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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, Ϸ28% of perfused aglycones are absorbed (Ϸ450 nmol/30 min). Both absorption and subsequent excretion of metabolites were rapid and site-dependent (p Ͻ 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 Ͻ 0.05). In addition, rat jejunal microsomes glucuronidated both flavonoids faster (p Ͻ 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 (Ͼ2.5 times for glucuronic acid, Ͼ4.5 times for sulfate; p Ͻ 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.

The purpose of this study was to determine the mechanisms responsible for intestinal disposition of apigenin in the human Caco-2 cell culture model. The results indicated that most of the absorbed apigenin (10 M) were conjugated and only a small fraction was transported intact. The amounts of conjugates excreted, especially that of the sulfate, were dependent on dayspost-seeding. Apical efflux of apigenin sulfate did not change with concentration of apigenin (4 to 40 M), whereas its basolateral efflux increased (p Ͻ 0.01) with concentration and plateaued at about 25 M. In contrast, sulfate formation rates in cell lysate increased with concentration and plateaued at 25 M and were 4 to 6 times faster than the corresponding excretion rates. Formation and polarized excretion rates of glucuronidated apigenin increased with apigenin concentration but formation rates were usually 2.5 to 6 times faster than the corresponding excretion rates. Inhibitors of multidrug resistance-related proteins (MRPs) such as leukotriene C 4 and MK-571, which inhibited glucuronidation of apigenin at a high concentration (Ն25 M), significantly decreased excretion of both apigenin conjugates, and higher concentrations of MK-571 increased the extent of inhibition. In contrast, an organic anion transporter (OAT) inhibitor estrone sulfate only inhibited excretion of apigenin sulfate. In conclusion, we have shown for the first time that intestinal efflux is the rate-limiting step in the intestinal excretion of phase II conjugates of flavones. Furthermore, MRP and OAT are involved in the intestinal efflux of these hydrophilic phase II conjugates.

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.

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 ϭ 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 Ͻ 0.05) than formononetin, whereas intestinal microsomes glucuronidated both isoflavones faster (p Ͻ 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.Red clover (Trifolium pratense L.) extracts are sold as dietary supplements in supermarkets and health food stores in both industrialized and developing countries. Several defined extracts of red clover are sold in the United States to treat women who suffer from menopausal-related symptoms (Rijke et al., 2001;Oleszek and Stochmal, 2002). The extracts are available as tablets (e.g., Promensil), capsules, tea, liquid preparations, and several other forms. Limited clinical studies have demonstrated the effectiveness of Promensil in the management of hot flashes (van de Weijer and Barentsen, 2002), although the efficacy of such products is not always consistent (Baber et al., 1999;St. Germain et al., 2001).Red clover extract contains many of the same isoflavones as soy (Lin et al., 2000). However, a majority of isoflavones in red clover are present in aglycone forms, whereas they are present as glucosides in soy and soy extracts. Furthermore, the principal isoflavones are biochanin A and formononetin in red clover but are genistein and daidzein in soy. Nevertheless, these isoflavones all have similar biological activities, and biochanin A and formononetin can be converted to genistein and daidzein by cytochrome P450 (Tolleson et al., 2002;Hu et al., 2003b). Moreover, daidzein can be metabolized to h...

ABSTRACT:Caco-2 cell lysate, and intestinal and liver microsomes derived from female humans and rats were used to compare and contrast the metabolism and disposition of raloxifene. In Caco-2 cell lysate, raloxifene 6-␤-glucuronide (M1) was the main metabolite, although raloxifene 4-␤-glucuronide (M2) was formed in comparable abundance (58% versus 42%). In rat liver and intestinal microsomes, M1 represented about 76 to 86% of glucuronidated metabolites. In contrast, raloxifene 4-␤-glucuronide (M2) was the predominant metabolite in expressed UGT1A10 (96%) and human intestinal (92%) microsomes. Intrinsic clearance for M2 (CL int, M2 ) in human intestinal microsomes was 33-to 72-fold higher than in rat microsomes, whereas intrinsic clearance for M1 (CL int, M1 ) was 3-to 4-fold lower. Taken together, total intrinsic clearance (CL int, M1 ؉ CL int, M2 ) in human intestinal microsomes was 3-to 6-fold higher than that in rat intestinal microsomes, but was similar in liver microsomes. In addition, intrinsic clearance in small intestinal microsomes was 2-to ϳ5-fold higher than that in hepatic microsomes, regardless of species. To account for the difference in species-and disposition model-dependent intestinal metabolism, we probed the presence of various UGT1A isoforms in Caco-2 cells using real-time reverse transcriptase-polymerase chain reaction and, as expected, detected no UGT1A10. In conclusion, the lack of UGT1A10 may explain why Caco-2 cell and rat intestinal microsomes metabolized raloxifene differently from human intestinal microsomes. The presence of human intestinal UGT1A10 and the higher overall intrinsic clearance value in the human intestine as the result of UGT1A10 expression could explain why raloxifene has much lower bioavailability in humans (2%) than in rats (39%).

The ferroptosis effect has been illuminated with a clear Fenton reaction mechanism that converts endogenous hydrogen peroxide (H 2 O 2 ) into highly oxidative hydroxyl radicals (•OH) in ROS-amplified tumor therapy. This ferroptosis-related oxidation effect was then further enhanced by the enzyme-like roles of cisplatin (CDDP). This CDDP-induced apoptosis was promoted in reverse by ferroptosis via the depletion of glutathione (GSH) and prevention of DNA damage repair. Here, we have developed degradable metallic complexes (PtH@FeP) containing an Fe(III)-polydopamine (FeP) core and HA-cross-linked CDDP (PtH) shell, exaggerating in situ toxic ROS production via the synergistic effect of CDDP and Fe(III). Taken together, the rationally designed PtH@FeP provided a new strategy for self-amplified synergistic chemotherapy/ferroptosis/photothermal therapy (PTT) antitumor effects with a reduced dosage that facilitates clinical safety.

1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;48:1069-1079.