Leukine (Sargramostim) for Parkinson's Disease

Parkinson's disease (PD) is a progressive and disabling neurological disorder involving the
nigrostriatum and for which no cure is known. Evidence suggests that inflammation
contributes, significantly, to the cause and/or progression of the disease. Studies in
postmortem human brain, animal models, and human peripheral blood support the idea that the
frequency and function of particular circulating T cell subsets are abnormal and worsen as
disease progresses. This abnormality is linked specifically to increases in numbers of T
effector neurodestructive cells (Teff) and to dysfunction of regulatory T cells (Treg) that
control Teff. This imbalance tips the homeostatic balance to a pro-inflammatory profile with
limited control. Nonetheless, whether such T cell deficits are a cause of PD or a reaction
to it remains unknown. If these deficits are causal or exacerbative, then correcting the
deficit could have significant positive effects on disease progression and could ameliorate
nigrostriatal degeneration or its sequelae. Granulocyte macrophage colony stimulating factor
(GM-CSF) is a potential immunomodulatory therapeutic for PD to increase Treg numbers or
function and reduce or transform proinflammatory Teff responses, leading to neuroprotection
of the nigrostriatum and improved clinical outcomes for disease. Recombinant human GM-CSF
(sargramostim) is available as Leukine®. The purpose of this study is to determine if
Leukine (sargramostim) can be safely administered to Parkinson's disease patients for an
extended period of time (56 days).

In a previous study conducted at the University of Nebraska Medical Center (UNMC) using
samples of whole blood, PD patients showed differences from caregiver controls according to
results from complete blood count (CBC) with white blood cell (WBC) differential,
fluorescence-activated cell sorting (FACS) analysis for T cell markers, and Treg functional
assays. These immune aberrations correlated with motor dysfunction as determined by
Parkinson's disease rating scale (UPDRS) part III assessments. In this pilot study, the
effect of Leukine treatment on immune deficits in PD patients will be monitored. Immune cell
analyses of whole blood, motor assessments, and physical examinations and blood analyses for
safety, will be conducted before, during, and after Leukine or placebo treatment of PD
patients, and using healthy controls during baseline data collection before drug treatment.
Initially, 16 PD patients and 16 controls will be recruited. Subjects will be drawn from the
previous study. Enrollment will be supplemented as needed with patients from Neurology
Consultants of Nebraska-PC, UNMC Neurological Sciences, or recruitment through the American
Parkinson's Disease Association. Enrollment will be staggered, and additional subjects will
be recruited as needed to replace any withdrawing participants, with the goal of 32
subjects, 16 PD patients and 16 controls, completing the study.

At enrollment and repeating again at two 4-week intervals, whole blood from PD patients and
controls will be obtained percutaneously using standard sterile techniques. CBC with WBC
differential, FACS analysis for T cell markers, and Treg functional assays will be performed
and used to calculate immune response profiles as a baseline for comparison after drug
treatment. Blood chemistries will be analyzed including liver enzymes, electrolytes, blood
urea nitrogen, creatinine, total protein, albumin and bilirubin levels, and anti-GM-CSF
antibodies will be assessed in PD patients. PD patients will be evaluated by physical and
clinical examinations, including UPDRS part III assessments for evaluation of motor
function.

PD patients will then be randomized into two groups, placebo (n=8) and Leukine (n=8), and
participation of the controls will end. Patients entered into the study will not have any
noted co-morbid conditions including infection, inflammatory or cancerous diseases and will
not be taking immunostimulatory or immunosuppressive medicines. Patients will continue to
take medicines prescribed for the general treatment of PD, including, but not limited to,
dopamine or dopamine agonists. There will be no history of prior surgeries linked to PD
treatment. Patients will receive training for self-injection, and the treatment group will
self-administer Leukine (6 µg/kg) by subcutaneous injection daily for eight weeks. The
placebo group will self-administer saline as placebo. Every two weeks after the start of
treatment and again 4 weeks after cessation of administration, all PD patients, receiving
drug or placebo, will be evaluated by physical and clinical examinations, including UPDRS
part III assessments. Every 2 weeks during Leukine treatment and again 4 weeks after
cessation of drug, whole blood from PD patients will be obtained percutaneously using
standard sterile techniques. At weeks 2, 4 and 8, results from CBC with WBC differential,
FACS analysis for T cell markers, and Treg functional assays will be used to calculate
immune response profiles and compared with baseline values. CBC with WBC differential, total
T cell count, and blood chemistries will be analyzed every 2 weeks for evidence of potential
toxicities, as well as 4 weeks after termination of Leukine treatment. Levels of anti-GM-CSF
antibodies will be assessed for PD patients at enrollment and at 4 weeks intervals during
drug treatment and 4 weeks after termination. Optional video recordings will be made at
baseline, end of drug/placebo treatment and at follow-up.

An additional component of the study includes the development of magnetoencephalography
(MEG) as a monitoring tool for PD. MEG provides a non-invasive method to study brain
activity. Currently, there are not adequate assessment tools for monitoring disease
progression or treatment protocols in PD. In preliminary experiments on PD patients and
their caregivers, MEG data have indicated strong beta synchronization during rest in PD and
milder beta desynchronization during movement preparation compared to age- and sex-matched
controls. In this study, the amplitude of this pathological beta
synchronization/desynchronization will be monitored to evaluate improvements in cortical
brain function of PD patients treated with Leukine. MEG will be performed on PD patients and
controls at the start of drug treatment, and on PD patients at the end of the drug treatment
period and 4 weeks after drug is stopped.

Among the first 8 patients enrolled in the study, we have observed unexpected and
statistically significant changes in the unified PD rating scale part III (UPDRS part III)
clinical evaluations for motor function in half of the patients tested. This test, however,
is viewed as subjective. To more objectively and analytically score motor functions,
biomechanical assessments of the subject's mobility, postural balance, upper extremity motor
control and resting state tremor will be performed on the remaining eight PD patients
receiving Leukine or placebo. These assessments will occur at baseline, between 7 and 8
weeks of drug treatment, and at 4-5 weeks after cessation of drug treatment. Motor function
tests are designed to provide an objective, computer generated score as measured by patient
performance on each test. To investigate a possible mechanism for changes in motor function
observed within two weeks of therapy, testing for plasma/serum neurotransmitters will be
performed to include glutamate, glutamine, serotonin, acetylcholine, gamma-aminobutyric acid
(GABA), norepinephrine and epinephrine. These measures will be performed on the last cohort
of PD patients of the study before initiation of treatment, 8 weeks after treatment is
initiated and at 4 weeks after cessation of drug treatment. The tests will be evaluated by
enzyme-linked immunosorbent assay (ELISA) or by ultra-performance liquid chromatography
(UPLC). A decision on which assay will be done for which neurotransmitter will be based on
sensitivity and standard curve measures. Relationships between neurotransmitter levels and
motor function will be determined. These tests are in addition to current ongoing and
approved laboratory tests. Eligibility requirements are unchanged from those currently
imposed. Measures of motor function and serum/plasma neurotransmitter concentrations will
also be completed 4-5 weeks after cessation of treatment. The notion of exploring
neurotransmitters in this study is based on role in motor function that include fatigue,
bowel, sexual dysfunction and mood changes that are also observed in PD as well as that
GM-CSF has been shown to affect neurotransmitter levels (96). This range of listed
neurotransmitters was not investigated in the first part of this study and will require
limited additional blood and assay resources for the last part of the investigation.

Inclusion Criteria:

PD Patients

- Onset of bradykinesia and 1 or both of the following: rest tremor and/or rigidity

- Asymmetric onset of clinical signs

- Progressive motor symptoms

- Age at onset 35-85 years

- Duration of PD symptoms of at least 3 years

- Female subjects must be either:

Not pregnant, not breastfeeding, and not planning on becoming pregnant during the study;
Not of childbearing potential, defined as one who has been postmenopausal for at least 1
year and with follicle stimulating hormone (FSH) levels in the laboratory defined
postmenopausal range, or has been surgically sterilized, or has had a hysterectomy at
least 3 months prior to the start of this trial; or If of childbearing potential, must
agree to use an effective method of avoiding pregnancy to the end of the trial and must
have a negative serum beta-human chorionic gonadotropin (β-HCG) test. Effective methods of
avoiding pregnancy are contraceptive methods used consistently and correctly (including
implantable contraceptives, injectable contraceptives, oral contraceptives, transdermal
contraceptives, intrauterine devices, diaphragm with spermicide, male or female condoms
with spermicide, or cervical cap), abstinence, or a sterile sexual partner

- Have the ability to comply with basic instructions and have the ability to sit still
comfortably inside the MEG

- Must be stage 4 or less according to the Hoehn and Yahr scale

- Caregiver, spouse, friend, or relative must agree to participate in the research
study

Control subjects:

- Age 35-85 years

- Caregiver, spouse, relative, or friend of eligible PD patient

- Female subjects must be either:

Not pregnant, not breastfeeding, and not planning on becoming pregnant during the study;
Not of childbearing potential, defined as one who has been postmenopausal for at least 1
year and with follicle stimulating hormone (FSH) levels in the laboratory defined
postmenopausal range, or has been surgically sterilized, or has had a hysterectomy at
least 3 months prior to the start of this trial; or If of childbearing potential, must
agree to use an effective method of avoiding pregnancy to the end of the trial and must
have a negative serum beta-human chorionic gonadotropin (β-HCG) test. Effective methods of
avoiding pregnancy are contraceptive methods used consistently and correctly (including
implantable contraceptives, injectable contraceptives, oral contraceptives, transdermal
contraceptives, intrauterine devices, diaphragm with spermicide, male or female condoms
with spermicide, or cervical cap), abstinence, or a sterile sexual partner

- Have the ability to comply with basic instructions and have the ability to sit still
comfortably inside the MEG

Exclusion Criteria:

PD Patients

- Atypical features indicative of a Parkinson-Plus disorder (Progressive Supranuclear
Palsy (PSP), Multiple System Atrophy (MSA), Corticobasal Degeneration (CBD))
including cerebellar signs, supranuclear gaze palsy, apraxia and other cortical
signs, or prominent autonomic failure

- Neuroleptic treatment at time of onset of parkinsonism

- Active treatment with a neuroleptic at time of study entry

- History of repeated strokes with stepwise progression of parkinsonism

- History of repeated head injury

- History of definite encephalitis

- More than one blood relative diagnosed with PD

- Prominent gait imbalance early in the course (< 5 years)

- Mini-mental state examination score <26

- Hematological malignancy or coagulopathy

- Abnormal blood analyses: hematocrit <30; WBC>11.5; clinically significant laboratory
data (e.g. alanine aminotransferase [ALT] or aspartate aminotransferase [AST] 3x the
upper limit of normal [ULN]), or any abnormal laboratory value that could interfere
with the assessment of safety in the judgment of the investigator; significant
abnormalities on the clinical examination, vital signs, and clinical chemistry or
hematology results (excluding findings of Parkinson's disease), that may interfere
with the study or present a safety risk for the subject as judged by the clinical
investigator charged in the care of study participants

- Serious medical illness or co-morbidity that may interfere with participation in the
study

- Brain surgery for parkinsonism (DBS, cell implantation, gene therapy)

- History of an autoimmune disorder or systemic inflammatory disorder

- Immunostimulatory or immunosuppressive treatment (including amphetamines or systemic
corticosteroids) within 90 days

- Exclusively unilateral parkinsonism for longer than 3 years

- Known hypersensitivity to GM-CSF, yeast-derived products or benzyl alcohol

- Current lithium treatment

- Individuals who have ferrous metal implanted in their body other than fillings

- Individuals with current diagnoses of alcohol or substance abuse/dependence

- Anyone who is not appropriate for participation in this research protocol as deemed
by the principal or co-investigator

Control subjects:

- Positive response to more than 3 items on the PD Screening Questionnaire

- More than one blood relative diagnosed with by PD

- Mini-mental state examination score <26

- Hematological malignancy or coagulopathy

- Abnormal blood analyses: hematocrit <30; WBC>11.5; clinically significant laboratory
data (e.g. alanine aminotransferase [ALT] or aspartate aminotransferase [AST] 3x the
upper limit of normal [ULN]), or any abnormal laboratory value that could interfere
with the assessment of safety in the judgment of the investigator; significant
abnormalities on the clinical examination, vital signs, and clinical chemistry or
hematology results that may interfere with the study or present a safety risk for the
subject as judged by the investigator

- Serious medical illness or comorbidity that may interfere with participation in the
study

- History of an autoimmune disorder or systemic inflammatory disorder

- Immunostimulatory or immunosuppressive treatment (including amphetamines or systemic
corticosteroids) within 90 days

- Individuals who have ferrous metal implanted in their body other than fillings

- Individuals with current diagnoses of alcohol or substance abuse/dependence

- Anyone who is not appropriate for participation in this research protocol as deemed
by the principal or co-investigator

PD Screening Questionnaire

- Do you have trouble arising from a chair?

- Is your handwriting smaller than it once was?

- Do people tell you that your voice is softer than it once was?

- Is your balance poor?

- Do your feet ever seem to get stuck to the floor?

- Do people tell you that your face seems less expressive than it once did?

- Do your arms and legs shake?

- Do you have trouble buttoning buttons?

- Do you shuffle your feet and/or take tiny steps when you walk?

- Has anyone ever told you that you have Parkinson's disease?

- Have you ever taken levodopa or Sinemet?