Determination of the main hydrolysis product of O-ethylS-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonic acid, in human serum

For the unequivocal proof of the use of a nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), a rapid, accurate and sensitive method which allows us to identify its main hydrolysis product ethyl methylphosphonic acid (EMPA) in human serum was explored by GC-MS. GC-MS analysis was performed after solvent extraction with acetonitrile in acidic conditions from the serum sample, which was previously deproteinized by micro-ultrafiltration, and subsequent tert.-butyldimethylsilyl derivatization with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) with 1% tert.-butyldimethylsilyl chloride (t-BDMSC). Linear calibration curves were obtained in the concentration range from 50 to 500 ng/ml for EMPA in the full-scan EI mode and from 5 to 50 ng/ml for EMPA in the SIM EI mode. The relative standard deviation obtained at a sample concentration of 50 ng/ml was 8.4% in the full-scan mode and 7.3% in the SIM mode. Upon applying the full-scan EI and CI mode, 40 ng/ml and 80 ng/ml were the detection limits. Using the SIM-EI mode, in which the ion at m/z 153 was chosen, the limit was 3 ng/ml.     

Percutaneous Exposure to VX: Clinical Signs, Effects on Brain Acetylcholine Levels and EEG

The nerve agent VX has a variable and delayed absorption through the skin, which may have implications for treatment regimens. In the present study, central and peripheral effects of percutaneous VX intoxication were investigated in hairless guinea pigs. Although onset times of clinical signs varied considerably, the relative onset times of signs of poisoning were shown to have a predictive value for survival time. All animals showed elevation of brain choline (Ch) levels. Only two of six animals demonstrated seizure activity on EEG, which was accompanied by acetylcholine (ACh) accumulation. The non-seizing animals displayed only marginal increases of ACh levels, but significant changes in all EEG bands. Acetylcholinesterase activity was highly inhibited in brain and diaphragm. The increases in Ch levels and EEG effects observed in non-seizing animals probably reflected those of ischemia induced by peripheral effects leading to cardiorespiratory compromise. In conclusion, clinical signs will mainly serve as indicators for the onset and maintenance of treatment in subsequent studies. Special issue article in honor of Dr. Frode Fonnum.     

Development of a sensitive method for quantitation of ABT-089 in plasma using fluorescence labeling with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole

A high-performance liquid chromatography method for the quantitation of ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine] (I), a new structural type of cholinergic channel modulators (ChCM), is described in this paper using 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescent-labeling reagent. The method combined an optimized liquid–liquid extraction from plasma followed by pre-column derivatization to yield a fluorescence product. The selectivity, sensitivity, and reproducibility of this method were found to be excellent. This method was applied to the determination of ng/ml plasma and tissue levels of ABT-089 and similar compounds in biological samples.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

Determination of (E)-5-(2-bromovinyl)-2′-deoxyuridine in plasma and urine by capillary electrophoresis

(E)-5-(2-Bromovinyl)-2′-deoxyuridine is an antiviral drug used for treatment of infections with Herpes simplex virus type 1 as well as Varicella zoster virus. Two fast methods for the determination of the drug and its metabolite in plasma and urine by capillary electrophoresis have been developed. The plasma method can be used for measurement of total as well as unbound drug and metabolite. Plasma and urine samples are prepared for measuring by liquid/liquid extraction resulting in a limit of quantification of 40 ng/ml for total and 10 ng/ml for free BVdU in plasma and 170 ng/ml in urine. Inter- as well as intra-day precision were found to be better than 10% and both methods have been used for drug monitoring of patients.     

Application of capillary electrophoresis to the separation of structurally diverse N-(substituted)-glycine–peptoid combinatorial mixtures

The capillary electrophoresis (CE)-based separation of five N-(substituted)-glycine (NSG)–peptoid mixtures with a wide range of physical and chemical properties was studied. A CE separation, initially developed using a single representative peptoid mixture, with a backround electrolyte (BGE) modified by the addition of both methyl-β-cyclodextrin and heptane sulfonic acid was found to provide good separations of most of the combinatorial mixtures investigated. For those mixtures not separated well by this procedure, the use of SDS micelles in conjunction with methyl-β-cyclodextrin resulted in dramatic improvements in the separation. While no single set of separation conditions proved sufficient for all of the NSG–peptoid combinatorial mixtures, the two methods were able to provide separation sufficient for characterization of a set of mixtures with a wide range of physical and chemical properties. The efficiency of the CE-based separation of the combinatorial mixtures studied was compared to a reversed-phase liquid chromatographic method using gradient elution.     

Mitochondrials complex I activity is reduced in latent adriamycin-induced cardiomyopathy of rat

We previously reported on the use of enzymatic analysis to impair fatty acid metabolism followed by reduced myocardial energy content, leading to severe heart failure in adriamycin (ADR)-treated rats. The aim of this study is to investigate whether impaired myocardial energy metabolism can also be detected by other methods; i.e. measuring mitochondrial complex I activity and myocardial ¹²⁵I-15-(p-iodophenyl)-3-(R,S)- methylpentadecanoic acid (BMIPP) accumulation in ADR-treated rats. Eight-week-old male Sprague-Dawley rats received 6 intraperitoneal injections of ADR (total 15 mg/kg: group ADR) or saline (control group) over 2 weeks. Left ventricular (LV) ejection fraction was assessed using echocardiography at 3- and 6-weeks after ADR injection (3 weeks and 6 weeks, respectively). Myocardial fatty acid utilization was assessed at 3 weeks and 6 weeks. The myocardial counts of BMIPP were measured after intravenous BMIPP (370 kBq) injection, and ¹²⁵I counts were measured to calculate the uptake ratio. The enzymatic activity of complex I was assessed by monitoring the oxidation of nicotinamide-adenine-dinucleotide-disodium-salt (NADH). In rats treated with ADR, significant decrease in LV ejection fraction was observed only at 6 weeks compared to control (72.5 vs. 84.5%, p < 0.01rpar;. LV ejection fraction at 3 weeks was identical between group ADR and control (81.8 vs. 84.4%). However, at 3 weeks, complex I activity was already reduced significantly in group ADR as compared to control group (p = 0.03), but the reduction in BMIPP accumulation was not (p = 0.15). Our data indicated that reduced complex I activity in a phenomenon occurred in early phase of ADR-induced cardiomyopathy, and it might play an important role in the progression of ADR-induced heart failure.     

High-performance liquid chromatographic determination of a new oral thrombin inhibitor in the blood of rats and dogs

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of a new oral thrombin inhibitor (compound I) in the blood of rats and dogs. The analyte was deproteinized with a 1.5 volume of methanol and a 0.5 volume of 10% zinc sulfate, and the supernatant was injected into a 5-μm Capcell Pak C₁₈ column (150×4.6 mm I.D.). The mobile phase was a mixture of acetonitrile and 0.2% triethylamine of pH 2.3 (31:69, v/v) with a flow-rate of 1.0 ml/min at UV 231 nm. The retention time of compound I was approximately 9.3 min. The calibration curve was linear over the concentration range of 0.05–100 mg/l for rat blood (r²>0.9995, n=6) and dog blood (r²>0.9993, n=6). The limit of quantitation was 0.05 mg/l for both bloods using a 100-μl sample. For the 5 concentrations (0.05, 0.1, 1, 10, and 100 mg/l), the within-day recovery (n=4) and precision (n=4) were 98.1–104.1% and 1.5–6.8% for rat blood and 95.4–105.7% and 1.4–5.3% for dog blood, respectively. The between-day recovery (n=6) and precision (n=6) were 99.8–105.3% and 3.7–12.6% for rat blood and 87.5–107.1% and 2.9–15.3% for dog blood, respectively. The absolute recoveries were 82.4–93.3%. No interferences from endogenous substances were observed. In conclusion, the presented simple, sensitive, and reproducible HPLC method proved and was used successfully for the determination of compound I in the preclinical pharmacokinetics.     

Progress in the development of enzyme-based nerve agent bioscavengers

Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers.     

Simultaneous determination of thioTEPA, TEPA and a novel, recently identified thioTEPA metabolite, monochloroTEPA, in urine using capillary gas chromatography

An assay for the simultaneous quantitative determination of thioTEPA, TEPA and the recently identified metabolite N,N′-diethylene-N″-2-chloroethylphosphoramide (monochloroTEPA) in human urine has been developed. MonochloroTEPA was synthesized by incubation of TEPA with sodium chloride at pH 8. Thus, with this assay monochloroTEPA is quantified as TEPA equivalents. Analysis of the three analytes in urine was performed using gas chromatography with selective nitrogen–phosphorous detection after extraction with a mixture of 1-propanol and chloroform from urine samples. Diphenylamine was used as internal standard. Recoveries ranged between 70 and 100% and both accuracy and precision were less than 15%. Linearity was accomplished in the range of 25–2500 ng/ml for monochloroTEPA and 25–5000 ng/ml for thioTEPA and TEPA. MonochloroTEPA proved to be stable in urine for at least 4 weeks at −80°C. ThioTEPA, TEPA and monochloroTEPA cummulative urinary excretion from two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples and that the excreted amount of monochloroTEPA exceeded that of thioTEPA on day 2 to 5 of urine collection.     

Enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-beta-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.     

The mPlrp2 and mClps genes are involved in the hydrolysis of retinyl esters in the mouse liver

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5'-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.     

Synthesis of 3-fluoro-6-S-(2-S-pyridyl) nucleosides as potential lead cytostatic agents

The 3-deoxy-3-fluoro-6-S-(2-S-pyridyl)-6-thio-β-d-glucopyranosyl nucleoside analogs 7 were prepared via two facile synthetic routes. Their precursors, 3-fluoro-6-thio-glucopyranosyl nucleosides 5a-e, were obtained by the sequence of deacetylation of 3-deoxy-3-fluoro-β-d-glucopyranosyl nucleosides 2a-e, selective tosylation of the primary OH of 3 and finally treatment with potassium thioacetate. The desired thiolpyridine protected analogs 7a-c,f,g were obtained by the sequence of deacetylation of 5a-c followed by thiopyridinylation and/or condensation of the corresponding heterocyclic bases with the newly synthesized peracetylated 6-S-(2-S-pyridyl) sugar precursor 13, which was obtained via a novel synthetic route from glycosyl donor 12. None of the compounds 6 and 7 showed antiviral activity, but the 5-fluorouracil derivative 7c and particularly the uracil derivative 7b were endowed with an interesting and selective cytostatic action against a variety of murine and human tumor cell cultures.     

Characterization of a RFamide-positive subset of ganglionic cells in the hydrozoan planular nerve net

Summary The complexity of the hydrozoan planular nervous system was examined. Using a whole-mount technique with indirect immunofluorescence, the spatial pattern of ganglionic cells showing RFamide-like immunoreactivity was visualized. RFamide antiserum bound a subset of ganglionic cells in the anterior and upper middle regions of the planula and a few ganglionic cells in the upper tail region. Labeled cells consisted of bipolar and multipolar neurons. Stained processes from these cells formed a three-dimensional nerve net that followed the contour of the mesoglea; such fibers were striking in terms of their large numbers, long lengths, and organization into distinct bundles. Labeled fibers were seen to contact other ganglionic cells, sensory cells, epitheliomuscle cells, the mesoglea, and the outside free surface. All stained cell bodies and fibers were found in the ectoderm. Using the same technique the reappearance of RFamide-positive ganglionic cells in epithelial tissue of chimeric grafts of planulae was observed. Interstitial cells capable of forming RFamide-positive ganglionic cells underwent extensive anterior-posterior migrations in the grafts, moved into the epithelial tissue, and differentiated into RFamide-positive ganglionic cells. Stained repopulated ganglionic cells always formed in the same position in the epithelial tissue as was observed in control planulae suggesting that the expression of RFamide-like substances may be position dependent in the planula.     

New cyclomaltoheptaose (beta-cyclodextrin) derivative 2-O-(2-hydroxybutyl)cyclomaltoheptaose: preparation and its application for the separation of enantiomers of drugs by capillary electrophoresis

A new soluble cyclomaltoheptaose (cyclodextrin) derivative, 2-O-(2-hydroxybutyl)cyclomaltoheptaose [2-O-(2-hydroxybutyl)-beta-cyclodextrin, 2-HB-beta-CD], was prepared and studied as an efficient chiral selector in the separation of racemic mixtures of drugs by capillary electrophoresis (CE). Results showed that 2-HB-beta-CD could provide higher separating capability than that of beta-CD and the similarly substituted 2-HP-beta-CD.     

Efficient hydrolysis of the chemical warfare nerve agent tabun by recombinant and purified human and rabbit serum paraoxonase 1

Paraoxonase 1 (PON1) has been described as an efficient catalytic bioscavenger due to its ability to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs). It is the future most promising candidate as prophylactic medical countermeasure against highly toxic OPs and CWNAs. Most of the studies conducted so far have been focused on the hydrolyzing potential of PON1 against nerve agents, sarin, soman, and VX. Here, we investigated the hydrolysis of tabun by PON1 with the objective of comparing the hydrolysis potential of human and rabbit serum purified and recombinant human PON1. The hydrolysis potential of PON1 against tabun, sarin, and soman was evaluated by using an acetylcholinesterase (AChE) back-titration Ellman method. Efficient hydrolysis of tabun (100 nM) was observed with ∼25-40 mU of PON1, while higher concentration (80-250 mU) of the enzyme was required for the complete hydrolysis of sarin (11 nM) and soman (3 nM). Our data indicate that tabun hydrolysis with PON1 was ∼30-60 times and ∼200-260 times more efficient than that with sarin and soman, respectively. Moreover, the catalytic activity of PON1 varies from source to source, which also reflects their efficiency of hydrolyzing different types of nerve agents. Thus, efficient hydrolysis of tabun by PON1 suggests its promising potential as a prophylactic treatment against tabun exposure.     

Determination of an arylacetamide antiarrhythmic in rat blood and tissues using reversed-phase high-performance liquid chromatography

A method was developed for quantification of (+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b- ]thiophene-4-acetamide (compound I), an antiarrhythmic drug, in rat whole blood, heart, brain, liver and skeletal muscle. Blood and tissue samples were homogenized and purified by chemical extraction. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography (HPLC) coupled with UV detection (215 nm). Drug recoveries from the extraction procedure ranged from 77 to 90%. Within- and between-day reproducibility of peak area (coefficient of variation) ranged from 1.1 to 15.7%. The detection limit was 80–200 ng/ml (in a 500-μl extracted solution) depending on the type of biological sample. This method was used in a pharmacokinetic study of compound I disposition in rats after a bolus intravenous dose of 3.1 mg/kg.     

Determination of the lipid peroxidation product (E)-4-hydroxy-2-nonenal in clinical samples by gas chromatography-negative-ion chemical ionisation mass spectrometry of the O-pnetafluorobenzyl oxime

(E)-4-Hydroxy-2-nonenal (HNE) is a highly reactive product of the free radical-stimulated lipid peroxidation of phospholipid-bound arachidonic acid in cellular membranes. We describe a sensitive and specific method for the determination of HNE in clinical samples. The method is based on the formation of the O-pentafluorobenzyl (O-PFB) oxime derivative of HNE, which is then extracted and cleaned up by solid-phase extraction. The HNE O-PFB oxime is then analysed without further derivatisation by capillary column gas chromatography-negative ion chemical ionisation mass spectrometry (GC-NICI-MS) using selected-monitoring. Concentrations down to the pmol range were achieved using deuterated HNE as an internal standard. The method was used to determine HNE in the cerebrospinal fluid and plasma of patients with Parkinson's disease, the plasma of patients with HIV-1 infection and AIDS and in inflamed mucosal biopsy specimens from patients with inflammatory bowel disease.     

Modulation by the ratio of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio can modulate phosphorylation of a specific protein.