Jun Chen

Washington State University, Pullman, WA, United States

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Publications (9)26.87 Total impact

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    ABSTRACT: The Caco-2 cell culture model is used to determine the absorption potentials of drug candidates and the transport and metabolism mechanisms of drugs and dietary chemicals. The Food and Drug Administration (FDA) recognized the model system as useful in classifying a compound’s absorption characteristics in the Biopharmaceutics Classification System. In addition to its usefulness as an absorption model, the Caco-2 cells are useful for studying the metabolism of drugs. More recently, they have been used to determine the efflux mechanisms of phase II conjugates of drugs and natural products. However, Caco-2 cells do not always express appropriate amounts of transporters or enzymes, which may introduce bias in the determination of their transport via a carriermediated process or their metabolism via a particular pathway. Additional genetic manipulation of the Caco-2 cells will be needed to further advance the utility of this model in the drug development process and to ultimately establish this model as the “gold standard” for studying intestinal disposition of drugs. Key WordsCaco-2–absorption screening–transporter–metabolism–efflux–transepithelial transport–Biopharmaceutics Classification System
    02/2008: pages 19-35;
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    ABSTRACT: The purpose of this study is to determine the importance of coupling of efflux transporters and metabolic enzymes in the intestinal disposition of six isoflavones (genistein, daidzein, formononetin, glycitein, biochanin A, and prunetin), and to determine how isoflavone structural differences affect the intestinal disposition. A rat intestinal perfusion model was used, together with rat intestinal and liver microsomes. In the intestinal perfusion model, significant absorption and excretion differences were found between isoflavones and their respective glucuronides (p <0.05), with prunetin being the most rapidly absorbed and formononetin glucuronides being the most excreted in the small intestine. In contrast, glucuronides were excreted very little in the colon. In an attempt to account for the differences, we measured the glucuronidation rates of six isoflavones in microsomes prepared from rat intestine and liver. Using multiple regression analysis, intrinsic clearance (CL(int)) and other enzyme kinetic parameters (V(max) and K(m)) were determined using appropriate kinetic models based on Akaike's information criterion. The kinetic parameters were dependent on the isoflavone used and the types of microsomes. To determine how metabolite excretion rates are controlled, we plotted excretion rates versus calculated microsomal rates (at 10 microM), CL(int) values, K(m) values, or V(max) values, and the results indicated that excretion rates were not controlled by any of the kinetic parameters. In conclusion, coupling of intestinal metabolic enzymes and efflux transporters affects the intestinal disposition of isoflavones, and structural differences of isoflavones, such as having methoxyl groups, significantly influenced their intestinal disposition.
    Drug Metabolism and Disposition 11/2006; 34(11):1837-48. · 3.36 Impact Factor
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    ABSTRACT: The purpose of this study was to compare intestinal versus hepatic disposition of six flavonoids to fully characterize their first-pass metabolism. The perfused rat intestinal model and microsomes prepared from rat liver, duodenum, jejunum, ileum, and colon were used. The results indicated that isoflavone (12.5 microM) glucuronidation was highly variable among different microsomes prepared from liver or intestine. Comparing to liver metabolism, the intestinal metabolism had higher K(m) values (>2-fold). Likewise, the hepatic intrinsic clearance (IC, or a ratio of V(max)/K(m)) values of isoflavones were generally higher than their intestinal IC values (200-2000% higher), except for prunetin, for which the jejunal IC value was 50% higher than its hepatic IC. When comparing intestinal metabolism, the results showed that intestinal metabolism rates and V(max) values of isoflavones were less when an additional A-ring electron-donating group was absent (i.e., daidzein and formononetin). In the rat perfusion model using the whole small intestine, genistein (10 microM) was well absorbed (77% or 352 nmol/120 min). The first-pass metabolism of genistein was extensive, with 40% of absorbed genistein excreted as conjugated metabolites into the intestinal lumen. In contrast, the bile excretion of genistein conjugates was much less (6.4% of absorbed genistein). In conclusion, intestinal glucuronidation is slower in isoflavones without an additional A-ring substitution. Perfusion studies suggest that intestine is the main organ for genistein glucuronide formation and excretion in rats and may serve as its main first-pass metabolism organ.
    Drug Metabolism and Disposition 01/2006; 33(12):1777-84. · 3.36 Impact Factor
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    Jun Chen, Huimin Lin, Ming Hu
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    ABSTRACT: The purposes of this study were to determine the effect of structural change on the intestinal disposition of isoflavones and to elucidate the mechanisms responsible for transport of phase II isoflavone conjugates. Transport and metabolism of six isoflavones (i.e., genistein, daidzein, glycitein, formononetin, biochanin A, and prunetin) were studied in the human intestinal Caco-2 model and mature Caco-2 cell lysate. Glucuronides were the main metabolites in intact Caco-2 cells for all isoflavones except prunetin, which was mainly sulfated. In addition, the 7-hydroxy group was the main site for glucuronidation whereas the 4'-hydroxy group was only one of the possible sites for sulfation. Glucuronidated isoflavones (except biochanin A) were preferably excreted to the basolateral side, whereas sulfated metabolites (except genistein and glycitein) were mainly excreted into the apical side. Polarized excretion of most isoflavone conjugates was inhibited by the multidrug resistance-related protein (MRP) inhibitor leukotriene C(4) (0.1 micro M) and the organic anion transporter (OAT) inhibitor estrone sulfate (10 micro M). When formation and excretion rates of isoflavones were determined simultaneously, the results showed that formation served as the rate-limiting step for all isoflavone conjugates (both glucuronides and sulfates) except for genistein glucuronide, which had comparable excretion and formation rates. In conclusion, the intestinal disposition of isoflavones was structurally dependent, polarized, and mediated by MRP and OAT. Formation generally served as the rate-limiting step in the cellular excretion of conjugated isoflavones in the Caco-2 cell culture model.
    Cancer Chemotherapy and Pharmacology 03/2005; 55(2):159-69. · 2.80 Impact Factor
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    ABSTRACT: To evaluate the labeling accuracy and product uniformity of available soy isoflavone products in the eastern Washington State region for 13 products and to perform a cost comparison based on the isoflavone content in milligrams for 11 products. Thirteen (13) isoflavone products (7 tablet and 6 capsule formulations) were randomly obtained from health food, grocery, and pharmacy outlets in eastern Washington State. Four different samples of the same product were extracted using 75% ethanol. Each sample was analyzed for isoflavone content using a gradient high-performance liquid chromatography (HPLC) method. Pure isoflavonoid standards were used to quantify the content of genistein, daidzein, and glycitein and their respective glycosides. The amount of total isoflavonoids per purchased product was then divided into its purchase price in order to make cost comparisons between the products based on a 50 mg/d dose. The weight variation within each product was generally small (<4%). However, there was significant variability in the composition of the products. Only 4 of the 13 products contained at least 90% of the isoflavone content claimed on the label and 2 of products contained impurities in the HPLC chromatogram that exceeded 40%. There was no difference in the total isoflavonoid contents of the 2 identical products made by the same manufacturer, but the percentages of the main components had changed significantly over time. Based on a 50-mg isoflavone per day dose, the cost of a 30-day supply ranged from $3.20 to $65.88. There is a lack of uniformity among the products tested and the majority (67%) of the products contained less than 90% of labeled amounts. There is significant variability in the compositions between the products and in the composition of the same product over time. Consumers cannot trust isoflavonoid product labels to represent the product's content accurately or that product pricing is a reflection of isoflavone content.
    The Journal of Alternative and Complementary Medicine 01/2005; 10(6):1053-60. · 1.46 Impact Factor
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    ABSTRACT: This study aims to evaluate a cytochrome P450-based tamoxifen-isoflavone interaction and to determine the mechanisms responsible for inhibitory effects of isoflavones (e.g., genistein) on the formation of alpha-hydroxytamoxifen. Metabolism studies were performed in vitro using female rat liver microsomes. The effects of genistein and an isoflavone mixture on tamoxifen metabolism and the inhibition mechanism were determined using standard kinetic analysis, preincubation, and selective chemical inhibitors of P450. Metabolism of tamoxifen was saturable with Km values of 4.9+/-0.6, 14.6+/-2.2, 25+/-5.9 microM and Vmax values of 34.7+/-1.4, 297.5+/-19.2, 1867+/-231 pmol min(-1) mg(-1) for a-hydroxylation, N-desmethylation, and N-oxidation, respectively. Genistein (25 microM) inhibited alpha-hydroxylation at 2.5 microM tamoxifen by 64% (p < 0.001) but did not affect the 4-hydroxylation, N-desmethylation, and N-oxidation. A combination of three (genistein, daidzein, and glycitein) to five isoflavones (plus biochanin A and formononetin) inhibited tamoxifen alpha-hydroxylation to a greater extent but did not decrease the formation of identified metabolites. The inhibition on alpha-hydroxylation by genistein was mixed-typed with a Ki, value of 10.6 microM. Studies using selective chemical inhibitors showed that tamoxifen alpha-hydroxylation was mainly mediated by rat CYP1A2 and CYP3A1/2 and that genistein 3'-hydroxylation was mainly mediated by rat CYP1A2, CYP2C6 and CYP2D1. Genistein and its isoflavone analogs have the potential to decrease side effects of tamoxifen through metabolic interactions that inhibit the formation of a-hydroxytamoxifen via inhibition of CYP1A2.
    Pharmaceutical Research 12/2004; 21(11):2095-104. · 4.74 Impact Factor
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    ABSTRACT: The purpose of this study was to continue our effort to determine how enzyme-transporter coupling affect disposition of flavonoids. The rat intestinal perfusion and Caco-2 cell models were used together with relevant microsomes. In perfusion model, isoflavone (i.e., formononetin and biochanin A) absorption and subsequent excretion of its metabolites were always site-dependent. Maximal amounts of intestinal and biliary conjugates excreted per 30 min were 31 and 51 nmol for formononetin, more than that for pure biochanin A (12 and 20 nmol). When a standardized red clover extract (biochanin A/formononetin = 10:7) was used, the results indicated that more metabolites of biochanin A than formononetin were found in the perfusate (36.9 versus 22.8 nmol) and bile (78 versus 51 nmol). In metabolism studies, rat intestinal and liver microsomes always glucuronidated biochanin A faster (p < 0.05) than formononetin, whereas intestinal microsomes glucuronidated both isoflavones faster (p < 0.05) than liver microsomes. However, rapid metabolism in the microsomes did not translate into more efficient excretion in either the rat perfusion model as shown previously or in the Caco-2 model. In the Caco-2 model, both isoflavones were rapidly absorbed, efficiently conjugated, and the conjugates excreted apically and basolaterally. More formononetin conjugates were excreted than biochanin A when used alone, but much more biochanin A conjugates were found when using the isoflavone mixture. In conclusion, efficiency of enzyme-transporter coupling controls the amounts of metabolites excreted by the intestine and liver and determines the relative contribution of enteric and enterohepatic recycling to the in vivo disposition of isoflavones.
    Journal of Pharmacology and Experimental Therapeutics 10/2004; 310(3):1103-13. · 3.89 Impact Factor
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    ABSTRACT: This study determined the cytochrome P450 (P450) isoforms responsible for metabolism of isoflavones using human liver microsomes (HLM) and expressed P450s. The primary metabolite of genistein is 3'-OH-genistein, as identified with an authentic chemically synthesized standard. CYP1A2 was predominantly responsible for 3'-OH-genistein formation since its formation was inhibited (>50%, p < 0.05) by a monoclonal antibody specific for CYP1A2, was correlated with CYP1A2 activities of HLM, and was catalyzed by expressed CYP1A2. In addition to CYP1A2, CYP2E1 also catalyzed, although to a lesser extent, its formation. The contribution of these P450s to the formation of 3'-OH-genistein was also confirmed with a panel of expressed enzymes. Methylated isoflavones biochanin A, prunetin, and formononetin (10-100 microM) were rapidly converted by HLM and expressed CYP1A2 to more active genistein and daidzein. The conversion of biochanin A to genistein appears to be mainly mediated by CYP1A2 because of the strong correlation between the conversion rates and CYP1A2 activities in HLM. Thus, CYP1A2 is an effective prodrug-converting enzyme for less active methylated isoflavones. CYP1A2-catalyzed conversion of biochanin A to genistein (Km, 7.80 microM; Vmax, 903 pmol/min/mg of protein; Vmax/Km, 116 microl/min/mg of protein) was much faster than 3'-hydroxylation of genistein (Km, 12.7 microM and Vmax, 109 pmol/min/mg of protein; Vmax/Km, 8.6 microl/min/mg of protein). The interaction studies showed that genistein inhibited formation of acetaminophen from phenacetin with an IC50 value of 16 microM. Additional studies showed that phenacetin and genistein were mutually inhibitory. In conclusion, CYP1A2 and CYP2E1 metabolized genistein and CYP1A2 acted as prodrug-converting enzymes for other less active methylated isoflavones.
    Drug Metabolism and Disposition 07/2003; 31(7):924-31. · 3.36 Impact Factor
  • Jun Chen, Huimin Lin, Ming Hu
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    ABSTRACT: The purpose of this study was to determine the importance of intestinal disposition in the first-pass metabolism of flavonoids. A four-site perfused rat intestinal model, rat liver and intestinal microsomes, Caco-2 cell microsomes, and the Caco-2 cell culture model were used. In the four-site model, approximately 28% of perfused aglycones are absorbed (approximately 450 nmol/30 min). Both absorption and subsequent excretion of metabolites were rapid and site-dependent (p < 0.05). Maximal amounts of intestinal conjugates excreted per 30 min were 61 and 150 nmol for genistein and apigenin, respectively. Maximal amounts of biliary conjugates excreted per 30 min were 50 and 30 nmol for genistein and apigenin, respectively. Microsomes, prepared from Caco-2 cells, rat intestine, and rat liver, always glucuronidated apigenin faster than genistein (p < 0.05). In addition, rat jejunal microsomes glucuronidated both flavonoids faster (p < 0.05) than rat intestinal microsomes prepared from other regions. When comparing glucuronidation in different organs, jejunal microsomes often but not always glucuronidated both flavonoids faster than liver microsomes. In the Caco-2 model, both flavonoids were rapidly absorbed and rapidly conjugated, and the conjugates were excreted apically and basolaterally. Similar to the four-site perfusion model, apigenin conjugates were excreted much faster than genistein conjugates (>2.5 times for glucuronic acid, >4.5 times for sulfate; p < 0.05). In conclusion, intestinal disposition may be more important than hepatic disposition in the first-pass metabolism of flavonoids such as apigenin. In conjunction with enterohepatic recycling, enteric recycling may be used to explain why flavonoids have poor systemic bioavailabilities.
    Journal of Pharmacology and Experimental Therapeutics 04/2003; 304(3):1228-35. · 3.89 Impact Factor

Publication Stats

316 Citations
26.87 Total Impact Points

Institutions

  • 2003–2008
    • Washington State University
      • Department of Pharmaceutical Sciences
      Pullman, WA, United States
  • 2006
    • University of Houston
      • Department of Pharmacological and Pharmaceutical Sciences
      Houston, TX, United States