(M0930-04-22) A Catalytic Aldicarb Bioscavenger as a Treatment for Aldicarb Poisoning

Purpose: As the most toxic carbamate pesticide – a potent, fast-acting inhibitor of acetylcholinesterase (AChE), aldicarb is readily absorbed from all routes of exposure, including oral and dermal exposure. In all species tested, the acute oral toxicities of aldicarb are similar. Due to its physical properties documented in literature,^1,2 aldicarb could be used by terrorists to cause mass casualty. Organophosphorus (OP) warfare/pesticides and carbamate pesticides have a common mode of action for their neurotoxicity as AChE inhibitors. Currently available options for treating this type of poisoning, such as administration of atropine or co-administration of atropine and pralidoxime (2-PAM), have limited efficacy. There have been extensive efforts in the development of improved options for treatment of OP poisoning. Relatively less studies have been carried out in the development of aldicarb poisoning treatment. Reported studies indicated that 2-PAM had neither positive nor negative effects on survival in animal studies on aldicarb intoxication. This outcome is understandable, as 2-PAM was developed to reactivate phosphorylated AChE (an intermediate formed from the inhibition reaction of AChE with an OP), but not carbamylated AChE (an intermediate formed from the reaction of AChE with a carbamate like aldicarb). It is highly desired to develop a new, effective post-exposure treatment option for aldicarb poisoning. In the present study, we focused on the identification of an enzyme (from an in-house library of enzymes³) capable of detoxifying aldicarb as a catalytic aldicarb bioscavenger for aldicarb poisoning.

Methods: Molecular modeling was carried out to screen an in-house library of enzymes (designed as potential cocaine hydrolases in our previous studies) for their potential interactions with aldicarb. Based on the molecular modeling, the most promising enzyme was evaluated in vitro and in vivo for its reactivity with aldicarb. For in vitro analysis, the enzyme was incubated with aldicarb at 37 °C at an initial enzyme concentration of 300 nM and an initial aldicarb concentration of 1,000 nM (1 mM). The enzyme activity was measured throughout the aldicarb inhibition and reactivation time course by determining the enzyme activity. Three measurement replicates were prepared per timepoint. Hydrolysis rates were compared to the corresponding measurements of the aldicarb-free enzyme preparations, allowing for calculation of relative activity and active enzyme concentration. The time-dependent active enzyme concentration data was analyzed by using the MatLab software to obtain the kinetic parameters. For in vivo activity, all mice (n=8/group) were injected (IP) with a lethal dose (0.75 mg/kg » 2 × LD₅₀) of aldicarb, followed by IV administration of the enzyme at varying dose (100, 50, or 25 mg/kg) or vehicle (as the control) at 1-2 min after the aldicarb injection. Mice were monitored for symptoms and survival. The times for occurring of all events in all mice were recorded for analysis.

Results: According to our molecular modeling, an enzyme (denoted as Aldicarb Hydrolase-1 or AH1 for convenience) may potently bind aldicarb and react with aldicarb. The chemical reaction of aldicarb with AH1 consists of two stages: carbamylation to form a carbamylated AH1 and decarbamylation which reactivates AH1. Based on the computational prediction, we first evaluated the actual reactivity of AH1 with aldicarb in vitro and confirmed that AH1 indeed can catalyze aldicarb hydrolysis. Based on the kinetic data obtained, the bimolecular rate constant (k) for the carbamylation of AH1 with aldicarb was estimated to be 5.13 × 10⁴ M^-1 min^-1, and the decarbamylation rate constant  was estimated to be 1.09 × 10^-2 min^-1. In further in vivo rescue experiment, we first tested AH1 at a dose of 100 mg/kg (IP) against aldicarb in male mice (n=8/group), demonstrating that post-treatment with 100 mg/kg aldicarb effectively rescued all mice that had been injected (via IP) with a lethal dose (0.75 mg/kg » 2 × LD₅₀) of aldicarb (LD₅₀ = 0.3 mg/kg for IP route); all mice in the control group died at an average time of 8.2 min after the aldicarb injection. Prior to the death, all mice in the control group exhibited hyperactivity and convulsion-like movements. In comparison, the mice with the post-exposure treatment using AH1 did not exhibit hyperactivity or convulsion-like behavior at all. So, we continued to do the rescue experiments with lower doses (50 and 25 mg/kg) of AH1. It turned out that 50 mg/kg AH1 also rescued all the mice that had been injected with 0.75 mg/kg aldicarb. 25 mg/kg AH1 rescued 5 out of 8 mice (62.5%) that had been injected with 0.75 mg/kg aldicarb.

Conclusion: AH1 can rapidly react with aldicarb and accelerate aldicarb hydrolysis. As a catalytic aldicarb bioscavenger, AH1 can powerfully rescue mice that had been injected intraperitoneally (IP) with a lethal dose (0.75 mg/kg » ~2 × LD₅₀) of aldicarb.

References: 1. Harp, P. R., Aldicarb, in Encyclopedia of Toxicology (Second Edition), Ed. by Philip Wexler, Page 64-66. 2005.
2. USEPA, Aldicarb (https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-098301_18-Nov-83_021.pdf).
3. Chen, X.; Xue, L.; Hou, S.; Jin, Z.; Zhang, T.; Zheng, F.; Zhan, C.-G., Long-acting cocaine hydrolase for addiction therapy. Proc. Natl. Acad. Sci. USA 2016, 113, 422-427.

Acknowledgements: Funding and conflict of interest statements:
This work was supported by the National Institutes of Health (NIH grants UG3/UH3 NS134920, U01 DA051079, UH2/UH3 DA041115, R01 DA056646, U18 DA052319, R01 DA035552, R01 DA032910, and R01 DA013930).