Protective efficacy of catalytic bioscavenger, paraoxonase 1 against sarin and soman exposure in guinea pigs

Human paraoxonase 1 (PON1) has been portrayed as a catalytic bioscavenger which can hydrolyze large amounts of chemical warfare nerve agents (CWNAs) and organophosphate (OP) pesticides compared to the stoichiometric bioscavengers such as butyrylcholinesterase. We evaluated the protective efficacy of purified human and rabbit serum PON1 against nerve agents sarin and soman in guinea pigs. Catalytically active PON1 purified from human and rabbit serum was intravenously injected to guinea pigs, which were 30 min later exposed to 1.2 × LCt50 sarin or soman using a microinstillation inhalation exposure technology. Pre-treatment with 5 units of purified human and rabbit serum PON1 showed mild to moderate increase in the activity of blood PON1, but significantly increased the survival rate with reduced symptoms of CWNA exposure. Although PON1 is expected to be catalytic, sarin and soman exposure resulted in a significant reduction in blood PON1 activity. However, the blood levels of PON1 in pre-treated animals after exposure to nerve agent were higher than that of untreated control animals. The activity of blood acetylcholinesterase and butyrylcholinesterase and brain acetylcholinesterase was significantly higher in PON1 pre-treated animals and were highly correlated with the survival rate. Blood O2 saturation, pulse rate and respiratory dynamics were normalized in animals treated with PON1 compared to controls. These results demonstrate that purified human and rabbit serum PON1 significantly protect against sarin and soman exposure in guinea pigs and support the development of PON1 as a catalytic bioscavenger for protection against lethal exposure to CWNAs

Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200 mg will be required to protect a human against 2 × LD50 of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ~24 h and had a mean residence time of 78–103 h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5 × LD50 of soman or 8 × LD50 of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents

Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide

Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200 mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2 x LD50 of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable, conformation

Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs

To explore the efficacy of paraoxonase 1 (PON1) as a catalytic bioscavenger, we evaluated human recombinant PON1 (rePON1) expressed in Trichoplusia ni larvae against sarin and soman toxicity using microinstillation inhalation exposure in guinea pigs. Animals were pretreated intravenously with catalytically active rePON1, followed by exposure to 1.2 X LCt50 sarin or soman. Administration of 5 units of rePON1 showed mild increase in the blood activity of the enzyme after 30 min, but protected the animals with a significant increase in survival rate along with minimal signs of nerve agent toxicity. Recombinant PON1 pretreated animals exposed to sarin or soman prevented the reduction of blood O2 saturation and pulse rate observed after nerve agent exposure. In addition, rePON1 pretreated animals showed significantly higher blood PON1, acetylcholinesterase (AChE), and butyrylcholinesterase activity after nerve agent exposure compared to the respective controls without treatments. AChE activity in different brain regions of rePON1 pretreated animals exposed to sarin or soman were also significantly higher than respective controls. The remaining activity of blood PON1, cholinesterases and brain AChE in PON1 pretreated animals after nerve agent exposure correlated with the survival rate. In summary, these data suggest that human rePON1 protects against sarin and soman exposure in guinea pigs

Enzymes of the Cholinesterase family

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Structure and Function of Cholinesterases and Related Proteins

The Sixth International Meeting on Cholinesterases and Related Proteins, Choli- nesterases '98, was organized by Palmer Taylor and his associates at the University of California-San Diego and convened in La Jolla, California, USA, in March of 1998. This was the first conference of the series to be held in the United States, let alone on the Pa- cific Rim. Nearly 200 delegates from twenty countries-from Asia, Australia, Europe, and North and South America-heard 75 oral presentations and viewed 90 posters on current research on cholinesterases and related proteins. The meeting framework was structured to include two days of plenary sessions, followed by two days of concurrent sessions and workshops in specific areas. Communication at the concurrent sessions was facilitated by the conference settings of the Martin Johnson House, on a scenic bluff overlooking the blue Pacific Ocean, and the San Diego Supercomputer Center, which enabled projection and rotation of protein structures in three dimensions for a large audience. This book is the compilation of the presentations at the Sixth International Meeting on Cholinesterases and Related Proteins into a volume that describes recent investigations on the structure, catalytic and non-catalytic functions of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and related proteins, as well as studies on the molecular and cellular biology of these enzymes and the genes that encode them