BMSCs overexpressed ISL1 reduces the apoptosis of islet cells through ANLN carrying exosome, INHBA, and caffeine.

Early apoptosis of grafted islets is one of the main factors affecting the efficacy of islet transplantation. The combined transplantation of islet cells and bone marrow mesenchymal stem cells (BMSCs) can significantly improve the survival rate of grafted islets. Transcription factor insulin gene enhancer binding protein 1 (ISL1) is shown to promote the angiogenesis of grafted islets and the paracrine function of mesenchymal stem cells during the co-transplantation, yet the regulatory mechanism remains unclear. By using ISL1-overexpressing BMSCs and the subtherapeutic doses of islets for co-transplantation, we managed to reduce the apoptosis and improve the survival rate of the grafts. Our metabolomics and proteomics data suggested that ISL1 upregulates aniline (ANLN) and Inhibin beta A chain (INHBA), and stimulated the release of caffeine in the BMSCs. We then demonstrated that the upregulation of ANLN and INHBA was achieved by the binding of ISL1 to the promoter regions of the two genes. In addition, ISL1 could also promote BMSCs to release exosomes with high expression of ANLN, secrete INHBA and caffeine, and reduce streptozocin (STZ)-induced islets apoptosis. Thus, our study provides mechanical insight into the islet/BMSCs co-transplantation and paves the foundation for using conditioned medium to mimic the ISL1-overexpressing BMSCs co-transplantation.

Co-Dissolved Isostructural Polyoxovanadates to Construct Single-Atom-Site Catalysts for Efficient CO2 Photoreduction.

We explored a co-dissolved strategy to embed mono-dispersed Pt center into V2 O5 support via dissolving [PtV9 O28 ]7- into [V10 O28 ]6- aqueous solution. The uniform dispersion of [PtV9 O28 ]7- in [V10 O28 ]6- solution allows [PtV9 O28 ]7- to be surrounded by [V10 O28 ]6- clusters via a freeze-drying process. The V centers in both [PtV9 O28 ]7- and [V10 O28 ]6- were converted into V2 O5 via a calcination process to stabilize Pt center. These double separations can effectively prevent the Pt center agglomeration during the high-temperature conversion process, and achieve 100 % utilization of Pt in [PtV9 O28 ]7- . The resulting Pt-V2 O5 single-atom-site catalysts exhibit a CH4 yield of 247.6 µmol g-1 h-1 , 25 times higher than that of Pt nanoparticle on the V2 O5 support, which was accompanied by the lactic acid photooxidation to form pyruvic acid. Systematical investigations on this unambiguous structure demonstrate an important role of Pt-O atomic pair synergy for highly efficient CO2 photoreduction.

Solvothermal Syntheses and Characterizations of Four Quaternary Copper Sulfides BaCu3MS4 (M = In, Ga) and BaCu2MS4 (M = Sn, Ge).

Hydro(solvo)thermal syntheses of quaternary copper sulfides containing alkaline earth metal ions remain a great challenge because of the low solubility of Cu-S compounds. Herein, a new facile solvothermal method was developed, and four quaternary copper sulfides, i.e., BaCu3InS4 (1), BaCu3GaS4 (2), BaCu2SnS4 (3), and BaCu2GeS4 (4), were prepared using excess sulfur as a mineralizer. Compound 1 possesses a novel three-dimensional (3D) anionic [Cu3InS4]2- framework constructed by an 8-membered ring of [Cu4S4] and [Cu2In2S4] alternatively. Compound 2 features a unique 3D anionic [Cu3GaS4]2- framework composed of [Cu3GaS10]n14n- anionic chains and 8-membered rings, in which [Cu4S4] and [Cu2Ga2S4] reside alternatively. Compounds 3 and 4 feature 3D anionic [Cu2MS4]2- (M = Sn, Ge) frameworks composed of CuS4 and MS4 tetrahedra with Ba2+ located in the channels. It is worth noting that different 3D Cu-S frameworks exist in the title crystal structures, in which main group ions are incorporated. This paper provides a new synthetic strategy for new quaternary sulfides.

Metabolism of c-Met Kinase Inhibitors Containing Quinoline by Aldehyde Oxidase, Electron Donating, and Steric Hindrance Effect.

Some quinoline-containing c-Met kinase inhibitors are aldehyde oxidase (AO) substrates. 3-Substituted quinoline triazolopyridine analogs were synthesized to understand the electron-donating and steric hindrance effects on AO-mediated metabolism. Metabolic stability studies for these quinoline analogs were carried out in liver cytosol from mice, rats, cynomolgus monkeys, and humans. Several 3-N-substituted analogs were found to be unstable in monkey liver cytosolic incubations (half-life, <10 minutes), and five of them (63, 53, 51, 11, and 71) were chosen for additional mechanistic studies. Mono-oxygenation on the quinoline ring was identified by liquid chromatography tandem mass spectrometry. Metabolite formation was inhibited by the AO inhibitors menadione and raloxifene, but not by the xanthine oxidase inhibitor allopurinol. It was found that small electron-donating groups at the 3-quinoline moiety made the analogs more susceptible to AO metabolism, whereas large 3-substituents could reverse the trend. Although species differences were observed, this trend was applicable to all species tested. Small electron-donating substituents at the 3-quinoline moiety increased both affinity (decreased Michaelis constant) and V max maximum velocity toward AO in kinetic studies, whereas large substituents decreased both parameters probably as a result of steric hindrance. Based on our analysis, a common structural feature with high AO liability was proposed. Our finding could provide useful information for chemists to minimize potential AO liability when designing quinoline analogs.

A high-nuclearity CuI/CuII nanocluster catalyst for phenol degradation.

Herein, we report a 54-nuclei copper nanocluster, [Cu54S13O6(tBuS)20(tBuSO3)12] (Cu54), which is the largest atom-precise CuI/CuII mix-valent cluster reported. The Cu54 nanoclusters supported by TiO2 exhibit decent photocatalytic activity for phenol degradation under visible light. This work provides a platform to explore the catalytic behaviors of CuI/CuII nanosystems.

Boosting Room Temperature Sensing Performances by Atomically Dispersed Pd Stabilized via Surface Coordination.

The urgent requirement of monitoring air pollution worldwide evokes intensive research interest in developing chemiresistive gas sensing techniques. To overcome the limits in sensitivity and selectivity of room temperature (RT) chemiresistive sensing materials, a new strategy using single-atom catalysts (SACs) via surface coordination is proposed. As a proof-of-concept, single Pd atoms on TiO2 (Pd1-TiO2) possess high efficiency in generating adsorbed O2- as well as high activity and selectivity in catalyzing CO oxidation at RT. As a result, Pd1-TiO2 shows record high sensitivity among the reported RT sensing materials, which is even comparable to those of the best materials working at high temperature. It also provides an approximately 1 order of magnitude lower limit of detection than the best CO sensing materials. Moreover, Pd1-TiO2 presents high selectivity toward 12 kinds of interference gases. This work not only paves a way to design high-performance RT gas sensing materials but also extends the application of SACs.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of daphnetin.

Daphnetin has been developed as an oral medicine for treatment of coagulation disorders and rheumatoid arthritis in China, but its in vitro metabolism remains unknown. In the present study, the UDP-glucuronosyltransferase (UGT) conjugation pathways of daphnetin were characterized. Two metabolites, 7-O-monoglucuronide daphnetin (M-1) and 8-O-monoglucuronide daphnetin (M-2), were identified by liquid chromatography/mass spectrometry and NMR when daphnetin was incubated, respectively, with liver microsomes from human (HLM), rat (RLM), and minipig (PLM) and human intestinal microsomes (HIM) in the presence of UDP-glucuronic acid. Screening assays with 12 human recombinant UGTs demonstrated that the formations of M-1 and M-2 were almost exclusively catalyzed by UGT1A9 and UGT1A6, whereas M-1 was formed to a minor extent by UGT1A3, 1A4, 1A7, 1A8, and 1A10 at a high substrate concentration. Kinetics studies, chemical inhibition, and correlation analysis were used to demonstrate that human UGT1A9 and UGT1A6 were major isoforms involved in the daphnetin glucuronidations in HLM and HIM. By in vitro-in vivo extrapolation of the kinetic data measured in HLM, the hepatic clearance and the corresponding hepatic extraction ratio were estimated to be 19.3 ml/min/kg b.wt. and 0.93, respectively, suggesting that human clearance of daphnetin via the glucuronidation is extensive. Chemical inhibition of daphnetin glucuronidation in HLM, RLM, and PLM showed large species differences although the metabolites were formed similarly among the species. In conclusion, the UGT conjugation pathways of daphnetin were fully elucidated and its C-8 phenol group was more selectively catalyzed by UGTs than by the C-7 phenol.

Glucuronidation, a new metabolic pathway for pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) possess significant hepatotoxicity to humans and animals after metabolic activation by liver P450 enzymes. Metabolism pathways of PAs have been studied for several decades, including metabolic activation, hydroxylation, N-oxidation, and hydrolysis. However, the glucuronidation of intact PAs has not been investigated, although glucuronidation plays an important role in the elimination and detoxication of xenobiotics. In this study, PAs glucuronidation was investigated, and three important points were found. First, we demonstrated that senecionine (SEN)-a representative hepatotoxic PA-could be conjugated by glucuronic acid via an N-glucuronidation reaction catalyzed by uridine diphosphate glucuronosyl transferase in human liver microsomes. Second, glucuronidation of SEN was catalyzed not only by human but also other animal species and showed significant species differences. Rabbits, cattle, sheep, pigs, and humans showed the significantly higher glucuronidation activity than mice, rats, dogs, and guinea pigs on SEN. Kinetics of SEN glucuronidation in humans, pigs, and rabbits followed the one-site binding model of the Michaelis-Menten equation, while cattle and sheep followed the two-sites binding model of the Michaelis-Menten equation. Third, besides SEN, other hepatotoxic PAs including monocrotaline, adonifoline, and isoline also underwent N-glucuronidation in humans and several animal species such as rabbits, cattle, sheep, and pigs.

Clinical effects of apatinib mesylate for treatment of multiple brain micrometastases: Two case reports.

BACKGROUND: Apatinib is a small-molecule multitargeted tyrosine kinase inhibitor. Apatinib has demonstrated encouraging antitumor activities. This study aimed to observe the efficacy and safety of apatinib for the treatment of multiple brain micrometastases. CASE SUMMARY: We report two patients with multiple brain micrometastases after failure of second-line treatment. Both patients had extracerebral metastases. When the patients took 250 mg/d apatinib orally, the intracerebral lesions disappeared. The extracerebral lesions were partially alleviated. Both patients had a progression-free survival of more than 12 mo and were still stable. The safety was good. The main adverse events (AEs) were mild hypertension and proteinuria, which could be controlled. CONCLUSION: Apatinib has clear efficacy and good tolerance in patients with multiple brain micrometastases after failure of second-line treatment.

Terahertz time-domain absorption spectra of Cu(I) complexes bearing tetraphosphine ligands: the bridge between the C-Hπ and ππ interactions and photoluminescence properties.

The synthesis of four heteroleptic dinuclear Cu(i) complexes bearing tetraphosphine and diimine ligands was reported. Complexes 1-3 were successfully obtained through microwave synthesis while complex 4 was synthesized through traditionally stirring at room temperature. These complexes are listed as follows: [Cu2(Dpq)2(dppeda)](ClO4)2·1.5CH2Cl2 (1), [Cu2(neo)2(dppeda)](ClO4)2·1.3CH2Cl2·1.7C4H10O (2), [Cu2(batho)2(dppeda)](ClO4)2·C4H10O (3), and [Cu2(batho)2(dpppda)](ClO4)2·3CH2Cl2 (4) {(Dpq = pyrazino[2,3-f][1,10]phenanthroline, batho = 4,7-diphenyl-1,10-phenanthroline, neo = 2,9-dimethyl-1,10-phenanthroline, dppeda = N1,N1,N2,N2-tetrakis[(diphenylphosphino)methyl]-1,2-ethanediamine, and dpppda = N1,N1,N4,N4-tetrakis[(diphenylphosphino)methyl]-1,4-benzenediamine}. Their crystal structures have been elucidated by X-ray crystallography and their photophysical properties have been investigated in detail. Photophysical studies and time domain density functional theory (TD-DFT) calculations show that the luminescence performance of these four complexes is ascribed to metal-to-ligand charge transfer (MLCT) mixed with ligand-to-ligand charge transfer (LLCT), and complex 2 shows green emission at 574 nm with the highest quantum yield of up to 52.80%. In addition, the research of photoluminescence properties under the guidance of terahertz spectroscopy technology leads to the preliminary discovery of a correlation between crystal packing and luminescence. It is found that the terahertz spectrum and absorption peak are strongly interdependent on C-Hπ and ππ interactions, and the external weak interactions have negative effects on the luminescence performance. Herein, we predict that the terahertz spectroscopy analysis establishes a bridge between weak interactions (C-Hπ and ππ interactions) and the photoluminescence properties, and puts forward a problem that should be noticed in designing Cu(i) complexes.

Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry.

AIM: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes. METHODS: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system. RESULTS: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 micromol/L and 35 pmol x min(-1) x mg(-1), respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin. CONCLUSION: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

Anti-androgen-independent prostate cancer effects of ginsenoside metabolites in vitro: mechanism and possible structure-activity relationship investigation.

Treatment of androgen-independent prostate cancer (AIPC) remains unsatisfactory. In our present experiment, natural occurring ginsenosides (NOGs) and intestinal bacterial metabolites (IBMs) were employed to investigate their anti-AIPC cell growth activity using PC-3 cells. Our results showed that the IBMs exerted more portent anti-AIPC activity than NOGs, by decreasing survival rate, inhibiting proliferation, inducing apoptosis, and leading to cell cycle arrest in AIPC PC-3 cells. The increase of LogP and decrease of C-6 steric hindrance, which were caused by deglycosylation by intestinal bacteria, may be the reason for the higher anti-AIPC activity of IBMs.

Semi-Rigid Molecular-Clip-Based Molecular Crystal Gearshift.

A new version of molecular clip, with a semi-rigid symmetrical crab-type architecture and flexible cavity size, has been successfully designed and synthesized via a one-pot Friedel-Crafts alkylation reaction. The X-ray single-crystal diffraction data provide a simple and intuitive explanation, not only for its well-preorganized and regulated conformation but also for its selective and tunable guest-binding capability. For the first time, the newly designed molecular clip was demonstrated to be not only a controllable variable-speed nonporous adsorption material in solution iodine capture, but also capable of on-off switching in volatile iodine capture. The presented new concept of molecular crystal gearshift directly from the molecular clip crystals represents an important advance in the development of synthetic receptor chemistry, which will exert a significant influence on small-molecule crystallography.

New sesquiterpenoids from the stems of Dendrobium nobile and their neuroprotective activities.

Three new sesquiterpenoids, (+)-(1R,2S,3R,4S,5R,6S,9R)-3,11,12-trihydroxypicrotoxane-2(15)-lactone (1), (-)-(1S,2R,3S,4R,5S,6R,9S,12R)-3,11,13-trihydroxypicrotoxane-2(15)-lactone (2), and (+)-(1R,5R,6S,8R,9R)-8,12-dihydroxy-copacamphan-3-en-2-one (3), together with five known compounds, were isolated from the n-butanol soluble fraction of a 95% EtOH extract of the stems of Dendrobium nobile. Their structures were determined by extensive spectroscopic analysis. Particularly, to solve difficult stereochemical problems, electronic circular dichroism calculations, NMR data calculations, and a single-crystal X-ray diffraction were performed. Interestingly, compounds 1 and 2 were picrotoxinin-type sesquiterpenoids with an unusual C15,2-lactone ring. All new sesquiterpenoids (1-3) showed a significant neuroprotective activity against H2O2-induced oxidative damage in PC12 cells. Notably, at 25 and 50 µM, compounds 1 and 2 showed the best protective effects, even better than the positive control (vitamin E).

Fabrication of silver chalcogenolate cluster hybrid membranes with enhanced structural stability and luminescence efficiency.

The present study reports the fabrication of a silver chalcogenolate cluster hybrid membrane (SCC membrane) through self-assembly of SCCs, and then covalent cross-linking of the modified SCC assembled materials. This strategy provides access to silver clusters with superior chemical stability and enhanced luminescence efficiency for practical applications.

In Vitro Metabolism by Aldehyde Oxidase Leads to Poor Pharmacokinetic Profile in Rats for c-Met Inhibitor MET401.

BACKGROUND AND OBJECTIVES: MET401 is a potent and selective c-Met inhibitor with a novel triazolopyrimidine scaffold. The aim of this study was to determine the pharmacokinetic profile of MET401 in preclinical species, and to identify the metabolic soft spot and enzyme involved, in order to help medicinal chemists to modify the compound to improve the pharmacokinetic profile. METHODS: A metabolite identification study was performed in different liver fractions from various species. Chemical inhibition with selective cytochrome P450 (CYP) and molybdenum hydroxylase inhibitors was carried out to identify the enzyme involved. The deuterium substitution strategy was adopted to reduce metabolism. Pharmacokinetic studies were performed in rats to confirm the effect. RESULTS: Although M-2 is a minor metabolite in liver microsomal incubations, it became the predominant metabolite in incubations with liver S9, cytosol, hepatocytes and rat pharmacokinetic study. M-2 was synthesized enzymatically and the structure was identified as a mono-oxidation on the triazolopyrimidine moiety. The M-2 formation was ascribed to aldehyde oxidase (AO)-mediated metabolism based on the following evidence-M-2 production was NADPH independent, pan-CYP inhibitor 1-aminobenzotriazole and xanthine oxidase inhibitor allopurinol did not inhibit M-2 formation, and AO inhibitors menadione and raloxifene inhibited M-2 formation. The deuterated analog MET763 demonstrated an improved pharmacokinetic profile with lower clearance, longer terminal half-life and double oral exposure compared with MET401 in rats. CONCLUSIONS: These results indicate that the main metabolic pathway of MET401 is AO-mediated metabolism, which leads to poor in vivo pharmacokinetic profiles in rodents. The deuterium substitution strategy could be used to reduce AO-mediated metabolism liability.

UDP-glucuronosyltransferase 1A6 is the major isozyme responsible for protocatechuic aldehyde glucuronidation in human liver microsomes.

Glucuronidation is an important pathway in the metabolism of protocatechuic aldehyde (3,4-dihydroxybenzaldehyde, PAL). However, the metabolites and primary UDP-glucuronosyltransferase (UGT) isozymes responsible for PAL glucuronidation remain to be determined in human. Here, we characterized PAL glucuronidation by human liver microsomes (HLMs), human intestine microsomes (HIMs), and 12 recombinant UGT (rUGT) isozymes to identify what kinds of metabolites are present and which human UGT isozymes are involved. Two metabolites (M-1 and M-2) were detected in reactions catalyzed by HLMs, HIMs, rUGT1A6, and rUGT1A9 and were identified as monoglucuronides by liquid chromatography-mass spectrometry. A kinetic study showed that PAL glucuronidation by rUGT1A6, rUGT1A9, HIMs, and HLMs followed Michaelis-Menten kinetics. The K(m) values of HLMs, HIMs, rUGT1A6, and rUGT1A9 for PAL glucuronidation were as follows: 432.7 +/- 24.5, 626.9 +/- 49.2, 367.5 +/- 25.1, and 379.9 +/- 42.5 microM for M-1 and 336.7 +/- 15.3, 494.3 +/- 48.7, 211.4 +/- 13.4, and 238.5 +/- 26.2 microM for M-2, respectively. The PAL glucuronidation activity was significantly correlated with UGT1A6 activity rather than with UGT1A9 activity from 15 individual HLMs. A chemical inhibition study showed that the IC(50) for phenylbutazone inhibition of PAL glucuronidation was similar in HLMs (61.9 +/- 7.9 microM) compared with rUGT1A6 (45.3 +/- 7.7 microM). In contrast, androsterone inhibited rUGT1A9-catalyzed and HLM-catalyzed PAL glucuronidation with IC(50) values of 27.1 +/- 3.8 and > 500 microM, respectively. In combination, we identified UGT1A6 as the major isozyme responsible for PAL glucuronidation in HLMs.

Taxane's substituents at C3' affect its regioselective metabolism: different in vitro metabolism of cephalomannine and paclitaxel.

To investigate how taxane's substituents at C3' affect its metabolism, we compared the metabolism of cephalomannine and paclitaxel, a pair of analogs that differ slightly at the C3' position. After cephalomannine was incubated with human liver microsomes in an NADPH-generating system, two monohydroxylated metabolites (M1 and M2) were detected by liquid chromatography/tandem mass spectrometry. C4'' (M1) and C6alpha (M2) were proposed as the possible hydroxylation sites, and the structure of M1 was confirmed by (1)H NMR. Chemical inhibition studies and assays with recombinant human cytochromes P450 (P450s) indicated that 4''-hydroxycephalomannine was generated predominantly by CYP3A4 and 6alpha-hydroxycephalomannine by CYP2C8. The overall biotransformation rate between paclitaxel and cephalomannine differed slightly (184 vs. 145 pmol/min/mg), but the average ratio of metabolites hydroxylated at the C13 side chain to C6alpha for paclitaxel and cephalomannine varied significantly (15:85 vs. 64:36) in five human liver samples. Compared with paclitaxel, the major hydroxylation site transferred from C6alpha to C4'', and the main metabolizing P450 changed from CYP2C8 to CYP3A4 for cephalomannine. In the incubation system with rat or minipig liver microsomes, only 4''-hydroxycephalomannine was detected, and its formation was inhibited by CYP3A inhibitors. Molecular docking by AutoDock suggested that cephalomannine adopted an orientation in favor of 4''-hydroxylation, whereas paclitaxel adopted an orientation favoring 3'-p-hydroxylation. Kinetic studies showed that CYP3A4 catalyzed cephalomannine more efficiently than paclitaxel due to an increased V(m). Our results demonstrate that relatively minor modification of taxane at C3' has major consequence on the metabolism.

Metabolic profiling and cytochrome P450 reaction phenotyping of medroxyprogesterone acetate.

Medroxyprogesterone acetate (MPA) is one of the most frequently prescribed progestins for conception, hormone replacement therapy, and adjuvant endocrine therapy. MPA has a low oral bioavailability because of extensive metabolism; however, its metabolism was poorly documented. This study was intended to profile the phase I metabolites of MPA and the cytochrome P450 (P450) isoforms involved. After MPA was incubated with human liver microsomes and the NADPH-generating system, five main metabolites (namely M-1, M-2, M-3, M-4, and M-5) were isolated by high-performance liquid chromatography. Three major metabolites (M-2, M-4, and M-3) were tentatively identified to be 6beta-, 2beta-, and 1beta-hydroxy MPA by liquid chromatography/mass spectrometry and (1)H nuclear magnetic resonance. By consecutive metabolism of purified M-2, M-3, and M-4, M-1 and M-5 were proposed to be 2beta-, 6beta-dihydroxy MPA, and 1,2-dehydro MPA, respectively. CYP3A4 was identified to be the isoform primarily involved in the formation of M-2, M-3, and M-4 in studies with specific P450 inhibitors, recombinant P450s, and correlation analysis. Rat and minipig liver microsomes were included evaluating species differences, and the results showed little difference among the species. In human liver microsomes, the K(m) values ranged from 10.0 to 11.2 muM, and the V(m) values ranged from 194 to 437 pmol/min/mg for M-2, M-3, and M-4. In conclusion, CYP3A4 was the major P450 isoform involved in MPA hydroxylation, with 6beta, 2beta, and 1beta being the possible hydroxylation sites. Minipig and rat could be the surrogate models for man in MPA pharmacokinetic studies.

Determination of UDP-glucuronosyltransferase UGT2B7 activity in human liver microsomes by ultra-performance liquid chromatography with MS detection.

A rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3'-azido-3'-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm x 2.1mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5-500 microM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.