Pharmacokinetics and efficacy of atropine sulfate/obidoxime chloride co-formulation against VX in a guinea pig model.

Nerve agent exposure is generally treated by an antidote formulation composed of a muscarinic antagonist, atropine sulfate (ATR), and a reactivator of acetylcholinesterase (AChE) such as pralidoxime, obidoxime (OBI), methoxime, trimedoxime or HI-6 and an anticonvulsant. Organophosphates (OPs) irreversibly inhibit AChE, the enzyme responsible for termination of acetylcholine signal transduction. Inhibition of AChE leads to overstimulation of the central and peripheral nervous system with convulsive seizures, respiratory distress and death as result. The present study evaluated the efficacy and pharmacokinetics (PK) of ATR/OBI following exposure to two different VX dose levels. The PK of ATR and OBI administered either as a single drug, combined treatment but separately injected, or administered as the ATR/OBI co-formulation, was determined in plasma of naïve guinea pigs and found to be similar for all formulations. Following subcutaneous VX exposure, ATR/OBI-treated animals showed significant improvement in survival rate and progression of clinical signs compared to untreated animals. Moreover, AChE activity after VX exposure in both blood and brain tissue was significantly higher in ATR/OBI-treated animals compared to vehicle-treated control. In conclusion, ATR/OBI has been proven to be efficacious against exposure to VX and there were no PK interactions between ATR and OBI when administered as a co-formulation.

Surface modification of pralidoxime chloride-loaded solid lipid nanoparticles for enhanced brain reactivation of organophosphorus-inhibited AChE: Pharmacokinetics in rat.

The nanotechnological approach is an innovative strategy of high potential to achieve reactivation of organophosphorus-inhibited acetylcholinesterase in central nervous system. It was previously shown that pralidoxime chloride-loaded solid lipid nanoparticles (2-PAM-SLNs) are able to protect the brain against pesticide (paraoxon) central toxicity. In the present work, we increased brain AChE reactivation efficacy by PEGylation of 2-PAM-SLNs using PEG-lipid N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) (DSPE-PEG2000) as a surface-modifier of SLNs. To perform pharmacokinetic study, a simple, sensitive (LLOQ 1.0 ng/mL) high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization by multiple reaction monitoring mode (HPLC-APCI-MS) was developed. The method was compared to mass spectrometry with electrospray ionization. The method was validated for linearity, accuracy, precision, extraction recovery, matrix effect and stability. Acetophenone oxime was used as the internal standard for the quantification of 2-PAM in rat plasma and brain tissue after intravenous administration. 2-PAM-DSPE-PEG2000-SLNs of mean size about 80 nm (PDI = 0.26), zeta-potential of -55 mV and of high in vitro stability, prolonged the elimination phase of 2-PAM from the bloodstream more than 3 times compared to free 2-PAM. An increase in reactivation of POX-inhibited human brain acetylcholinesterase up to 36.08 ± 4.3 % after intravenous administration of 2-PAM-DSPE-PEG2000-SLNs (dose of 2-PAM is 5 mg/kg) was achieved. The result is one of the first examples where this level of brain acetylcholinesterase reactivation was achieved. Thus, the implementation of different approaches for targeting and modifying nanoparticles' surface gives hope for improving the antidotal treatment of organophosphorus poisoning by marketed reactivators.

A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Behavioral, Pharmacokinetic, and Cardiovascular Evaluation of Candidate Oximes in African Green Monkeys.

Oxime reactivators are critical antidotes after organophosphate pesticide or nerve agent poisoning, directly restoring the function of inhibited acetylcholinesterase. In the continuing search for more broad-spectrum acetylcholinesterase reactivators, this study evaluated one of the leading next-generation oxime reactivators: methoxime, (1,1'-trimethylene bis[4-(hydroxyimino)methyl]pyridinium dichloride (MMB-4). The pharmacokinetics of both salts of MMB-4 (dichloride [2Cl] and dimethanesulphonate [DMS]) were characterized across a range of relevant doses (19, 58, and 116 µmol/kg, intramuscular) in a nonhuman primate model (male African green monkeys), and only subtle differences were observed between the salts. Additionally, the behavioral and cardiovascular safety of these MMB-4 salts was compared directly to other available oximes (HI-6 2Cl, HI-6 DMS, and pyridine-2-aldoxime chloride (2-PAM Cl)) at comparable projected doses. Automated operant behavioral tests were used to examine attention, motivation, visual discrimination, concept execution, and fine motor coordination after high doses of all oxime salts, and of all oximes studied, only the highest dose of 2-PAM Cl (447 µmol/kg) disrupted behavioral performance. Likewise, the effects of a range of doses of MMB-4 2Cl or DMS, HI-6 2Cl or DMS, or 2-PAM Cl on cardiovascular parameters were measured in African green monkeys implanted with telemetry devices. Only a small transient decrease in pulse pressure was observed following administration of the highest dose of MMB-4 DMS (116 µmol/kg). Thus, MMB-4 salts, up to the 9× equivalent of a projected autoinjector dose in humans, did not produce behavioral or cardiovascular toxicity in African green monkeys in the current study, and the pharmacokinetic parameters were orderly and predictable.

Pharmacokinetic profile of promising acetylcholinesterase reactivators K027 and K203 in experimental pigs.

Standard treatment of organophosphorus compounds (OPs) poisoning includes administration of an anti-muscarinic (atropine), anticonvulsive (diazepam) and acetylcholinesterase reactivator (oxime). From a wide group of newly synthesized oximes, oxime K027 and oxime K203 seem to be perspective compounds in some specific OPs intoxication. The available in vitro and in vivo preclinical data indicate that both oximes may be considered for potential human use. The main aim of this study was to establish plasmatic concentration curves of both oximes after intramuscular (i.m.) and intragastric (i.g.) application with subsequent pharmacokinetic analysis and study distribution after (i.m.) application on a non-rodent animal model (experimental pigs; 1500mg/animal). According to the results, both oximes had similar Cmax (K027: 106±19µg/mL and K203: 111±8µg/mL) in Tmax 19±5min, respectively, in 22±3min. Bioavailability of oxime K027 calculated as AUCtotal (8389±1024minµg/mL) was halved compared to oxime K203 (16938±795minµg/mL). The highest concentration from peripheral tissues was found in the kidney and lung, but the brain concentrations stay very low, the plasma/brain ratio being approximately 1%. The applied doses were derived from the recommendation where it is possible to use three autoinjectors to save human life. The results provide us with knowledge about the pharmacokinetics and distribution of these new oximes and may help us to better estimate the human pharmacokinetic profile.

An easy method for the determination of active concentrations of cholinesterase reactivators in blood samples: Application to the efficacy assessment of non quaternary reactivators compared to HI-6 and pralidoxime in VX-poisoned mice.

Organophosphorus nerve agents, like VX, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). AChE inhibited by VX can be reactivated using powerful nucleophilic molecules, most commonly oximes, which are one major component of the emergency treatment in case of nerve agent intoxication. We present here a comparative in vivo study on Swiss mice of four reactivators: HI-6, pralidoxime and two uncharged derivatives of 3-hydroxy-2-pyridinaldoxime that should more easily cross the blood-brain barrier and display a significant central nervous system activity. The reactivability kinetic profile of the oximes is established following intraperitoneal injection in healthy mice, using an original and fast enzymatic method based on the reactivation potential of oxime-containing plasma samples. HI-6 displays the highest reactivation potential whatever the conditions, followed by pralidoxime and the two non quaternary reactivators at the dose of 50 mg/kg bw. But these three last reactivators display equivalent reactivation potential at the same dose of 100 µmol/kg bw. Maximal reactivation potential closely correlates to surviving test results of VX intoxicated mice.

Acetylcholinesterase reactivators (HI-6, obidoxime, trimedoxime, K027, K075, K127, K203, K282): structural evaluation of human serum albumin binding and absorption kinetics.

Acetylcholinesterase (AChE) reactivators (oximes) are compounds predominantly targeting the active site of the enzyme. Toxic effects of organophosphates nerve agents (OPNAs) are primarily related to their covalent binding to AChE and butyrylcholinesterase (BChE), critical detoxification enzymes in the blood and in the central nervous system (CNS). After exposure to OPNAs, accumulation of acetylcholine (ACh) overstimulates receptors and blocks neuromuscular junction transmission resulting in CNS toxicity. Current efforts at treatments for OPNA exposure are focused on non-quaternary reactivators, monoisonitrosoacetone oximes (MINA), and diacylmonoxime reactivators (DAM). However, so far only quaternary oximes have been approved for use in cases of OPNA intoxication. Five acetylcholinesterase reactivator candidates (K027, K075, K127, K203, K282) are presented here, together with pharmacokinetic data (plasma concentration, human serum albumin binding potency). Pharmacokinetic curves based on intramuscular application of the tested compounds are given, with binding information and an evaluation of structural relationships. Human Serum Albumin (HSA) binding studies have not yet been performed on any acetylcholinesterase reactivators, and correlations between structure, concentration curves and binding are vital for further development. HSA bindings of the tested compounds were 1% (HI-6), 7% (obidoxime), 6% (trimedoxime), and 5%, 10%, 4%, 15%, and 12% for K027, K075, K127, K203, and K282, respectively.

Organochlorine compound levels in fertile and infertile women from Rio de Janeiro, Brazil.

OBJECTIVE: The aim of the study was quantify organochlorine compounds in women seeking for infertility treatment (n = 15) and in spontaneously pregnant ones (n = 21). MATERIALS AND METHODS: A questionnaire was applied regarding lifestyle, occupational and reproductive history. Blood samples were collected from both groups. RESULTS: From the pesticides studied, pp'DDE was detected in 100% of infertile women, at higher mean levels than in pregnant women (3.02 mcg/L vs. 0.88 mcg/L; p = 0.001; power of 69%), without correlation with the etiology of infertility. Levels of the polychlorinated biphenyls (PCBs) were low, with positive samples in 100% in the infertile women for PCBs 138, 153, 180, while in pregnant women, they were 85.7% for congeners 138 and 153. Only PCB180 showed significance, with frequency of 71.4% (p = 0.019). CONCLUSIONS: The risk factors for female infertility were: age, consumption of untreated water and of canned foods. Exposure to the most prevalent organochlorine compounds described in literature was confirmed in the study, indicating that pp'DDE may adversely influence female fertility.

Organochlorine compound levels in fertile and infertile women from Rio de Janeiro, Brazil / Níveis de substâncias organocloradas em mulheres férteis e inférteis do Rio de Janeiro, Brasil

OBJECTIVE: The aim of the study was quantify organochlorine compounds in women seeking for infertility treatment (n = 15) and in spontaneously pregnant ones (n = 21). MATERIALS AND METHODS: A questionnaire was applied regarding lifestyle, occupational and reproductive history. Blood samples were collected from both groups. RESULTS: From the pesticides studied, pp'DDE was detected in 100% of infertile women, at higher mean levels than in pregnant women (3.02 mcg/L vs. 0.88 mcg/L; p = 0.001; power of 69%), without correlation with the etiology of infertility. Levels of the polychlorinated biphenyls (PCBs) were low, with positive samples in 100% in the infertile women for PCBs 138, 153, 180, while in pregnant women, they were 85.7% for congeners 138 and 153. Only PCB180 showed significance, with frequency of 71.4% (p = 0.019). CONCLUSIONS: The risk factors for female infertility were: age, consumption of untreated water and of canned foods. Exposure to the most prevalent organochlorine compounds described in literature was confirmed in the study, indicating that pp'DDE may adversely influence female fertility.

OBJETIVO: O estudo teve como objetivo quantificar as substâncias organocloradas em mulheres buscando tratamento para infertilidade (n = 15) e que espontaneamente engravidaram (n = 21). MATERIAIS E MÉTODOS: Foi aplicado questionário considerando estilo de vida, história ocupacional e reprodutiva. Amostras de sangue foram obtidas em ambos os grupos. RESULTADOS: Dos pesticidas, pp'DDE foi detectado em 100% das inférteis, com níveis maiores que nas grávidas (3,02 mcg/L vs. 0,88 mcg/L; p = 0,001; poder 69%), sem correlação na etiologia da infertilidade. Os níveis de detecção das bifenilas policloradas (PCBs) foram baixos, com 100% de positividade das amostras nas inférteis para os PCBs 138, 153, 180, e de 85,7% nas grávidas para os congêneres 138 e 153. Apenas PCB180 mostrou significância na frequência de 71,4% (p = 0,019). CONCLUSÕES: Os fatores de risco para infertilidade feminina foram: idade, consumo de água não tratada e alimentos enlatados. A exposição aos organoclorados mais prevalentes descritos na literatura foi confirmada no estudo, indicando que pp'DDE pode influenciar adversamente a fertilidade feminina.

Pharmacokinetic study of two acetylcholinesterase reactivators, trimedoxime and newly synthesized oxime K027, in rat plasma.

K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane dibromide] is a promising new reactivator of organophosphate- or organophosphonate-inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg(-1)) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed-phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R(2) > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1-100 µg ml(-1). Near-identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of C(max) (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml(-1), P = 0.72) and AUC(0-180min) (2290 ± 304 vs 2269 ± 197 min µg ml(-1), P = 0.84). However, the percentage coefficient of variation of the first-order rate constant of absorption (k(a)) was 3-fold higher (P < 0.01) providing evidence for more erratic absorption of intramuscular trimedoxime as compared with K027. In conclusion, oxime K027 might have superior pK properties that may be translated in its faster absorption and subsequent tissue distribution.

Asoxime (HI-6) impact on dogs after one and tenfold therapeutic doses: assessment of adverse effects, distribution, and oxidative stress.

Asoxime (HI-6) is a well known oxime reactivator used for counteracting intoxication by nerve agents. It is able to reactivate acetylcholinesterase (AChE) inhibited even by sarin or soman. The present experiment was aimed to determine markers of oxidative stress represented by thiobarbituric acid reactive substances and antioxidants represented by ferric reducing antioxidant power, reduced and oxidized glutathione in a Beagle dog model. Two groups of dogs were intramuscularly exposed to single (11.4 mg/kg.b.wt.) or tenfold (114 mg/kg.b.wt.) human therapeutically doses of HI-6. HI-6 affinity for AChE in vitro was evaluated in a separate experiment. Complete serum biochemistry and pharmacokinetics were also performed with significant alteration in blood urea nitrogen, creatine phosphokinase, glucose and triglycerides. Blood samples were collected before HI-6 application and after 30, 60, and 120 min. The overall HI-6 impact on organism is discussed.

Organophosphate hydrolases as catalytic bioscavengers of organophosphorus nerve agents.

Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection. Thus, different strategies have been envisioned. The most straightforward consists in injecting a large dose of highly purified natural hBChE to increase the amount of bioscavenger in the bloodstream. This proved to be successful for protection against lethal doses of soman and VX but remains expensive. An improved strategy is to regenerate prophylactic cholinesterases (ChE) by administration of reactivators after exposure. But broad-spectrum efficient reactivators are still lacking, especially for inhibited hBChE. Cholinesterase mutants capable of reactivating spontaneously are another option. The G117H hBChE mutant has been a prototype. We present here the Y124H/Y72D mutant of human acetylcholinesterase; its spontaneous reactivation rate after V-agent inhibition is increased up to 110 fold. Catalytic bioscavengers, enzymes capable of hydrolyzing OP, present the best alternative. Mesophilic bacterial phosphotriesterase (PTE) is a candidate with good catalytic efficiency. Its enantioselectivity has been enhanced against the most potent OP isomers by rational design. We show that PEGylation of this enzyme improves its mean residence time in the rat blood stream 24-fold and its bioavailability 120-fold. Immunogenic issues remain to be solved. Human paraoxonase 1 (hPON1) is another promising candidate. However, its main drawback is that its phosphotriesterase activity is highly dependent on its environment. Recent progress has been made using a mammalian chimera of PON1, but we provide here additional data showing that this chimera is biochemically different from hPON1. Besides, the chimera is expected to suffer from immunogenic issues. Thus, we stress that interest for hPON1 must not fade away, and in particular, the 3D structure of the hPON1 eventually in complex with OP has to be solved.

Partition of bispyridinium oximes (trimedoxime and K074) administered in therapeutic doses into different parts of the rat brain.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood-brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood-brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD(50). The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

Pharmacokinetics of obidoxime in patients poisoned with organophosphorus compounds.

OBJECTIVES: Reactivation of inhibited acetylcholinesterase (AChE) with oximes is a causal therapy of intoxication with organophosphorus compounds (OPs). Maximal oxime effects are expected when effective doses are administered as soon as possible and as long as reactivation can be anticipated. An obidoxime plasma level in the range of 10-20 microM was estimated as appropriate. The achievement of this target was assessed in 34 severely OP-poisoned patients. METHODS: After admission to the intensive care unit (ICU) the obidoxime regimen (250 mg i.v. as bolus, followed by 750 mg/24h) was started and maintained as long as reactivation was possible. Plasma concentrations of obidoxime were determined by HPLC. RESULTS: A total amount of 2269+/-1726 mg obidoxime was infused over 65 h+/-55 h resulting in a steady state plasma concentration of 14.5+/-7.3 microM. Obidoxime was eliminated with t(1/2(1)) 2.2 and t(1/2(2)) 14 h. The volumes of distribution amounted to 0.32+/-0.1L/kg (V((1))) and 0.28+/-0.12 (V((2)))L/kg. Postmortem examination of tissue in one patient showed obidoxime accumulation in cartilage, kidney and liver and pointed to brain concentrations similar to plasma concentration. CONCLUSIONS: Using the suggested obidoxime regimen, the targeted plasma concentration could be achieved. Obidoxime was eliminated biphasically and was well tolerated. This result allows the recommendation of using this definite regimen for adults also in case of mass casualties.

Development and validation of a sensitive HPLC method for the quantification of HI-6 in guinea pig plasma and evaluated in domestic swine.

A rapid and small volume assay to quantify HI-6 in plasma was developed to further the development and licensing of an intravenous formulation of HI-6. The objective of this method was to develop a sensitive and rapid assay that clearly resolved HI-6 and an internal standard in saline and plasma matrices. A fully validated method using ion-pair HPLC and 2-PAM as the internal standard fulfilled these requirements. Small plasma samples of 35 microL were extracted using acidification, filtration and neutralization. Linearity was shown for over 4 microg/mL to 1mg/mL with accuracy and precision within 6% relative error at the lower limit of detection. This method was utilized in the pharmacokinetic analysis HI-6 dichloride (2Cl) and HI-6 dimethane sulfonate (DMS) in anaesthetized guinea pigs and domestic swine following an intravenous bolus administration. From the resultant pharmacokinetic parameters a target plasma concentration of 100 microM was established and maintained in guinea pigs receiving an intravenous infusion. This validated method allows for the analysis of low volume samples, increased sample numbers and is applicable to the determination of pharmacokinetic profiles and parameters.

Time-dependent changes in concentration of two clinically used acetylcholinesterase reactivators (HI-6 and obidoxime) in rat plasma determined by HPLC techniques after in vivo administration.

A simple and reliable HPLC method for determination of rat plasma levels of clinically used acetylcholinesterase (AChE) reactivators (HI-6 and obidoxime) is presented in our study. Separation was carried out by HPLC using an octadecyl silica stationary phase and a mobile phase consisting of 24% acetonitrile and containing 5 mM sodium octanesulfonate and 5 mM tetramethylammonium chloride (pH 2.3). Following intramuscular administration of equimolar doses of both oximes (22.23 mg/kg), the maximum of HI-6 concentration in rat plasma was reached in about 20 min giving 15.26 +/- 1.71 microg/mL. The distribution of obidoxime was fast; the single maximum 23.62 +/- 3.563 microg/mL was recorded at about 10 min. HPLC with UV detection presented in our study is a general method which could be applied for quick measurements of bisquaternary AChE reactivators in rat plasma.

A liquid chromatographic-mass spectrometric (LC-MS) method for the analysis of the bis-pyridinium oxime ICD-585 in plasma: application in a guinea pig model.

In recent animal studies, several novel oxime compounds that are better than 2-PAM as antidotes against selected organophosphate (OP) nerve agents have been identified. The purpose of this study was to develop and validate a liquid chromatographic-mass spectrometric (LC-MS) method for analysis of the bis-pyridinium oxime ICD-585 (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-propane) in plasma and to establish the utility of the method in a guinea pig model. Calibration curves were prepared using ICD-585-spiked plasma at concentrations from 0.156 to 10 microg/ml. Curves were run over a 1-month time frame and a total of 13 (n=13) were generated. The lower limit of quantification (LLOQ) was determined to be 0.216 microg/ml. Intra- and inter-day variability was assessed by studying precision and accuracy. For intra-day studies, data from the precision determinations indicated that the % CV's ranged from 4.28 to 14.98%. The % error in the accuracy assessments ranged from -8.73 to 4.61%. For inter-day studies, precision data ranged from 3.53 to 13.20%. The % error in the accuracy assessments ranged from 0.39 to 13.77%. Room temperature, freeze-thaw and autosampler stability was also examined. For all 3 stability studies, the compound remained within +/-15% of the initial analysis. Application of the method was demonstrated by analyzing samples from guinea pigs challenged with sarin (GB) or cyclosarin (GF) (1x LD(50)) followed with intramuscular ICD-585 (58 microM/kg, 21.8 mg/kg). At 55 min after oxime administration, mean (+/-SD) plasma concentrations were 15.98 (+/-4.88)microg/ml and 14.57 (+/-3.70) microg/ml in GB- and GF-exposed animals, respectively. In summary, studies have been carried out to verify the sensitivity, precision and accuracy of the assay as well as the stability of the analyte under various conditions. The method has been demonstrated to be applicable to the analysis of plasma from nerve agent-exposed guinea pigs.

In vitro and in vivo metabolisms of K-48.

Metabolic pathways of the oxime K-48 have been elucidated by means of in vitro and in vivo experiments. K-48 exposure to rat liver microsomal fraction resulted in the formation of a hydroxylated derivative, in addition to a small molecular fragment. The in vivo metabolism in rats was investigated after intramuscular administration of 50 mumol oxime. K-48 was present in the rat serum in unchanged form. Similarly, the analysis of rat cerebrospinal fluid indicated the sole occurrence of unchanged K-48. In contrast, unchanged K-48 was not found in the rat urine, where only the metabolite generated by epoxidation on the alkyl chain connecting the two pyridinium rings was present. The presence of unchanged K-48 in the serum and cerebrospinal fluid facilitates quantitative determination using HPLC separation and ultraviolet absorbance detection.

HPLC analysis of K-48 concentration in plasma.

K-48 is a new oxime-type compound to be used as an enzyme reactivator in the treatment of exposure to organophosphorous compounds. Plasma concentration of K-48 can be determined using reversed-phase HPLC. Analysis using octyl silica stationary phase and ultraviolet-absorbance detection is fast and simple. K-48 displays a relatively high dose-normalized area under the curve as compared to pralidoxime, which might be beneficial for an antidote. After i.m. administration of 50 mumol K-48, the time course of the concentration can be approximated by a straight line between 15 and 120 min meaning the elimination follows zero-order kinetics.