There is mounting evidence from human epidemiological, animal
in vivo, and
in vitro studies to suggest beneficial effects related to the consumption of quercetin and its glucosides. However, there is ...limited knowledge on the oral bioavailability of these natural products. This study examined the intestinal epithelial membrane transport of quercetin, quercetin 4′-glucoside, and quercetin 3,4′-diglucoside, using the Caco-2 human colonic cell line, a model of human intestinal absorption. The apparent permeability (P
app) of each agent was measured in both apical to basal and basal to apical directions. The apical to basolateral flux of quercetin, P
app 5.8 ± 1.1 × 10
−6 cm · sec
−1 (mean ± SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.48 ± 0.09 × 10
−6 cm · sec
−1 (
P < 0.01). Under identical conditions, the P
app for the transcellular marker propranolol was about 5-fold higher than for quercetin (
P < 0.001). Interestingly, the reverse, basolateral to apical, flux of quercetin (P
app 11.1 ± 1.2 × 10
−6 cm · sec
−1) was almost 2-fold higher than the apical to basolateral flux (
P < 0.001). In similar experiments, quercetin 4′-glucoside demonstrated no absorption, P
app < 0.02 × 10
−6 cm · sec
−1 in the apical to basal direction, but did demonstrate basal to apical flux, P
app 1.6 ± 0.2 × 10
−6 cm · sec
−1. Quercetin 3,4′-diglucoside showed a low apical to basolateral transport (P
app 0.09 ± 0.03 × 10
−6 cm · sec
−1); its reverse, basolateral to apical, transport was, however, 4-fold higher (
P < 0.05). In these cells, glucose was actively transported with an apical to basolateral P
app of 36.8 ± 1.1 × 10
−6 cm · sec
−1. These observations suggest facile absorption of quercetin through the human intestinal epithelium, but contrary to a previous proposal, they do not support an active transport process for quercetin glucosides.
Although it has been suggested that the intestinal glucose transporter may actively absorb dietary flavonoid glucosides, there is a lack of direct evidence for their transport by this system. In ...fact, our previous studies with the human Caco-2 cell model of intestinal absorption demonstrated that a major dietary flavonoid, quercetin 4'-beta-glucoside, is effluxed by apically expressed multidrug resistance-associated protein-2, potentially masking evidence for active absorption. The objective of this study was to test the hypothesis that quercetin 4'-beta-glucoside is a substrate for the intestinal sodium-dependent D-glucose cotransporter SGLT1. Cellular uptake of quercetin 4'-beta-glucoside was examined with Caco-2 cells and SGLT1 stably transfected Chinese hamster ovary cells (G6D3 cells). Although quercetin 4'-beta-glucoside is not absorbed across Caco-2 cell monolayers, examination of the cells by indirect fluorescent microscopy as well as by HPLC analysis of cellular content revealed cellular accumulation of this glucoside after apical loading. Consistent with previous observations, the accumulation of quercetin 4'-beta-glucoside in both Caco-2 and G6D3 cells was markedly enhanced in the presence of multidrug resistance-associated protein inhibition. Uptake of quercetin 4'-beta-glucoside was greater in SGLT1-transfected cells than in parental Chinese hamster ovary cells. Uptake of the glucoside by Caco-2 and G6D3 cells was sodium-dependent and was inhibited by the monovalent ionophore nystatin. In both Caco-2 and G6D3 cells, quercetin 4'-beta-glucoside uptake was inhibited by 30 mM glucose and 0.5 mM phloridzin. These results demonstrate for the first time that quercetin 4'-beta-glucoside is transported by SGLT1 across the apical membrane of enterocytes.
Although there is strong evidence to suggest that flavonoid consumption is beneficial to human health, the extent to which flavonoids are absorbed and the mechanisms involved are controversial. ...Contrary to common dogma, we previously demonstrated that quercetin 4'-beta-glucoside, the predominant form of the most abundant dietary flavonoid, quercetin, was not absorbed across Caco-2 cell monolayers. The aim of this study was to test the hypothesis that a specific efflux transporter is responsible for this lack of absorption. Transport of quercetin 4'-beta-glucoside, alone or with inhibitors, was examined with Caco-2 cell monolayers. In addition, subcellular localization of the multidrug resistance-associated proteins MRP1 and MRP2 was examined by immunofluorescent confocal microscopy. Efflux of quercetin 4'-beta-glucoside, a saturable process, was not altered by verapamil, a P-glycoprotein inhibitor, but was competitively inhibited by MK-571, an MRP inhibitor. These data in combination with immunofluorescent localization of MRP2 to the apical membrane support a role for MRP2 in the intestinal transcellular efflux of quercetin 4'-beta-glucoside. These results suggest a role for MRP2 in the transport of a new class of agents, dietary glucosides.
Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is ...an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.
Genetic inheritance plays a significant role in the interindividual variability of drug response. The field of pharmacogenomics seeks to identify genetic factors that influence drug response, ...including both those that are inherited and those that arise within tumors, and use this information to improve drug therapy. Candidate gene approaches have led to clinical tests for toxicity avoidance (eg, TPMT, UGT1A1) and efficacy prediction (eg, epidermal growth factor receptor-activating mutations). However, the "right" genes are not known for most anticancer drugs. Strategies for uncovering pharmacogenomic associations vary widely from monogenic candidate gene approaches to polygenic genome-wide approaches. This review will place in context clinically relevant pharmacogenomic discovery approaches, including the relative strengths and weaknesses and the challenges inherent with achieving the goal of individualized therapy.
The Caco‐2 cell monolayer has become an increasingly useful in‐vitro model of human intestinal absorption. In this study we have determined the effect of plasma on the basolateral side on the ...absorption as well as exsorption of several drugs that are highly bound to plasma proteins. The drugs used included propranolol and quercetin, which both use the transcellular route of absorption, and taxol and oestradiol 17 β‐D‐glucuronide, which are thought to undergo efflux by P‐glycoprotein and the multidrug resistance protein MRP, respectively. All experiments were carried out under sink conditions to mimic normal absorption. It was necessary to use heparin anticoagulation for generation of the plasma, as EDTA was found to make the monolayers very leaky.
The apparent permeability (Papp) values for absorption were 1.54×10−6 cms−1 for oestradiol 17 β‐D‐glucuronide, 3.33×10−6 cms−1 for taxol, 20.8×10−6 cms−1 for quercetin, and 35.3×10−6 cms−1 for propranolol. For these four compounds, plasma on the basolateral side had no influence on absorption. However, plasma on the basolateral side significantly reduced the efflux of oestradiol 17 β‐D‐glucuronide by 66%, taxol by 75%, propranolol by 82%, and quercetin by 94%.
Failure to consider the effect of plasma binding can result in an overestimate of basolateral to apical efflux and result in misleading net flux calculations.
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