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Malignant pleural mesothelioma (MPM) is a devastating malignancy with a prognosis of12 months. Even with bans on the use of asbestos in most Western countries, the incidence is still increasing due to the long latency periods between exposure and development of the disease. Diagnosis is often delayed due to invasive biopsies and lack of distinguishable markers. Patients frequently present with pleural effusions months to years before a radiologically detectable mass appears. This study aimed to investigate the proteome of pleural effusions taken from patients with MPM, adenocarcinoma and benign conditions in an attempt to identify a biomarker for early diagnosis. We identified several proteins that may be possible targets and warrant further investigation. Due to the predominance of up regulated proteins involved in VEGF signalling in MPM, we analysed VEGFA levels in effusions and found a strong correlation between VEGFA levels and survival in MPM.

Background/aim The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model. Materials and methods Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice. The mice were treated with 200 μl/ml MET and/or 0.4% (w/v) VPA diluted in drinking water, or with vehicle control, and were monitored until the tumor volume reached 2,000 mm3 Evaluation of toxicity of the drug combination was determined in liver and kidney by histology. Results In both LNCaP and PC-3 xenografts, MET combined with VPA significantly reduced tumor growth during the first 4 weeks following treatment, and delayed the time-to-tumor volume of 2,000 mm3 by 90 days, as compared to MET or to VPA alone, and to vehicle control. There was no significant difference in total mouse weight, liver or kidney morphology in response to combination treatment (MET+VPA) compared to MET or VPA alone and vehicle control. Conclusion The combination treatment of MET with VPA is more effective at slowing prostate tumor growth in vivo compared to either drug alone, in mouse xenografts. These pre-clinical results support previous in vitro data and also demonstrate the low toxicity of the combination of these drugs, suggesting that this may be a potential new therapy to be investigated in clinical trials for prostate cancer.

Curcumin, a polyphenol derived from turmeric, has a wide variety of therapeutic benefits including antiinflammatory, antioxidative, and chemopreventative effects. Oral gavage is widely performed to administer curcumin in laboratory rodents in several experimental models. Although effective, this method can increase stress in the animal, potentially influencing experimental results. Moreover, oral gavage can result in mortality due to accidental instillation of fluid into the lungs, serious mechanical damage, and gavage-related reflux. Here we describe a method for the administration of fixed dosages of curcumin to rats through voluntary consumption of peanut butter, to reduce gavage-related morbidity and distress to animals and to provide environmental enrichment. Fischer 344 (n = 6) rats received 1100 mg/kg of a commercial curcumin product (equivalent to approximately 200 mg/kg of curcumin) in 8 g/kg of peanut butter daily for 5 wk. Curcumin concentrations in rat plasma were measured by using UPLC-MS at 2 to 4 h after administration. All rats voluntarily consumed the peanut butter-curcumin mixture consistently over the 5-wk period. Total curcumin concentrations in plasma samples collected 2 to 4 h after curcumin consumption were 171 ± 48.4 ng/mL (mean ± 1 SD; range, 103 to 240 ng/mL). This noninvasive curcumin delivery method was effective, eliminated the stress caused by daily oral gavage, and added environmental enrichment.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is responsible for metabolism of an endogenous inhibitor of nitric oxide synthase (NOS), asymmetric dimethylarginine (ADMA), which plays a key role in modulating angiogenesis. In addition to angiogenesis, tumours can establish a vascular network by forming vessel-like structures from tumour cells; a process termed vasculogenic mimicry (VM). Here, we identified over-expression of DDAH1 in aggressive MDA-MB-231, MDA-MB-453 and BT549 breast cancer cell lines when compared to normal mammary epithelial cells. DDAH1 expression was inversely correlated with the microRNA miR-193b. In DDAH1+ MDA-MB-231 cells, ectopic expression of miR-193b reduced DDAH1 expression and the conversion of ADMA to citrulline. In DDAH1− MCF7 cells, inhibition of miR-193b elevated DDAH1 expression. Luciferase reporter assays demonstrated DDAH1 as a direct target of miR-193b. MDA-MB-231 cells organised into tube structures in an in vitro assay of VM, which was significantly inhibited by DDAH1 knockdown or miR-193b expression. Mechanistically, we found miR-193b regulates cell proliferation and migration of MDA-MB-231 cells, whilst DDAH1 knockdown inhibited cell migration. These studies represent the first evidence for DDAH1 expression, regulation and function in breast cancer cells, and highlights that targeting DDAH1 expression and/or enzymatic activity may be a valid option in the treatment of aggressive breast cancers.

Background: Methotrexate (MTX) treatment in rheumatoid arthritis (RA) has been associated with lower cardiovascular risk compared to other disease-modifying antirheumatic drugs (DMARDs). We sought to identify whether the MTX-associated cardioprotection involves changes in blood pressure (BP) and/or arterial function. Methods: Clinic and 24-hour peripheral and central systolic and diastolic BP (SBP and DBP), augmentation index (AIx), pulse wave velocity (PWV) and plasma asymmetric dimethylarginine (ADMA) were assessed in RA patients on stable treatment with either MTX ± other DMARDs (MTX group, n = 56, age 61 ± 13 years, 70% females) or other DMARDs (non-MTX group, n = 30, age 63 ± 12 years, 76% females). Measurements were performed at baseline and after 8 months. Results: After adjusting for visit, age, gender, body mass index, folic acid use and 28-joint disease activity score, the MTX group had significantly lower clinic peripheral SBP (−7.7 mmHg, 95% CI −13.2 to −2.3, p = 0.006) and DBP (−6.1 mmHg, 95% CI −9.8 to −2.4, p = 0.001) and clinic central SBP (−7.8 mmHg, 95% CI −13.1 to −2.6, p = 0.003) and DBP (−5.4 mmHg, 95% CI −9.1 to −1.6, p = 0.005) versus the non-MTX group. Furthermore, the MTX group had significantly lower 24-hour peripheral and central SBP and DBP and PWV versus the non-MTX group ( p < 0.01 for all comparisons). By contrast, there were no significant between-group differences in AIx and ADMA. Conclusions: RA patients on MTX treatment had significantly lower clinic and 24-hour peripheral and central BP compared to those who did not take MTX. The lower BP with MTX may be related to differences in PWV, but not in AIx or ADMA concentrations. Further longitudinal studies including randomized controlled trials are warranted to confirm these findings, to identify other possible mechanisms responsible for the effects of MTX on BP and PWV, and to establish whether these effects might account for the reduced cardiovascular risk with MTX.

Given that the peculiar redox behavior of ergothioneine involves a rapid regeneration process, the measurement of its precursor and redox metabolite hercynine could be particularly useful in assessing its role in oxidative stress or other biological processes. Thus, a LC-MS/MS method for the determination of hercynine concentrations in whole blood was developed. After lysis of red blood cells by cold water, samples were filtered on micro concentrators at a controlled temperature of 4 &deg, C. The clear filtered fluid was then treated with diethylpyrocarbonate to derivatize hercynine for the analysis by LC-MS/MS. The derivatized analyte was isocratically separated as a carbethoxy derivative on a C18 column with a mobile phase of an aqueous 0.1% v/v formic acid and acetonitrile (95:5). Effluents were monitored by MRM transitions at m/z 270.28&rarr, 95 and 273.21&rarr, 95 for hercynine and its deuterated counterpart, respectively. No cross-talk between MRM transitions was observed and a good linearity was found within a range of 35&ndash, 1120 nmol/L. The LOD and LOQ were, respectively, 10.30 and 31.21 nmol/L with an intraday and intermediate precision below 7%. The average hercynine concentration in whole blood from 30 healthy male volunteers (aged 77 &plusmn, 12 years) was 178.5 &plusmn, 118.1 nmol/L. Overall, the method is easy to perform, allowing a rapid and accurate assessment of whole blood concentrations of hercynine.

Background/Aim: The hypoglycemic drug metformin (MET) and the anti-epileptic drug valproic acid (VPA) have individually shown anti-tumor effects in prostate cancer in vitro. The present study intended to investigate the efficacy of the combination of MET and VPA in prostate cancer treatment in a pre-clinical xenograft model. Materials and Methods: Prostate cancer cell lines (LNCaP and PC-3) were inoculated under the skin of BALB/c nude mice. The mice were treated with 200 μl/ml MET and/or 0.4% (w/v) VPA diluted in drinking water, or with vehicle control, and were monitored until the tumor volume reached 2,000 mm(3). Evaluation of toxicity of the drug combination was determined in liver and kidney by histology. Results: In both LNCaP and PC-3 xenografts, MET combined with VPA significantly reduced tumor growth during the first 4 weeks following treatment, and delayed the time-to-tumor volume of 2,000 mm(3) by 90 days, as compared to MET or to VPA alone, and to vehicle control. There was no significant difference in total mouse weight, liver or kidney morphology in response to combination treatment (MET+VPA) compared to MET or VPA alone and vehicle control. Conclusion. The combination treatment of MET with VPA is more effective at slowing prostate tumor growth in vivo compared to either drug alone, in mouse xenografts. These pre-clinical results support previous in vitro data and also demonstrate the low toxicity of the combination of these drugs, suggesting that this may be a potential new therapy to be investigated in clinical trials for prostate cancer.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a key enzyme involved in the metabolism of the endogenous nitric oxide synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA). Increased DDAH1 expression and subsequent increased NO production have been recently linked to cancer. Specifically, DDAH1 is implicated in establishment of a vascular network by tumour cells, vasculogenic mimicry (VM), which is strongly associated with tumour progression and poor patient prognosis. The use of DDAH1 inhibitors as potential therapeutic agents thus represents a growing field of interest. Here we describe a UPLC-MS assay to quantify stability and intracellular concentration of two small molecule DDAH1 inhibitors synthesised by our group, ZST316 and ZST152, following incubation with MDA-MB-231 breast cancer cells. In an in vitro assay of VM, both DDAH1 inhibitors significantly attenuated formation of capillary-like tube structures in a dose-dependent fashion. This was not due to cell toxicity or altered cell proliferation, but may be due in part to inhibition of cell migration. Mechanistically, we demonstrate significant modulation of the endogenous DDAH/ADMA/NO pathway following exposure of 100 μM ZST316 or ZST152: a 40% increase in the DDAH1 substrate ADMA, and a 38% decrease in the DDAH1 product l-citrulline. This study represents the first evidence for therapeutic inhibition of DDAH1 by small molecules in breast cancer.

The inhibition of arginase, resulting in higher arginine (ARG) availability for nitric oxide synthesis, may account for the putative protective effect of homoarginine (HOMOARG) against atherosclerosis and cardiovascular disease. However, uncertainty exists regarding the significance of HOMOARG-induced arginase inhibition in vivo. A novel UPLC-MS method, measuring the conversion of ARG to ornithine (ORN), was developed to determine arginase 1 and arginase 2 inhibition by HOMOARG, lysine (LYS), proline (PRO), agmatine (AG), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and NG-Monomethyl-L-arginine (L-NMMA). Plasma HOMOARG, ARG and ORN concentrations were further measured in 50 healthy older adults >65 years (27 males and 23 females). HOMOARG inhibited arginase 1 with IC50 and Ki values of 8.14 ± 0.52 mM and 6.1 ± 0.50 mM, and arginase 2 with IC50 and Ki values of 2.52 ± 0.01 mM and 1.73 ± 0.10 mM, respectively. Both arginase isoforms retained 90% activity vs. control when physiological HOMOARG concentrations (1–10 µM) were used. In partial correlation analysis, plasma HOMOARG was not associated with ARG (P = 0.38) or ARG/ORN ratio (P = 0.73) in older adults. Our results suggest that arginase inhibition is unlikely to play a significant role in the reported cardio-protective effects of HOMOARG.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Objectives The current study was designed to explore the associations between L-arginine metabolites and muscle mass and function in old age, which are largely unknown. Design The study used a randomised, double-blind, placebo-controlled design. Setting The study was carried out in a laboratory setting. Participants 50 healthy older adults [median age 70 years (IQR 67-73); 27 males]. Intervention Participants undertook an 18-week resistance exercise program, and a nutritional intervention (fish oil vs. placebo). Measurements Serum homoarginine, ornithine, citrulline, asymmetric dimethylarginine (ADMA), NG-monomethyl-L-arginine (L-NMMA), and symmetric dimethylarginine (SDMA), maximal voluntary contraction (MVC) and isokinetic torque of the knee extensors at 30° s-1 (MIT), muscle cross sectional area (MCSA) and quality (MQ) were measured at baseline and after the intervention. Results No significant exercise-induced changes were observed in metabolite concentrations. There were significant sex differences in the associations between metabolites and muscle parameters. After adjusting for age, glomerular filtration rate and fish oil intervention, citrulline (P=0.002) and ornithine (P=0.022) were negatively associated with MCSA at baseline in males but not females. However, baseline citrulline was negatively correlated with exercise-induced changes in MVC (P=0.043) and MQ (P=0.026) amongst females. Furthermore, amongst males, baseline homoarginine was positively associated with exercise-induced changes in MVC (P=0.026), ADMA was negatively associated with changes in MIT (P=0.026), L-NMMA (p=0.048) and ornithine (P

Proton pump inhibitor (PPI)-induced inhibition of dimethylarginine dimethylaminohydrolase 1 (DDAH1), with consequent accumulation of the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA), might explain the increased cardiovascular risk with PPI use. However, uncertainty exists regarding whether clinical PPI concentrations significantly inhibit DDAH1 under linear initial rate conditions, and whether PPI-induced DDAH1 inhibition significantly increases ADMA in humans. DDAH1 inhibition by esomeprazole, omeprazole, pantoprazole, lansoprazole and rabeprazole was determined by quantifying DDAH1-mediated L-citrulline formation in vitro. Plasma ADMA was measured in PPI users (n = 134) and non-users (n = 489) in the Hunter Community Study (HCS). At clinical PPI concentrations (0.1–10 μmol/L), DDAH1 retained >80% activity vs. baseline. A significant, reversible, time-dependent inhibition was observed with lansoprazole (66% activity at 240 min, P = 0.034) and rabeprazole (25% activity at 240 min, P in vitro. The lack of significant associations between PPIs and ADMA in HCS participants questions the significance of DDAH1 inhibition as a mechanism explaining the increased cardiovascular risk reported with PPI use.

A capillary electrophoresis coupled to tandem mass spectrometry (CE–MS/MS) has been used to make a qualitative determination of hercynine—the main precursor of l-ergothioneine biosynthesis—in some key human biological specimens, such as urine, whole blood, plasma, and saliva. From semiquantitative analysis results, the highest concentrations of hercynine were detected in saliva and whole blood, whereas much lower concentrations were measured in urine and plasma. Whole blood was the biological matrix with the highest concentration of l-ergothioneine followed by plasma, saliva, and urine. The antioxidant effects attributed to l-ergothioneine, along with its peculiar antioxidant mechanism, offer a possible explanation for the presence of the hercynine, as well as its concentration, in the considered biological matrices. "Fondazione Banco di Sardegna”, Sassari, Italy, and by the “Ministero dell’Università e della Ricerca”, Italy

1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant. Refereed/Peer-reviewed

Background Transcripts, which have been subject to Post-transcriptional exon shuffling (PTES), have an exon order inconsistent with the underlying genomic sequence. These have been identified in a wide variety of tissues and cell types from many eukaryotes, and are now known to be mostly circular, cytoplasmic, and non-coding. Although there is no uniformly ascribed function, several have been shown to be involved in gene regulation. Accurate identification of these transcripts can, however, be difficult due to artefacts from a wide variety of sources. Results Here, we present a computational method, PTESFinder, to identify these transcripts from high throughput RNAseq data. Uniquely, it systematically excludes potential artefacts emanating from pseudogenes, segmental duplications, and template switching, and outputs both PTES and canonical exon junction counts to facilitate comparative analyses. In comparison with four existing methods, PTESFinder achieves highest specificity and comparable sensitivity at a variety of read depths. PTESFinder also identifies between 13 % and 41.6 % more structures, compared to publicly available methods recently used to identify human circular RNAs. Conclusions With high sensitivity and specificity, user-adjustable filters that target known sources of false positives, and tailored output to facilitate comparison of transcript levels, PTESFinder will facilitate the discovery and analysis of these poorly understood transcripts. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-0881-4) contains supplementary material, which is available to authorized users.

Long-chain unsaturated fatty acids inhibit several cytochrome P450 and UDP-glucuronosyltransferase (UGT) enzymes involved in drug metabolism, including CYP2C8, CYP2C9, UGT1A9, UGT2B4, and UGT2B7. Bovine serum albumin (BSA) enhances these cytochrome P450 and UGT activities by sequestering fatty acids that are released from membranes, especially with human liver microsomes (HLM) as the enzyme source. Here, we report the effects of BSA on CYP1A2-catalyzed phenacetin (PHEN) O-deethylation and lidocaine (LID) N-deethylation using HLM and Escherichia coli-expressed recombinant human CYP1A2 (rCYP1A2) as the enzyme sources. BSA (2% w/v) reduced (p < 0.05) the K(m) values of the high-affinity components of human liver microsomal PHEN and LID deethylation by approximately 70%, without affecting V(max). The K(m) (or S(50)) values for PHEN and LID deethylation by rCYP1A2 were reduced to a similar extent. A fatty acid mixture, comprising 3 μM concentrations each of oleic acid and linoleic acid plus 1.5 μM arachidonic acid, doubled the K(m) value for PHEN O-deethylation by rCYP1A2. Inhibition was reversed by the addition of BSA. K(i) values for the individual fatty acids ranged from 4.7 to 16.7 μM. Single-point in vitro-in vivo extrapolation (IV-IVE) based on the human liver microsomal kinetic parameters obtained in the presence, but not absence, of BSA predicted in vivo hepatic clearances of PHEN O-deethylation and LID N-deethylation that were comparable to values reported in humans, although in vivo intrinsic clearances were underpredicted. Prediction of the in vivo clearances of the CYP1A2 substrates observed here represents an improvement on other experimental systems used for IV-IVE.

Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50-100 μM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with K(i) values ranging from 0.10 to 0.40 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (K(i) range 14-18 μM), whereas competitive inhibition (K(i) 62 μM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 μM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean V(max) for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 μM NFA. Mean K(m) values decreased in parallel with V(max), although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

The human UDP glycosyltransferase (UGT) 3A family is one of three families involved in the metabolism of small lipophilic compounds. Members of these families catalyze the addition of sugar residues to chemicals, which enhances their excretion from the body. The UGT1 and UGT2 family members primarily use UDP glucuronic acid to glucuronidate numerous compounds, such as steroids, bile acids, and therapeutic drugs. We showed recently that UGT3A1, the first member of the UGT3 family to be characterized, is unusual in using UDP N-acetylglucosamine as sugar donor, rather than UDP glucuronic acid or other UDP sugar nucleotides (J Biol Chem 283:36205-36210, 2008). Here, we report the cloning, expression, and characterization of UGT3A2, the second member of the UGT3 family. Like UGT3A1, UGT3A2 is inactive with UDP glucuronic acid as sugar donor. However, in contrast to UGT3A1, UGT3A2 uses both UDP glucose and UDP xylose but not UDP N-acetylglucosamine to glycosidate a broad range of substrates including 4-methylumbelliferone, 1-hydroxypyrene, bioflavones, and estrogens. It has low activity toward bile acids and androgens. UGT3A2 transcripts are found in the thymus, testis, and kidney but are barely detectable in the liver and gastrointestinal tract. The low expression of UGT3A2 in the latter, which are the main organs of drug metabolism, suggests that UGT3A2 has a more selective role in protecting the organs in which it is expressed against toxic insult rather than a more generalized role in drug metabolism. The broad substrate and novel UDP sugar specificity of UGT3A2 would be advantageous for such a function.

Because codeine (COD) is eliminated primarily via glucuronidation, factors that alter COD glucuronide formation potentially affect the proportion of the dose converted to the pharmacologically active metabolite morphine. Thus, in vitro-in vivo extrapolation approaches were used to identify potential drug-drug interactions arising from inhibition of COD glucuronidation in humans. Initial studies characterized the kinetics of COD-6-glucuronide (C6G) formation by human liver microsomes (HLM) and demonstrated an 88% reduction in the Michaelis constant (K(m)) (0.29 versus 2.32 mM) for incubations performed in the presence of 2% bovine serum albumin (BSA). Of 13 recombinant UDP-glucuronosyltransferase (UGT) enzymes screened for COD glucuronidation activity, only UGT2B4 and UGT2B7 exhibited activity. The respective S(50) values (0.32 and 0.27 mM) generated in the presence of BSA were comparable with the mean K(m) observed in HLM. Known inhibitors of UGT2B7 activity in vitro or in vivo and drugs marketed as compound formulations with COD were investigated for inhibition of C6G formation by HLM. Inhibition screening identified potential interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone. Inhibitor constant values generated for dextropropoxyphene (3.5 microM), fluconazole (202 microM), ketoconazole (0.66 microM), and methadone (0.32 microM) predicted 1.60- to 3.66-fold increases in the area under the drug plasma concentration-time curve ratio for COD in vivo. Whereas fluconazole and ketoconazole inhibited UGT2B4- and UGT2B7-catalyzed COD glucuronidation to a similar extent, inhibition by dextropropoxyphene and methadone resulted largely from an effect on UGT2B4. Interactions with dextropropoxyphene, fluconazole, ketoconazole, and methadone potentially affect the intensity and duration of COD analgesia.

To evaluate zolpidem as a mechanism-based inactivator of human CYP3A in vitro, and to assess its metabolic interaction potential with CYP3A drugs (in vitro-in vivo extrapolation; IV-IVE). A co- vs. pre-incubation strategy was used to quantify time-dependent inhibition of human liver microsomal (HLM) and recombinant CYP3A4 (rCYP3A4) by zolpidem. Experiments involving a 10-fold dilution step were employed to determine the kinetic constants of inactivation (K I and k inact) and to assess the in vitro mechanism-based inactivation (MBI) criteria. Inactivation data were entered into the Simcyp population-based ADME simulator to predict the increase in the area under the plasma concentration-time curve (AUC) for orally administered midazolam. Consistent with MBI, the inhibitory potency of zolpidem toward CYP3A was increased following pre-incubation. In HLMs, the concentration required for half maximal inactivation (K I) was 122 µM and the maximal rate of inactivation (k inact) was 0.094 min−1. In comparison, K I and k inact values with rCYP3A4 were 50 µM and 0.229 min−1, respectively. Zolpidem fulfilled all other in vitro MBI criteria, including irreversible inhibition. The mean oral AUC for midazolam in healthy volunteers was predicted to increase 1.1- to 1.7-fold due to the inhibition of metabolic clearance by zolpidem. Elderly subjects were more sensitive to the interaction, with mean increases in midazolam AUC of 1.2- and 2.2-fold for HLM IV-IVE and rCYP3A4 IV-IVE, respectively. Zolpidem is a relatively weak mechanism-based inactivator of human CYP3A in vitro. Zolpidem is unlikely to act as a significant perpetrator of metabolic interactions involving CYP3A.

UGT1A1 coding region mutations, including UGT1A1*6 (G71R), UGT1A1*7 (Y486D), UGT1A1*27 (P229Q) and UGT1A1*62 (F83L), have been linked to Gilbert syndrome in Asian populations, whereas homozygosity for UGT1A1*7 is associated with the Crigler-Najjar syndrome type II. This work compared the effects of (a) the individual UGT1A1 mutations on the glucuronidation kinetics bilirubin, beta-estradiol, 4-methylumbelliferone (4MU) and 1-naphthol (1NP), and (b) the Y486 mutation, which occurs in the conserved carboxyl terminal domain of UGT1A enzymes, on 4MU, 1NP and naproxen glucuronidation by UGT1A3, UGT1A6 and UGT1A10.Mutant UGT1A cDNAs were generated by site-directed mutagenesis and the encoded proteins were expressed in HEK293 cells. The glucuronidation kinetics of each substrate with each enzyme were characterized using specific high-performance liquid chromatography (HPLC) methods.Compared with wild-type UGT1A1, in-vitro clearances for bilirubin, beta-estradiol, 4MU and 1NP glucuronidation by UGT1A1*6 and UGT1A1*27 were reduced by 34-74%, most commonly as a result of a reduction in Vmax. However, the magnitude of the decrease in the in-vitro clearances varied from substrate to substrate with each mutant. The glucuronidation activities of UGT1A1*7 and UGT1A1*62 were reduced by95%. Introduction of the Y486D mutation essentially abolished UGT1A6 and UGT1A10 activities, and resulted in 60-90% reductions in UGT1A3 in-vitro clearances.The glucuronidation of all UGT1A1 substrates is likely to be impaired in subjects carrying the UGT1A1*6 and UGT1A1*62 alleles, although the reduction in metabolic clearance might vary with the substrate. The Y486D mutation appears to greatly reduce most, but not all, UGT1A activities.

Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the planar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4-MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glucuronides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged between UGT1A3 and UGT1A4 to generate UGT1A3-4((45-535)), UGT1A3-4((45-154))-3, UGT1A4-3((45-535)), and UGT1A4-3((45-154))-4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, UGT1A4-3((45-535)) and UGT1A4-3((45-154))-4 [but not UGT1A3-4((45-535)) and UGT1A3-4((45-154))-3] retained the capacity to glucuronidate LTG and TFP. Likewise, UGT1A3-4((45-535)) and UGT1A3-4((45-154))-3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3((45-535)) and UGT1A4-3((45-154))-4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. "Reciprocal" mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 reduced in vitro clearances for LTG and TFP glucuronidation by >90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronidation by >90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glucuronidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover.

Despite the important role of UDP-glucuronosyltransferases (UGT) in the metabolism of drugs, environmental chemicals and endogenous compounds, the structural features of these enzymes responsible for substrate binding and selectivity remain poorly understood. Since UGT2B7 and UGT2B15 exhibit distinct, but overlapping, substrate selectivities, UGT2B7-UGT2B15 chimeras were constructed here to identify substrate binding domains. A UGT2B7-15-7 chimera that incorporated amino acids 61-194 of UGT2B15 glucuronidated the UGT2B15 substrates testosterone and phenolphthalein, but not the UGT2B7 substrates zidovudine and 11alpha-hydroxyprogesterone. Derived apparent K(m) values for testosterone and phenolphthalein glucuronidation by UGT2B7-15((61-194))-7 were similar in magnitude to those determined for UGT2B15. Moreover, glucuronidation of the non-selective substrate 4-methylumbelliferone (4MU) by UGT2B7-15((61-194))-7 and UGT2B15 followed Michaelis-Menten and weak substrate inhibition kinetics, respectively, whereas 4MU glucuronidation by UGT2B7 exhibited sigmoidal kinetics characteristic of autoactivation. Six UGT2B7-15-7 chimeras that incorporated smaller domains of UGT2B15 were subsequently generated. Of these, UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 glucuronidated 4MU, but activity towards the other substrates investigated here was not detected. Like UGT2B7, the UGT2B7-15((61-157))-7, UGT2B7-15((91-157))-7 and UGT2B7-15((61-91))-7 chimeras exhibited sigmoidal 4MU glucuronidation kinetics. The sigmoidal 4MU kinetic data were well modelled using both the Hill equation and the expression for a two-site model that assumes the simultaneous binding of two substrate molecules at equivalent sites. It may be concluded that residues 61-194 of UGT2B15 are responsible for substrate binding and for conferring the unique substrate selectivity of UGT2B15, while residues 158-194 of UGT2B7 appear to facilitate the binding of multiple 4MU molecules within the active site.

Studies were performed to elucidate the mechanism responsible for the reduction in Km values of UDP-glucuronosyltransferase 2B7 (UGT2B7) substrates observed for incubations conducted in the presence of albumin. Addition of bovine serum albumin (BSA) and fatty acid-free human serum albumin (HSA-FAF), but not "crude" HSA, resulted in an approximate 90% reduction in the Km values for the glucuronidation of zidovudine (AZT) by human liver microsomes (HLM) and UGT2B7 and a 50 to 75% reduction in the S50 for 4-methylumbelliferone (4MU) glucuronidation by UGT2B7, without affecting Vmax. Oleic, linoleic, and arachidonic acids were shown to be the most abundant unsaturated long-chain fatty acids present in crude HSA and in the membranes of HLM and human embryonic kidney (HEK)293 cells, and it was demonstrated that these and other unsaturated long-chain fatty acids were UGT2B7 substrates. Glucuronides with Rf (retention factor) values corresponding to the glucuronides of linoleic and arachidonic acid were detected when HLM and HEK293 cell lysates were incubated with radiolabeled cofactor, and the intensity of the bands was modulated by the presence of crude HSA (increased) and BSA or HSA-FAF (decreased). Oleic, linoleic, and arachidonic acid inhibited AZT and 4MU glucuronidation by HLM and/or UGT2B7, due to an increase in Km/S50 without a change in Vmax. Addition of BSA and HSA-FAF reversed the inhibition. Likewise, coexpression of UGT2B7 and HSA in HEK293 cells reduced the Km/S50 values of these substrates. It is postulated that BSA and HSA-FAF sequester inhibitory fatty acids released during incubations, and the apparent high Km values observed for UGT2B7 substrates arise from the presence of these endogenous inhibitors.

Aims Using the fluconazole–zidovudine (AZT) interaction as a model, to determine whether inhibition of UDP–glucuronosyltransferase (UGT) catalysed drug metabolism in vivo could be predicted quantitatively from in vitro kinetic data generated in the presence and absence bovine serum albumin (BSA).

Household surveys are infrequently used for the estimation of international migration. The European Union (EU) Labour Force Surveys (LFS), however, compulsorily include a question on place of residence one year before. Potentially this provides a consistent definition across EU countries for estimating immigration from outside the EU, and for estimating a complete system of intra-EU migration. This potential is evaluated here for all (EU and non-EU) immigrants to the United Kingdom (UK) and for immigrants from the UK to continental EU countries. Evaluation is by comparison with estimates from the UK's port survey, the International Passenger Survey, and from the 1991 and 2001 UK Censuses. The UK LFS estimates the geographical distribution and time trend of total annual immigrants well. It underestimates the level of total annual immigration by 15 to 25 per cent, due mainly to its underestimating immigration of non-EU citizens. In both the UK and continental EU LFSs, returning citizen migrants are better estimated than are new migrants. The continental EU's LFSs exhibit considerable heterogeneity in their capturing of migrants from the UK. Aggregating them therefore results in poor estimation of both the total migrants to continental Europe and their distribution by country of destination.

Selected active site residues in substrate recognition sites (SRS) 1 and 5 of cytochrome P450 2C8 (CYP2C8) were mutated to the corresponding amino acids present in CYP2C9 to investigate the contribution of these positions to the unique substrate selectivity and regioselectivity of CYP2C8. The effects of mutations, singly and in combination, were assessed from changes in the kinetics of paclitaxel 6alpha-hydroxylation, a CYP2C8-specific pathway, and the tolylmethyl and ring hydroxylations of torsemide, a mixed CYP2C9/CYP2C8 substrate. Within SRS1, the single mutation S114F abolished paclitaxel 6alpha-hydroxylation, while the I113V substitution resulted in modest parallel reductions in K(m) and V(max). Mutations in SRS5 (viz., V362L, G365S, and V366L) reduced paclitaxel intrinsic clearance (V(max)/K(m)) by 88-100%. Torsemide is preferentially metabolized by CYP2C9, and it was anticipated that the mutations in CYP2C8 might increase activity. However, methyl and ring hydroxylation intrinsic clearances were either unchanged or decreased by the mutations, although hydroxylation regioselectivity was often altered relative to wild-type CYP2C8. The mutations significantly increased (28-968%) K(m) values for both torsemide methyl and ring hydroxylation but had variable effects on V(max). The effects of the combined mutations in SRS1, SRS5, and SRS1 plus SRS5 were generally consistent with the changes produced by the separate mutations. Mutation of CYP2C8 at position 359 (S359I), a site of genetic polymorphism in CYP2C9, resulted in relatively minor changes in paclitaxel- and torsemide-hydroxylase activities. The results are consistent with multiple substrate binding orientations within the CYP2C8 active site and a differential contribution of active site residues to paclitaxel and torsemide binding and turnover.

Morphine 3-β-D-glucuronide (M3G) and morphine 6-β-D-glucuronide (M6G) are the major metabolites of morphine in humans. More recently, morphine-3-β-d-glucoside (M-3-glucoside) was identified in the urine of patients treated with morphine. Kinetic and inhibition studies using human liver microsomes (HLM) and recombinant UGTs as enzyme sources along with molecular modeling were used here to characterize the relationship between morphine glucuronidation and glucosidation. The M3G to M6G intrinsic clearance (C(Lint)) ratio (∼5.5) from HLM supplemented with UDP-glucuronic acid (UDP-GlcUA) alone was consistent with the relative formation of these metabolites in humans. The mean C(Lint) values observed for M-3-glucoside by incubations of HLM with UDP-glucose (UDP-Glc) as cofactor were approximately twice those for M6G formation. However, although the M3G-to-M6G C(Lint) ratio remained close to 5.5 when human liver microsomal kinetic studies were performed in the presence of a 1:1 mixture of cofactors, the mean C(Lint) value for M-3-glucoside formation was less than that of M6G. Studies with UGT enzyme-selective inhibitors and recombinant UGT enzymes, along with effects of BSA on morphine glycosidation kinetics, were consistent with a major role of UGT2B7 in both morphine glucuronidation and glucosidation. Molecular modeling identified key amino acids involved in the binding of UDP-GlcUA and UDP-Glc to UGT2B7. Mutagenesis of these residues abolished morphine glucuronidation and glucosidation. Overall, the data indicate that morphine glucuronidation and glucosidation occur as complementary metabolic pathways catalyzed by a common enzyme (UGT2B7). Glucuronidation is the dominant metabolic pathway because the binding affinity of UDP-GlcUA to UGT2B7 is higher than that of UDP-Glc.

This review details the methods used in the production of a range of Q-state semiconductor and metal particles in Langmuir–Blodgett films, and describes some of the many methods used to characterise the particle-films. Chalcogenide based semiconductor particles can be grown in Langmuir–Blodgett films by cycles of immersion in a solution containing the appropriate metal cation and exposure to a chalcogenide gas. The preparation of metal chalcogenide (MX, where X=O, S, Se, Te) particles is discussed in terms of mechanistic insights into particle formation and growth. In particular, research investigating the role of the metal ion coordination sphere in the rate of particle formation and the roles of water and chemical capping in particle growth are presented. Various analytical techniques have been used to characterise the films including UV/visible absorption and grazing angle FTIR spectroscopies, atomic force microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, surface plasmon resonance and quartz crystal microbalance gravimetry. Methods for manipulating the optical properties of the particle-films, such as the production of core-shell particles and co-existent particles of different composition are presented. The formation of Q-state metal particles by CO or hydrazine reduction is referred to as an analogous technique. Finally, the results of photoelectrochemical and photobleaching measurements, are discussed in light of their potential usage as components in photoactivated devices.

Langmuir–Blodgett (LB) films were constructed on hydrophobic quartz substrates from monolayers of behenic acid on subphases containing only Cd 2+ or Hg 2+ and containing a mixture of Cd 2+ and Hg 2+ ions. The reaction of these films with H 2 S to form metal sulphide particles was investigated using UV/visible spectroscopy and X-ray photoelectron spectroscopy (XPS). For the mixed metal ion system it was concluded that reaction with H 2 S results in individual CdS and HgS particles forming rather than a mixed cadmium mercury sulphide. In order to investigate the kinetics of the H 2 S reaction, the frequencies of MBe (M=Cd or Hg) coated quartz crystal microbalances were followed as a function of H 2 S exposure time. Immersion of CdS particles into a Hg 2+ solution resulted in a change in the UV–visible absorbance spectrum attributed to the exchange of Cd 2+ ions in the particles for Hg 2+ ions most likely resulting in a HgS layer around the CdS core.

Langmuir monolayers of arachidic acid on a subphase of 0.3 mM CuSO 4 /17 mM NH 3 were transferred to hydrophobic glass and mica substrates to give Langmuir–Blodgett films of cupric arachidate (CuAr). Transfer ratios of close to 1 were observed and regular “Y”-type deposition was demonstrated with hydrophobic glass. With mica as the substrate, “Z”-type deposition occurred. The films were exposed to H 2 S and hydrazine, and evidence for the formation of copper sulfide and metallic copper particles, respectively, was obtained by ultraviolet/visible spectrophotometry, X-ray photoelectron spectroscopy and atomic force microscopy.

Langmuir-Blodgett films incorporating complex ions of gold(III) have been fabricated from air-water monolayers of arachidic acid, dimethyldioctadecylammonium bromide and dihexadecyl phosphate. The films, and their reactions with reducing agents to produce colloidal gold particles, have been characterised by surface pressure/area per molecule (π-A) isotherms, UV-vis. and XPS spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). Colloidal gold particles, produced in films exposed to carbon monoxide, were examined by AFM and TEM. The gold particles, as determined by TEM, were of a mean size of 10 nm and were randomly distributed in the films. Solvent immersion studies were also conducted in order to investigate the effect of the dielectric medium on the position of the gold colloid plasmon band. The position of the wavelength maximum of the gold plasmon band of the gold-LB film in air was not consistent with a just a layer of surfactant surrounding the particles. From the position of the surface plasmon band peak an effective refractive index of 1.35 is calculated for the LB film, which is considerably lower than that of a dense surfactant film. Also, the band maximum did not shift to any measurable extent when the films were immersed in water. We suggest that the reason for these observations is that air is trapped in the film and this lowers the refractive index of the film and prevents water penetration into the film. Immersion of a gold-LB film in the high refractive index liquid CS2 resulted in a redshift in the gold plasmon band, which was a consequence of the aggregation of the gold particles due to the dissolution of the surfactant film in the surrounding liquid.

A quartz crystal microbalance (QCM) was used to measure the in situ binding of lead (II) ions to a Langmuir monolayer of arachidic acid (ArH) at the air-water interface. A horizontally oriented QCM was lowered onto an ArH monolayer and an analiquot of a Pb 2+ solution was injected beneath the monolayer. A decrease in the QCM frequency was observed, consistent with Pb 2+ binding to the ArH monolayer. Models for a fatty acid to divalent metal ion ratio ( F : M ) of 2 and 1 were considered. The experimental data was consistent with 2:1 binding.

Surface pressure/area per molecule (π-A) isotherms of arachidic acid monolayers on subphases containing various Pt II and Pd II amine complexes were measured. Metal arachidate LB films derived from these monolayers were deposited on gold-coated glass and mica substrates. The reactions of these films with H 2 S were investigated using UV-visible, grazing angle Fourier transform infrared, and X-ray photoelectron spectroscopic techniques and atomic force microscopy. The absorbance onsets of the H 2 S exposed films are dramatically blue shifted relative to the bulk bandgaps of PtS and PdS consistent with the formation of particles in the Q state size regime. Atomic force microscopy images of palladium arachidate films on mica exposed to H 2 S revealed particles with an average diameter of 4.8 nm.

Langmuir-Blodgett (LB) films of mercury(II) arachidate (HgAr) and mercury(II) behenate (HgBe) were deposited on hydrophobed quartz and mica using airwater monolayers of the respective fatty acids spread on subphases containing Hg(OOCCH 3 ) 2 . The reactions of HgAr and HgBe films with H 2 S were investigated using ultraviolet-visible-near infrared spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy (AFM), and quartz crystal microbalance techniques. It was concluded that HgS forms quantitatively in the LB films. Additional HgS can be introduced into the film after gassing by immersion of the films in a Hg 2+ solution and regassing with H 2 S. Absorbance data do not provide information as to the nature and size of the HgS aggregates in the film. AFM measurements, however, indicate that individual particles are formed in the size range 2–10 nm. Agglomerates of individual particles were also observed.

Cytochrome P4501A2 (CYP1A2) is responsible for the metabolism of a diverse range of clinically used drugs and dietary and environmental chemicals (including many procarcinogens). CYP1A2 expression is influenced by numerous factors, and hence wide interindividual variability is a characteristic feature of this enzyme in humans. Phenacetin represents a convenient probe for the assessment of human CYP1A2 activity in vitro (hepatic microsomes and recombinant enzyme). It is a relatively high-turnover substrate that forms only one major primary metabolite, the O-deethylated derivative acetaminophen. Acetaminophen formation in incubations of phenacetin with a CYP1A2 source is readily measured by HPLC with UV detection. The assay has a low requirement for human liver microsomes or recombinant enzyme, and is both selective and sensitive without the requirement for a solvent extraction step. Overall assay reproducibility is excellent, with coefficients of variation

The fluorescence of 1-anilinonaphthalene-8-sulfonate (ANS) in the presence of human liver microsomes (HLMs) is altered by drugs that bind nonspecifically to the lipid bilayer. The present study characterized the relationship between the nonspecific binding (NSB) of drugs to HLMs as measured by equilibrium dialysis and the magnitude of the change in baseline ANS fluorescence. Fraction unbound in incubations of HLMs (f(u(mic))) was determined for 16 drugs (12 bases, 3 acids, and 1 neutral) with log P values in the range 0.1 to 6.7 at three concentrations (100, 200, and 500 μM). Changes in ANS fluorescence induced by each of the drugs in the presence of HLMs were measured by spectrofluorometry. Values of f(u(mic)) determined by equilibrium dialysis ranged from 0.08 to 1.0. Although NSB of the basic drugs tended to increase with increasing log P, exceptions occurred. Basic drugs generally caused an increase in ANS fluorescence, whereas the acidic and neutral drugs resulted in a decrease in ANS fluorescence. There were highly significant (p < 0.001) linear relationships between the modulus (absolute value) of the increment/decrement in ANS fluorescence and both f(u(mic)) (r = 0.90 to 0.96) and log(1 - f(u(mic))/f(u(mic))) (r = 0.85 to 0.92) at the three drug concentrations. Agreement between measured f(u(mic)) and that predicted by ANS fluorescence was very good (

The complex [CoRh(CO) 3 (μ-dppm) 2 ] (1) is easily protonated to give [CoRh(μ-H)(CO) 3 (μ-dppm) 2 ] + (2). The structure of 2[BF 4 ] has been determined crystallographically [space group P1, a=12.673 (2) A, b=19.833 (3) A, c=11.992 (3) A, α=94.29 (2) o , β=1 17.08 (2) o , γ=107.93 (1) o , Z=2, R=0.0519, R w =0.0558] and it is shown that the Co-Rh bond is shorter in 2 than in 1

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • Carboxylic acid NSAIDs are extensively glucuronidated as either the parent drug or hydroxylated metabolites and UGT2B7 is ranked highest in terms of NSAID-glucuronidation activity. • NSAIDs cause adverse renal effects including sodium and water retention and hyperkalaemia. • In human kidney the mineralocorticoid aldosterone is glucuronidated directly to form aldosterone 18β-glucuronide. WHAT THIS STUDY ADDS • Human liver and kidney microsomes and UGT1A10 and UGT2B7 catalyze aldosterone18β-glucuronidation. • Non-selective NSAIDs inhibit renal and hepatic aldosterone18β-glucuronidation and in vivo this may lead to elevated intra-renal concentrations of this hormone. • Common involvement of UGT2B7 in NSAID and aldosterone glucuronidation predicates an intra-renal NSAID-aldosterone interaction that may explain in part the clinical observations of variable effects of NSAIDs on electrolytes, fluid retention and blood pressure. AIMS To characterize: i) the kinetics of aldosterone (ALDO) 18β-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18β-glucuronidation and ii) the inhibition of ALDO 18β-glucuronidation by non-selective NSAIDs. METHODS Using HPLC and LC-MS methods, ALDO 18β-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18β-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC50) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen. RESULTS ALDO 18β-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (±SD) Km, Vmax and CLint values for HLM and HKCM were 509 ± 137 and 367 ± 170 µm, 1075 ± 429 and 1110 ± 522 pmol min−1 mg−1, and 2.36 ± 1.12 and 3.91 ± 2.35 µl min−1 mg−1, respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18β-glucuronide. All NSAIDs investigated inhibited ALDO 18β-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC50) of renal and hepatic ALDO 18β-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates. CONCLUSION A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.

In order to gain insights into the renal and hepatic glucuronidation of frusemide (FSM), this study: (i) characterised the kinetics of FSM glucuronidation by human liver microsomes (HLM) and human kidney cortical- (HKCM) and medullary- (HKMM) microsomes, and (ii) identified the human UDP-glucuronosyltransferase enzyme(s) involved in this pathway. HLM, HKCM and HLMM efficiently glucuronidated FSM. FSM glucuronide (FSMG) formation followed Michaelis-Menten kinetics in all tissues. While the mean K(m) for FSMG formation by HKMM (386 +/- 68 microM) was lower than the K(m) values for HLM (988 +/- 271 microM) and HKCM (704 +/- 278 microM), mean V(max)/K(m) values were comparable for the three tissues. A panel of recombinant UGT enzymes was screened for the capacity to glucuronidate FSM. UGT 1A1, 1A3, 1A6, 1A7, 1A9, 1A10 and 2B7 metabolised FSM. Of the renally and hepatically expressed enzymes, comparison of kinetic parameters suggests a predominant role of UGT1A9 in FSM glucuronidation, although UGT1A1 may also contribute to FSMG formation by HLM. Consistent with these observations, the UGT1A selective inhibitors phenylbutazone and sulfinpyrazone decreased FSMG formation by HLM, HKCM and HKMM by 60-80%, whereas the UGT2B7 selective inhibitor fluconazole reduced FSM glucuronidation byor =20%. The ability of HKCM and HKMM to form FSMG supports the proposition that the kidney is the main organ involved in FSM glucuronidation in vivo, although a role for hepatic metabolism remains a possibility in renal dysfunction. The data further demonstrate the potential importance of both the medulla and cortex in renal drug metabolism and detoxification.

This study characterized the mechanism by which bovine serum albumin (BSA) reduces the K(m) for phenytoin (PHY) hydroxylation and the implications of the "albumin effect" for in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates. BSA and essentially fatty acid-free human serum albumin (HSA-FAF) reduced the K(m) values for PHY hydroxylation (based on unbound substrate concentration) by human liver microsomes (HLMs) and recombinant CYP2C9 by approximately 75%, with only a minor effect on V(max). In contrast, crude human serum albumin increased the K(m) with both enzyme sources. Mass spectrometric analysis of incubations containing HLMs was consistent with the hypothesis that BSA sequesters long-chain unsaturated acids (arachidonic, linoleic, oleic) released from membranes. A mixture of arachidonic, linoleic and oleic acids, at a concentration corresponding to 1/20 of the content of HLMs, doubled the K(m) for PHY hydroxylation by CYP2C9, without affecting V(max). This effect was reversed by addition of BSA to incubations. K(i) values for arachidonic acid inhibition of human liver microsomal- and CYP2C9-catalyzed PHY hydroxylation were 3.8 and 1.6 microM, respectively. Similar effects were observed with heptadecanoic acid, the most abundant long-chain unsaturated acid present in Escherichia coli membranes. Extrapolation of intrinsic clearance (CL(int)) values for each enzyme source determined in the presence of BSA and HSA-FAF accurately predicted the known CL(int) for PHY hydroxylation in vivo. The results indicate that previously determined in vitro K(m) values for CYP2C9 substrates are almost certainly overestimates, and accurate in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates is achievable.

What is already known about this subject Gliclazide is a widely used oral hypoglycaemic agent. The major metabolites of gliclazide formed in vivo have been identified. However, the cytochrome P450 enzymes catalysing the rate-limiting pathways of gliclazide elimination are unknown. What this study adds CYP2C9 is the major enzyme involved in the various hydroxylation pathways of gliclazide, although a contribution of CYP2C19 to tolymethylhydroxylation, the major metabolic route, cannot be discounted. Factors known to influence CYP2C9 activity will provide the main source of variability in gliclazide pharmacokinetics. To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6 beta-hydroxy (6 beta-OHGz), 7 beta-hydroxy (7 beta-OHGz) and hydroxymethyl (MeOH-Gz) metabolites of gliclizide (Gz). 6 beta-OHGz, 7 beta-OHGz and MeOH-Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high-performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. Microsomes from six human livers converted Gz to its 6 beta-OHGz, 7 beta-OHGz, and MeOH-Gz metabolites, with respective mean (+/- SD) K-m values of 461 +/- 139, 404 +/- 143 and 334 +/- 75 mu m and mean V-max values of 130 +/- 55, 82 +/- 31 and 268 +/- 115 pmol min(-1) mg(-1), respectively. V-max/K-m ratios for the microsomal reactions parallelled relative metabolite formation in vivo. Sulfaphenazole inhibited microsomal 6 beta-OHGz, 7 beta-OHGz and MeOH-Gz formation by 87, 83 and 64%, respectively, whereas S-mephenytoin caused significant inhibition (48%) of only MeOH-Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6 beta-OHGz and 7 beta-OHGz. Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH-Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics.

The structural characterization of the title complex, [Cu2(OH)(C2H302)(C 8 H 19 N 3 ))2(H 2 O)](ClO 4 )2, provides only the third example of a binuclear copper complex containing a bridging water molecule. The three bridging groups (aqua, acetato and hydroxo) are all different, a rare feature for spontaneous self-assembly reactions. The resulting geometry about the two Cu atoms is distorted octahedral, with the Cu-O(H) distance of 1.963 (2) A distinguished from the Cu-O(H 2 ) distance of 2.737 (5) A. The cation has crystallographically imposed mirror symmetry.

The uricosuric agent sulfinpyrazone (SFZ) is metabolized via C-glucuronidation, an uncommon metabolic pathway, in humans. The present study aimed to characterize SFZ glucuronidation by human liver microsomes (HLMs) and identify the hepatic forms of UDP-glucuronosyltransferase responsible for this pathway. Incubations of SFZ with HLMs formed a single glucuronide that was resistant to beta-glucuronidase and acid hydrolysis, consistent with formation of a C-glucuronide. Mass spectral analysis confirmed the identity of the metabolite as SFZ glucuronide (sulfinpyrazone beta-D-glucuronide; SFZG). SFZ C-glucuronidation by HLMs exhibited Michaelis-Menten kinetics, with mean (+/- S.D.) Km and Vmax values of 51 +/- 21 microM and 2.6 +/- 0.6 pmol/min . mg, respectively. Fifteen recombinant human UDP-glucuronosyltransferases (UGTs), expressed in HEK293 cells, were screened for their capacity to catalyze SFZ C-glucuronidation. Of the hepatically expressed enzymes, only UGT1A9 formed SFZG. UGTs 1A7 and 1A10, which are expressed in the gastrointestinal tract, also metabolized SFZ, but rates of metabolism were low compared with UGT1A9. SFZ glucuronidation by UGT1A9 exhibited "weak" negative cooperative kinetics, which was modeled by the Hill equation (S50 16 microM). The data indicate that UGT1A9 is the enzyme responsible for hepatic SFZ C-glucuronidation and that SFZ may be used as a substrate "probe" for UGT1A9 activity in HLMs.