Thalibealine, a novel tetrahydroprotoberberine--aporphine dimeric alkaloid from Thalictrum wangii.

A novel tetrahydroprotoberberine-aporphine dimeric alkaloid, (-)-thalibealine (1), was isolated from the roots of Thalictrum wangiii, and its structure established via spectroscopic analysis. Three other alkaloids were isolated, including the benzyltetrahydroisoquinoline-aporphine dimer (+)-thalmelatidine, the aporphine (+)-magnoflorine, and the protoberberine berberine. This is the first reported isolation of a tetrahydroprotoberberine-aporphine dimer from nature, as well as the first reported isolation of constituents from Thalictrum wangii.

Synthesis of thalprzewalskiinone, a revision of structure.

A direct comparison of the spectral data for synthetic 2-methyl-6,7-dimethoxy-3'-methoxy-4'-hydroxyoxobenzylisoquinoline iodide (1) and its positional isomer 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2) with the data obtained for the oxobenzylisoquinoline alkaloid thalprzewalskiinone revealed that the original structural assignment of the alkaloid as 1 was in error. These results mandate the revision of structure of thalprzewalskiinone to 2-methyl-6,7-dimethoxy-3'-hydroxy-4'-methoxyoxobenzylisoquinoline iodide (2).

Simplified procedure for measurement of serum dehydroepiandrosterone and its sulfate with gas chromatography-ion trap mass spectrometry and selected reaction monitoring.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are endogenous steroids that have recently been widely publicized as potential treatments for many disorders. This paper describes a gas chromatographic-ion trap mass spectrophotometric assay with selected reaction monitoring for measurement of DHEA and DHEAS levels. The hormones and internal standard (5-androsten-3beta-ol-16-one methyl ester) are extracted from serum with Oasis solid-phase extraction tubes. The extracted steroids are dissolved in methanol and injected into a Finnigan GCQ ion trap mass spectrometer. In the selected reaction mode, both DHEA and DHEAS can be identified and quantified in a single injection. No derivatization or expensive deuterated internal standards are required.

Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.

Galiposin: a new beta-hydroxychalcone from Galipea granulosa.

A novel beta-hydroxychalcone, galiposin, was isolated from the bark of Galipea granulosa. The structure of galiposin was established via spectroscopic analysis, including high resolution one- and two-dimensional nuclear magnetic resonance spectrometry, as well as mass spectroscopy.

Granulosin, a new chromone from Galipea granulosa.

A novel chromone, granulosin (1), has been isolated from the bark of Galipea granulosa. The extract of the bark, as well as granulosin (1), exhibited lethality in the brine shrimp test. The structure of granulosin (1) as 2-propyl-7,8-(methylenedioxy)chromone was established via spectroscopic analysis.

Determination of plasma mexiletine levels with gas chromatography-mass spectrometry and selected-ion monitoring.

Mexiletine, 1-(2,6-dimethylphenoxy)-2-aminopropane (Mexitil), is an orally effective agent useful in the treatment of serious ventricular arrhythmias. This paper describes a gas chromatographic-mass spectrophotometric assay with selected-ion monitoring for the measurement of plasma or serum mexiletine levels. The drug and internal standard (p-chlorophenylalanine methyl ester) were extracted from plasma into ethyl acetate-hexane-methanol (60:40:1, v/v). After separation and evaporation of the organic phase, the drug and internal standard were derivatized to their pentafluoropropyl derivatives prior to analysis. The reproducibility of the daily standard curve yielded mean inter- and intra-day coefficients of variability from 0.7 to 11.0%. The coefficients of variability for control plasma samples (0.5 and 1.0 micrograms/ml) ranged from 2.6 to 5.0% and the accuracy of the assay was 106 +/- 6 and 100 +/- 5% for the low and high level controls respectively. The limit of quantitation for the assay was 0.1 micrograms/ml. No interfering peaks were detected in any patient samples. This method can be used as a primary analytical method to measure mexiletine plasma levels or can serve as a convenient back-up method to HPLC procedures when contaminating peaks coelute with mexiletine.

FK 506 (Tacrolimus) metabolism by rat liver microsomes and its inhibition by other drugs.

The in vitro metabolism of FK 506 and its inhibition by other drugs was studied with hepatic microsomes from rats pre-treated with dexamethasone, a selective cytochrome P-450 IIIA inducer. Nonspecific inhibitors of cytochrome P-450, such as ketoconazole, itraconazole, fluconazole and SKF 525 A, and most of the cytochrome P-450 IIIA specific substrates used in this study significantly inhibited FK 506 metabolism. Although cyclosporine is a known substrate of cytochrome P-450 IIIA, it had no effect on FK 506 metabolism. Cytochrome P-450 II substrates had minimal but significant effect on FK 506 metabolism. This data supports our earlier observations that FK 506 metabolism is mediated predominantly by the steroid inducible cytochrome P-450 IIIA enzyme subfamily. The results of this study indicate that in transplant patients there is a potential for an interaction of FK 506 with other drugs that are metabolized by the cytochrome P-450 IIIA subfamily or those that alter the activity of cytochrome P-450 IIIA subfamily. Careful monitoring and FK 506 dosing adjustment may be necessary to maintain therapeutic concentration and minimize toxicity in patients receiving this agent.

Reductive metabolism of the anticonvulsant agent zonisamide, a 1,2-benzisoxazole derivative.

1. The metabolism of zonisamide in vitro was characterized through aerobic and anaerobic incubations with rat liver subcellular fractions and cultured gastrointestinal microflora. 2. Zonisamide reacted with rat hepatic microsomal cytochrome P-450 and exhibited a Type I binding spectrum. 3. Metabolism of zonisamide in vitro by hepatic subcellular fractions and cultured gastrointestinal flora produced a single metabolite, 2-(sulphamoylacetyl)-phenol (2-SMAP), by reductive cleavage of the 1,2-benzisoxazole ring. 4. The reductive metabolism of zonisamide was primarily mediated by microsomal cytochrome P-450. The soluble fraction enhanced reduction when combined with the microsomal fraction but itself possessed only weak reductive activity. 5. Reduction of zonisamide by the most enzymically active liver fractions required NADPH, was stimulated by FMN and SKF-525A, and was inhibited by CO or air, as well as by n-octylamine. 6. Unlike their involvement in the reduction of numerous nitro, azo, and N-oxide compounds, cultured aerobic and anaerobic intestinal flora were not principally involved in the reduction of zonisamide.

New high-performance liquid chromatographic method for the determination of alprazolam and its metabolites in serum: instability of 4-hydroxyalprazolam.

A high-performance liquid chromatographic method was developed for the determination of alprazolam (ALP) and its active metabolites, alpha-hydroxyalprazolam (AOH) and 4-hydroxyalprazolam (4OH) in human serum. During assay development, the instability of 4OH was revealed. Factors affecting stability of 4OH were then investigated. In this report, the assay methodology for the determination of ALP and AOH, the instability of 4OH, subsequent interference of 4OH breakdown products with AOH quantification, and factors affecting 4OH stability are described. The clinical significance of our findings are reported.

Metabolism of the anticonvulsant agent zonisamide in the rat.

The metabolism of zonisamide [3-(sulfamoylmethyl)-1,2-benzisoxazole], a new anticonvulsant, has been studied. In rats dosed with [14C]zonisamide (100 mg/kg, ip) 86.5% of the radioactive dose was excreted in the urine over 72 hr. The remainder of the radioactive dose (13.5%) was excreted in the feces over the same time period. Unchanged drug and eight metabolites were isolated from the urine, and the structures of five metabolites were assigned by physicochemical methods. metabolism of zonisamide primarily involves reductive and conjugative mechanisms, with oxidation of this compound being of minor metabolic significance. The percentage of urinary radioactivity accounted for by unmetabolized zonisamide and metabolites is as follows: unmetabolized zonisamide (metabolite 9), 32.8%; metabolite 8 [N-acetyl-3-(sulfamoylmethyl)-1,2-benzisoxazole], 7.7%; unidentified metabolite 7, 2.4%; metabolite 6 (zonisamide glucuronide), 7.6%; metabolite 5 [3-(carboxy)-1,2-benzisoxazole], 5.4%; unidentified metabolite 4, 13.1%; metabolite 3 [2-(sulfamoylacetyl)-phenol glucuronide], 12.6%; unidentified metabolite 2, 3.8%; and metabolite 1 (2-[1-(amino)sulfamoylethyl]phenol sulfate), 2.3%. A total of 87.7% of the 0-24 hr urinary radioactivity was accounted for by unchanged zonisamide and metabolites.

A comparison of the effects of cyclosporine (CsA) on hepatic microsomal drug metabolism in three different strains of rat.

1. Male albino Wistar (W), Sprague-Dawley (SD) and Fischer-344 (F344) rats, Rattus norvegicus, were dosed orally once daily with cyclosporine (CsA), 25 mg/kg/day, for 14 days. Control and treated rats were then sacrificed and selected assays were performed to measure differences in hepatic microsomal enzyme activity including: protein (PROT), cytochrome P-450 (CYT-P450) and cytochrome b5 (CYT-b5) levels, NADPH-cytochrome c reductase (CYT-c) activity, ethylmorphine N-demethylase (ETM) and aniline hydroxylase (ANL) activities. 2. Two-way analysis of variance (ANOVA) indicated that the effect of treatment alone showed significant strain-related differences in all parameters examined except CYT-b5 levels, while the effect of strain alone indicated significant differences in PROT, CYT-B5, CYT-c, and ANL. 3. The interaction component of the two-way ANOVA (treatment x strain) indicated strain-related differences in substrate metabolism only (ETM and ANL). 4. F344 rats were found to be significantly different from either Sprague-Dawley or Wistar rats in terms of the effect of CsA on ETM and ANL metabolism.

Biopharmaceutical aspects of FK-506.

FK is a potent immunosuppressive agent. FK can be analyzed in biologic fluids by EIA. The oral absorption of FK is rapid but variable in dogs. After intramuscular administration, FK is slowly and continuously absorbed. FK is primarily eliminated by metabolism. Less than 1% of the administered dose is excreted in the bile or the urine. After chronic intramuscular administration FK inhibits drug metabolism. Monitoring of FK levels in plasma is essential for the proper interpretation of efficacy and toxicity studies.

3,4-Dichlorobenzyloxyacetic acid is extensively metabolized to a taurine conjugate in rats.

The metabolism of the antisickling agent 3,4-dichlorobenzyloxyacetic acid (3,4-DCBAA) was examined after ip administration to rats. Within 5 days after administration of radiolabeled 3,4-DCBAA, 77.4 +/- 4.6% of the dose was recovered in the urine and only 3.2 +/- 0.5% was recovered in the feces. Metabolites in the urine were isolated and characterized by HPLC, electron impact MS, and LC/MS, and their identities were confirmed by comparing their spectra with those of synthetic standards. Quantitation of these urinary metabolites revealed that the majority of the radioactive dose was excreted as a taurine conjugate (60.1 +/- 4.4%), while lesser amounts were excreted as 3,4-dichlorohippurate, unchanged 3,4-DCBAA, the glycine conjugate of 3,4-DCBAA, and a polar unknown which is believed to be glycolic acid. A pathway involving an initial O-dealkylation at the benzyl carbon of 3,4-DCBAA and proceeding through the glycine conjugation of 3,4-dichlorobenzoic acid has been proposed to explain the formation of 3,4-dichlorohippurate and the polar unknown. The extensive conjugation of 3,4-DCBAA with taurine is an unprecedented observation in rats, which usually utilize glycine for amino acid conjugation reactions. Further studies with 3,4-DCBAA may provide insight into the enzymatic mechanisms of taurine conjugation, which are not well defined at this time.

The effects of ethylene oxide (EO) exposure on tissue glutathione levels in rats and mice.

Male Fischer 344 rats and male Swiss-Webster mice were exposed to different atmospheric concentrations of ethylene oxide (EO) for 4 hours. In mice sacrificed immediately after exposure to 100, 450 or 900 ppm EO, there was a concentration related decrease in the GSH levels of all tissues examined. Similar findings were obtained in rats immediately after exposure to 100, 600 or 1200 ppm EO except that blood GSH levels were not affected at any exposure concentration. In both species, lung and liver GSH levels were depressed at all exposure concentrations. Twenty-four hours after exposure to 1200 ppm EO, the GSH concentrations of rat bone marrow and testis had not returned to control levels. Only blood GSH levels remained depressed in mice 48 hours after exposure to 900 ppm EO. The results indicate a marked species difference between rats and mice regarding the effects of EO exposure on blood GSH levels which may have important toxicological implications.

The effects of cyclosporin A (CsA) on hepatic microsomal drug metabolism in the rat.

The effects of cyclosporin A (CsA), a powerful immunosuppressant, on the hepatic microsomal mixed function oxidase (MFO) system was studied in male rats. Difference spectroscopy studies indicated that CsA binds to cytochrome P-450 producing a type I spectral change. To investigate potential interactions with the MFO system, CsA was administered orally at doses of either 25 mg/kg or 50 mg/kg once daily for 9 days. Various metabolic parameters were examined, including: levels of microsomal protein, cytochrome P-450, and cytochrome b5, NADPH-cytochrome c reductase activity, N-demethylation of ethylmorphine (ETM), and p-hydroxylation of aniline (ANL). Rats treated with 50 mg/kg showed a 25% or greater decrease over controls in all parameters examined except microsomal protein and cytochrome b5 levels. Rats treated with 25 mg/kg showed a 28% or greater decrease in all parameters except microsomal protein, cytochrome b5, and cytochrome P-450. Kinetic studies of ETM-N-demethylase and ANL-hydroxylase activities were conducted either with microsomes prepared from CsA-treated animals (50 mg/kg/day for 5 days) or with pooled microsomes prepared from untreated animals to which CsA was added directly. Enzyme reaction velocities were measured and apparent KM and apparent Vmax were determined. Studies with CsA-treated animals revealed a 57% decrease in both KM and Vmax for ETM-N-demethylase, and a 46% decrease in KM and a 32% decrease in Vmax for ANL-hydroxylase. Studies involving direct addition of CsA to microsomes at final concentrations of 0.01 mM and 0.10 mM revealed no significant changes in apparent KM or Vmax for either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive metabolism of furazolidone by Escherichia coli and rat liver in vitro.

The metabolism of furazolidone by rat liver and Escherichia coli was characterized in vitro under aerobic and anaerobic incubation conditions. Rat liver 9000g supernatant rapidly metabolized 14C-furazo-lidone to more polar metabolites in the presence or absence of oxygen when NADPH was provided as a cofactor. At least five polar radiolabeled metabolites were detected in these incubations by high pressure liquid chromatography. Moreover, a significant (30-40%) proportion of the total radiolabeled metabolites remained tightly associated with liver protein despite repeated organic solvent extractions of the tissue. The major solvent-extractable metabolites produced under aerobic and anaerobic incubation conditions were isolated and analyzed by mass spectrometry. The mass spectra indicated that these derivatives possessed the same chemical structure. Subsequently, this metabolite was unequivocally identified as 3-(4-cyano-2-oxobutylideneamino)-2-oxazolidinone, an end product of reductive metabolism of the nitro group of furazolidone. The formation of the reduced metabolite under aerobic conditions indicated that this metabolic pathway was markedly less sensitive to oxygen than many previously studied nitroreduction reactions catalyzed by mammalian enzymes. This NADPH-dependent, oxygen-insensitive nitroreductase activity was further localized to the microsomal fraction of rat liver. E. coli also rapidly metabolized furazolidone (FZN) to a complex series of metabolites, including the reduced cyano metabolite, under both aerobic and anaerobic conditions. Sonic lysis of the bacteria released an NADPH-dependent, oxygen-insensitive nitroreductase which converted FZN to the cyano metabolite and other unidentified derivatives. The complete reduction of FZN by the solubilized bacterial enzyme was strongly inhibited by the addition of the thiol nucleophile glutathione to the incubation medium.

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver.

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24-36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulse labeled by an intravenous injection of [3H]delta-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic delta-aminolevulinic acid synthetase (delta-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.