Zhong-Ze Fang

CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes

Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), 1H-nuclear magnetic resonance (NMR), and 13C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin’s metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

New insights into the risk of phthalates: Inhibition of UDP-glucuronosyltransferases

Wide utilization of phthalates-containing products results in the significant exposure of humans to these compounds. Many adverse effects of phthalates have been documented in rodent models, but their effects in humans exposed to these chemicals remain unclear until more mechanistic studies on phthalate toxicities can be carried out. To provide new insights to predict the potential adverse effects of phthalates in humans, the recent study investigated the inhibition of representative phthalates di-n-octyl ortho-phthalate (DNOP) and diphenyl phthalate (DPhP) towards the important xenobiotic and endobiotic-metabolizing UDP-glucuronosyltransferases (UGTs). An in vitro UGTs incubation system was employed to study the inhibition of DNOP and DPhP towards UGT isoforms. DPhP and DNOP weakly inhibited the activities of UGT1A1, UGT1A7, and UGT1A8. 100 µM of DNOP inhibited the activities of UGT1A3, UGT1A9, and UGT2B7 by 41.8% (p < 0.01), 45.6% (p < 0.01), and 48.8% (p < 0.01), respectively. 100 µM of DPhP inhibited the activity of UGT1A3, UGT1A6, and UGT1A9 by 81.8 (p < 0.001), 49.1% (p < 0.05), and 76.4% (p < 0.001), respectively. In silico analysis was used to explain the stronger inhibition of DPhP than DNOP towards UGT1A3 activity. Kinetics studies were carried our to determine mechanism of inhibition of UGT1A3 by DPhP. Both Dixon and Lineweaver-Burk plots showed the competitive inhibition of DPhP towards UGT1A3. The inhibition kinetic parameter (Ki) was calculated to be 0.89 µM. Based on the [I]/Ki standard ([I]/Ki < 0.1, low possibility; 1>[I]/Ki > 0.1, medium possibility; [I]/Ki > 1, high possibility), these studies predicted in vivo drug-drug interaction might occur when the plasma concentration of DPhP was above 0.089 µM. Taken together, this study reveales the potential for adverse effects of phthalates DNOP and DPhP as a result of UGT inhibition.

Regulation profile of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) components towards UDP-glucuronosyltransferases (UGTs) isoforms

Abstract 1.u2002Endogenous compounds have been reported to be the regulators of UDP-glucuronosyltransferases (UGTs) isoforms. This study aims to investigate the regulatory effects of the activity of UGT isoforms by two important lipid components phosphatidylcholine (PC) and lysophosphatidylcholines (LPC) using in vitro incubation system. 2.u2002UGTs supersomes-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as the probe reaction to evaluate the inhibition of compounds towards UGT isoforms except UGT1A4, and UGT1A4-catalyzed trifluoperazine (TFP) glucuronidation reaction was utilized to phenotype the activity of UGT1A4. 3.u2002About 50u2009μM of LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0 exhibited inhibition towards more than 90% activity of UGT isoforms, and other LPC and PC components showed negligible inhibitory potential towards all the UGT isoforms. UGT1A6 and UGT1A8 were identified to be the most sensitive UGT isoforms susceptible for the inhibition by LPC15:0, LPC16:0, LPC17:0, LPC18:0, LPC18:1 and PC16:0, 2:0, indicating the strong influence of these LPC and PC components towards UGT1A6 and UGT1A8-catalyzed metabolic reaction when the concentrations of these components increased.

A dried blood spot mass spectrometry metabolomic approach for rapid breast cancer detection

Objective Breast cancer (BC) is still a lethal threat to women worldwide. An accurate screening and diagnosis strategy performed in an easy-to-operate manner is highly warranted in clinical perspective. Besides the routinely focused protein markers, blood is full of small molecular metabolites with diverse structures and properties. This study aimed to screen metabolite markers with BC diagnosis potentials. Methods A dried blood spot-based direct infusion mass spectrometry (MS) metabolomic analysis was conducted for BC and non-BC differentiation. The targeted analytes included 23 amino acids and 26 acylcarnitines. Results Multivariate analysis screened out 21 BC-related metabolites in the blood. Regression analysis generated a diagnosis model consisting of parameters Pip, Asn, Pro, C14:1/C16, Phe/Tyr, and Gly/Ala. Tested with another set of BC and non-BC samples, this model showed a sensitivity of 92.2% and a specificity of 84.4%. Compared to the routinely used protein markers, this model exhibited distinct advantage with its higher sensitivity. Conclusion Blood metabolites screening is a more plausible approach for BC detection. Furthermore, this direct MS analysis could be finished within few minutes, which means that its throughput is higher than the currently used imaging techniques.

Inhibition of UDP-Glucuronosyltransferases (UGTs) Activity by constituents of Schisandra chinensis.

Structure–activity relationship for the inhibition of Schisandra chinensiss ingredients toward (Uridine‐Diphosphate) UDP‐glucuronosyltransferases (UGTs) activity was performed in the present study. In vitro incubation system was employed to screen the inhibition capability of S.u2009chinensiss ingredients, and in silico molecular docking method was carried out to explain possible mechanisms. At 100u2009μM of compounds, the activity of UGTs was inhibited by less than 90% by schisandrol A, schisandrol B, schisandrin, schisandrin C, schisantherin A, gomisin D, and gomisin G. Schisandrin A exerted strong inhibition toward UGT1A1 and UGT1A3, with the residual activity to be 7.9% and 0% of control activity. Schisanhenol exhibited strong inhibition toward UGT2B7, with the residual activity to be 7.9% of control activity. Gomisin J of 100u2009μM inhibited 91.8% and 93.1% of activity of UGT1A1 and UGT1A9, respectively. Molecular docking prediction indicated different hydrogen bonds interaction resulted in the different inhibition potential induced by subtle structure alteration among schisandrin A, schisandrin, and schisandrin C toward UGT1A1 and UGT1A3: schisandrin Au2009>u2009schisandrinu2009>u2009schisandrin C. The detailed inhibition kinetic evaluation showed the strong inhibition of gomisin J toward UGT1A9 with the inhibition kinetic parameter (Ki) to be 0.7u2009μM. Based on the concentrations of gomisin J in the plasma of the rats given with S.u2009chinensis, high herb–drug interaction existed between S.u2009chinensis and drugs mainly undergoing UGT1A9‐mediated metabolism. In conclusion, in silico‐in vitro method was used to give the inhibition information and possible inhibition mechanism for S.u2009chinensiss components toward UGTs, which guide the clinical application of S.u2009chinensis. Copyright

Enantioselective Inhibition of Carprofen Towards UDP-glucuronosyltransferase (UGT) 2B7

UDP-glucuronosyltransferases (UGTs)-catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms-catalyzed 4-methylumbelliferone (4-MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)-carprofen and (S)-carprofen towards multiple UGT isoforms. The results showed that (S)-carprofen exhibited stronger inhibition potential than (R)-carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)-carprofen and (S)-carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)-carprofen and (R)-carprofen towards UGT2B7. A Lineweaver-Burk plot showed that both (S)-carprofen and (R)-carprofen exhibited competitive inhibition towards UGT2B7-catalyzed 4-MU glucuronidation. The inhibition kinetic parameter (Ki ) was calculated to be 7.0u2009μM and 31.1u2009μM for (S)-carprofen and (R)-carprofen, respectively. Based on the standard for drug-drug interaction, the threshold for (S)-carprofen and (R)-carprofen to induce a drug-drug interaction is 0.7u2009μM and 3.1u2009μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP-glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)-carprofen and (R)-carprofen to possibly induce the drug-drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)-carprofen is more important than (R)-carprofen to avoid a possible drug-drug interaction between carprofen and the drugs mainly undergoing UGT2B7-catalyzed metabolism.

Evaluation and Comparison of the Inhibition Effect of Astragaloside IV and Aglycone Cycloastragenol on Various UDP-Glucuronosyltransferase (UGT) Isoforms.

As one of the main active ingredients from Radix Astragali (RA), orally dosed astragaloside IV (AST) is easily transformed to sapogenin-cycloastragenol (CAG) by deglycosylation in the gastrointestinal tract. Because the potential adverse effects of AST and CAG remain unclear, the present study in this article was carried out to investigate the inhibition effects of AST and CAG on UDP-glucuronosyltransferases (UGTs) to explore potential clinical toxicity. An in vitro UGTs incubation mixture was employed to study the inhibition of AST and CAG towards UGT isoforms. Concentrations of 100 μM for each compound were used to initially screen the inhibitory efficiency. Deglycosylation of AST to CAG could strongly increase the inhibitory effects towards almost all of the tested UGT isoforms, with an IC50 of 0.84 μM and 11.28 μM for UGT1A8 and UGT2B7, respectively. Ulteriorly, the inhibition type and kinetics of CAG towards UGT1A8 and UGT2B7 were evaluated depending on the initial screening results. Data fitting using Dixon and Lineweaver–Burk plots demonstrated that CAG competitively inhibited UGT1A8 and noncompetitively inhibited UGT2B7. From the second plot drawn with the slopes from the Lineweaver–Burk plot versus the concentrations of CAG, the inhibition constant (Ki) was calculated to be 0.034 μM and 20.98 μM for the inhibition of UGT1A8 and UGT2B7, respectively. Based on the [I]/Ki standard ([I]/Ki < 0.1, low possibility; 1 > [I]/Ki > 0.1, medium possibility; [I]/Ki > 1, high possibility), it was successfully predicted here that an in vivo herb–drug interaction between AST/CAG and drugs mainly undergoing UGT1A8- or UGT2B7-catalyzed metabolism might occur when the plasma concentration of CAG is above 0.034 μM and 20.98 μM, respectively.

In Vitro Comparative Study of the Inhibitory Effects of Mangiferin and Its Aglycone Norathyriol towards UDP-Glucuronosyl Transferase (UGT) Isoforms

Mangiferin (MGF), the predominant constituent of extracts of the mango plant Mangifera Indica L., has been investigated extensively because of its remarkable pharmacological effects. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to investigate the inhibition of mangiferin and aglycone norathyriol towards various isoforms of UGTs in our study, which evaluated the inhibitory capacity of MGF and its aglycone norathyriol (NTR) towards UDP-glucuronosyltransferase (UGT) isoforms. Initial screening experiment showed that deglycosylation of MGF into NTR strongly increased the inhibitory effects towards almost all the tested UGT isoforms at a concentration of 100 μM. Kinetic experiments were performed to further characterize the inhibition of UGT1A3, UGT1A7 and UGT1A9 by NTR. NTR competitively inhibited UGT1A3, UGT1A7 and UGT1A9, with an IC50 value of 8.2, 4.4, and 12.3 μM, and a Ki value of 1.6, 2.0, and 2.8 μM, respectively. In silico docking showed that only NTR could dock into the activity cavity of UGT1A3, UGT1A7 and UGT1A9. The binding free energy of NTR to UGT1A3, 1A7, 1A9 were −7.4, −7.9 and −4.0 kcal/mol, respectively. Based on the inhibition evaluation standard ([I]/Ki < 0.1, low possibility; 0.1 < [I]/Ki < 1, medium possibility; [I]/Ki > 1, high possibility), an in vivo herb–drug interaction between MGF/NTR and drugs mainly undergoing UGT1A3-, UGT1A7- or UGT1A9-catalyzed metabolism might occur when the plasma concentration of NTR is above 1.6, 2.0 and 2.8 μM, respectively.

Rapid differentiating colorectal cancer and colorectal polyp using dried blood spot mass spectrometry metabolomic approach

Colorectal cancer (CRC) is the third leading causes of cancer mortality, and the early‐stage detection could significantly enhance survival rates. Cancer influences the important metabolic pathways and the changes in metabolite levels had been used in many studies as the potential biomarkers. This study is aimed at screening metabolite biomarkers with CRC diagnosis potentials. The direct infusion mass spectrometry (MS) metabolomic analysis based on dried blood spot was used to distinguish CRC from polyp. The target metabolites were composed of 23 amino acids and 26 acylcarnitines. The 21 metabolites in blood were selected via multivariate analysis. A regression model was established based on parameters C16, Arg, C4/C8, C5/C3, Val, Phe/Tyr, Ala, C4/C3. Tenfold cross validation (CV) method was used to test this model and showed sensitivity of 81.18% and specificity of 83.95%. The metabolomic analysis is a practicable method for CRC detection. The use of direct MS analysis in metabolite screening could be finished in several minutes and served as a higher‐throughput method to distinguish CRC and polyps.

Role of the lipid-regulated NF-κB/IL-6/STAT3 axis in alpha-naphthyl isothiocyanate-induced liver injury

Alpha-naphthyl isothiocyanate (ANIT)-induced liver damage is regarded as a useful model to study drug-induced cholestatic hepatitis. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry (UPLC-ESI-QTOF MS)-based metabolomics revealed clues to the mechanism of ANIT-induced liver injury, which facilitates the elucidation of drug-induced liver toxicity. 1-Stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:0) and 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 18:1) were significantly increased in serum from ANIT-treated mice, and this increase resulted from altered expression of genes encoding the lipid metabolism enzymes Chka and Scd1. ANIT also increased NF-κB/IL-6/STAT3 signaling, and in vitro luciferase reporter gene assays revealed that LPC 18:0 and LPC 18:1 can activate NF-κB in a concentration-dependent manner. Activation of PPARα through feeding mice a Wy-14,643-containing diet (0.1%) reduced ANIT-induced liver injury, as indicated by lowered ALT and AST levels, and liver histology. In conclusion, the present study demonstrated a role for the lipid-regulated NF-κB/IL-6/STAT3 axis in ANIT-induced hepatotoxicity, and that PPARα may be a potential therapeutic target for the prevention of drug-induced cholestatic liver injury.