Date: December 10, 2005
To: Maryland Pao, MD, Chair, IRB, NIMH
Recommended by: Robert B. Innis MD, PhD, Chief, Molecular Imaging Branch, NIMH
Protocol Title: PET imaging of brain peripheral type benzodiazepine receptors
Identifying Words: Pharmacokinetics, compartmental analysis, distribution volume, identifiability
Principal Investigator:
Masahiro Fujita, MD, PhD, NIMH
Associate Investigators:
Masao Imaizumi, MD, PhD, NIMH
Janet Sangare, MSN, C-RNP; NIMH
Yong Hoon Ryu, MD, PhD; NIMH
Robert B. Innis, MD, PhD; NIMH
Estimated Duration of Study: two years
Study Subjects
Healthy controls
Number
15
Off Site Project: NO
Project uses ionizing radiation: YES
Project uses Durable Power of Attorney: NO
Page 1 of 22
Sex
M&F
Age Range
18-40
Table of Contents
I. PRECIS ................................................................................................................................................................ 4
II. INTRODUCTION .............................................................................................................................................. 4
A. Type of Protocol............................................................................................................................................. 4
B. Background .................................................................................................................................................... 4
C. Research question ........................................................................................................................................... 9
D. Background of Approach ............................................................................................................................... 9
E. Qualifications of investigators ........................................................................................................................ 9
III. STUDY DESIGN AND METHODS................................................................................................................ 9
A. Study design ................................................................................................................................................... 9
B. Overview ........................................................................................................................................................ 9
C. Study phases ................................................................................................................................................. 10
D. Sample stratification .................................................................................................................................... 10
E. Sample size justification ............................................................................................................................... 10
F. Data analysis ................................................................................................................................................. 10
G. Justification for the use of placebo, medication washout, or provocative stimuli ....................................... 11
IV. SUBJECT ENROLLMENT ........................................................................................................................... 11
A. Recruitment - sample composition and characteristics ................................................................................ 11
B. Inclusion criteria ........................................................................................................................................... 11
C. Exclusion criteria.......................................................................................................................................... 11
D. Study initiation and screening methods ....................................................................................................... 11
V. PROCEDURES................................................................................................................................................ 11
A. Details of method ......................................................................................................................................... 11
B. Details of assessment by study phase ........................................................................................................... 13
C. Details of secondary procedures .................................................................................................................. 13
D. Relationship to other studies proposed ........................................................................................................ 13
VI. PROVISION OF CARE TO RESEARCH SUBJECTS ................................................................................. 13
A. Concomitant clinical care............................................................................................................................. 13
B. After care ...................................................................................................................................................... 13
C. Reasons for discontinuation from study ....................................................................................................... 13
D. Toxicity criteria ............................................................................................................................................ 14
VII. HUMAN SUBJECT RISKS AND PROTECTIONS .................................................................................... 14
A. Consent and assent procedures .................................................................................................................... 14
B. Risks of study participation and minimization of risks ................................................................................ 14
C. Benefits of study participation ..................................................................................................................... 16
D. Investigator conflicts of interest ................................................................................................................... 16
E. Privacy and confidentiality provisions ......................................................................................................... 16
F. Adverse event reporting ................................................................................................................................ 16
G. Data and safety monitoring processes .......................................................................................................... 16
H. Subject compensation .................................................................................................................................. 16
VIII. PHARMACEUTICAL, BIOLOGIC AND/OR DEVICE INFORMATION ............................................... 16
A. Source........................................................................................................................................................... 16
B. Relevant pharmacology ................................................................................................................................ 17
C. Toxicity ........................................................................................................................................................ 17
D. Formulation and preparation ........................................................................................................................ 17
E. Stability and storage ..................................................................................................................................... 17
F. Incompatibilities ........................................................................................................................................... 17
Page 2 of 22
G. Administration procedures ........................................................................................................................... 17
IX: REFERENCES ............................................................................................................................................... 18
X. APPENDIX: REIMBURSEMENT SCHEDULE ............................................................................................ 22
Page 3 of 22
I. PRECIS
The peripheral benzodiazepine receptor (PBR) is distinct from central benzodiazepine
receptors associated with GABAA receptors. Although PBR was initially identified in peripheral
organs such as kidneys, endocrine glands and lungs, later studies identified PBR in the central
nervous system. In normal conditions, PBR is expressed in low levels in some neurons and glial
cells. PBR can be a clinically useful marker to detect neuroinflammation because activated
microglial cells in inflammatory areas express much greater levels of PBR than in microglial
cells in resting conditions.
PBR has been imaged with positron emission tomography (PET) using [11C]1-(2chlorophenyl-N-methylpropyl)-3-isoquinoline carboxamide (PK11195). However, this classical
ligand provides only low levels of specific signals and is not sensitive to detect changes occurred
in vivo. Recently we developed a new ligand, N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5pyridinamine [11C]PBR28, which showed much greater specific signals than [11C]PK11195 in
non-human primates. In the present protocol, we plan to perform a kinetic brain imaging study
in healthy human subjects to measure PBR in brain regions with [11C]PBR28. Successful
development of a PET ligand to image PBR will have a strong impact on clinical management of
brain disorders with inflammation such as multiple sclerosis and ischemia and neurodegenerative
disorders such as Alzheimer’s and Parkinson’s disease where inflammation is involved in the
disease progression.
II. INTRODUCTION
A. Type of Protocol
Healthy subjects will be studied to measure PBR in brain by performing brain PET
imaging studies with [11C]PBR28. This study will be performed with an intravenous injection of
up to 20 mCi of [11C]PBR28 and imaging for 2 – 3 h.
B. Background
Since their discovery in the late 1950’s, benzodiazepines have been widely used as
anxiolytics, anticonvulsants and sedative medications (Sternbach 1983). In the last decades, two
pharmacologically distinct subclasses of benzodiazepine binding site have been demonstrated.
One class, the central benzodiazepine receptor (CBR) is mainly localized on the extracellular
domain of the γ-aminobutyric acid (GABA)A receptor and regulates the chloride channel of
GABAA receptors in the central nervous system (CNS) (Tallman et al 1978).
The second class of benzodiazepine receptor was initially identified in peripheral tissue
and was called the peripheral benzodiazepine receptor (PBR). PBR is located on the
mitochondrial outer membrane in several organs including the kidney, nasal epithelium, lung,
heart and endocrine organs such as the adrenal gland, testis and pituitary gland (Anholt et al
1985; Anholt et al 1986; Braestrup et al 1977; Gavish et al 1999). Contrary to the original
nomenclature of “peripheral” benzodiazepine receptors, later studies demonstrated the presence
of PBR in the CNS (Schoemaker et al 1981; Weissman et al 1984; Zisterer and Williams 1997).
In mitochondria, PBR belongs to the mitochondrial permeability transition pore (Anholt et al
1985; Bernassau et al 1993; McEnery et al 1992), where it is intimately associated in trimeric
complex with adenosine nucleotide translocase and the voltage-dependent anion channel. This
complex is a megachannel located on the inner and outer mitochondrial membrane contact sites.
Although the physiological role of PBR in still unclear, PBR has been implicated in various
functions such as neurosteroid synthesis (Culty et al 1999; Papadopoulos et al 1997),
immunomodulation (Zavala 1997), cell proliferation (Okuyama et al 1999; Schlichter et al 2000;
Verma et al 1998) and apoptosis (Bono et al 1999). In the normal brain, PBRs are mainly found
in glial cells and are especially highly localized to the ependyma lining of the ventricles,
choroids plexus, olfactory bulb (Anholt et al 1984; Benavides et al 1983; Cymerman et al 1986;
Page 4 of 22
Schoemaker et al 1983). The expression of PBR in vivo is reported to be increased in microglia
activated by brain injury (Banati 2002; Banati 2003), and this increased has been used as an
indicator of neuronal injury and neurodegenerative disease (Benavides et al 1987; Cagnin et al
2002).
Several compounds are able to cross the blood brain barrier and specifically bind to PBR
in vivo. The most widely used selective ligand is 1-(2-Chlorophenyl-N-methylpropyl)-3isoquinoline carboxamide (PK11195), which has been labeled with [11C] for positron emission
tomography (PET) studies. Various neurologic disorders have been clinically investigated by
using [11C]PK11195such as Alzheimer’s disease (Cagnin et al 2001a; Groom et al 1995),
multiple sclerosis (Banati et al 2000; Debruyne et al 2003), stroke (Pappata et al 2000),
Rasmussen’s encephalitis (Banati et al 1999), herpes encephalitis (Cagnin et al 2001b),
amyotrophic lateral sclerosis (Turner et al 2004) and multiple system atrophy (Gerhard et al
2003). However, the brain uptake is very low (only about the same as the average activity in the
entire body) with low ratios of specific binding to nondisplaceable radioactivity (less than 20%),
which is not high enough for stable quantitative analysis (Pappata et al 1991; Pappata et al 2000;
Sauvageau et al 2002).
In the last several years, a new class of high affinity PBR ligands has been radiosynthesized based on aryloxyanilides (Okuyama et al 1999), and some promising PET
radioligands have already been developed from this class (Maeda et al 2004; Zhang et al 2003).
We have also sought to develop PET ligands with high brain uptake and high ratios of specific
binding to nondisplaceable radioactivity based on aryloxyanilides. We successfully developed a
new PET ligand ([11C]PBR28) with high affinity ([11C]PBR28; IC50 = 0.6 nM measured with
[3H]PK11195) and selectivity. In addition, in monkeys, these PET ligands showed high brain
uptake and high ratios of specific binding to nondisplaceable radioactivity (Briard et al 2005).
The objective of this study was to fully characterize pharmacokinetics of this PET ligand
by performing compartmental analysis with arterial input function and also by analyzing the
composition of radioactive chemicals in the rat brain with high performance liquid
chromatography (HPLC).
Studies in Nonhuman Primates.
Two rhesus monkeys (Macacca mulatta, body weight) were used (Table 1). Anesthesia
was initiated with i.m. injection of ketamine (10 mg/kg) and then maintained under anesthesia
with 1.6% isoflurane and 98.4% O2. The electrocardiograph (ECG), body temperature, heart and
respiration rates were measured throughout the experiment. Body temperature was maintained at
37.0-37.5°C.
All of PET scans were performed on a GE Advance scanner (General Electric Medical
Systems, Waukesha, WI), with reconstructed resolution of 6 mm full-width half-maximum in all
directions in 3D mode by applying scatter correction. The high resolution research tomograph
(HRRT, Siemens/CPS, Knoxville, TN, USA) scanner (Schmand et al 1998; Wienhard 2002). All
scans of HRRT were acquired in 64-bit list mode format. Data were reconstructed into a
256x256x207 image matrix with pre-determined frame schedule using a list mode OSEM
algorithm (Carson et al 2003), resulting in an image resolution of 2.5 mm FWHM. No scatter
correction was applied. After a transmission scan, the radiopharmaceutical (dose: 4.17±1.36
mCi, specific activity: 1530±580 mCi/μmol) was intravenously injected. Coronal slices covering
the whole brain were obtained. PET scans were acquired for 120-180 min (33-45 frames with
longer scan duration at later time points). To measure plasma concentration of [11C]PBR28 and
the metabolites, a second line, intra-arterial, in the contralateral limb was used to obtain 14 blood
samples. Eight samples (0.5mL each) were drawn at 15 s intervals until 2 min, followed with 1mL samples at 3, 5, 10, 30, 60, 90, 120min in heparin-treated syringes. Each blood sample was
separated into plasma and blood cell fraction by centrifugation.
Page 5 of 22
The tomographic images were analyzed with PMOD 2.65 (pixelwise modeling computer
software; PMOD Group, Zurich, Switzerland) (Burger et al 1998). All frames of the original
reconstructed PET data were summed, and this summed image was coregistered to a T1weighted magnetic resonance (MR) image acquired separately on a GE 1.5 T Signa MR scanner
(SPGR, TR/TE/flip angle = 13.1 ms/5.8 ms/45°, 0.4 x 0.4 x 1.5 mm and coronal acquisition on a
256 x 256 x 60 matrix) (GE Medical Systems,Waukesha, WI) using SPM2 (Wellcome
Department of Cognitive Neurology, London, U.K.), and regions of interest were defined on the
frontal, temporal , parietal and occipital cortices, cerebellum, putamen, thalamus, 3rd ventricle
and 4th ventricle of the MRI. To normalize brain uptake relative to the injection dose and the
body weight, standardized uptake values (SUVs) were determined as (% Injected activity/ g
brain) × g body weight.
Estimation of distribution volume with arterial input function:
One-tissue (1C) and unconstrained two-tissue compartment (2C) models were applied.
Rate constants (K1, k2, k2', k3, and k4) were defined as described previously (Laruelle et al 1994).
In the 1C,
VT=K1 / k2'f1
where VT is the distribution volume for the single tissue compartment.
In the 2C, VT is described separately by the distribution volumes in nondisplaceable (VN) and
specific binding compartments (VS).
VN = K1/k2'f1
VS = K1k3/k2k4f1 = Bmax'/Kd
VT = K1(1+ k3/k4) k2f1
where Bmax' is unoccupied binding site density. Under tracer conditions, Bmax' = Bmax. In
these two models, although the definition of K1 is the same, that of k2 and k2'is different. That is,
k2 is transfer rate to the vascular compartment from the nondisplaceable compartment in the 2C,
and k2'refers to the transfer from the total tissue compartment (Laruelle et al 1994).
Vi' is defined as
Vi' = f1Vi
These definitions indicate that Vi values are expressed relative to the free fraction of radioligand
in plasma and that VT' values are expressed relative to the total (free plus protein-bound)
concentration of radioligand in plasma. Non-linear least-squares analysis was performed on the
VOI-generated time-activity data using PMOD 2.65. Parameters were estimated using the
Marquardt algorithm (Bevington and Robinson 2003) with constraints restricting parameters to
positive values.
Page 6 of 22
Statistical analysis
Goodness-of-fit by nonlinear least squares analysis was evaluated using the model
selection criterion (MSC), which is a modification of the Akaike information criterion (AIC)
(Akaike 1974). MSC gives greater values for better fitting. Goodness-of-fit by 1C and 2C was
compared with F statistics (Hawkins et al 1986). The standard errors (SEs) of non-linear least
squares estimation for rate constants were given by the diagonal of the covariance matrix
(Carson 1986) and expressed as a percentage of the rate constants (coefficient of variation,
%COV). In addition, %COV of VT' was calculated from the covariance matrix using the
generalized form of error propagation equation (Bevington and Robinson 2003), where
correlations among parameters were taken into account. A value of P<0.05 was considered
significant.
Table1. List of nonhuman primate [11C]PBR28 PET imaging
Study #
PBR_M10
PBR_M11
PBR_M18
PBR_M21
PBR_M22
Blocking
Agent
Dose
(mg/kg)
Blocking
Agents
Cold PBR28
-
3
-
Monkey
P41
P41
P41
R16
P41
Wt.
(kg)
16.1
16.1
15.3
9.6
14.2
Specific
Activity
(mCi/µmol)
1122
1260
1285
2549
1435
Injected
Dose
(mCi)
4.23
5.47
1.95
5.06
4.15
Mass dose
(µg)
1.31
1.51
0.53
0.69
1.01
Brain Uptake of [11C]PBR28
[11C]PBR28 showed high brain uptake of approximately 300-500 SUV%. The
distribution of brain uptake for the [11C]PBR28 were consistent with the distribution of PBRs.
That is, regions with high receptor densities (e.g., 4th Ventricle) had highest levels of activities at
late time points of the scan (Fig.1). The brain uptake of [11C]PBR28 was blocked with a receptor
saturating dose of non-radiolabeled PBR28 (3 mg/kg i.v., injection at the same time of [11C]
PBR28 administration; Fig.1).
Baseline Study
Blocking Study
500
700
Frontal
Temporal
Parietal
Occipital
Cerebellar
Putamen
Thalamus
4th_Ventricular
600
400
%SUV
%SUV
500
300
200
400
300
200
100
100
0
0
0
25
50
75
100
Time (min)
125
150
175
0
25
50
75
100
125
Time (min)
Fig. 1 Time activity curves of [11C]PBR28 baseline and blocking studies.
Page 7 of 22
Arterial Plasma Analysis
Activity of total activity and parent radioligand in plasma was determined from arterial
samples over multiple time points after injection. The radioligand was quickly metabolized and
represented 86.2±6.8%, 20.2±4.0% and 8.8%±4.7% of total plasma activity at 5, 30 and 60 min,
respectively. Plasma activity of [11C]PBR28 peaked approximately 1 min and decreased rapidly
to 65.2±5.3%, 18.1%±4.4%, 6.9±1.5% and 3.1±0.9% at 2, 10, 30 and 60 min, respectively
(Fig.2).
120
%SUV
100
Non-metabolite ratio
(%)
Total
Parent
80
60
40
20
100
Non-Metabolite ratio (%)
80
60
40
20
0
0
0
20
40
60
80
100
0
20
Time (min)
40
60
80
100
Time (min)
Fig. 2 Time-dependent change of radioactivity and non-metabolite ratio of [11C]PBR28 in
plasma
Nonlinear least squares compartmental analyses
Convergence was achieved in all regions (n=6) of all studies (n=3) with both 1C and 2C.
2C did not provide significantly better fitting than 1C. The difference between 1C and 2C fitting
was not significant by F-test in all regions of all animals. Average MSC values were 3.88±0.23
and 3.74±0.24 for the 1C and 2C fits. VT' estimated by 1C was consistent among 3 experiments
performed using the same animal with COV values less than 10%.
After 100 min, VT' of [11C]PBR28 by 1C and 2C became independent of scan length in
cerebellum and 4th Ventricle (Fig.3). The changes of VT' between 100 and 120 min by 1C and 2C
were less than 1.5%. Therefore, the binding of [11C]PBR28 was accurately measured with 1C
using arterial input function.
2 Tissue Compartment Model
1 Tissue Compartment Model
125
100
75
50
150
Cerebellum
4th Ventricle
VT' (mL/cm3)
VT' (mL/cm3)
150
70
90
110
130
150
Cerebellum
4th Ventricle
125
100
75
50
70
90
110
Time (min)
Time (min)
Page 8 of 22
130
150
Fig. 3 The change of VT’ by 1 tissue compartment and 2 tissue compartment model of
[11C]PBR28
Pharmacological Effects in Nonhuman Primates.
A total of 4 PET scans were performed in monkeys: all involved injection of tracer doses
of [ C]PBR28. One of the studies was involved with blockade with non-radiolabeled PBR28 (3
mg/kg i.v.). In these PET scans, the average injected mass dose of [11C]PBR28 was 1.0 µg for
an average body weight of 14.0 kg (corresponding to 0.07 µg/kg). In all cases (for injection of
both radiolabeled and non-radiolabeled PBR28), the difference of measurement between pre- and
post-injection were: < 15 mmHg for systemic blood pressure, < 10/min for pulse, < 5/min for
respiratory rate and < 0.3 ºC for temperature.
11
C. Research question
The protocol seeks to measure PBR in brain regions with [11C]PBR28.
D. Background of Approach
This protocol describes the use of a new PET radiotracer and follows all aspects of
protocol “PET imaging of brain 5-HT1A receptors using [11C](-)-RWAY” (PI: Xiang-Yang
Zhang, MD), which has been approved by CSRP. The current and the protocol on [11C](-)RWAY follow some but not all aspects of the previously approved template for initial human
use. The prior template had two components: whole body imaging for measurement of organ
dosimetry and kinetic imaging of the brain. Whole body imaging has been deleted from this
study, because of the new Guidance from the FDA on the so-called “Exploratory IND”
(http://www.fda.gov/cder/guidance/6384dft.htm). By this new path for PET radiotracers, the
initial human studies should evaluate whether the tracer is useful in a “limited” number of human
subjects. If it is, then additional studies are justified. If not, then no further studies will be done,
and the whole body dosimetry studies will have been avoided.
On July 28, 2005, the NIH Radiation Safety Committee reviewed the Guidance on
Exploratory IND and supported this approach. In addition, the NIMH Council at its February
and May 2005 meetings has reviewed ways to reduce the regulatory barrier for introduction of
new radiotracers in man (http://www.nimh.nih.gov/council/pastmeetings.cfm). The Council has
supported reduced barriers such as the exploratory IND, written a letter to the FDA to this effect,
and had the FDA Director of Medical Imaging (George Mills, MD) speak at its May 2005
meeting.
E. Qualifications of investigators
Investigators listed in this protocol are experienced in the execution of neuroreceptor PET
imaging studies in humans.
III. STUDY DESIGN AND METHODS
A. Study design
Fifteen healthy controls will have one PET scan with [11C]PBR28 and one MRI scan.
B. Overview
This protocol entails three components: Evaluation, MRI and PET. The MRI will be
obtained within one year of the PET scan, i.e., up to one year before or one year after the PET
Page 9 of 22
scan. All subjects will have an initial visit(s) for evaluation: physical/history, laboratory
screening tests. Evaluation, MRI and PET sessions will take approximately 3 h, 1 h, and 4 h,
respectively. A timetable for these visits is given below.
1st Visit
–
Screen
Time - weeks 0
Informed
consent
Physical
Exam
Neurological
Exam
Pregnancy
test (female
≤ 55)
MRI**
Brain PET
Blood and
urine tests
2nd
Visit
3rd
Visit
< 24
< 24



*





*A pregnancy test will be done within 24 h prior to the PET ligand administration.
**Depending on the availability of PET and MRI scanners, an MRI scan may be scheduled at
any point in time for this protocol, either before or after the PET procedures. MRI scans will be
repeated on another day if the image quality is not adequate due to subject’s movement or other
reasons.
C. Study phases
This is a baseline study.
D. Sample stratification
Fifteen healthy controls will be studied. All subjects must meet the inclusion and
exclusion criteria listed in Section IV – Subject Enrollment.
E. Sample size justification
We plan to measure PBR in brain regions by evaluating identifiability of distribution
volumes from volumes of interest (VOI’s) and in each pixel using arterial input function.
Because of intersubject variability in pharmacokinetics and metabolism of PET ligands, sample
size of approximately 10 is required in such studies (Fujita et al 1999; Ichise et al 2003; Koeppe
et al 1999). By taking into account possible withdrawal in the study due to difficulties obtaining
arterial blood, we request permission to study 15 subjects.
F. Data analysis
Data from VOI’s and each voxel will be analyzed with compartmental nonlinear least
squares analyses and non-compartmental linear and multilinear regression analyses using arterial
input function. Distribution volume (Bmax/Kd plus radioactivity not specifically bound to PBR)
will be calculated in various brain regions. Because the half-life of C-11 is 20 min, it is critical
Page 10 of 22
that the kinetics of the PET ligand is fast enough to provide accurate measurement of distribution
volume before noise of the data increases significantly due to the radioactive decay. Necessary
length of data acquisition for accurate measurement of distribution volume will be examined by
its identifiability and intersubject variability. Compartmental nonlinear least squares analyses
and non-compartmental linear and multilinear regression analyses will be performed using PMD
(http://www.pmod.com/technologies/index.html).
G. Justification for the use of placebo, medication washout, or provocative stimuli
This protocol will not involve the use of placebo, medication washout or provocative
stimuli.
IV. SUBJECT ENROLLMENT
A. Recruitment - sample composition and characteristics
We will select healthy adult female and male volunteers (age 18–40 years old) in this
protocol. The subjects will be screened under another protocol (01-M-0254). We will exclude
children or minors because this study involves radiation exposure. The proportion of ethnic
minorities (vs. Caucasians) in the total sample will approximately be consistent with the overall
U.S. population proportions.
B. Inclusion criteria
All subjects must be healthy and aged 18–40 years.
C. Exclusion criteria
1. Current psychiatric illness, substance abuse or severe systemic disease based on history
and physical exam.
2. ECG with clinically significant abnormalities. Any existing physical exam and ECG
within one year will be reviewed and if none already exists in the chart, these will be
obtained and reviewed.
3. Laboratory tests with clinically significant abnormalities.
4. More than moderate hypertension (see below for details).
5. Prior participation in other research protocols or clinical care in the last year such that
radiation exposure would exceed the annual limits.
6. Pregnancy and breast feeding.
7. Claustrophobia.
8. Presence of ferromagnetic metal in the body or heart pacemaker.
9. Positive HIV test.
10. A history of brain disease.
D. Study initiation and screening methods
We will initiate the study within 1–2 months of final approval. Healthy controls meeting
inclusion and exclusion criteria (above) will be recruited from the community and NIH through
advertisements in newspaper and newsletter, private physicians and social service agencies. We
will obtain informed consent from all healthy controls.
V. PROCEDURES
A. Details of method
1. Evaluation
Except as described below, all subjects will undergo a physical examination to ascertain
general good health. All subjects will have a 12-lead ECG and will be asked to provide blood
and urine samples for a battery of laboratory screening tests such as complete blood count (CBC)
Page 11 of 22
with diff., chemistries (Na, K, Cl, HCO3, BUN, Cr, glucose, Ca, PO4, SGOT, SGPT, LDH,
alkaline phosphatase, CPK, bilirubin, total protein, albumin), VDRL, urinalysis, and urine drug
screen. All women with child bearing potential will have a blood or a urine pregnancy test.
Subjects will be excluded if they have more than moderate hypertension. Subjects may
be on anti-hypertensive medications, however, the initial screening must show no more than
moderate hypertension – i.e. <160/95. In addition, the subject must have normal laboratory
values (e.g., BUN, creatinine, urinalysis, and ECG) to document lack of end organ damage. On
baseline evaluation on the day of the scan (i.e., before injection of tracer), the subject must be
asymptomatic (no headache, dizziness, neurological symptoms, or blurred vision) AND have
sustained BP < 180/100. After PET ligand administration, the scan will be discontinued if BP
remains greater than 180/100 continuously for more than 5 min. The subject will then be asked
to relax. If BP continues to be greater than 180/100 for more than 15–30 min, a cardiology
consult will be ordered STAT.
The exception to the general plans above has to do with the timing of the physical exam
and laboratory tests, for example, the subject has been seen at NIH previously, the physical exam
and ECG have been performed, and in the chart, anytime within the prior year. Furthermore, the
laboratory tests (SMA-20, CBC with diff, urinalysis, and thyroid function test) can be performed
on the morning of the scan, but must be reviewed and meet criteria, prior to injection of the
radiotracer.
2. PET Procedure
The NIMH Radiochemistry Laboratory (Dir., Victor Pike, PhD) will synthesize and
perform quality control (QC) for [11C]PBR28.
All women with child bearing potential will have a blood or a urine pregnancy test again
within 24 h of each PET tracer injection. The screening laboratory tests described above will be
repeated on the day before PET tracer injection to compare with the results after the injection.
PET dynamic brain scanning will be performed using the GE Advance or HRRT at the
PET Department. Subjects will be placed on the scanner bed with his/her head held firmly in
place with a thermoplastic mask fixed to the bed. One antecubital venous and one radial arterial
catheters will be placed. One venous catheter is for radioligand injection and the arterial catheter
is for blood sampling. Another additional antecubital venous catheter may be placed for blood
sampling. Just prior to the PET scanning, a transmission scan will be performed with a 68Ge
rotating pin source to provide a measured attenuation correction. The radiation-absorbed dose
from a transmission scan was estimated to be 0.05 rad to the red marrow, lens of eyes, thyroid,
bone surfaces, skin, and the brain (based on measurement by M. Daube-Witherspoon, PhD,
memo of Nov 29, 1994). The radioligand (20 mCi of [11C]PBR28, maximum mass dose: 10 g)
will be injected intravenously as a bolus injection. The dosage of 20 mCi of [11C]PBR28 was
selected for a few reasons. First, it is quite safe from a radiation safety perspective and would
cause an estimated effective dose of 0.82 rem. In addition, this is an exploratory IND with the
purpose of determining whether the tracer is useful for PBR imaging. For this purpose, we plan
to scan for 2 h – i.e., six half-lives. Thus, little activity will remain at the end of the study. The
latter portions of the brain time activity curve (90 – 120 min) will be particularly important to
examine for the presence of radiolabeled metabolites (which would be evidenced by an
increasing distribution volume). Finally, we hope to obtain plasma measurements of parent
radiotracer and metabolites for the entire period. We need to start with adequate activity to have
measurable amounts at the end of the experiment.
PET images will be acquired in the three-dimensional mode with increasing length of
frame for a total duration of 2 – 3 h. To measure input function of the radioligand, blood
samples will be obtained frequently from the arterial line. Several venous samples may also be
Page 12 of 22
obtained at several time points to estimate arterial input function using venous blood data. Blood
sampling volume will be no more than 200 mL.
3. Safety monitoring of subjects
The pulse rate, temperature, blood pressure, respiratory rate and 12-lead ECG will be
recorded within 3 h before tracer injection, and again at about 15, 30, 90 and 120 min after tracer
injection. Neurobehavioral assessment will also be recorded within 3 h before tracer injection,
and repeated at about 30 and 120 min after tracer injection. The screening laboratory tests
including complete blood count (CBC) with diff. (excluding pregnancy test) described above
will be repeated ~24 h after tracer injection. If the subjects cannot come at the above mentioned
time point, the blood and urine samples will be taken right after completing the PET scan. We
will also call the subject for any adverse events ~24 h after injection. In addition, the adverse
events will be assessed at every study visit.
4. MRI procedure
Subjects will have an MRI scan for anatomical localization by coregistering onto PET
image. MRI scanning will be done on a 1.5 Tesla scanner located at the NIH Clinical Center.
Structural transaxial and coronal scans will be acquired. MRI will take up to 1 h.
If subjects become anxious during a scan, diazepam (Valium 2–4 mg) or lorazepam
(Ativan 0.5–1mg) may be administered orally. If these medications are given, there will be at
least a one week interval between MRI and PET scans.
B. Details of assessment by study phase
Subjects will undergo one PET and one MRI scan. No other assessments will be made in
this study.
C. Details of secondary procedures
There are no secondary procedures in this protocol.
D. Relationship to other studies proposed
None.
VI. PROVISION OF CARE TO RESEARCH SUBJECTS
A. Concomitant clinical care
As described in section V.A.4, if necessary, Valium or Ativan will be administered
orally for subjects becoming anxious in the MRI scanner.
B. After care
After participation in this study, subjects will not receive after care in this protocol.
C. Reasons for discontinuation from study
Scanning procedures will be stopped for any subject who asks to stop for any reason at
any time. Subjects will be asked if they wish to continue the rest of the study. Subjects have the
right to withdraw from this study at any time for any reason.
Subjects will be excluded if they have more than moderate hypertension, i.e. <160/95.
On baseline evaluation on the day of the scan (i.e., before injection of tracer), the subject must be
asymptomatic (no headache, dizziness, neurological symptoms, or blurred vision) AND have
sustained BP < 180/100. After PET tracer administration, the scan will be discontinued if BP
remains greater than 180/100 continuously for more than 5 min.
Page 13 of 22
D. Toxicity criteria
There is no expected toxicity in this study.
VII. HUMAN SUBJECT RISKS AND PROTECTIONS
A. Consent and assent procedures
From one of the investigators, each subject will receive an oral and written explanation of
the purposes and potential risks of participation in this protocol. Specifically, they will be told
that (a) the information derived may eventually lead to better understanding of brain chemistry
and behavior; (b) PET imaging as used in this study is a research tool, hence no diagnostic
interpretation will be given; (c) a confidential code number will be used to ensure that
information cannot be linked or traced to any person or family; (d) data will be treated to group
statistical analyses only; and (e) subjects will be given ample opportunity to ask questions of the
investigators.
If the subject shows clinically significant abnormalities in lab tests or MRI, the
abnormalities will be notified to the subject.
For women of child bearing potential, a pregnancy test will be conducted within 24 h
before the PET scan. Finally, when the laboratory tests and medical examination show
significant abnormalities, appropriate referrals will be made to address their health problems.
Consent will be obtained by the Principal Investigator or one of the Associate Investigators.
B. Risks of study participation and minimization of risks
This is a more than minimal risk study. Potential risks from this study include those
associated with: 1) medical examinations including laboratory testing that may reveal previously
undiagnosed medical disorders, 2) radiation exposure from the PET and transmission scans, 3)
PET scanning, and 4) placement of arterial and venous line and blood sampling, 5) blood
sampling and 6) MRI.
1. Medical Examination and Laboratory Testing
The potential risks of a medical examination are small but do include the detection of an
otherwise undiagnosed disorder. We will first explain and familiarize the subjects with the
laboratory testing to minimize discomfort, if any, during testing. In the present protocol, all
healthy normal volunteers are expected to undergo recruitment and assessment procedures
without any difficulties. However, if, in the opinion of the study staff, PI, or subject, the study
participation is adversely affecting the subject's emotional and or physical well-being, the
individual circumstances will be reviewed to determine what additional steps should be taken,
such as termination of the study and making appropriate referrals to address their underlying
health problems. If the subject desires not to proceed further with testing, we will end these
sessions at any time point. Blood tests may lead to the formation of a small subcutaneous
hematoma caused by blood leaking from a punctured blood vessel. This hematoma causes only
minor discomfort. It is not dangerous and requires no treatment other than reassuring the patient.
There is also a small risk of infection at the site of the needle puncture, which can be readily
treated with antibiotic therapy. Approximately 25 mL of blood will be withdrawn for screening
purpose.
2. Radiation Exposure Risks
Radiation exposure in this protocol will be from [11C]PBR28 and the 68Ge transmission
scan. The radiation-absorbed dose from a transmission scan is based on the measurement by M.
Page 14 of 22
Daube-Witherspoon, PhD, (memo of Nov 29, 1994). Based on whole body imaging of rhesus
monkeys performed at Molecular Imaging Branch, MIB, effective dose from the current study is
0.83 rem, which is well below the NIH RSC annual guideline of 5 rem.
All subjects will be asked about any prior research participation involving radiation
exposure so that the total exposure, in combination with the present study, will not exceed an
effective dose of 2.5 rem per 12 months. This limit of 2.5 rem is half that typically used for
screening purposes at NIH (i.e., 5 rem). This reduced screening limit incorporates a safety factor
in light of not having human biodistribution and dosimetry data for this specific PET tracer.
3. PET Scanning
PET scanning, which detects injected radioactivity within the body, is associated with no
known physical hazards to the subject lying on the table. We routinely use a series of procedures
to minimize the risk for discomfort during scanning sessions. Namely, the procedures are
conducted in the presence of trained health professionals to whom subjects will have ready
access, should they experience any problems. Subjects can communicate with the trained health
professionals whilst in the scanner and can withdraw from the study at any time if they wish to
do so.
Occasionally subjects become anxious during the scan. In that case, subjects can request
the operator of the PET to stop the scan.
4. Arterial/Venous line Placement
Arterial catheterization has been shown to be a generally safe and reliable method to
obtain arterial blood samples (Lockwood 1985). Placement of a radial arterial catheter may
cause bruising or infection. There is also a risk of occlusion and microemboli. In the past, over
3,000 arterial catheters have been placed for PET studies at NIH. Of these, two complications
requiring physician’s care were reported. In the first case, a small radial artery aneurysm
developed several months later, which was successfully repaired surgically. In the second case,
a radial artery thrombosis developed 28 days later, which was also successfully repaired
surgically. The arterial line will be placed by a member of the anesthesiology staff after
confirming normal double circulation (both radial and ulnar arteries).
Venous catheter insertion, which is less invasive than arterial catheterization, can also be
associated with bruising, infection, or clot formation. Using proper placement techniques will
minimize these risks.
The needle stick and the insertion of arterial and venous catheters cause temporal
discomfort. To minimize the discomfort, for the arterial line placement, the insertion area will be
anesthetized with local injections of Novocain or an equivalent medication. Venous line
placement causes lower levels of discomfort and local anesthesia is not required.
5. Blood Sampling
Subjects will have no more than 250 mL blood sampling including that for lab tests.
Careful screening of health status and CBC will be done prior to the enrollment in the study.
Subjects will be asked not to donate blood for a period of eight weeks after the participation is
completed. The risks are associated with the arterial or venous catheterization as described in the
previous section.
6. MRI
MRI is not associated with any known deleterious biological effects. 1.5 Tesla MRI is
also widely used as a clinical imaging tool. Subjects will be screened and excluded for the
presence of any metallic prostheses both at the time of recruitment and just prior to MR imaging.
Subjects will wear ear-plugs to minimize exposure to excessively loud noises. Occasionally
Page 15 of 22
subjects become anxious during the scan. In that case, Valium® 2-4 mg or Ativan® 0.5-1 mg
may be given per oral before MRI upon a request by subjects. Subjects can also request the
operator to stop the scan.
Claustrophobic subjects find it difficult be scanned on MRI and subjects with this
condition will be excluded at the time of recruitment.
C. Benefits of study participation
There is no direct benefit to subjects participating in this protocol.
D. Investigator conflicts of interest
There are no investigator conflicts of interest in this protocol.
E. Privacy and confidentiality provisions
Every necessary step will be taken to prevent identification of study participants and
other violations of subject confidentiality. Information will be stored using a confidential case
number, and no identifiers (name, address, phone number, etc.) will be used that could allow
direct linking of database information to individual subjects. Where temporary linking of
information with identifiers is needed, such identifiers will be temporarily attached to the data,
and will be removed after information has been encoded. Secured e-mail will be used for all
electronic communications of subject information between investigators.
F. Adverse event reporting
The PI will report immediately all serious adverse events to the NIMH Clinical Director
verbally and the NIMH IRB and RSC verbally and in writing within the guidelines set by the
NIH Standards for intramural clinical research.
G. Data and safety monitoring processes
Demographic and clinical data will be archived in EXCEL on a PC server. Imaging and
blood data will be offloaded from the scanner/blood analyzing device to a PC or a SUN
workstation after each imaging session has been completed. Clinical Safety Monitoring data will
be archived together with other data. Laboratory test results will be stored on the CRIS.
H. Subject compensation
Reimbursement is based on NIH standards for time devoted to the research project.
Subjects will be paid for each portion of the study they have completed whether or not they opt
for early withdrawal from participation. (Please refer to Appendix I for breakdown of payment
schedule.)
VIII. PHARMACEUTICAL, BIOLOGIC AND/OR DEVICE INFORMATION
A. Source
The NIMH Radiochemistry Laboratory will synthesize and perform QC on the PET
tracer [11C]PBR28 in accordance with IND. As for other IND agents synthesized at the CC PET
Department, the FDA grants authority for such synthesis and QC. Furthermore, the FDA may
inspect the process at any time.
Page 16 of 22
B. Relevant pharmacology
PBR28 is an analog of an agonist at PBR, (N-5-fluoro-2-phenoxyphenyl)-N-(2,5dimethoxybenzyl) acetamide (DAA1106) (Okuyama et al 1999). Because of the tracer doses, no
pharmacological effects are expected with [11C]PBR28.
C. Toxicity
No effects, side effects or toxicity is expected from this radioligand, since it is
administered at tracer doses.
D. Formulation and preparation
[11C]PBR28 will be synthesized at the radiochemistry lab of MIB/NIMH by simple
methylation of its O-desmethyl analog with [11C]iodomethane followed by reverse phase HPLC
purification and formulation in normal saline.
E. Stability and storage
[11C]PBR28 will be administered within 60 minutes after synthesis of the radioligand.
The radioligand is stable during this period.
F. Incompatibilities
[11C]PBR28 will be administered at a tracer dose and no other medication will be
involved in this protocol. Therefore, we do not expect pharmacological effects or interactions
with concomitant medication the subject is taking.
G. Administration procedures
The radioligand is administered via an indwelling intravenous catheter over ~1 min.
Page 17 of 22
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Page 21 of 22
X. APPENDIX: REIMBURSEMENT SCHEDULE
Inconvenience Pay for
Time
Units
inconvenience (h)
(1)
Pay for
time (2)
Total Pay (1
+ 2)
7
$70
2
$30
$100
Visit 2 to NIH (outpatient)
MRI
9
$90
1
$20
$110
Visit 3 (outpatient)
PET scanning
Arterial catheter
Antecubital venous catheters
Pregnancy test
Movement restriction
10
6
3
1
1
$100
$60
$30
$10
$10
4
$50
$150
$60
$30
$10
$10
Visit 4 (outpatient)
Blood test and urinalysis
8
$80
1
$20
$50
Visit 1 (Outpatient)
History taking, physical exams,
blood test, and urinalysis
Total
$570
Page 22 of 22