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The aerial crop dusting and spraying of fields with the phosphorothioate insecticide parathion in the late 1900s, significantly improved crop yields but resulted in high levels of occupational toxicity in handlers and agricultural workers, as well as cases of intentional self-harm poisoning, culminating in its banning in many western countries by early 2000s. However because of the low solubility and volatility of parathion, most available products were formulated using organic solvents e.g. xylene, to increase the efficacy of the aerosols and dusts. In the present study, the toxicity of parathion was assessed when formulated in an aqueous solvents (ethanol/PBS (1:9)), and delivered to macaques as an aerosol. Doses of 780 μg/kg and 1.56 mg/kg were delivered one day apart, using a modified nebulizer calculated to result in lung deposition of ∼480 μg/kg with a similar or larger amount being swallowed; these doses being similar to the estimated lethal oral dose 286ug/kg - 1.43 mg/kg of formulated parathion in humans. Surprisingly, this dose (a combined amount of ∼14 mg) caused only low AChE inhibition and moderate BChE inhibition with no clinical symptoms, indicating that the use of organic solvents may have previously played a critical role in the severity of parathion toxicity following inhalation exposure. In addition, unlike constitutively toxic OPs, which are highly toxic when inhaled, these results are consistent with the idea that phosphorothioate insecticides appear to be more intoxicating following oral than inhalation exposure. However, this still remains uncertain because the presence of organic solvents in the ingested parathion studies was not always known., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The recent identification of human monoclonal antibodies with broad and potent neutralizing activity against HIV-1 (bnAbs) has resulted in substantial efforts to develop these molecules for clinical use in the prevention and treatment of HIV-1 infection. As with any protein therapeutic drug product, it is imperative to have qualified assays that can accurately detect and quantify anti-drug antibodies (ADA) that may develop in patients receiving passive administration of HIV-1 bnAbs. Here, we have optimized and qualified a functional assay to assess the potential of ADA to inhibit the neutralizing function of HIV-1 bnAbs. Using a modified version of the validated TZM-bl HIV-1 neutralization assay, murine anti-idiotype antibodies were utilized to optimize and evaluate parameters of linearity, range, limit of detection, specificity, and precision for measuring inhibitory ADA activity against multiple HIV-1 bnAbs that are in clinical development. We further demonstrate the utility of this assay for detecting naturally occurring ADA responses in non-human primates receiving passive administration of human bnAbs. This functional assay format complements binding-antibody ADA strategies being developed for HIV-1 bnAbs, and when utilized together, will support a multi-tiered approach for ADA testing that is compliant with Good Clinical Laboratory Practice (GCLP) procedures and FDA guidance., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoxime e.g. pralidoxime, to reactivate the OP-inhibited AChE. However, commonly used oximes inefficiently cross the bloodbrain barrier and are rapidly cleared and their benefit is debated. Recent findings have demonstrated the ability of a novel zwitterionic, centrally acting, brain penetrating oxime (RS194B) to reverse severe symptoms and rapidly reactivate sarin-inhibited AChE in macaques, but it has not been tested following OP pesticide poisoning. In the present study, the symptoms following a lethal dose of inhaled paraoxon (100ug/kg), were shown to mimic those in insecticide poisoned individuals and were also rapidly reversed in macaques by post-exposure IM administration of 80mg/kg of RS194B. This occurred with a concomitant reactivation of AChE to 40-100% in<1hr and BChE (40% in 8h). These findings will be used to develop a macaque model with RS194B as a post-exposure treatment for insecticide poisoning and generate efficacy data for approval under the FDA Animal rule., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 μg/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recombinant (r) and native butyrylcholinesterse (BChE) are potent bioscavengers of organophosphates (OPs) such as nerve agents and pesticides and are undergoing development as antidotal treatments for OP-induced toxicity. Because of the lethal properties of such agents, regulatory approval will require extensive testing under the Animal Rule. However, human (Hu) glycoprotein biologicals, such as BChE, present a challenge for assessing immunogenicity and efficacy in heterologous animal models since any immune responses to the small species differences in amino acids or glycans between the host and biologic may alter pharmacodynamics and preclude accurate efficacy testing; possibly underestimating their potential protective value in humans. To establish accurate pharmacokinetic and efficacy data, an homologous animal model has been developed in which native and PEGylated forms of CHO-derived rMaBChE were multiply injected into homologous macaques with no induction of antibody. These now serve as controls for assessing the pharmacokinetics and immunogenicity in macaques of multiple administrations of PEGylated and unmodified human rBChE (rHuBChE) by both intravenous (IV) and pulmonary routes. The results indicate that, except for maximal concentration (Cmax), the pharmacokinetic parameters following IV injection with heterologous PEG-rHuBChE were greatly reduced even after the first injection compared with homologous PEG-rMaBChE. Anti-HuBChE antibody responses were induced in all monkeys after the second and third administrations regardless of the route of delivery; impacting rates of clearance and usually resulting in reduced endogenous MaBChE activity. These data highlight the difficulties inherent in assessing pharmacokinetics and immunogenicity in animal models, but bode well for the efficacy and safety of rHuBChE pretreatments in homologous humans., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Recombinant (r) butyrylcholinesterase (rBChE) produced in CHO cells is being developed as a prophylactic countermeasure against neurotoxicity resulting from exposure to organophosphates (OPs) in the form of pesticides and nerve agents. To evaluate the efficacy of a parenteral pretreatment, a PEGylated macaque (Ma) form of rBChE was administered into homologous animals to ensure good plasma retention without immunogenicity. Thus, macaques were administered PEG-rMaBChE at either 5 or 7mg/kg intravenously (i.v.) and exposed subcutaneously to 12μg/kg of the potent pesticide paraoxon (Px) at 1h or at 1 and 72h, respectively. Protection was measured by the ability of rBChE prophylaxis to prevent the inhibition of circulating acetylcholinesterase on red blood cells (RBC-AChE). In rBChE-pretreated animals, no inhibition of RBC-AChE activity after the first Px exposure and only a 10-20% reduction after the second exposure were observed as compared to a 75% RBC-AChE inhibition usually obtained without pretreatment. In addition, these studies raised other interesting issues. The lipophilic nature of Px, appears to result in early and transient inhibition of RBC-AChE as a result of transfer of OP bound to RBC even in BChE-pretreated animals. The protection by a single injection of rBChE against two administrations of Px represents the first example of protection by an i.v. rBChE pretreatment against a pesticide such as Px and bodes well for a parenteral rHuBChE pretreatment as an OP countermeasure in humans., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Butyrylcholinesterase (BChE) is the leading pretreatment candidate against exposure to organophosphates (OPs), which pose an ever increasing public and military health. Since respiratory failure is the primary cause of death following acute OP poisoning, an inhaled BChE therapeutic could prove highly efficacious in preventing acute toxicity as well as the associated delayed neuropathy. To address this, studies have been performed in mice and macaques using Chinese Hamster Ovary cells (CHO)-derived recombinant (r) BChE delivered by the pulmonary route, to examine whether the deposition of both macaque (Ma) and human (Hu) rBChE administered as aerosols (aer) favored the creation and retention of an efficient protective "pulmonary bioshield" that could scavenge incoming (inhaled) OPs in situ thereby preventing entry into the circulation and inhibition of plasma BChE and AChE on red blood cells (RBC-AChE) and in cholinergic synapses. In contrast to parenteral delivery of rBChE, which currently requires posttranslational modification for good plasma stability, an unmodified aer-rBChE pretreatment given 1-40 h prior to >1 LD50 of aer-paraoxon (Px) was able to prevent inhibition of circulating cholinesterase in a dose-dependent manner. These studies are the first to show protection by rBChE against a pesticide such as paraoxon when delivered directly into the lung and bode well for the use of a non-invasive and consumer friendly method of rHuBChE delivery as a human treatment to counteract OP toxicity., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Human serum and recombinant butyrylcholinesterase (rHuBChE) are the most advanced prophylactics against organophosphate (OP) toxicity due to nerve agent or insecticide exposure. For ethical reasons, such potential multi-use treatments cannot be tested in humans and will require extensive testing in animal models and the "Animal Rule" 21 (21 CFR 601.90) for regulatory approval. This will involve multiple injections of rHuBChE into heterologous animals, e.g. macaques, rodents with inevitable immunogenicity and subsequent elimination of the enzyme on repeat injections. In order to accurately assess pharmacokinetics, efficacy and safety of a candidate rBChE in an "antibody free" system, a homologous macaque (Ma) model has been developed. In these studies, macaques received single or multiple intravenous injections of native MaBChE as well as unmodified or PEG-conjugated forms of rMaBChE produced in CHO cells. Compared to the poor plasma retention of unmodified rBChE (MRT: <10h), three injections of 1.5-2.3mg/kg of PEG-conjugated tetrameric rBChE resulted in high circulatory stability (MRT: >134h) and lack of immunogenicity similar to native MaBChE. PEG-conjugation of the monomeric rMaBChE form also exhibited pharmacokinetic profiles comparable to the tetrameric form (MRT: >113h). However, despite the increased bioavailability of PEG-rBChE, antigenicity studies using sandwich ELISA showed that while macaque BChE was not immunogenic in macaques, PEGylation of rMaBChE did not prevent binding to anti-BChE antibodies, suggesting PEGylation may not be sufficient to mask non-human epitopes on rBChE. This homologous model can provide necessary preclinical protection data for the use of PEG-rHuBChE in humans and bodes well for a safe and efficacious CHO-derived rHuBChE therapeutic., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)