MATERIALS AND METHODS
MRI
The anatomy of all monkey brains was examined by structural Magnetic Resonance Imaging
(sMRI) using a 1.5 T General Electrics Signa (GE Milwaukee, WI) system. A spoiled gradient
recalled (SPGR) sequence was acquired in the coronal plane with the following parameters:
TR = 21 ms; TE = 4 ms; flip angle = 35º; slice thickness = 1.0 mm; FOV = 12.8 cm; NEX = 2;
voxel size = 0.5  0.5  1 mm3.
Image Preprocessing and Regions of Interests
Before delineation of regions of interest (ROIs), the orientation of the monkey brain was
spatially normalized by reorienting the high resolution T1-weighted MR images so that the
line defined by the anterior and posterior commissures was parallel to the horizontal plane and
the inter-hemispheric plane parallel to the sagittal plane. The delineations of anatomical brain
regions were made manually on the reoriented MR images using the Human Brain Atlas
adapted for nonhuman primates (Roland et al., 1993). ROIs for the whole brain and
cerebellum were delineated on the horizontal projections. Brain MRIs were coregistered to the
averaged brain positron emission tomography (PET) images using SPM5 (Wellcome
Department of Imaging Neuroscience). The spatially transformed ROIs were displayed on the
corresponding PET images and pooled for each anatomical region. The radioactivity
concentration in each brain ROI was calculated for each sequential frame, corrected for
radioactive decay, and plotted vs time.
Materials
O-desmethyl sulpiride was obtained from lgc standards AB, N-desmethyl doxepin was
obtained from PharmaSynth AS, N-desmethyl citalopram was obtained from TLC
PharmaChem Inc., and N-desmethyl sertraline was obtained from Toronto Research
Chemicals Inc. Other drug standards, precursors, and chemicals were obtained from SigmaAldrich and used without further purification.
Radiochemistry and Quality Control
[11C]Methane was produced at the Karolinska University Hospital with a General Electrics
Medical Systems PETtrace cyclotron using 16 MeV protons in the 14N(p.α)11C reaction on
nitrogen gas. [11C]Methyl iodide was synthesized by recirculating [11C]methane through
heated iodine in an in-house developed apparatus as described elsewhere (Larsen et al.,
1997). [11C]Methyl triflate was in its turn prepared by sweeping [11C]methyl iodide vapor
through a heated glass column containing silver-triflate impregnated on graphitized carbon,
as previously described (Jewett et al., 1997).
Radiolabeling of all 13 reference drugs was achieved by trapping the alkylating
agent (11C-labeled methyl iodide or triflate) at room temperature in a reaction vessel
containing the appropriate reaction mixture. After completed entrapment and reaction,
radiolabeled drugs were purified using semipreparative high performance liquid
chromatography. During separations, the high performance liquid chromatography column
outlet was connected to an absorbance detector ( = 254 nm) in series with a GM-tube for
radiation detection. The product fraction was collected and isolated by on-line evaporation
using a carburetor or by solid phase extraction. Radiolabeled drugs were formulated in a 5%
solution of ethanol in phosphate buffered saline (pH 7.4) or in a mixture of phosphate
buffered saline and 30% propylene glycol in ethanol (5:3, vol/vol). Finally, the product
formulation was sterilized via membrane filtration through a 0.22-µm Millipore filter
(Waters).
The radiochemical purity of each product was determined by reverse phase
HPLC equipped with an absorbance detector ( = 254 nm) in series with a -flow detector
(Beckman) for radiation detection. The radiochemical identity of each radiolabeled drug was
determined prior to administration by co-injection with the authentic drug standard. The
chemical identity of the radiolabeled product was confirmed after the product had decayed by
ultra-performance tandem mass spectrometry analysis of the carrier associated with the
radioactive product.
Drug Formulations
Drugs were formulated for intravenous administration in their appropriate vehicles and
sterilized through membrane filtration (Millipore 0.22 µm, Waters) on the day of the
experiment. Drug retention on the sterile filter was assessed by HPLC analysis of drug
concentration in the formulation prior and post filtration.
REFERENCES
Jewett DM, Kilbourn MR, Lee LC (1997) A simple synthesis of [11C]dihydrotetrabenazine
(DTBZ). Nucl Med Biol 24:197–199.
Larsen P, Ulin J, Dahlstrom K, Jensen M (1997) Synthesis of [C-11]iodomethane by
iodination of [C-11]methane. Applied Radiation and Isotopes 48:153–157.
Roland PE, Graufelds CJ, Wǎhlin J, Ingelman L, Andersson M, Ledberg A, Pedersen J,
Åkerman S, Dabringhaus A, Zilles K (1993) Human brain atlas: for high-resolution
functional and anatomical mapping. Human Brain Mapping 1:173–184.
Reference drug
Caffeine
Citalopram
Clomipramine
Clozapine
Diazepam
Doxepin
Morphine
Nicotine
Pharmacological target
Adenosine receptor
Serotonin transporter
Serotonin transporter
Multiple
Gamma-aminobutyric acid receptor
Histamine H1 receptor
Opioid receptors
Nicotinic acetyl choline receptor
Functional activity
Antagonist
Inhibitor
Inhibitor
Antagonist. agonist
Positive modulator
Antagonist
Agonist
Agonist
Action
CNS stimulant
Antidepressant
Antidepressant
Antipsychotic
Anxiolytic
Anxiolytic. antidepressant
Analgesic
CNS stimulant
Brønstedt class
Base
Base
Base
Neutral
Neutral
Base
Base
Base
Seligiline
Sertraline
Sulpiride
Monoamine oxidase type B
Serotonin transporter
Dopamine D2/D3 receptors
Irreversible inhibitor
Inhibitor
Antagonist
Anti-parkinson drug
Antidepressant
Antipsychotic
Base
Base
Base
Venlafaxine
Serotonin/norepinephrine transporter
Inhibitor
Antidepressant
Base
Verapamil
Calcium channel
Blocker
Antiarrhythmic
Base
SUPPLEMENTAL TABLE 1. Reference drugs for PET microdosing. Brønstedt class refers to the propensity of the drug to be a proton acceptor
or donor at physiologic pH 7.4.
SUPPLEMENTAL FIGURE 1. Two-tissue compartment model for the disposition of
radioactivity in brain following administration of a microdose of radiolabeled drug. K1 is
expressed in mL cm3-1 min-1 and k2, k3 and k4 are expressed in min-1.
Drug
[11C]Caffeine
[11C]Citalopram
[11C]Clomipramine
[11C]Clozapine
[11C]Diazepam
[11C]Doxepin
[11C]Morphine
[11C]Nicotine
[11C]Selegiline
[11C]Sertraline
[11C]Sulpiride
[11C]Venlafaxine
[11C]Verapamil
Cmax, %ID
Cmax, SUV
brain
brain
(%ID)
(SUV)
5.0
4.6
4.0
3.0
3.2
2.8
2.9
2.7
3.6
2.7
3.7
3.4
5.2
3.9
3.5
3.7
2.5
2.2
4.2
3.1
5.6
5.7
5.5
5.3
1.1
1.0
1.2
1.1
6.2
5.7
5.1
4.7
3.7
2.7
4.8
4.5
4.8
4.9
4.3
4.1
1.4
1.3
1.3
1.2
2.0
1.8
3.9
2.9
1.3
1.2
1.0
1.0
Tmax
brain
(min)
1.3
0.8
35
54
42
42
32
54
3.5
1.8
36
42
0.17
0.17
2.5
7.5
NA
2.5
90
102
0.2
0.2
11
17
0.75
0.75
SUPPLEMENTAL TABLE 2. Pharmacokinetic parameters for intravenously injected
microdoses of 11C-labeled drugs in two different cynomolgus monkeys.
AUC brain0-90min/AUCplasma0Dose
Drug
(mg/kg)
11
[ C]Caffeine
micro
2.5
11
[ C]Citalopram
micro
3
11
[ C]Clomipramine micro
0.8
11
[ C]Clozapine
micro
1.5
11
[ C]Diazepam
micro
2
11
[ C]Doxepin
micro
0.1
11
[ C]Morphine
micro
0.5
11
[ C]Nicotine
micro
0.12
11
[ C]Selegiline
micro
0.5
11
[ C]Sertraline
micro
1
11
[ C]Sulpiride
micro
2
11
[ C]Venlafaxine micro
2
11
[ C]Verapamil
micro
0.3
90 min
(KP)
0.75
0.76
11.7
11.7
20.1
20.7
11.9
9.6
2.0
1.7
25.5
20.9
0.36
0.19
5.5
6.3
11.2
5.9
24.6
25.5
0.0029
0.0022
8.0
8.3
3.2
2.7
VT Difference relative to
(KP)
VT (%)
0.77
-2.7
0.80
-4.6
21.6
-46
17.4
-33
41.8
-52
35.4
-42
16.5
-27
13.0
-27
2.6
-23
1.8
-2.3
46
-44
29.1
-28
NA
NA
NA
NA
8.4
-34
8.8
-29
NA
NA
12.2
-51
68.4
-64
66.0
-61
NA
NA
NA
NA
10.3
-22
11.4
-27
6.5
-50
2.8
-1.0
SUPPLEMENTAL TABLE 3. Comparison of brain/plasma partition coefficients obtained
using the AUC ratio method (AUC brain0-90min/AUCplasma0-90 min) and kinetic modeling (VT).
Drug
Condition
parent
fraction parent
(2.5
fraction
min)
(45min)
Caffeine
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
Microdose
Pharmacologic dose
0.98
0.98
0.83
0.78
0.84
0.92
0.97
0.97
0.94
0.91
0.96
0.96
0.13
0.16
0.95
0.91
0.92
0.88
0.96
0.95
0.99
0.99
0.97
0.92
0.96
0.98
Citalopram
Clomipramine
Clozapine
Diazepam
Doxepin
Morphine
Nicotine
Selegiline
Sertraline
Sulpiride
Venlafaxine
Verapamil
0.97
0.963
0.45
0.45
0.16
0.15
0.44
0.43
0.2
0.25
0.17
0.14
0.04
0.05
0.13
0.11
0.14
0.11
0.32
0.32
0.89
0.83
0.19
0.18
0.16
0.19
SUPPLEMENTAL TABLE 4. Data on parent 11C-labeled drug in plasma at two time-points
after intravenous injection into rhesus monkeys.