Synthesis of the acetylcholinesterase inhibitor paraoxon (POX) as a carbon-11 positron emission tomography tracer ([(11)C]POX) and profiling in live rats is reported. Naive rats intravenously injected with [(11)C]POX showed a rapid decrease in parent tracer to ~ 1% with an increase in radiolabeled serum proteins to 87%, and red blood cells (RBCs) to 9%. Protein and RBC leveled over 60 min reflecting covalent modification of proteins by [(11)C]POX. Ex vivo biodistribution and imaging profiles in naive rats had the highest radioactivity levels in lung followed by heart and kidney, and brain and liver the lowest. Brain radioactivity levels were low but observed immediately after injection, and persisted over the 60 min experiment. This showed for the first-time that even low POX exposures (~200 ng of tracer) can rapidly enter brain. Rats given an LD(50) dose of non-radioactive paraoxon at the LD(50) 20 or 60 min prior to [(11)C]POX tracer revealed protein pools were blocked. Blood radioactivity at 20 min were markedly lower than naive levels due to rapid protein modification by non-radioactive POX, however by 60 min, the blood radioactivity returned to near naive levels. Live rat tissue imaging-derived radioactivity values were 10-37% of naive levels in non-radioactive POX pretreated rats at 20 min, but by 60 min the AUC values had recovered to 25-80% of naive. The live rat imaging supported blockade by non-radioactive POX pretreatment at 20 min and recovery of proteins by 60-min. Significance Statement Paraoxon (POX) is an organophosphorus (OP) compound and a powerful prototype and substitute for OP chemical warfare agents (CWAs) such as sarin, VX etc. To study the distribution and penetration of POX into the CNS and other tissues, a positron emission tomography (PET) tracer analog, [(11)C]POX, was prepared. Blood and tissue radioactivity levels in live rats demonstrated immediate penetration into the CNS and persistent radioactivity levels in tissues indicative of covalent target modification.
Organophosphorus esters (OPs) were originally developed as pesticides but were repurposed as easily manufactured, inexpensive, and highly toxic chemical warfare agents. Acute OP toxicity is primarily due to inhibition of acetylcholinesterase (AChE), an enzyme in the central and peripheral nervous system. OP inhibition of AChE can be reversed using oxime reactivators but many show poor CNS penetration, indicating a need for new clinically viable reactivators. However, challenges exist on how to best measure restored AChE activity in vivo and assess the reactivating agent efficacy. This work reports the development of molecular imaging tools using radiolabeled OP analog tracers that are less toxic to handle in the laboratory, yet inhibit AChE in a similar fashion to the actual OPs. Carbon-11 and fluorine-18 radiolabeled analog tracers of VX and sarin OP agents were prepared. Following intravenous injection in normal Sprague-Dawley rats (n = 3-4/tracer), the tracers were evaluated and compared using noninvasive microPET/CT imaging, biodistribution assay, and arterial blood analyses. All showed rapid uptake and stable retention in brain, heart, liver, and kidney tissues determined by imaging and biodistribution. Lung uptake of the sarin analog tracers was elevated, 2-fold and 4-fold higher uptake at 5 and 30 min, respectively, compared to that for the VX analog tracers. All tracers rapidly bound to red blood cells (RBC) and blood proteins as measured in the biodistribution and arterial blood samples. Analysis of the plasma soluble activity (nonprotein/cell bound activity) showed only 1-6% parent tracer and 88-95% of the activity in the combined solid fractions (RBC and protein bound) as early as 0.5 min post injection. Multivariate analysis of tracer production yield, molar activity, brain uptake, brain area under the curve over 0-15 min, and the amount of parent tracer in the plasma at 5 min revealed the [(18)F]VX analog tracer had the most favorable values for each metric. This tracer was considered the more optimal tracer relative to the other tracers studied and suitable for future in vivo OP exposure and reactivation studies.
Oxime antidotes regenerate organophosphate-inhibited acetylcholinesterase (AChE). Although they share a common mechanism of AChE reactivation, the rate and amount of oxime that enters the brain are critical to the efficacy, a process linked to the oxime structure and charge. Using a platform based on the organophosphate [(18) F]-VXS as a positron emission tomography tracer for active AChE, the in vivo distribution of [(18) F]-VXS was evaluated after an LD50 dose (250 mug/kg) of the organophosphate paraoxon (POX) and following oximes as antidotes. Rats given [(18) F]-VXS tracer alone had significantly higher radioactivity (two- to threefold) in the heart and lung than rats given LD50 POX at 20 or 60 min prior to [(18) F]-VXS. When rats were given LD50 POX followed by 2-PAM (cationic), RS194b (ionizable), or monoisonitrosoacetone (MINA) (neutral), central nervous system (CNS) radioactivity returned to levels at or above untreated naive rats (no POX), whereas CNS radioactivity did not increase in rats given the dication oximes HI-6 or MMB-4. MINA showed a significant, pairwise increase in CNS brain radioactivity compared with POX-treated rats. This new in vivo dynamic platform using [(18) F]-VXS tracer measures and quantifies peripheral and CNS relative changes in AChE availability after POX exposure and is suitable for comparing oxime delivery and AChE reactivation in rats.
Title: Divergent synthesis of organophosphate [(11)C]VX- and [(11)C]Sarin-surrogates from a common set of starting materials Hayes TR, Blecha JE, Thompson CM, Gerdes JM, VanBrocklin HF Ref: Appl Radiat Isot, 151:182, 2019 : PubMed
Radiolabeled 1-[(11)C]ethyl, 4-nitrophenyl methylphosphonate (VX surrogate) and 2-[(11)C]-propanyl, 4-nitrophenyl methylphosphonate (sarin surrogate) were developed as organophosphate (OP) tracers. The [(11)C]ethyl- and [(11)C]isopropyl-iodide radiolabeled synthons were obtained by temperature controlled, in loop reactions of [(11)C]CO2 with MeMgBr followed by reduction with LiAlH4, then reaction with HI. Distillation of the [(11)C]alkyl iodides into a solution of hydrogen (4-nitrophenyl)methylphosphonate and cesium carbonate afforded the desired tracers in >95% radiochemical purity, yields from [(11)C]CO2 of 1-3% and 1.7-15.1 GBq/mmol molar activities.
Title: Radiosynthesis of O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl)methylphosphonate: A novel PET tracer surrogate of sarin Hayes TR, Thompson CM, Blecha JE, Gerdes JM, VanBrocklin HF Ref: J Labelled Comp Radiopharm, 61:1089, 2018 : PubMed
O-(1-Fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate is a reactive organophosphate ester (OP) developed as a surrogate of the chemical warfare agent sarin that forms a similar covalent adduct at the active site serine of acetylcholinesterase. The radiolabeled O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate ([(18) F] fluorosarin surrogate) has not been previously prepared. In this paper, we report the first radiosynthesis of this tracer from the reaction of bis-(4-nitrophenyl) methylphosphonate with 1-[(18) F]fluoro-2-propanol in the presence of DBU. The 1-[(18) F]fluoro-2-propanol was prepared by reaction of propylene sulfite with Kryptofix 2.2.2 and [(18) F] fluoride ion. The desired tracer O-(1-[(18) F]fluoropropan-2-yl)-O-(4-nitrophenyl) methylphosphonate was obtained in a >98% radiochemical purity with a 2.4% +/- 0.6% yield (n = 5, 65 minutes from start of synthesis) based on starting [(18) F] fluoride ion and a molar activity of 49.9 GBq/mumol (1.349 +/- 0.329 Ci/mumol, n = 3). This new facile radiosynthesis routinely affords sufficient quantities of [(18) F] fluorosarin surrogate in high radiochemical purity, which will further enable the tracer development as a novel radiolabeled OP acetylcholinesterase inhibitor for assessment of OP modes of action with PET imaging in vivo.
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 [(11)C]CH3I provided the first-in-class [(11)C]2-PAM tracer in 3.5% decay corrected radiochemical yield from [(11)C]CH3I, >99% radiochemical purity, and 4831 Ci/mmol molar activity. [(11)C]2-PAM tracer distribution was evaluated by ex vivo biodistribution and in vivo dynamic positron emission tomography (PET) imaging in naive (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 approximately 82% parent [(11)C]2-PAM tracer. The imaging and biodistribution data are now established, enabling future comparisons to outcomes acquired in OP intoxicated rodent models.
Title: An improved radiosynthesis of O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer Neumann KD, Thompson CM, Blecha JE, Gerdes JM, VanBrocklin HF Ref: J Labelled Comp Radiopharm, 60:337, 2017 : PubMed
O-(2-Fluoroethyl)-O-(p-nitrophenyl) methylphosphonate 1 is an organophosphate cholinesterase inhibitor that creates a phosphonyl-serine covalent adduct at the enzyme active site blocking cholinesterase activity in vivo. The corresponding radiolabeled O-(2-[18 F]fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [18 F]1, has been previously prepared and found to be an excellent positron emission tomography imaging tracer for assessment of cholinesterases in live brain, peripheral tissues, and blood. However, the previously reported [18 F]1 tracer synthesis was slow even with microwave acceleration, required high-performance liquid chromatography separation of the tracer from impurities, and gave less optimal radiochemical yields. In this paper, we report a new synthetic approach to circumvent these shortcomings that is reliant on the facile reactivity of bis-(O,O-p-nitrophenyl) methylphosphonate, 2, with 2-fluoroethanol in the presence of DBU. The cold synthesis was successfully translated to provide a more robust radiosynthesis. Using this new strategy, the desired tracer, [18 F]1, was obtained in a non-decay-corrected radiochemical yield of 8 +/- 2% (n = 7) in >99% radiochemical and >95% chemical purity with a specific activity of 3174 +/- 345 Ci/mmol (EOS). This new facile radiosynthesis routinely affords highly pure quantities of [18 F]1, which will further enable tracer development of OP cholinesterase inhibitors and their evaluation in vivo.
