Radiosynthesis, ex Vivo Biodistribution, and in Vivo Positron Emission Tomography Imaging Evaluations of [11C]2-Pyridinealdoxime Methiodide ([11C]2-PAM): A First-In-Class Antidote Tracer for Organophosphate Intoxication

Article abstract of DOI:10.1021/acschemneuro.8b00212

2-Pyridinealdoxime methiodide (2-PAM) is a widely used antidote for the treatment of organophosphorus (OP) exposure that reactivates the target protein acetylcholinesterase. Carbon-11 2-PAM was prepared to more fully understand the in vivo mode of action, distribution, and dynamic qualities of this important countermeasure. Alkylation of 2-pyridinealdoxime with [¹¹C]CH₃I provided the first-in-class [¹¹C]2-PAM tracer in 3.5% decay corrected radiochemical yield from [¹¹C]CH₃I, >99% radiochemical purity, and 4831 Ci/mmol molar activity. [¹¹C]2-PAM tracer distribution was evaluated by ex vivo biodistribution and in vivo dynamic positron emission tomography (PET) imaging in na?ve (OP exposure deficient) rats. Tracer alone and tracer coinjected with a body mass-scaled human therapeutic dose of 30 mg/kg nonradioactive 2-PAM demonstrated statistically similar tissue and blood distribution profiles with the greatest uptake in kidney and significantly lower levels in liver, heart, and lung with lesser amounts in blood and brain. The imaging and biodistribution data show that radioactivity uptake in brain and peripheral organs is rapid and characterized by differential tissue radioactivity washout profiles. Analysis of arterial blood samples taken 5 min after injection showed ～82% parent [¹¹C]2-PAM tracer. The imaging and biodistribution data are now established, enabling future comparisons to outcomes acquired in OP intoxicated rodent models.

Full text of DOI:10.1021/acschemneuro.8b00212

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Article
Radiosynthesis, Ex Vivo Biodistribution, and In Vivo Positron
Emission Tomography (PET) Imaging Evaluations of [11C]2-
Pyridinealdoxime Methiodide ([11C]2-PAM): A First-In-
Class Antidote Tracer for Organophosphate Intoxication
Keil D. Neumann, Joseph E Blecha, Thomas R Hayes, Tony Huynh, Chih-Kai Chao, Nicolas
Guilloteau, Kurt R Zinn, Henry F. VanBrocklin, Charles Mark Thompson, and John M. Gerdes
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00212 • Publication Date (Web): 02 Aug 2018
Downloaded from http://pubs.acs.org on August 7, 2018
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Radiosynthesis, Ex Vivo Biodistribution, and In Vivo Positron Emission
Tomography (PET) Imaging Evaluations of [11C]2ꢀPyridinealdoxime Methiodide
([11C]2ꢀPAM): A FirstꢀInꢀClass Antidote Tracer for Organophosphate Intoxication.
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Kiel D. Neumann,† Joseph E. Blecha,† Thomas R. Hayes,† Tony Huynh,† Chihꢀ
Kai Chao,‡ Nicolas Guilloteau,‡ Kurt R. Zinn,§ Henry F. VanBrocklin,† Charles M.
Thompson,‡ and John M. Gerdes*‡
†Department of Radiology and Biomedical Imaging; University of California, San
Francisco; San Francisco, California, 94143, United States
‡Department of Biomedical and Pharmaceutical Sciences, University of Montana,
Missoula, Montana, 59812, United States
§ Departments of Radiology and Biomedical Engineering, Michigan State
University, East Lansing, Michigan, 48824, United States
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ABSTRACT
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2ꢀPyridinealdoxime methiodide (2ꢀPAM) is a widely used antidote for the
treatment of organophosphorus (OP) exposure that reactivates the target protein
acetylcholinesterase. Carbonꢀ11 2ꢀPAM was prepared to more fully understand
the in vivo mode of action, distribution and dynamic qualities of this important
countermeasure. Alkylation of 2ꢀpyridinealdoxime with [11C]CH3I provided the
firstꢀinꢀclass [11C]2ꢀPAM tracer in 3.5 % decay corrected radiochemical yield from
[11C]CH3I , >99% radiochemical purity and 4,831 Ci/mmol molar activity. [11C]2ꢀ
PAM tracer distribution was evaluated by ex vivo biodistribution and in vivo
dynamic positron emission tomography (PET) imaging in naïve (OP exposure
deficient) rats. Tracer alone and tracer coꢀinjected with a body massꢀscaled
human therapeutic dose of 30 mg/kg nonꢀradioactive 2ꢀPAM, demonstrated
statistically similar tissue and blood distribution profiles with the greatest uptake in
kidney with significantly lower levels in liver, heart and lung with lesser amounts in
blood and brain. The imaging and biodistribution data show that radioactivity
uptake in brain and peripheral organs is rapid and characterized by differential
tissue radioactivity washout profiles. Analysis of arterial blood samples taken 5
min after injection showed ~82% parent [11C]2ꢀPAM tracer. The imaging and
biodistribution data are now established enabling future comparisons to outcomes
acquired in OP intoxicated rodent models.
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KEYWORDS
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2ꢀpyridinealdoxime methiodide (2ꢀPAM)
antidote
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biodistribution
carbonꢀ11
organophosphate
PET imaging
INTRODUCTION
The therapeutic agent 2ꢀpyridinealdoxime methiodide (2ꢀPAM, pralidoxime;
Figure 1) is widely accepted as an antidote therapy in the US for the reactivation
of acetylcholinesterase (AChE) that has been inhibited by organophosphorus
compounds such as chemical warfare agents and certain insecticide oxons
(Scheme 1).1-3 The combination of 2ꢀPAM with other centrallyꢀacting drugs such
as a muscarinic blocker (e.g., atropine4) and antiꢀseizure medication (midazolam5,
6) along with other supportive regimens define the standard of care for OP
exposures, including selfꢀpoisoning.7
Figure 1. Chemical structures of the pyridine aldoximes.
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Scheme 1. Inhibition of acetylcholinesterase by organophosphorus compounds
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and reactivation of the OPꢀAChE adduct by 2ꢀPAM.
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The mechanism by which 2ꢀPAM or P2S (the analogous methanesulfonate
salt), in addition to other select oximes, regenerates acetylcholinesterase
enzymatic activity occurs via nucleophilic displacement at the phosphorus atom of
the serineꢀOꢀphosphoryl linkage (Scheme 1) by the oxime C=NꢀOH moiety. The
charged pyridinium ring of 2ꢀPAM plays a supportive role in this mechanism as it
is believed to initially guide the antidote to the cationꢀattracting, peripheral binding
domain of AChE and aids docking in the active site similar to that achieved by the
natural substrate acetylcholine that also bears a quaternary amine. As an
antidote therapeutic to OP intoxication, 2ꢀPAM reacts selectively with OPꢀmodified
AChE and other OPꢀadducted esterases. In the absence of OPꢀAChE and
esterase modifications, 2ꢀPAM is without a specific high affinity in vivo target.
Despite the favorable role of the cation moiety in the enzyme activity
restoration mechanism, it is believed that cationic 2ꢀPAM is primarily distributed to
peripheral tissues because the blood–brain barrier (BBB) impedes facile diffusion
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of the cationic antidote into the central nervous system (CNS).8, 9 Diminished CNS
access by charged therapeutic agents such as 2ꢀPAM limits their usefulness.
However, select studies reported that 2ꢀPAM does reactivate OPꢀmodified AChE
in rat brain.10 In support of that work, Sakurada and colleagues reported that 2ꢀ
PAM enters rat brain in a doseꢀdependent manner in the absence of OP treatment
using microdialysis measures.11 Additionally, it has been shown that [14C]2ꢀPAM
enters brain more readily following exposure to the OP trichlofon (Dipterex; O,Oꢀ
dimethyl, 2,2,2ꢀtrichloroꢀ1ꢀhydroxyethyl phosphonate).10, 12 Although low
concentrations of 2ꢀPAM can enter brain, it remains less clear how brain levels
might vary over time within the first hour of 2ꢀPAM administration, as a function of
the absence or presence of OP exposure. In an effort to further interrogate 2ꢀPAM
brain penetration under these and related conditions, we sought to generate and
initially evaluate carbonꢀ11 (t1/2 = 20.4 min) radiolabeled 2ꢀPAM ([11C]2ꢀPAM)
tracer that would enable facile ex vivo and in vivo CNS and peripheral tissue
determinations.
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The [11C]2ꢀPAM tracer was considered ideal for preliminary assessments of
pharmacokinetic (PK) properties in rat CNS and peripheral tissues by ex vivo
biodistribution (bioꢀd) and in vivo positron emission tomography (PET) imaging
evaluations. The acquired tracer data would ultimately enable correlation to
previously established 2ꢀPAM biochemical and toxicological study outcomes.1-3, 7,
11-14
In this study, we report the radiosynthesis of a firstꢀinꢀclass [11C]2ꢀPAM
tracer and initial evaluations of the tracer using naïve rats that have not
undergone OP exposure. The assessments include ex vivo bioꢀd measures,
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blood metabolite assessment and in vivo PET imaging determinations after
administration of tracer alone (baseline profiles). Additionally, we describe select
experiments, in which a therapeutic dose level of nonꢀradioactive (cold) 2ꢀPAM
was coꢀadministered with the tracer. Together, the naïve rat [11C]2ꢀPAM tracer
profiles serve as fundamental data for comparisons to related future tracer
measures in OP intoxicated rats.
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RESULTS AND DISCUSSION
The high molar activity radiosynthesis of [11C]2ꢀPAM was developed based
on literature precedent15 from the readily accessible radiolabelling synthon
[11C]methyl iodide ([11C]CH3I)16 and 2ꢀpyridinealdoxime, such that the tracer could
be generated by a oneꢀstep alkylation procedure (Scheme 2). Special
considerations were taken using the [11C]CH3I approach since most cold
preparations of Nꢀmethyl pyridinium ions use a large surplus of the alkyl iodide
and long reaction times relying on precipitation of the salt for isolation. Preliminary
cold chemistry studies using a large inverse ratio of 10ꢀ to 50ꢀfold 2ꢀ
pyridinealdoxime relative to methyl iodide revealed that a high conversion (> 80%
based on methyl iodide as the limiting reagent) to 2ꢀPAM could be accomplished
in less than 5 min at 120 ˚C in dimethylformamide (DMF). Acetonitrile (CH3CN)
was also found as a suitable solvent and led to ~ 40% conversion to cold product
under similar conditions. Methylation in other solvents such as DMSO, Nꢀ
methylpyrrolidine, and dimethoxyethane (DME) were found to be less effective
than DMF or CH3CN when a surplus of 2ꢀpyridinealdoxime was used. The nonꢀ
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radioactive reaction yield in either DMSO or CH3CN was not improved at
temperatures below 90 ˚C nor above 120 ˚C (sealed tube conditions).
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Scheme 2. Radiosynthesis of tracer [11C]2ꢀPAM..
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Adaptation of the modified cold synthetic route to the corresponding
radiosynthesis required [11C]CH3I, which was prepared by an established gasꢀ
phase automated method.16 In this approach, [11C]CH3I was bubbled into a
solution of 2ꢀpyridinealdoxime (1 mg) in CH3CN, the reaction vessel was sealed
and heated at 120 ˚C for 8 min, then cooled briefly, diluted with saline and the
tracer was purified by reversedꢀphase high performance liquid chromatography
(HPLC). The rapid formation of [11C]2ꢀPAM occurred in a decayꢀcorrected
radiochemical yield of 3.5 ± 0.9 % (n = 8), relative to [11C]CH3I with a measured
molar activity of 4,831 ± 911 Ci/mmol (n = 8), >99% radiochemical purity, and in a
total synthesis time of 40 min from delivery of [11C]CH3I. A similar protocol using
DMF as the reaction solvent at 140 ˚C afforded a higher decayꢀcorrected tracer
yield (16.5 ± 6.5%, n = 3) relative to [11C]CH3I; yet, purification by HPLC was
difficult when DMF was used as reaction solvent, resulting in a lower tracer
radiochemical purity (85ꢀ90%, see: Supporting Information). A limited series of
reactions was conducted in an attempt to increase the yield of [11C]2ꢀPAM using
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CH3CN as the reaction solvent. Adjusting temperature (80ꢀ120˚C) and mass of
precursor (5ꢀ10 mg) did not afford an improved yield of [11C]2ꢀPAM (per
Supporting Information, Additional Labeling Studies for [11C]2ꢀPAM Preparation,
Table S1). Since the use of CH3CN solvent was superior for gaining higher final
tracer purity, albeit affording tracer in lower radiochemical yield, the CH3CN
protocol was used for the reported preclinical studies. Tracer doses, formulated in
~ 1 mL sterile isotonic saline, were found to be stable for > 2 h at room
temperature by HPLC analysis and up to 10 mCi (nonꢀdecay corrected) of final
formulated [11C]2ꢀPAM tracer was typically achieved.
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To initially appraise CNS and peripheral tissue tracer uptake profiles, bioꢀd
determinations and PET imaging studies were performed using 250ꢀ400 g (mean
body weight 305 g) male SpragueꢀDawley rats (n = 3 per group) that were
administered [11C]2ꢀPAM by tail vein intravenous (i.v.) injection under light
isoflurane (1ꢀ2%) anesthesia. Biodistribution experiments used 100ꢀ200 µCi tracer
doses (1.0ꢀ1.5 mL) where the initial studies were carried out with tracer alone
(baseline conditions). Baseline ex vivo biodistribution radioactivity profiles (blood
and tissues) were sampled at select 2ꢀ60 min time points after tracer injection
(Figure 2), for blood, brain, liver, heart, kidney and bone (femur). The samples
were counted affording data as decayꢀcorrected percent injected dose
radioactivity per gram tissue values (% ID/g) ± standard error measures (SEM).
The bioꢀd radioactivity profiles reveal highest uptake in kidney with significantly
diminished amounts in liver, lower uptake in lung and heart, and the lowest
radioactivity in blood, brain and bone. For all samples, timeꢀdependent
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radioactivity uptake and washout profiles were observed for the tissues and blood
over 60 min. The rapid radioactivity uptake and washout seen within naïve rat
tissues (i.e., lack of an OPꢀAChE modification) are consistent with the absence of
a high affinity 2ꢀPAM target within the tissues and blood.
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The elevated radioactivity in kidney at 2 and 5 minutes was anticipated for
a cationic, highly water soluble compound and thought to be associated with
clearance.17, 18 Limited brain radioractivity uptake observed at all times is
considered to be a function of the diminished ability of cationic [11C]2ꢀPAM (and/or
related species) to diffuse across the BBB.11, 12 Applying the averageꢀsized rat
brain blood volume of ~ 30ꢀ35 µL/g of tissue19 for the blood and brain % injected
dose (ID) per gram profiles over time (Figure 2), indicates that ~ 8ꢀ15 % of the
brain radioactivity detected over the 2ꢀ60 minutes is contributed from the cerebral
blood pool radioactivity within brain tissue. Thus, the majority of the brain
radioactivity (Figure 2) is associated with tissue (see Methods section and
Supporting Information).
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Figure 2. Mean baseline (tracer alone, n = 3, ± SEM) biodistribution (2ꢀ60 min) of
decayꢀcorrected radioactivity after [11C]2ꢀPAM tracer i.v. injection in naïve rats.
Tracer metabolism events are thought to contribute to the liver radioactivity
levels (Figure 2).12, 20 Since the highest liver radioactivity was observed at 5 min,
evaluation of blood nonꢀproteinꢀbound fraction components was conducted to
assess the amount of parent tracer activity relative to metabolites. Tail artery
blood sampling at 5 minutes, blood sample processing and chromatographic
profiling against nonꢀradioactive (cold) chemical metabolite standards afforded a
distribution of radioactivity components (Figure 3). At 5 minutes after tracer
injection, the nonꢀproteinꢀbound fraction showed 82.3% parent [11C]2ꢀPAM tracer
and a mixture of metabolites including 4.4% Nꢀmethylꢀ2ꢀ pyridinecarboxaldehyde,
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4.7% of a mixture of Nꢀmethylꢀ2ꢀ pyridinecarboxamide, Nꢀmethylꢀ2ꢀ
pyridinecarboxylic acid, Nꢀmethylꢀ2ꢀpyridinecarbonitrile, and 8.6% Nꢀmethylꢀ2ꢀ
pyridone (Figure 3). The metabolite structures and relative distribution are
consistent with 2ꢀPAM metabolites reported previously,12, 15, 20 albeit with some
minor differences that could result from dose sizes and rat strains. The Figure 3
metabolism results provide insight into the maximal 5 min liver bioꢀd profile. At
present it remains unclear whether uptake of the metabolites into brain occurs,
are produced in brain, or both.
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Parent Tracer
Metabolites
N
N
OH
N
CHO
N
R
N
O
[11C]CH3
[11C]CH3
[11C]CH3
[11C]CH3
R = CONH2,
CO2H, CN
82.3%
4.4%
4.7%
8.6%
Figure 3. Plasma freeꢀfraction relative radioactive components determined by
chromatographic separation and counting at 5 min after tracer injection.
We sought to further interrogate the 5 min bioꢀd time point since previous
studies with 2ꢀPAM have shown a doseꢀdependent increase in brain
penentration.11 Our initial appraisal used tracer that was coꢀinjected with a 30
mg/kg of cold 2ꢀPAM dose that is akin to a human 30 mg/kg therapeutic 2ꢀPAM
dose,1, 3, 11 where the total mass of cold 2ꢀPAM injected was not allometrically
scaled. The experiment was designed to compare the presence vs. absence of
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cold 2ꢀPAM in relation to activity uptake and washout profiles in naïve rats in the
absence of an OPꢀAChE modification. Further, differential tracer tissue
interactions associated with metabolism, clearance or other processes might also
be detected in the presence of cold therapeutic agent. The results are shown in
Figure 4 with baseline (tracer alone) versus 30 mg/kg cold 2ꢀPAM dose coꢀ
injected with tracer. Oneꢀway ANOVA analyses show no significant differences
between the two tracer dose regimens, [11C]2ꢀPAM only (baseline) versus tracer
in the presence of 30 mg/kg cold 2ꢀPAM, in most of tissue activity distributions at
5 min except the liver (P = 0.0085). The significant elevated liver activity in the
presence of cold 2ꢀPAM is thought to be a result of altered tracer metabolic
processes. No significant difference in 2ꢀPAM brain radioactivity uptake between
tracer alone and tracer plus therapeutic cold 2ꢀPAM dose experiments were
found, suggesting that the uptake was not effected by mass dose level. With the
exception of liver and in the absence of OPꢀAChE modification, the naïve rat
activity biodistribution PK profiles resulting from [11C]2ꢀPAM tracer low mass
doses (Figure 4) are thought to be similar to those of higher mass doses of nonꢀ
radioactive 2ꢀPAM.17, 18
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Figure 4. Comparison of the mean (n = 3, ± SEM) radioactivity (decayꢀ corrected)
biodistributions of baseline [11C]2ꢀPAM (tracer alone, aqua) and [11C]2ꢀPAM coꢀ
injected with 30 mg/kg cold 2ꢀPAM (purple) at 5 min after respective tracer i.v.
injections in naïve rats.
To distinguish the in vivo radioactivity tissue distributions in naïve rats, 30
min dynamic in vivo PET scanning (5 min frames) was employed that used 0.5ꢀ
1.0 mCi tracer i.v. doses. The 30 min scan time was based on the rapid activity
uptake and washout defined by the bioꢀd profiles (Figure 2). The PET scans were
carried out to afford in vivo data for comparison to the ex vivo biodistribution
profiles. The PET imaging was carried out in parallel with microꢀcomputed axial
tomography (CT) and magnetic resonance (MR) scanning for the sake of
anatomical tissue information and data coꢀregistration allowing for conservatively
defined volumetric threeꢀdimensional regions of interest (ROIs) relative to
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established rat brain and peripheral tissue atlas designations.21-23 Regional
radioactivity signals were determined as standardized uptake values (SUV, per
Methods section)24 that permitted imaging evaluations across rats for
determinations of the mean (n = 3) SUV values ± SD per ROI vs. time (min). The
resultant tracer baseline and tracer in the presence of cold 2ꢀPAM timeꢀactivity
curves (Figure 5) and expanded the brain TAC data (Figure 6) were obtained. A
typical sagittal PET view of summed radioactivity coꢀregistered with CT data, after
baseline [11C]2ꢀPAM tracer injection is depicted in Figure 7.
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Figure 5. The mean values (n = 3, ± SD) PET imaging detected radioactivity
(decay corrected and subject normalized as SUV) vs. time (min) curves in naïve
rats after [11C]2ꢀPAM tracer: Panel A, tracer alone (baseline) and Panel B, tracer
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coꢀinjected with 30 mg/kg cold 2ꢀPAM (see Supporting Information for expanded
plots of these curves).
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Figure 6. Expanded view PET imaging brain timeꢀactivity curves of mean SUV
(n = 3, ± SD) vs. time (min) for [11C]2ꢀPAM tracer alone (baseline; open circles
and solid line) and tracer coꢀinjected with 30 mg/kg cold 2ꢀPAM (solid points and
dashed line).
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Figure 7. A PETꢀCT sagittal (cranial, rostral left; caudal right) view near midꢀline
(MR data omitted for clarity) of 30 min summed activity (SUV color bar) after
injection of 0.95 mCi of [11C]2ꢀPAM tracer alone (baseline) in a male 330 g rat.
Baseline PET imaging TAC plots (Figure 5, Panel A) show high
radioactivity in kidney, less in liver, low levels in heart and lung and lower level
activity in whole brain. These relative in vivo SUV profiles are similar to the ex vivo
biodistribution radioactivity determinations (Figure 2). An analogous assessment
was made when [11C]2ꢀPAM was coꢀinjected with 30 mg/kg of cold 2ꢀPAM (Figure
5, Panel B) showing the similarity (Panel A vs. Panel B) and significant amounts
of radioactivity washout by 25 min after tracer injection. The relative distributed
amounts of radioactivity detected (Figure 5, Panel A and B) are found similar to
the ex vivo bioꢀd profiles of Figure 2; that is, radioactivity distribution profiles as
kidney >> liver > heart, lung > brain. The elevated radioactivity found in kidney
and liver as compared to heart and lung (Figure 5) likely represents greater tissue
reservoirs for 2ꢀPAM (and related radiolabeled species) associated with clearance
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and metabolism, respectively. The Figure 5 mean radioactivity liver values at five
minutes after tracer injection do not clearly show a distinction between the Panel A (tracer
alone) vs. Panel B (tracer in the presence of 30 mg/kg cold 2ꢀPAM), whereas differences
are found from the similar liver bioꢀd determinations as shown in Figure 4. The reasons
for the differences remain unclear. The expanded naïve rat brain TAC plots (Figure
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6) for baseline and the coꢀinjection of cold 2ꢀPAM experiments reveal that the
curves are not significantly different from each other. Radioactivity enters brain
rapidly, albeit at low levels, and reaches a steady concentration in brain after 5
min. Low brain radioactivity uptake was anticipated based on previous
biodistribution reports using naïve rats.11, 12
The PET imaging results demonstrate that the use of the short carbonꢀ11
halfꢀlife [11C]2ꢀPAM tracer within naïve rats is suitable for quantitatively detecting
radioactivity PK uptake and washout changes in tissues of interest. The baseline
PET data reveals radioactivity distributions that are relatively uniform relative to
the ex vivo timeꢀactivity biodistribution determinations. Within naïve rat tissues,
the PET TAC data from the coꢀinjection of nonꢀradioactive 2ꢀPAM is similar to
baseline profiles, which we consider consistent for the naïve rat condition that is
devoid of an OPꢀAChE adduct (Scheme 1) thereby lacking a high affinity 2ꢀPAM
target.
CONCLUSIONS
To gain deeper insights into the pharmacokinetic properties of the
therapeutic antidote 2ꢀPAM, the firstꢀinꢀclass [11C]2ꢀPAM tracer was selected for
study initially within naïve (OP exposure deficient) rats that lack a high affinity in
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vivo target for the tracer. The tracer was prepared by a straightforward approach
using a oneꢀstep alkylation reaction with [11C]CH3I followed by HPLC purification,
providing stable (2 h) salineꢀformulated dose preparations that were administered
intravenously. Initial uptake and washout radioactivity PK assessments within
CNS and peripheral tissues, and also blood, were made by ex vivo biodistribution
profiling over 60 min. Radioactivity distributions were realized for baseline (tracer
alone) and at the selected 5 min time with tracer coꢀinjected with cold 2ꢀPAM
(human therapeutic dose level, 30 mg/kg). A range of high to low radioactivity was
found in blood and tissues; respectively, kidney >> liver > lung and heart >>
blood, brain and bone. The high kidney radioactivity was anitcipated for a water
soluble cationic compound, and similarly, the low brain activity was expected
based on the diminished ability of cationic species to diffuse across the BBB.
Realizing the liver activity maxima at 5 min could be related to previously
described 2ꢀPAM metabolic transformations, and that this activity level was
affected by cold 2ꢀPAM coꢀadministration, arterial blood collection under baseline
conditions at 5 min and subsequent processing revealed plasma freeꢀfraction
activity components as 82.3% parent [11C]2ꢀPAM tracer and just 17.7% as
cognate metabolites previously described in the literature.
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Dynamic in vivo PET imaging for 30 min using [11C]2ꢀPAM tracer under
baseline condtions and also tracer coꢀinjected with 30 mg/kg cold 2ꢀPAM revealed
CNS and peripheral tissue radioactivity distributions that were relatively similar to
the timeꢀactivity profiles established by the ex vivo biodistribution experiments.
The nonꢀradioactive 2ꢀPAM plus tracer conditions were found without significant
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differences on brain radioactivity profiles over 30 min, which was expected for
naïve rats that lack a highꢀaffinity OPꢀAChE adduct target. The PET imaging
confirms low radioactivity uptake into brain as previously described in the
literature for nonꢀradioactive 2ꢀPAM. In summary, the naïve rat ex vivo
biodistribution and in vivo PET imaging timeꢀactivity measures after [11C]2ꢀPAM
tracer i.v. injection serve as fundamental correlated data sets. This requisite data
is now poised to enable meaningful comparisons relative to tracer determinations
in OPꢀintoxicated rats in which the detection of [11C]2ꢀPAM participation in the
critical therapeutic regeneration of acetylcholinesterase activity will be probed.
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METHODS
General. Reagents (e.g., 2ꢀpyridinealdoxime, iodomethane, et al.), solvents such
as anhydrous dimethylformamide (DMF), anhydrous acetonitrile (CH3CN), and 2ꢀ
pyridinealdoxime methiodide were reagent grade or better, used without any
additional purification, and were purchased from Sigma Aldrich Chemical
Company (Milwaukee, WI). USPꢀgrade sterile isotonic saline was also purchased
from SigmaꢀAldrich. 2ꢀPyridinecarboxaldehyde, 2ꢀpyridinecarboxamide, 2ꢀ
pyridinecarboxylic acid and 2ꢀpyridinecarbonitrile were converted into the Nꢀ
methyl pyridium metabolite standards per literature methods.25, 26 Nuclear
magnetic resonance (NMR) data were recorded in CDCl3 on a Varian Avance
400ꢀMHz spectrometer. High performance liquid chromatography (HPLC) was
performed with a Waters 590 system (Milford, MA) coupled to a Shimadzu SPD
UVꢀVisible detector (Columbia, MD) and a gamma counting inꢀline radiation flow
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detector (Model 105s, CRA; Berkeley, CA). The HPLC data was collected with a
SRI Peaksimple, model 304, data system (Torrance, CA). Counting of tissue and
blood samples utilized a Hidex automated gamma counter (Turku, Finland).
Male SpragueꢀDawley rats (250ꢀ400 g; Charles River, Inc., Skokie, IL)
were used for the [11C]2ꢀPAM biodistribution, blood and PET imaging studies. The
animals were cared for and used at the University of California, San Francisco
(UCSF) facilities that are accredited by the American Association for Accreditation
of Laboratory Animal Care (AAALAC). The animal studies adhered to UCSF
IACUC approved protocols that satisfied NIH guidelines and institutional
regulations. Prior to injection of [11C]2ꢀPAM, rats were lightly anesthetized (~1ꢀ2%
isoflurane) and lateral tail vein catheters were installed in the lower tail portion.
The catheter line was flushed with 200 ꢁL of saline and capped. Thereafter, the
catheter cap was removed and tracer was injected as a bolus and then the
catheter was flushed with 0.3 mL saline.
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Synthesis of 2- pyridinealdoxime methiodide (2-PAM) under limiting methyl
iodide reagent conditions. 2ꢀPyridinealdoxime (10 mg; 0.082 mmol) was
dissolved in 200 µL DMF at 125 °C or CH3CN at 80 °C and added drop wise to a
solution of methyl iodide (2.0 µL) in 50 µL DMF or CH3CN over 5 min. After the
addition, the reaction was kept at the indicated temperature for 5 min. The
percent conversion to 2ꢀPAM was 80ꢀ90% (DMF) and 35ꢀ40% (CH3CN) as
determined by NMR integration of aldoxime CH=N peaks or by the ratio of the Nꢀ
CH3 to aromatic region in d6ꢀDMSO. Conversion to 2ꢀPAM in CH3CN was 40 ±
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4% (using 2ꢀPAM as internal standard) as measured by reversedꢀphase HPLC
(UV at 290 nm) following evaporation of the solvent in vacuo using a Sonoma
C18(2) 5µ 100Å 15cm x 4.6mm (ES Industries; West Berlin, NJ) with a mobile
phase of 10% C2H5OH/10 mM 2ꢀ(Nꢀmorpholino)ethanesulfonic acid in H2O at 1.0
mL/min.
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Radiosynthesis of [11C]2-PAM. 2ꢀPyridinealdoxime (1ꢀ10 mg) is dissolved in
300 ꢁL of anhydrous CH3CN in a 4 mL 1ꢀdram vial and sealed with a Teflonꢀlined
silicone septum screwꢀcap. Utilizing a gasꢀphase method16 and a GE Tracerlab
FX C Pro radiosynthesis box, [11C]CH3I was produced then bubbled into the
CH3CN solution and the reactor vial was sealed. The mixture was heated at 80ꢀ
140 °C for 8 min on an aluminum block heater. The reactor vial was removed from
the heating source and allowed to cool for 60 seconds. The cap was removed, the
CH3CN solution was diluted with 2.5 mL of saline, and the crude material was
injected onto a 10 x 250 mm, 10 ꢁm Hamilton PRPꢀ1 HPLC column. The HPLC
purification used saline as mobile phase at a flow rate of 3 mL/min, with a retention time
of [11C]2ꢀPAM at 8.5 min (see Supporting Information, Chromatograms section for
representative HPLC elution profiles, and the Additional Radiolabeling Studies for [11C]2ꢀ
PAM Production section).
Dose Formulation. Doses of [11C]2PAM were formulated in sterile isotonic saline
(pH ~ 7) as 100ꢀ200 µCi in 1.0ꢀ1.5 mL for the biodistribution and blood
determinations, and also 0.5ꢀ1.0 mCi in 0.5ꢀ1.0 mL for the PET imaging
evaluations; in which the average dose volume across both sets of experiments
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was ~ 1.0 mL. Before animal dosing, quality control of the dosing was
accomplished by analytical HPLC (7µm Hamilton PRPꢀ1 4.6 x 250 mm flow rate
1mL/min) demonstrating >99% radiochemical purity and >95% chemical purity. In
separate experiments, doses were allowed to remain at room temperature for 2 h,
and then subjected to reversedꢀphase analytical HPLC analysis (see Supporting
Information).
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Rat Biodistribution Studies. Rats received a bolus injection of 100ꢀ200 ꢁCi of
[11C]2ꢀPAM via tail vein catheter port followed by a 0.3 mL saline flush. Rats were
euthanized by lateral chest puncture under anesthesia and dissected at 2, 5, 10,
30, and 60 min after injection (n = 3 per time point). Blood was collected by
cardiac puncture immediately prior to euthanasia at each time point. Whole
organs of brain, liver, heart, kidneys, lungs, bone (femur) and blood were
collected into 20 mL preꢀweighed glass scintillation vials and capped, where they
were placed into a Hidex automated gamma counter (Hidex AMG) to be counted
and weighed. Decay corrected data was exported from the Hidex instrument to
Excel (Microsoft Office) software and plotted as decay corrected percent injected
dose per gram (% ID/g) ± SEM versus time (min). To compare the % ID/g values
of the two tracer dose regimens ( i.e., tracer alone and tracer in the presence of
30 mg/kg of cold 2ꢀPAM) oneꢀway ANOVA analyses were performed for each
tissue and blood using R software (version 3.0 or later).
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Calculated Estimates of Blood Activity in Biodistribution Rat Brain Tissues.
Rat blood and brain tissue biodistribution radioactivity data from 2ꢀ60 min were
analyzed to estimate the amount of the percent contribution of blood signal in
brain tissue. Everett19 previously reported that averageꢀsized rat brain blood
volume is ~30ꢀ35 µL/g of tissue. Using a midꢀrange brain blood volume of 32.5
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µL/g (equal to 0.0325 mL/g), and blood and brain % ID/g values per time point
(Figure 2; per Supporting Information, Numerical Blood and Brain Data) with a
blood density of 1 g/mL afforded the following relationships (Eqn 1ꢀ2) and ratio
(Eqn 3), where t = time point measured (for example at t1):
blood at t1 (% ID/g) = blood % ID/g at at t1 x 1 g/1mL x 0.0325 mL/g
brain at t1 (% ID/g) = brain % ID/g at t1
(Eqn 1)
(Eqn 2)
% of blood in brain tissue at t1 (% ID/g) =
blood at t1 (% ID/g) / brain at t1 (% ID/g) x 100
(Eqn 3)
Rat Arterial Blood Metabolites. Rats were installed with a tail artery catheter at
the upper tail region for blood sampling and the catheter was flushed with saline
heparin solution prior to blood collection. Tracer (1ꢀ3 mCi) was injected via a lower
tail vein catheter and then 5 min later arterial blood (100 ꢁL) was collected by
heparin treated syringe. Blood samples were placed in heparin treated plastic 1.5
mL snapꢀlid vials containing 10 µL of 10 mg/mL citric acid, centrifuged at 13,200 g
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for 1 min and remaining supernatant was removed and placed in a second tube.
The supernatant was treated with 100 µL of acetonitrile to precipitate protein, the
protein pellet was removed after centrifugation and the serum supernatant was
collected. Chromatographic separation of serum radioactive components was
conducted by preparative thin layer chromatography (TLC, 3.5 x 10 cm aluminumꢀ
backed silica with fluorescent indicator (254 nm), mobile phase = nꢀbutanol:acetic
acid:water (5:1:3)) to accommodate the carbonꢀ11 halfꢀlife. The chromatography
was run in parallel with known cold chemical metabolite standards12, 15, 20 that
were prepared in house by literature methods.25, 26 Coꢀmigration of standards with
radioactive spots were excised with scissors, placed in 20 mL glass scintillation
vials and counted with the Hidex gamma counter. The data was exported from the
Hidex instrument to Excel (Microsoft Office) software and reported as relative % of
total radioactivity, defined as the decay corrected ratio of component radioactivity
over the sum of all radioactivity counted across the samples x 100.
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Rat PET-CT and MR Imaging Studies. Tracer [11C]2ꢀPAM i.v. bolus doses of
0.5ꢀ2.0 mCi as formulated in 0.5ꢀ2.0 mL of saline were administered. The cold 2ꢀ
PAM PET imaging experiments were accomplished by coꢀinjecting 2ꢀPAM at 30
mg/kg, saline (1ꢀ2 mL) with tracer. The PET, CT, and MR imaging were performed
northermic (37 ˚C) under isoflurane anesthesia (1ꢀ1.5 %). The PET and CT
imaging data were acquired with a Siemens Inveon microPET/CT scanner system
(ca. 1.5 mm PET imaging spatial resolution). Dynamic PET imaging data were
acquired over 30 min beginning ~0.5 min after the time of injection of tracer. The
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PET data were reconstructed with using a Siemens Inveon reconstruction
program suite, including OSEM2D; as 6 frames, 300 seconds per frame for the 30
min scans, decay time corrected, and quantified with a radiation phantom
instrument calibration factor. A partial volume correction was not applied, and
conservative ROI definitions were used as described below. The CT data were
acquired in standard rat mode: 80 kVp, 225 mA; 400 ms exposure, 194 steps x
194 degrees, and 97ꢀmicron isotropic resolution. The MR data were acquired with
a Bruker Biospin 7ꢀTesla magnet, using a multiꢀslice 2D FLASH protocol with the
following parameters: T2*ꢀweighted gradient recall echo, TR = 1528.3 msec, TE =
7 msec, and 256 x 256 x 50 voxels, affording 16 µm3 resolution.
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The reconstructed MR, CT and PET imaging data files were processed
with AMIDE open source software27 (SourceForge), version 1.0.4 (or later
versions). The MR and CT images were oriented as defined by Paxinos21, 22 and
Walker23. Cranial landmarks of bregma and lambda were identified from the CT
images. The X, Y, Z coordinates of imaging views were centered at bregma,
equivalent to the origin of the first scan, then consistent landmark structures were
iteratively coꢀregistered and template fit against the cranial structures of the first
scan landmarks. Subsequently, the landmarks were correlated with cerebral soft
tissues from the MR scans.
The coꢀregistered imaging data permitted regions of interest (ROIs)
(Figures 5ꢀ7) to be defined within their ROI volume size limits and locations, and
against established stereotaxic threeꢀdimensional locations.21-23 The ROIs were
defined as follows: whole brain, heart, lung, kidney and liver. PET scan regional
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tissue radioactivity is reported as Standardized Uptake Value (SUV) defined as:
[activity concentration in the tissue region of interest (MBq/cc) / decay corrected
injected dose at time = 0 (MBq)] x body weight of the rat as gram (g).24 ROI PET
scan statistics (SUV ± SEM) collected from time points at midꢀframe were
exported to Excel (Mircrosoft Corp.) and plots of SUV versus time were generated
using either Excel or GraphPad Prism (La Jolla, CA) software.
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ASSOCIATED CONTENT
Supporting Information
Chromatograms of the reversedꢀphase HPLC UV and radioactivity detection
profiles of the semiꢀpreparative crude material tracer purification and similar
analytical HPLC profiles are shown. A limited series of pyridoꢀ2ꢀaldoxime
precursor, solvent (DMF, CH3CN), temperature and time radiolabeling trials
reporting radiochemical yield and purity is given. Enhanced views of the Figure 5,
baseline (tracer alone) and tracer coꢀinjected with 30 mg/kg cold 2ꢀPAM, timeꢀ
activity curves in rat are provided. Numerical data from the baseline blood and
brain tissue biodistribution experiments (Figure 2) are given. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
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*Mailing address: Department of Biomedical and Pharmaceutical Sciences,
University of Montana, Missoula. MT, 59812ꢀ1552. Phone: 406ꢀ243ꢀ4984. Eꢀmail:
john.gerdes@umontana.edu
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Author Contributions
C.M.T. and J.M.G designed the tracer. C.M.T. and N.G. performed the cold
alkylation studies, and prepared the nonꢀradioactive tracer and metabolite
standards. K.D.N., J.E.B. and H.F.V. developed the radiosynthesis, K.D.N.,J.E.B.
and T.R.H produced [11C]2ꢀPAM, and performed the rodent imaging,
biodistribution studies and blood metabolite analyses with T.H . CꢀK.C. with
J.M.G. performed the PET imaging analyses; J.M.G., and C.M.T, wrote the
manuscript with contributions provided from CꢀK. C., K.D.N., T.R.H. and H.V.B. All
authors reviewed the manuscript.
Funding
Research reported in this publication was supported by the National Institute Of
Neurological Disorders And Stroke of the National Institutes of Health under
Award Number U01NS092495. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the National
Institutes of Health.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
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We are grateful for support from the NIH and the expert radiochemical technical
assistance of Ms. Salma Jivan of the Department of Radiology and Biomedical
Imaging at the University of California, San Francisco.
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ABBREVIATIONS
ACh, acetylcholine; AChE, acetylcholinesterase; ANOVA, analysis of variance;
bioꢀd, biodistribution; BBB, bloodꢀbrain barrier; CH3CN, acetonitrile; CNS, central
nervous sytem; CT, microꢀcomputed axial tomography; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; HPLC, reversedꢀphase high performance liquid
chromatography; ID, injected dose; i.v., intravenous; MR, magnetic resonance;
min, minutes; NMR, nuclear magnetic resonance; 2ꢀPAM, 2ꢀpyridinealdoxime
methiodide; P2S, 2ꢀpyridinealdoxime methyl methanesulfonate; PET, positron
emission tomography; PK, pharmacokinetic; OP, organophosphate; OPꢀAChE
adduct, phosphorylated acetylcholinesterase; RCY, radiochemical yield; ROI,
region of interest; SD, standard deviation; SEM, standard error measures; SUV,
standardized uptake value; TAC, timeꢀactivity curve; TLC, thinꢀlayer
chromatography; UV, utlraviolet.
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