Development and validation of an LC-MS/MS method for pharmacokinetic study of methoxyamine in phase I clinical trial.

Methoxyamine (MX) is the first DNA base-excision-repair (BER) inhibitor evaluated in humans. This work described the development and validation of an LC-MS/MS method for quantitative determination of MX in human plasma. In this method, MX and its stable isotope methoxyl-d(3)-amine (MX-d3 as internal standard) were directly derivatized in human plasma with 4-(N,N-diethylamino)benzaldehyde. The derivatized MX and IS were extracted by methyl-tert-butyl ether, and separated isocratically on a Xterra C18 column (2.1 mm × 100 mm) using an aqueous mobile phase containing 45% acetonitrile and 0.4% formic acid at a flow rate of 0.200 ml/min. Quantitation of MX was carried out by multiple-reaction-monitoring (MRM) mode of positive turbo-ion-spray tandem mass spectrometry. This method has been validated according to FDA guidelines for bioanalytical method. The linear calibration range for MX was 1.25-500 ng/ml in human plasma with a correlation coefficient≥0.9993. The intra- and inter-assay precision (%CV) at three concentration levels (3.50, 45.0 and 450 ng/ml) ranged 0.9-1% and 0.8-3%, respectively. The stability studies showed that MX met the acceptable criteria under all tested conditions. The method developed had been applied to the determination of plasma MX concentrations in the first-in-human phase I clinical trial, and PK data were presented.

A quinoline antibiotic from Rhodococcus erythropolis JCM 6824.

A new quinoline antibiotic, aurachin RE, was isolated and identified from a culture broth of Rhodococcus erythropolis JCM 6824. The aurachin RE structure was determined based on NMR and mass spectrometric analysis. The structure is similar to that of aurachin C antibiotics that have been identified from Stigmatella aurantiaca. Compared to aurachin C, however, aurachin RE exhibits a wide and strong antimicrobial spectrum against both high- and low-GC Gram-positive bacteria.

A new class of bacterial RNA polymerase inhibitor affects nucleotide addition.

RNA polymerase (RNAP) is the central enzyme of gene expression. Despite availability of crystal structures, details of its nucleotide addition cycle remain obscure. We describe bacterial RNAP inhibitors (the CBR703 series) whose properties illuminate this mechanism. These compounds inhibit known catalytic activities of RNAP (nucleotide addition, pyrophosphorolysis, and Gre-stimulated transcript cleavage) but not translocation of RNA or DNA when translocation is uncoupled from catalysis. CBR703-resistance substitutions occur on an outside surface of RNAP opposite its internal active site. We propose that CBR703 compounds inhibit nucleotide addition allosterically by hindering movements of active site structures that are linked to the CBR703 binding site through a bridge helix.

Comparison of methods for extraction, storage, and silylation of pentafluorobenzyl derivatives of carbonyl compounds and multi-functional carbonyl compounds.

The employment of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) derivatization along with bis-(trimethylsilyl)trifluoroacetamide (BSTFA) or N, N-( tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) derivatization is a popular method for measurement of oxygenated organics in environmental and biological samples. Most notably, the derivatization method enables the measurement of atmospheric photooxidation products not detected by using other methods. PFBHA derivatization is often conducted in an aqueous solution. Accordingly, experiments were performed to compare the efficiency of hexane, methyl- tert-butyl ether (MTBE), and dichloromethane (CH(2)Cl(2)) for extraction of O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) derivatives of carbonyl compounds from water. Further, the stability of these compounds when stored at 4 degrees C in CH(2)Cl(2) was determined, and commonly used methods for silylation of -OH and -COOH groups on the PFBHA derivatives were compared. Overall, CH(2)Cl(2)was the most efficient solvent for extraction of PFBHA derivatives of hydroxycarbonyl compounds, dicarbonyl compounds, and keto-acids from water. Derivatives of carbonyl compounds that do not have secondary functional groups were extracted with approximately equal efficiency by each of the three solvents examined. The PFBHA derivatives of aromatic and saturated aliphatic carbonyl compounds and hydroxycarbonyl compounds were stable in CH(2)Cl(2) at 4 degrees C for > or = 66 days whereas the derivatives of keto-acids and unsaturated aliphatic aldehydes begin to degrade after approximately 38 days. Comparison of four procedures for bis-(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization of -OH and -COOH groups on PFBHA derivatives revealed that primary -OH groups react efficiently in 20-100% BSTFA in CH(2)Cl(2), and do not require a catalyst. Secondary -OH groups also react efficiently in 20-100% BSTFA, but the reaction yield improves slightly when trimethylchlorosilane (TMCS) is added as a catalyst. Reaction of tertiary -OH groups with BSTFA was very inefficient, but improved with addition of 10% TMCS to the BSTFA solution. Finally, -COOH groups seemed to react most efficiently and consistently in 100% BSTFA, without catalyst.

Capture-ROMP-release: application for the synthesis of O-alkylhydroxylamines.

[reaction: see text] A new capture-ROMP-release method for chromatography-free purification of N-hydroxysuccinimde Mitsunobu reactions is described. The Mitsunobu reaction captures a variety of alcohols onto a norbornenyl N-hydroxysuccinimide monomer. Subjection of the resulting crude reaction mixture to ROM-polymerization generates a polymer that can be precipitated with methanol and filtered from the Mitsunobu byproducts. Treatment of the polymer with hydrazine releases the substrate from the water-soluble polymer, producing a variety of O-alkylhydroxylamines with good purity.

Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions.

A previous report that the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) allows DEPMPO radical cation formation to be detected via the production of a carbon-centred radical adduct (assigned as the cis-hydroxyethyl species, formed by an intramolecular process) is shown to be incorrect. Rather, this and other paramagnetic species arise from the facile oxidation of trace hydroxylamine impurities present in commercial DEPMPO samples. As a result, techniques for the detection and elimination of such hydroxylamine impurities from DEPMPO solutions were developed and are described; these should prove to be of general use in EPR spin trapping experiments.

Evidence for a new and major metabolic pathway clenbuterol involving in vivo formation of an N-hydroxyarylamine.

Clenbuterol is a beta-adrenergic agonist widely but illegally used in cattle as a growth promoter. The metabolic fate of this drug remains unknown in the main target species, i.e. the bovine, and only limited data have been published concerning its biotransformations in laboratory animals. A metabolic study has been carried out in the rat using 3H- and 14C-labeled clenbuterol. Urine appeared to be the major excretion pathway. Using a soft technique for urine preparation, extraction, and purification, as well as adequate analytical tools in order to preserve labile metabolites, N-oxidation products of the parental drug on the primary amine function were identified for the first time. Clenbuterol hydroxylamine was the major compound, but 4-nitroclenbuterol was also detected. The metabolic pathway leading to the formation of clenbuterol hydroxylamine prevails at high dosages. Clenbuterol hydroxylamine (but not 4-nitroclenbuterol) was also formed extensively when the drug was incubated with rat liver microsomal fractions in aerobic conditions. It is concluded that oxido reduction during urine preparation have previously impaired the identification of this toxicologically important clenbuterol metabolic route.

Characterization of mutagenic activity in instant hot beverage powders.

Extracts of several grain-based coffee-substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee-substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain-based coffee-substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines.

[Electron transport chain in a thermophilic methane-oxidizing culture of Methylococcus thermophilus]. / Tsep' transporta élektronov u termofil'noi metanokisliaiushchei kul'tury Methylococus thermophilus.

The electron transport chain was studied in the obligate methane oxidizing culture of Methylococcus thermophilus during the oxidation of methanol (the source of carbon) which is an oxidized derivative of methane as well as during the oxidation of hydroxylamine which is an intermediate in the oxidation of ammonium (the source of nitrogen) by Mc. thermophilus cells. Cytochromes a, b and c are involved in electron transport. Cytochrome cco and cytochrome c554 have been isolated from the cell-free extract of Mc. thermophilus and purified. A scheme for electron transport operating in the oxidation of methanol and hydroxylamine is suggested on the basis of studying the characteristics of these cytochromes. Cytochrome a was shown to be a component of terminal oxidase. Cytochromes b are connected with membranes and also found in the composition of hydroxylamine oxidase. Cytochrome cco and, possibly, terminal oxidase (cytochromes a) are involved, in the oxidation of CH3OH by methanol dehydrogenase, in electron transport; cytochrome c554 as well as cytochrome b and c in the composition of hydroxylamine oxidase participate in electron transport in the oxidation of NH2OH by hydroxylamine oxidase. The characteristics of the electron transport system in Mc. thermophilus are discussed.