kept at the pharmacy, laboratories, and 
medico-technical departments involved in the clinical trial. Access to source data will be limited to 
members of the research team who have undergone appropriate regulatory training and who are listed 
on the IRB protocol for this study. 
To ensure that anyone who would access the patient medical record has adequate knowledge that the 
patient is participating in a clinical trial, the study coordinator will enter a note into each consented 
patient’s Duke medical record indicating that the patient has been consented into this study. 
 
12 QUALITY ASSURANCE AND QUALITY CONTROL 
The principle investigator and study team will ensure that this clinical trial is conducted according to 
written SOPS and that data are generated, documented (recorded), and reported in compliance with 
the protocol, GCP, and the applicable regulatory requirements (e.g., Good Laboratory Practices, 
Good Manufacturing Practices). 
 
The study team will provide direct access to all trial-related sites, source data/documents, and reports 
for the purpose of monitoring and auditing by local and regulatory authorities. 
 
The clinical trial personnel will receive training in maintenance of all of the following: 
1. 
Personnel signature log 
2. 
Correspondence log (e-mail and phone) 
3. 
Trial protocol and amendments signed by investigator 
Copy of IRB/EC-approval 
Copy of IRB/EC-approved ICF and amendments 
Copy of laboratory certification 
Copy of investigator’s curriculum vita 
Copy of IRB/EC membership that approved the trial and name of the chair (if 
possible) 
Copy of training and audit reports 
4. 
Updated subject enrollment/completed log 
5.  File with the identification numbers of case report forms (CRFs) corresponding to 
serum/CSF samples 
6. 
File of completed CRFs and signed ICFs 
 
Case report forms will be locked in the PI’s office, the offices of Clinical Anesthesiology Research 
Endeavors staff, or stored on Duke University Health System (DUHS) or Department of 
Anesthesiology computer servers within the Duke firewall. 


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Quality control procedures will be implemented beginning with the data entry system, and data QC 
checks that will be run on the database will be generated. Any missing data or data anomalies will be 
communicated to the site for clarification/resolution. 
 
The accuracy of data analysis will be verified by each investigator reviewing the primary data and its 
analysis. The accuracy and precision of laboratory assays will be assured with the measurement of 
appropriate controls for each assay. Investigators conducting the laboratory assays will be blinded as 
to a participant’s (and sample’s) trial arm assignment, thus eliminating the possibility of investigator 
bias while conducting these assays. 
 
13 ETHICS/PROTECTION OF HUMAN SUBJECTS 
13.1 
Ethical Standard 
 
The investigator will ensure that this trial is conducted in full conformity with regulations for the 
protection of human subjects of research codified in 45 Code of Federal Regulations [CFR] Part 46, 
21 CFR Part 50, 21 CFR Part 56, and/or the International Council on Harmonisation E6. 
 
13.2 
Institutional Review Board 
 
The protocol, ICF, recruitment materials, and all participant materials will be submitted to the IRB 
for review and approval. Approval of both the protocol and the consent form must be obtained before 
any participant is enrolled. Any amendment to the protocol will require review and approval by the 
IRB before the changes are implemented to the trial. All changes to the consent form will be IRB 
approved; a determination will be made regarding whether previously consented participants need to 
be reconsented. 
 
13.3 
Informed Consent Process 
 
13.3.1 
Consent and Other Informational Documents Provided to Participants 
 
Consent forms describing in detail the study agent, trial procedures, and risks are given to the 
participant, and written documentation of informed consent is required before administering study 
product. 
 
13.3.2 
Consent Procedures and Documentation 
 
Patients will be consented by one of the trial investigators or coordinators, all of whom are trained in 
participant enrollment. If the patient consents to participate in the trial, the trial coordinator will then 
set up an appointment for the patient to have preoperative or baseline cognitive testing. Furthermore, 
given concerns over COVID-19 transmission and safety for both research participants and study staff, 
when possible we use electronic consent (eConsent) for patients using the REDCap (Research 
Electronic Data Capture), which is a secure, web-based application designed to support data capture. 
Participants are sent the currently approved version of the consent via email, at which point the study 
staff can go over this with the patient (either in person or over a virtual platform like Zoom). If the 
consent is done using the electronic consent,  the patient provides a signature at the end (using their 
finger or a mouse/cursor), and once they accept, the ICF is saved in the REDCap database and a copy 
is emailed to the patient. Duke implemented eConsent through REDCap last year, and the files live 
behind Duke’s firewall on Duke servers.    


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Informed consent will be obtained from each participant himself or herself whenever possible. In 
those situations in which a participant is not competent to give informed consent, then the LAR will 
provide the consent on behalf of the participant. We will initially use consent of the LAR for 
participants deemed not to have medical decision-making capacity; however, should these 
participants regain medical decision-making capacity after enrollment, they will be re-consented 
before continuation of participation in trial. The participant’s capacity to consent will be assessed at 
each research visit by a qualified licensed person per DUHS policy. 
 
If a participant is able to give informed consent to participate in the trial but physically cannot sign 
the consent form (due to blindness, hand injury, or other physical impairment), the participant’s LAR 
may sign the consent form on behalf of and at the direction of the participant. In such cases, the 
participant must be able to understand the potential risks, benefits, and alternatives to trial 
participation, and all questions and concerns of the participant will be addressed. Participants or their 
LARs will sign all consent paperwork before taking part in any trial procedures or cognitive testing. 
 
13.4 
Participant and Data Confidentiality 
 
Participant confidentiality is strictly held in trust by the participating investigators, their staff, and the 
sponsor and their agents. This confidentiality is extended to cover testing of biological samples in 
addition to the clinical information relating to participants. Therefore, the trial protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the trial or the data will be released to any unauthorized third party without 
previous written approval of the sponsor. 
 
Representatives of the IRB or pharmaceutical company supplying study product may inspect all 
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 participants in this trial. The 
clinical trial site will permit access to such records. 
 
The trial participant’s contact information will be securely stored for internal use during the trial. At 
the end of the trial, all records will continue to be kept in a secure location for as long a period as 
dictated by the Duke IRB and institutional regulations. 
 
Trial participant research data, which is for purposes of statistical analysis and scientific reporting, 
will be kept in the Duke Anesthesiology department. This will not include the participant’s contact or 
identifying information. Rather, individual participants and their research data will be identified by a 
unique trial identification number. The trial data entry and trial management systems used by clinical 
sites and by research staff will be secured and password protected. At the end of the trial, all trial 
databases will be de-identified and archived in the Duke Anesthesiology Department. 
 
13.5 
Research Use of Stored Human Samples, Specimens, or Data 
 
Data collected for this trial will be analyzed and stored at the Duke Anesthesiology Clinical 
Anesthesia Research Endeavors offices or other Duke Anesthesiology Department offices, as 
appropriate. After the trial is completed, the de-identified, archived data will be stored for use by 
other researchers, including those outside of the trial. 
Permission to transmit data to this data repository will be included in the informed consent. 
 


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51 
 
 
With the participant’s approval and as approved by local IRBs, de-identified biological samples will 
be stored at Duke with the same goal as the sharing of data with collaborating investigators. These 
samples could be used for research into the causes of POCD and delirium, as well as AD, their 
complications and other conditions for which older surgical patients are at increased risk, and to 
improve treatment. Our repository will also be provided with a code-link that will allow linking the 
biological specimens with the phenotypic data from each participant, maintaining the masking of the 
identity of the participant. 
 
During the conduct of the trial, an individual participant can choose to withdraw consent to have 
biological specimens stored for future research. However, withdrawal of consent with regard to 
biosample storage will not be possible after the trial is completed. 
 
When the trial is completed, access to trial data and/or samples will be controlled by the Duke 
Neurologic Outcomes Research Group. 
 
 
14 DATA HANDLING AND RECORDKEEPING 
14.1 
Data Collection and Management Responsibilities 
 
Forms describing the collection of CSF and plasma aliquots will be completed at every sample draw. 
Case report forms will be completed at the time of enrollment and during the operative procedure. 
Data collected will incorporate variables reported in the subject’s medical record and anesthetic 
record as dictated by standard of care practices and will include, but not be limited to, imaging 
studies, neurological exam, records of any postoperative infections or antibiotic usage, 
microbiological culture data, records of any delayed wound healing or other wound healing issues, 
medications, presence of mechanical ventilation and/or tracheostomy, laboratory profiles, ECGs, and 
type of diet. 
 
14.2 
Trial Records Retention 
 
Trial documents will be retained for a minimum of 2 years after a marketing application is approved 
for the drug, or, if an application is not approved for the drug, until 2 years after shipment and 
delivery of the drug for investigational use is discontinued and FDA has been notified. Per Duke IRB 
regulations, all trial documents will be retained for 6 years after the completion of the study. 
 
14.3 
Protocol Deviations 
 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or manual of 
procedures requirements. The noncompliance may be on the part of either the participant, the 
investigator, or the trial site staff. As a result of deviations, 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. 
It is the responsibility of the study team to use continuous vigilance to identify and promptly report 


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PI: Miles Berger, MD, PhD 
52 
 
 
deviations to the IRB. All deviations must be addressed in trial source documents. The site PI/trial 
staff is responsible for knowing and adhering to their IRB requirements. 
 
14.4 
Publication and Data Sharing Policy 
 
This trial will comply with the NIH public access policy, which ensures that the public has access to 
the published results of NIH-funded research. It requires scientists to submit final peer- reviewed 
journal manuscripts that arise from NIH funds to the digital archive PubMed Central upon 
acceptance for publication. 
 
FDAAA mandates that a "responsible party" (i.e., the sponsor or designated PI) register and report 
results of certain "applicable clinical trials". Since this is an applicable clinical trial, the study team 
will register the trial with clinicaltrials.gov and will report all results as required. 
 
 
15 CONFLICT OF INTEREST POLICY 
The independence of this trial from any actual or perceived influence, such as by the pharmaceutical 
industry, is critical. Therefore, any actual conflict of interest of persons who have a role in the 
design, conduct, analysis, publication, or any aspect of this trial will be disclosed and managed. 
Furthermore, persons who have a perceived conflict of interest will be required to have such conflicts 
managed in a way that is appropriate to their participation in the trial. The trial leadership has 
established policies and procedures for all trial group members to disclose all conflicts of interest and 
will establish a mechanism for the management of all reported dualities of interest. 
 
This is a blinded study (which should reduce bias), and the study PI (Dr. Berger) himself has no COI 
with respect to this study, CN-105 itself, or AEGIS CN. Nonetheless since Duke University itself 
owns the IP on CN-105 (which was discovered and developed at Duke), Duke itself has a conflict of 
interest with respect to this CN-105 trial. Because of Duke’s conflict of interest, the study PI (Dr. 
Berger) has met with the Duke University Research Integrity Office and the Conflict of Interest 
Committee to discuss this. The Duke Research Integrity Office and this Committee have agreed that 
this study can proceed at Duke under the approval of an External Institutional Review Board and the 
monitoring of an external DSMB plus, provided that the DSMB plus is comprised of qualified 
individuals from outside Duke who themselves have no other conflicts of interest, and that the study 
is reviewed and approved by an external IRB. 
 
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