Investigation on the urinary excretion kinetics of three atractylenolides from crude and processed Atractylodis rhizoma extracts in rats by UPLC-MS/MS.

Atractylodis rhizoma is a frequently-used traditional Chinese medicine in clinical practice, which have the effect of eliminating dampness and tonifying spleen. And after being processed with wheat bran, the dryness of A. rhizoma is reduced, and the function of tonifying spleen is enhanced. Atractylenolides are the major bioactive components of A. rhizoma, including atractylenolide I (AI), atractylenolide Ⅱ (AⅡ) and atractylenolide Ⅲ (AⅢ). The present study aimed to develope a new UPLC-MS/MS method for simultaneous quantification of three atractylenolides in rat urine, and applied to the excretory kinetics in Sprague-Dawley rats after oral administration of crude and processed A. rhizoma extracts. Analytes and internal standard were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The excretory kinetics parameters were calculated by a urine drug analysis model of drug and statistics (DAS) 3.2.8 software. The t1/2 and Ke of three atractylenolides had no significant difference between crude and processed A. rhizoma, but the recovery accumulative excretion of them in processed A. rhizoma were apparently higher than the crude ones (p<0.05, p<0.01). The results showed that only a small amount of atractylenolides excreted in urine and processing A. rhizoma with wheat bran by stir frying could promote the urinary excretion of them.

Human Gut Microbiota Metabolism of Dietary Sesquiterpene Lactones: Untargeted Metabolomics Study of Lactucopicrin and Lactucin Conversion In Vitro and In Vivo.

SCOPE: Gut microbiota converts dietary phytochemicals into metabolites and modulates their health effects. The microbial metabolism of dietary terpenoids, as the sesquiterpene lactones of leafy vegetables, is unknown. METHODS AND RESULTS: In vitro fermentation of lactucopicrin, lactucin, and romaine lettuce with gut microbiota from independent donors, show their extensive metabolism through untargeted metabolomics of the fecal incubations. Dehydroxylations and double bond hydrogenations are the main catabolic reactions. Isomers of dihydrolactucopicrin, tetrahydrolactucopicrin, and deoxylactucin, are observed after lactucopicrin metabolism. Tetrahydrolactucin and hexahydrolactucin are also found after lactucin metabolism. Lettuce fermentation shows similar metabolic conversions. Phase II conjugates of most of these metabolites are detected in the urine of healthy volunteers after escarole salad intake. Glucuronides, and sulfates, of dihydrolactucopicrin, tetrahydrolactucopicrin, dihydrolactucin, and deoxylactucin, are detected in the urine although with large inter-subject variability. CONCLUSION: This is the first report on the gut microbiota metabolism of sesquiterpene lactones in humans, and one of the first reports to describe that dietary terpenoids of widely consumed leafy vegetables are extensively catabolized by human gut microbiota. A large inter-subject variation in the metabolism of sesquiterpene lactones also reflects differences in gut microbiota composition. It suggests that inter-individual differences in their health effects should be expected.

Untargeted UPLC-MS metabolomics reveals multiple changes of urine composition in healthy adult volunteers after consumption of curcuma longa L. extract.

Curcuma longa L. is used as food supplement to prevent diseases, although limited studies have been performed on healthy subjects up to now. In the present work, an untargeted UPLC-MS metabolomics approach was applied to study the changes of 24-hours urinary composition on healthy volunteers due to a 28-days daily consumption of a dried C. longa extract containing a standardized amount of curcuminoids. Changes in the excretion of different metabolites were observed after supplementation. Curcumin and two metabolic derivatives (hexahydrocurcumin and dihydrocurcumin) were detected in urine, indicating the absorption of the main curcuminoid from the extract and its further metabolism by liver and gut microbiota. For the first time ar-turmerone, the main apolar constituent of curcuma, was detected in urine in intact form, and its presence was confirmed by a targeted GC-MS analysis. The increase of tetranor-PGJM and tetranor-PGDM, two prostaglandin-D2 metabolites, was observed, being related to the anti-inflammatory effect exerted by curcuma. The variation of the amounts of HPAG, PAG, proline-betaine and hydroxyphenyllactic acid indicate that the supplementation induced changes to the activity of gut microbiota. Finally, the reduced excretion of niacin metabolites (nicotinuric acid, trigonelline and 2PY) and medium- and short-chain acylcarnitines suggests that curcuma could induce the mitochondrial ß-oxidation of fatty acids for energy production in healthy subjects. Overall, the results indicate that a prolonged daily consumption of a dried curcuma extract exerts multiple effects on healthy subjects, furthermore they show the opportunity offered by untargeted metabolomics for the study of the bioactivity of natural extracts in healthy human volunteers.

Fate of ptaquiloside-A bracken fern toxin-In cattle.

Ptaquiloside is a natural toxin present in bracken ferns (Pteridium sp.). Cattle ingesting bracken may develop bladder tumours and excrete genotoxins in meat and milk. However, the fate of ptaquiloside in cattle and the link between ptaquiloside and cattle carcinogenesis is unresolved. Here, we present the toxicokinetic profile of ptaquiloside in plasma and urine after intravenous administration of ptaquiloside and after oral administration of bracken. Administered intravenously ptaquiloside, revealed a volume of distribution of 1.3 L kg-1 with a mean residence-time of 4 hours. A large fraction of ptaquiloside was converted to non-toxic pterosin B in the blood stream. Both ptaquiloside and pterosin B were excreted in urine (up to 41% of the dose). Oral administration of ptaquiloside via bracken extract or dried ferns did not result in observations of ptaquiloside in body fluids, indicating deglycosolidation in the rumen. Pterosin B was detected in both plasma and urine after oral administration. Hence, transport of carcinogenic ptaquiloside metabolites over the rumen membrane is indicated. Pterosin B recovered from urine counted for 7% of the dose given intravenously. Heifers exposed to bracken for 7 days (2 mg ptaquiloside kg-1) developed preneoplastic lesions in the urinary bladder most likely caused by genotoxic ptaquiloside metabolites.

[Determination of coriatin and corianin in plasma and urine using ultra-performance liquid chromatography-triple quadrupole mass spectrometry].

An ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) method has been developed for the determination of coriatin and corianin in plasma and urine, which are the biomarkers of poisoning caused by Coriaria sinica Maxim. Plasma and urine samples were extracted and purified using a solid supported liquid/liquid extraction method. Chromatographic separation was performed on a Cortecs C18 column (100 mm×2.1 mm, 1.6 µm) using a gradient elution of methanol and water. Coriatin and corianin were detected using negative electrospray ionization tandem mass spectrometry in multiple reaction monitoring (MRM) mode and quantified via a matrix working standard curve internal standard method; florfenicol was used as the internal standard. The assay was linear in the calibration range of 0.03-5.0 µg/L for coriatin and 0.3-50 µg/L for corianin in plasma, and 0.1-10 µg/L and 1-100 µg/L for coriatin and corianin in urine, respectively. The average recoveries were 86.2%-110% for coriatin and corianin in plasma and urine with relative standard deviations of 5.1%-14.6% (n=6). The limits of detection (S/N=3) for coriatin and corianin were 0.01 µg/L and 0.1 µg/L in plasma, and 0.03 µg/L and 0.3 µg/L in urine, respectively. The method is simple, sensitive and accurate for the determination of coriatin and corianin in plasma and urine for toxicological purposes.

Evaluation of the in vitro and in vivo metabolic pathway and cytochrome P450 inhibition/induction profile of Huperzine A.

Huperzine A (HupA), one of the reversible and selective acetylcholinesterase inhibitors derived from Chinese herb Huperzia Serrata, possesses affirmative action of ameliorating cognitive dysfunction of Alzheimer's disease. Up to now, the effects of HupA on human cytochrome P450s (CYPs) have not been fully elucidated. The purpose of the present study was to clarify the metabolic pathway of HupA in vitro and in vivo, and to evaluate the CYPs inhibition/induction profile of HupA in vitro. The catalytic activity of CYP enzymes (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4) was measured by the quantification of specific enzyme substrates using validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods. The in vivo metabolic pathway evaluation was performed in an open, single-dose pharmacokinetic study of HupA in fourteen elderly subjects, with urine collecting at certain intervals. In human liver microsomes, HupA (10 ng/mL) was not metabolized within 90 min, and it showed negligible inhibition against these CYP isoforms within 0.2-100 ng/mL. In human liver hepatocytes, the activities of CYP1A2 and CYP3A4 were not significantly altered when incubated at 2 or 20 ng/mL of HupA. After oral administration of 0.1 mg HupA, the total proportion of HupA excreted through urine was relatively high, accounting to 35± 9% at the limited time period of 48 h. These results suggest that HupA is substantially excreted by kidney unchanged rather than metabolized by human liver, and is unlikely to cause clinically relevant drug-drug interaction (DDI) when co-administrated with drugs that are metabolized by CYP isoenzyme system.

Individuality and Transgenerational Inheritance of Social Dominance and Sex Pheromones in Isogenic Male Mice.

Phenotypic variation and its epigenetic regulations within the inbred isogenic mice have long intrigued biologists. Here, we used inbred C57BL/6 mice to examine the individual differences and the inheritance of social dominance and male pheromones, expecting to create a model for studying the underlying epigenetic mechanisms for the evolution of these traits. We used a repeated male-male contest paradigm to form stable dominance-submission relationships between paired males and make superior or inferior quality manifest. Females showed olfactory preferences for the urine of dominant males to that of subordinate opponents. Gas chromatography-mass spectrometer analysis revealed that dominance-related or superior quality related pheromones were actually exaggerated male pheromone components (e.g., E-ß-farnesene, hexadecanol, and 1-hexadecanol acetate) of preputial gland origin. Although the socially naïve sons of both dominant and subordinate males elicited the same female attraction when reaching adulthood, the former could dominated over the latter during undergoing the male-male competition and then gained more attraction of females. Our results demonstrated that social dominance or superior quality and the related pheromones were heritable and could be expressed through the interaction between aggression-related epigenotypes and male-male contests. It suggested that the evolution of sexually selected traits could be epigenetically determined and promoted through female mate choice. The epigenetic mechanisms driving the individual differences in behavior and male pheromones deserve further studies.

In vivo absorption and disposition of α-cedrene, a sesquiterpene constituent of cedarwood oil, in female and male rats.

This study aimed to evaluate the potential of α-cedrene as a new anti-obesity drug by characterizing absorption, metabolism and pharmacokinetics in rats. α-Cedrene was administered intravenously (10 and 20 mg/kg) and orally (50 and 100 mg/kg) to female and male Sprague-Dawley rats. Blood, tissues, urine, and feces were collected at predetermined times. α-Cedrene concentrations were determined by a validated gas chromatography-tandem mass spectrometry (GC-MS/MS). A gas chromatography-mass selective detection (GC-MSD) method was used to identify the major metabolite. After i.v. injection, α-cedrene exhibited a rapid clearance (98.4-120.3 ml/min/kg), a large distribution volume (35.9-56.5 l/kg), and a relatively long half-life (4.0-6.4 h). Upon oral administration, it was slowly absorbed (Tmax = 4.4 h) with bioavailability of 48.7-84.8%. No gender differences were found in its pharmacokinetics. Upon oral administration, α-cedrene was highly distributed to tissues, with the tissue-to-plasma partition coefficients (Kp) far greater than unity for all tissues. In particular, its distribution to lipid was notably high (Kp = 132.0) compared to other tissues. A mono-hydroxylated metabolite was identified as a preliminary metabolite in rat plasma. These results suggest that α-cedrene has the favorable pharmacokinetic characteristics to be further tested as an anti-obesity drug in clinical studies.

Identification of metabolites of rupestonic acid in rat urine by liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Rupestonic acid, a potential anti-influenza agent, is an important and characteristic compound in Artemisia rupestris L., a well-known traditional Uighur medicine for the treatment of colds. In the present study, high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry was used to detect and identify the metabolites in rat urine after oral administration of rupestonic acid. A total of 10 metabolites were identified or partially characterized. The structure elucidations of the metabolites were performed by comparing the changes in accurate molecular masses and fragment ions with those of the parent compound. The results showed that the main metabolites of rupestonic acid in rat urine were formed by oxidation, hydrogenation and glucuronidation. A metabolism pathway was proposed for the first time based on the characterized structures. This metabolism study can provide essential information for drug discovery, design and clinical application of rupestonic acid.

Metabolism of (2S)-pterosin A: identification of the phase I and phase II metabolites in rat urine.

The metabolic profile of the potent hypoglycemic agent, (2S)-pterosin A (1), in rat urine via intragastrical oral administration was investigated. In total, 19 metabolites (M1-M19) were identified. Among these, 16 metabolites were characterized by high-performance liquid chromatography solid-phase extraction-tube transfer-NMR, and seven metabolites were further isolated from the treated urine to enable further structural determination. Twelve of these are new compounds. The phase I metabolites of 1 were formed via various oxidations at positions C-3, C-10, C-12, C-13, or C-1 followed by decarboxylation of C-10 or C-14, and lactonization at C-12/C-14 or C-14/C-12. The phase II metabolites were glucuronide conjugates from the parent compound or phase I metabolites. The major metabolites were found to be (2S)-14-O-glucuronylpterosin A (M9), (2S)-2-hydroxymethylpterosin E (M14), and (±)-pterosin B (M19). Quantitative HPLC analysis of metabolites, based on similar UV absorption and use of the regression equation of 1, indicated that ∼71% 1 was excreted as metabolites in rat urine.

Pharmacokinetics, tissue distribution, and excretion of buagafuran in rats.

The pharmacokinetics, tissue distribution, and excretion of buagafuran (BF, 4-butyl-α-agarofuran), a promising antianxiety drug isolated from Gharu-wood (Aquilaria agallocha Roxb), were investigated in rats. BF plasma concentration was determined in rats after oral and intravenous doses by GC-TOF-MS. BF showed nonlinear pharmacokinetics after oral and intravenous administration of 4, 16, and 64 mg/kg. The AUC(0-∞) and C(max) did not increase proportionally with doses, indicating the saturation in absorption kinetics of BF in rats after oral dosage. BF absorption was extremely poor with an absolute bioavailability below 9.5%. After oral administration of (3)H-BF (4 mg/kg) to rats, radioactivity was well distributed to the tissues examined. The highest radioactivity was found in gastrointestinal tract, followed by liver and kidney. Radioactivity in brain, as a target organ, was about 20-40% of that in plasma at all time points. Total mean percent recovery of radioactive dose was about 80% in rats (51.2% in urine; 28.7% in feces). Bile elimination was also the major excretion route of BF, and 45.4% of the radioactive dose was recovered in bile.

Isolation and identification of phase 1 metabolites of curcumol in rats.

Curcumol is one of the major components of the essential oil of Curcuma wenyujin with the structure of a guaiane-type sesquiterpenoid hemiketal. It exhibits clear antitumor, antihepatic fibrosis, antioxidant, and antimicrobial activities. In this article, the metabolism of curcumol in rats was investigated by characterizing metabolites excreted into urine. Sixteen phase 1 metabolites of curcumol were isolated from the urine of rats after an oral dose of 40 mg/kg, and their structures were elucidated on the basis of spectroscopic data. The metabolites were characterized as 2α-hydroxycurcumol (M-1), (11αH)-3α-hydroxy-9-en-8,13-epoxycurcumol (M-2), (11αH)-14-hydroxy-9-en-8,13-epoxycurcumol (M-3), (11ßH)-14-hydroxy-9-en-8,12-epoxycurcumol (M-4), 10α,14-dihydroxy-(1αH,7ßH)-guai-4-en-3,8-dione (M-5), 10ß,14-dihydroxy-(1αH,7ßH)-guai-4-en-3,8-dione (M-6), 10ß-hydroxy-(1αH,7ßH,11αH)-guai-8(13),8(14)-diepoxy-4-en-3-one (M-7), 10ß-hydroxy-(1αH,7ßH,11ßH)-guai-8(12),8(14)-diepoxy-4-en-3-one (M-8), 10α-hydroxy-(1αH,7ßH,11αH)-guai-8(13), 8(14)-diepoxy-4-en-3-one (M-9), 10α-hydroxy-(1αH,7ßH,11ßH)-guai-8(12),8(14)-diepoxy-4-en-3-one (M-10), 10α,14,15-trihydroxy-(1αH,7ßH)-guai-4-en-3,8-dione (M-11), 10ß-hydroxy-(1αH,7ßH)-guai-4-en-3,8-dioxo-13-oic acid (M-12), (1αH,7ßH)-guai-4,10(14)-dien-3, 8-dioxo-13-oic acid (M-13), 5ß,10ß-dihydroxy-(1αH,7ßH,11αH)-guai-8(13),8(14)-diepoxide (M-14), 10ß,14-dihydroxycurcumol (M-15), and 5ß,10ß,14-trihydroxy-(1αH,7ßH)-guai-8-one (M-16). All were newly identified compounds, among which M-3 and M-4, M-5 and M-6, and M-7, M-8, M-9, and M-10 are three groups of epimers. On the basis of the metabolite profile, the possible metabolic pathways of curcumol in rats are proposed. This is the first study of the metabolites of guaiane-type sesquiterpene in animals.

Characterization of human urinary metabolites of the antimalarial piperaquine.

Five metabolites of the antimalarial piperaquine (PQ) (1,3-bis-[4-(7-chloroquinolyl-4)-piperazinyl-1]-propane) have been identified and their molecular structures characterized. After a p.o. dose of dihydroartemisinin-piperaquine, urine collected over 16 h from two healthy subjects was analyzed using liquid chromatography (LC)/UV, LC/tandem mass spectrometry (MS/MS), Fourier transform ion cyclotron resonance (FTICR)/MS, and H NMR. Five different peaks were recognized as possible metabolites [M1, 320 m/z; M2, M3, and M4, 551 m/z (PQ + 16 m/z); and M5, 567 m/z (PQ + 32 m/z)] using LC/MS/MS with gradient elution. The proposed carboxylic M1 has a theoretical monoisotopic molecular mass of 320.1166 m/z, which is in accordance with the FTICR/MS (320.1168 m/z) findings. The LC/MS/MS results also showed a 551 m/z metabolite (M2) with a distinct difference both in polarity and fragmentation pattern compared with PQ, 7-hydroxypiperaquine, and the other 551 m/z metabolites. We suggest that this is caused by N-oxidation of PQ. The results showed two metabolites (M3 and M4) with a molecular ion at 551 m/z and similar fragmentation pattern as both PQ and 7-hydroxypiperaquine; therefore, they are likely to be hydroxylated PQ metabolites. The molecular structures of M1 and M2 were also confirmed using H NMR. Urinary excretion rate in one subject suggested a terminal elimination half-life of about 53 days for M1. Assuming formation rate-limiting kinetics, this would support recent findings that the terminal elimination half-life of PQ has been underestimated previously.

Pharmacokinetics, metabolism, and routes of excretion of intravenous irofulven in patients with advanced solid tumors.

Irofulven is currently in Phase 2 clinical trials against a wide variety of solid tumors and has demonstrated activity in ovarian, prostate, gastrointestinal, and non-small cell lung cancer. The objectives of this study were to determine its pharmacokinetics and route of excretion and to characterize its metabolites in human plasma and urine samples after a 30-min i.v. infusion at a dose of 0.55 mg/kg in patients with advanced solid tumors. Three patients were administered i.v. 100 microCi of [14C]irofulven over a 30-min infusion on day 1 of cycle 1. Serial blood and plasma samples were drawn at 0 (before irofulven infusion) and up to 144 h after the start of infusion. Urine and fecal samples were collected for up to 144 h after the start of infusion. The mean urinary and fecal excretion of radioactivity up to 144 h were 71.2 and 2.9%, respectively, indicating renal excretion was the major route of elimination of [14C]irofulven. The C(max), AUC(0-infinity), and terminal half-life values for total radioactivity were 1130 ng-Eq/ml, 24,400 ng-Eq . h/ml, and 116.5 h, respectively, and the corresponding values for irofulven were 82.7 ng/ml, 65.5 ng . h/ml, and 0.3 h, respectively, suggesting that the total radioactivity in human plasma was a result of the metabolites. Twelve metabolites of irofulven were detected in human urine and plasma by electrospray ionization/tandem mass spectrometry. Among these metabolites, the cyclopropane ring-opened metabolite (M2) of irofulven was found, and seven others were proposed as glucuronide and glutathione conjugates.

Insect pheromones and precursors in female African elephant urine.

Using automated solid-phase dynamic extraction and gas chromatography-mass spectrometry, our search for urinary chemical signals from ovulatory female African elephants (Loxodonta africana) has revealed the bark beetle aggregation pheromones frontalin, exo-brevicomin, and endo-brevicomin, as well as their precursors and the aphid alarm pheromones (E,E)-alpha-farnesene and (E)-beta-farnesene. Enantiomeric ratios for brevicomins have been determined. Prior discovery of common insect/elephant pheromones in Asian elephants, namely, (Z)-7-dodecenyl acetate and frontalin, suggests that the present findings may yield valuable insights into chemical communication among African elephants.

Melanocortin-5 receptor deficiency reduces a pheromonal signal for aggression in male mice.

Mice lacking the melanocortin-5 receptor (MC5R) exhibit decreased sensitivity to the stimulatory effects of systemic melanocortin injections on aggressive behavior. Because the pheromone-producing preputial gland expresses the MC5R, we tested the hypothesis that decreases in preputial pheromones underlie the behavioral deficit. Here we show that MC5R deficiency decreases preputial and urine levels of the sex pheromones, alpha- and beta-farnesene, relative to wild-type mice. We also demonstrate that farnesenes potently stimulate aggression in mice. Moreover, farnesene-stimulated aggression is reduced in MC5R-deficient mice, relative to wild-type mice. Our results suggest that activation of the MC5R promotes aggression by increasing farnesene signaling.

Glucuronidation of dihydroartemisinin in vivo and by human liver microsomes and expressed UDP-glucuronosyltransferases.

The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with [12-(3)H]DHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified alpha-DHA-beta-glucuronide (alpha-DHA-G) and a product characterized as the tetrahydrofuran isomer of alpha-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, alpha-DHA-G, was highly variable (median 0.75; range 0.09-64). Nevertheless, alpha-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from alpha-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found (V(max) 177 +/- 47 pmol min(-1) mg(-1), K(m) 90 +/- 16 microM). Alpha-DHA-G was formed in incubations of DHA with expressed UGT1A9 (K(m) 32 microM, V(max) 8.9 pmol min(-1) mg(-1)) or UGT2B7 (K(m) 438 microM, V(max) 10.9 pmol mg(-1) min(-1)) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that alpha-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.

Voltammetric determination of parthenolide in spiked human plasma and urine.

The voltammetric behaviour of parthenolide, a biologically active sesquiterpene lactone, was studied using direct current (DCt), alternating current and differential-pulse polarography (DPP). Parthenolide developed well-defined cathodic waves over the whole pH range in Britton-Robinson buffers. At pH 10 the diffusion current constant was 3.54 +/- 0.08 (+/- standard deviation; n = 8). The current vs concentration plots were rectilinear over the range 4-36 and 1-28 micrograms/mL in the DCt and DPP modes, respectively, with a minimum detectability of 0.06 microgram/mL (about 1 x 10(-7) M) using the latter technique. The waves were characterised as being diffusion controlled, although adsorption phenomenon played a limited role in the electrode process. The described analytical method was applied to the determination of parthenolide in spiked human urine and plasma; the percentage recoveries were 95.72 +/- 0.22 and 94.0 +/- 0.13 (+/- standard deviation; n = 9), respectively.

Efficient preparations of the beta-glucuronides of dihydroartemisinin and structural confirmation of the human glucuronide metabolite.

New and greatly improved preparations of the 12alpha,1'beta- (5) and 12beta,1'beta- (6) glucuronides of dihydroartemisinin (DHA, 2) are reported using anomeric hydroxy and imidate glucuronate intermediates. Comparison of the synthetic and natural materials shows that the human metabolite of DHA is the 12alpha-epimer 5.

Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl) fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi's sarcoma.

The pharmacokinetics of TNP-470 and its major metabolites were investigated in AIDS patients enrolled in a phase I dose escalation trial for the treatment of Kaposi's sarcoma. The patients received TNP-470 by 1-h intravenous infusion in dose cohorts of 10, 20, 30, 40, 50 and 70 mg/m2. The parent drug and metabolites, MII and MIV, were measured by high-performance liquid chromatography/mass spectrometry (HPLC/MS) in plasma samples collected during and out to 168 h after the beginning of the infusion. Both metabolites were detected in all patients' plasma, while the parent drug was undetectable at time-points as early as 5 min after the end of infusion for some patients. A large interpatient variability of pharmacokinetic parameters among the dosing cohorts was observed for TNP-470, with a mean (+/- SD) plasma elimination half-life (t1/2) of 0.06 +/- 0.04 h, plasma clearance (CL) of 1487 +/- 1216 l/h and an area under the concentration versus time curve (AUC) of 49.9 +/- 35.8 ng/ml x h. Time to maximum plasma concentration (Tmax) typically occurred before the end of the infusion. The predominant plasma metabolite was MII with a t1/2 of 1.21 +/- 0.43 h, AUC of 1226 +/- 2303 l/h and a Tmax occurring between 5 and 15 min after infusion. The reported active metabolite MIV had a t1/2 of 0.24 +/- 0.13 h, AUC of 24.9 +/- 32.6 ng/ml x h and a Tmax occurring between the midpoint of the infusion and 15 min after infusion. The parent drug was undetectable by HPLC/MS/MS in urine samples collected and pooled between 0-6 and 6-24 h from the beginning of drug administration. Metabolite MIV was present in the 0-6-h urine pool of two patients enrolled in the highest dosing cohorts, equivalent to 0.4% of the administered dose. Metabolite MII was present in all 0-6-h samples analyzed and represented 1.12 +/- 0.9% of the administered dose. Renal clearance (CLR) for MII was 140 +/- 70 ml/h.