Conditions: Alzheimer's Disease
Keywords: Alzheimer's disease neuropsychological assessment Granulocyte-Macrophage Colony-Stimulating Factor Leukine
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 44
Healthy volunteers: no

Brief summary:
A medicine that is FDA-approved for bone marrow stimulation (called Leukine) will be tested for its ability to be tolerated by Alzheimer's disease patients and potentially to improve their memory.

Detailed description:
Preliminary preclinical results demonstrated that GM-CSF (Granulocyte macrophage colony-stimulating factor, e.g. Leukine®/Sargramostim) rapidly reduced cerebral amyloid deposition and completely reversed memory deficits in transgenic mouse models of Alzheimer's Disease (AD). To assess the efficacy of GM-CSF in humans, the investigators performed a retrospective analysis of a cognition study of human patients undergoing hematopoietic cell transplantation for cancer and who garner cognitive impairments from the chemotherapy or irradiation. In the patients that received a colony-stimulating factor (CSF) to stimulate the bone marrow and recover immune system function, the investigators found that those who received GM-CSF (Leukine®/Sargramostim) plus G-CSF (Filigrastim) significantly improved in cognitive function as compared to those who received G-CSF alone. These findings combined with over two decades of accrued safety data using recombinant human GM-CSF, Leukine®/Sargramostim, in elderly leukopenic patients, suggested that Leukine® should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

Arms:
arm 1: 5 subjects 250 mcg /m2/day Leukine subcutaneously for 5 days/week for three weeks. Data and Safety Monitoring Board will then review data and recommend whether to continue at the same current recommended dose for additional subjects or to reduce the dose by half if excessive leukocytosis occurs arm 2: Saline -- placebo comparator. Given as a subcutaneous injection.

Interventions: intervention 1: Sagramostim intervention 2: Saline -- placebo comparator
Intervention descriptions: intervention 1: 5 subjects 250 mcg /m2/day Leukine subcutaneously for 5 days/week for three weeks. Data and Safety Monitoring Board will then review data and recommend whether to continue at the same current recommended dose for additional subjects or to reduce the dose by half if excessive leukocytosis occurs intervention 2: subcutaneous injection

Protocol document:
Prot_SAP_000.pdf: 
 
1 
COVER PAGE 
 
 
 
 
 
 
Pilot Phase 2 Trial of the Safety & Efficacy of GM-CSF (Leukine®) 
in the Treatment of Alzheimer’s Disease 
 
 
ClinicalTrials.gov Identifier: NCT01409915  
 
 
 
Version January 23, 2020 
COMIRB # 12-1273 
 
 


 
2 
Pilot Phase 2 Trial of the Safety & Efficacy of GM-CSF (Leukine®) 
in the Treatment of Alzheimer’s Disease 
 
 
Principal Investigator:  Huntington Potter, PhD 
Co-Principal Investigator:  Jonathan Woodcock, MD 
 
 
Department of Neurology 
University of Colorado School of Medicine 
 
 
 
Version January 23, 2020 
COMIRB # 12-1273 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
3 
TABLE OF CONTENTS 
 
Introductory Statement 
 
 
 
 
 
 
 
 
3 
Specific Aims 
 
 
 
 
 
 
 
 
 
7 
Background and Research Rationale 
 
 
 
 
 
 
8 
Preliminary Preclinical Results 
 
 
 
 
 
 
 
9 
Intrahippocampal injections of colony-stimulating factors 
 
 
9 
Daily subcutaneous injection of GM-CSF 
 
 
 
 
10 
Preliminary Clinical Results 
 
 
 
 
 
 
 
12 
Experimental Design and Methods 
 
 
 
 
 
 
14 
Study Events   
 
 
 
 
 
 
 
 
 
20 
Cognitive Tests 
 
 
 
 
 
 
 
 
 
21 
Labs 
 
 
 
 
 
 
 
 
 
 
22                             
MRI Procedure 
 
 
 
 
 
 
 
 
 
22 
PET Procedure 
 
 
 
 
 
 
 
 
 
23 
Randomization and Maintenance of Blind  
 
 
 
 
 
25 
Data Safety Monitoring Plan and Protection of Human Subjects 
 
 
 
25 
Control of Conflict of Interest 
 
 
 
 
 
 
 
29 
Available Resources 
 
 
 
 
 
 
 
 
30 
References 
 
 
 
 
 
 
 
 
 
32 
Study Schedule 
 
 
 
 
 
 
 
 
 
35 
Pharmacology, Toxicology, and Previous Human Experience 
 
 
 
36 
 
Appendix A: Principal Investigator Education, Training, and Experience 
 
 
Curriculum Vitae: Huntington Potter, Ph.D. 
 
Curriculum Vitae and Medical License: Jonathan Woodcock, MD 
 
Appendix B: GM-CSF Upregulated in Rheumatoid Arthritis Reverses Cognitive 
Impairment and Amyloidosis in Alzheimer Mice 
Appendix C: Granulocyte Macrophage Colony Stimulating Factor Treatment is 
Associated with Improved Cognition in Cancer Patients 
Appendix D: Leukine® (Sargramostim) Product Monograph and full Prescribing 
Information 
Appendix E: Briefing Package – FDA Advisory Committee Meeting May 3, 2013; 
Study Drug: Leukine® (Sargramostim) 
Appendix F: Common Terminology Criteria for Adverse Events (CTCAE) 
 


 
4 
INTRODUCTORY STATEMENT  
 
Trial Background: 
GM-CSF (Granulocyte-Macrophage Colony-Simulating Factor) is a hematopoietic growth factor which 
stimulates the proliferation and differentiation of hematopoietic progenitor cells. Recombinant human 
GM-CSF, Leukine® (Sargramostim), has been FDA-approved since 1991 and safely used worldwide   
for leukopenia, in multiple stem cell transplantation settings, and following induction chemotherapy in 
patients 55 years and older with Acute Myelogenous Leukemia (AML) to shorten time to neutrophil 
recovery and to reduce the incidence of severe and life-threatening infections. Specifically,   
recombinant human GM-CSF, Leukine® is a leukocyte growth factor indicated for use following 
induction chemotherapy in AML, for use in mobilization and following transplantation of autologous 
Peripheral Blood Progenitor Cells, for use in myeloid reconstitution after autologous bone marrow 
transplantation, for use in myeloid reconstitution after allogeneic bone marrow transplantation, and for 
use in bone marrow transplantation failure or engraftment delay. Any adverse events are usually rare 
mild-to-moderate pyrogenic effects that subside upon reducing the dosage of Leukine® by half or by 
halting administration. Leukine® has not been withdrawn from investigation or marketing in any 
country for any reason related to safety or effectiveness. In fact, in a briefing package, composed by 
Genzyme Corporation in May 2013 for an FDA Advisory Committee Meeting, it states that “a large 
body of safety data exists with Leukine®, in both approved and investigational therapeutic settings, 
representing over 21 years of post-marketing experience,” that “approximately 470,000 patients have 
received Leukine treatment in the post-marketing setting from the time of product launch in March 
1991 through December 2012,” and that “the safety profile observed to date has been consistent and 
predictable across multiple indications and across special patient populations (healthy volunteers, 
pediatric and geriatric subjects)” ([1], attached as Appendix E). 
Our preliminary preclinical results demonstrated that GM-CSF rapidly reduced cerebral 
amyloid deposition and completely reversed memory deficits in transgenic mouse models of  
Alzheimer’s Disease (AD) ([2], attached as Appendix B). To assess the efficacy of GM-CSF in  
humans, we performed a retrospective analysis of a cognition study of human patients undergoing 
hematopoietic cell transplantation for cancer and who garner cognitive impairments from the 
chemotherapy or irradiation. In the patients that received a colony-stimulating factor (CSF) to 
stimulate the bone marrow and recover immune system function, we found that those who received 
GM-CSF (Leukine®/Sargramostim) plus G-CSF (Filigrastim) significantly improved in cognitive 
function as compared to those who received G-CSF alone ([3], attached as Appendix C). These 
findings combined with over two decades of accrued safety data using recombinant human GM-CSF, 
Leukine®/Sargramostim, in elderly leukopenic patients, suggested that Leukine® should be tested as a 
treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.  
 
Trial History: 
Based upon the above findings, we initiated an ongoing clinical trial using Leukine® in mild-to-
moderate AD subjects (NCT01409915), which began at the University of South Florida (USF) and 
which received an exemption from the IND regulations in October 2010.  At USF, the trial progressed 
favorably, with 14 subjects completing the trial, and there were  no incidences of any serious adverse 
events (AE’s). In July 2012, Principal Investigator, Dr. Huntington Potter, relocated to the University 
of Colorado Denver (UCD), and the trial was halted while UCD Institutional Review Board approval 
was sought. 
In July 2013, we submitted a revision to the NCT01409915 clinical trial protocol, so that a 
second clinical site, additional experimentation, and additional study subjects could be added, and the 
revised protocol received exemption from IND regulations on August 8, 2013 (Re: IND 119282, 
Reference ID: 3351332). In that revised protocol, we noted that the trial will now be based at UCD, 
and that subjects will continue to be recruited and enrolled at both the UCD and USF site. The Data 


 
5 
Safety Monitoring Board (DSMB) was also expanded to include safety oversight from individuals at 
both institutions. Additionally, our secondary endpoint of efficacy within our original 2010 protocol 
was divided into multiple separate secondary endpoints, so that our analysis plan could include details 
about how each secondary endpoint and its hypothesis will be tested, including the specific metric that 
will be compared between groups, the time point at which the comparison will be made, and any 
adjustments needed for multiple comparisons. Furthermore, in that revised protocol, we also added Dr. 
Jonathan Woodcock, MD, as a Co-Principal Investigator at the UCD site, and noted that Dr. Balebail 
Ashok Raj, MD will remain the Co-Principal Investigator at the USF site. Although we received 
exemption from IND regulations on August 8, 2013 (Re: IND 119282, Reference ID: 3351332), the 
revised protocol for the study at that time contained the usage of the Pittsburgh Compound B (11C-PiB), 
a Positron Emission Tomography (PET) radioactive diagnostic agent that is used to estimate the β-
amyloid neuritic plaque density in the brain of Alzheimer’s disease subjects. However, it was 
subsequently determined by the Colorado Multiple Institutional Review Board that the 11C-PiB 
compound was unable to be  synthesized for use in this trial, and also that Dr. Peter Smith-Jones, the 
Sub-Investigator who would have been involved in the synthesis of 11C-PiB for this trial, had 
announced his intent to resign from the University of Colorado. Therefore, we revised the protocol 
again to remove the 11C-PiB- PET imaging and to remove Dr. Peter Smith-Jones from the list of Sub-
Investigators. However, the rest of the protocol remained exactly the same as the protocol which 
received the Exemption from IND regulations on August 8, 2013. Thereafter, we resubmitted the IND 
and received Exemption again on November 15, 2013 (Re: IND 120445, Reference ID: 3407661). 
Specifically, the November 15, 2013 revised protocol proposed to treat 20 mild-to-moderate AD 
subjects with Leukine® for five days a week for three weeks and to compare their clinical and cognitive          
responses to 20 untreated mild-to-moderate AD subjects, in order to determine the safety and potential 
efficacy of Leukine® for an indication of cognitive decline. There was proposed to be 10 treatment 
subjects and 10 placebo subjects recruited at each of the UCD and USF sites. Before this amended 
protocol, there had been no serious adverse events observed in either the half or full dose groups that had 
already completed the study at the University of South Florida, and the original DSMB had authorized 
the full FDA-recommended dosage of Leukine® for subsequent subjects (i.e. 250 μg /m2/day/SC). 
Therefore the November 15, 2013 revised protocol proposed to continue at the full recommended dose 
of Leukine®. The DSMB was scheduled to review the data at the 3 week time point of the first 5 
Leukine®-treated and 5 placebo subjects, and decide whether to continue at the same dose for each 
subsequent 5 Leukine®-treated and 5 placebo subjects. Following receipt of informed consent, a medical 
history, physical exam, complete blood cell count (CBC) with differential, complete metabolic panel 
(CMP), glomerular filtration rate (GFR) test, electrocardiogram (ECG), cognitive testing (MMSE only), 
and an MRI to determine medial temporal lobe atrophy (MTA) was scheduled to occur at screening. 
Following enrollment, blood biomarker assays, CBC with differential, and cognitive testing (all cognitive 
tests) was scheduled to be performed at baseline (i.e. Day 1 of treatment phase). The CBC with 
differential was scheduled to be performed approximately every three days during the treatment phase, 
and the CMP was scheduled to be performed again once per week. Vital signs, injection site review, and 
adverse event monitoring was scheduled to occur on each treatment day. A physical exam, CBC with 
differential, CMP, biomarker assays, MRI, and all cognitive tests was scheduled to be administered at 
the end of the treatment phase. A physical exam, CMP, biomarker assays, and all cognitive tests was 
scheduled to be administered again at the first (6-Week/45 day) follow-up visit. At the second (3-
month/90 day) follow-up visit, a physical exam, CMP, biomarker assays, and all cognitive tests was 
again scheduled to be administered. Any adverse events throughout the complete study time was to be 
reported to the DSMB immediately. 
On February 12, 2016, the treatment phase for the first cohort of randomized participants at the 
UCD site (5 Leukine treated at the 250 mcg/m2/day/SQ and 5 placebo controls) was completed, in 
addition to 3 randomized participants from the USF site who had completed the trial.  The DSMB 


 
6 
reviewed the data for all of these participants and recommended the following changes to the protocol 
going forward: 
 
“1)   Based on review of the safety data reported to the DSMB, the current study dosing 
protocol does not present safety concerns and we have no recommended changes to dosing. 
2)   We recommend that the investigators establish specific criteria for terminating subjects 
from the study due to uncontrolled hypertension, in order to ensure standardized practices 
across subjects and treatment groups. 
3)  We recommend that with all scheduled DSMB data reviews in the future, the following data 
be included for review: 1) all vital sign data for each participant, 2) location of injection 
sites for each treatment and recorded outcomes from injection site review at each treatment 
visit, and 3) a tabulated list of the dates of treatment for each participant. 
4)  We request a copy at this point in time of the randomization assignment procedure currently 
being used by the University of Colorado site. 
5)  We recommend against altering the randomization procedure in any manner unless a 
protocol amendment with justification is approved by the IRB. This recommendation is 
prompted by the discovery that an investigator requested that the randomization assignment 
given to an enrolled participant who withdrew from the study be given to the next 
consecutively enrolled participant (i.e. that the same study drug be assigned to the next 
participant as had been given to the subject who withdrew). 
6)  The current protocol has no additional blood draws for complete blood count with 
differential (CBC with diff) beyond treatment day 15. We recommend a protocol change to 
include an additional CBC with diff on follow up day 90, at the same time as the day 90 
scheduled blood draw for biomarkers and comprehensive metabolic panel. We recommend 
this change for all participants currently enrolled who have not reached day 90 as of April 
7, 2016. 
7)  We recommend a correction to the protocol on page 19, to state that serious adverse events 
will be reported to the DSMB instead of all adverse events. In other locations it is specified 
that only serious adverse events will be reported to the DSMB, and we do not recommend 
that this be changed to all adverse events based on our review of the safety data.” 
 
A revised protocol came after the completion of treatment phase for the first cohort of 
randomized participants (5 Leukine®-treated at 250 μg /m2/day/SC, 5 placebo control), plus three 
participants from the University of South Florida (USF) site. The amendments within the revised 
protocol were in response to the recommendations of the April 6, 2016 Data Safety Monitoring Board 
(DSMB) Summary Report. The first DSMB Recommendation stated that “Based on review of the safety 
data reported to the DSMB, the current study dosing protocol does not present safety concerns and we 
have no recommended changes to dosing.” This very positive conclusion gave us optimism for the 
successful completion of this trial, especially considering that there were no incidences in these 
participants of Amyloid-Related Imaging Abnormalities (ARIAs, including ARIA-E indicating 
vasogenic edema, and ARIA-H indicating hemorrhage), which have plagued all of the industry 
Alzheimer’s disease (AD) clinical trials that have used antibodies against amyloid beta (Aβ) species or 
amyloid plaques.  
The other DSMB Recommendations pertained to specific suggestions for the improvement of 
the trial’s protocol. Specifically, the revised protocol removed USF as a site for this trial, due to the 
retirement of Dr. Balebail Ashok Raj, MD who was the Co-Principal Investigator at the USF site, and 
also because of the low recruitment rate at USF. We had already obtained all of the records and 
biological samples that were collected at USF during this trial, and the USF pharmacy had 
confidentially communicated only to the UCD pharmacy, who received Leukine and who received 
placebo amongst the 3 participants that completed the trial at UCD. The revised protocol also included 
new criteria for participants who presented with uncontrolled hypertension, improved scheduling of and 


 
7 
reporting to the DSMB, additional blood draws for complete blood count with differential (CBC with 
diff) on follow-up visits, and to request increased blood samples that will allow us to perform additional 
biomarker testing. The remaining amendments to the protocol pertained to clarifying and removing text 
related to the USF site, and to make other grammatical corrections (i.e. page numbers, correct tense, 
etc.). 
Because our first cohort of randomized participants did not show any evidence of ARIA, and 
because our preliminary preclinical results had demonstrated that GM-CSF rapidly reduced cerebral 
amyloid deposition in transgenic mouse models of  AD ([2], attached as Appendix B), we then needed to 
determine whether Leukine was indeed affecting amyloid load in the trial’s remaining two cohorts of 
participants (10 Leukine®-treated and 10 placebo).  We also needed to be certain that the determination 
of mild-to-moderate cognitive impairment and subsequent enrollment of potential study participants 
was due to Alzheimer’s disease with cerebral amyloid deposition, and not due to other neurological 
disease. Amyloid Positron Emission Tomography (PET) observations from recent AD phase 3 trials of 
bapineuzumab and solanezumab had illuminated that 16% to 22% of enrolled subjects diagnosed with 
mild-to-moderate AD based on clinical criteria, were misclassified and did not have evidence of 
abnormal Aβ pathology on retrospective analyses of PET imaging (Sevigny et al., Alzheimer Dis Assoc 
Discord, 2016; 30:1-7).  Therefore, we revised the protocol again to perform PET imaging at screening 
visit, as a new inclusion criterion, and at the first follow-up visit, or within a week thereof depending 
upon scheduling availability, utilizing the FDA-approved amyloid-binding PET imaging agent, 
Forbetapir F18 (Amyvid®). However, no treatment decisions were declared would be made based on 
the outcome of the follow-up PET scan. Additionally, we included another brain MRI at the first 
follow-up visit, to further assess any evidence of ARIA at that time point. Because all of the other AD 
clinical trials had shown the ability to reduce cerebral amyloid, but had also shown the very serious 
adverse effects of ARIAs, it was imperative that we determine whether Leukine was indeed affecting 
amyloid load and if so, whether it was doing so safely and without evidence of ARIAs. Thus, we 
submitted to the FDA a revised protocol using Amyvid® PET imaging and received Exemption from 
IND regulations on July 15, 2016 (IND 131238). 
 
Update from last revision and current trial amendment: 
Since the last amendment, we have completed another cohort of 10 randomized participants (i.e. 5 
Leukine®-treated at 250 μg /m2/day/SC, and 5 placebo control).  This brings the total number of 
participants to 30, since we relocated to Colorado  and who have completed the trial at the full FDA-
recommended dose of Leukine® (except for the 30th participant who still has their second follow-up 
visit remaining). Additionally to date, there still have NOT been any study-related Serious Adverse 
Events, especially NO evidence of onset of ARIA’s among any trial participants. The DSMB has again 
convened after the 30th participant finished their treatment phase, and the DSMB has since approved the 
continuation of the trial at the current dosage of Leukine®. However,  before we could resume 
enrollment after this last DSMB meeting, we received a letter from our regional representative at 
PETNET Solutions, Inc., a Siemens Medical Solutions, Inc.-owned subsidiary that is contracted with 
Eli Lilly/Avid Radiopharmaceuticals to manufacture and distribute Amyvid® in Colorado. In this letter, 
Eli Lilly stated that it had decided to terminate production of Amyvid® by the end of December, 2017 in 
4 geographical locations within the United States, which includes Denver, Colorado. Because Amyvid® 
is not available to be made elsewhere and transported to Denver, CO, and because the other two FDA-
approved PET tracers for imaging cerebral amyloid load (i.e. GE’s Vizamyl® and Piramal’s 
NeuroCeq®) are also not commercially made within Colorado, we have decided to replace the use of 
Amyvid® in this trial with 11C-PiB.  We currently make and use 11C-PiB here at UCD for another 
ongoing study (IND 131985, COMIRB # 16-2064). 
The replacement of Amyvid® with 11C-PiB, along with an update of the Conflict of Interest 
section, are the only changes that we are proposing within this amended protocol.  
 


 
8 
SPECIFIC AIMS: 
 
SPECIFIC AIM 1.  To assess tolerability and safety of recombinant human GM-CSF, Leukine®, 
in mild-to-moderate AD patients (Primary Endpoint).  
Effects of Leukine® on mobilization of white blood cells will be determined by CBC with differential at 
screening, baseline Day 1 and on days 4, 6, 9, 11 and 15 of the treatment schedule (i.e. placebo or 
Leukine® at 250 mcg /m2/day/SC for 5 days/ week for three weeks), and at the 45 and 90 day follow-up 
visits. Vital signs, injection site review, and adverse event monitoring will be performed at each 
treatment visit. Complete metabolic panels (CMP) will be performed at screen, and on days 4, 9, 15, 45, 
and 90 days after initiation of GM-CSF treatment (Table 1 below summarizes the time-table of 
procedures for the entire study).  The follow up MRI at the end of the treatment phase will provide 
information on any adverse changes in the brain, such as vasogenic edema and microhemorrhage. The 
DSMB will review all available data after each group of 5 Leukine®-treated and 5 placebo subjects have 
finished the treatment phase and determine whether the remainder of the subjects (5 Leukine®-treated 
and 5 placebo) will continue to receive the same full recommended dose (250 mcg/m2/day/SC).   
  
HYPOTHESIS 1.  Recombinant human GM-CSF, Leukine®, will be tolerated by, and safe to 
use in mild-to-moderate AD patients. 
 
SPECIFIC AIM 2.  To test the hypothesis that Leukine® treatment will improve cognition in 
mild-to-moderate AD patients (Secondary Endpoint).  
A battery of cognitive tests will be performed at baseline and at approximately 15, 45, and 90 days after 
initiation of Leukine® treatment to determine whether any AD features reverse during or after Leukine® 
treatment compared to subjects receiving placebo. Primary cognitive tests will include the Mini Mental 
State Examination (MMSE) and the ADAS-cog (Alzheimer's Disease Assessment Scale-cognitive 
subscale).  
 
HYPOTHESIS 2.  Leukine® treatment in adults with mild-to-moderate AD will improve 
cognition as measured primarily by MMSE total score and ADAS-cog total score.  
 
SPECIFIC AIM 3.  To investigate whether Leukine® treatment has an effect on Medial Temporal 
Lobe Atrophy (MTA) in mild-to-moderate AD patients (Tertiary Endpoint). 
Atrophy in the Entorhinal cortex (ERC) and Hippocampus (HPC), measured on MRI scans, predicts 
future cognitive decline and conversion to AD among individuals with Mild Cognitive Impairment 
(MCI).  Severity of MTA, assessed with MRI scans, is strongly associated with severity of medial 
temporal lobe degenerative pathology, especially the severity of neurofibrillary pathology, at autopsy.   
 
HYPOTHESIS 3.  Leukine® treatment in adults with mild-to-moderate AD will reverse or stop 
the progression of MTA. 
 
EXPLORATORY AIM 1.  To assess whether Leukine® treatment will improve specific cognitive 
domains in mild-to-moderate AD patients. 
A battery of cognitive tests will be performed at baseline Day 1 and at approximately 15, 45, and 90 
days after initiation of Leukine® treatment to determine whether any AD features reverse during or after 
Leukine® treatment compared to subjects receiving placebo. Secondary cognitive domain tests will 
include the ADCS-ADL (Alzheimer's Disease Cooperative Study Activities of Daily Living Inventory),  
Trail making tests (TRAILS) A, TRAILS B, Clinical Dementia Rating (CDR), and the Mohs 
Cancellation Task. 
 
EXPLORATORY HYPOTHESIS 1.  Leukine® treatment in adults with mild-to-moderate 
AD will improve specific cognitive domains as measured by the ADCS-ADL, TRAILS A, 


 
9 
TRAILS B, CDR, and the Mohs Cancellation Task. 
 
EXPLORATORY AIM 2.  To examine the effects of Leukine® treatment in mild-to-moderate AD 
patients on biomarkers associated with AD .  
Biomarkers from peripheral blood collection include various forms of the hallmark proteins that form 
amyloid plaques in AD (i.e. amyloid-beta [Aβ] peptides), as well as other proteins which levels are 
known to be altered in AD (hyper-phosphorylated Tau and various inflammation-associated cytokines 
and chemokines). 
 
EXPLORATORY HYPOTHESIS 2.  Leukine® treatment in adults with mild-to-moderate 
AD will induce changes in blood biomarkers toward published control levels. 
 
EXPLORATORY AIM 3. To determine if Leukine® treatment will change amyloid load in 
mild-to-moderate AD patients.  
Amyloid PET scans will be performed using Pittsburgh Compound B (11C-PiB), produced at the UCD-
Anschutz Medical Campus (AMC) cyclotron laboratory, which allows for amyloid-beta neuritic plaque 
density imaging, and will be used to test the hypothesis that Leukine® treatment of mild-to-moderate 
AD subjects reduces cerebral amyloid load. 
 
EXPLORATORY  HYPOTHESIS 3.  Leukine® treatment in adults with mild-to-moderate 
AD will change amyloid load. 
 
BACKGROUND AND RESEARCH RATIONALE: 
 
Alzheimer’s Disease (AD) is a major cause of death and disability in the elderly, affecting about 12% 
of those over age 65 and about 40-50% of those over age 85. Although treatments slowing the course of 
the disease, such as cholinesterase inhibitors or a glutamate receptor agonist are available, their benefits 
are transient, are only effective for about half of the patients that take them, and they do not attack the 
etiology of the AD pathogenic pathway. It is therefore imperative that new pharmacological 
interventions be developed that can prevent or reverse the disease and the associated cognitive decline. 
Most treatments that are currently under investigation are designed to attack the formation of the Aβ 
peptide or its polymerization into neurotoxic oligomers/fibrils. Following encouraging results in 
animals, human trials have thus far not been very successful.  Evidently a completely new approach 
may be necessary.  
 
Amyloid deposition and cognitive decline in transgenic animals, perhaps because it develops 
over a relatively short period of time and in the absence of clear neuronal cell death, is fundamentally 
different from that which leads to AD in humans, and this concern always overshadows the use of 
animal models for developing AD therapies. We therefore sought a new approach that would identify a 
new mechanism that would provide protection against AD. Our investigation is based on the 
epidemiological evidence that the vast majority of people with Rheumatoid arthritis do not tend to 
develop onset of AD. 
Rheumatoid arthritis (RA) is an autoimmune disease in which inflamed synovial tissue and 
highly vascularized pannus form and irreparably damage the cartilage and bone. In this inflammatory 
pannus, leukocyte populations are greatly expanded, perhaps as an endogenous, but ineffective attempt 
to remove the autoantibody-mediated inflammatory insult. As a result, many other proinflammatory 
factors are produced that work together in feed-forward mechanisms to further increase leukocytosis, 
cytokine/chemokine release, osteoclastogenesis, angiogenesis, and continued autoantibody production 
(rheumatoid factors and anti-citrullinated protein antibodies) [4,5]. Additionally, the adaptive immune 
system presents a Th17 phenotype within CD4+ lymphocytes, with ultimate production of interleukin 
17 (IL-17) which is then responsible for inducing much of the pro-inflammatory effects [6,7]. Further 
enhancements of leukocyte populations come from increased expression of structurally-unrelated 


 
10 
colony-stimulating factors (CSFs): M-CSF (macrophage), G-CSF (granulocyte), and GM-CSF 
(granulocyte-macrophage) [8-11]. 
While it has been commonly assumed that RA patients’ usage of non-steroidal anti-
inflammatory drugs (NSAIDs) was protective against the onset and progression of AD, clinical trials 
with NSAIDs have proven unsuccessful in AD patients. We therefore reversed the approach and 
concluded that intrinsic factors within RA pathogenesis itself may underlie RA’s protective effect. 
When, we examined the RA literature for evidence for circulating molecules that might enter or affect 
the brain and interfere with the Alzheimer pathogenic pathway, we were particularly struck by reports 
that several colony stimulating factors that induce phagocytic and other immune cells are released 
during RA. Therefore, we investigated the activity of these colony-stimulating factors on the pathology 
and behavior of transgenic AD mice ([2], attached as Appendix B).  
To investigate the interplay of the innate immune system and AD, we studied the effects on AD 
pathology of the three hematopoietic colony-stimulating factors (macrophage, granulocyte, and 
granulocyte-macrophage colony-stimulating factors; M-CSF, G-CSF, and GM-CSF), which are 
up-regulated during RA pathogenesis. Those CSFs enhance the survival and function of their respective 
leukocytes and drive their proliferation and differentiation from myeloid lineage precursors. GM-CSF 
induces dendritic cells, macrophages, and granulocytes (neutrophils, basophils, and eosinophils), while 
M-CSF and G-CSF respectively induce the macrophage and granulocyte subsets of the innate immune 
system. These innate immune cells have the ability to diapedese from the circulatory system and to 
differentiate further into various specialized immune cells within organs (microglia, Langerhan’s cells, 
etc.). GM-CSF and G-CSF are also known to also be involved in erythropoiesis, and GM-CSF and 
erythropoietin act synergistically in the maturation and proliferation of the burst-forming and colony-
forming erythroid units to the normoblast stage of erythropoiesis [12]. Moreover, circulating Aβ42, the 
β-sheet-prone misfolding pathogenic peptide that primarily comprises cerebral amyloid plaques in AD 
[13], binds to complement opsonin C3b in an antibody-independent fashion, and C3b-opsonized 
particles bind to the complement receptor, CR1, on erythrocytes and to CR1g on liver-resident kupfer 
macrophages, in classical immune adherence mechanisms that are key steps in the removal from the 
bloodstream of pathogens and foreign proteins, and wherein complement-opsonized pathogens and 
proteins bound to erythrocyte CR1 are stripped off and degraded by sessile macrophages in the liver 
[14,15]. Thus GM-CSF could potentially function in both, the peripheral clearance of toxic Aβ and in 
bone marrow-derived microglial activity, because it is involved in erythrocyte proliferation  and also in 
the proliferation, differentiation, and maintenance of most innate immune system leukocytes.  
 
PRELIMINARY PRECLINICAL RESULTS: 
  
Intrahippocampal injections of colony-stimulating factors:  
In the first experiment, 5µg bolus injections of M-CSF, G-CSF, and GM-CSF were injected into the 
hippocampus of the ipsilateral brain hemisphere with vehicle, artificial cerebrospinal fluid (aCSF), 
injected into the contralateral hippocampus as a control in aged cognitively-impaired transgenic AD 
(PS/APP) mice [2]. The mice were then sacrificed one week later and analyzed. While M-CSF injection 
resulted in significant hyperplasia to the treatment hemisphere and no effect on amyloid deposition, 
G-CSF showed a modest reduction in amyloid deposition in the injected side. This outcome was 
confirmed by colleagues using daily peripheral G-CSF injections, which also led to reduced 
amyloidosis and cognitive deficits in the Radial Arm and Morris Water Mazes [16]. In contrast to the 
mild G-CSF effect, our GM-CSF injections, demonstrated pronounced decreases in amyloid deposition, 
as compared to control hemispheres (Fig 1a). Anterior to posterior quantification of amyloid plaques 
revealed significant reductions within individual mice and overall significant reductions for all plaque 
parameters measured (Fig 1b). 
 


 
11 
Figure 1. Intrahippocampal injection of GM-CSF on left and artificial cerebrospinal fluid (aCSF) 
on right.  (a) Representative coronal tissue cryo-sectioned at 14 µm and stained with MabTech 
α-Aβ/Alexa 546. Image is a montage of about 145 pictures taken at 10X. (b) Overall plaque reductions 
seen in all 4 plaque parameters measured from 5 quantified sections per mouse (n = 4 mice). Area and 
Perimeter data were calculated from the total number of plaque values in each hemisphere per section, 
and Feret Diameter and Integrated Density were calculated from the average values of the plaques 
measured in each hemisphere per section. (Area: p < 1.11E-07; Perimeter: p < 1.41E-06; Feret 
Diameter: p < 2.36E-09; Integrated Density: p < 1.11E-07) 
 
 
Daily subcutaneous injection of GM-CSF: 
Based upon the remarkably-positive results of GM-CSF from only 7 days after intrahippocampal 
injections, we next investigated the effect of subcutaneous GM-CSF injection on AD pathology and 
cognitive function. Prior to GM-CSF treatment, both APPsw transgenic mice (Tg) were first confirmed 
by RAWM testing to be cognitively-impaired for working memory. Both, the non-transgenic control 
mice (NT) and the Tg mice were then sub-divided into two cognitively-balanced groups. Twenty days 
of daily subcutaneous injection of 5µg of GM-CSF (the most amyloid-reducing CSF in the bolus 
experiment) were administered to one of the cognitively balanced cohorts of AD mice. RAWM testing 
post-GM-CSF treatment re-confirmed that Tg control mice were substantially impaired compared to 
NT control mice. This impairment was evident in individual blocks of testing, as well as over all 4 days 
of testing (Fig 2a). In sharp contrast, GM-CSF-treated Tg mice now performed equally well or better 
than NT control mice, during individual blocks and overall. Even the GM-CSF-treated NT mice 
performed as well as, or slightly better than, NT controls (Fig 2a). An interference cognitive test that 
assessed the ability of the mice to switch from one water maze to another with different cues, also 
showed that GM-CSF restored normal cognitive function to the APP and PS/APP animals (not shown). 
Subsequent histological analysis of brains from the APPsw mice of this study revealed that GM-CSF 
treatment induced large reductions in amyloid burdens within Entorhinal cortex (↓55%) and the 
Hippocampus (↓57%) as compared to control Tg mice (Fig 2b). 
 


 
12 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2. Behavioral and pathology analysis following daily subcutaneous GM-CSF injections. 
(a) Tg control mice (n = 8) show substantial impairment on working memory trials T4 and T5 of 
the Radial Arm Water Maze compared to NT control mice (n = 8) in individual blocks of testing 
(upper), and over all 4 days of testing (lower). GM-CSF-treated Tg mice (n = 7) performed as well 
as or better than NT control mice on working memory trials T4 and T5 during individual blocks 
and over all. (b) Percent of amyloid burden from the average of five 5-µm sections (150-µm apart) 
through both anatomic regions of interest (hippocampus and entorhinal cortex) per mouse of GM-
CSF-treated (n = 5) versus saline- treated (n = 6).  (c) Percent of Iba1 burden from the average of 
five 5-µm sections (150 µm apart) through both anatomic regions of interest (H and EC) per mouse 
of GM-CSF-treated (n = 5) versus saline-treated (n = 6). 
 
The improved cognitive function and reduced cortical amyloidosis of GM-CSF-treated Tg mice were 
paralleled by increased microglial density as compared to saline-treated Tg mice (Fig 2c), implying an 
augmented ability to bind and remove amyloid deposition. Similarly, the GM-CSF-treated Tg mice 
demonstrated increased synaptophysin immunoreactivity in both CA1 and CA3 regions (Fig 3), 
indicating increased synaptic density in these hippocampal areas.   
  


 
13 
 
 
Figure 3. Synaptophysin immunostaining in subcutaneous 
GM-CSF-injected mice. Percent of synaptophysin 
immunoreactivity from the average of 5 sections per mouse of 
GM-CSF-treated (n = 5) versus saline control-treated (n = 6). 
(CA1(p  < 0.0013), CA3(p  < 0.0023). 
 
 
Although our experimental mouse models did not allow for 
examination of other mechanistic effects from GM-CSF 
treatment, that may also help explain GM-CSF’s reversal of cognitive impairment, GM-CSF has been 
shown in other studies to induce the proliferation and differentiation of neural stem cells, which are 
known to migrate to areas of damage in the brain [17,18].  It is also known from several stroke models 
that GM-CSF increases cerebral angiogenesis and is neuroprotective in the neurons and 
oligodendrocytes within the penumbras surrounding infarcts [19-22].  Neurogenesis and angiogenesis 
are known to occur together, with some of the same repulsive and attractive neuronal guidance factors 
that direct axonal growth, also directing migration of endothelial cells [23].  Because the Aβ peptide 
itself is anti-angiogenic [24], it is imperative that the amyloid plaques must be removed before any 
neovascularization and neurogenesis/synaptogenesis can occur. Studies have shown that truncated and 
occluded microvasculature surround amyloid plaques [25], and that cerebral amyloid angiopathy is 
found in almost all Alzheimer’s disease patients [26].  Immunotherapy strategies that have been used to 
remove cerebral amyloid have all failed thus far in large randomized clinical trials, and they have also 
all been plagued with the induction of vasogenic edema and microhemorrhage [27,28]. These serious 
adverse events can be logically explained by the fact that these immunotherapies prevent the induction 
of bone marrow-derived leukocytes, and thus do not induce the conditions for neovascularization, 
which is necessary while simultaneously removing the amyloid and vascular-deposited Aβ. The 
mobilization from the bone marrow of peripheral blood progenitor cells is a critical step for any 
angiogenesis to occur, due to the production of endothelial progenitor cells [29], immature dendritic 
cells, myeloid-derived suppressor cells, and monocytes [30].  Recombinant human GM-CSF 
(Leukine®) has been FDA-approved and safely used over the last 20 years as a peripheral blood 
progenitor cell mobilizer, and a ongoing Phase II trial (NCT01041417) of GM-CSF in subjects with 
peripheral arterial disease (PAD) has already produced results showing it to be safe and that it is 
“associated with mobilization of progenitor cells, improvement of endothelial dysfunction, and exercise 
capacity” [31].  Since our peripheral administration of GM-CSF rapidly removed amyloid, while also 
increasing synaptic area and microglial density, we suspect that indeed neovascularization did occur. 
Furthermore, a subsequent study has found that targeted ablation of GM-CSF signaling in the 
hippocampus and amygdala results in spatial and fear memory deficits, and that the mice, carrying a 
null allele of the GM-CSF gene, have hippocampal neurons with significantly less dendritic tree 
arborization and reductions in spine densities and mature spines [32].  Combined, these pre-clinical 
(mouse) findings and ongoing PAD clinical trial suggest that improving cognition might be a new 
indication and function for GM-CSF.   
 
PRELIMINARY CLINICAL RESULTS: 
 
Moffitt Cancer Center:   
Together with Dr. Heather Jim of the Moffitt Cancer Center, we performed a retrospecitive 
analysis of the data that was gathered from a study assessing cognition in Bone Marrow 
Transplant patients, who acquire cognitive deficits from the chemotherapy or irradiation 
procedures ([3], attached as Appendix C;[33]).  Their cognitive function was measured 


 
14 
before treatments and then 6 months and 12 months after transplant and associated 
treatment, with a combination of either GM-CSF plus G-CSF or G-CSF alone.  The 
neurological measures used included some of the cognitive measures that are used routinely 
to assess AD patients.   
 
Total neuropsychological performance z scores (TNP) were calculated by summarizing the 
cognitive domains of memory, executive functioning (i.e., complex cognition), and 
attention. Scores indicate change in TNP from pre-transplant baseline. Kruskal-Wallis one-
way analyses of variance were conducted at six months and 12 months after baseline 
assessment and hematopoietic cell transplantation (HCT), using all available data to 
compare between-group changes in TNP by receipt of GM-CSF.  Wilcoxon signed rank 
tests were conducted using all available data to examine within-group changes in TNP by 
receipt of GM-CSF. Despite a high level of education (average of 13.89 years), the subjects 
displayed a statistically significant cognitive deficit at baseline.   
 
The preliminary results show that patients who received GM-CSF plus G-CSF improved 
their cognitive functions (neuro minus motor features) between base line and six months, 
whereas subjects receiving only G-CSF did not improve (Figure 4 below). This result 
validates in humans that GM-CSF can improve cognition in an at-risk population using the 
standard recommended, FDA-approved dosage that we propose to also use in this protocol. 
This human study also showed that a small number (less than 20) of patients only receiving 
part GM-CSF at the standard FDA-approved dose was sufficient to reveal that GM-CSF led 
to improved cognitive measures 6 months after HCT and CSF treatment, compared to the 
much larger group receiving G-CSF alone.  
 
University of South Florida:   
Before initiation of the current trial at the UCD site,  fourteen participants enrolled in a 
preliminary study at USF.  Of these 14 participants, 9 completed the entire study, including 
both follow-up visits; and the other 5 completed all treatment visits and at least one of the 
follow-up visits.  Also, 3 of the 14 participants may have received the full Leukine® dose, 
but this number cannot be definite due to blinding of the study team at USF.  Finally, the 
only AE’s reported include injection site reactions seen in 6 patients (redness, raised, 
itching, bruising).  No serious AE’s were reported in these 14 participants.   
 
The current trial (COMIRB #12-1273) was originally approved as a multi-site trial with 
recruitment efforts again, at USF, in Tampa, FL.  USF’s recruitment yielded 5 subjects who 
were enrolled into the current trial.  Two of these subjects were screen failures and three 
completed the trial at USF.  USF’s last subject officially completed the trial on June 12th, 
2015.  However, due to USF’s low recruitment, the decision was made to amend the 
protocol and close USF as a site.  This amendment was approved by COMIRB on July 25th, 
2016.  Since the closure of the USF site, the research team at UCD has received all pertinent 
study data and study samples via secure HIPAA compliant practices.   
 
 
 


 
15 
 
 
Figure 4.  Total Neuropsychological Performance in Hematopoietic Cell Transplant (HCT) 
Recipients Receiving GM-CSF+G-CSF versus G-CSF Only. 
The GM-CSF+GCSF group performed significantly better at 6 months post-HCT (p=.04), but there 
were no group differences at 12 months post-HCT (p=.24). The GM-CSF+G-CSF group improved 
significantly from baseline to 6 months post-HCT (p=.01) and from baseline to 12 months post-HCT 
(p<.01). The G-CSF group demonstrated no change from baseline to 6 months post-HCT (p=.33) but 
significant improvement from baseline to 12 months post-HCT (p<.01). Note: No patients received 
GM-CSF only. (GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor; G-CSF: Granulocyte 
Colony-Stimulating Factor) 
 
 
EXPERIMENTAL DESIGN AND METHODS:  
 
Recruitment:  
For recruitment at the UCD site, this pilot clinical trial of Leukine® in AD will utilize subjects with 
mild-to-moderate AD who are patients in the Memory and Dementia Clinic at the University of 
Colorado Hospital (UCH), of which the Co-Principal Investigator, Dr. Woodcock is the Clinical 
Director.  Subjects will also be recruited from the Community. For recruitment at the USF site, this pilot 
clinical trial of GM-CSF utilized subjects with mild-to-moderate AD from the USF Byrd Alzheimer 
Institute Clinic patients and from the Tampa Bay Community. Recombinant human GM-CSF is 
commercially available from Sanofi/Genzyme Corporation as a recombinant molecule, i.e. 
Leukine®/Sargramostim, which is FDA-approved for use in elderly patients (> 55 years old) and has an 
outstanding safety record. To recruit from the community we will utilize study flyers posted at 
Anschutz Medical Campus, Clinicaltrials.gov and the UCD Clinical Trials Website. The consent 
process will emphasize the voluntary nature of the research study and the fact that declining 
participation will not alter clinical care.  As help towards travel costs, patients will be informed that 
they will be receiving a $75.00 gift card at the end of the three week treatment period even if they 
cannot or choose not to complete the study, and will receive a $25.00 gift card after each of the follow 
up visits for a total of $125.00.  
 
 


 
16 
Inclusion/exclusion criteria: 
Subjects must be  
1. age 55 to 85 years; 
2. should have a mild-to-moderate AD diagnosis (MMSE 10-26 inclusive); 
3. should have qualitative evidence of elevated cortical amyloid by PET imaging. 
4. if on anti-dementia treatment should be on stable treatment for at least 2 months (i.e. 
cholinesterase inhibitor and/or Memantine or Axona); 
5. stable on all other medications for at least 30 days prior to screen; 
6. should be fluent in English;  
7. should be physically able to participate by medical history, clinical exam and tests;  
8. should have a study partner to accompany them to scheduled visits. 
Exclusion criteria are  
1. clinically relevant arrhythmias or uncontrolled hypertension;  
2. a resting pulse less than 50; 
3. active cancer other than non-melanoma skin cancers; 
4. use of another investigatory drug within 2 months of screening;  
5. significant stroke or head trauma by history or MRI; 
6. Contraindication for having a MRI; 
7. DSM-IV criteria for a current major psychiatric disorder;  
8. sensitivity to yeast or yeast products; 
9. impaired kidney function as measured by a Glomerular Filtration Rate less than 60Ml/min; 
10. preexisting fluid retention, pulmonary infiltrates, or congestive heart failure; 
11. history of moderate-to-severe lung disease; 
12. history of moderate-to-severe liver disease; 
13. pregnant women, any women who feel they are likely to become pregnant during the study; 
14. and prisoners 
 
Leukine safety information and criteria for removal of trial subjects for cardiovascular events: 
Leukine® (sargramostim) is indicated for the following uses: (i) following induction 
chemotherapy in older adult patients with acute myelogenous leukemia (AML) to shorten time 
to neutrophil recovery; (ii) for mobilization and following transplantation of autologous 
peripheral blood progenitor cells; (iii) for myeloid reconstitution after autologous or allogeneic 
bone marrow transplantation (BMT); (iv) for use in bone marrow transplantation failure or 
engraftment delay. Sanofi summarizes the studies on their website that led to FDA-approval 
for these indications. Of these indications, only two list reports of Cardiovascular System 
events:  
 
For AML (http://www.leukine.com/hcp-leukine-in-aml), Leukine  is indicated for use 
following induction chemotherapy in older adult patients with acute myelogenous 
leukemia (AML) to shorten time to neutrophil recovery and to reduce the incidence of 
severe and life-threatening infections and infections resulting in death. The safety and 
efficacy of Leukine have not been assessed in patients with AML under 55 years of 
age. In a Phase III clinical trail, the safety and efficacy of Leukine® in the treatment of 
AML were evaluated in a multi-center, randomized, double-blind placebo-controlled 
trial of 99 newly diagnosed adult patients, 55–70 years of age, receiving induction with 
or without consolidation (52 Leukine®, 47 placebo). The percent of AML patients 
reporting cardiovascular system events of hypertension were 25% of the  Leukine® 
group and 32% of the placebo group. Other cardiovascular system events reported in 


 
17 
each group include hypotension (13% Leukine®, 26% placebo), cardiac (23% 
Leukine®, 32% placebo), and hemorrhage (29% Leukine®, 43% placebo). 
 
For the indication of Leukine® in the acceleration of myeloid recovery in patients 
undergoing allogeneic BMT from HLA-matched related donors 
(http://www.leukine.com/hcp-leukine-in-allogenic-bone-marrow-transplantation), 
Leukine® was found to be safe and effective in accelerating myeloid engraftment, 
reducing the incidence of bacteremia and other culture positive infections, and 
shortening the median duration of hospitalization. In a multi-center, randomized, 
placebo-controlled, and double-blinded study,  a total of 109 patients (53 Leukine®, 56 
placebo) were enrolled to evaluate the safety and efficacy of Leukine® for promoting 
hematopoietic reconstitution following allogeneic BMT. Hypertension was reported in  
18 subjects receiving Leukine® (34%), and in 18 of the placebo subjects (32%).  
 
On Sanofi’s website under Important Safety Information for Leukine® (sargramostim), it is 
stated that “Leukine® should be used with caution and monitored in patients with preexisting 
fluid retention, pulmonary infiltrates, or congestive heart failure, respiratory symptoms or 
disease; cardiac symptoms or disease; and renal or hepatic dysfunction.” Sanofi’s website 
states specifically for Cardiovascular Symptom that “Occasional transient supraventricular 
arrhythmia has been reported in uncontrolled studies during Leukine® administration, 
particularly in patients with a previous history of cardiac arrhythmia. However, these 
arrhythmias have been reversible after discontinuation of Leukine®. Leukine® should be used 
with caution in patients with preexisting cardiac disease.”  
 
Due to safety concerns regarding the potential of cardiac symptoms or disease, the Co-
Principal Investigator and/or the DSMB may determine that a trial subject should no longer 
remain in the trial if there is evidence of uncontrolled hypertension. For an individual who has 
consented to volunteer for the trial, meets inclusion criteria and is enrolled, then begins the 
treatment phase of the trial, but who subsequently is determined to exhibit uncontrolled 
hypertension, the DSMB will be immediately notified, adverse events reporting protocols will 
be followed, and the following criteria will be used to determine whether the subject needs to 
be removed from the trial: 
 
Inclusion Screening Criteria for hypertension: 
• Should the potential subject have a screening blood pressure (BP) of more than 150 
systolic or 95 diastolic, they will need to be under the care of a primary care 
provider for management of hypertension. 
• The BP will have been well-controlled on a stable regimen for at least two months 
prior to study entry. 
• The screening BP will be no greater than 160 systolic or 95 diastolic.  (This limit 
will apply to all and any BP measurements during the screening process.) 
 
Management Criteria during the treatment phase of the study: 
• Should blood pressure rise above 165 systolic or 95 diastolic, the patient will be 
removed from the study. 


 
18 
• The subject’s hypertensive regimen may be adjusted by the primary care provider 
during the study as needed. 
 
Sample size considerations:  
Due to the well documented safety history of Leukine® ([1], attached as Appendix D) combined with 
additional patient information from the USF site, we believe that a sample size of 20 patients per 
research arm is adequate to assess our Primary Endpoint of measuring safety. Although the Moffitt 
BMT study was not specifically designed to distinguish the cognition of patients receiving GM-CSF 
plus G-CSF from those receiving G-CSF alone, it was possible to glean conclusions about the cognitive 
benefit of GM-CSF or G-CSF treatment. The study found highly significant improvement (p<0.01) in 
the scores of 19 patients receiving GM-CSF plus G-CSF, compared to patients receiving only G-CSF.  
 
A Power analysis showed the following results: 
 
Because the effect size of cognition improvement was so high with the BMT patients, that analysis 
provides clinical information as a reference to choose an appropriate sample size of AD subjects to 
receive GM-CSF as a potential cognition enhancer (one of our Secondary Endpoints).  Essentially, only 
3-4 subjects are needed for a power of 0.6. Therefore, the November 15, 2013 protocol proposed to 
recruit a total of 40 mild-to-moderate AD patients of either sex in a placebo-controlled protocol, with 20 
subjects (10 Leukine®-treated and 10 placebo) recruited at the UCD site and 20 subjects (10 Leukine®-
treated and 10 placebo) recruited at the USF site. However, with the revisions from the April 6, 2016 
DSMB report, the revised protocol also removes active recruiting from the USF site, due to the 
retirement of Dr. Ashok Raj, and the remaining trial participants will be recruited at the UCD site.  
The study will have two arms with subjects assigned in permuted blocks examining placebo 
and full recommended Leukine® dose. As mentioned above, in the trial subjects who have thus far 
received Leukine® at the University of South Florida, there were no serious adverse events reported by 
patients receiving either half dose or full dose Leukine® using the same methods proposed by this 
protocol.  Therefore we will be starting patients at the full FDA-recommended Leukine® dose 
(250mcg/m2/day SC). To assure that safety concerns of treating a novel population (AD patients) with 
GM-CSF are addressed, initially 5 subjects will be treated for 5 days/week for 3 weeks with Leukine® 
and 5 participants will be given placebo, followed by a DSMB review of the CBC+diff blood data, the 
MRI data, and the physical exam and adverse event monitoring at the end of the treatment phase. The 
DSMB will then recommend whether the dosage should be continued for the subsequent 5 Leukine® 
treatment subjects and 5 placebo controls or whether the Leukine® should be adjusted to a half dose.  
Recruitment and treatment initiation will be timed to allow for the DSMB interim assessments before 
further patients are treated, thus assuring maximum early knowledge of any safety concerns. Up to an 
additional 30 patients will be recruited at each study site, assuming that some screen failures and 
dropouts will occur, which will still allow us to treat 20 subjects with Leukine® and 20 with placebo, 
combined for a total of 40 mild-to-moderate AD subjects enrolled from both the UCD and the USF 
sites.  
  
Data Analysis Plan:  
Primary Endpoint: Generally, descriptive statistics will be utilized to examine safety of 
participants who received Leukine® injections compared to placebo injections. Safety will be assessed 
by measuring the toxicity grades of AE’s.  These toxicity grades have been standardized by the 


 
19 
National Cancer Institute (NCI) Common Toxicity Criteria for Adverse Events (CTCAE Version 4.03 
attached as Appendix E).  Differences in toxicity grades will be compared between treatments using a 
Wilcoxon test for ordered outcome.   
Secondary Endpoint and Exploratory Aim 1:  Neuropsychological assessments will be graded 
and scored using the standardized scoring methods associated with each of the neuropsychological 
tests.  Our primary measures for cognitive assessments will be based on the participants’ MMSE total 
score and ADAS-cog total scores.  All other neuropsychometry tests will be used secondarily to 
examine changes in specific cognitive domains.  Due to the multitude of both the neuropsychometry 
tests and the different time points these tests are administered, a Bonferroi correction method will be 
used to counteract the problem of multiple comparisons. 
Tertiary Endpoint:  Medial Temporal Lobe Atrophy (MTA) will be assessed using an 
automated rating system to grade the severity of atrophy in the entire medial temporal region will be 
employed using the FreeSurfer software automated rating system of individual medial temporal 
structures, i.e., the Hippocampus (HPC), Entorhinal cortex (ERC) and Perirhinal cortex (PRC), which 
are specific regions of interest (ROI) in AD pathogenesis.  FreeSurfer automatically computes cortical 
reconstruction and volumetric segmentation, including segmentation of the subcortical white matter and 
deep gray matter volumetric structures [34] and parcellation of the cortical surface [35] according to a 
previously published parcellation scheme [36]. This labels cortical sulci and gyri, and thickness values 
are calculated in the ROIs. 
Group comparisons of changes in regional volumes and thicknesses will be analyzed using a 
series of one-way analyses of variance (ANOVAs). The Scheffé post hoc procedure will be used to 
examine differences between means; Pearson product–moment correlation coefficients evaluated the 
strength of relationships between of changes in regional volumes and thicknesses and cognitive 
measures. Receiver operator curve analyses will determine sensitivity and specificity of various MTA 
cut points for distinguishing between diagnostic groups. Hazard ratios (HRs) for specific predictors of 
MTA progression and regression will be calculated using a three-state Markov model in continuous 
time. The proportional intensity model will be used to analyze effects of covariates on HRs.  
Exploratory Aim 2:  Levels of AD-associated biomarkers in blood will be compared between 
placebo controls and Leukine® treatment groups.  The identification of circulating biomarkers in AD is 
a relatively new field, with inconsistent biomarker level results reported as increased, decreased, or 
unchanged between studies [37].  These mixed results mean that standardized levels for AD-associated 
circulating biomarkers have yet to be established.  Therefore, we will compare observed biomarker 
levels from our project to other published levels to explore whether any similarities are observed.  This 
comparison, as well as our own results, will add to the AD-associated blood biomarker field of 
literature.  
 
Exploratory Aim 3:  Amyloid PET scans will be performed using Pittsburgh Compound B 
(11C-PiB), produced at UCD-AMC, which allows for amyloid-beta neuritic plaque density imaging, and 
will be used to test the hypothesis that Leukine® treatment of mild-to-moderate AD subjects reduces 
cerebral amyloid load. 11C-PiB PET scans will be performed at screening and at the first follow-up visit 
(+/- 7 days depending upon scheduling availability). For quantification of the brain amyloid load, 
assessments will evaluate the screening 11C-PiB scan before the baseline visit and another 11C-PiB scan 
within +/- 7 days of the first follow-up visit after the end of treatment with Leukine® or placebo. The 
change from the screening 11C-PiB scan’s SUVR to follow-up SUVR will be analyzed by a contracted 
central reading facility (i.e., BioClinica, a specialty clinical trial services provider) using the subject's 
MRI to reduce white matter contamination, thereby increasing the SUVR signal change by eliminating 
counts in the region that do not change (white matter). The 11C-PiB PET scans and accompanying MRI 
scans will be stripped of any personal identifying information, containing only the trial’s randomized 
subject ID number and date.  Data will be compared using parametric (SPM) and non-parametric 
(SnPM) statistical methods with family-wise error (FWE) and false discovery rate (FDR) corrections.  
Comparisons of  SUVRs will be made between the first and second PET for each individual study 
participant.  These differences will then be compared between placebo and treatments groups to 


 
20 
determine if Leukine® treatment changes amyloid load. The results of the 11C-PiB PET quantitative 
analyses will not be used to make any treatment decisions. 
 
Important Note: Full-dose data from the two sites will be combined and analyzed together for 
both safety and cognitive outcomes.  Also, other statistical tests that may be utilized include: 
independent t-tests to examine differences between means; the Chi-square test to assess 
association between variables; and logistical regression to calculate the ratio of the odds of an 
event occurring in one group compared to another group. 
 
Dosage:  
Recombinant human GM-CSF (Leukine®) is currently marketed in the US by Genzyme Corporation, a 
subsidiary of Sanofi.  Leukine® has been FDA-approved since 1991 and safely used worldwide for 
leukopenia, in multiple stem cell transplantation settings, and following induction chemotherapy in 
patients 55 years and older with Acute Myelogenous Leukemia (AML) to shorten time to neutrophil 
recovery and to reduce the incidence of severe and life-threatening infections.  Specifically, Leukine® is 
a leukocyte growth factor indicated for use following induction chemotherapy in AML, for use in 
mobilization and following transplantation of autologous Peripheral Blood Progenitor Cells, for use in 
myeloid reconstitution after autologous bone marrow transplantation, for use in myeloid reconstitution 
after allogeneic bone marrow transplantation, and for use in bone marrow transplantation failure or 
engraftment delay. ([38], attached as Appendix D). The FDA-approved recommended dosage is 250 
mcg/m2/day SC for up to 42 days until absolute neutrophil count (ANC) reaches 1500 cells/mm3 for 
three consecutive days.  Although it is not possible to directly translate the dosage used in mice to that 
to be used in humans for cognitive enhancement, the dosage of GM-CSF administered to transgenic AD 
mice which resulted in cognitive improvement and amyloid plaque reduction was about twice the 
recommended dosage of Leukine® (~500 mcg/m2/day SC or ~167 mcg/kg/day) for a total of 20 
injections (cumulative amount per mouse = 100 mcg).  However, several clinical trials with Leukine® 
have shown no serious adverse events at a dosages of  >500 mcg/m2/day [39-41], and the maximum 
amount of Leukine® that can safely by administered by single or multiple dose has yet to be determined. 
Doses up to 100 mcg/kg/day (4000 mcg/m2/day or 16 times the recommended dose) were administered 
to 4 patients in a Phase I uncontrolled study by continuous intravenous infusion for 7 to 18 days and 
with WBC reaching up to 200,000 cells/mm3. All adverse events (dyspnea, malaise, nausea, fever, rash, 
sinus tachycardia, headache and chills) were reversible upon discontinuation of the drug, as discussed 
above ([38], attached as Appendix D). These and other results, from the approximately 470,000 
subjects that have received Leukine® during its post-marketing setting ([1]; attached as Appendix E), 
indicate that Leukine® has an extremely good safety profile.   
More importantly, the Moffitt Cancer Center study, shown in the Preliminary Clinical Results 
section above, used the recommended FDA-approved dosage (250 mcg/m2/day) and found highly 
significant (p<0.01) cognitive enhancement in patients treated with GM-CSF plus G-CSF compared to 
G-CSF alone ([3], attached as Appendix C).  This allowed us to calculate the Power analysis presented 
above.   
Finally, as mentioned, in our current Leukine® Safety Trial at the University of South Florida 
there were no serious adverse events reported by patients receiving either half dose or full dose 
Leukine® and using the same methods proposed by this study.  Therefore we will be starting patients at 
the full FDA-approved recommended Leukine® dose. Nonetheless because of safety concerns, the go-
slow approach of the proposed trial of assessing the subjects regularly and providing interim DSMB 
analyses (after each 10 subjects: 5 Leukine®-treated and 5 placebo) will allow us to determine quickly if 
there are any unexpected toxic effects and for the DSMB to halt the trial as per protocol. Each subject’s 
first injection will be administered at the time of baseline (randomization) visit in the Clinical and 
Translational Research Center (CTRC) Outpatient Clinic on the 3rd floor of the UCH Leprino Building 
for the UCD site or at the USF Health Byrd Alzheimer Center clinic for the USF site. Subsequent 


 
21 
injections will also be administered in the CTRC Outpatient Clinic or at the USF Health Byrd  
Alzheimer Center clinic.  
 
Data Storage:   
All data collected at UCH will be stored in a REDCap, a HIPPA compliant database.  Data collected 
will include general patient information (age, phone, address etc.), background health information, and 
research outcomes. The Principal Investigator is responsible for the individually identifiable private 
health information. Access to the REDCap database key will be restricted to the research team 
approved on the application in order to minimize risk of unintentional disclosure.  All affiliated/local 
principal investigators, co-investigators, and research coordinators involved in consenting and carrying 
out the proposed study will complete Collaborative Institutional Training Initiative an online basic 
Course in Human Subject Protections and the HIPAA research course prior to submitting a protocol.   
For the general manipulation of data, the clinical data will be transformed into a de-identified data set 
using the unique study IDs.  This patient clinic data will be stored in a REDCap, HIPPA compliant 
database.  Any paper record of the data will be kept under lock and key with the Co-Principal 
Investigator at the University of Colorado Hospital or with the Co-Principal Investigator at the USF 
Health Byrd Alzheimer Center. The co-investigators at the University of South Florida only had access 
to the de-identified clinic data.  The statistician at the University of South Florida will also have access 
to the de-identified clinic data to conduct statistical analysis. 
 
 
Specimen Storage:   
The blood samples collected will only be used for this study and will not be provided to any third party.   
Samples will be stored for at least 7 years, or the entire course of this study.  Excess sample will be 
added to biobanks for future studies, but only with consent of the subject. These biobanks will be 
located in the Linda Crnic Institute for Down Syndrome and the Colorado Intellectual & 
Developmental Disabilities Research Center.  If a participant withdraws from the study, their samples 
will be discarded at the written request of the subject or appropriate consenting party. To confirm 
destruction of the samples at the Colorado Intellectual & Developmental Disabilities Research Center 
site, the site will send the research team written documentation confirming disposal of the sample. 
Otherwise, the sample will be stored at the Linda Crnic Institute for Down Syndrome and the Colorado 
Intellectual & Developmental Disabilities Research Center. The blood samples collected from the USF 
Health Byrd Alzheimer Center were de-identified and only coded with the unique study ID, and shipped 
for analysis and storage to the Linda Crnic Institute for Down Syndrome and the Colorado Intellectual & 
Developmental Disabilities Research Center. 
 
STUDY EVENTS: 
   
A summary of all study events are presented in Table 1.  All interviews with subjects and potential 
subjects will be carried out in a private room. At the initial visit (screen), after informed consent is 
obtained, the MMSE is performed.  If the subject qualifies on the MMSE entry criteria then vital signs 
are recorded, an ECG and a physical exam are performed, and 8 ml of blood is drawn for a CBC with 
differential, and a complete metabolic panel (CMP). A brain MRI will also be done during the 
screening period to exclude other causes of dementia and to assess for Medial Temporal Lobe Atrophy 
(MTA). Also, the first positron emission tomography (PET) scan, using 11C-PiB, will be performed 
during the screening period to assess evidence of cerebral amyloid load. If the PET scan is performed 
on the same day as the MRI scan, the PET scan will be performed after the MRI scan. A qualitative 
read of the 11C-PiB PET scan will be made to determine evidence of β-amyloid neuritic plaques. For 
women enrolled, who feel that there is a chance they may be pregnant, a pregnancy test will be 
administered.  Clinically, if the patient cannot name their last menstrual period or is uncertain whether 
they are pregnant or not, a urine dipstick test is performed.  The creatinine level from the CMP will also 


 
22 
be used to assess the Glomular Filtration Rate (GFR).  Also a list of all patient medications will be 
recorded.  Patients will have to meet the inclusion/exclusion criteria (stability on AD medications for 2 
months prior to screen and all other meds must be stable 30 days prior to screen).  Particular caution 
will be paid to individuals taking Lithium or corticosteroids, but they will not be excluded from the 
study. 
Once the results are available, if the subject continues to meet entrance criteria they will return 
for their baseline Day 1 (randomization) visit.  This visit takes place about 1 to 4 weeks after the screen 
visit.  At this visit, vital signs are recorded, 33 ml of blood will be drawn for a CBC with differential, a 
complete metabolic panel (CMP), and for biomarker assays (for example, GM-CSF, IFN-γ, IL-1β, IL-2, 
IL-6, IL-8, IL-10, IL-12p70, TNF-α, Eotaxin, IP-10, MCP-1, MCP-4, MIP-1β, TARC, P-Tau, Total 
Tau, and Aβ species), and tests of cognition, such as the MMSE, ADAS-cog, the ADCS-ADL scale, 
Trails A, Trails B, CDR, and the Mohs Cancellation task, are administered. The test article will then 
delivered by the unblinded pharmacist, administered subcutaneously by the (blinded) study nurse or 
clinician, and the subject then goes home.  
Subsequent injections will also be administered at the clinic.  About 4 ml of blood will be 
drawn for CBC plus differential approximately every three days during the treatment phase to monitor 
for leukocytosis (see safety monitoring plan),and an addition 4 ml of blood will be drawn for CMPs 
once every week of the treatment phase. Vital signs, injection site review, and adverse events will be 
recorded at each visit.  At the last treatment visit approximately three weeks later, we will record vitals, 
perform a physical exam, review for adverse events, administer cognitive tests and draw 33 ml blood 
for a CBC with differential, a CMP, and biomarker assays. A brain MRI will be done for comparison 
with the initial screen MRI to assess for any changes in the Medial Temporal lobe and for any incidence 
of vasogenic edema and microhemorrhage.  
The first follow-up visit is about 6-weeks/45 days after the last treatment phase visit. At this 
visit the vital signs are recorded, 33 ml of blood for a CBC plus differential, CMP and biomarkers is 
drawn, a physical exam is performed, adverse events are reviewed, and cognitive tests are administered. 
Also, a brain MRI and second 11C-PiB PET scan will be performed at the 6-weeks/45 day follow-up 
visit, or within a week before or after, depending upon scheduling availability. If the PET scan is 
performed on the same day as the MRI scan, the PET scan will be performed after the MRI scan. No 
treatment decisions will be made based on the outcome of the follow-up 11C-PiB PET scan. The end of 
study and the second follow-up visit is performed approximately 6-weeks/45 days after the first follow-
up visit (e.g. 3 months after the end of the treatment phase).  At this visit, vital signs are recorded, 
adverse events are reviewed, a physical exam is performed, 33 ml of blood is drawn for a CBC plus 
differential, CMP and biomarker assays, and cognitive tests are administered.   
 
COGNITIVE TESTS: 
 
MMSE (Mini-Mental State Exam)  
Education corrected Folstein Mini-Mental State Exam is a standard cognitive assessment used by all 
ADRCs including the FADRC to identify and monitor AD subjects. 
  
ADAS-cog (Alzheimer’s Disease Assessment Scale-cognitive subscale)  
ADAS was designed to measure the severity of the most important symptoms of AD. Its subscale 
ADAS‐cog is the most popular cognitive testing instrument used in clinical trials of nootropics (drugs 
or agents that improve cognitive function). It consists of 11 tasks measuring the disturbances of 
memory, language, praxis, attention and other cognitive abilities which are often referred to as the core 
symptoms of AD.  
 
 
ADCS/ADL (Alzheimer’s Disease Cooperative Study Activities of Daily Living Inventory)  


 
23 
ADCS-ADL is a caregiver rated questionnaire of 23 items, with possible scores over a range of 0-78, 
where 78 implies full functioning with no impairment. The ADCS-ADL assesses functional capacity 
across a wide spectrum of severity and will be the primary tool for collecting ADL data. 
 
TRAILS A 
Psychomotor speed will be assessed by the Trail Making Test-A, a timed test in which subjects must 
connect a series of numbers randomly placed on a page. 
 
TRAILS B 
Executive function will be assessed using Trail Making Test-B, a sensitive test of cognitive  
flexibility and psychomotor speed in which subjects must connect a series of alternating numbers and 
letters placed randomly on a page. 
 
Mohs Cancellation Task 
 Psychomotor speed and cognitive vigilance will be assessed using Mohs Cancellation Task, a timed 
task involving cancellation of target stimuli in a larger array of distractors. 
 
CDR (Clinical Dementia Rating) 
The CDR is a caregiver and subject based interview to assess changes in domains such as memory, 
orientation, judgment and problem solving, community affairs, home and hobbies, and personal care. 
Each domain is rated as 0 (no dementia), 0.5 (uncertain dementia), 1 (mild dementia), 2 (moderate 
dementia), or 3 (severe dementia). All raters in this trial will have completed the Washington University 
Alzheimer’s Disease Research Center CDR Training. 
 
LABS: 
 
Drawing blood may cause mild pain at the site of needle insertion and occasionally local ecchymosis 
formation.  With phlebotomy, rarely people may experience vasovagal reactions that are typically 
limited and resolve spontaneously.  The total amount of blood (cumulative amount for research and 
clinical procedures) drawn will not exceed five percent of estimated total blood volume over a 24-hour 
time period.  At the UCD site, phlebotomy will be performed by the University of Colorado Hospital 
Clinical and Translational Research Center (CTRC) nurses and at the USF site, by the USF Health Byrd 
Alzheimer Center clinic nurses.  
 
Complete blood count (CBC) with differential and complete metabolic panel (CMP):   
These labs will processed by an outside independent test center. 
 
Biomarkers:   
This testing will be performed in Dr. Potter’s lab.   
 
MRI PROCEDURE: 
 
Atrophy in the Entorhinal cortex (ERC) and Hippocampus (HPC), measured on MRI scans, predict 
future cognitive decline and conversion among individuals with Mild Cognitive Impairment (MCI) to 
AD [42,43]. Severity of MTA, assessed with MRI scans, is strongly associated with severity of medial 
temporal lobe degenerative pathology, especially the severity of neurofibrillary pathology, at autopsy 
[44]. Because of the high prevalence of AD in the elderly, 85-90% of all degenerative pathology in the 
medial temporal lobe, either alone or in combination with other diseases, is AD pathology [45]. 
An automated rating system to grade the severity of atrophy in the entire medial temporal 
region (MTA) will be employed using the FreeSurfer software automated rating system of individual 
medial temporal structures, i.e., the HPC, ERC and perirhinal cortex (PRC), which are specific regions 


 
24 
of interest (ROI) in AD pathogenesis.  We have shown that these individual and summed MTA scores 
distinguish AD and acute MCI subjects from normal elderly control subjects and predict progression 
from acute MCI to dementia/AD [46,47].  
Brain MRI scans will be obtained on a 3.0-tesla MRI machine using proprietary three-
dimensional magnetization-prepared rapid-acquisition gradient echo or the three-dimensional spoiled 
gradient recalled echo sequences; MRI scans will be acquired with isotropic resolution, and contiguous 
slices with thickness of 1.5 mm or less will be reconstructed.  Multiple MRI scans without contrast are 
safe as they do not involve exposure to radiation.  Some subjects may have difficulty lying motionless 
during the scan or may experience anxiety while in the scanner.  This can be mitigated by administering 
a sedative prior to scanning. 
 
Brain MRI scans at the UCD trial site will be performed in Building 400 on the Anschutz 
Medical Campus (AMC) at the University of Colorado and Denver VA Medical Center Brain Imaging 
Center, a component of the AMC’s comprehensive Colorado Translational Research Imaging Center 
(C-TRIC). Scans may also be done in the University of Colorado Hospital Anschutz Outpatient 
Pavilion radiology department. Brain MRI scans at the USF trial site were performed at University 
Diagnostic Institute on the campus of the University of South Florida. 
 
PET PROCEDURE – Pittsburgh Compound B (11C-PiB): 
  
The amyloid PET procedure will utilize the C-TRIC research PET scanner for human imaging studies. 
The Philips Gemini 64TF imaging system is located in Building 400 on the Anschutz Medical Campus 
at the University of Colorado and shares patient handling facilities with the Department of Psychiatry.  
 
Pittsburgh Compound B (11C-PiB) is a commonly used radiotracer that binds to β-amyloid plaques in 
the brain, as a biological marker of Alzheimer’s disease.  For evidence of β-amyloid neuritic plaques 
for inclusion into the trial, subjects will receive a screening 11C-PiB PET scan before the baseline visit 
that will be assessed by a Nuclear Medicine Radiologist (Michael Mueller, PhD) within the University 
of Colorado School of Medicine. The Nuclear Medicine Radiologist will remain blinded to subject 
identity and randomized test article group, and qualitative results, including any incidental findings, 
will reported to the Study Coordinator and Study Physician. If the PET scan is performed on the same 
day as the MRI scan, the PET scan will be performed after the MRI scan. The PET scans will be 
performed at an outpatient visit at C-TRIC (located in Building 400 at CU-AMC). For quantification of 
the brain amyloid load, assessments will evaluate the screening 11C-PiB scan before the baseline visit 
and another 11C-PiB scan within +/- 7 days of the first follow-up visit after the end of treatment with 
Leukine® or placebo. The change from the screening 11C-PiB scan’s SUVR to follow-up SUVR will be 
analyzed by a contracted central reading facility (i.e., BioClinica, a specialty clinical trial services 
provider) using the subject's MRI to reduce white matter contamination, thereby increasing the SUVR 
signal change by eliminating counts in the region that do not change (white matter). The 11C-PiB PET 
scans and accompanying MRI scans will be stripped of any personal identifying information, 
containing only the trial’s randomized subject ID number and date. The results of the 11C-PiB PET 
quantitative analyses will not be revealed to the trial participants or used to make any treatment 
decisions. MRI scans scans will also be performed in conjunction with the 11C-PiB PET imaging, so that any 
evidence of ARIA can be determined. 
 
PET Imaging  
Prior to the visit, subjects will be asked to abstain from food or drinks for 4 hours before the procedure. 
These procedures will be performed on a Philips Gemini 64TF scanner at the Colorado Translational 
Research Imaging Center (C-TRIC). Each study visit will take approximately 2 hours. The subject will 
be comfortably positioned in a chair. An intravenous catheter will be inserted using standard aseptic 
technique. A 10-15 mCi intravenous injection of 11C-PiB will be administered. The subject will be 
placed on the scanning table and moved into the scanner. A preliminary, 3-5 minute head CT scan will 


 
25 
precede the PET scan to aid in alignment and calibration, and adjust for attenuation effects. 11C-PiB 
PET images will be acquired for approximately 20 minutes. The IV catheter will be removed at the 
conclusion of the study and materials will be disposed of in biohazard containers or decay chambers 
until they can be safely disposed.  
 
Indications and Usage 
Pittsburgh Compound B (11C-PiB) is a radioactive imaging agent for Positron Emission Tomography 
(PET) imaging of the brain to estimate β-amyloid neuritic plaque density in adult patients with 
cognitive impairment who are being evaluated for Alzheimer’s Disease (AD) and other causes of 
cognitive decline. A negative 11C-PiB scan indicates sparse to no neuritic plaques, and is inconsistent 
with a neuropathological diagnosis of AD at the time of image acquisition; a negative scan result 
reduces the likelihood that a patient’s cognitive impairment is due to AD. A positive 11C-PiB scan 
indicates moderate to frequent amyloid neuritic plaques; neuropathological examination has shown this 
amount of amyloid neuritic plaque is present in patients with AD, but may also be present in patients 
with other types of neurologic conditions as well as older people with normal cognition. The use of 
Pittsburgh Compound B has become a widely accepted amyloid imaging method in human subjects 
research. As of 2008, PiB imaging was being performed at over 38 sites worldwide and had become a 
part of the Alzheimer’s Disease Neuroimaging Initiative (48). Therefore, it has substantiated potential 
as a quantitative imaging biological marker for detecting amyloid plaques in subjects. 
 
Mechanism of Action 
Pittsburgh Compound B (11C-PiB, or N-methyl-[11C]-2-(4′-methylaminophenyl)-6-hydroxy-
benzothiazole) is an 11C-labeled positron emission tomography (PET) imaging agent that binds with 
high affinity to the amyloid-β (Aβ) peptide fibrils that constitute amyloid plaques, and maps on to 
amyloid deposition in post-mortem studies (49). Klunk and colleagues developed a number of 
derivatives of Congo Red, a known beta-sheet binding molecule (50). These derivatives were made 
lipophilic so as to be able to cross the blood brain barrier after intravenous injection and could be 
shown to be non-toxic. Similarly, uncharged derivatives of thioflavin T were also shown to bind 
amyloid in situ (51). Klunk and colleagues were able to demonstrate strong target engagement of the 
radiotracer (i.e. sensitivity), and cross-labeling studies further demonstrated that 11C-PiB does not 
label structures that do not also preferentially label amyloid specific dye (i.e. specificity). 
Comprehensive  toxicology tests were conducted on pre-clinical models, including tests of acute 
vascular irritation in rabbits, bacterial reverse mutation assay, in vitro mammalian cell gene 
mutation test, mammalian erythrocyte micronucleus test, in vitro mammalian chromosome 
aberration test, expanded acute intravenous toxicity study in rats, an expanded acute toxicity study 
in dogs, an evaluation of the cardiovascular and pulmonary effects in dogs, a bridging study on the 
acute intravenous toxicity of 11C-PiB in rats, and a bridging study on the cardiovascular and 
pulmonary effects in dogs. Based on these studies, no deleterious toxicology results have been 
noted in animals. 
Early clinical research studies with humans demonstrated significant and consistent  11C-PiB 
retention in frontal cortex, precuneus/posterior  cingulate, striatal, temporal, and parietal cortices in 
the brains of patients diagnosed with AD dementia relative to healthy controls. 11C-PiB was reported 
in 2004 as successfully able to bind to amyloid and thus remain in the brain of AD patients, while 
quickly clearing the brain of normal subjects (49). These findings  were subsequently confirmed in 
other studies (52-54), with the retention pattern consistent with the cortical distribution of 
neuropathological findings of Aβ plaques on autopsy (55), suggesting that in vivo imaging of 
amyloid deposition parallels the underlying neuropathology. Since then, many studies have reported 
on the use of PiB for safely imaging amyloid in AD brain by PET, and the compound has become the 
method of choice for longitudinal studies of amyloid development in AD and for demonstrating and 
quantitating the efficacy of amyloid-reducing therapies (56).  
 


 
26 
Radiation Exposure 
Any exposure to radiation carries a very small risk of causing damage to tissues and the possibility of 
triggering a new cancer. However, the amount of radiation that each subject will be exposed to per (11C-
PiB) PET scan is similar to the amount received from natural sources, such as the sun (background 
radiation) over the course of living in Denver for approximately 1 year. We will perform PET imaging 
at the screening visit and at the first follow-up visit, or within a week thereof, depending upon 
scheduling availability. Our administered dose of (11C-PiB) will be 10-15 mCi. For a (11C-PiB) dose of 
15 mCi (555 MBq), the expected whole body effective dose is 2.9 mSv. In addition, the effective dose 
for a lose dose head CT performed for localization and attenuation correction as part of the standard 
brain PET/CT protocol is 0.6 mSv. Thus, with two (11C-PiB) scans, we expect the trial subjects to be 
exposed to about 7 mSv, well below the 50 mSv maximum annual dose of radiation exposure. At the 
first screening visit, subjects and/or subject’s partner/caregiver will be asked to provide information 
whether the subject has been exposed to radiation during the past year (e.g. prior X-ray, CT, PET scans, 
etc.), so that the Study Physician and/or the Nuclear Medicine Imaging Professional can determine 
whether there will be a risk of the subject exceeding maximum allowed levels of radiation exposure.  
 
 
 
RANDOMIZATION AND MAINTENACE OF BLIND 
 
The drug will be purchased with funds for this study and study participants will not be billed for study 
related costs.  At the UCD site, the Leukine® and the placebo samples for injection will be prepared by 
the UCH Pharmacy, which has policies and procedures in place that describe randomization and 
blinding. Briefly, the pharmacy has built and implemented an Excel spreadsheet that produces random 
number sets which may be used for simple, weighted, block (two, four, or six sized), and stratified 
randomization. For blinding, the pharmacy staff includes the pharmacist manager and technician. The 
pharmacy staff has been involved in several clinical trials which require double blinding, and double 
dummy. The pharmacy is an independent entity, and has the ability to ensure and maintain blinding in 
any regard so that the staff at the Alzheimer’s Clinic will not know which study participants are 
receiving the study drug or the placebo. 
 
 At the USF site, the Leukine® and the placebo samples for injection were randomized, 
prepared, and supplied to the USF Health Byrd Alzheimer’s Institute’s Comprehensive Clinic by the 
USF College of Pharmacy Manager of the Investigational Research Pharmacy, which is located within 
the Carol and Frank Morsani Center for Advanced Healthcare, and which is a component of USF’s 
Clinical and Translational Research Institute (CTSI). 
 
DATA SAFETY MONITORING PLAN AND PROTECTION OF HUMAN SUBJECTS 
 
Because of the extensive safety history of Leukine® in elderly patients, we do not anticipate any serious 
safety problems, but we must be vigilant in assessing safety.  A review of published adverse events for 
GM-CSF and the Genzyme Leukine® Product Insert and the papers it references will be used to guide 
the safety monitoring protocol ([38], attached as Appendix D). The greatest safety concern noted with 
Leukine® is excessive leukocytosis.  Therefore, as advised by Sanofi/Genzyme Corporation, we will 
obtain a CBC with differential twice a week during treatment and the dosage will be reduced by half if 
total ANC rises above 20,000/mm3. In addition, the DSMB described below will monitor laboratory 
results, the MRI and the cognitive assessments for any indications of toxicity.  Following are the 
abbreviated categories of the National Cancer Institute’s (NCI) graded toxicity protocol used by Daud 
et al., 2008  for assessing safety of Leukine® [57], although the latest version of the NCI-CTCAE 
(Version 4.03) will also be referenced for all AE’s (attached as Appendix F): 
 


 
27 
Anemia  
Lymphopenia  
Pain  
Fever Flu-like symptoms  
Arthralgia/myalgia  
Injection-site reaction  
Fatigue  
Abdominal cramps  
GERD/indigestion Insomnia  
Cough  
Sweating 
 
Data and Safety Monitoring Board (DSMB):   
 
The DSMB will be comprised of members of relevant Departments at UCH, USF and the 
Moffitt Cancer Center.  The DSMB will be charged with overseeing patient safety and 
compliance.  An initial meeting of the DSMB, via teleconference or video-conference, will be 
held before dual enrollment occurs between the two sites in order for the members to review 
the study protocol and the study/participant termination guidelines.  Data will be reviewed 
immediately if a serious adverse event is reported.  Otherwise, all data will be reviewed after 
the first 10 participants (5 Leukine®-treatment, 5 placebo control) have completed the initial 
treatment phase, and again, after each next 10 participants (5 Leukine®-treatment, 5 placebo 
control) have completed the initial treatment phase from both UCD and USF clinical 
sites.  Additionally, review of data will occur in a similar manner as above at 45 and 90 days 
after treatment for each group of 10 participants (5 Leukine®-treatment, 5 placebo control). 
Furthermore, because this was a multi-center study, serious AE’s will be reported immediately 
to the DSMB, which includes members from both UCD and USF.  If necessary, DSMB 
members from both sites will convene via teleconference or video-conference to discuss the 
serious AE’s and to decide whether the study should continue.  Of note, if serious AE’s Grade 
3 or 4 occur in 20% of subjects between the two sites following Leukine® administration the 
study will be stopped.  All DSMB meetings will be documented. 
 
 
The members of the DSMB include: 
DSMB Members:  
 
Neda Rasouli, MD 
Professor of Medicine & Clinical Pharmacy 
Division of Endocrinology, Metabolism and Diabetes  
Phone: 303-724-4651 
Director, CU Diabetes and Endocrinology Clinical Trial Program 
University of Colorado School of Medicine 
12631 East 17th Avenue Mail Stop 8106 
Aurora, CO 80045 
Phone: 303-724-4651 
Fax:  303-399-8020 
Email: neda.rasouli@CUAnschutz.edu 
 


 
28 
Dr. Rasouli is Professor of Medicine at the University of Colorado. She received her M.D. from the 
University of Tehran and did her Internship and Residency at Michigan State University. Dr. Rasouli’s 
expertise is in endocrinology and she heads many clinical trials in diabetes, obesity and heart failure 
and she is a member of the DSMB for “Neuroendocrine dysfunction after TBI” at the Craig 
Rehabilitation Hospital, CO. 
 
Donald Leung, MD, PhD 
Allergist-Immunologist 
Professor and Division Head of Pediatric Allergy and Immunology 
Department of Pediatrics 
National Jewish Health 
National Jewish Health Main Campus 
1400 Jackson Street 
Denver, CO 80206 
Phone: 877-225-5654 
Fax: 303-270-2182 
Email: leungd@njhealth.org 
 
Dr. Leung is Professor of Pediatrics at the University of Colorado. He received his Ph.D. and M.D. 
from the University of Chicago and is an expert on allergy and clinical immunology, which is very 
appropriate expertise for membership on the DSMB of a trial on an immune modulator, GM-CSF. He 
has also served on multiple NIH advisory panels, has given many national and international invited 
lectures and was Editor in Chief of the Journal of Allergy and Clinical immunology form 1998-2015. 
He is currently Chair of the Aimmune Peanut Immunotherapy DSMB. 
 
Theresa Zesiewicz, MD 
Neurologist, Director of the USF Ataxia Research Center 
Professor, University of South Florida, College of Medicine 
12901 Bruce B. Downs Blvd, MDC-55 
Tampa, FL 33612 
Phone: 813-974-5909 
Fax: 813-974-8032 
Email: tzesiewi@health.usf.edu 
 
Dr. Zesiewicz received her M.D. from the University of Medicine and Dentistry of New Jersey, is 
Professor of Neurology at the University of South Florida, and is Director of the USF Ataxia Research 
Center. She has served on the DSMB for the ‘Pilot phase 2 Trial of the Safety and Efficacy of GM-CSF 
(Leukine) in the Treatment of Mild-Moderate Alzheimer’s Disease’ since its inception at the University 
of South Florida through its new IND and IRB application at the University of Colorado Anschutz 
Medical Campus. Thus, she is well aware of the safety data of this drug in this trial. She has also great 
experience leading and/or participating in multiple safety and efficacy trials of interventions in 
Friedreich Ataxia (i.e. PMID: 30656180, 30051753, 29624723). 
 
Heather Jim, PhD (Statistician) 
Behavioral Oncology Researcher 
Moffitt Cancer Center 
12902 USF Magnolia Drive 
Tampa, FL 33612 
Phone: 813-745-6369 
Email: Heather.Jim@moffitt.org 
 


 
29 
Dr. Heather Jim is a medical researcher of psychosocial and behavioral aspects of cancer control at the 
Moffitt Cancer Center in Tampa, FL, with research that specifically focuses on the adjustment to 
diagnosis and treatment, management of symptoms and side effects, and quality of life. Because of her 
interest in assessment, measurement issues are a recurrent theme in her research, which is augmented 
by an expertise in biostatistics. She has recently been involved in the investigation of cognitive 
functioning following treatment for breast and prostate cancer. Thus, due to her strong background in 
statistical analyses, cognitive testing, and immunology, Dr. Jim was recruited and has agreed to be the 
statistician for this DSMB. 
Assurance of Independence of Judgment: Members of the DSMB are employed by several different 
departments and institutions.  
 
 
Data Monitoring Plan: 
 
Data Recording: These functions will be carried out by the study staff and transcribed into the case 
report forms (adverse events page).  The Co-Principal Investigator, Dr. Woodcock, will review, edit and 
submit the AE forms at the UCD site and the Co- Principal Investigator, Dr. Raj, reviewed, edited and 
submitted the AE forms at the USF site. Laboratory results and data from case report forms will be 
entered into a computerized data registry.  
 
Data Verification: Dr. Woodcock and Dr. Raj will discuss the AE’s with the study coordinators, and 
verify AE data entry. 
 
Data to be monitored, assessed, and reported to/by the DSMB: 
Common but non-life threatening side effects include: 
• 
Fatigue or asthenia  
• 
Malaise  
• 
Rash  
• 
Itching (pruritis)  
• 
Vomiting. 
• 
Diarrhea  
• 
Abdominal pain  
• 
Sore throat (pharyngitis)  
• 
Chills  
• 
Bone pains  
• 
Joint pains  
• 
Weight loss or gain 
• 
Elevations in kidney or liver enzymes  
• 
Fluid retention (peripheral edema)  
• 
Injection site irritation 
• 
Insomnia 
• 
Headaches 
• 
Transient Fevers 
 
Less common but potentially serious side effects include: 
• 
Bleeding into the eye  
• 
Allergic reactions 
• 
Excessive increase in the white blood cell count.   
• 
Fluid retention in the lung lining (pleura) and in the heart lining (pericardium). 


 
30 
• 
Shortness of breath. 
• 
Occasional transient heart rhythm changes (supraventricular tachycardia) 
• 
Sweating, tachycardia, claustrophobia or anxiety induced by the MRI procedure 
 
Serious Adverse Events (AE’s) will be determined according to the most recent National Cancer 
Institute (NCI) Common Toxicity Criteria for Adverse Event (CTCAE) Version 4.03 (attached as 
Appendix E). 
 
Frequency: An initial meeting of the DSMB, via teleconference or video-conference, will be held 
before dual enrollment occurs between the two sites in order for the members to review the study 
protocol and study/participant termination guidelines.  Data will be reviewed immediately if a 
serious adverse event is reported.  Otherwise, all data will be reviewed after the first 10 participants 
(5 Leukine®-treatment, 5 placebo control) have completed the initial treatment phase, and again, 
after each next 10 participants (5 Leukine®-treatment, 5 placebo control) have completed the initial 
treatment phase from both UCD and USF clinical sites.  Additionally, review of data will occur in a 
similar manner as above at 45 and 90 days  after treatment for each 10 participants (5 Leukine®-
treatment, 5 placebo control) from both UCD and USF clinical sites.  Furthermore, because this was 
a multi-center study, serious AE’s will be reported immediately to the DSMB, which includes 
members from both UCD and USF. 
 
Report of Unexpected Problems to the DSMB:  The study coordinator will notify the Co-Principal 
Investigator if there are any unexpected problems. The Co-Principal Investigator will call the DSMB 
when unexpected problems arise that require immediate action.  
 
Criteria for Action: 
 
Specific triggers that will dictate when a specified action (such as reporting or stopping) is 
required. Any serious adverse event will be reported by the Co-Principal Investigator, Dr. Woodcock, 
to COMIRB using the on-line reporting system. If 2 of 5 subjects experience serious AE’s, the FDA 
will also be notified.   
 
Stopping rules: If serious AE’s Grade 3 or 4 occur in 20% of subjects following GM-CSF 
administration the study will be stopped. For individual subjects, the DSMB will discontinue any 
subject that reaches grade 3 or 4 in any category, or if they decline more than 25% in any of the 
cognitive tests from baseline. Subjects, are, of course free to withdraw from the study at any time for 
any or no reason. 
 
Reporting: 
  
Timeframe for reporting: Serious AE’s will be reported immediately to the DSMB for review and, 
within 48 hrs (2 business days) to COMIRB. A persistent or significant disability will be reported 
within 5 business days. The FDA will be notified by written report within one or two weeks if 2 of 5 
subjects experience serious AE’s.  
 
Reporting mechanisms: AE forms will be completed and submitted on-line using the UCH AE 
reporting system. The report will include a description of the event, judgment as to whether or not it 
was related to study medication and the outcome of the AE.  
 
Responsibility for preparing and submitting reports: The study coordinator will prepare the AE 
report and the Co-Prinicipal Investigator will review/edit as appropriate and submit the AE.  
 


 
31 
Confidentiality Procedures:   
The meetings of the DSMB will be closed to respect the privacy of the research participants. Data will 
be kept in case report binders in a locked cabinet when not in use. Data entered into the computerized 
data registry will only be accessible to those members of the research team. 
 
Control of Conflict of Interest: 
 
Dr. Potter was one of the conceivers of the Arthritis/GM-CSF/G-CSF approach to AD therapy and, 
along with Dr. Timothy Boyd, is one of the inventors on a University of South Florida patent on the use 
of GM-CSF in AD (US Patent 9,132,168) that is subject to the standard University of South Florida 
royalty sharing agreement. According to that agreement and although there are no current or 
immediately expected financial benefits that would attend the results of the proposed research, the 
University of Colorado Anschutz Medical Campus Conflict of Interest Committee (UCD-AMC COIC) 
has determined that Drs. Huntington Potter and Timothy Boyd each have a significant financial interest 
due to the issuance of the patent, and the UCD-AMC COIC has approved management plans, which 
Drs. Potter and Boyd will be glad to share upon request, and that allow the continued involvement of 
Drs. Potter and Boyd in the clinical trials of Leukine. Additionally, the conduct of this trial will be 
further managed as follows:  The Co-Principal Investigators, Dr. Woodcock and Dr. Raj, who each 
have no real or perceived conflict, will carry out the clinical assessments of the subjects.  Furthermore, 
the following steps will be used to help manage any potential conflict of interest: 
 
1)  The investigators will be blinded to the treatment group. 
2)  Endpoints are quantitative/objective (improvement in cognition by assessment with a battery 
of neuropsychological and clinical assessments used in the UCH clinic and changes in blood 
biomarkers towards normal). 
3)  If interested investigator(s) do participate in data collection and recording of data, they will be 
blinded and will not be the sole evaluator of the results/endpoints. 
4)  Financial interest has not been licensed and has no current commercial value. 
5)  Additional controlled studies would be done if outcome of study indicates results that would 
be favorable to the financial interest. 
6)  An independent DSMB has been established to oversee patient safety. 
7)  There are numerous other non-interested investigators involved in the study who will be 
collecting data. 
8)  Notification of Dr. Potter’s and Dr. Boyd’s financial interests in the study will be made to 
editors of journals publishing study results and to the public in the context of communication of 
any research results. 
  
AVAILABLE RESOURCES: 
 
University of Colorado Denver: 
 
Laboratory: The Principal Investigator, Dr. Potter, has 1800  square feet of space in the 
Linda Crnic Institute for Down Syndrome where the biomarker levels of cytokines, 
chemokines, Tau and Aβ peptides from the plasma of the trial’s participants will be 
assessed. 
 
Clinic – UCH: The Clinical and Translational Research Center (CTRC) Outpatient Clinic 
on the 3rd floor of the UCH Leprino Building will be the location for subject evaluation in 
private rooms. The clinic contains 11 Exam Rooms, 3 Metabolic Testing Rooms, an 
Echocardiogram Testing Room with ECG services, a Cognitive Testing Room, a 5-Chair 


 
32 
Infustion Center, and a Phlebotomy Room. There is also a patient registration area and 
waiting room.  
 
Computer: The laboratories and clinic are adequately equipped with a PC and  Apple 
computers with access to the Internet and literature search programs. All staff members also 
have computers running Windows XP with necessary software, printer and internet access. 
The server is administered and backed-up daily by UCH IT. 
 
 
Office: Dr. Potter and Dr. Woodcock each have 100-200 Sq. Ft. offices  with telephone, fax 
access, and voice mail. The study coordinator has office space in the LCI and in the 
Academic Office 1 Building, and the  psychometrist will utilize space in the Cognitive 
Testing Room of the CTRC Outpatient Clinic. Within each office are PC’s, telephones, fax 
access, and voice mail.Various large and small conference/lecture rooms are available for 
reservation for meetings, seminars and outreach events. All conference space is equipped 
with tele-communication equipment for tele-conferencing and audio visual equipment and 
marker boards for presentations. 
 
Equipment: 
• 
Two -80C freezers, plus other refigerators and -20C freezers,  
• 
Cryo Locator 6 liquid nitrogen storage sytem, 
• 
Cell culture room with BSL2 tissue culture and sample hood, 
• 
CO2 incubators, 
• 
A new Zeiss flourescence microscope with Z stage and ccd cameras for analyzing 
immunostained and immunofuorescent samples, 
• 
A Meso Scale Discovery system with ccd camera for imaging 
electrochemiluminescent immunoassays, 
• 
Preparative and ultra centrifuges with refrigeration and rotors for plates, conicals 
and ultrafuge tubes, 
• 
Real-time PCR machine,  
• 
Autoclave, 
• 
Spectrophotometer,  
• 
Biotek microtitre plate reader with luminometer, and plate washing system,   
• 
Electroporation and electrophoretic power supplies and associated equipment, 
• 
Stereo microscope for array QC, 
• 
Designated sets of drummond and micropipettors. 
 
University of South Florida: 
 
Clinic – USF Health Byrd Alzheimer’s Center: The USF Health Byrd Alzheimer’s Institute’s 
Comprehensive Clinic, will be the location for subject evaluation in private rooms. The clinic 
comprises over 8000 square feet. There are 6 exam rooms, 5 patient consult rooms and 4 offices 
shared                by associated clinical staff. There is also a patient registration area, waiting room 
and patient library. 
 
Computer: The laboratories and clinic are adequately equipped with a PC and Apple computers with 
access to the Internet and literature search programs. All staff members also have computers running 
Windows operating systems with necessary software, printer and internet access. The server is 
administered and backed-up daily by USF Health IT. 
 
Office: Dr Raj has 100-200 Sq. Ft. office with telephone, fax access, and voice mail. The study 


 
33 
coordinator and the psychometrist, each have offices within the Byrd Institute. Within each office 
are PC’s, telephones, fax access, and voice mail.Various large and small conference/lecture rooms 
are available for reservation for meetings, seminars and outreach events. All conference space is 
equipped with tele-communication equipment for tele-conferencing and audio visual equipment 
and marker boards for presentations. 
 
 
Equipment: 
• 
Preparative and ultra-centrifuges, 
• 
-80C freezer, plus other refigerators and -20C freezers, 
• 
Cell culture room with BSL2 tissue culture and sample hood. 


 
34 
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amyloidosis in Alzheimer mice. J Alzheimers Dis, 2010. 21(2): p. 507-18. (attached as Appendix B) 
3. Jim HS, Boyd TD, Booth-Jones M, Pidala J, Potter H. Granulocyte Macrophage Colony 
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26. Jellinger, K.A., Prevalence and impact of cerebrovascular lesions in Alzheimer and lewy body 
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27. Holmes, C., et al., Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of 
a randomised, placebo-controlled phase I trial. Lancet, 2008. 372(9634): p. 216-23. 
28. Uro-Coste, E., et al., Cerebral amyloid angiopathy and microhemorrhages after amyloid beta 
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30. Murdoch, C., et al., The role of myeloid cells in the promotion of tumour angiogenesis. Nat Rev 
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31. Subramaniyam  V., et al., Bone marrow mobilization with granulocyte macrophage colony-
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important for cognitive functions. Sci Rep. 2012;2:697. 
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human cerebral cortex. Cereb Cortex.  2004. 14,11-22. 
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Corporation, a Sanofi Company. (attached as Appendix D) 
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50. Klunk WE, Bacskai BJ, Mathis CA, Kajdasz ST, McLellan ME, Frosch MP, Debnath ML, Holt 
DP, Wang Y, Hyman BT. Imaging Abeta plaques in living transgenic mice with multiphoton 
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51. Klunk WE, Wang Y, Huang GF, Debnath ML, Holt DP, Mathis CA. Uncharged thioflavin-T 
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2001;69(13):1471-84. PubMed PMID: 11554609.  
52. Ziolko SK, et al. Evaluation of voxel-based methods for the statistical analysis of PIS PET 
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57. Daud et al., Phenotypic and Functional Analysis of Dendritic Cells and Clinical Outcome in 
Patients With High-Risk Melanoma Treated With Adjuvant Granulocyte Macrophage Colony-
Stimulating Factor. J. Clinica Oncology. 2008; 26: 3236-3241.


 
37 
STUDY SCHEDULE 
 
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
  
  
  
VISITS SCREEN 
TREATMENT PHASE 
  
Follow 
up 1 
End of 
study 
DAYS 
7-28 
1 2 3 4 5 
 
 
6 7 
8 
9 
1
0 
 
 
1
1 
1
2 
1
3 
1
4 
1
5 
45 days 
post (PET 
+/- 7 days) 
90 days 
post 
Informed 
consent 
X 
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Pregnancy test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
GFR 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MMSE 
X 
X   
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
X 
X 
X 
Medical history 
X 
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
  
  
  
concomitant 
meds 
X 
X X X X X     X X X X X 
  
  
X X X X X 
X 
X 
Adverse events 
  
X X X X X     X X X X X 
  
  
X X X X X 
X 
X 
Vital signs 
X 
X X X X X     X X X X X 
  
  
X X X X X 
X 
X 
Physical exam 
X 
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
X 
X 
X 
Injection site 
review 
 
X X X X X     X X X X X 
  
  
X X X X X 
 
 
ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
MRI 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
X 
 
PET 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
LABS 
  
  
  
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Biomarkers 
 
X   
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
X 
X 
X 
Quest CBC+diff  
X 
X   
  
X   
    X   
  
X 
  
  
  
X 
  
  
  
X 
X  
X  
CMP  
X  
  
  
  
X   
    
  
  
  
X 
  
  
  
  
  
  
  
X 
X  
X  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Cognitive testing 
  
X   
  
  
  
    
  
  
  
  
  
  
  
  
  
  
  
X 
X 
X 
Study drug 
injections 
  
X X X X X     X X X X X 
  
  
X X X X X 
  
  


 
38 
 
PHARMACOLOGY, TOXICOLOGY, AND PREVIOUS HUMAN EXPERIENCE 
 
 
 


 
39 
 
 
 
 


 
40 
 
 
 


 
41 
 
 
 


 
42 
 
 
 


 
43 
 
 
 


 
44 
 
 


 
45 
 
 
 


 
46 
 
 
 


 
47