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Hepatoenteric recycling is a new disposition mechanism for orally administered phenolic drugs and phytochemicals in rats

  1. Yifan Tu
  2. Lu Wang
  3. Yi Rong
  4. Vincent Tam
  5. Taijun Yin
  6. Song Gao
  7. Rashim Singh
  8. Ming Hu  Is a corresponding author
  1. Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, United States
  2. Department of Pharmaceutical Sciences, College of Pharmacy, Texas Southern University, United States
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Cite this article as: eLife 2021;10:e58820 doi: 10.7554/eLife.58820

Abstract

Many orally administered phenolic drugs undergo enterohepatic recycling (EHR), presumably mediated by the hepatic phase II enzymes. However, the disposition of extrahepatically generated phase II metabolites is unclear. This paper aims to determine the new roles of liver and intestine in the disposition of oral phenolics. Sixteen representative phenolics were tested using direct portal vein infusion and/or intestinal perfusion. The results showed that certain glucuronides were efficiently recycled by liver. OATP1B1/1B3/2B1 were the responsible uptake transporters. Hepatic uptake is the rate-limiting step in hepatic recycling. Our findings showed that the disposition of many oral phenolics is mediated by intestinal glucuronidation and hepatic recycling. A new disposition mechanism ‘Hepatoenteric Recycling (HER)”, where intestine is the metabolic organ and liver is the recycling organ, was revealed. Further investigations focusing on HER should help interpret how intestinal aliments or co-administered drugs that alter gut enzymes (e.g. UGTs) expression/activities will impact the disposition of phenolics.

Introduction

Enterohepatic recirculation/recycling (i.e. EHR) refers to the recirculation/recycling of endogenous (produced within the body, like biliary acids and bilirubin) and exogenous (e.g. drugs or other substances) compounds. EHR involves the excretion of substances from the liver to the small intestine via bile, followed by absorption into enterocytes, which then enters the liver again via portal vein (Lennartsson et al., 2012). EHR is important in the disposition of both endogenous and exogenous compounds. For endogenous compounds (e.g. bile acids), the recirculation through EHR keeps them from being eliminated into the feces and reduces the requirement of daily biosynthesis. For exogenous compounds, EHR prolonged the in vivo exposure and increased their apparent half-life. In other words, EHR could greatly affect the efficacy of drugs and the toxicity of xenobiotics (or exogenous compounds).

EHR was first conceptualized in the 1950s for bile acid recirculation (Norman and Sjovall, 1958). The recirculation of bile acids requires only the re-absorption of the intact bile acids in the intestine without any change. In the 1960s, this concept was expanded into the disposition of drugs and other xenobiotics that undergo phase II metabolism via enzymes such as uridine 5'-diphospho-glucuronosyltransferase (UGTs) and sulfotransferases (SULTs) primarily expressed in the small intestine and liver. Currently, a variety of drugs are known to undergo EHR, including anticancer drugs (e.g. sorafenib, SN-38), anti-cholesterol drugs (e.g. ezetimibe), anti-osteoporosis drugs (e.g. raloxifene), analgesics (e.g. morphine) Gårdmark et al., 1993, and others (e.g. mycophenolic acid) (Trdan Lušin et al., 2012; Oswald et al., 2008; Ando and Hasegawa, 2005; Vasilyeva et al., 2015; Hasselström and Säwe, 1993). In the case of exogenous compounds, the R in EHR was ‘recycling’ not ‘recirculation,’ as the enzymes of the microflora present in the intestine are required to hydrolyze the phase II metabolites and regenerate the aglycones (the parent form of the phase II metabolites) for reabsorption (Williams et al., 1965). The current concept of EHR does not define or specify the sources of phase II metabolites (i.e. where they are formed) that excreted in bile. Previously, it is postulated that the phase II metabolites (especially glucuronide metabolites) found in bile are mainly produced by the liver because bile acids are biosynthesized in the liver.

EHR of bile acids led most investigators, ourselves included, to believe that liver is the major organ contributing to the formation of conjugated metabolites. Faster in vitro microsomal conjugation rates observed in the liver as compared to intestinal microsomes for many compounds also supported this assumption (Hu et al., 2014; Zhou et al., 2010). However, we recently found that certain phenolic glucuronides formed in the intestine were efficiently taken up by hepatocytes and then rapidly excreted into the bile (Zeng et al., 2016). These phenomena suggest that glucuronides formed extrahepatically (such as in the intestine), available for hepatic uptake transporters (e.g. Organic Anion Transporting Polypeptides) and subsequently excreted by efflux transporters (e.g. Multidrug Resistance-associated Protein 2), could be recycled by the liver. This led us to hypothesize a new recycling phenomenon, 'Hepatoenteric Recycling (HER)'.

EHR differs from HER in the predominant organs for formation and transport of phase II metabolites. We believe that it is important to delineate HER from EHR since the disposition of phase II metabolites in different metabolic organs (such as liver and intestine) could greatly change with age, gender, and disease conditions (Bolling et al., 2011; Bhatt et al., 2019; Liu et al., 2013; Zhou et al., 2013; Pimentel et al., 2019; Langmann et al., 2004). These conditions often dictate the organ-level expression and activities of phase II enzymes and transporters (both efflux and uptake) that could significantly impact the EHR vs HER-mediated disposition of glucuronides. The changes in HER-mediated disposition can lead to changes in systemic and intestinal exposure of drugs and their glucuronides by altering the hepatic uptake and biliary excretion of glucuronides, which could significantly affect the drug efficacy and toxicity, both locally (intestinal lumen) and systemically.

However, EHR redefinition, which could explain the disposition of extrahepatically derived phase II metabolites, requires a systemic study to provide initial evidence that is based on the sources of biliary phenolic glucuronides. The current study provides answers to the three key questions: (1) how efficiently extrahepatically derived phenolic glucuronides are recycled by liver; (2) how these glucuronides are taken up by the liver cells; and (3) whether the aglycone or glucuronides entering the liver is better source of biliary excreted glucuronides. By answering these questions, we aim to elucidate the mechanism of HER, and the significance of distinguishing it from traditional EHR.

Through the analysis of our study results, a new disposition mechanism called ‘Hepatoenteric Recycling (HER)’ mainly applied to the disposition of drugs and their glucuronides, with significant intestinal metabolism, is proposed. The new mechanism distinguish from the enterohepatic recirculation/recycling (Figure 1), which is mainly applied to compounds which undergo recirculation in intact form or are metabolized by the liver. In HER, intestine is the organ for glucuronide formation and liver is the organ for its recycling (Figure 1A) as opposed to in EHR, where liver is the organ for glucuronide formation and intestine is the organ for its recycling (Figure 1B). The newly proposed HER concept delineates more clearly the disposition mechanism of phenolic drugs and phytochemicals such as flavonoids and polyphenols, which are significantly metabolized in the human intestine (up to ~70% or more) before entering the liver (Patrick et al., 2002; Yang et al., 2012; Strassburg et al., 1998; Kokawa et al., 2013; Sun et al., 2013; Kemp et al., 2002; Kosoglou et al., 2005; Teeter and Meyerhoff, 2002).

The pictorial representation of the newly proposed mechanism of hepatoenteric (HER) recycling and conventional mechanism of enterohepatic recycling/recirculation (EHR).

(A). HER starts from intestinal glucuronidation of orally dosed aglycones. The glucuronides are taken up into the hepatocytes via portal vein using hepatic uptake transporter OATPs, and then excreted back into bile by apical hepatic efflux transporters, allowing them to return to the small intestine. For biliary glucuronides, the gut microflora β-glucuronides (GUS) will hydrolyze them back into the aglycone form, which are then reabsorbed in colon to complete the recycling. (B) EHR starts from the hepatic metabolism of an aglycone entering liver (from intestinal absorption or blood circulation) into its phase II metabolites (mostly glucuronides). The glucuronides formed in the liver are excreted into bile by the apical hepatic efflux transporters,returned to the small intestine and then moved to colon, where the gut microflora GUS hydrolyze them back into the aglycone form. The aglycone is then re-absorbed from colon and reached the liver again to complete the EHR.

Results

Portal vein infusion

The portal vein infusion uses a direct method to assess the recirculation efficiency of a phenolic glucuronide by the liver using liver recycle efficiency% (LRE%, defined as steady-state biliary secretion rate divided by the hepatic infusion rate). Sixteen phenolic glucuronides, including four we studied previously (Zeng et al., 2016) and several of them with matching aglycones, were used to study this recirculating phenomenon.

Effects of Won-7-G concentrations on LRE%

The biliary secretion and blood concentrations of Won-7-G reached steady state after 1 hr of infusion (Figure 2A1 and A) and remained steady until 2.5 hr. With increased Won-7-G concentrations, both biliary secretion amount (Figure 2A1) and blood concentrations (Figure 2A2) increased. LRE%, calculated by Equation 1, were significantly lower (p<0.05) at higher infused concentration (Figure 2A3). LRE% were also plotted against steady state blood concentrations (Figure 2A4). It showed that at lower blood concentrations (<100 μM) of wogonoside, its biliary excretion rates increased linearly. At higher blood concentrations (>=100 μM), biliary excretion rates showed a saturation trend, indicative of transporter-mediated excretion (Figure 2A4).

Figure 2 with 3 supplements see all
Effect of the glucuronide concentrations, aglycone structures, and aglycone concentrations on the biliary secretion, systemic exposure and liver recycling efficiency (LRE%) in a rat portal vein infusion model.

Following portal vein infusion of Won-7-G at various concentrations (2–1000 μM), amounts of glucuronide excreted into bile (A1), concentrations of glucuronide in blood (A2) and LRE% (A3) were determined, and then the biliary secretion rates of glucuronides were plotted against their steady-state blood concentrations (A4). After the hepatic infusion of seven different aglycones [Won; Api; Bai; Lut; Ral; and Eze] and their corresponding glucuronides [Won-7-G; Api-7-G; Bai-7-G; Lut-3’-G; Ral-6-G; Ral-4’-G and Eze-4’-G] at 10 μM concentration, amount of the glucuronides secreted in bile during portal vein infusion of aglycones versus glucuronides were determined (B1–B7). Following the portal vein infusion of Won at 2 μM for the first 2.5 hr and at 100 μM for next 2 hr, the amount of Won-7-G secreted in bile (C1), the accumulated amount of Won-7-G secreted in bile (C2), and LRE% (C3) at low (2 μM) and high (100 μM) concentrations of Won were compared to study the effect of protein binding of Won on its liver uptake. The liver concentrations of Won and Won-7-G could be found in Appendix 2—table 2. 4. Four male Wistar rats were used in each experimental group. Statistical significance was calculated using student t test ('*', '**', and '***' indicates p<0.05, p<0.01, and p<0.001, respectively).

Comparison between aglycones and their corresponding glucuronides

Lower LRE% were found for six aglycone compounds (including four flavonoids, and two drugs raloxifene and ezetimibe, Figure 2B1–B7) when comparing to their corresponding glucuronides. All seven glucuronides (raloxifene has two glucuronides) showed a significantly higher (p<0.05) LRE% than their corresponding aglycones (Table 1, Figure 2B1–B7). Among the aglycones, wogonin had highest LRE% (41%) when recycled in the form of Won-7-G. Other aglycones showed even lower LRE% (<10%), also in the form of their glucuronides. The generally lower LRE% of aglycones in comparison with their glucuronides were not expected since glucuronides are highly hydrophilic. To rule out the possibility that slow aglycone uptake was the reason for lower LRE%, an uptake comparison between Won and Won-7-G in three cell lines were also conducted (Appendix 2—table 2 and Appendix 2—table 3). The results indicated that the uptake of Won was not influenced in the presence of specific transporter inhibitor, while Won-7-G was greatly inhibited, suggesting that aglycone uptake was mainly by passive diffusion, and often faster than their corresponding glucuronides.

Table 1
Comparison of bililary secretion rates and liver recycling efficiency (%) of glucuronides following hepatic glucuronide infusion, hepatic aglycone infusion, and small intestinal aglycone perfusion.

The rate of hepatic infusion was 20 nmol/hr and the rate of intestinal perfusion was 24 nmol/hr.

Dosing methodHepatic glucuronide infusionHepatic aglycone infusionSmall intestine aglycone perfusion
Infused compoundsEze-4'-GEzetimibe (Eze)Ezetimibe (Eze)
Won-7-GWongonin (Won)Wongonin (Won)
Ral-6-GRaloxifene (Ral)Raloxifene (Ral)
Api-7-GApigenin (Api)Apigenin (Api)
Bai-7-GBaicalein (Bai)Baicalein (Bai)
Measured compoundBile secretion rate(nmol/hr)
Eze-4'-G19.31 ± 1.852.93 ± 0.41***,†21.94 ± 5.29
Won-7-G16.30 ± 4.288.46 ± 3.93***,†17.90 ± 11.96
Ral-6-G10.53 ± 1.513.91 ± 0.82***4.00 ± 0.68
Api-7-G10.64 ± 4.491.22 ± 0.76***,†11.32 ± 4.08
Bai-7-G0.69 ± 0.290.09 ± 0.07***,†ND
Measured compoundLRE(%)
Eze-4'-G96.54 ± 9.2314.65 ± 0.02***,†91.42 ± 22.04
Won-7-G81.5 ± 21.4142.30 ± 0.20***,†74.58 ± 49.83
Ral-6-G52.64 ± 7.5419.55 ± 0.04***,†16.67 ± 2.83
Api-7-G53.21 ± 22.446.10 ± 0.04***,†47.17 ± 17.00
Bai-7-G3.43 ± 1.460.45 ± 0.35***,†ND
  1. *Significant difference between hepatic glucuronide infusion and hepatic aglycone infusion, p<0.01.

    Significant difference between hepatic aglycone infusion and small intestinal perfusion , p<0.01.

  2. Not determined due to below quantification limit.

Effects of protein binding

It was reported that the protein binding of Won could be as high as 90% (Talbi et al., 2014); therefore, it is possible that lower LRE% values associated with aglycones was due to extensive binding to plasma protein (i.e. free aglycones were not readily available for liver uptake). To confirm this, we used higher concentration of aglycone (Won) that could saturate the plasma protein and more unbound aglycones could be available for hepatic uptake. However, the results indicated that LRE% at a higher concentration (100 μM) was significantly lower (p<0.001) than at a lower concentration (2 μM) (Figure 2C3), ruling out the impact of extensive protein binding on the lower LRE% of aglycones. Liver tissue concentrations of wogonin was at least five times higher than won-7-G concentrations (Appendix 2—table 4), which indicated that the slow metabolism in liver was the likely reason for the lower LRE%.

Effect of phenolic and phenolic glucuronide structures on LRE%

We determined the LRE% of 16 phenolic-O-glucuronides, derived from dietary phenolics and phenolic drugs (Appendix 2—table 5). The results showed that LRE% was highly variable, ranging from 95% to 5%. In a study of nine 7-O-glucuronides of flavonoids, we were able to show that glucuronidation at 7-position alone is not sufficient to determine if a glucuronide will have high LRE%, suggesting that the aglycone structures are important determinant of its LRE%. This is rather interesting since there is only small structural differences between these flavonoid aglycones. On the other hand, glucuronides of drug molecules showed similarly large difference in their LRE%, and their structures are quite different.

Effect of sex differences on glucuronide LRE%

We are interested in determining the effects of sex difference on the OATP liver expression levels, because the expression level of Oatp in rat liver was reported to be significantly higher in males than in females (Hou et al., 2014). Also, E2G is a classical OATP substrate that has a drastically higher blood concentrations in females than males. The results indicated that there was not sex related differences in the LRE% of Won-7-G and Lut-3’-G. (Appendix 2—table 6). Interestingly, the LRE% of Bai-7-G increased from ~4% (male rats) to ~10% (female rats) (p<0.05).

Effect of cassette dosing on glucuronide LRE%

To rapidly evaluate LRE% of glucuronides, we determined if cassette dosing of several glucuronides impacted their excretion when they were infused together. In order to avoid any potential competitive inhibition among glucuronides, we choose to infuse them at low concentration (10 μM). We found that when given at a low concentration (10 μM), cassette dosing did not significantly impact LRE% (Figure 2—figure supplement 1A to D). The results indicated that the hepatic recirculating system is capable of handling multiple glucuronides simultaneously at 10 μM concentration. This is because compounds infused into the portal vein were immediately diluted by ~200 folds therefore not expected to interact with each other (Davies and Morris, 1993).

Cellular uptake with OATP overexpressing cells

We chose to focus on hepatic OATP transporters, because OATPs mediated the uptake of hormone glucuronides such as E2G (Lin et al., 2012). In addition, our earlier study had used glucuronides of isoflavones, which are phytoestrogens (similar to estrogens). OATP 1B1/1B3/2B1 overexpressing cells are used because they are expressed on the basolateral side (sinusoidal) membrane of hepatocytes.

Structural effects on glucuronide uptake in OATP 1B1/1B3/2B1 cells

We found that uptake by three OATP cells varied greatly (p<.005) between glucuronides (Figure 3A). In addition, for a particular phenolic glucuronide, their uptake was also significantly different (p<.005) between three OATP-expressing cells. Moreover, most glucuronides with high LRE% was taken up rapidly in these cells. However, Lut-3’-G were found to have low LRE% (Figure 2B4) even though it was rapidly taken up by the OATP1B1 cells. The discrepancy can be explained by the fact that Lut-3’-G could be further metabolized into di-glucuronides of Lut in vivo (Kemp et al., 2002). A liver microsome-mediated metabolism of Lut-3’-G confirmed the formation of Lut di-glucuronides by LC-MS (Figure 2—figure supplement 2A and B). Moreover, the peaks of Lut-3’-G and Lut-di-G both decreased upon incubation with β-glucuronidases (Figure 2—figure supplement 2C).

The OATP uptake kinetics, and the effect of glucuronide structures and uptake inhibitors on the hepatic uptake of glucuronides by OATP1B1/1B3/2B1 in over-expressed cell lines.

Intracellular concentration of glucuronides obtained using 1 μM of 10 different glucuronides [Won-7-G; E2G; Bai-7-G; Que-3-G; Scu-7-G; Api-7-G; Lut-7-G; Lut-3’-G; Eze-4’-G; and E2S] (A) was determined (10 μM was used in OATP 2B1 but results were normalized to 1 μM). Uptake kinetics of Won-7-G (B1), Api-7-G (B2), Lut-3’-G (B3) and Eze-4’-G (B4) in the concentration range of 0.5–50 µM in OATP1B1/1B3/2B1 over-expressed cell lines were determined. Km and Vmax values were calculated using Michaelis-Menten kinetics and summarized in Appendix 2—table 7. Effect of OATP inhibitors (50 μM rifampicin as OATP1B1/1B3 inhibitor and 50 μM erlotinib as OATP2B1 inhibitor) on the cellular uptake of five different glucuronides (E2G for OATP1B1/1B3 and E1S for OATP2B1 as positive controls) in OATP1B1 (C1), OATP1B3 (C2), and OATP2B1 (C3) over-expressed cell lines was studied at 10 μM substrate concentration (Figure 3C1–C3). Cross-over uptake in OATP1B1 cells using 1 μM of Won-7-G, E2G, Lut-3’-G as substrates was studied and intracellular concentrations of the glucuronides were determined in absence and presence of 25 μM of other aglycones and glucuronides as inhibitors. Each experiment was run in triplicate using substrates solutions in HBSS buffer (pH 7.4) at 37°C and the incubation lasted for 20 min. Statistical significance was calculated using student t test in Figure 3B1–B3 and one-way ANOVA in Figure 3A and C1–C ('*', '**', and '***' indicates p<0.05, p<0.01, and p<0.001, respectively).

Concentration effects on glucuronide uptake

We determined the Km and Vmax values of four phenolic glucuronides (Figure 2B1–2B4 and Table 1), representative of glucuronides with high (Won-7-G and Eze-4’-G), medium (Api-7-G), or low (Lut-3’-G) LRE%. We found that these glucuronides have good affinity to OATP1B1, 1B3, or 2B1 with Km values in the several micro molar range (Appendix 2—table 7), well within their in vivo exposure levels reported in the literature (Mo et al., 2019; Wang et al., 2019).

Effects of OATP inhibitors on glucuronide uptake

Rifampicin (25 μM) was shown to be potent inhibitor (>90% inhibition) of the uptake of OATP1B1 and OATP 1B3 substrate E2G, Won-7-G, Lut-3’-G (all at 1 μM) (Figure 3C1–C3), consistent with literature-reports that rifampicin shown strong pharmacokinetic interactions with atorvastatin and provastatin (classical OATPs substrate) in healthy volunteers (Kashani et al., 2009; Riveros et al., 2009). In addition to rifampicin, telmisartan was also shown to be a potent inhibitor (>90%) of the uptake of OATP 2B1 substrate E1S and Lut-3’-G (Figure 3C3). The results indicated that the uptake of these glucuronides was inhibited by specific OATP inhibitors. It supported our hypothesis that the uptake of these glucuronides was via a transporter-mediated process.

Cross-inhibition of uptake between glucuronides and aglycones

To establish a fast screening criterion that determines whether a phenolic compound was likely to undergo HER as an OATP substrate, we performed the uptake cross-inhibition experiments using several known substrates and non-substrates of OATP1B1. The results showed that if a compound was not an inhibitor of prototypical OATP substrate, it was not a substrate of that OATP (Figure 3D1–D3), but the reverse was not true. In contrast, substrates of OATP1B1 were able to significantly inhibited (p<.005) the uptake of glucuronides (Won-7-G, E2G, and Lut-3’-G) that are good substrates of a specific OATP. Interestingly, aglycones were better inhibitors than their corresponding glucuronides, even though the corresponding glucuronides had higher LRE%.

Correlation of LRE% and cell uptake

A total of 66 combinations with different OATP weightings were applied in Fisher exact test (Appendix 4—table 1). OATP 1B1 is the most abundant OATP expressed in livers (Appendix 2—table 1; Gårdmark et al., 1993) and also considered the most important among all OATPs. OATP 1B3 is grouped with OATP1B1 in both localization and substrates, but the contribution of OATP 1B3 is considered to be always smaller than OATP 1B1. Therefore, combinations which assigned the highest weightings to OATP1B3 were considered not physiologically relevant and eliminated from the results. Of all the results from the Fisher exact test (Appendix 4—table 2), 22 combinations were eliminated based on this criterion. Of the remaining 44 combinations, 41 out of 44 results (93.2%) showed statistical significance. Taken together, these results demonstrated there were solid correlation between cellular uptake and LRE%. Thus, we showed that cellular uptake of glucuronides is the rate-limiting step in HER. In addition, this correlation (Figure 4) could be mathematically described by an Emax model (Emax86 ± 17%) with an EC50 value of 42 ± 33 nM.

Figure 4 with 3 supplements see all
Correlation of liver recycling efficiency (LRE%) and intracellular concentration of glucuronides.

LRE% of 16 glucuronides calculated based on rat portal vein infusion experiment were plotted against the intracellular concentrations calculated as the sum of the individual measured intracellular concentrations in OATP1B1, 1B3, and 2B1 cells in the OATP uptake studies, weighted by their protein expression levels in human liver using Emax model. The Emax and EC50 parameter values were estimated and summarized in the table below the graph.

Impact of LRE% on the pharmacokinetic properties of phenolic compounds

The pharmacokinetic profiles of Won-7-G, Bai-7-G, Ace-G, and Eze-4’-G (Figure 2—figure supplement 3A to 3D) were fitted to a two-compartment model. Cmax values ranged from 41 nM (Eze-4’-G) to 572 nM (Ace-G), Tmax values ranged from ~2 hr (Ace-G) to ~8 hr (Eze-4’-G), and the half-lives ranged from 4.2 hr (Ace-G) to 52 hr (Won-7-G) (Figure 2—figure supplement 1). The elimination half-life was plotted against LRE% (Figure 4; Figure 4—figure supplement 1). Compound with a higher LRE% displayed a longer elimination half-life, which is expected since recycling will inevitably lead to a longer exposure.

Biliary secretion of five phenolic glucuronides in the intestine perfusion versus portal vein infusion model

The results indicated that the biliary excretion rates of five phenolic glucuronides were similar when five aglycones (Wog, Bai, Api, Eze, Ral) were given using the small intestine aglycone perfusion or when their corresponding glucuronides were given via direct portal vein infusion. Moreover, these rates were significantly higher than those observed with direct hepatic infusion of aglycones (Table 1), except for Ral. In intestinal perfusion study, the hepatic portal vein blood concentrations of glucuronides are also significantly higher (p<0.01) than tail vein blood (Figure 5). These results indicated that small intestine serves as a major metabolism organ for most of the perfused aglycones. Ral behaved differently in this study because Ral-6-G are formed both in liver and small intestine. The formation of Ral-6-G in liver is also fast. Taken together, the biliary excretion for most of phenolic glucuronides generated in small intestine was more efficient than direct portal vein administration of aglycones for four out of five phenolics.

Differences in the portal vein and systemic concentrations of glucuronides after the intestinal perfusion of aglycone.

five aglycones [Eze; Won; Ral; Api and Bai] were perfused in the rat small intestine (duodenum and jejunum, approximate length = 15 cm) individually at the rate of 24 nmol/hr for 2.5 hr in male Wistar rats (n = 4 per experimental group). Bile and intestinal perfusate samples were collected for every 30 min. Blood samples from tail vein and portal vein were collected at the end of the study and analyzed for the concentration of respective glucuronides. The absorption percentage of each aglycone were calculated and tabulated below the graph in the figure. The biliary secretion of the glucuronides was summarized in Table 1. Statistical significance was calculated using student t-test ('*', '**', and '***' indicates p<0.05, p<0.01, and p<0.001, respectively).

Effects of OATP inhibition on LRE% of phenolic glucuronides

In the presence of OATP transporter inhibitors (rifampicin, telmisartan, E2G and E1S at 1000 μM each with 1 hr pretreatment as well), the LRE% value of Won-7-G decreased from over ~80% to~40% and that of Lut-3’-G from ~7% to ~3% (Figure 6A–D). The results indicated that OATP transporters played the major role in the hepatic recycling of Won-7-G and Lut-3’-G, which is consistent with the results in the cell uptake study. According to the Fisher exact test, we claimed that hepatic uptake is the rate-limiting step in the recycling of these glucuronides. However, since we could not inhibit the LRE% by more than 80%, other uptake transporters could also participate in the recirculation of the glucuronides.

Effect of hepatic uptake inhibitors on the biliary excretion of glucuronides.

A combination of four hepatic uptake inhibitors (rifampicin, telmisartan, estradiol-17β-glucuronide and estrone-3-sulfate) at 1 mM concentration were infused in rat portal vein for an hour as pretreatment, followed by co-infusion of inhibitors (at 1 mM) and glucuronide substrate [Won-7-G or Lut-3’-G] (at 10 µM) in the rat portal vein at the rate of 2 ml/hr for 2.5 hr in male Wistar rats (n = 4 per group). The bile amounts of Won-7-G (A), Lut-3’-G (B) and the accumulated bile amounts of Won-7-G (C), Lut-3’-G (D) with or without the uptake inhibitors were determined. Statistical significance was calculated by student t test (‘**” indicates p<0.01).

Blood stability test of Won-7-G, Api-7-G, and Bai-7-G

After 2.5 hr of incubation, the relative stability of Won-7-G, Api-7-G, and Bai-7-G remained over 80% in 2 μM, 10 μM, and 25 μM (Figure 4—figure supplement 2). No significant changes were observed compared to the 0 hr time point. The results indicated that these glucuronide remained stable for the duration of the experiment time.

Discussion

We have defined a new disposition process as ‘Hepatoenteric Recycling (HER)’ for phenolic drugs and phytochemicals (e.g. flavonoids) as well as their glucuronides (Figure 1). HER (Figure 1A) differs significantly from EHR (Figure 1B) associated with bile acids and certain drugs, in that intestine is the main metabolite forming organ and liver is the recycling organ. Previously, EHR of bile acids and various drugs (Nielsen et al., 2009) is associated with metabolites that are generated in liver and recycled in intestine. This new definition for the first-pass metabolism of phenolic drugs and phytochemicals pinpoints where the metabolites are formed and subsequently recycled. In other words, the roles of liver and intestine are reversed in the new HER vs the classical EHR. The word ‘Recycling’ refers to the fact that many extrahepatically generated phenolic glucuronides were able to achieve hepatic recycling in HER. The word ‘Recycling’ is also important in HER because biliary excreted glucuronides are not well absorbed in the intestine. They must be reactivated to aglycones, by the microflora β-glucuronidases, before reabsorption can occur (Figure 1A).

The new definition is needed because: (1) unlike EHR of bile acids, intestine, not liver, is the source of the recycled metabolites (i.e. phenolic glucuronides); (2) unlike EHR of bile acids, liver, not the intestine, is the recycling organ for glucuronides; and (3) unlike EHR of bile acids, hepatic (e.g. OATPs) but not intestinal uptake transporters (e.g. ASBT) appears to be the rate-limiting step in determining the LRE% in HER. Due to the presence of HER, hepatic transporters (e.g. OATPs) will often determine the plasma concentrations of glucuronides, and play a much more important role than the relevant hepatic enzymes (e.g. UGTs) in determining how much glucuronides are excreted into the bile and participate in recycling. For orally administered dietary phenolics such as wogonin and drugs such as ezetimibe with high LRE%, double peaks caused by HER were observed in their plasma profiles (Figure 4—figure supplement 3A to 3D) even though biliary metabolites are not generated by the liver. For phenolics with more moderate LRE%, the role of metabolite generating organ or recycling organ is not mutually exclusive in HER vs EHR, in that biliary glucuronides may come from metabolites generated in the liver as well as the intestine, with variable contributions depending on the structure of aglycones.

How significant is this new disposition process called HER in determining how phenolics and their glucuronides are handled in vivo? We believe that HER is critically important in determining an orally administered phenolic’s pharmacokinetic profile with extensive intestinal metabolism. A high LRE% means that a phenolic could have double peaks in their plasma profile and a longer terminal half-life. This could mean high plasma levels of aglycone if glucuronides are readily hydrolyzed back into aglycones (Zhang et al., 2010). In other words, for extrahepatically generated glucuronides, HER is critically important because almost every glucuronides we tested were more likely to be excreted into bile if they are given as glucuronides vs corresponding aglycones into the portal vein (Figure 2B1–B7). If HER is critical, how important is EHR in the recycling process? We believe that EHR is not that important here for compounds with high LRE%, since these compounds were nearly fully (>80%) metabolized after the aglycones were absorbed in small intestine (Figure 5). Therefore, we believe that HER is much more important than EHR for orally administered phenolics whose major metabolic pathway is rapid intestinal glucuronidation. The importance of HER can be further manifested in disease conditions. For example, in chemically (e.g. trinitrobenzene sulfonic acid) induced colitis model, small intestinal UGTs were significantly downregulated but hepatic UGTs were not (Zhou et al., 2013). These differences in UGTs regulation indicated that HER would likely to be impacted, whereas EHR might not, since changes in metabolism only occurs in the intestine. On the other hand, in hepatitis C, where hepatic CYPs, OATPs, OCTs (Nakai et al., 2008) and UGTs (Smolders et al., 2016) were all downregulated, EHR might be affected more than HER. However, for compounds that were metabolized both in the intestine and liver, the situation might be more convoluted.

We have proposed the new HER mechanism to delineate more clearly the significance and the need to describe the recycling of phenolics using a terminology different from EHR. How would HER impact the beneficial effects of dietary phenolics, therapeutic efficacy of phenolic drugs, and toxicities of phenolic toxins that are taken orally, produced by microbiota or formed as the result of phase II metabolism in the liver? The possible answers are made up of two parts, and subsequently combined to argue for the importance of HER in determining the disposition of orally administered phenolics with mainly extrahepatic glucuronidation.

Firstly, HER will shuttle more phenolic compounds and their metabolites, both beneficial and toxic to the colon. This is because there is no known mechanism that can rapidly take up glucuronides in the small intestine. For compounds with beneficial effects, including therapeutic efficacy, this new disposition mechanism can be useful in preventing or treating diseases in colon. The prevention or treatment effects can be achieved via a direct effect (e.g. suppress inflammation) or indirect effect (e.g. production of beneficial microbial metabolites that suppress inflammation). In contrary, some of the toxic phenolic metabolites can be further biotransformed by the microbial enzymes to more potent toxins (Koppel et al., 2017). Taken together, the presence of HER for an oral phenolic could substantially enhance its colonic impact and maintenance of a healthy colon microbiota (i.e. a desirable colon microbiome homeostasis) that is essential for our health. Therefore, the use of phenolics to maintain and improve the health of microbiota is an important future research goal. On the other hand, HER could promote the recycling of toxic phenolic metabolites, enhancing its exposure and toxicities, and this should be limited to improve human health.

Second, it may provide the theoretical foundation to understand a myriad phenomenon when there are drug-drug interactions involving glucuronidation. For example, if we only recognize the importance of EHR, if would be difficult to understand why inhibition of hepatic UGTs does not always increase aglycone concentration and decrease glucuronide concentration in the blood. If we recognize that a phenolic is mainly processed by HER, an inhibitor that only inhibit liver UGTs is not expected to affect plasma concentrations of orally administered aglycone and its glucuronides as long as intestine is the metabolite forming organ. In contrast, the emerging role of OATPs means that the plasma concentrations of both an aglycone and its glucuronide (if both are substrates of OATPs) or just glucuronide (if only glucuronide is substrate of OATPs), could increase by a drug that did not affect intestinal or liver UGT enzymes. Furthermore, one can propose a variety of drug-drug interaction schemes that can be explained by both HER and EHR. Therefore, the proposed HER mechanism should help us determine the dominating mechanism by which drug-drug interactions occur to influence the plasma concentrations of a phenolic aglycone and its glucuronides that are formed in the intestine. This influence is more significant when the glucuronides are pharmacologically active (i.e. ezetimibe and dabigatran [Jia and Zhao, 2011; Jiang and Li, 2010]) or can inhibit CYP enzymes (i.e. gemfibrozil and clopidogrel [Sivapathasekaran et al., 2010; Mutwil et al., 2010]).

When combined together, the new HER definition could help predict the potential drug-drug interactions under pathological conditions. Since small intestinal UGTs play a significant role in the HER of the compounds, factors that alter the UGT expression in small intestine will significantly influence the recycling of the compounds and their in vivo exposure. Searching in Google Scholar by using key words combination ‘UGT + intestine + disease’, yielded a total of 14,500 hits. Among the top 50 results ranked by relevance, 15 of them reported different causes leading to change in intestinal UGTs including diseases (colitis, Gilbert’s disease etc.), genetic polymorphism and drug-drug interactions. The results indicated that pathological conditions will greatly influence the intestinal UGTs expression level and thus change the in vivo exposure of a phenolic whose disposition is controlled by a HER process. Similar conclusion could be made on hepatic OATPs, since OATPs mediate the hepatic uptake step of glucuronides generated from intestinal metabolism. Using ‘OATP +liver + disease’ as key words combination, a total of 14,000 hits were found and 21 out of top 50 results ranked by relevance reported the hepatic OATPs expression changes due to disease like cholestasis, liver hepatitis, and carcinoma.

We have obtained several lines of evidence in support of new HER definition, especially for oral phenolics such as wogonin and ezetimibe. First, our data showed that when infused directly into the portal vein, the vast majority of their glucuronides reached bile, displaying a highly efficient recycling (LRE%) (Appendix 2—table 5). For those compounds, portal vein concentrations of glucuronides were much higher than the corresponding aglycones in the rat intestinal perfusion studies (Figure 5). Second, direct portal vein infusion of aglycone was substantially less effective in producing biliary excretion of glucuronides than their corresponding glucuronides (Figure 2B1–B7). Third, hepatic OATPs took up these glucuronides in a saturable process that can be inhibited by OATP inhibitors (Figure 3B, Figure 3C1–C3). Fourth, the structural differences between phenolic-glucuronides had a substantial impact on the LRE%. Taken together, these results showed clearly that intestine is the glucuronide forming organ, and liver is the glucuronide recycling organ for these two compounds. Hence, the term HER properly describes the determining disposition processes of these two compounds, a naturally occurring polyphenol flavonoid and a prescription drug, which share minimal structural similarity. The latter suggest that HER could be broadly applicable to other phenolic phytochemicals and drugs.

The new HER disposition process could explain the pharmacokinetic behaviors of many phenolic drugs, including ezetimibe, raloxifene and diclofenac. All three drugs were rapidly glucuronidated by intestinal microsomes and had a double peak PK profiles (Watanabe et al., 1983; Herz and Bläsig, 1974). For diclofenac, extensive intestinal glucuronidation by microsomes (Bläsig et al., 1973) with >95% glucuronides formation (Herz and Bläsig, 1974), are consistent with it undergoing through HER instead of EHR. In addition, the recycle properties of ezetimibe reported in human (Christoffersen et al., 2015) was consistent with the proposed HER hypothesis. As over 80% of ezetimibe was converted into glucuronides in intestine (Christoffersen et al., 2015) with a second peak in its PK profile attributable to hepatic recycling. Therefore, for compounds with extensive intestinal glucuronidation and hepatic recycling, HER will fully explain their pharmacokinetic behaviors.

Although many glucuronides with rapid uptake by OATPs are correlated with higher LRE% (Figure 4), two glucuronides (i.e. Lut-G and Ica-G) (Wu et al., 2016; Wu et al., 2015) did not. We found that these two glucuronides were further metabolized in liver, forming di-glucuronides of Lut and di-glucuronides of Ica, respectively, rendering the original glucuronides unavailable for biliary excretion. Whereas, we have obtained large amount of evidence in support of roles of OATP uptake transporters in enabling HER (Figure 4), we could not rule out the contribution of other transporters. This is because we could only achieve a maximum of 50% inhibition of Won-7-G and Lut-3’-G LRE% in portal vein infusion experiment (Figure 6). It is likely that transporters belong to the OAT3 subfamily contributed to their liver uptake, since several flavonoid glucuronides were shown to be substrates of OAT3 (Rees et al., 2012), which is also expressed in the liver (Nema et al., 2011).

Lastly, we have significant but not overwhelming structural diversity in our phenolic glucuronides, and as such, we could not predict which structure will have high LRE%. The empirical evidence supports the hypothesis that good uptake of glucuronides by the OATPs will correlate with high LRE%, as long as the glucuronides taken up by the hepatocytes are not further metabolized. Because the direct portal vein infusion method is convenient to use with cassette dosing (Figure 2—figure supplement 1), HER potential can be readily estimated using OATP overexpressing cells and confirmed using the portal vein perfusion model. The latter should allow medicinal chemists to design new compounds with primarily intestinal glucuronidation that are tailored to treat intestinal, especially colonic diseases. In addition to the involvement of multiple uptake transporters, the role of efflux transporters may further complicate the disposition behaviors of phenolics and their glucuronides, but that is beyond the scope of this paper.

In conclusion, a new disposition terminology HER has been proposed to describe and delineate more clearly the disposition of dietary phenolics and phenolic drugs that are taken orally with intestine as their major metabolism (i.e. glucuronidation) organ and liver as their major recirculating organ. The new terminology more accurately captures the recycling of relevant phenolics, similar to the use of ‘EHR’ to capture the recycling of bile acids, where the liver is the metabolism organ and intestine as the recycling organ. This new HER more accurately captures the disposition of oral phenolics and can be used to better understand why this class of compounds may have larger than expected effects in the colon for human health and diseases. It may also help to delineate drug-drug interaction mechanisms involving intestinal UGT enzymes and hepatic transporters of glucuronides, a major challenge that we are still facing today because of all the complexity involved.

Materials and methods

Materials and reagents

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Apigenin-7-O-glucuronide (Api-7-G) was purchased from HWI Analytik GmbH (Rheinzaberner, Rülzheim, German). Wogonin-7-glucuronide or wogonoside (Won-7-G), quercetin (Que-3-G), scutellarin (Scu-7-G), luteolin-3’-glucuronide (Lue-3’-G), luteolin-7-glucuronide (Lue-7-G), wogonin (Won), luteolin (Lue), icaritin (Ica), icaritin-3-glucuronide (Ica-3-G), and icaritin-7-glucuronide (Ica-7-G) were purchased from Meilunebio (Dalian, China). Estradiol-17β-glucuronide (E2G) and estrone-3-sulfate (E1S) were purchased from Steraloids Company (RI,USA). Raloxifene (Ral), raloxifene-4’-glucuronide (Ral-4’-G), raloxifene-6-glucuronide (Ral-6-G), ezetimibe (Eze), and ezetimibe-4’-phenoxy-glucuronide (Eze-4’-G) were purchased from Toronto Research Chemical (Toronto, Canada). Apigenin (Api), baicalein (Bai) and baicalein-7-glucuronide (Bai-7-G) was purchased from Indofine (NJ, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (MO,USA). Acetonitrile (ACN) and methanol (MeOH) were purchased from Omni Solv (CA, US). ORA-Plus suspending vehicle was purchased from Perrigo (MI, USA). Hanks balanced salt solution (HBSS) was purchased from Sigma-Aldrich (MO, USA). All other chemicals were of reagent grade or better, which were purchased from reputable commercial suppliers.

Animals

Experiments were performed on adult male and female Wistar rats weighing 280–330 g (male) or 220–270 g (female) at the time of the experiment. The rats were fasted for approximately 16 hr before the experiment. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Houston.

OATP-overexpressing cells

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OATP1B1 and OATP1B3 over-expressed HEK-293 cells, described in a publication (Musshoff and Daldrup, 1993), were kindly provided by Dr. Yue Wei from University of Oklahoma Health Science Center. OATP2B1 over-expressed HEK-293 cell, first described in a publication (Gjerde et al., 1991), was kindly provided by Dr. Per Artursson’s lab from Uppsala University (Uppsala, Sweden). Cell line authentication was conducted by Core lab in MD Anderson. All cell lines have been compared with the STR profiles in CCLC database and no contamination detected.

Methods

Bio-synthesis of flavonoid glucuronides

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Selected flavonoid glucuronides were bio-synthesized using Hela-UGT1A9-MRP3 cells modified from the Hela-UGT1A9 cells developed in our lab (Dougherty et al., 1990). The concentration of glucuronide was quantified by UPLC using a previously published method (Pérez-Gandía et al., 2010).

Portal vein infusion

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The procedure of portal vein infusion was the same as our publication (Zeng et al., 2016). Briefly, rats were anesthetized by i.p. injection of 50% urethane at the dose of 1.875 g/kg. The portal vein and bile duct was catheterized after the anesthesia. The phenolic compounds or their corresponding glucuronides were prepared in HBSS buffer (pH = 7.4) and infused from portal vein catheterization at the rate of 2 ml/hr. Bile samples were collected from bile duct catheterization and blood samples were collected by snipping the tail. The infusion lasted for 2.5 hr and samples were collected every 0.5 hr. The liver recycling efficiency (LRE %) was calculated to evaluate the recycling efficiency using the following equation:

(1) LRE(Liverrecyclingefficiency)%=SteadystatebiliaryglucuronideexcretionratePortalveininfusionrate

The steady-state biliary glucuronide excretion rate was calculated by the linear regression analysis of accumulated amount excreted via bile vs. time curve.

This LRE%, derived from portal vein infusion experiments, was used to evaluate the effects of concentrations, inhibitors, structures of glucuronides on the liver recycling. Additional studies were used to determine the effects on LRE%, of aglycones vs glucuronides, protein binding, cassette dosing, and sex (male vs female).

Effect of infusion concentrations on the LRE% of glucuronide using Won-7-G
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Won-7-G was selected as a model compound. Won-7-G was prepared at 2, 10, 25, 100, and 1000 μM and infused into hepatic portal vein.

Comparison of LRE% between aglycones and their corresponding glucuronides
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Won, Lue, Api, Bai, Eze, and Ral and their glucuronides were selected and prepared at 10 μM concentration for the infusion experiments.

Effect of aglycone concentrations on the LRE% of their corresponding glucuronides
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The infusion study was conducted as described previously with minor modifications. After 2.5 hr of 2 μM Won infusion, the infusion solution was switched to 100 μM Won and infused for another 2 hr.

Effect of structures on the LRE% of glucuronides 
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Thirteen glucuronides: Won-7-G, Api-7-G, Bai-7-G, Lut-3’-G, Que-3-G, Scu-7-G, Ica-3’-G, Ica-7-G, Lue-7-G, Eze-4’-G, Ace-G, Ral-4’-G, and Ral-6-G, prepared at 10 μM concentration were used in the infusion experiment to determine the structure activity relation of glucuronides and their recycle ratio.

Effect of cassette dosing on the LRE% of glucuronides 
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A mixture of four glucuronides (Won-7-G, Api-7-G, Lut-3’-G, and Bai-7-G) were prepared at 10 μM together and infused simultaneously.

Effect of sex differences on the LRE% of glucuronides
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Won-7-G, Bai-7-G, and Lut-3’-G were prepared at 10 μM concentration. Prepared working solutions were further infused with female rats.

Cellular uptake

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Cellular uptake of phenolic glucuronides by OATP over-expressed cell lines
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Three OATP over-expressed cell lines (HEK 293 OATP 1B1/1B3/2B1 over-expressed cell lines) were used in the uptake study because OATPs are the most important hepatic uptake transporters for xenobiotics (Fahrmayr et al., 2010). Briefly, cells were seeded into 24 well plate 3 days before the experiment. Selected phenolic glucuronides were prepared in HBSS buffer (pH = 7.4) at required concentrations as working solution. Before incubation, cell culture medium was removed and cell was washed with 400 μl 37°C HBSS buffer (pH = 7.4) twice. Working solution was incubated with cells at 37°C for 20 min, when the intracellular concentrations reached steady state. After incubation, cells were washed with 400 μl ice-cold HBSS buffer (pH = 7.4) twice and cell pellet was collected in 200 μl of HBSS buffer. The cell pellet was further sonicated for 30 min. 150 μl of suspensions was collected and 150 μl of acetonitrile (contain 0.2 μM rutin as internal standard) was added into the pellet suspension. The suspension was centrifuged at 15,000 rpm for 15 min and supernatant was collected for analysis to measure the intracellular concentration of glucuronides.

Effect of specific inhibitors on the cell uptake 
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Fifty μM OATP inhibitors (rifampicin for OATP 1B1/1B3 and erlotinib or telmisartan for OATP 2B1) were prepared in working solutions. Working solutions without inhibitors were set as control group. Cells were incubated with their corresponding working solutions (containing different inhibitors) for 20 min.

Effect of substrate concentration on the cell uptake
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Selected phenolic glucuronides were prepared at 0.5, 1, 5, 10, 25, and 50 μM as working solutions. Cells were incubated with working solutions for 20 min. The Km and Vmax values were calculated by using the Michaelis-Menten equation.

Cross inhibition on OATP1B1 cells using glucuronides and their corresponding aglycones
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Several known substrates and non-substrates of OATP1B1 was applied as an inhibitor to inhibit the uptake of three selected substrates (Won-7-G, E2G, and Lut-3’-G). Substrate concentration was 1 μM while inhibitor concentration was 25 μM. Cells incubated with substrate without inhibitor were set as control group (100% uptake). Relative uptake percentage was calculated by comparing intracellular concentrations of all experiment groups with the control group.

Effects of OATP inhibition on the hepatic recycling of glucuronides

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A cocktail consists of 1 mM rifampicin (OATP 1B1/1B3 inhibitor), 1 mM telmisartan (OATP 2B1 inhibitor), 1 mM E2G (OATP 1B1/1B3 substrate), and 1 mM E1S (OATP 2B1 substrate) were prepare in HBSS buffer (pH = 7.4) and used in the experiment. The experiment procedure was the same as we described earlier with minor modifications. To achieve a better inhibition effect, rats were infused with the inhibitor cocktail for 1 hr before the beginning of glucuronides infusion. After 1 hr of treatment, selected phenolic glucuronides were infused with inhibitor cocktail for 2.5 hr.

Small intestine perfusion

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To investigate the impact of intestinal metabolism on biliary excretion of phase II metabolites, small intestine perfusion study was performed as our previous publication with minor modifications (Jia et al., 2004). Briefly, one segment of small intestine was perfused. The perfusion solution started from the beginning segment duodenum and went through 15 cm length of down-stream intestine after the inlet segment. 2 μM of flavonoid aglycones (Won, Api, Bai) and phenolic drugs (Eze, Ral) were prepared together in HBSS buffer (pH = 7.4) as perfusion solution. The inlet cannulate was insulated and kept warm by a 37°C circulating water bath. The perfusion rate was set at 0.193 ml/min. The perfusion lasted for 2.5 hr. Bile samples and perfusate were collected. At the last time point, blood samples from tail vein and from hepatic portal vein were also collected and analyzed. The absorption (%) was calculated as below:

(2) Absorption (\% )=PerfusedamountofaglyconeamountofaglyconeinperfusatePerfusedamountofaglycone

Evaluation of LRE% on the pharmacokinetic profiles of selected phenolics

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To investigate whether the HER potentials have an impact on pharmacokinetic profiles of phenolic compounds, a pharmacokinetic experiment was conducted (n = 4). Briefly, Won and Bai were prepared together in oral suspension with ORA-Plus at the concentration of 50 mg/ml. Drugs were given to animals at the dose of 30 mg/kg. Eze/Ace were prepared and dosed the same way as described above for the other two flavonoids. Blood samples were taken at 0, 0.5, 1, 2, 4, 6, 8, and 24 hr after dosing. Samples were analyzed with LC/MS method after sample processing as detailed below.

LC/MS analysis of blood, bile, liver tissue, cell, and perfusate samples

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Blood, bile, liver tissue, perfusate, and cell samples were analyzed by LC/MS after sample process. Detailed sample process methods could be found in Supplement: Bioanalysis Method.

Blood stability test of Won-7-G, Api-7-G, and Bai-7-G

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The stability of Won-7-G, Api-7-G, and Bai-7-G in blood matrix were evaluated. The stability test was conducted for 2.5 hr at three concentrations (2, 10, and 25 μM, n = 3).

Correlation between LRE% and cell uptake

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The LRE% obtained from rat infusion experiment were correlated with cell uptake results. Since cell studies were conducted using human OATPs over-expressed cell lines, the analysis was performed with Fisher exact test (Blevins and McDonald, 1985), the details of which is in Supplement: Fisher Exact Test of Correlation Analysis.

An Emax model were used to describe the correlation since LRE% has a maximum of 100% with the LRE% and intracellular concentrations to describe the correlation. The relative expression level close to human liver OATP isoform expression level (Appendix 2—table 1) was applied in the calculation of intracellular concentrations. The elimination half-life of Won/Bai and Eze/Ace were calculated using WinNolin and plotted against their LRE% value.

Appendix 1

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugApigenin-7-O-glucuronide (Api-7-G)HWI Analytik GmbHLot#: 0449059
Chemical compound, drugWogonoside (Won-7-G)Meilun bioMB6662
Chemical compound, drugQuercetin (Que-3-G)Sigma-Aldrich00310590
Chemical compound, drugScutellarin (Scu-7-G)MeilunebioMB7004-S
Chemical compound, drugLuteolin-3’-glucuronide (Lue-3’-G)Chengdu Alfa Biotechnology Co.,Ltd.AF8025306
Chemical compound, drugLuteolin-7-glucuronide (Lue-7-G)Chengdu Alfa Biotechnology Co.,Ltd.Af7022398
Chemical compound, drugWogonin (Won)MeilunebioMB6663
Chemical compound, drugLuteolin (Lue)MeilunebioMB6799
Chemical compound, drugIcaritin (Ica)MeilunebioMB7035
Chemical compound, drugIcaritin-3-glucuronide (Ica-3-G)This compound was synthesized by our cooperative lab.
Chemical compound, drugIcaritin-7-glucuronide (Ica-7-G)This compound was synthesized by our cooperative lab.
Chemical compound, drugEstradiol-17β-glucuronide (E2G)Steraloids CompanyE1073-000
Chemical compound, drugEstrone-3-sulfate (E1S)Steraloids CompanyE2335-000
Chemical compound, drugRaloxifene (Ral)Toronto Research ChemicalR099995
Chemical compound, drugRaloxifene-4’-glucuronide (Ral-4’-G)Toronto Research ChemicalR100020
Chemical compound, drugRaloxifene-6-glucuronide (Ral-6-G)Toronto Research ChemicalR100025
Chemical compound, drugEzetimibe (Eze)Toronto Research ChemicalE975000
Chemical compound, drugEzetimibe-4’-phenoxy-glucuronide (Eze-4’-G)Toronto Research ChemicalE975030
Chemical compound, drugApigenin (Api)IndofineA-002
Chemical compound, drugBaicalein (Bai)IndofineB-101
Chemical compound, drugBaicalein-7-glucuronide (Bai-7-G)Indofine06–012
Chemical compound, drugDimethyl sulfoxide (DMSO)Sigma-Aldrich276855
Chemical compound, drugHanks balanced salt solution (HBSS)Sigma-AldrichH1387
Chemical compound, drugAcetonitrile (ACN)Omni SolvAX0149
Chemical compound, drugmethanol (MeOH)Omni SolvMX0486
Chemical compound, drugORA-Plus suspending vehiclePerrigo0574-0303-16
Cell line (Homo-sapiens)HEK-293 OATP1B1/1B3 over-expressed cell lineDr.Yue Wei’s LabThe related publication could be searched by using PMID:29538325
Cell line (Homo-sapiens)HEK-293 OATP2B1 over-expressed cell lineDr. Per Artursson’s LabThe related publication could be searched by using
PMID:24799396
Software, algorithmGraphPad Prism 6GraphPad Software IncRRID:SCR_002798

Reagents

Chemicals and reagents used in this manuscript were summarized below. Detailed supplier information could also be found in this section.

Materials and reagents

Apigenin-7-O-glucuronide (Api-7-G) was purchased from HWI Analytik GmbH (Rheinzaberner, Rülzheim, German). Wogonin-7-glucuronide or wogonoside (Won-7-G), quercetin (Que-3-G), scutellarin (Scu-7-G), luteolin-3’-glucuronide (Lue-3’-G), luteolin-7-glucuronide (Lue-7-G), wogonin (Won), luteolin (Lue), icaritin (Ica), icaritin-3-glucuronide (Ica-3-G), and icaritin-7-glucuronide (Ica-7-G) were purchased from Meilunebio (Dalian, China). Estradiol-17β-glucuronide (E2G) and estrone-3-sulfate (E1S) were purchased from Steraloids Company (RI,USA). Raloxifene (Ral), raloxifene-4’-glucuronide (Ral-4’-G), raloxifene-6-glucuronide (Ral-6-G), ezetimibe (Eze), and ezetimibe-4’-phenoxy-glucuronide (Eze-4’-G) were purchased from Toronto Research Chemical (Toronto, Canada). Apigenin (Api), baicalein (Bai) and baicalein-7-glucuronide (Bai-7-G) were purchased from Indofine (NJ, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (MO,USA). Acetonitrile (ACN) and methanol (MeOH) were purchased from Omni Solv (CA, US). ORA-Plus suspending vehicle was purchased from Perrigo (MI, USA). Hanks balanced salt solution (HBSS) was purchased from Sigma-Aldrich (MO, USA). All other chemicals were of reagent grade or better, which were purchased from reputable commercial suppliers.

Appendix 2

Methods

Detailed descriptions of experiment design and operation were summarized in this section. All the experiments including animal infusion/perfusion experiment, cell uptake experiment and PK experiments were summarized below.

1. Portal vein infusion

Portal vein infusion 

The procedure of portal vein infusion was the same as those described previously (Zeng et al., 2016). Briefly, rats were anesthetized by i.p. injection of 50% urethane at the dose of 1.875 g/kg. The portal vein and bile duct were catheterized after the anesthesia. The phenolic compounds or their corresponding glucuronides were prepared in HBSS buffer (pH = 7.4) and infused from portal vein catheterization at the rate of 2 ml/hr. Bile samples were collected from bile duct catheterization and blood samples were collected by snipping the tail. The infusion lasted for 2.5 hr and samples were collected every 0.5 hr. The liver recycling efficiency (LRE %) was calculated to evaluate the recycling efficiency using the following equation:

(A2-1) LRE(Liverrecyclingefficiency)%=SteadystatebiliaryglucuronideexcretionratePortalveininfusionrate

The glucuronide secretion rate at the steady state was calculated by the linear regression of accumulated amount excreted in bile vs. time curve.

Effect of infusion concentrations on the LRE% of glucuronide using Won-7-G

Won-7-G was selected as a model compound. Won-7-G was prepared at 2, 10, 25, 100, and 1000 μM and infused into hepatic portal vein.

Comparison of LRE% between aglycones and their corresponding glucuronides

Won, Lue, Api, Bai, Eze, and Ral and their glucuronides were selected and prepared at 10 μM concentration for the infusion experiments.

Effect of aglycone concentrations on the LRE% of their corresponding glucuronides

The infusion study was conducted as described previously with minor modifications. After 2.5 hr of 2 μM Won infusion, the infusion solution was switched to 100 μM Won and infused for another 2 hr.

Effect of structures on the LRER% of glucuronides

Thirteen glucuronides: Won-7-G, Api-7-G, Bai-7-G, Lut-3’-G, Que-3-G, Scu-7-G, Ica-3’-G, Ica-7-G, Lue-7-G, Eze-4’-G, Ace-G, Ral-4’-G, and Ral-6-G, prepared at 10 μM concentration were used in the infusion experiment to determine the structure activity relation of glucuronides and their liver recycling efficiency.

Effect of cassette dosing on the LRER% of glucuronides

A mixture of 4 glucuronides (Won-7-G, Api-7-G, Lut-3’-G, and Bai-7-G) were prepared at 10 μM together and infused simultaneously.

Effect of gender differences on the LRE% of glucuronides

Won-7-G, Bai-7-G, and Lut-3’-G were prepared at 10 μM concentration. Prepared working solutions were further infused with female rats.

2. Cellular uptake

Cellular uptake of flavonoid glucuronides and their aglycones by OATP over-expressed cell lines

Three OATP over-expressed cell lines (HEK 293 OATP 1B1/1B3/2B1 over-expressed cell lines) were used in the uptake study because they are the most important hepatic uptake transporters for xenobiotics (Sun et al., 2013). Briefly, cells were seeded into 24-well plate 3 days before the experiment. Selected phenolic glucuronides were prepared in HBSS buffer (pH = 7.4) at required concentrations as working solution. Before incubation, cell culture medium was removed and cell was washed with 400 μl 37°C HBSS buffer (pH = 7.4) twice. Working solution was incubated with cells at 37°C for 20 min, when the intracellular concentrations reached steady state. After incubation, cells were washed with 400 μl ice-cold HBSS buffer (pH = 7.4) twice and cell pellet was collected in 200 μl of HBSS buffer. The cell pellet was further sonicated for 30 min. A total of 150 μl of suspensions was collected and 150 μl of acetonitrile (contain 0.2 μM rutin as internal standard) was added into the pellet suspension. The suspension was centrifuged at 15,000 rpm for 15 min and supernatant was collected for analysis to measure the intracellular concentration of glucuronides.

Effect of specific inhibitors on the cell uptake

Fifty μM OATP inhibitors (rifampicin for OATP 1B1/1B3 and erlotinib or telmisartan for OATP 2B1) were prepared in working solutions. Working solutions without inhibitors were set as control group. Cells were incubated with their corresponding working solutions (containing different inhibitors) for 20 min.

Effect of substrate concentration on the cell uptake

Selected phenolic glucuronides were prepared at 0.5, 1, 5, 10, 25, and 50 μM as working solutions. Cells were incubated with working solutions for 20 min. The Km and Vmax values were calculated by using the Michaelis-Menten equation.

Cross inhibition on OATP1B1 cells using glucuronides and their corresponding aglycones

Several known substrates and non-substrates of OATP1B1 was applied as an inhibitor to inhibit the uptake of three selected substrates (Won-7-G, E2G and Lut-3’-G). Substrate concentration was 1 μM while inhibitor concentration was 25 μM. Cells incubated with substrate without inhibitor were set as control group (100% uptake). Relative uptake percentage was calculated by comparing intracellular concentrations of all experiment groups with the control group.

3. Portal vein infusion inhibition

Determine the effect of OATP inhibition on the recycle of phenolic glucuronides

A cocktail consists of 1 mM rifampicin (OATP 1B1/1B3 inhibitor), 1 mM telmisartan (OATP 2B1 inhibitor), 1 mM E2G (OATP 1B1/1B3 substrate), and 1 mM E1S (OATP 2B1 substrate) were prepare in HBSS buffer (pH = 7.4) and used in the experiment. The experiment procedure was the same as we described earlier with minor modifications. To achieve a better inhibition effect, rats were infused with the inhibitor cocktail for 1 hr before the beginning of glucuronides infusion. After 1 hr of treatment, selected phenolic glucuronides were infused with inhibitor cocktail for 2.5 hr.

4. Small intestine perfusion

Small intestine perfusion

To investigate the impact of intestinal metabolism on biliary excretion of phase II metabolites, small intestine perfusion study was performed as our previous publication with minor modifications (Kemp et al., 2002). Briefly, one segment of small intestine was perfused. The perfusion solution started from the beginning segment duodenum and went through 15 cm length of down-stream intestine after the inlet segment. Two μM of flavonoid aglycones (Won, Api, Bai) and phenolic drugs (Eze, Ral) were prepared together in HBSS buffer (pH = 7.4) as perfusion solution. The inlet cannulate was insulated and kept warm by a 37°C circulating water bath. The perfusion rate was set at 0.193 ml/min. The perfusion lasted for 2.5 hr. Bile samples and perfusate were collected. At the last time point, blood samples from tail vein and from hepatic portal vein were also collected and analyzed. The absorption (%) was calculated as below:

(A2-2) Absorption (\% )=PerfusedamountofaglyconeamountofaglyconeinperfusatePerfusedamountofaglycone

5. Pharmacokinetic experiment

Evaluation of LRE% on the pharmacokinetic profiles of phenolic compounds

To investigate whether the HER potentials have an impact on pharmacokinetic profiles of phenolic compounds, a pharmacokinetic experiment was conducted (n = 4). Briefly, Won and Bai were prepared together in oral suspension with ORA-Plus at the concentration of 50 mg/ml. Drugs were given to animals at the dose of 30 mg/kg. Eze/Ace were prepared and dosed the same way as described above for the other two flavonoids. Blood samples were taken at 0, 0.5, 1, 2, 4, 6, 8, and 24 hr after dosing. Samples were analyzed with LC/MS method after sample processing as detailed below.

Appendix 2—table 1
The hepatic expression level of OATP 1B1/1B3/2B1.

Expression level was presented as average ± SD. Data was obtained from Pubmed Gene database.

TransporterOATP 1B1*OATP 1B1OATP 2B1
Expression (RPKM)119.3 ± 20.830.2 ± 3.944.0 ± 4.1
Relative expression (%)61.715.622.7
Appendix 2—table 2
The uptake of wogonin in four different cell lines.

The incubation concentration, time, condition and intracellular concentrations were summarized in the table. Inhibitor(s) were changed in different cell line. For OATP1B1 and OATP1B3 cell line, inhibitor was 50 μM rifampicin. For OATP 2B1 cell line, inhibitor was 50 μM telmisartan. For MDCK cell line, inhibitors were 50 μM rifampicin and 50 μM telmisartan. Student t test was applied to calculate the p values. No significant differences observed when wogonin was incubated with or without inhibitor.

Cell typeIncubation concentration(μM)Incubation time(min)InhibitorIntracellular concentration(nM)
MDCK MRP3 over-expressed cell line5120Yes150.33 ± 37.87
5120No163.67 ± 21.57
HEK-293 OATP1B1 over- expressed cell line1020Yes442.00 ± 309.56
1020No365.67 ± 168.67
HEK-293 OATP1B3 over- expressed cell line1060Yes333.33 ± 33.08
1060No381.33 ± 128.82
HEK-293 OATP2B1 over- expressed cell line1020Yes782.33 ± 115.42
1020No897.00 ± 409.17
Appendix 2—table 3
The uptake of wogonin and wogonin-7-G (wogonoside) in three different cell lines.

The incubation concentration, time, condition and intracellular concentrations were summarized iin the table. Inhibitor(s) were changed in different cell line. For OATP1B1 and OATP1B3 cell line, inhibitor was 50 μM rifampicin. For OATP 2B1 cell line, inhibitor was 50 μM telmisartan. Student t test was applied to calculate the p values. Significant differences were observed when wogonoside incubated with or without inhibitors in OATP 1B1 and 1B3 cell lines. No significant differences observed when wogonin was incubated with or without inhibitor.

Cell typeIncubation concentration (μM)Incubation time (min)InhibitorWogonoside intracellular concentration
(nM)
Wogonin intracellular concentration
(nM)
HEK-293 OATP1B1 over- expressed cell line1020Yes0.01 ± 0.01442.00 ± 309.56
1020No401.00 ± 29.60††365.67 ± 168.67
HEK-293 OATP1B3 over- expressed cell line1060Yes48.33 ± 9.12333.33 ± 33.08
1060No58.03 ± 16.60381.33 ± 128.82
HEK-293 OATP2B1 over- expressed cell line1020Yes34.57 ± 21.26782.33 ± 115.42
1020No82.87 ± 2.45*897.00 ± 409.17
  1. *p< 0.05.

    p<0.01.

Appendix 2—table 4
The liver concentration of wogonin/wogonin-7-G (n = 3).
CompoundWogoninWogonin-7-G (Wogonoside)
Liver concentration
(nmol/g)
0.51 ± 0.28<0.1*
Ratio (Wogonin/Wogonoside)5 >\
  1. * The concentration was below quantification limit (4 nM).

Appendix 2—table 5
Biliary glucuronide secretion rate and liver recycling efficiency of 16 different glucuronides.
Infused compound (10 μM)Secretion rate (nmol/hr)*LRE%†
Ezetimibe-4'-G19.31 ± 1.8596.5 ± 9.2
Wogonin-7-G(wogonoside)16.3 ± 4.2881.5 ± 21.4
Genestein-7-G9.78 ‡59.3 ‡
Raloxifene-4'-G11.48 ± 2.3257.4 ± 11.6
Apigenin-7-G10.64 ± 4.4953.2 ± 22.4
Raloxifene-6-G10.53 ± 1.5152.6 ± 7.5
Chrysin-7-G9.86 ‡49.3 ‡
Icaritin-7-G7.46 ± 0.7237.3 ± 3.6
Biochanin A-G3.28 ‡27.8 ‡
Icaritin-3-G5.35 ± 1.1226.8 ± 5.6
Scuttelarin4.34 ± 1.1821.7 ± 5.9
Luteolin-3’-G1.33 ± 0.346.7 ± 1.7
Baicalin0.69 ± 0.303.4 ± 1.5
Luteolin-7-glycoside0.67 ± 0.563.4 ± 2.8
Quercetin-3-G0.43 ± 0.572.2 ± 2.8
Ace-G0.10 ± 0.060.5 ± 0.3
  1. *The glucuronide secretion rate at the steady state was calculated by the linear regression of accumulated amount secreted in bile vs. time.

    † LRE (liver recycling efficiency) % = Excretion rate at steady state/infusion rate.

  2. ‡ Data from previous published study (Zeng et al., 2016).

Appendix 2—table 6
Gender differences in bile secretion rates and liver recycling efficiency (LRE%).
Infusion compounds
(10 μM)
Secretion rate (nmol/hr)LRE(%)
MaleFemaleMaleFemale
Won-7-G16.30 ± 4.2815.06 ± 0.7081.5 ± 21.475.3 ± 3.5
Bai-7-G0.69 ± 0.291.78 ± 0.423.4 ± 1.58.9 ± 2.1
Lut-3'-G1.33 ± 0.341.67 ± 0.326.7 ± 1.78.3 ± 1.6
Appendix 2—table 7
Kinetic parameters of uptake (Km and Vmax) of selected glucuronides wogonoside, luteolin-3’-glucuronide (Lut-3’-glu) and apigenin-7-glucuronide (Api-7-glu) in OATP1B1/1B3/2B1 over-expressed cells.
Compound1B11B32B1
Km(μM)Vmax
(pmol/min)
Km(μM)Vmax
(pmol/min)
Km(μM)Vmax
(pmol/min)
Won-7-G27.6853.37>5011.72>5055.26
Lut-3’-G4.4622.79>509.620.894.07
Api-7-G>50>200>50>2009.725.09
Eze-4'-G>5011.4517.641.678.551.61

Appendix 3

Bioanalysis methods

Methods applied in the analysis of biological samples were summarized in this section. The sample process methods and parameters of LC/MS methods were also presented below.

1.Sample process

Blood

 A volume of 10 μl blood was mixed with 10 μl of water and 200 μl of acetonitrile (ACN) (with 0.2 μM rutin as internal standard) was added into the mixture. After vortexing for 1 min, the mixture was centrifuged at 15,000 rpm for 15 min. The supernatant was dried under air at room temperature and reconstitute with 100 μl of 20% ACN for analysis. Standard samples of known concentrations were prepared in the same way.

Bile

A volume of 5 μl bile was mixed with 5 μl of water and further dilute into 1 ml of mixture (containing 0.1 μM rutin as internal standard). The mixture was further loaded on an OasisR HLB SPE cartridge and eluted by 1 ml of water, 3 ml of 40% methanol. One milliliter of MeOH was used as final elution fluid and sample was dried under air blowing at room temperature after elution. The residue was reconstituted with 200 μl 20% ACN for analysis.

Cells

Cell samples were processed by adding 150 μl ACN (containing 0.2 μM rutin as internal standard) into 150 μl of cell pellet. After centrifuge at 15,000 rpm for 15 min, the supernatant was collected for analysis.

Perfusate

A volume of 50 μl perfusate sample was mixed with 50 μl of water and 100 μl of ACN was (with 0.2 μM rutin as internal standard) added into the mixture. After vortex for 1 min, the mixture was centrifuged at 15,000 rpm for 15 min. 100 μl of supernatant was taken for analysis.

2. LC/MS methods

UPLC conditions

UPLC condition: system, Waters Acquity with diode array; column, Restek Ultra BPh (5 μm, 100 mm × 2.1 mm); mobile phase, A 0.1% formic acid in water, mobile phase B acetonitrile; gradient, 10% B (0–0.5 min), 10% B-34% B (0.5–1.0 min), 34% B-60% B (1.0–2.5 min), 60% B-95% B (2.5–6.0 min), 95% B-10% B (6.0–7.0 min); flow rate,0.45 ml/min; column temperature,45 °C; injection volume, 10 μL.

Mass spectrometry conditions

The MS analysis was performed on a Sciex 5500 triple quadrupole mass spectrometer (AB Sciex LLC, Framingham, MA) equipped with an ESI source. The detection was conducted using MRM scan type in positive ion mode. The instrument dependent parameters were: ion-spray voltage, 5.5 kV; ion source temperature, 400°C; nebulizer gas (gas 1), nitrogen, 20 psi; turbo gas (gas 2), nitrogen 20 psi; curtain gas, nitrogen 20 psi. Unit mass resolution was set in both mass-resolving quadruples Q1 and Q3. The compound dependent parameters were summarized in Appendix 3—table 1.

Appendix 3—table 1
Compound-dependent parameter of the analytes and I.S.
CompoundQ1/Q3DPCEEPCXP
Api-7-G445.0/269.0−90−34−10−23
Ace-G326.0/150.0−100−34−10−15
Bai-7-G445.0/269.0−120−30−10−19
E1S349.0/269.0−45−52−10−19
E2G447.0/271.0−170−40−10−19
Eze-4’-G584.0/271.0−154−42−10−15
Ica-3-G543.1/367.1−90−33−10−10
Ica-7-G543.1/352.0−90−53−10−10
Lut-3’-G461.2/285.2−87−29−10−15
Que-3-G477.0/301.0−110−32−10−11
Ral-4’-G649.3/473.3−80−44−10−13
Ral-6-G649.3/473.3−80−44−10−13
Scu-7-G461.0/285.0−70−34−10−13
Won-7-G459.0/283.0−87−21−10−15
Rutin (I.S.)609.0/300.0−87−54−10−21

Blood stability test

Blood stability test of Won-7-G, Api-7-G, and Bai-7-G

The stability of Won-7-G, Api-7-G, and Bai-7-G in blood matrix were evaluated by adding standard compounds into rat blood. Stock solutions of glucuronides were added into 1 ml of rat blood to reach the concentrations at 2, 10, and 25 μM (n = 3). Samples were further incubated in water bath at 37°C. Fifty μl of samples were collected every 0.5 hr. The incubation lasted for 2.5 hr. Samples collected at 0 hr point were set as control (100%, no degradation). Samples were further analyzed after processing as described above. Relative stability was calculated by comparing blood concentrations at each sampling time point to blood concentration at the ‘0 hr’ time point. The results of blood stability test could be found in Figure 2—figure supplement 3.

Appendix 4

Fisher exact test of correlation analysis

1.Intracellular concentration

The intracellular concentrations were calculated by adding the uptake results in 3 OATP isoforms (OATP1B1, 1B3 and 2B1) together with weightings. The intracellular concentrations were calculated as the equation presented below:

Total intracellular concentration =
X10*OATP1B1 concentration +Y10*OATP1B3 concentration +Z10* OATP2B1 concentration

Concentrations are intracellular concentration of phenolic glucuronide in 3 cell lines generated from cell uptake experiment. X, Y, Z are integers and are weightings of individual isoform, which follows the description below:

X + Y + Z = 10, 0 ≤ X ≤ 10, 0 ≤ Y ≤ 10, 0 ≤ Z ≤ 10.

Different X, Y, and Z numbers indicated different relative expression levels of individual OATP isoform. It also describes the contribution of specific isoform in the hepatic uptake of the substrates.

2. Fisher exact test

The liver recycling efficiency (LRE%) obtained from rat infusion experiment were correlated with the intracellular concentration obtained from cell uptake results. Since cell studies were conducted using human OATPs over-expressed cell lines, it was important to determine if there is a statistical correlation between these two parameters by using Fisher exact test (Kosoglou et al., 2005).

To conduct Fisher exact test, all compounds were classified into four groups by their LRE% (high and low) and intracellular concentrations (high and low). LRE% higher than 50% are considered ‘high recycling’ and concentrations higher than the average values are considered ‘high uptake’. The same definition was applied in ‘low recycling’ and ‘low uptake’.

The number of substrates that belongs to the classified groups (e.g. high recycle and high uptake, high recycle and low uptake) was counted. A 2*two table was generated from the classification. An example of the classification was presented below:

Then Fisher exact test was applied to test the statistically significant correlation of two parameters (liver recycling efficiency and intracellular concentration) influencing the distribution of the classified numbers. p<0.05 was considered of significant correlation. Different X, Y, Z weighting combinations were assigned and individual p values were calculated (Appendix 4—table 2).

OATP 1B1 is the most abundant uptake transporter expressed in liver (Appendix 2 table S4). It is also the most important uptake transporter due to its substrate spectrum. OATP 1B3 is grouped with OATP1B1 in both localization and substrates, but the contribution of OATP 1B31 is smaller than OATP 1B1. Therefore, combinations which assigned the highest weightings to OATP1B3 were considered not physiologically relevant and eliminated from the results.

Appendix 4—table 1
Example of fisher exact test generated by the methods described above.
Liver recycling efficiency (%)
HighLow
Intracellular concentrations (nM)High41
Low04
Appendix 4—table 2
The results of Fisher exact test in different weightings were summarized.

p Values of different OATP weightings were calculated and summarized in this table. p<0.05 indicated significant correlation found between recycle ratios and cell uptake. Results marked in red were eliminated.

WeightingpWeightingp
OATP1B1OATP1B3 OATP2B1valueOATP1B1OATP1B3 OATP2B1value
23 50.007971 20.0476
22 60.007970 30.0476
21 70.007982 00.0476
20 80.007980 20.0476
32 50.007981 10.0476
31 60.007991 00.0476
30 70.007990 10.0476
41 50.0079100 00.0476
40 60.0079010 00.1667
51 40.007909 10.1667
50 50.007908 20.1667
60 40.007907 30.1667
03 70.047606 40.1667
02 80.047605 50.1667
01 90.047604 60.1667
00 100.047619 00.1667
13 60.047618 10.1667
12 70.047617 20.1667
11 80.047616 30.1667
10 90.047615 40.1667
33 40.047614 50.1667
44 20.047628 00.1667
43 30.047627 10.1667
42 40.047626 20.1667
54 10.047625 30.1667
53 20.047624 40.1667
52 30.047637 00.1667
64 00.047636 10.1667
63 10.047635 20.1667
62 20.047634 30.1667
61 30.047646 00.1667
73 00.047645 10.1667
72 10.047655 00.1667

Appendix 5

Microsome incubation

1.Microsome incubation

40 µM L3’G is incubated with male F344 rat liver microsome (final concentration of 0.053 mg of protein per mL mixture) for 2 hr. The reaction is stopped by adding acetonitrile of same volume of the mixture. By analyzing the microsome samples with UPLC and a UV detector, a peak is observed at 1.79 min with similar bands in UV spectra (Figure 1.B1 and 1.B2). We presume the peak consists of one or more di-glucuronide(s) of luteolin. To confirm the peak is glucuronide metabolite(s) of L3’G, we performed hydrolysis study with beta-glucuronidase, which converts glucuronides into aglycones.

2.Hydrolysis study of microsome samples

Samples from microsome study are incubated with beta-glucuronidase (at a final concentration of 900 unit/mL) for 2 hr. Both the postulated di-glucuronide peak and L3’G peak disappeared while a luteolin peak appear at 3.02 min (Figure 2.C1 and 2 .C2).

To further investigate the hydrolysis process, a separate time-dependent hydrolysis study is performed. A final concentration of 180 unit/mL beta-glucuronidase is added in 100 µL microsome samples. The reaction is stopped at 5 min and 30 min. From the UPLC-UV results, the postulated di-glucuronide peak and L3’G peak both decrease with time, and the luteolin peak increases with time (Figure 2.A1 and 2.B1).

Based on the microsome incubation results, we confirm that luteolin 3’-glucuronide can be further metabolized in rat liver microsome.

Appendix 5—table 1
Peak area of postulated di-glucuronide of luteolin, L3’G, and luteolin from UPLC chromatograph (n = 2).
Retention time/minBefore hydrolysis5 min hydrolysis30 min hydrolysis
Postulated di-glucuronide1.792854326070.518922
L3'G2.6778905.56900642460.5
Luteolin3.02-1728966341
sum107448.5112365.5127723.5

Data availability

All datasets associated with the published data will be made freely and widely available in the most useful formats, and according to the relevant reporting standards. All data generated or analysed during this study are included in the manuscript and supporting files and has been uploaded into Dryad Digital Repository. The files included the original version of the figures and tables included in the manuscript, appendix and supplement documents.

The following data sets were generated

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Decision letter

  1. Arduino A Mangoni
    Reviewing Editor; Flinders Medical Centre, Australia
  2. Olga Boudker
    Senior Editor; Weill Cornell Medicine, United States
  3. Noelle S Williams
    Reviewer; University of Texas Southwestern Medical Center, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Tu and colleagues present an interesting body of work describing what they feel is a novel method of recycling of glucuronidated metabolites, specifically those of phenolic compounds. Rather than relying on the traditionally held pathway wherein a compound is metabolized in the liver and then recycled in the intestine after enzymes of the gut microbiota remove the glucuronide (enterohepatic recirculation/recycling – EHR), this mechanism involves glucuronidation of the compound in the small intestine, uptake of the glucuronidated metabolites into the liver by OATP1 transporters and then transport back out of the liver into the bile and subsequently into the small intestine where the glucuronide is recycled into its aglycone form by gut microbiota enzymes (hepatoenteric recycling -HER).

Decision letter after peer review:

Thank you for submitting your article "Hepatoenteric Recycling Is A New Disposition Mechanism for Orally Administered Phenolic Drugs and Phytochemicals" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Olga Boudker as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Noelle S Williams (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

Tu and colleagues present a body of work describing a novel method of recycling of glucuronidated metabolites, specifically those of phenolic compounds. Rather than relying on the traditionally held pathway wherein a compound is metabolized in the liver and then recycled in the intestine after enzymes of the gut microbiota remove the glucuronide (enterohepatic recirculation/recycling – EHR), this mechanism involves glucuronidation of the compound in the small intestine, uptake of the glucuronidated metabolites into the liver by OATP1 transporters and then transport back out of the liver into the bile and subsequently into the small intestine where the glucuronide is recycled into its aglycone form by gut microbiota enzymes (hepatoenteric recycling -HER). The authors argue that this has relevance for drug-drug interactions involving the OATP transporters and as a means to shuttle glucuronidated metabolites to the colon since the metabolism of phenolics by intestinal cells to glucuronidated forms as well as subsequent recycling means there should be large amounts of these metabolites in the colon.

Essential revisions:

– The introduction could be more directed to the general audience.

– In describing Figure 2A4, the authors claim that RR% is plotted against steady state blood concentration which "…displayed a saturating trend, indicative of transporter-mediated excretion." First, excretion rate not RR% is plotted and although these values tend to trend together the authors should be more precise in their description and better explain their rationale for making the claim that this indicates transport. It would also be helpful if there were more data in between 100 and 1000 uM.

– In Figure 2B1-7, the authors show decreased recycling of the glucuronide relative to the aglycone form by monitoring appearance of the glucuronide in the bile after hepatic infusion. While the difference is dramatic, it is hard to know the relative roles of metabolism of the aglycone to the glucuronide versus uptake and transport of the aglycone form. It might be helpful to evaluate levels of the aglycone and glucuronide inside the liver after this experiment.

– The authors say they are examining the role of protein binding in section 3.1.3 by evaluating a higher concentration of Won aglycone, making the assumption that this would saturate protein binding. This seems somewhat arbitrary and if they truly wanted to assess the role of saturable protein binding, should do a protein binding experiment. It seems it would have been better to directly evaluate uptake of the aglycone form as previously suggested and just eliminate this evaluation.

– In section 3.1.4, the authors evaluate different flavonoids to make a comment on the specific role of glucuronidation at the 7-position in RR%. This doesn't make sense. One should evaluate the same compound that is glucuronidated at multiple sites to evaluate the role of glucuronidation location – and correlate this with cell based studies with the transporters. It was also unclear why the 7 location was so relevant. The authors should elaborate.

– The experiments describing sex differences and cassette dosing on glucuronide RR% seems insufficient and unnecessary for this manuscript.

– In section 3.2.1 when discussing the outcome for Lut-3'-G it would be helpful if the authors could refer to the data for this point in figure 2B4 and to show the evidence for additional metabolism in supplementary data.

– In Section 3.2.4 where the authors make the claim that substrates of the transporters make better inhibitors, it would again be helpful to evaluate uptake of the aglycone forms rather than just looking at accumulation of glucuronides.

– The authors need to elaborate further in section 3.4 on the lack of physiological relevance for the 18/25 examples where there was no correlation between cellular uptake and RR% and the 7 which were not relevant with respect to relative expression of OATPs in human liver. Does the latter have something to do with Table S4? If so, that table should be referenced here.

– Please describe the relevance of the blood stability test. This seems something included to satisfy a previous reviewer without any reference to the relevance of the finding to the authors' overall conclusion.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Hepatoenteric Recycling Is A New Disposition Mechanism for Orally Administered Phenolic Drugs and Phytochemicals in Rat" for further consideration by eLife. Your revised article has been evaluated by Olga Boudker (Senior Editor) and a Reviewing Editor.

Tu and colleagues present an interesting body of work describing what they feel is a novel method of recycling of glucuronidated metabolites, specifically those of phenolic compounds. Rather than relying on the traditionally held pathway wherein a compound is metabolized in the liver and then recycled in the intestine after enzymes of the gut microbiota remove the glucuronide (enterohepatic recirculation/recycling – EHR), this mechanism involves glucuronidation of the compound in the small intestine, uptake of the glucuronidated metabolites into the liver by OATP1 transporters and then transport back out of the liver into the bile and subsequently into the small intestine where the glucuronide is recycled into its aglycone form by gut microbiota enzymes (hepatoenteric recycling -HER).

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Essential revisions:

1) Please provide a reference for the statement in the last sentence of the introduction on line 135.

2) Please define meaning of "LRE%" abbreviation upon its first use on line 140.

3) Please change recycle rate to LRE% on line 554.

https://doi.org/10.7554/eLife.58820.sa1

Author response

Summary:

Tu and colleagues present a body of work describing a novel method of recycling of glucuronidated metabolites, specifically those of phenolic compounds. Rather than relying on the traditionally held pathway wherein a compound is metabolized in the liver and then recycled in the intestine after enzymes of the gut microbiota remove the glucuronide (enterohepatic recirculation/recycling – EHR), this mechanism involves glucuronidation of the compound in the small intestine, uptake of the glucuronidated metabolites into the liver by OATP1 transporters and then transport back out of the liver into the bile and subsequently into the small intestine where the glucuronide is recycled into its aglycone form by gut microbiota enzymes (hepatoenteric recycling -HER). The authors argue that this has relevance for drug-drug interactions involving the OATP transporters and as a means to shuttle glucuronidated metabolites to the colon since the metabolism of phenolics by intestinal cells to glucuronidated forms as well as subsequent recycling means there should be large amounts of these metabolites in the colon.

In addition to the responses to the reviewers’ comments, we also made several changes in the terminology to better clarify the concept of liver recycling efficiency, recirculation and recycling. For example, we change the recirculation ratio (RR%) to liver recycling efficiency (LRE%), since the results we calculated did not actually reflect the whole process of HER. Only the recycling ratio of liver was determined. In addition, “recirculation” has been replaced by “recycling” at several places of the manuscript.

Lastly, because editors and reviewers were concerned about making our topic more general and as such we inserted the following paragraph into the “Introduction” to better address this concern.

“EHR differs from HER in the organs for formation and disposition of phase II metabolites. We believe It is important to delineate HER from EHR since the formation and disposition of phase II metabolites could be greatly different in disease condition related to different organs that could lead to differences in drug efficacy and toxicity both in systemic circulation and intestinal lumen. We believe that the differences in phase II enzymes, efflux and uptake transporters in different metabolic organs (such as liver and intestine) based on gender and disease conditions plays a significant role in determining the impact of EHR vs HER on the drug disposition.”

Essential revisions:

– The introduction could be more directed to the general audience.

We have modified the introduction as suggested by the reviewers. The contents are now made more directed to general introduction. (see above)

We have added the clinical relevance for understanding the difference between EHR and HER recycling processes with references. (see above)

We also got the feedback from colleagues not in the same area of research on the introduction and incorporate their comments in the introduction.

– In describing Figure 2A4, the authors claim that RR% is plotted against steady state blood concentration which "…displayed a saturating trend, indicative of transporter-mediated excretion." First, excretion rate not RR% is plotted and although these values tend to trend together the authors should be more precise in their description and better explain their rationale for making the claim that this indicates transport. It would also be helpful if there were more data in between 100 and 1000 uM.

The reviewer is correct that we did not plot LRE% against the steady-state blood concentration. Instead we plotted the excretion rates against steady-state blood concentration. The saturation in the excretion rate as a function of blood concentration is expected if the blood concentration is correlated with the biliary excretion of wogonoside. This means that a higher blood concentration will result in a higher rate of excretion until the transporter is saturated. We also agreed that having more concentrations between 100 and 1000 μM would be valuable, but that was not done. However, the current data are enough to show that the transporter is involved because process is saturable.

Another reason we believe that the uptake/efflux transporters are involved in the recycling process is because of the hydrophilicity of glucuronides (precluding significant passive diffusion). The following is the revised changes along with page and paragraph information.

Page 17, paragraph 2:

“It showed that at lower blood concentrations (<100μM) of wogonoside, its biliary excretion rates increased linearly. At higher blood concentrations (>=100μM), biliary excretion rates showed a saturation trend, indicative of transporter-mediated excretion (Figure 2A4).”

– In Figure 2B1-7, the authors show decreased recycling of the glucuronide relative to the aglycone form by monitoring appearance of the glucuronide in the bile after hepatic infusion. While the difference is dramatic, it is hard to know the relative roles of metabolism of the aglycone to the glucuronide versus uptake and transport of the aglycone form. It might be helpful to evaluate levels of the aglycone and glucuronide inside the liver after this experiment.

We do not disagree with the suggestion that “It might be helpful to evaluate levels of the aglycone and glucuronide inside the liver after this experiment”. Our recent measurement of liver concentration of Wogonin and Wogonoside showed a high ratio (> 5) of aglycone/glucuronide (Appendix 2 Table S4), providing evidence that metabolism of aglycone is slow. Furthermore, our uptake studies in the several OATP-overexpressed cell lines (Appendix 2 Table S2 and table S3) indicated that uptake/transport of aglycone was always equal to or faster than corresponding glucuronides. Also, there was no significant reduction in the uptake of Wogonin in the presence of OATP inhibitors, suggesting that the hepatic uptake of aglycone is primarily by passive diffusion and not active uptake. Lastly, in Caco-2 cells studies, flavonoid aglycones always displayed high permeability [1-3]. Therefore, there is no evidence that the hepatic uptake of phenolic aglycones would be slow and become a rate-limiting step in the biliary excretion of glucuronides, suggesting that a low LRE% for the aglycones meant slow metabolism in the liver rather than slow uptake of aglycones.

The following is the revised changes along with page and paragraph information.

Page 18, paragraph 2:

“Liver tissue concentrations of wogonin was at least 5 times higher than won-7-G concentrations (Appendix 2 supplement Table S4), which indicated that slow metabolism in liver was the likely reason for the lower LRE%.”

Page 18, paragraph 1:

“To rule out the possibility that slow aglycone uptake was the reason for lower LRE%, an uptake comparison between Won and Won-7-G in 3 cell lines were also conducted (Appendix 2 supplement Table S2 and Table S3). The results indicated that the uptake of Won was not influenced in the presence of specific transporter inhibitor, while Won-7-G was greatly inhibited, suggesting that aglycone uptake was mainly by passive diffusion, and often faster than their corresponding glucuronides.”

– The authors say they are examining the role of protein binding in section 3.1.3 by evaluating a higher concentration of Won aglycone, making the assumption that this would saturate protein binding. This seems somewhat arbitrary and if they truly wanted to assess the role of saturable protein binding, should do a protein binding experiment. It seems it would have been better to directly evaluate uptake of the aglycone form as previously suggested and just eliminate this evaluation.

As stated in the answer to the question #3, there is no evidence to suggest that uptake of flavonoid aglycones is slow, and therefore become the rate-limiting step in hepatic glucuronide formation. Based on the uptake experiments performed in our lab, we believe that uptake of aglycones into the hepatocytes is equal to or faster than the corresponding glucuronides. Therefore, apart from the slow metabolism, another possible explanation for low LRE% could be the plasma protein binding of phenolic aglycone, which impedes its hepatic transport by decreasing the effective concentration available to the liver.

We hypothesize that if protein binding plays a significant role, LRE% would be higher at a higher aglycone concentration once the protein binding becomes saturated. But our results showed the opposite, indicating that protein binding was not the major reason for low LRE%. Therefore, we are quite certain that decrease in LRE% is not the result of protein binding but possibly saturation of metabolic enzymes. This is supported by the ratio of liver concentration of wogonin and wogonoside (Table S1) when aglycone is infused in portal vein. It indicated that the decrease of LRE% was due to the slow metabolism.

The following is the revised changes along with page and paragraph information.

Page 18, paragraph 2:

“However, the results indicated that LRE% at a higher concentration (100μM) was significantly lower (P<.001) than at a lower concentration (2μM) (Figure 2C3), ruling out the impact of extensive protein binding on the lower LRE% of aglycones. Liver tissue concentrations of wogonin was at least 5 times higher than won-7-G concentrations (Appendix 2 supplement Table S4), which indicated that the slow metabolism in liver was the likely reason for the lower LRE%.”

– In section 3.1.4, the authors evaluate different flavonoids to make a comment on the specific role of glucuronidation at the 7-position in RR%. This doesn't make sense. One should evaluate the same compound that is glucuronidated at multiple sites to evaluate the role of glucuronidation location – and correlate this with cell based studies with the transporters. It was also unclear why the 7 location was so relevant. The authors should elaborate.

The 7-position is highly relevant because major dietary important flavonoids have a hydroxyl group at 7 position, and several UGTs are shown to have region-selective glucuronidation at this position [4]. For flavonoid compounds such as genistein [5], chrysin [6], apigenin [7], wogonin [8], and baicalein [9], at least one of their main glucuronides are conjugated at the 7position. We agreed with the reviewer that it would be better to study this problem with a flavonoids that have 3-5 OH groups and then make synthetic glucuronide for each of these OH positions, but this was not scope of the current study as it requires huge amount of efforts to purify mono-glucuronides from a mixture of other mono-glucuronides. Multiple monoglucuronides are expected if we use biosynthesis method, and chemical synthesis of these mono-glucuronides is beyond the scope of this work. However, we have turned down the importance of this 7-position in our discussion.

– The experiments describing sex differences and cassette dosing on glucuronide RR% seems insufficient and unnecessary for this manuscript.

For sex differences:

OATPs were identified as the major transporters determining LRE%. Since Oatps were reported to be male-predominant in rat liver [10], we hypothesized that sex differences could cause the changes of OATP expression levels and required investigating. In addition, E2G, a precursor for female sex hormone and a classical OATP substrate, has a drastically different blood concentrations in male and female. Therefore, we tested some of our model compounds to verify whether the sex difference matters.

For cassette dosing:

It’s a labor intensive and animal intensive experiments to determine LRE% of glucuronides. In order to improve the throughput of the experimental determination of LRE% and save the number of animal usage (IACUC 3R’s principle), we infused multiple compounds simultaneously. We chose to use a substrate concentration of 10μM because at concentrations lower than 10μM, many glucuronides cannot be detected in bile samples. Whereas experiment at higher concentration requires a much large quantities of glucuronides, which are generally not commercially available and hard to obtain via biosynthesis.

Another reason we did not use higher concentration because it is possible that these glucuronides would have competitive inhibition when infused together. To rule out the possibility that they may still compete at 10μM, we compared the single and cassette dosing of two model compounds (high recycle and low recycle compounds) and found no significant differences between single compound infusion and cassette infusion.

The following is the revised changes along with page and paragraph information.

Page 19, paragraph 2:

“We are interested in determining the effects of sex difference on the OATP liver expression levels, because the expression level of Oatp in rat liver was reported to be significantly higher in males than in females [20]. Also, E2G is a classical OATP substrate that has a drastically higher blood concentrations in females than males. The results indicated that there was not sex related differences in the LRE% of Won-7-G and Lut-3’-G. (Appendix 2 supplement Table S6).

Interestingly, the LRE% of Bai-7-G increased from ~4% (male rats) to ~10% (female rats) (P<.05).”

– In section 3.2.1 when discussing the outcome for Lut-3'-G it would be helpful if the authors could refer to the data for this point in figure 2B4 and to show the evidence for additional metabolism in supplementary data.

As per reviewer’s suggestion, we have added additional reference in section 3.2.1. We have also added additional evidence of metabolite formation using in vitro glucuronidation of Lut-3'-G in rat liver microsomes (Figure 2 supplement Figure S1A-S1C) that may explain the low LRE% of Lut-3’-G. Our results showed that Lut-3’-G could be further metabolized to Lut-di-G (di-glucuronides), which may provide an explanation why Lut-3’-G was present in bile at much lower than expected concentration. Luteolin was previously reported to be metabolized into Lut-di-G, which is consistent with our observation [11].

The following is the revised changes along with page and paragraph information.

Page 20, paragraph 3:

“The discrepancy can be explained by the fact that Lut-3’-G could be further metabolized into diglucuronides of Lut in vivo [23]. A liver microsome-mediated metabolism of Lut-3’-G confirmed the formation of Lut di-glucuronides by LC-MS (Figure 2 supplement Figure S2A and Figure S2B). Moreover, the peaks of Lut-3’-G and Lut-di-G both decreased upon incubation with glucuronidases (Figure 2 supplement Figure S2C).”

– In Section 3.2.4 where the authors make the claim that substrates of the transporters make better inhibitors, it would again be helpful to evaluate uptake of the aglycone forms rather than just looking at accumulation of glucuronides.

We agreed that we did not articulate that clearly. We did not claim “that substrates of the transporters make better inhibitors”. Rather, we wanted to state that “phenolic glucuronides that do not inhibit the uptake of prototypical substrates are not substrates of OATP.” We expected to use this characteristic as a fast screening tool to determine whether a glucuronide is not a substrate of a specific OATP. The latter will save our time to develop an analysis method to measure the glucuronide. This has been updated in the section 3.2.4.

Page 22, paragraph 1:

“The results showed that if a compound was not an inhibitor of prototypical OATP substrate, it was not a substrate of that OATP (Figure 3D1 to 3D3), but the reverse was not true. In contrast, substrates of OATP1B1 were able to significantly inhibited (P<.005) the uptake of glucuronides (Won-7-G, E2G and Lut-3’-G) that are good substrates of a specific OATP. Interestingly, aglycones were better inhibitors than their corresponding glucuronides, even though the corresponding glucuronides had higher LRE%.”

– The authors need to elaborate further in section 3.4 on the lack of physiological relevance for the 18/25 examples where there was no correlation between cellular uptake and RR% and the 7 which were not relevant with respect to relative expression of OATPs in human liver. Does the latter have something to do with Table S4? If so, that table should be referenced here.

We modified section 3.4 to further elaborate our statistical analysis methods to explain the physiological relevance as suggested by the reviewer.

The following is the revised changes along with page and paragraph information.

Page 23, paragraph 1:

“A total of 66 combinations with different OATP weightings were applied in Fisher exact test (Appendix 4 supplement table S1). OATP 1B1 is the most abundant OATP expressed in livers (Appendix 2 Supplement Table S1) [28] and also considered the most important among all OATPs.

OATP 1B3 is grouped with OATP1B1 in both localization and substrates, but the contribution of OATP 1B3 is considered to be always smaller than OATP 1B1. Therefore, combinations which assigned the highest weightings to OATP1B3 were considered not physiologically relevant and eliminated from the results. Of all the results from the Fisher exact test (Appendix 4 supplement Table S1), 22 combinations were eliminated based on this criterion. Of the remaining 44 combinations, 41 out of 44 results (93.2%) showed statistical significance. Taken together, these results demonstrated there were solid correlation between cellular uptake and LRE%. Thus, we showed that cellular uptake of glucuronides is the rate-limiting step in HER. In addition, this correlation (Figure 5) could be mathematically described by an Emax model (Emax 86%±17%) with an EC50 value of 42±33 nM.”

Reference

1. Fang, Y., et al., Study of Structure and Permeability Relationship of Flavonoids in Caco2 Cells. Nutrients, 2017. 9(12).

2. Tian, X.J., et al., Studies of intestinal permeability of 36 flavonoids using Caco-2 cell monolayer model. Int J Pharm, 2009. 367(1-2): p. 58-64.

3. Ahmed, I., et al., Caco-2 Cell Permeability of Flavonoids and Saponins from Gynostemma pentaphyllum: the Immortal Herb. ACS Omega, 2020. 5(34): p. 2156121569.

4. Singh, R., et al., Uridine diphosphate glucuronosyltransferase isoform-dependent regiospecificity of glucuronidation of flavonoids. J Agric Food Chem, 2011. 59(13): p. 7452-64.

5. Yuan, B., et al., Role of metabolism in the effects of genistein and its phase II conjugates on the growth of human breast cell lines. AAPS J, 2012. 14(2): p. 329-44.

6. Mohos, V., et al., Effects of Chrysin and Its Major Conjugated Metabolites Chrysin-7Sulfate and Chrysin-7-Glucuronide on Cytochrome P450 Enzymes and on OATP, P-gp, BCRP, and MRP2 Transporters. Drug Metab Dispos, 2020. 48(10): p. 1064-1073.

7. Griffiths, L.A. and G.E. Smith, Metabolism of apigenin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro. Biochem J, 1972. 128(4): p. 901-11.

8. Chen, X., et al., Quantitation of the flavonoid wogonin and its major metabolite wogonin7 β-D-glucuronide in rat plasma by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2002. 775(2): p. 169-78.

9. Zhang, L., et al., Hepatic metabolism and disposition of baicalein via the coupling of conjugation enzymes and transporters-in vitro and in vivo evidences. AAPS J, 2011. 13(3): p. 378-89.

10. Hou, W.Y., et al., Age- and sex-related differences of organic anion-transporting polypeptide gene expression in livers of rats. Toxicol Appl Pharmacol, 2014. 280(2): p. 370-7.

11. Wang, L., et al., Metabolic Disposition of Luteolin Is Mediated by the Interplay of UDPGlucuronosyltransferases and Catechol-O-Methyltransferases in Rats. Drug Metab Dispos, 2017. 45(3): p. 306-315.

– Please describe the relevance of the blood stability test. This seems something included to satisfy a previous reviewer without any reference to the relevance of the finding to the authors' overall conclusion.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1) Please provide a reference for the statement in the last sentence of the introduction on line 135.

The statement is based on results shown in two papers. We used these references because in paper 1, it stated that raloxifene “is excreted almost exclusively as glucuronide conjugates via the feces of patients” 1[]. Since the metabolites of raloxifene are made of Ral-6-G and Ral-4’-G, with the latter represents 70% of its metabolites 2[], we made the statement that phenolic drugs are “significantly metabolized in the human intestine (up to ~70% or more) before entering the liver…”

The reason why the major metabolite of raloxifene Ral-4’-G is only formed in the human small intestine is because UGT isoforms UGT1A8 and UGT1A10 are responsible for its formation 34[, ], and both isoforms are predominantly expressed in the human enterocytes 5[]. Hence, small intestine was considered as being responsible for up to 70% of raloxifene metabolism in vivo.

Similar results showed that 78% of orally dosed ezetimibe could be found in feces of patients 6[]. Since intestine is the major metabolism organ of ezetimibe 7[], we believed that a large portion of that 78% (found in feces) was due to metabolism in the small intestine and subsequent excretion via HER. Our data in this paper also confirmed that ezetimibe was extensively metabolized in rat small intestine and underwent HER almost exclusively.

Genistein, a dietary flavonoid, is another compound extensively metabolized in the small intestine, and in humans only 10~30% of the dose was found in urine, suggesting 70-90% of the dose could be eliminated via feces in humans 8[].

2) Please define meaning of "LRE%" abbreviation upon its first use on line 140.

The content has been edited as showed below:

“The portal vein infusion uses a direct method to assess the recirculation efficiency of a phenolic glucuronide by the liver using liver recycle efficiency% (LRE%, defined as steady-state biliary secretion rate divided by the hepatic infusion rate).”

3) Please change recycle rate to LRE% on line 554.

We found an incorrect term on line 548, and changed the text from “RR%” to “LRE%”. However the term “excretion rate at the steady-state” on line 548 is not as clear as it should be, and therefore we replaced it with “steady-state glucuronide excretion rate”, which is the same term used in Equation 1.

References.

1. Teeter, J.S. and R.D. Meyerhoff, Environmental fate and chemistry of raloxifene hydrochloride. Environ Toxicol Chem, 2002. 21(4): p. 729-36.

2. Sun, D., et al., Characterization of raloxifene glucuronidation: potential role of UGT1A8 genotype on raloxifene metabolism in vivo. Cancer Prev Res (Phila), 2013. 6(7): p. 719-30.

3. Kemp, D.C., P.W. Fan, and J.C. Stevens, Characterization of raloxifene glucuronidation in vitro: contribution of intestinal metabolism to presystemic clearance. Drug Metab Dispos, 2002. 30(6): p. 694-700.

4. Kokawa, Y., et al., Effect of UDP-glucuronosyltransferase 1A8 polymorphism on raloxifene glucuronidation. Eur J Pharm Sci, 2013. 49(2): p. 199-205.

5. Strassburg, C.P., M.P. Manns, and R.H. Tukey, Expression of the UDP-glucuronosyltransferase 1A locus in human colon. Identification and characterization of the novel extrahepatic UGT1A8. J Biol Chem, 1998. 273(15): p. 8719-26.

6. Patrick, J.E., et al., Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects. Drug Metab Dispos, 2002. 30(4): p. 430-7.

7. Kosoglou, T., et al., Ezetimibe: a review of its metabolism, pharmacokinetics and drug interactions. Clin Pharmacokinet, 2005. 44(5): p. 467-94.

8. Yang, Z., et al., Bioavailability and pharmacokinetics of genistein: mechanistic studies on its ADME. Anticancer Agents Med Chem, 2012. 12(10): p. 1264-80.

https://doi.org/10.7554/eLife.58820.sa2

Article and author information

Author details

  1. Yifan Tu

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2728-6753
  2. Lu Wang

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8643-6790
  3. Yi Rong

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Resources, Data curation, Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Vincent Tam

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Data curation, Formal analysis, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  5. Taijun Yin

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Resources, Supervision, Investigation, Methodology
    Competing interests
    No competing interests declared
  6. Song Gao

    Department of Pharmaceutical Sciences, College of Pharmacy, Texas Southern University, Houston, United States
    Contribution
    Supervision, Validation, Investigation, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
  7. Rashim Singh

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Resources, Formal analysis, Writing - review and editing
    Competing interests
    No competing interests declared
  8. Ming Hu

    Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    mhu@uh.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2744-6294

Funding

National Institutes of Health (GM070377)

  • Ming Hu

National Institutes of Health (CA246209)

  • Ming Hu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work is supported by NIH GM 070377 and CA246209.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations by the Institutional Animal Care and Use Committee (IACUC) at the University of Houston. The animal protocol (TR201800017) was approved by the IACUC at the University of Houston. All procedures were approved by the IACUC at the University of Houston. All surgery was performed under sodium pentobarbital anesthesia, and every effort was made to minimize suffering.

Senior Editor

  1. Olga Boudker, Weill Cornell Medicine, United States

Reviewing Editor

  1. Arduino A Mangoni, Flinders Medical Centre, Australia

Reviewer

  1. Noelle S Williams, University of Texas Southwestern Medical Center, United States

Publication history

  1. Received: May 12, 2020
  2. Accepted: June 16, 2021
  3. Version of Record published: July 1, 2021 (version 1)
  4. Version of Record updated: July 12, 2021 (version 2)

Copyright

© 2021, Tu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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    Julia Stokes et al.
    Research Article Updated

    Volatile anesthetics (VAs) are widely used in medicine, but the mechanisms underlying their effects remain ill-defined. Though routine anesthesia is safe in healthy individuals, instances of sensitivity are well documented, and there has been significant concern regarding the impact of VAs on neonatal brain development. Evidence indicates that VAs have multiple targets, with anesthetic and non-anesthetic effects mediated by neuroreceptors, ion channels, and the mitochondrial electron transport chain. Here, we characterize an unexpected metabolic effect of VAs in neonatal mice. Neonatal blood β-hydroxybutarate (β-HB) is rapidly depleted by VAs at concentrations well below those necessary for anesthesia. β-HB in adults, including animals in dietary ketosis, is unaffected. Depletion of β-HB is mediated by citrate accumulation, malonyl-CoA production by acetyl-CoA carboxylase, and inhibition of fatty acid oxidation. Adults show similar significant changes to citrate and malonyl-CoA, but are insensitive to malonyl-CoA, displaying reduced metabolic flexibility compared to younger animals.