Novel oximes as blood–brain barrier penetrating cholinesterase reactivators

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Abstract

The US Army utilizes pralidoxime (2-PAM) for the reactivation of OP-inhibited AChE. While 2-PAM effectively reactivates acetylcholinesterase (AChE) in the body, it does not cross the blood–brain barrier (BBB) at therapeutically relevant levels. To address this problem of central nervous system AChE reactivation, novel sugar–oxime conjugates were utilized. These ‘sugar–oximes’ would potentially be transported across the BBB because they contain a sugar moiety which would be recognized by the facilitative glucose transporters. Eight previously reported, but understudied sugar–oximes, as well as six novel sugar–oximes were synthesized, and their ability to reactivate both human red blood cell AChE and plasma butyrylcholinesterase poisoned with DFP, paraoxon, sarin and VX were tested. The results show that the novel sugar–oxime 13c was more active than the other compounds with a reactivation potential similar to 2-PAM. The sugar–oxime 8b had low toxicity with a LD50 of 1590 mg/kg from a single IM dose in the guinea pig and >2000 mg/kg IP in the mouse. Histopathological analysis showed that there were no apparent differences in hippocampus, heart, liver, kidney sciatic nerve, or skeletal muscle between treated and untreated animals. These results show that sugar–oximes can be effective reactivators and suggest that high treatment doses may be possible.

Introduction

Organophosphate (OP) nerve agents are highly toxic compounds. Their toxicity is correlated with their inhibition of acetylcholinesterases (AChEs). This inhibition leads to the over-stimulation of postsynaptic cholinergic neurons. The cause of death is usually by perturbation of breathing from loss of diaphragm control. OPs inhibit acetylcholinesterases (AChEs) by reaction of the phosphorous with the AChE active-site serine, yielding a phosphonylated AChE. This reaction is reversible until dealkylation. The dealkylation process is known as ‘aging’. However, prior to aging, a nucleophilic attack on the OP phosphorous can displace the OP from the serine, and the AChE is reactivated.

The US Army utilizes 2-pralidoxime (2-PAM) as an oxime reactivator, while NATO allies use the oxime HI-6. These oximes are effective in reactivating the AChE in the body but are not useful in reactivating AChE in the central nervous system (CNS). HI-6 does not readily penetrate into the CNS [1] and some controversy exists regarding the passage of 2-PAM across the BBB, but separate studies have concluded that its penetration rate is low, less than 10% [2], [3]. Therefore, current oximes in use provide little to no protection against the neurological effects of OP-exposure, which includes seizures, convulsions, behavioral and psychological changes. These latter changes were most recently demonstrated in the Tokyo subway nerve agent bombings in 1995, where it has been reported that the survivors have experienced neurological effects of OP-exposure: headache, weakness, fasciculation, numbness of extremities, decreased consciousness level, vertigo, and convulsions [3]. Therefore protection and recovery of CNS AChE are essential and thus a major goal of this research. The CNS is protected by the blood–brain barrier (BBB). The BBB is comprised of an endothelial cell layer that is nearly impenetrable to proteins and polar molecules [4]. The barrier properties are due to the cell membranes, tight intercellular junctions, and limited vesicle transport from pinocytic activity [5]. However, substances such as glucose, the primary energy source for the brain, and certain amino acids, i.e. glutamate, are all polar molecules cross this barrier. These types of compounds have specific facilitative transporters that actively move compounds in response to their concentration gradients.

The transport of glucose is mediated by facilitative transporters such as the glucose transporter protein type 1 (GLUT1). There are two isoforms of this protein found in the brain, both of which are derived from the same gene [6]. They differ in the degree of glycosylation. The 55 kDa form is found on the luminal and abluminal membranes of brain capillary endothelial cells while the 44 kDa form is found in neuronal cells. The GLUT1 transporter is relatively non-specific and transports an assortment of hexoses and Glc-conjugates across the BBB. A growing body of evidence has shown that glucose conjugates have pharmacological activity in the CNS when the parent compounds do not. The compounds are a diverse array that includes conjugates to dopa, l-dopa, 7-chlorokynurenic acid, enkephalins, and the prodrugs ketoprofen and indomethacin [7], [8], [9], [10], [11].

Although the GLUT1 was not cloned until 1986, a study was published earlier by Rachman et al. [12] in an effort to improve the bio-availability of oximes by conjugating oximes to glucose. This promising work demonstrated that sugar–oximes could be effective reactivators of AChE inhibited with tetraethylpyrophosphate (TEPP), protecting mice exposed to 2.5 LD50 of TEPP. This work was followed with somewhat more extensive studies by Heldman et al. [13], where they showed for the first time that sugar–oximes appear to be penetrating the BBB. This was demonstrated by the attenuation of paraoxon-induced hypothermia in rats by the sugar–oximes. They included a minimal pharmacokinetic study of one relevant sugar–oxime in mice showing that Glc-conjugation increased the time in which half the maximal dose was detectable in serum and present in the blood relative to the parent oximes (T1/2 = 45 min vs. 15 min for parent oxime). Sugar–oximes were also less toxic with ratios of sugar–oxime/parent oxime greater than three [12], [13], [14]. Several trends became apparent for the new oximes from the protective ratios (PR) of VX and paraoxon-ethyl (PX) exposed mice when compared to the parent compounds: (1) propyl-bridge linked sugar–oximes had higher PR than direct-linked ones; and (2) deacetylation of the Glc moiety in direct-linked sugar–oximes increased the PR relative to the acetylated direct-link sugar–oximes. Altogether, these studies indicate that sugar–oximes have the following desirable characteristics as effective CNS AChE reactivators: (1) sugar–oximes cross the BBB, becoming available to CNS tissue; (2) the sugar–oximes could be more effective than their parent compounds for reactivating AChE; and (3) the new compounds were less toxic than the parent compounds. These results indicate that these compounds maybe useful as therapeutics for OP poisoning and to protect AChE within the CNS. Therefore to expand the understanding of these compounds we synthesized 14 sugar–oximes and tested them for reactivation of human AChE (Hu ACHE) and BChE (Hu BChE) inhibited with four OP. Also, we performed toxicity studies with one of the most promising reactivators on mice and guinea pigs and present the parameters for the important animal model, guinea pig, typically used for OP efficacy studies.

Section snippets

Sugar–oxime synthesis

Starting materials and other reagents were purchased from Sigma–Aldrich (St. Louis, MO) or Acros Organics (Geel, Belgium). Nuclear Magnetic Resonance spectra were acquired on a Bruker Advance 300 MHz NMR. Elemental analysis data was purchased from Atlantic Microlabs (Atlanta, Georgia). The previously reported compounds were synthesized as described in the literature [12], [13], [15]. The procedures and synthetic detail for the novel sugar–oximes 12ac and 13ac are provided.

10, tetraacetyl-β-d

Oxime selection

The structures of the sugar–oximes synthesized for this study are shown in Fig. 1 schemes A–C. Six previously reported but understudied sugar–oximes were synthesized to confirm and expand our knowledge of these compounds [12], [13]. These 6 sugar–oximes were denoted (a, 3-aldoxime; b, 4-aldoxime derivatives): 3a, 3b; 4a, 4b; 6a, 6b; and 8a, 8b were initially synthesized. The 3 series are of the direct linkage type with acetylated Glc. The 4 series is the direct linkage type with deacetylated

Discussion

Pyridinium based oximes are very useful compounds for reactivating OP-inhibited AChE. The BBB however, is a difficult barrier to penetrate with drugs that have a significant charge character such as the pyridinium oximes. In light of the new emphasis to protect CNS AChE, new therapeutics that penetrate the BBB are necessary. We proposed to evaluate the glucose transporter system as a means to move oximes across the BBB as sugar–oxime conjugates. The transporters are present in high density in

Conflicts of interest

None.

Acknowledgements

This work was supported by the Defense Threat Reduction Agency (grant number 1.E005_08_WR). The opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in

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