Pro-2-PAM therapy for central and peripheral cholinesterases

Abstract

Novel therapeutics to overcome the toxic effects of organophosphorus (OP) chemical agents are needed due to the documented use of OPs in warfare (e.g. 1980-1988 Iran/Iraq war) and terrorism (e.g. 1995 Tokyo subway attacks). Standard OP exposure therapy in the United States consists of atropine sulfate (to block muscarinic receptors), the acetylcholinesterase (AChE) reactivator (oxime) pralidoxime chloride (2-PAM), and a benzodiazepine anticonvulsant to ameliorate seizures. A major disadvantage is that quaternary nitrogen charged oximes, including 2-PAM, do not cross the blood brain barrier (BBB) to treat brain AChE. Therefore, we have synthesized and evaluated pro-2-PAM (a lipid permeable 2-PAM derivative) that can enter the brain and reactivate CNS AChE, preventing seizures in guinea pigs after exposure to OPs. The protective effects of the pro-2-PAM after OP exposure were shown using (a) surgically implanted radiotelemetry probes for electroencephalogram (EEG), (b) neurohistopathology of brain, (c) cholinesterase activities in the PNS and CNS, and (d) survivability. The PNS oxime 2-PAM was ineffective at reducing seizures/status epilepticus (SE) in diisopropylfluorophosphate (DFP)-exposed animals. In contrast, pro-2-PAM significantly suppressed and then eliminated seizure activity. In OP-exposed guinea pigs, there was a significant reduction in neurological damage with pro-2-PAM but not 2-PAM. Distinct regional areas of the brains showed significantly higher AChE activity 1.5h after OP exposure in pro-2-PAM treated animals compared to the 2-PAM treated ones. However, blood and diaphragm showed similar AChE activities in animals treated with either oxime, as both 2-PAM and pro-2-PAM are PNS active oximes. In conclusion, pro-2-PAM can cross the BBB, is rapidly metabolized inside the brain to 2-PAM, and protects against OP-induced SE through restoration of brain AChE activity. Pro-2-PAM represents the first non-invasive means of administering a CNS therapeutic for the deleterious effects of OP poisoning by reactivating CNS AChE.

Figure 1

Pro-2-PAM synthesis and in vivo distribution in comparison to 2-PAM. On the lower left, synthesis of pro-2-PAM from 2-PAM [11]. Pro-2-PAM, a pro-drug form of 2-PAM, exhibits lipid solubility due to its reduced charge, readily penetrates the blood brain barrier to the CNS as well as the PNS. Inside the brain, Pro-2-PAM is rapidly converted to 2-PAM [13] (modified from [12]). 2-PAM, due to its quaternary charged pyridine ring, cannot cross the BBB. Right, structures of 2-PAM, Pro-2-PAM, MMB-4, and HI-6.

Fig. 2

Radiotelemetry probe implantation and recorder instrumentation. Top panels: Left, surgical implantation of a radiotelemetry probe (inset). The guinea pig’s nose is held in an isoflurane delivery cone. On the right, EEG leads are tunneled to screws in the skull. Probe body is sutured underneath the skin of the back. Bottom panels: Left, radiotelemetry recorder set up on animal rack with 2 holding cages in place. Right, schematic drawing of the radiotelemetry recorder. The pads receive the probe’s radio signal, permitting untethered animal movement, which is processed, stored, and visualized on the computer.

Fig. 3

Representative EEG traces for Control, DFP, DFP then 2-PAM, and DFP then Pro-2-PAM treated guinea pigs. Each block shown is 6 h of EEG recording, with a continuous trace to 24 h for each treatment. Animals received pyridostigmine bromide (0.026 mg/kg, i.p.) then 20 min later DFP (8 mg/kg, s.c; black circle) followed at 1 min by atropine methylbromide (2 mg/kg) and 1.5 human auto-injector equivalents of each oxime (13 mg/kg, i.m.). 2-PAM was injected at 1 min post-DFP exposure, whereas pro-2-PAM treatment was delayed in this example by 15 min. The 19–24 h EEG panels show the efficacy of pro-2-PAM, but not 2-PAM, to alleviate SE.

Fig. 4

AChE activity in guinea pig blood (left panel; U/ml) and brain frontal cortex (right panel; mU/mg) at 1.5 h post-treatment with saline (control; diamond), PB and atropine (inverted-triangle), DFP then pro-2-PAM (triangle), DFP then 2-PAM (square), and DFP without oxime (half-filled circle); number of animals = 2, 1, 4, 4, and 1, respectively.

Fig. 5

Fluoro-jade staining of representative guinea pig brains (40× magnification) for the hippocampus pyramidal neuron layer (lower CA1–CA2 region) at 24 h. White arrows point to the hippocampus neuron layer. Apoptotic cells, highlighted by fluorescence above background, are distinctly seen in the DFP and DFP then 2-PAM treatment 1 min after OP exposure.

Fig. 6

Twenty-four h survival outcome for animals exposed to GD and 2 auto-injectors of pro-2-PAM 1 min later. Animals used for each treatment are shown above the corresponding bars. The diagonal striped bar shows the LD₅₀ for untreated control animals (11 mg/kg GD). The checkered bar represents the LD₅₀ of GD (66 mg/kg) observed for treatment with 3 auto-injectors of 2-PAM 1 min after exposure. In contrast, the cross-hatched bar shows the LD₅₀ of GD (118 mg/kg) that was obtained for 2 auto-injectors of pro-2-PAM treatment 1 min after exposure. Dotted lines are the 95% confidence intervals for the survival curve.