Oxime Therapy for Brain AChE Reactivation and Neuroprotection after Organophosphate Poisoning

Abstract

One of the main problems in the treatment of poisoning with organophosphorus (OPs) inhibitors of acetylcholinesterase (AChE) is low ability of existing reactivators of AChE that are used as antidotes to cross the blood-brain barrier (BBB). In this work, modified cationic liposomes were developed that can penetrate through the BBB and deliver the reactivator of AChE pralidoxime chloride (2-PAM) into the brain. Liposomes were obtained on the basis of phosphatidylcholine and imidazolium surfactants. To obtain the composition optimized in terms of charge, stability, and toxicity, the molar ratio of surfactant/lipid was varied. For the systems, physicochemical parameters, release profiles of the substrates (rhodamine B, 2-PAM), hemolytic activity and ability to cause hemagglutination were evaluated. Screening of liposome penetration through the BBB, analysis of 2-PAM pharmacokinetics, and in vivo AChE reactivation showed that modified liposomes readily pass into the brain and reactivate brain AChE in rats poisoned with paraoxon (POX) by 25%. For the first time, an assessment was made of the ability of imidazolium liposomes loaded with 2-PAM to reduce the death of neurons in the brains of mice. It was shown that intravenous administration of liposomal 2-PAM can significantly reduce POX-induced neuronal death in the hippocampus.

Keywords: acetylcholinesterase reactivation; blood-brain barrier; cationic liposome; imidazolium surfactant; targeted drug delivery.

Figure 1

Chemical structure of compounds used in the work.

Figure 2

TEM and DLS images of the IA-16/PC modified liposomes with a molar ratio of 0.029/1, 25 °C.

Figure 3

In vitro rhodamine B release from mixed liposomes at various surfactant/lipid molar ratios: (a) IA-10/PC; (b) IA-16/PC; phosphate buffer (0.025 M), pH 7.4, 37 °C.

Figure 4

Rat brain slices: (a) after administration of free rhodamine B (2.5 mg/kg); (b) after intravenous injection of rhodamine B (2.5 mg/kg) in 20 mM IA-16/PC (0.029/1) liposomes.

Figure 5

In vitro 2-PAM release from IA-16/PC modified liposomes using the dialysis bag method (n = 3); C (2-PAM) = 10 mg/mL, phosphate buffer (0.025 M), pH = 7.4, 37 °C.

Figure 6

Photograph of microplate wells containing blood samples with addition of free 2-PAM and IA-16/PC formulation, C (2-PAM) = 10 mg/mL.

Figure 7

(a) Effect of cationic liposomes IA-16/PC (surfactant/lipid molar ratio of 0.029/1) on agglutination of erythrocytes was observed by fluorescent microscopy in phase contrast mode in liposome solutions with the highest lipid concentration; (b) in mixture of type A (II) and B (III) erythrocytes—positive agglutination control.

Figure 8

The study of brain AChE inhibition (a) and reactivation (b) in vivo. Mean AChE activity in brain homogenates was measured in a control group of rats (taken as 100%) after poisoning by POX and after injection of free 2-PAM (dose of 2-PAM was 25 mg/kg) and 2-PAM-loaded IA-16/PC liposomes (dose of 2-PAM was 25 mg/kg). Each point represents the mean percent of AChE inhibition in each experimental group. Data presented as mean ± standard error from 5 brain samples; * indicates significant difference by the Mann–Whitney test (p < 0.05) compared with the level of brain AChE inhibition detected 1 hour after POX injection.

Figure 9

Concentration of 2-PAM in blood plasma (a) and brains (b) of rats after intravenous injection of 2-PAM in IA-16/PC liposomes (25 mg/kg), determined by HPLC-ESI-MS. Each point represents the mean ± standard deviation for five rats.

Figure 10

Representative microphotographs of Fluoro-Jade B staining showing the neuroprotective effect of liposomes loaded with 2-PAM following POX poisoning in the dentate gyrus, CA1, and CA3 areas of the hippocampus and in the entorhinal cortex of mice. Scale bar 100 μm.

Scheme 1

Mechanism of IA-16/PC liposome penetration of the blood–brain barrier and reactivation of the inhibited AChE.