No abstract

A rapid and sensitive assay for pyridinium oximes in plasma and tissue was developed. The method was suitable for the analysis of mono- and di-pyridinium oximes and utilizes ultrafiltration followed by cation-exchange high-performance liquid chromatography with UV detection. The assay was originally developed for the measurement of the oxime MMB-4 in plasma for which the lower limit of detection was 0.0005 pg and the limit of quantitation was 0.001 to 2.5 microg. The assay required as little as 50 microL of whole blood or 30 pL of tissue homogenate, and it was used for a pharmacokinetic study from a single intramuscular injection of MMB-4 (dichloride or dimethylsulfonate salt) in the guinea pig. Both salts were found to have similar pharmacokinetic properties in the plasma with a T1/2 of about 34 to 42 min and the area-under-the-curve values increased dose dependently. MMB-4 tissue concentrations were much lower than the plasma. The tissue levels peaked at 5-20 min depending on the tissue. A rank of concentration was diaphragm > heart > thigh muscle.

To develop a prophylactic for organophosphorus (OP) poisoning utilizing catalytic bioscavengers, the circulatory stability of the enzymes needs to be increased. One strategy for increasing the bioavailability of OP bioscavengers is to target them to the surface of red blood cells (RBCs). Given the circulatory lifespan of 120 days for human RBCs, this strategy has the potential for creating a persistent pool of bioscavenger. Here we report the development of fusion proteins with a single chain variable fragment (scFv) of Ter119, a molecule that associates with glycophorin A on the surface of RBCs, and the VIID11 variant of paraoxonase 1 (scFv-PON1). We show that scFv-PON1 variants expressed by Trichoplusia ni larvae are catalytically active and that one variant in particular can successfully bind to the surface of murine RBCs both in vitro and in vivo. This study represents a proof of concept for targeting catalytic bioscavengers to the surface of RBCs and is an early step in developing catalytic bioscavengers that can remain in circulation for an extended period of time.

Huperzine A (Hup A) is under investigation as a treatment of Alzheimer's disease because of its properties of reversible and specific AChE inhibition. It has additional interesting pharmacological effects such as the protection of primary neuronal cells isolated from embryonic rat brains from glutamate-induced toxicity. We have isolated a new compound which has similar absorbance characteristics as Hup A from blood of rats administered Hup A. Monitoring the effluent from reversed-phase high-performance liquid chromatography (RP-HPLC) of blood collected 60 min after Hup A treatment at an absorbance of 308 nm (lambdamax for Hup A), yielded a peak height and area for this compound that was approximately 1.4-fold the initial Hup A peak. The compound was isolated from RP-HPLC fractions from blood and liver for analysis by mass spectrometry and nuclear magnetic resonance (NMR). The compound gave an (M+H)+ ion with m/z 259 in positive ion mode, yielding a molecular weight (MW) of 258. If derived from Hup A (MW 242), the change in MW indicates a mass gain of 16. This would be consistent with the addition of a single oxygen or a hydroxylation. To determine the location of the modification, it was examined by 1H NMR, and it was found that the added mass was due to a single epoxidation yielding 13,14-epoxy Hup-A.

Huperzines are a class a of compounds extractable alkaloids from the club moss Huperzia serretia. Moss extract, as a tea, has been used in China to improve cognitive abilities. (‐) Huperzine A (HUP) is a potent reversible acetylcholinesterase inhibitor that can cross the blood‐brain barrier. It is under investigation as an Alzheimer's treatment. These properties may also be useful for a protection against organophosphate (OP) nerve agent poisoning of peripheral and CNS acetylcholinesterase. The guinea pig is the rodent model of choice for OP studies. It has low circulating levels of OP hydrolyzing carboxylesterase, like humans, but unlike rats or mice with high levels that can complicate protection studies. For HUP guinea pig studies we developed an ultrahigh‐pressure liquid chromatography (UHPLC)‐MS method with solid phase extraction (SPE) clean up. HUP recovery from SPE was > 90% for range 0.5 to 20 ng/mL. The UHPLC‐MS was sensitive with an LOD limit of 1 pg. The limits LOQ were 0.32 to 1000 ng/mL (highest tested) with the LLOQ 5 x S/N. Here we demonstrate assay utility in pilot ADME studies in guinea pig plasma from single I.M. doses of 3, 9, and 18 mg/kg. This work was supported by the Defense Threat Reduction Agency.