Reactivator Report for: MINA

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). Current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. In vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatograph-tandem mass spectrometric method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 mumol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 mumol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). Current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. In vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatograph-tandem mass spectrometric method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.

Organophosphates (OP) inhibit acetylcholinesterase (AChE, EC 3.1.1.7), both in peripheral tissues and central nervous system (CNS), causing adverse and sometimes fatal effects due to the accumulation of neurotransmitter acetylcholine (ACh). The currently used therapy, focusing on the reactivation of inhibited AChE, is limited to peripheral tissues because commonly used quaternary pyridinium oxime reactivators do not cross the blood brain barrier (BBB) at therapeutically relevant levels. A directed library of thirty uncharged oximes that contain tertiary amine or imidazole protonable functional groups that should cross the BBB as unionized species was tested as tabun-hAChE conjugate reactivators along with three reference oximes: DAM (diacetylmonoxime), MINA (monoisonitrosoacetone), and 2-PAM. The oxime RS150D [N-((1-(3-(2-((hydroxyimino)methyl)-1H-imidazol-1-yl)propyl)-1H-1,2,3-triazol-4-y l)methyl)benzamide] was highlighted as the most promising reactivator of the tabun-hAChE conjugate. We also observed that oximes RS194B [N-(2-(azepan-1-yl)ethyl)-2-(hydroxyimino)acetamide] and RS41A [2-(hydroxyimino)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide], which emerged as lead uncharged reactivators of phosphylated hAChE with other OPs (sarin, cyclosarin and VX), exhibited only moderate reactivation potency for tabun inhibited hAChE. This implies that geometry of oxime access to the phosphorus atom conjugated to the active serine is an important criterion for efficient reactivation, along with the chemical nature of the conjugated moiety: phosphorate, phosphonate, or phosphoramidate. Moreover, modification of the active center through mutagenesis enhances the rates of reactivation. The phosphoramidated-hAChE choline-binding site mutant Y337A showed three-times enhanced reactivation capacity with non-triazole imidazole containing aldoximes (RS113B, RS113A and RS115A) and acetamide derivative (RS194B) than with 2PAM.

A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 mumol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 mumol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

We describe here the synthesis and activity of a new series of oxime reactivators of cholinesterases (ChEs) that contain tertiary amine or imidazole protonatable functional groups. Equilibration between the neutral and protonated species at physiological pH enables the reactivators to cross the blood-brain barrier and distribute in the CNS aqueous space as dictated by interstitial and cellular pH values. Our structure-activity analysis of 134 novel compounds considers primarily imidazole aldoximes and N-substituted 2-hydroxyiminoacetamides. Reactivation capacities of novel oximes are rank ordered by their relative reactivation rate constants at 0.67 mm compared with 2-pyridinealdoxime methiodide for reactivation of four organophosphate (sarin, cyclosarin, VX, and paraoxon) conjugates of human acetylcholinesterase (hAChE). Rank order of the rates differs for reactivation of human butyrylcholinesterase (hBChE) conjugates. The 10 best reactivating oximes, predominantly hydroxyimino acetamide derivatives (for hAChE) and imidazole-containing aldoximes (for hBChE) also exhibited reasonable activity in the reactivation of tabun conjugates. Reactivation kinetics of the lead hydroxyimino acetamide reactivator of hAChE, when analyzed in terms of apparent affinity (1/K(ox)) and maximum reactivation rate (k(2)), is superior to the reference uncharged reactivators monoisonitrosoacetone and 2,3-butanedione monoxime and shows potential for further refinement. The disparate pH dependences for reactivation of ChE and the general base-catalyzed oximolysis of acetylthiocholine reveal that distinct reactivator ionization states are involved in the reactivation of ChE conjugates and in conferring nucleophilic reactivity of the oxime group.

The capability of the tertiary oximes, monoisonitrosoacetone (MINA) and diacetylmonoxime (DAM), to reactivate acetylcholinesterase (AChE) inhibited by sarin (GB) in the blood, brain, and peripheral tissues of guinea pigs was compared with that of the quaternary oximes 2-PAM, HLo7, and MMB-4. Animals were injected subcutaneously (s.c.) with 1.0 x LD(50) of GB and treated intramuscularly (i.m.) 5 min later with one of these oximes. Sixty minutes after GB exposure, tissues were collected for AChE analysis. At low doses, MINA and DAM produced significant increases in AChE activity in all brain areas examined, but no significant AChE reactivation in peripheral tissues or blood. At higher doses, MINA and DAM increased AChE activity in the brain, peripheral tissues, and blood. In contrast, the quaternary oximes produced significant reactivation in peripheral tissues and blood AChE, but no significant reactivation of brain AChE. In another study, animals were pretreated i.m. with pyridostigmine 30 min prior to s.c. challenge with 2.0 x LD(50) of GB and treated i.m. 1 min later with atropine sulfate (2.0 mg/kg), plus a varied dose of oximes. MINA and DAM prevented or terminated GB-induced seizure activity and protected against GB lethality in a dose-dependent fashion. In contrast, none of the quaternary oximes prevented or stopped GB-induced seizures. Thus, tertiary oximes reactivated AChE in the brain, improved survival, and terminated seizures following GB intoxication.

Current oxime therapies do not readily cross the blood-brain barrier to reactivate organophosphorus nerve agent-inhibited cholinesterase (ChE) within the CNS. We investigated the ability of monoisonitrosoacetone (MINA), a tertiary oxime, to reactivate ChE inhibited by the nerve agent sarin (GB), cyclosarin (GF), or VX, in peripheral tissues and brain of guinea pigs and determined whether reactivation in the CNS will enhance protection against the lethal effects of these three agents. In the reactivation experiment, animals were pretreated with atropine methylnitrate (1.0mg/kg, i.m.) 15 min prior to subcutaneous (s.c.) challenge with 1.0 x LD(50) of GB, GF, or VX. Fifteen minutes later animals were treated intramuscularly (i.m.) with MINA (ranging from 22.1 to 139.3mg/kg) or 2-PAM (25.0mg/kg). At 60 min after nerve agent, CNS (brainstem, cerebellum, cortex, hippocampus, midbrain, spinal cord, and striatum) and peripheral (blood, diaphragm, heart, and skeletal muscle) tissues were collected for ChE analysis. MINA reactivated nerve agent-inhibited ChE in the CNS and peripheral tissues in a dose-dependent manner in the following order of potency: GB>GF>VX. In a survival experiment, animals were injected i.m. with atropine sulfate (0.5mg/kg), 2-PAM (25.0mg/kg), or MINA (35.0, 60.0, or 100.0mg/kg) alone or in combination 1 min after challenge with varying s.c. doses of GB, GF, or VX to determine the level of protection. The rank order of MINA's efficacy in guinea pigs against nerve agent lethality was the same as for reactivation of inhibited ChE in the CNS. These data show that MINA is capable of reactivating nerve agent-inhibited ChE and that the extent of ChE reactivation within the CNS strongly relates to its therapeutic efficacy.

The effects of monoisonitrosoacetone (MINA), diacetylmonoxime (DAM) and pyridine-2-aldoxime methiodide (P2AM) upon the cholinesterase of sarin poisoned rats have been studied. Monoisonitrosoacetone and diacetylmonoxime given before sarin protected blood and brain cholinesterase from inhibition. Monoisonitrosoacetone given after the appearance of signs of poisoning caused a rapid reactivation of brain cholinesterase. Diacetylmonoxime, at an equimolar dose, produced only a slight increase in enzyme activity, and pyridine-2-aldoxime methiodide, the best reactivator in vitro, reactivated blood but not brain cholinesterase. There is a relationship between protection and reactivation of brain cholinesterase and prevention and alleviation of signs of poisoning.

Three oximes, monoisonitrosoacetone (MINA), pyridine-2-aldoxime methiodide (PAM) and diacetylmonoxime (DAM), have been examined in combination with atropine as antidotes in sarin poisoning. When treatment was administered 15 min. before sarin, atropine enhanced the protective effect of MINA and DAM 2 to 3 times and of PAM 9 to 10 times in mice and rats. In mice, rats, and guinea-pigs, atropine increased by no more than 2 times the protective effect of all three oximes when given 30 sec. after sarin. Atropine given to monkeys 1 min. after sarin raised the LD50 approximately 3 times. When given in conjunction with MINA or DAM, the LD50 of sarin was raised 7 to 14 times.