[Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin].

This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 µL·min~(-1)·mg~(-1),respectively,while those for G1 were 543. 3,and 75. 9 µL·min~(-1)·mg~(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 µL·min~(-1)·mg~(-1)),CYP2 C8( 88. 0 µL·min~(-1)·mg~(-1)),CYP2 C19( 166. 7 µL·min~(-1)·mg~(-1)),and CYP2 D6( 9. 1 µL·min~(-1)·mg~(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 µL·min~(-1)·mg~(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 µL·min~(-1)·mg~(-1)),UGT1 A7( 922. 8 µL·min~(-1)·mg~(-1)),UGT1 A8( 133. 0 µL·min~(-1)·mg~(-1)),UGT1 A9( 348. 6 µL·min~(-1)·mg~(-1)) and UGT2 B7( 118. 7 µL·min~(-1)·mg~(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 µL·min~(-1)·mg~(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

Effect of pterostilbene on in vitro drug metabolizing enzyme activity.

Pterostilbene is a natural polyphenol compound found in small berries that is related to resveratrol, but has better bioavailability and a longer half-life. The purpose of this study was to assess the potential inhibitory effect of pterostilbene on in vitro drug metabolism. The effect of pterostilbene on cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzyme activities were studied using the enzyme-selective substrates amodiaquine (CYP2C8), midazolam (CYP3A4), estradiol (UGT1A1), serotonin (UGT1A6) and mycophenolic acid (UGT1A8/9/10). The IC50 value was used to express the strength of inhibition. Further, a volume per dose index (VDI) was used to estimate the potential for in vivo interactions. Pterostilbene significantly inhibited CYP2C8 and UGT1A6 activities. The IC50 (mean ±â€¯SE) values for CYP2C8 and UGT1A6 inhibition were 3.0 ±â€¯0.4 µM and 15.1 ±â€¯2.8 µM, respectively; the VDI exceeded the predefined threshold of 5 L/dose for both CYP2C8 and UGT1A6, suggesting a potential for interaction in vivo. Pterostilbene did not inhibit the metabolism of the other enzyme-selective substrates. The results of this study indicate that pterostilbene inhibits CYP2C8 and UTG1A6 activity in vitro and may inhibit metabolism by these enzymes in vivo. Clinical studies are warranted to evaluate the in vivo relevance of these interactions.

Glucuronidation of mono(2-ethylhexyl) phthalate in humans: roles of hepatic and intestinal UDP-glucuronosyltransferases.

Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP), which is an endocrine-disrupting chemical. In the present study, MEHP glucuronidation in humans was studied using recombinant UDP-glucuronosyltransferases (UGTs) and microsomes of the liver and intestine. Among the recombinant UGTs examined, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, and UGT2B7 glucuronidated MEHP. The kinetics of MEHP glucuronidation by UGT1A3, UGT1A7, UGT1A8, UGT1A10, UGT2B4, and UGT2B7 followed the Michaelis-Menten model, whereas that by UGT1A9 fit the negative allosteric model. CLint values were in the order of UGT1A9 > UGT2B7 > UGT1A7 > UGT1A8 ≥ UGT1A10 > UGT1A3 > UGT2B4. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model. Diclofenac (20 µM) and raloxifene (20 µM) decreased CLint values to 43 and 36 % that of native microsomes, respectively. The kinetics of MEHP glucuronidation by intestine microsomes fit the biphasic model. Diclofenac (150 and 450 µM) decreased CLint values to 32 and 13 % that of native microsomes for the high-affinity phase, and to 28 and 21 % for the low-affinity phase, respectively. Raloxifene (2.5 and 7.0 µM) decreased CLint values to 35 and 4.1 % that of native microsomes for the high-affinity phase, and to 48 and 53 % for the low-affinity phase, respectively. These results suggest that MEHP glucuronidation in humans is catalyzed by UGT1A3, UGT1A9, UGT2B4, and/or UGT2B7 in the liver, and by UGT1A7, UGT1A8, UGT1A9, UGT1A10, and/or UGT2B7 in the intestine, and also that these UGT isoforms play important and characteristic roles in the detoxification of DEHP.

Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 µM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 µM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions.

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3.

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.

Human intestine and liver microsomal metabolic differences between C19-diester and monoester diterpenoid alkaloids from the roots of Aconitum carmichaelii Debx.

The roots of Aconitum carmichaelii Debx. show excellent effects against rheumatism and cardiovascular diseases, but the effective compounds, C19-diester and monoester diterpenoid alkaloids (DDAs and MDAs) are toxic for their narrow therapeutic windows. It is noteworthy to investigate intestinal metabolism of these toxic compounds mainly by oral administration, because gut also express drug-metabolizing enzymes as well as liver. This study initially focused on phase I and phase II metabolism of DDAs (including aconitine, mesaconitine and hypaconitine) and MDAs (including benzoylaconine, benzoylmesaconine and benzoylhypaconine) in human intestine microsomes (HIM), with comparison of metabolism in human liver microsomes (HLM). CYP3A in HIM mainly catalyzed phase I metabolism of DDAs and MDAs, with polarity increased. The intestinal metabolic profiles of DDAs were more various than MDAs. No glucuronide metabolites were detected, which was in agreement with HLM metabolism. The results showed that gut played an important role in detoxification of DDAs and MDAs by oral administration. Moreover, eight new metabolites from DDAs were identified in microsomes incubations by UPLC-Q-TOF-HRMS/MS. Our findings provided reference to the detoxification of DDAs and MDAs in the intestine in vivo and supplemented the phase I metabolic pathways for DDAs.

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

OBJECTIVES: This study aimed to characterize the glucuronidation pathway of arctigenin (AR) in human liver microsomes (HLM) and human intestine microsomes (HIM). METHODS: HLM and HIM incubation systems were employed to catalyse the formation of AR glucuronide. The glucuronidation activity of commercially recombinant UGT isoforms towards AR was screened. A combination of chemical inhibition assay and kinetic analysis was used to determine the UGT isoforms involved in the glucuronidation of AR in HLM and HIM. KEY FINDINGS: AR could be extensively metabolized to one mono-glucuronide in HLM and HIM. The mono-glucuronide was biosynthesized and characterized as 4'-O-glucuronide. UGT1A1, 1A3, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7 and 2B17 participated in the formation of 4'-O-G, while UGT2B17 demonstrated the highest catalytic activity in this biotransformation. Both kinetic analysis and chemical inhibition assays demonstrated that UGT1A9, UGT2B7 and UGT2B17 played important roles in AR-4'-O-glucuronidation in HLM. Furthermore, HIM demonstrated moderate efficiency for AR-4'-O-glucuronidation, implying that AR may undergo a first-pass metabolism during the absorption process. CONCLUSION: UGT1A9, UGT2B7 and UGT2B17 were the major isoforms responsible for the 4'-O-glucuronidation of AR in HLM, while UGT2B7 and UGT2B17 were the major contributors to this biotransformation in HIM.