TransportâGlucuronidation Classification System and PBPK Modeling

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Transport-Glucuronidation Classification System and PBPK Modeling: New Approach to Predict the Impact of Transporters on Disposition of Glucuronides Shufan Ge, Yingjie Wei, Taijun Yin, Beibei Xu, Song Gao, and Ming Hu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00941 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

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Molecular Pharmaceutics

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Transport-Glucuronidation Classification System and PBPK Modeling: New

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Approach to Predict the Impact of Transporters on Disposition of Glucuronides

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Shufan Ge1, Yingjie Wei2, Taijun Yin1, Beibei Xu1, Song Gao1, and Ming Hu1*

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1. Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy,

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The University of Houston, 1441 Moursund Street, Houston, TX, 77030, USA

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2. Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu

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Provincial Academy of Chinese Medicine, 100 Shizi Street, Nanjing 210028, China

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*Address correspondence to: Ming Hu, Ph.D. 1441 Moursund Street Department of Pharmacological and Pharmaceutical Sciences College of Pharmacy, University of Houston Houston, TX77030 Tel: (713)-795-8320 E-mail: [email protected]

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1 ACS Paragon Plus Environment


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Abstract

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Glucuronide metabolites require the action of efflux transporters to exit cells due to their

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hydrophilic properties. In this study, we proposed a transport-glucuronidation

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classification system and developed a PBPK model to predict the impact of BCRP on

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systemic exposure of glucuronides. The clearance by UGTs in S9 fractions and the efflux

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clearance of glucuronides by BCRP in human UGT1A9-overexpressing HeLa cells were

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incorporated in the classification system and PBPK model. Based on simulations for

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glucuronide AUC for theoretical compounds in the classification system, it was indicated

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that BCRP was more important for compounds with greater efflux clearance of their

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glucuronides by BCRP regardless of differences in clearance by UGTs. Pharmacokinetic

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studies were performed in WT and Bcrp1 (-/-) mice for 8 compounds to verify our

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predictions. Among eight compounds, the glucuronide AUC of daidzein and genistein

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increased significantly in Bcrp1 (-/-) mice, while only slight increases in systemic

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exposure were observed for other glucuronides. The results from pharmacokinetic studies

34

were in agreement with the predictions except for resveratrol, which was effluxed

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predominantly by transporters other than BCRP. Therefore, for glucuronides that were

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predominantly mediated by BCRP, this study provided a useful approach in predicting

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the impact of BCRP on its disposition and the potential DDIs involving BCRP.

38 39

Keywords: Glucuronide disposition, BCRP, Transport-glucuronidation classification

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system, PBPK modeling

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Introduction

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UDP-glucuronosyltransferase (UGT) enzymes are the second most important family of

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enzymes for drug metabolism when comparing to cytochrome P450 (CYP). For drugs

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cleared by metabolism, it was reported that UGTs contribute to clearance of about one-

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seventh of the top 200 drugs prescribed in the United States 1.

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metabolism often significantly limits drugs’ oral bioavailability. For example, raloxifene

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undergoes extensive in vivo glucuronidation, which results in very low bioavailability 2.

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Most glucuronides are considered inactive. However, for active glucuronide metabolites

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such as ezetimibe glucuronide, which accounts for more than 80% of the total ezetimibe

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species in plasma 3-5, glucuronidation is a critical bioactivation pathway. Other important

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drugs undergoing glucuronidation includes sorafenib, mycophenolic acid, morphine, etc

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6

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drug candidates. For example, darexaban, a novel oral factor Xa inhibitor with

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antithrombotic potency, is metabolized mainly by UGT1A9. Its major metabolite is

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darexaban glucuronide, which accounted for about 90% of total radioactivity in plasma,

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was demonstrated to have inhibitory activity similar to that of darexaban in vitro and

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therefore determine the pharmacological effect of darexaban after oral administration in

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humans 7-9.

UGT-mediated

. Glucuronidation is also an important metabolic pathway for some newly developed

62 63

In regards to metabolically based drug-drug interaction (DDI) studies, most efforts were

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focused on the inhibition or the induction of CYP enzymes. According to FDA guidelines

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for drug interaction studies

10

, in vivo studies using appropriate inhibitors or inducers of



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enzymes are required when the contribution of metabolism pathway is 25% or more of

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the drug’s overall elimination. While many DDIs have been attributed to the induction or

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inhibition of CYPs

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observed. CYP-mediated drug interactions can lead to several to more than 35-fold

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changes in drug exposure measured by area under the plasma concentration versus time

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curve (AUC) 13-15, whereas the changes in the drug exposure were usually less than two-

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fold in DDIs involving UGTs. This is mainly because UGT substrates are typically

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metabolized by multiple UGTs and have higher Km values

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noticed that for almost all of the reported UGT-based drug interactions, the systemic

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exposure of parent drug and glucuronide metabolite was altered through multiple

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pathways instead of being only influenced by interacting with UGTs. For example,

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probenecid, which was commonly used as an in vivo inhibitor of UGT enzymes, could

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interfere with glucuronide transport in liver and kidney 16, 20, 21.

11, 12

, much fewer DDIs involving the regulation of UGTs were

1, 16-19

. In addition, it was

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On the other hand, greater changes in systemic exposure of parent compound or

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glucuronide metabolites were observed in studies performed in transporter knockout

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animals, indicating the critical role of transporters in disposition of glucuronide

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conjugates and the potential transporter-mediated DDIs for drugs undergoing

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glucuronidation. Enterocytes and hepatocytes are the most important sites for

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glucuronidation. Due to increased polarity and decreased permeability of glucuronide

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conjugates, they need the actions of efflux or uptake transporters to leave or enter the

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cells

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genistein and genistein glucuronide were observed after oral administration of genistein

22, 23

. In our previous studies, ~2-fold and ~18-fold increases in exposures of


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in Bcrp1 (-/-) mice. In the absence of Bcrp1, more genistein glucuronide was distributed

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to blood, where deglucuronidation took place leading to increased levels of aglycone

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(genistein)

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models were also performed by other groups. 17α-ethinylestradiol-3-O-glucuronide

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(EEG), acetaminophen glucuronide (APAP-G), diclofenac acyl glucuronide, and a few

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other glucuronides experienced altered disposition and systemic exposure in transporter

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(BCRP, MRP2, or MRP3) deficient animals. In particular, the levels of EEG in blood

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increased more than 100-fold in Mrp2 (-/-) rats, while the concentration of parent EE did

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not change much 25-30.

24

. Studies of glucuronide in transporter knockout animals or in vitro cell

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In this study, we focused on the role of Breast Cancer Resistance Protein (BCRP) in

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disposition of glucuronide conjugates and the potential BCRP-mediated DDIs involving

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glucuronides. BCRP expressed on the apical membrane of enterocytes and canalicular

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membrane of hepatocytes acts as an efflux pump for intracellularly formed glucuronide

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conjugates. A number of glucuronides including 17β-estradiol 17-(β-D-glucuronide)

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(E217βG),

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diclofenac glucuronide, and various glucuronide metabolites of flavonoid compounds

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were demonstrated to be substrates of BCRP

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glucuronidation and the transport of glucuronide were studied separately, we proposed a

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novel transport-glucuronidation classification system which describes the role of

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transporters for disposition of drugs with different glucuronidation clearance. In the

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present study, BCRP was selected as the first transporter to establish an approach to study

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the classification system.

7-Ethyl-10-hydroxycamptothecin

glucuronide

30-33

(SN-38-glucuronide),

. Unlike the previous studies where



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The complexities of glucuronide metabolites disposition make physiologically based

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pharmacokinetic (PBPK) model an appropriate tool to study the impact of different

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factors on glucuronide disposition. To apply the newly developed transport-

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glucuronidation classification system to delineate the impact of BCRP on systemic

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exposure and disposition of compounds with different properties, we developed a PBPK

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model that integrates glucuronidation, glucuronide transport, glucuronide hydrolysis, and

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enterohepatic recirculation. The model was used to generate hypothesis on how the rate

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of being effluxed by BCRP and metabolized by UGT affect the role of BCRP in systemic

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exposure of parent compounds (aglycones) and their glucuronides. In addition,

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pharmacokinetic studies of a series of compounds in wild-type (WT) and Bcrp1 (-/-) mice

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were performed to test the hypothesis. The transport-glucuronidation classification

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system and PBPK model developed here not only provide a new approach to understand

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the impact of BCRP on disposition of compounds undergoing glucuronidation, but also

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have major implications in predicting BCRP-mediated DDIs.

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Materials and Methods

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Chemicals and Reagents

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Genistein, chrysin, daidzein, and 3,6-dihydroxyflavone (3,6-DHF) were purchased from

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Indofine Chemicals (Somerville, NJ). Resveratrol, sorafenib, sorafenib tosylate, was

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obtained from LKT Laboratories (St. Paul, MN). Maackiain was bought from Ruicong

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Ltd (Shanghai, China). Formononetin and curcumin were bought from LC Laboratories

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(Woburn, MA). Mycophenolate mofetil (MMF), mycophenolic acid (MPA), β-


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glucuronidases, uridine diphosphoglucuronic acid (UDPGA), alamethicin, D-saccharic-

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1,4-lactone monohydrate, magnesium chloride, Hanks’ balanced salt solution (powder

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form), TPCK (L-1-tosylamido-2-phenylethyl chloromethyl ketone)-treated trypsin from

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bovine pancreas, ammonium bicarbonate, dithiothreitol, and iodoacetamide were

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purchased from Sigma–Aldrich (St. Louis, MO). Mem-PERTM Plus membrane protein

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extraction kit, BCA protein assay kit, synthesized signature peptide-1 (ENLQFSAALR,

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Pep-1, purity > 95%) and signature peptide-2 (ENLQFSAAL [13C6, 15N1] R, Pep-2, purity

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> 95%) were purchased from Thermo Scientific (Rockford, IL). Ora-Plus oral suspending

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vehicle was made by Paddock Laboratories Inc (Minneapolis, MN).

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Cell Culture

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Human UGT1A9-overexpressing HeLa cells were cultured and maintained as described

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previously. Before experiments, the engineered cells were grown on 6-well plates (2 ×

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105 cells / well) for 3 days. The expression of UGT1A9 and BCRP in these cells were

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characterized in previous studies 34.

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Animals

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Male wild-type (WT) FVB mice (6~8 weeks) were purchased from Envigo (Madison,

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MI). Male Bcrp1 (-/-) mice (6~8 weeks) with an FVB genetic background were from

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Taconic (Hudson, NY). Animals were kept in an environmentally controlled room

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(temperature: 25±2℃, humidity: 50±5%, 12 h dark-light cycle) for at least 1 week before

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the experiments.

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Glucuronidation in Mouse Hepatic and Intestinal S9 Fractions

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Glucuronidation Assay Hepatic and intestinal S9 fractions were prepared using the

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same procedure described in our previous studies. Mice were fasted overnight with

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access to water only and euthanized. Intestine and liver were cut out and washed with ice-

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cold saline containing 1mM dithiothreitol. Mouse livers were washed and then perfused

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with ice-cold saline and minced and suspended in homogenization buffer (consisted of

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10mM pH 7.4 KH2PO4, 250mM sucrose, 1mM EDTA, and 0.04 mg/ml PMSF). The

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segments of intestine were then pooled and washed twice with washing solution

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(consisted of 8 mM KH2PO4, 5.6 mM Na2HPO4, 1.5 mM KCl, 96 mM NaCl, 27 mM

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sodium citrate, and 0.04 mg/ml phenylmethylsulfonyl fluoride or PMSF). The intestinal

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strips were cut open lengthwise and mucosal cells were scraped off from intestine and

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washed with homogenization buffer. Mucosal cells and hepatic cells were collected and

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homogenized at 4°C. After 15min centrifugation in 9000 × g at 4°C, the fat layer was

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discarded and the supernatant was collected, aliquoted, and stored at -80°C until use. The

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protein concentration in S9 fraction was quantified using a BCA protein assay kit.

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Glucuronidation of eight compounds (daidzein, chrysin, maackiain, 3,6-DHF, resveratrol,

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genistein, sorafenib, MPA) in liver and intestinal S9 fractions were performed.

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Compounds at a series of concentrations were incubated with S9 fractions,

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saccharolactone, alamethicin, magnesium chloride, and UDPGA in potassium phosphate

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buffer at 37°C. The substrate concentrations varied from 50nM to 60 µM depending on

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different solubilities. The reaction was stopped by adding stop solution (94% acetonitrile


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/ 6% acetic acid containing formononetin as an internal standard). Samples were ready

181

for UPLC analysis after centrifugation.

182 183

Glucuronidation Kinetics

184

Glucuronide metabolites were identified by hydrolysis to their aglycone by treatment

185

with β-glucuronidase and the comparison of the UV spectra and MS fragment. Kinetic

186

parameters (Vmax, Km) were estimated by fitting the Michaelis-Menten equations to the

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substrate concentrations and initial rates (confirmed also by the linear behavior of the

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Eadie-Hofstee plot). Data analysis were performed with Microsoft Excel add-in program

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for modeling steady-state enzyme kinetics, the goodness of fit was evaluated on the basis

190

of coefficient of determination (R2), Akaike's information criterion (AIC), weighted sum

191

of squared residual (WSS), and the rule of parsimony was applied. Intrinsic

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glucuronidation clearance (CLint,UGT) was calculated from Vmax,UGT and Km (CLint,UGT =

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Vmax,UGT / Km,UGT). For 3,6-DHF and MPA, simple linear regression model was applied

194

due to solubility limitations of substrates. The value of CLint,UGT was obtained from the

195

slope of the line.

196 197

Excretion Studies in Human UGT1A9-overexpressing HeLa cells

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The cellular excretion kinetics of glucuronide conjugates were determined in human

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UGT1A9-overexpressing HeLa cells. Briefly, HeLa-UGT1A9 cells were washed with

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HBSS buffer before experiments. Then the cells were incubated with one of the eight

201

compounds (daidzein, chrysin, maackiain, 3,6-DHF, resveratrol, genistein, sorafenib,

202

MPA) at a series of concentrations in HBSS buffer (loading solution) at 37°C. The



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sampling times were selected to ensure that the amounts of glucuronide excreted versus

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time plots were in the linear range. At each time point, 200 µl of incubating media was

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collected and equal volume of loading solutions was added to replenish each well. At the

206

end of experiment, cells were washed with ice-cold HBSS buffer and then removed and

207

collected in 200 µl HBSS buffer. Then cells were sonicated at 4°C, and the cell lysate

208

was collected to determine intracellular concentration. The cell volume was estimated to

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be 4 µl / mg protein. All samples were mixed with stop solution (94% acetonitrile / 6%

210

acetic acid containing formononetin as an internal standard) and ready for UPLC analysis

211

after centrifugation. All experiments were performed in triplicates.

212 213

The kinetic parameters of BCRP-mediated efflux were derived from correlation plots

214

between efflux rates and intracellular concentrations. Different kinetic models were used

215

to fit the data based on the shape of Eadie-Hofstee plots. Simple linear regression model

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was applied for the excretion of sorafenib glucuronide due to solubility limitations of

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sorafenib. Biphasic two-site model was used to fit excretion kinetic data of resveratrol

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glucuronide. For all the other compounds, the efflux rates and intracellular concentrations

219

were fit to Michaelis-Menten equation. Data analysis was performed with Microsoft

220

Excel add-in program. The calculated intrinsic clearance was calculated (CLint,BCRP =

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Vmax,BCRP / Km,BCRP, or CLint,BCRP = VBCRP / Ci) and used as an indicator of the efficiency

222

of efflux transporter.

223 224 225


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Pharmacokinetic Studies in Wild-type and Bcrp1 (-/-) Mice

227

The animal protocols used in this study were approved by the University of Houston’s

228

Institutional Animal Care and Uses Committee. Pharmacokinetic studies of daidzein,

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maackiain, 3,6-DHF, MPA, and sorafenib were performed in WT and Bcrp1 (-/-) mice.

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Compound was dispersed in oral suspending vehicle and given to mice at the dose of

231

20mg/kg orally. The blood sample collection and processing were performed as described

232

previously with minor modifications 35. Briefly, about 10 µl of blood was collected from

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mice at 15, 30, 60, 120, 240, 360, 480, and 1440 min. Acetonitrile (using a 1 : 5 blood /

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acetonitrile ratio) was used as the precipitation agent. After centrifugation, the

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supernatant was dried and reconstituted in 100 µl of 15% acetonitrile aqueous solution for

236

analysis.

237 238

Pharmacokinetic parameters including Cmax (maximum blood concentration), Tmax (the

239

time that Cmax happens), t1/2 (half-life), and AUC0-t (area under the blood concentration

240

curve from time 0 to t) of parent compounds and their glucuronides were obtained by

241

using the noncompartmental model in WinNonlin 3.3 (Pharsight, Mountain View, CA).

242

The criteria adopted in previous studies were also applied in reporting the values of t1/2 in

243

this study 24. The values of t1/2 were not reported when they were longer than 12 h or the

244

extrapolated AUC was more than 30% of AUCinf or the fitting goodness (r2) in the

245

terminal phase was less than 0.8.

246 247 248



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249

Sample Analysis

250

The Waters ACQUITY UPLC (Ultra performance liquid chromatography) system

251

(Waters, Milford, MA) was used to analyze the parent compounds and the formed

252

glucuronides from glucuronidation assay in S9 fractions and cellular excretion studies.

253

For sorafenib and MPA, sample analysis were performed on LC-MS/MS due to low

254

concentrations used in these studies. All blood samples from pharmacokinetic studies

255

were analyzed using LC-MS/MS. LC-MS/MS analysis was performed on an API 5500

256

Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDS SCIEX, Foster

257

City, California) with a TurboIonSprayTM source coupled to a Waters UPLC system.

258

The LC conditions for analyzing the parent compounds and their glucuronides were:

259

column, 1.7µm C18 column (2.1×50mm) (AcQuity, BEH C18, CA); mobile phase A:

260

2.5mM ammonium acetate (PH7.4) in water (for daidzein, maackiain, genistein,

261

resveratrol, chrysin and 3,6-DHF) or 0.1% formic acid in water (for sorafenib and MPA);

262

mobile phase B: acetonitrile (for daidzein, maackiain, genistein, resveratrol, chrysin and

263

3,6-DHF) or 0.1% formic acid in acetonitrile (for sorafenib and MPA). The compound-

264

dependent parameters for mass spectrum were shown in Supplemental Table S1.

265

Quantification was performed by MRM (Multiple Reaction Monitoring) method in the

266

negative (for daidzein, maackiain, genistein, resveratrol, chrysin and 3,6-DHF) or

267

positive ion mode (for sorafenib and MPA). Data were processed using MultiQuantTM

268

2.0.2 Software.

269 270 271


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272

Absolute Protein Quantification of Bcrp

273

Absolute protein quantification of Bcrp was performed using LC-MS/MS. Membrane

274

protein from mouse liver, intestine, and kidney was extracted by using Mem-PERTM Plus

275

membrane protein extraction kit from Thermo Scientific. The membrane protein samples

276

were then processed following previous protocol with some modifications

277

protein samples were denatured and reduced by heating at 95°C for 10min in the presence

278

of dithiothreitol, which was followed by alkylation with iodoacetamide. The samples

279

were then digested with trypsin at a 1:50 protease-to-protein ratio at 37°C for 12h. At the

280

end of digestion, samples were acidified with equal amount of 50% acetonitrile in water

281

containing 0.1% of formic acid and centrifuged at 5000 rpm for 20 min prior to LC-

282

MS/MS analysis. A previously used peptide (ENLQFSAALR) was selected as the

283

signature peptide for quantitative analysis of Bcrp. The stable isotope labeled (SIL)

284

peptide (ENLQFSAAL [13C6,

285

mobile phase A is water with 0.1% of formic acid, whereas mobile phase B is acetonitrile

286

with 0.1% of formic acid. The instrument and compound dependent parameters for mass

287

spectrometry conditions for analyzing BCRP were shown in Supplemental Table S2-1.

15

36

. Briefly,

N1] R) was serving as internal standard (IS)

36

. The

288 289

PBPK Model Development

290

A whole-body PBPK model describing parent compound and glucuronide metabolites

291

was constructed and simulated using the Simbiology toolbox of Matlab ® (The

292

MathWorks, Inc., Natick, MA). The model contained seven organ compartments

293

connected by arterial and venous blood supplies (Fig.1). Each compartment was defined

294

by a tissue volume and tissue blood flow rate. Mass balance equations were constructed



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295

on the basis of flow of blood between compartments. The model was built and validated

296

based on the following four basic assumptions: (i) Each tissue is a single, well-stirred

297

compartment with uniform concentration of the compound within it; (ii) This model

298

assume perfusion rate-limited kinetics with the liver, intestine, and kidney being the sites

299

of clearance; (iii) The distribution and elimination of all the compounds and their

300

glucuronides follow first-order linear kinetics; and (iv) In Bcrp-deficient animals, the

301

clearance of glucuronides by other efflux transporters remains the same as they were

302

estimated in WT animals.

303 304

For non-eliminating tissues, the following equation was used:

305 306

= ∗ − ∗ / (eq. 1)

307 308

Mass balance equations of glucuronide in intestine, liver, and kidney tissue compartments

309

were as follows:

_ = _ ∗ _ + _ ∗ _ ∗ !"#$%%_ − &'(% )$*+ ∗ _ / _ − ( _,- + _&.$'_$// ) ∗ _ ∗ !"#$%%_ (eq. 2)

310 %*)$'

%*)$'_ = _%*)$' ∗ _ + &'(% )$*+ ∗ _ / _ + %*)$'_ ∗ %*)$'_ ∗ !"#$%%_ − 1_%*)$' ∗ %*)$'_ / %*)$'_ − ( %*)$'_,+ %*)$'_&.$'_$// ) ∗ %*)$'_ ∗ !"#$%%_ (eq. 3)

311


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3*+$4

3*+$4_ = _3*+$4 ∗ _ − 1_3*+$4 ∗ 3*+$4_ / 3*+$4_ − ( 3*+$4_,- + 3*+$4_&.$'_$// ) ∗ 3*+$4_ ∗ !"#$%%_ (56. 4)

312

Where V, C, Q and CL represent tissue volume, concentration, blood flow rate and

313

clearance, respectively. G and P are abbreviations of glucuronide and parent drugs. Kp is

314

the tissue partition coefficient, and fucell is the unbound fraction in cells. CLtissue_other_eff is

315

the clearance by transporters other than BCRP.

316 317

PBPK-Model Parameterization

318

The parameters used in PBPK model are either physiological or compound dependent.

319

The physiological parameters that were used to implement the model were obtained from

320

the literature and presented in Supplemental Table S3-1 37.

321 322

Organ:plasma partition coefficients were obtained from literature or predicted using the

323

approach of Poulin

324

calculated using equations in previous study. The glucuronidation clearance of genistein

325

and sorafenib in liver and intestine (CLliver_ugt; CLintestine_ugt) was scaled from in vitro

326

CLint,UGT based on S9 fraction yield and average tissue weight (Supplemental Table S3-

327

2). The amount of Bcrp expressed in liver, intestine, and kidney was scaled to the whole

328

organ based on membrane protein extraction efficiency and presented in Supplemental

329

Table S2-2. The clearance of genistein glucuronide by BCRP in mouse intestine

330

(CLintestine_BCRP) was obtained from intestinal perfusion studies in WT and Bcrp1 (-/-)

331

mice，assuming the efflux of genistein glucuronide by other transporters do not change

332

in Bcrp deficient mice

38

. The unbound drug fraction in enterocyte and hepatocyte was

39

. For sorafenib glucuronide, the clearance by BCRP was 15 ACS Paragon Plus Environment


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333

calculated by using the ratio of its clearance in UGT1A9-overexpressing HeLa cells to

334

that of genistein glucuronide. The clearance of glucuronide by BCRP in liver and kidney

335

was derived from CLintestine_bcrp using the ratio of expression levels of Bcrp in different

336

tissues, assuming that the relative activities of Bcrp can be represented by the expression

337

levels of Bcrp in tissues (Supplemental Table S3-3).

338 339

Data Fitting and Simulations.

340

The pharmacokinetic data in WT mice after oral administration of genistein or sorafenib

341

at the dose of 20mg/kg were used to estimate the rest of the parameters. The sorafenib PK

342

data were from the current study, while the genistein PK data were described previously

343

24

344

Additional sets of PK data were used to validate the model. Briefly, the concentration-

345

time profiles of genistein after intravenous administration in WT mice and oral

346

administration in Bcrp1 (-/-) mice were simulated based on the same parameters. For

347

Bcrp1 (-/-) mice, the efflux clearance by BCRP was set to 0. Similarly, the concentration-

348

time profile of sorafenib after oral administration in Bcrp1 (-/-) mice was also simulated.

349

All the simulated blood concentrations were compared with the corresponding observed

350

data, and the coefficient of determination (R2) was calculated. In addition, the AUC

351

values of simulated blood concentration-time profiles were derived by integrating the

352

instantaneous concentrations over time, and then compared with the experimental data.

. The estimation method was Isqnonlin (constrained nonlinear least-squares regression).

353 354 355


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356

Statistical Analysis.

357

Data were presented as means ± S.D., if not specified otherwise. Analysis of variance or

358

Student’s t test was used to analyze data. The level of significance was set at p

TransportâGlucuronidation Classification System and PBPK Modeling

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