Rapid and complete bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning in minipigs after intraosseous administration.

STUDY OBJECTIVE: Management of chemical weapon casualties includes the timely administration of antidotes without contamination of rescuers. Personal protective equipment makes intravenous access difficult but does not prevent intraosseous drug administration. We therefore measured the systemic bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning when administered by the intraosseous, intravenous, and intramuscular routes in a small study of Göttingen minipigs. METHODS: Animals were randomly allocated to sequentially receive atropine (0.12 mg/kg by rapid injection), pralidoxime (25 mg/kg by injection during 2 minutes), and hydroxocobalamin (75 mg/kg during 10 minutes) by the intravenous or intraosseous route, or atropine and pralidoxime by the intramuscular route. Plasma concentrations were measured for 6 hours to characterize the antidote concentration-time profiles for each route. RESULTS: Maximum plasma concentrations of atropine and pralidoxime occurred within 2 minutes when administered by the intraosseous route compared with 8 minutes by the intramuscular route. Maximum plasma hydroxocobalamin concentration occurred at the end of the infusion when administered by the intraosseous route. The mean area under the concentration-time curve by the intraosseous route was similar to the intravenous route for all 3 drugs and similar to the intramuscular route for atropine and pralidoxime. CONCLUSION: This study showed rapid and substantial antidote bioavailability after intraosseous administration that appeared similar to that of the intravenous route. The intraosseous route of antidote administration should be considered when intravenous access is difficult.

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.

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.

Acute renal failure alters the kinetics of pralidoxime in rats.

There is a trend towards increasing doses of pralidoxime to treat human organophosphate poisonings that may have relevance in subpopulations. Indeed, pralidoxime is eliminated unchanged by the renal route. This study assesses the effect of renal failure on the kinetics of pralidoxime in a rat model of acute renal failure induced by potassium dichromate administration. On the first day, Sprague-Dawley rats received subcutaneously potassium dichromate (study) or saline (control). Forty-eight hours post-injection, animals received pralidoxime methylsulfate (50mg/kg of pralidoxime base) intramuscularly. Blood specimens were sampled during 180min after the injection. Urine was collected daily during the 3 days of the study. Plasma pralidoxime concentrations were measured by liquid chromatography with electrochemical detection. There was a 2-fold increase in mean elimination half-life and a 2.5-fold increase in mean area under the curve in the study compared to the control group. The mean total body clearance was halved in the study compared to the control group. Our study showed acute renal failure does not modify the distribution of pralidoxime but significantly alters its elimination from plasma. These results suggest that dosages of pralidoxime should be adjusted in organophosphate-poisoned humans with renal failure when using high dosage regimen of pralidoxime.

High-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of diazepam, atropine and pralidoxime in human plasma.

A high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS) procedure for the simultaneous determination of diazepam from avizafone, atropine and pralidoxime in human plasma is described. Sample pretreatment consisted of protein precipitation from 100microl of plasma using acetonitrile containing the internal standard (diazepam D5). Chromatographic separation was performed on a X-Terra MS C8 column (100mmx2.1mm, i.d. 3.5microm), with a quick stepwise gradient using a formate buffer (pH 3, 2mM) and acetonitrile at a flow rate of 0.2ml/min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges of 1-500ng/ml for diazepam, 0.25-50ng/ml for atropine and 5-1000ng/ml for pralidoxime. The coefficients of variation were always <15% for both intra-day and inter-day precision for each analyte. Mean accuracies were also within +/-15%. This method has been successfully applied to a pharmacokinetic study of the three compounds after intramuscular injection of an avizafone-atropine-pralidoxime combination, in healthy subjects.

Measurement of serum pralidoxime methylsulfate (Contrathion) by high-performance liquid chromatography with electrochemical detection.

Pralidoxime methylsulfate (Contrathion) is widely used to treat organophosphate poisoning. Despite animal and human studies, the usefulness of Contrathion therapy remains a matter of debate. Therapeutic dosage regimens need to be clarified and availability of a reliable method for plasma pralidoxime quantification would be helpful in this process. We here describe a high-performance liquid chromatography technique with electrochemical detection to measure pralidoxime concentrations in human serum using guanosine as an internal standard. The assay was linear between 0.25 and 50 microg mL(-1) with a quantification limit of 0.2 microg mL(-1). The analytical precision was satisfactory, with variation coefficients lower 10%. This assay was applied to the analysis of a serum from an organophosphorate poisoned patient and treated by Contrathion infusions (100 and 200 mg h(-1)) after a loading dose (400 mg).

[Determination of pralidoxime chloride in rat plasma by high performance liquid chromatography].

OBJECTIVE: To provide a reference for clinically curing organophosphrous compounds Poisoning. A high performance liquid chromatography method has been developed for determination of pralidoxime chloride in rat plasma. BECKMAN ODS C18 column, Waters Model 510 HPLC pump and 996 photodiode Detector were used. The mobile phase consisted of 7.5% acetonitrile and 92.5% (20nmol/L NaH2PO4, 0.2% C8H17SO3 Na, pH3.0, adjusted by H3PO4 Solution) the flow rate was 1.0mol/min. detection wavelength was set at 296. The samples were pretreated with acetonitrite. The results show a good liner correlation between pralidoxime chloride concentration(from 1.0 - 5.0 microg/ml) and absorption intensity. The detection limit is 0.5 microg/ml with signal to noise ratio of 2. The intra-assay and inter-assay coefficients of variation were 1.35% and 2.73%. The recoveries for plasma were in ranges of 76% - 84%.

Effect of intravenous thiamine on pralidoxime kinetics.

Subjects were given pralidoxime chloride (5 mg/kg, intravenously) alone and again while they were receiving an infusion of thiamine hydrochloride. After the addition of thiamine: (1) overall, the urinary excretion of oxime was the same but the amount excreted in the first three hours was smaller; (2) the plasma half-life of oxime lengthened; (3) the plasma concentrations of oxime rose; and (4) the intercompartmental clearances and rate constant for elimination for oxime fell. These changes suggest that thiamine and oxime compete for a common renal secretory mechanism or that thiamine alters the membrane transport of oxime.

Improved delivery through biological membranes. 2. Distribution, excretion, and metabolism of N-methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride, a pro-drug of N-methylpyridinium-2-carbaldoxime chloride.

N-Methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride, the pro-drug of 2-PAM, was found to be converted in vivo to 2-PAM, rapidly and quantitatively. The significantly changed properties of the pro-2-PAM resulted in a longer biological half-life and a favorable distribution of 2-PAM formed upon its oxidation. No new metabolite was found when pro-2-PAM was administered intravenously; however, a new metabolic product was formed when the pro-drug was given by oral route.

Pharmacokinetics of pralidoxime chloride in the rat.

The pharmacokinetics of pralidoxime chloride (2-PAM) was studied in rats. Different groups of rats were given an intramuscular injection of 2-PAM at one of three doses (20, 40, or 80 mg/kg). This range of doses is used commonly in studies concerned with the efficacy of 2-PAM against poisoning by potent organophosphorus inhibitors of cholinesterase enzyme. Individual, sequential blood samples were collected during the course of the experiment. From these blood samples the plasma concentrations of 2-PAM were determined over time for each animal. Next the relationship of plasma concentration to time was expressed in terms of a standard pharmacokinetic model. Estimates of various pharmacokinetic parameters were calculated using an open, one-compartment model: volume of distribution (Vd), maximal plasma concentration (Cmax), elimination rate constant (k10), absorption rate constant (k01), area under the curve (AUC) and clearance (CL). Of the pharmacokinetic estimates, only Cmax and AUC were found to be statistically significant (p less than 0.0001) when compared across all the doses; these pharmacokinetic estimates were highly correlated with doses with r = 0.998 and r = 0.997, respectively. However, when AUC and Cmax were normalized by dividing through by dose, no significant differences were found in the transformed data. The results of this study in rat indicate that the pharmacokinetics of 2-PAM is linearly related to dose in a range employed in therapeutic studies of 2-PAM.

Sarin intoxication elevates plasma pralidoxime.

Groups of guinea pigs were injected with a range of dosages for sarin (0, 140, 279, 557 micrograms/kg) followed by pralidoxime (2-PAM) and atropine sulfate (16 mg/kg). Poisoning by sarin in these animals elevated plasma pralidoxime content in a dose-dependent manner within 10 min of intoxication. Plasma levels after administration of 3.12 mg/kg of 2-PAM were elevated from a control mean of 6.18 micrograms/ml to a maximum of 13.78 micrograms/ml in animals given 557 micrograms/kg of sarin at 2 min after the injection of the therapeutic compounds. This suggests that pathophysiological changes following intoxication by potent inhibitors of cholinesterase result in a decrease in the rate and extent of distribution of therapeutic compounds. This effect is most likely a consequence of changes in cardiovascular functions influencing blood flow to various organs.

Intramuscular kinetics and dosage regimens for pralidoxime in buffalo calves (Bubalus bubalis).

The plasma levels, disposition kinetics and a dosage regimen for pralidoxime (2-PAM) were investigated in male buffalo calves following single intramuscular administration (15 or 30 mg/kg). The effects of 2-PAM on various blood enzymes were also determined. The absorption half-life, elimination half-life, apparent volume of distribution and total body clearance of 2-PAM were 1.08 +/- 0.19 h, 3.14-3.19 h, 0.83-1.01 L/kg and 184.9-252.1 ml/(kg h), respectively. At doses of 15 and 30 mg/kg body weight, a plasma concentration > or = 4 microg/ml was maintained for up to 4 and 6 h, respectively. Pralidoxime significantly lowered the serum level of transferases, phosphatases and lactate dehydrogenase but did not influence the acetylcholinesterase and carboxylesterase enzymes. The most appropriate dosage regimen for 2-PAM in the treatment of organophosphate toxicity in buffaloes would be 25 mg/kg followed by 22 mg/kg at 8 h intervals.