Metabolism of xanthohumol and isoxanthohumol, prenylated flavonoids from hops (Humulus lupulus L.), by human liver microsomes

The female flowers of hops (Humulus lupulus L.) used to flavor beer contain the prenylated flavonoids xanthohumol (XN) and isoxanthohumol (IX). IX is moderately estrogenic in vitro and XN has pharmacological properties that might make it useful as a cancer chemopreventive agent. The metabolism of these dietary flavonoids was investigated in vitro using human liver microsomes. Hydroxylation of a prenyl methyl group was the primary route of oxidative metabolism forming either cis or trans hydroxylated metabolites of IX but only the trans isomer of XN. The double bond on the prenyl group of both compounds formed an epoxide which was opened by an intramolecular reaction with the neighboring hydroxyl group. The potent phytoestrogen 8-prenylnaringenin (8-PN) was detected as a demethylation product of IX. However, the analogous demethylation reaction was not observed for XN. Since XN can be converted to IX through acid-catalyzed cyclization in the stomach, XN might contribute to the in vivo levels of estrogenic 8-PN following consumption of hops extracts.     

In vitro metabolism of specific CYP2D and CYP3A opioid substrates using rat liver S9 fractions and mass spectrometry reveal a severe metabolic impairment with increasing age

Codeine and oxycodone are opioids used to alleviate pain. The outcome of the treatment is ultimately related to their metabolism by Cytochromes P450 (CYPs). Depending on the drugs used, alterations in the metabolism of drugs by CYPs can lead to severe consequences including alterations in their efficacy, safety and toxicity. The objectives of this study were to develop a novel HPLC–MS/MS method capable of quantifying codeine and oxycodone along with specific metabolites using an isotopic dilution strategy and study the rate of formation of morphine (CYP2D), norcodeine (CYP3A), oxymorphone (CYP2D) and noroxycodone (CYP3A). The chromatographic separation was achieved using a Biobasic C₁₈ 100 × 1 mm column combined with an isocratic mobile phase composed of methanol and 10 mm ammonium acetate (40:60) at a flow rate of 75 μL/min. The mass spectrometer was operating in scan mode MS/MS and the analytical range was set at 10–10 000 nm . The precision (RSD) and accuracy (RE) observed were 4.4–11.5 and −9.1–6.1% respectively. Liver S9 fractions from 3‐, 6‐, 12‐ and 18‐month‐old male Sprague–Dawley rats were prepared and Michaelis–Menten parameters were determined. The derived maximum enzyme velocity suggested a rapid saturation of the CYP2D and CYP3A active sites in the liver S9 fractions of 18‐month‐old rats. Moreover, metabolic stabilities of codeine and oxycodone in rat liver S9 fractions were significantly greater for the 18‐month‐old rats. This study suggests that there is an impairment of CYP2D and CYP3A metabolism in aging rats.     

Synthesis,anticancer evaluation,and docking studies of some novel azo chromene derivatives

We have described a simple, convenient, and high-yielding one-pot synthesis of novel azo chromene derivatives via a three-component reaction of various azo aldehydes with dimedone and malononitrile using 10 mol% of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as catalyst and ethanol as solvent at reflux condition. All the synthesized compounds have been characterized using Fourier-transform infrared spectroscopy (FT-IR), ¹H NMR, ¹³C NMR, and HR-MS spectra and molecular docking was performed to explore new inhibitors of human placental aromatase cytochrome P450 and cyclooxygenase-2 enzymes. Of all the compounds docked, compound (E)-2-amino-4-(4,4-dimethyl-2,6-dioxocyclohexyl)-6-((3-methoxyphenyl)diazenyl-4H-chromene-3-carbonitrile ( 4o ) showed good binding affinity with the active site of human placental aromatase cytochrome P450 enzyme (PDB: 3EQM) with inhibition constant (Ki) 1.66 nM and compound 4o also showed good binding affinity with the active site of cyclooxygenase-2 enzyme (PDB: 6COX) with inhibition constant (Ki) 367.17 pM. In vitro anti-cancer activity studies against MCF-7 cells were also performed for compounds 4o , anastrozole and celecoxib. Compound 4o showed an effective cytotoxicity at 19.8 μg/ml compared to anastrozole and celecoxib (24.7 and 26.2 μg/ml).     

A sensitive and high‐throughput LC‐MS/MS method for inhibition assay of seven major cytochrome P450s in human liver microsomes using an in vitro cocktail of probe substrates

A sensitive and high‐throughput LC‐MS/MS method was established and validated for the simultaneous quantification of seven probe substrate‐derived metabolites (cocktail assay) for assessing the in vitro inhibition of cytochrome P450 (CYP) enzymes in pooled human liver microsomes. The metabolites acetaminophen (CYP1A2), hydroxy‐bupropion (CYP2B6), n‐desethyl‐amodiaquine (CYP2C8), 4′‐hydroxy‐diclofenac (CYP2C9), 4′‐hydroxy‐mephenytoin (CYP2C19), dextrorphan (CYP2D6) and 1′‐hydroxy‐midazolam (CYP3A4/5), together with the internal standard verapamil, were eluted on an Agilent 1200 series liquid chromatograph in <7 min. All metabolites were detected by an Agilent 6410B tandem mass spectrometer. The concentration of each probe substrate was selected by substrate inhibition assay that reduced potential substrate interactions. CYP inhibition of seven well‐known inhibitors was confirmed by comparing a single probe substrate assay with cocktail assay. The IC₅₀ values of these inhibitors determined on this cocktail assay were highly correlated (R² > 0.99 for each individual probe substrate) with those on single assay. The method was selective and showed good accuracy (85.89–113.35%) and between‐day (RSD <13.95%) and within‐day (RSD <9.90%) precision. The sample incubation extracts were stable at 25 °C for 48 h and after three freeze–thaw cycles. This seven‐CYP inhibition cocktail assay significantly increased the efficiency of accurately assessing compounds’ potential inhibition of the seven major CYPs in drug development settings. Copyright © 2014 John Wiley & Sons, Ltd.     

In vitro ketamine CYP3A‐mediated metabolism study using mammalian liver S9 fractions,cDNA expressed enzymes and liquid chromatography tandem mass spectrometry

Ketamine is widely used in medicine in combination with several benzodiazepines, including midazolam. The objectives of this study were to develop a novel HPLC‐MS/selected reaction monitoring (SRM) method capable of quantifying ketamine and norketamine using an isotopic dilution strategy in biological matrices and study the formation of norketamine, the principal metabolite of ketamine with and without the presence of midazolam, a well‐known CYP3A substrate. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 μL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 μm . The precision (CV) and accuracy (NOM) observed were 3.9–7.8 and 95.9–111.1% respectively. The initial rate of formation of norketamine was determined using various ketamine concentrations and K_m values of 18.4, 13.8 and 30.8 μm for rat, dog and human liver S9 fractions were observed, respectively. The metabolic stability of ketamine on liver S9 fractions was significantly higher in human (T_1/2 = 159.4 min) compared with rat (T_1/2 = 12.6 min) and dog (T_1/2 = 7.3 min) liver S9 fractions. Moreover significantly lower IC₅₀ and K_i values observed in human compared with rat and dog liver S9 fractions. Experiments with cDNA expressed CYP3A enzymes showed that the formation of norketamine is mediated by CYP3A but results suggest an important contribution from other isoenzymes, most likely CYP2C particularly in rat. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and validation of a LC‐MS/MS method for the in vitro analysis of 1‐hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1‐hydroxymidazolam in plasma are often determined with liquid chromatography–quadrupole mass spectrometry (LC‐MS/MS). However, validated LC‐MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1‐hydroxymidazolam. Therefore, the aim was to validate and optimize an LC‐MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid–liquid extraction with tert‐butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed‐phase chromatography consisting of a Zorbax Extend‐C₁₈ column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC‐MS/MS method was validated over linear range of 1.0–500 nm for 1‐hydroxymidazolam. The results revealed good inter‐assay accuracy (≥85% and ≤115%) and within‐day and between‐day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β‐carotene, green tea, milk thistle and St. John's wort) was determined. Copyright © 2013 John Wiley & Sons, Ltd.