Characterization of asymmetric fluorogenic phosphonates as probes for developing organophosphorus hydrolases with broader stereoselectivity.

Organophosphorus hydrolases (OPH) such as mammalian plama paraoxonase (PON1) detoxify asymmetric toxic organophosphorus (OP) nerve agents by preferentially hydrolyzing the less toxic P(+) optical isomer. In order to develop new OPHs with broader stereoselectivity we have prepared a series of asymmetric fluorogenic organophosphonates (Flu-OPs). Such Flu-OPs may serve as molecular probes for screening large libraries of OP hydrolases during directed evolution. Flu-OPs were prepared as methylphosphonates (MPs) diesters containing either ethyl (E), isopropyl (I), cyclohexyl (C) or pinacolyl (P) groups that are structural congeners of the nerve agents VX, sarin, cyclosarin and soman, respectively. The second ester bond was formed with fluorescent moieties that are either 3-cyano-4-methyl-7-hydroxy coumarin (MeCyC) or 1,3-dichloro-7-hydroxy 9,9-dimethyl-9H-acridin-2-one (DDAO). To further characterize the Flu-OPs as surrogates of their respective nerve agents, we have studied the reactivation of Flu-OP-inhibited AChE using 2-PAM and toxogonin (TOX). AChE was 90-95% inhibited by all Flu-OPs (0.36-0.9(M) and then was reactivated by either 2-PAM or TOX. TOX caused a more rapid reactivation than 2-PAM with the following rank order; EMP>IMP>CMP. TOX was also shown to be a better reactivator than 2-PAM for AChE inhibited by the nerve agents VX and cyclosarin. PMP-AChE could not be reactivated by either TOX or 2-PAM, similarly to aging of PMP-AChE formed by inhibition with soman. Racemic CMP-MeCyC was used for screening two new PON1 variants from a neutral library of PON1. These multiple mutation variants include replacement of active site amino acid residues. Neither mutation in these new variants appeared in PON1 variants previously discovered by directed evolution using symmetric Flu-OP. Detoxification rate of cylcosarin by these new PON1 variants was rather slow indicating the need to further screen PON1 clones using optically active Flu-OPs. Therefore, we have separated enzymatically the P(-) enantiomer of CMP-MeCyC and determined its 98% purity using chiral HPLC.

A transfected L-myc gene can substitute for c-myc in blocking murine erythroleukemia differentiation.

We investigated the ability of the proto-oncogene L-myc to substitute for c-myc in blocking murine erythroleukemia differentiation. Murine erythroleukemia cells (line C19) were transfected with recombinant plasmids containing genomic and cDNA fragments of the L-myc gene driven by a Moloney murine leukemia virus long terminal repeat. Clones expressing constitutive high levels of L-myc failed to differentiate in response to the chemical inducer N,N'-hexamethylene bisacetamide (HMBA). The block to differentiation correlated with the level of L-myc expression. Furthermore, transfected clones grown in the presence of inducer for an extended period of time showed an increased level of L-myc expression. These results suggest that functional domains of the c-myc gene involved in differentiation are located in the discrete regions of homology between the c- and L-myc genes.

Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18.

Interleukin-18 (IL-18) is a pleiotropic proinflammatory cytokine that plays an important role in interferon gamma (IFN-gamma) production and IL-12-driven Th1 phenotype polarization. Increased expression of IL-18 has been observed in several autoimmune diseases. In this study we have analyzed the role of IL-18 in an antibody-mediated autoimmune disease and elucidated the mechanisms involved in disease suppression mediated by blockade of IL-18, using experimental autoimmune myasthenia gravis (EAMG) as a model. EAMG is a T cell-regulated, antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1- and Th2-type responses are both implicated in EAMG development. We show that treatment by anti-IL-18 during ongoing EAMG suppresses disease progression. The protective effect can be adoptively transferred to naive recipients and is mediated by increased levels of the immunosuppressive Th3-type cytokine TGF-beta and decreased AChR-specific Th1-type cellular responses. Suppression of EAMG is accompanied by down-regulation of the costimulatory factor CD40L and up-regulation of CTLA-4, a key negative immunomodulator. Our results suggest that IL-18 blockade may potentially be applied for immunointervention in myasthenia gravis.

Characterization of soman-binding antibodies raised against soman analogs.

The production of antibodies against the highly toxic organophosphorus compound soman (GD) has been undertaken. Monoclonal antibodies were raised against two structural analogs of soman which served as haptens for immunization. In these soman analogs the chemically active P-F bond of the soman molecule was substituted by a P-OH group (which is ionized to P-O- under physiological conditions) or a P-H bond, creating compounds which we have named GDOH and GDH, respectively. These soman analogs were linked to carrier proteins through a short linker extending from the pinacolyl group. Monoclonal antibodies were selected according to their ability to bind to the immunizing hapten, and their specificities were determined by competitive inhibition assays. Out of total of 103 anti-GDOH antibodies 22 bound soman, whereas no binding was achieved with 62 anti-GDH antibodies. The two groups of monoclonal antibodies differed also in their structural specificity as demonstrated by different reactivities against a variety of soman analogs and substituted derivatives. These studies indicate that in order to achieve further improvement in anti-soman reactivity with protective potential, other groups (which resemble the OH group) have to be substituted for the F atom in the soman molecule.

Enzymes as pretreatment drugs for organophosphate toxicity.

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

In vitro and in vivo protection of acetylcholinesterase against organophosphate poisoning by pretreatment with a novel derivative of 1,3,2-dioxaphosphorinane 2-oxide.

Covalent molecular combinations of a cyclic phosphate (dioxaphosphorinane) and a potential leaving group, such as 3-(trimethylammonio)phenol iodide (TMPH), suggested the synthesis of O-[3-(trimethylammonio)phenyl]-1,3,2-dioxaphosphorinane 2-oxide iodide (TDPI). TDPI inhibited acetylcholinesterase (AChE) (ki = 8.4 x 10(3) M-1 min-1) via the formation of an unstable covalent intermediate. TDPI-inhibited AChE hydrolyzed spontaneously with t1/2 approximately equal to 10 min. Butyrylcholinesterase (BuChE) was also inhibited by TDPI (ki = 1.8 x 10(4) M-1 min-1), but the inhibited BuChE was more stable (greater than 10 times) than the corresponding AchE-TDPI conjugate. Pretreatment of mice with TDPI conferred protection against 22 LD50's of paraoxon and 5 LD50's of soman, provided that treatment with anticholinergics and an oxime followed administration of these anticholinesterase poisons. Correlation between in vitro and in vivo observations suggests that the main protection of AChE conferred by TDPI results from temporary masking of the active site of the enzyme. The acute toxicity of TDPI was found to be 444 mg/kg (sc, mice), whereas analogous carbamates and a noncyclic phosphate also displaying antidotal properties are greater than 170 times more toxic.

Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice.

Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization.

Butyrylcholinesterase purified from human plasma (HuBChE) was evaluated both in vitro and in vivo in mice and rats as a single prophylactic antidote against the lethal effects of highly toxic organophosphates (OP). The variation among the bimolecular rate constants for the inhibition of HuBChE by tabun, VX, sarin, and soman was 10-fold (0.47 to 5.12 x 10(7) M-1 min-1; pH 8.0, 26 degrees). The half-life of HuBChE in blood after its i.v. administration in mice and rats was 21 and 46 hr, respectively. The peak blood-enzyme level was obtained in both species approximately 9-13 hr following i.m. injection of HuBChE, and the fraction of the enzyme activity absorbed into the blood was 0.9 and 0.54 for rats and mice, respectively. The stoichiometry of the in vivo sequestration of the anti-cholinesterase toxicants was consistent with the HuBChE/OP ratio of the molar concentration required to inhibit 100% enzyme activity in vitro. Linear correlation was demonstrated between the blood level of HuBChE and the extent of protection conferred against the toxicity of nerve agents. Pretreatment with HuBChE alone was sufficient not only to increase survivability following exposure to multiple median lethal doses of a wide range of potent OPs, but also to alleviate manifestation of toxic symptoms in mice and rats without the need for additional post-exposure therapy. It appeared that in order to confer protection against lethality nerve agents had to be scavenged to a level below their median lethal dose LD50 within less than one blood circulation time. Since the high rate of sequestration of nerve agents by HuBChE is expected to underlie the activity of the scavenger in other species as well, a reliable extrapolation of its efficacy from experimental animals to humans can be made.

Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice.

Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.

Protection against tabun toxicity in mice by prophylaxis with an enzyme hydrolyzing organophosphate esters.

We demonstrate here the correlation between protection afforded by pretreatment alone with parathion hydrolase purified from Pseudomonas sp. against tabun toxicity in mice and the kinetic parameters which are assumed to determine the in vivo detoxification of tabun by the same enzyme. Results show that 15 and 22 micrograms of parathion hydrolase per animal conferred a protective ratio of 3.94 and 5.65 respectively, against tabun toxicity, without post-exposure treatment.

Amplification of the effectiveness of acetylcholinesterase for detoxification of organophosphorus compounds by bis-quaternary oximes.

Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.

Prophylaxis against soman inhalation toxicity in guinea pigs by pretreatment alone with human serum butyrylcholinesterase.

Human butyrylcholinesterase (HuBChE) has previously been shown to protect mice, rats, and monkeys against multiple lethal toxic doses of organophosphorus (OP) anticholinesterases that were challenged by i.v. bolus injections. This study examines the concept of using a cholinesterase scavenger as a prophylactic measure against inhalation toxicity, which is the more realistic simulation of exposure to volatile OPs. HuBChE-treated awake guinea pigs were exposed to controlled concentration of soman vapors ranging from 417 to 430 micrograms/liter, for 45 to 70 s. The correlation between the inhibition of circulating HuBChE and the dose of soman administered by sequential i.v. injections and by respiratory exposure indicated that the fraction of the inhaled dose of soman that reached the blood was 0.29. HuBChE to soman molar ratio of 0.11 was sufficient to prevent the manifestation of toxic signs in guinea pigs following exposure to 2.17x the inhaled LD50 dose of soman (ILD50, 101 micrograms/kg). A slight increase in HuBChE:soman ratio (0.15) produced sign-free animals after two sequential respiratory exposures with a cumulative dose of 4.5x ILD50. Protection was exceptionally high and far superior to the currently used traditional approach that consisted of pretreatment with pyridostigmine and postexposure combined administration of atropine, benactyzine, and an oxime reactivator. Quantitative analysis of the results suggests that in vivo sequestration of soman, and presumably other OPs, by exogenously administered HuBChE, is independent of the species used or the route of challenge entry. This assuring conclusion significantly expands the database of the bioscavenger strategy that now offers a dependable extrapolation from animals to human.

Huperzine A as a pretreatment candidate drug against nerve agent toxicity.

Huperzine A (HUP) is a naturally-occurring, potent, reversible inhibitor of acetylcholinesterase (AChE) that crosses the blood-brain barrier. To examine its ability to protect against nerve agent poisoning, HUP was administered i.p. to mice, and the s.c. LD50 of soman was determined at various time intervals after pretreatment. Results were compared to those obtained for animals treated with physostigmine. A protective ratio of approximately 2 was maintained for at least 6 hr after a single injection of HUP, without the need for any post-challenge drug therapy. By contrast, pretreatment with physostigmine increased the LD50 of soman by 1.4- to 1.5-fold for only up to 90 min. The long-lasting antidotal efficacy displayed by HUP correlated with the time course of the blood-AChE inhibition. The results suggest that the protection of animals by HUP from soman poisoning was achieved by temporarily sequestering the active site region of the physiologically important AChE.

Prophylaxis against organophosphate poisoning by an enzyme hydrolysing organophosphorus compounds in mice.

Parathion hydrolase purified from Pseudomonas sp. was injected i.v. into mice to demonstrate the feasibility of using organophosphorus acid anhydride (OPA) hydrolases as pretreatment against organophosphates (OP) poisoning. Results show that exogenous administration of as low as 7 to 26 micrograms of parathion hydrolase conferred protection against challenge with multiple median lethal doses (LD50) of diethyl p-nitrophenyl phosphate (paraoxon; 3.8-7.3 x LD50) and diethylfluorophosphate (DEFP; 2.9 x LD50) without administration of supportive drugs. The extent of protection observed was consistent with blood-parathion hydrolase levels and the kinetic constants of the enzymatic hydrolysis of paraoxon and DEFP by parathion hydrolase. OPA hydrolases not only appear to be potential prophylactic drugs capable of increasing survival ratio following OP intoxication but also to alleviate post-exposure symptoms.

The involvement of the NMDA receptor complex in the protective effect of anticholinergic drugs against soman poisoning.

Organophosphate poisoning is associated with adverse effects on the central nervous system such as seizure/convulsive activity and long term changes in neuronal networks. This study reports on investigations designed to assess the consequences of soman exposure on excitatory amino acids receptors in the rat brain. In addition, the protective effects of caramiphen which acts at these receptors, and scopolamine, which does not, was determined on soman-induced alteration in rat brain functions. Administration of soman (1xLD50) to pyridostigmine pretreated rats produced seizure activity (measured by EEG monitoring) in all animals tested. Estimation of [3H]MK-801 binding to brain membranes from intoxicated rats revealed a marked decrease in Bmax value 24 but not 2 hrs following soman administration. The specific nature of these effects of soman was demonstrated by the findings that [3H]flunitrazepam binding to central benzodiazepine receptors remained unchanged in soman-poisoned rat brain membranes. Both scopolamine and caramiphen, when used prophylactically prevented the lethal effect of soman and completely blocked the development of electrographic seizure activity (EGSA). In contrast, only caramiphen abolished soman-induced modifications in NMDA/ion channel characteristics. Caramiphen displaced [3H]MK-801 bound to the NMDA/ion channel complex, possibly by interacting with the Zn2+ site whereas scopolamine did not. Moreover, caramiphen, but not scopolamine, partially protected mice from NMDA-induced lethality. Thus, it is suggested that an important component of the protective efficacy of caramiphen against organophosphate poisoning might be attributed to its ability to modulate NMDA receptors in addition to its anticholinergic properties.

Prevention of soman-induced cognitive deficits by pretreatment with human butyrylcholinesterase in rats.

This study examined the ability of pretreatment with human serum butyrylcholinesterase (HuBChE) to prevent soman-induced cognitive impairments. Behavioral testing was carried out using the Morris water maze task evaluating learning, memory, and reversal learning processes. Pretreatment with HuBChE significantly prevented the memory and reversal learning impairments induced by soman. A small deficiency in performance was observed only during part of the learning period in HuBChE-treated rats after administration of soman. Results support the contention that pretreatment alone with HuBChE is sufficient to increase survival and to prevent impairment in cognitive functioning following exposure to soman.

Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug.

Butyrylcholinesterase from human plasma (HuBChE) is a potential drug candidate for detoxification of certain harmful chemicals that contain carboxylic or phosphoric acid ester bonds. Large quantities of purified HuBChE, displaying a high stability upon long-term storage, are required for the evaluation of its therapeutic capacity and its pharmaceutical properties. Several modifications of a previously reported procedure enabled us to purify the enzyme > 15,000-fold from pools of up to 100 1 of human plasma. The three-step procedure is based on precipitation of plasma proteins by ammonium sulfate (step I) and batch adsorption of HuBChE on procainamide-Sepharose 4B gel (step II). Ammonium sulfate was also employed in the third stage to fractionate the final product from procainamide-containing HuBChE solution. The overall yield (63%) of electrophoretically pure enzyme was significantly higher than that previously reported (34%) for the purification of HuBChE from 12.5 1 of plasma or from 5 kg of Cohn fraction IV-4. Purified HuBChE was stored at 5 degrees C in 10 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.02% NaN3. The specific activity, protein migration on gel electrophoresis, thermostability at 54 degrees C and the mean residence time in the circulation of mice remained essentially constant for at least 46 months. The modifications introduced can provide large quantities of purified enzyme that maintains its activity and bioavailability properties for several years.

Sugar conjugates of pyridinium aldoximes as antidotes against organophosphate poisoning.

A series of pyridinium aldoximes having a sugar conjugated to the pyridine ring has been prepared as potential antidotes against organophosphate poisoning. The sugar residue was attached either directly through C-1 or C-6 of the pyranose ring or through a C3 bridge between the glycosyl group and the nitrogen atom of the pyridine moiety. Attachment of a sugar group to the oxime derivative seems to increase the bioavailability of the antidote. The clearance rate of the sugar conjugates was significantly lower than that of their non-sugar analogs and thus they were retained longer in the blood circulation. The sugar derivatives were more potent in decreasing paraoxon-induced hypothermia (which is regulated within the central nervous system) than N-methyl-2-pyridiniumaldoxime methanesulfonate, one of the most commonly used mono-oximes. The sugar analogs were also less toxic than the non-sugar analogs; some also displayed higher efficacy. The mechanism underlying the improved features of the sugar oximes, and the structural requirements in relation to the sugar attachment to the oxime function, are discussed.

Sarin-induced neuropathology in rats.

Sarin, a highly toxic cholinesterase (ChE) inhibitor, administered at near 1 LD50 dose causes severe signs of toxic cholinergic hyperactivity in both the peripheral and central nervous systems (CNS). The present study evaluated acute and long-term neuropathology following exposure to a single LD50 dose of sarin and compared it to lesions caused by equipotent doses of soman described previously. Rats surviving 1 LD50 dose of sarin (95 micrograms/kg; IM), were sacrificed at different time intervals post exposure (4 h-90 days) and their brains were taken for histological and morphometric study. Lesions of varying degrees of severity were found in about 70% of the animals, mainly in the hippocampus, piriform cortex, and thalamus. The damage was exacerbated with time and at three months post exposure, it extended to regions which were not initially affected. Morphometric analysis revealed a significant decline in the area of CA1 and CA3 hippocampal cells as well as in the number of CA1 cells. The neuropathological findings, although generally similar to those described following 1 LD50 soman, differed in some features, unique to each compound, for example, frontal cortex damage was specific to soman poisoning. It is concluded that sarin has a potent acute and long-term central neurotoxicity, which must be considered in the design of therapeutic regimes.

Aging does not alter the binding characteristics to voltage dependent calcium channels in the brain of CW1 mice.

Structural age-related changes in cholinergic regions within the central nervous system of CW1 mice have been described previously. Since elevated calcium concentration has been suggested to play a role in the brain aging processes, the possible involvement of voltage dependent calcium channels in this degeneration was investigated. The binding characteristics of the calcium channel antagonist [3H](+)PN 200-110 to brains of aged CW1 mice were studied. This ligand exhibited high affinity binding (Kd values in the range of 50-70 pM) to a single type of sites with a density (Bmax) of 150-200 fmol/mg protein. No significant differences were observed between the binding parameters measured in young (3 months), mid-aged (9 and 15 months) and old (20 months) mice. Autoradiographic study confirmed these results and extended them to specific brain regions. It is concluded that age-dependent degeneration processes, observed histologically in specific brain regions of these mice, are probably not related to alterations in voltage-dependent calcium channels.