Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET

User menu

  • My alerts
  • Log in
  • My Cart

Search

  • Advanced search
Drug Metabolism & Disposition
  • Other Publications
    • Drug Metabolism and Disposition
    • Journal of Pharmacology and Experimental Therapeutics
    • Molecular Pharmacology
    • Pharmacological Reviews
    • Pharmacology Research & Perspectives
    • ASPET
  • My alerts
  • Log in
  • My Cart
Drug Metabolism & Disposition

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Fast Forward
    • Latest Articles
    • Special Sections
    • Archive
  • Information
    • Instructions to Authors
    • Submit a Manuscript
    • FAQs
    • For Subscribers
    • Terms & Conditions of Use
    • Permissions
  • Editorial Board
  • Alerts
    • Alerts
    • RSS Feeds
  • Virtual Issues
  • Feedback
  • Submit
  • Visit dmd on Facebook
  • Follow dmd on Twitter
  • Follow ASPET on LinkedIn
Research ArticleArticle

Detection of Weak Organic Anion–Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans

Yueping Zhang, Vinay K. Holenarsipur, Hamza Kandoussi, Jianing Zeng, T. Thanga Mariappan, Michael Sinz and Hong Shen
Drug Metabolism and Disposition October 2020, 48 (10) 841-848; DOI: https://doi.org/10.1124/dmd.120.000076
Yueping Zhang
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Vinay K. Holenarsipur
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hamza Kandoussi
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jianing Zeng
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
T. Thanga Mariappan
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Sinz
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hong Shen
Departments of Metabolism and Pharmacokinetics (Y.Z., M.S., H.S.) and Bioanalytical Sciences (H.K., J.Z.), Bristol Myers Squibb Company, Princeton, New Jersey; and Departments of Metabolism and Pharmacokinetics (V.K.H., T.T.M.), Biocon Bristol Myers Squibb R&D Centre (BBRC), Syngene International Ltd., Biocon Park, Bommasandra IV Phase, Bangalore, India
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF
Loading

Abstract

Probenecid (PROB) is a clinical probe inhibitor of renal organic anion transporter (OAT) 1 and OAT3 that inhibits in vitro activity of hepatic drug transporters OATP1B1 and OATP1B3. It was hypothesized that PROB could potentially affect the disposition of OATP1B drug substrates. The plasma levels of the OATP1B endogenous biomarker candidates, including coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA), were examined in 14 healthy subjects treated with PROB. After oral administration with 1000 mg PROB alone and in combination with furosemide (FSM), AUC(0–24 h) values were 1.39 ± 0.21-fold and 1.57 ± 0.41-fold higher than predose levels for CPI and 1.34 ± 0.16-fold and 1.45 ± 0.57-fold higher for CPIII. Despite increased systemic exposures, no decreases in CPI and CPIII renal clearance were observed (0.97 ± 0.38-fold and 1.16 ± 0.51-fold for CPI, and 1.34 ± 0.53-fold and 1.50 ± 0.69-fold for CPIII, respectively). These results suggest that the increase of CP systemic exposure is caused by OATP1B inhibition. Consistent with this hypothesis, PROB inhibited OATP1B1- and OATP1B3-mediated transport of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively, in transporter-overexpressing human embryonic kidney cell assay. The inhibition potential was further confirmed by CPI and CPIII hepatocyte uptake experiments. In contrast, administration of PROB alone did not change AUC(0–24 h) of HDA and TDA relative to prestudy levels, although the administration of PROB in combination with FSM increased HDA and TDA levels compared with FSM alone (1.02 ± 0.18-fold and 0.90 ± 0.20-fold vs. 1.71 ± 0.43-fold and 1.62 ± 0.40-fold). Taken together, these findings indicate that PROB displays weak OATP1B inhibitory effects in vivo and that coproporphyrin is a sensitive endogenous probe of OATP1B inhibition. This study provides an explanation for the heretofore unknown mechanism responsible for PROB’s interaction with other xenobiotics.

SIGNIFICANCE STATEMENT This study suggested that PROB is a weak clinical inhibitor of OATP1B based on the totality of evidence from the clinical interaction between PROB and CP and the in vitro inhibitory effect of PROB on OATP1B-mediated CP uptake. It demonstrates a new methodology of utilizing endogenous biomarkers to evaluate complex drug-drug interaction, providing explanation for the heretofore unknown mechanism responsible for PROB’s inhibition. It provides evidence to strengthen the claim that CP is a sensitive circulating endogenous biomarker of OATP1B inhibition.

Introduction

It is well established that membrane-bound transport systems are primarily critical determinants in the cellular traffic and disposition of exogenous and endogenous compounds. Notably, organic anion–transporting polypeptide (OATP) 1B1 and OATP1B3 can govern the disposition of drug substrates, including the widely used 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). Moreover, it is increasingly recognized that endogenous biomarkers of OATP1B1 and OATP1B3—such as coproporphyrin I (CPI), CPIII, hexadecanedioate (HDA), and tetradecanedioate (TDA)—and glycochenodeoxycholate-3-sulfate are useful tools in elucidating the role of OATP1B in complex drug-drug interaction (DDI) in healthy subjects and patients (Lai et al., 2016; Shen et al., 2016, 2017, 2018, Takehara et al., 2018, Barnett et al., 2019; Yee et al., 2019; Jones et al., 2020; Mori et al., 2020).

Probenecid (PROB) is widely used as a uricosuric agent (Cunningham et al., 1981). In addition, PROB has been used as an adjunct to enhance blood levels of antibiotics such as penicillins and cephalosporins because of an inhibitory effect on the renal transporters OAT1 and OAT3. In agreement, most of the DDIs involving PROB are due to its inhibition on the kidney transport of acidic drugs such as cefaclor (Welling et al., 1979), cefonicid (Pitkin et al., 1981), cefoxitin (Vlasses et al., 1980), cephradine (Welling et al., 1979), dicloxacillin (Beringer et al., 2008), famotidine (Inotsume et al., 1990), and furosemide (Vree et al., 1995; Shen et al., 2019a). As a result, the US Food and Drug Administration (FDA) suggests PROB as an index inhibitor to assess OAT1 and OAT3 in clinical DDI studies (https://www.fda.gov/drugs/drug-interactions-labeling/drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers). However, knowledge of the transporters involved in PROB inhibition could be incomplete. For example, some studies showed that the action of PROB at the renal level was not sufficient to account for severalfold serum level elevations encountered for drugs such as fexofenadine, statins, and methotrexate in humans and animals (Gewirtz et al., 1984; Yasui-Furukori et al., 2005; Liu et al., 2008; Kosa et al., 2018). As an organic acid, probenecid increased the area under plasma concentration–time curve (AUC) of fexofenadine approximately 1.5-fold by interfering with secretion of fexofenadine by the renal tubules in humans (Yasui-Furukori et al., 2005; Liu et al., 2008). The total and renal clearance (CLR) values of fexofenadine were 16.0 and 6.2 L/h (Lappin et al., 2010). Given the fact that fexofenadine is 60%–70% bound to plasma proteins (Molimard et al., 2004), the active tubular secretion contributes to approximately half of the CLR. As a result, inhibition of OAT3-mediated renal secretion clearance alone, which resulted in a decreased amount of dose excreted in urine from 11%–12% to 6%–8%, cannot explain the observed AUC change (Yasui-Furukori et al., 2005; Liu et al., 2008). Moreover, studies in cynomolgus monkeys and rats have demonstrated that probenecid decreased the clearance of statins and methotrexate by the hepatic route as well (Gewirtz et al., 1984; Kosa et al., 2018), contributing to prolonged elevation of circulating statins and methotrexate levels. The reduction in hepatic uptake of statins in monkey hepatocytes and biliary secretion of methotrexate in rats is thought to arise from inhibition of hepatic uptake mediated by OATP in the presence of PROB. However, a direct interaction between PROB and OATP1B in humans has not been fully tested.

Recent bioanalytical methodologies, especially metabolomics, have identified a number of endogenous biomarker candidates for the transporter proteins expressed in the organs of importance in drug disposition, including liver and kidney (Chu et al., 2018; Muller et al., 2018; Rodrigues et al., 2018; Shen, 2018). Changes in the levels of such endogenous probes were able to phenotype the mechanism of complex DDIs involving multiple elimination pathways. For example, a clinical DDI study was conducted to assess the inhibition potential of fenebrutinib using midazolam (CYP3A), simvastatin (CYP3A and OATP1B), and rosuvastatin (Breast cancer resistance protein and OATP1B) as drug substrates (Jones et al., 2020). Fenebrutinib increased the AUC values of all three probes 2- to 3-fold. However, there was no change in the plasma levels of CPI and CPIII, endogenous biomarkers of OATP1B1, suggesting that the DDIs were due to the inhibition of CYP3A and breast cancer resistance protein rather than OATP1B (Jones et al., 2020). The present studies were designed to evaluate the inhibitory effects of PROB on human OATP1B1 and OTP1B3 by analyzing OATP1B biomarker levels in a clinical study and evaluating coproporphyrin uptake in transfected cell lines and hepatocytes in the presence of PROB to determine whether PROB modulates OATP1B activity.

Materials and Methods

Reagents and Materials.

CPI and CPIII were purchased as dihydrochloride salts from Frontier Scientific (Logan, UT). HDA, TDA, carbamazepine, and high-performance liquid chromatography–grade methanol, acetonitrile, and water were purchased from Sigma Aldrich (St. Louis, MO). Rosuvastatin (RSV), estradiol-17β-d-glucuronide (E17βG), cholecystokinin octapeptide (CCK-8), CPI-15N4 sodium bisulfate salt, HDA-d4, TDA-d4, and rifampicin SV (RIF SV) were purchased from Toronto Research Chemicals (North York, ON, Canada). Water-soluble probenecid sodium salt was purchased from Life Technologies (Carlsbad, CA). CPIII-d8 was synthesized at Bristol Myers Squibb Company (Princeton, NJ). [3H]E17βG (38.8 Ci/mmol) and [3H]CCK-8 (92.5mCi/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA). Charcoal stripped plasma was obtained from Bioreclamation IVT (Westbury, NY). Cryopreserved human hepatocytes from two female donors were purchased from Celsis In Vitro Technologies (Baltimore, MD) (lot NRJ) and Bioreclamation IVT (lot BXW). Human plasma containing dipotassium EDTA was obtained from Biologic Specialty Corporation (Colmar, PA). Cell culture reagents were purchased from Corning (Manassas, VA) and Life Technologies Corporation. All other reagents and chemicals used were of the highest grade commercially available.

Clinical Drug Interaction Study between PROB and FSM.

Plasma and urine samples were collected from an open-label, single-dose, three-treatment, three-period clinical DDI study between PROB and FSM reported previously (Shen et al., 2019a). Briefly, 14 male healthy Indian subjects who had a normal medical history and physical examination participated in the study. Each subject received 1000 mg PROB alone (period 1), 40 mg FSM alone (period 2), and 40 mg FSM at 1 hour after administration of 1000 mg PROB (period 3) with a 1-week washout between treatments. Subjects fasted the night before and until at least 4 hours after administration of the drugs. PROB and FSM were administered orally with 240 ml of water. Blood samples (3 ml each) for determination of drug and transporter endogenous biomarker concentrations were obtained at predose and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 hours after dosing. Urine samples were collected during 0–8, 8–16, and 16–24 hours postdose. Subjects were housed in a clinical facility 36 hours prior to dosing in period 1, and blood and urine samples were also collected over 24 hours prior to dosing to obtain the baseline levels of transporter endogenous biomarkers in the absence of drug. Plasma was separated immediately, and the plasma and urine samples were prepared into two aliquots and kept at −70°C until analysis using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Shen et al., 2019a).

Quantification of CPI and CPIII in Plasma and Urine by LC-MS/MS.

Plasma and urine concentrations of CPI and CPIII were quantitated by using the methods developed previously (Kandoussi et al., 2018).

Quantification of HDA and TDA in Plasma by LC-MS/MS.

The bioanalytical analyses of HDA and TDA in plasma were performed as described in detail by Santockyte et al. (2018).

Inhibition Studies in OATP1B1- and OATP1B3-Expressing HEK293 Cells.

The in vitro uptake experiments were repeated in at least two independent experiments. There are three replicates per experiment for each condition (n = 3). The stable OATP1B1- and OATP1B3-transfected and mock cells generated by Bristol Myers Squibb were cultured at 37°C in an atmosphere of 95% air/5% CO2 and subcultured once per week (Han et al., 2010; Shen et al., 2013). OATP1B1- and OATP1B3-expressing cells used in the current study were passaged fewer than 30 times to retain consistent transporter expression and functional activity. To assess the inhibition potential of PROB toward OATP1B1 and OATP1B3, the inhibitory effect was evaluated on the uptake of CPI and radiolabeled probe substrates in the presence of increasing concentrations of PROB in transporter-expressing cells using a protocol described previously (Shen et al., 2017; Panfen et al., 2019). In brief, transporter-expressing cells were grown to confluence in poly-d-lysine–coated 24-well plates (BD Biosciences, San Jose, CA). At confluence, medium was aspirated, and cells were rinsed twice with 2 ml of prewarmed Hanks’ balanced salt solution (HBSS) (catalog number 21-023-CM; Mediatech, Manassas, VA) and preincubated with 200 µl of the transport buffer (HBSS with 10 mM HEPES, pH 7.4) containing PROB at concentrations ranging from 1 to 10,000 µM for 30 minutes at 37°C. Uptake was then initiated by the addition of 200 µl of the prewarmed transport buffer containing CPI (0.2 µM) or radiolabeled probe substrate (1 µM [3H]E17βG and 0.1 µM [3H]CCK-8 for OATP1B1 and OATP1B3, respectively). The probe substrate concentrations are well below the Km values. Incubation proceeded for 2 minutes at 37°C to ensure linearity with time (Supplemental Fig. 1). After 2 minutes, the uptake buffer was then removed, and the cell monolayers were immediately washed three times with 1.5 ml ice-cold HBSS buffer to terminate the uptake process. To analyze the concentrations of radiolabeled compounds, cells in the dried plate were lysed with 300 µl 0.1% Triton X-100. Aliquots of 200 and 20 µl cell lysate were used for radioactivity counting and protein concentration analysis, respectively. After the addition of 5 ml of scintillation cocktail, radioactivity was measured on a dual-channel liquid scintillation counter, Tri-Carb 3100TR liquid scintillation counter (PerkinElmer Life Sciences, Boston, MA). To analyze the concentrations of CPI, cells in the dried plate were lysed in 300 µl 2:1 (v/v) ratio of acetonitrile and 1 M formic acid with CPI-15N4 (internal standard). The contents were filtered through a 96-well filter plate (0.45-µm low-binding hydrophilic polytetrafluoroethylene), and the filtrate was dried under nitrogen gas. The dried contents were reconstituted in 80 µl of 1 M formic acid/acetonitrile (20:80, v/v), and the concentration of CPI was measured by LC-MS/MS. Throughout the entire process, appropriate precautions were taken to minimize sample exposure to ambient light. Protein concentration of cell lysates was determined with the bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). Cellular uptake in OATP1B1- and OATP1B3-HEK cells was normalized based on the protein amount in each well.

Inhibition of CP Uptake in Human Hepatocytes by PROB.

To further assess the inhibitory effect of PROB on OATP1B, the uptake of CPI and CPIII in human hepatocytes in the presence of PROB was examined using a protocol described previously, with some modification (Shen et al., 2016; Zhang et al., 2019). Briefly, transporter-qualified cryopreserved human hepatocytes from two female donors were thawed according to the vendor’s instructions (Bioreclamation IVT). The hepatocytes were then pooled by resuspending the cell pellet in Krebs-Henseleit buffer to give a cell density of 2 million viable cells per milliliter, which was determined by trypan blue staining (≥80% post-thaw viability). After cell suspensions were prewarmed to 37°C, the uptake of CPI in hepatocytes in the presence and absence of an inhibitor (final concentration of 100, 300, and 1000 µM PROB and 200 µM RIF SV) was determined. The uptake study was initiated by adding an equal volume of CP solution to the hepatocyte suspensions at 37°C, resulting in a final cell density of 1 million viable cells per milliliter and a CP or RSV concentration of 1 µM. There was a 3-minute preincubation with the inhibitors or control buffer with hepatocytes at 37°C before the initiation of uptake. At 0.25, 1, 1.5, and 5 minutes, 100-µl reaction mixtures were removed and overlaid onto prepared 0.4-ml microcentrifuge tubes containing 50 ml 2 M ammonium acetate (bottom layer) and 100 µl filtration oil (top layer; 84.5:15.5 silicon oil–mineral oil mix, final density of 1.015). Samples were centrifuged immediately at 10,000g for 15 seconds using a benchtop centrifuge to pellet the cells. The tubes were placed on dry ice and then cut, and the cell pellet was digested in a 2:1 (v/v) ratio of acetonitrile and 1 M formic acid containing the internal standard at room temperature. The contents were filtered through a 96-well filter plate (0.45-µm low-binding hydrophilic polytetrafluoroethylene), and the filtrate was dried under nitrogen gas. Finally, the dried contents were reconstituted in 80 µl of 1 M formic acid/acetonitrile (20:80, v/v) for LC-MS/MS analysis. Throughout the entire process, appropriate precautions were taken to minimize sample exposure to ambient light.

LC-MS/MS Analysis of CPI and CPIII in Cell Lysates.

Cell lysate concentrations of CPI, CPIII, and RSV were measured by LC-MS/MS as described previously (Lai et al., 2016; Shen et al., 2016).

Pharmacokinetic and Transport Analyses.

The pharmacokinetic parameters of CPI, CPIII, HDA, and TDA were derived using Phoenix WinNonlin, version 8.1 (Certara, Princeton, NJ). Maximum plasma concentration (Cmax) and area under the concentration-time curve from time 0 to 24 hours [AUC(0–24 h)] were obtained from plasma concentrations versus time data by performing a noncompartmental analysis with mixed log-linear trapezoidal method. CLR was estimated by the following equation:Embedded Imagewhere Ae(0–24 h) is the cumulative amount excreted in the urine during the time interval from 0 to 24 hours.

The concentrations required to inhibit transport by 50% (IC50) of PROB toward OATP1B1- and OATP1B3-mediated uptake of CPI and radiolabeled probes were estimated by fitting the uptake data to the following equation using the nonlinear regression approach (Phoenix WinNonlin; Certara):Embedded Imagewhere V is the mean transporter-mediated uptake of substrate at 2 minutes observed at the given PROB testing concentration (I), V0 is the uptake in the absence of PROB, and h is the Hill slope factor.

The hepatic uptake rate in the absence and presence of inhibitor was calculated based on the initial rate of uptake during the linear phase (up to 1.0 or 1.5 minutes). The uptake clearance (CLu) was calculated by dividing the uptake rate by the initial substrate concentration. Percent CLu inhibition was calculated using the following equation:Embedded Image

Statistical Analyses.

The results are expressed as means ± S.D. in the text and tables. To test for statistically significant differences in Cmax, AUC(0–24 h), and CLR among treatments (predose control, PROB, FSM, and PROB with FSM) in the clinical study, repeated measures one-way ANOVA was performed. When the F ratio showed that there were significant differences among treatments, the Tukey’s method of multiple comparisons was used to determine which treatments differ. All statistical analyses were carried out using GraphPad Prism version 8 (GraphPad Software, San Diego, CA), and a P value of less than 0.05 was considered statistically significant.

Results

Effects of Administration of PROB on Plasma Levels of CPI and CPIII.

The effect of PROB on the pharmacokinetics of CPI and CPIII, endogenous biomarkers of OATP1B1 and OATP1B3, was evaluated in both plasma and urine after administration of PROB alone, furosemide alone, and probenecid in combination with FSM in 14 subjects. The systemic exposures and CLR of CPI and CPIII are shown in Fig. 1 and Table 1. The table also presents the statistical comparisons for each treatment relative to predose baseline. The administration of 1000 mg PROB alone and in combination with 40 mg FSM significantly increased Cmax and AUC(0–24 h) of CPI and CPIII compared with the baseline (1.34- to 1.62-fold) (Table 1) (P < 0.05). In contrast, the Cmax and AUC(0–24 h) values were similar to the basal levels after administration of FSM alone (1.02- to 1.15-fold). These changes were not statistically significant (P > 0.05). Moreover, the CLR values of CPI and CPIII were not significantly changed in the presence of PROB compared with the baseline control, although the mean CLR of CPIII increased by 1.34- to 1.50-fold (Table 1) (P > 0.05), indicating no significant inhibition of the CLR of CPI and CPIII during PROB treatments.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

Effect of 1000 mg PROB and 40 mg FSM doses on plasma concentration and CLR of CP. The plasma concentration–time profiles of CPI (A) and CPIII (B) are shown as the mean and S.D. values obtained from 14 healthy subjects before dose (open squares) and after a single oral dose of PROB (open circles), FSM (open triangles), and coadministration of PROB and FSM (open diamonds). The CLR of CPI (C) and CPIII (D) before and after the indicated treatment.

View this table:
  • View inline
  • View popup
TABLE 1

Comparison of pharmacokinetic parameters of CPI and CPIII in healthy subjects after administration of PROB (1000 mg), FSM (40 mg), and coadministration of PROB and FSM

Data represent the means and S.D. from 9 to 14 subjects (n = 9–14).

Effects of Administration of PROB on Plasma Levels of HDA and TDA.

We also examined the effects of a single dose of 1000 mg PROB, either alone or in combination with 40 mg FSM, on the plasma concentrations of HDA and TDA in healthy subjects. Figure 2 shows the arithmetic mean plasma concentrations ± S.D. of HDA and TDA before the first dose of the study (predose) and during the three treatment phases. Table 2 shows the arithmetic mean ± S.D. of Cmax, AUC(0–24 h), and the ratios of HDA and TDA. Although pretreatment with PROB alone did not significantly alter the Cmax or AUC(0–24 h) of HDA and TDA compared with baseline (0.73- to 1.02-fold) (Table 1) (P > 0.05), the coadministration of PROB with FSM significantly increased the systemic exposures of HDA and TDA by 1.62- and 2.27-fold compared with FSM alone (Table 2) (P < 0.05). In addition, administration of a single oral dose of 40 mg FSM alone resulted in less than a 1% change in the Cmax and AUC(0–24 h) of HDA compared with the baseline controls (P > 0.05). In contrast, administration of FSM alone significantly decreased the Cmax and AUC(0–24 h) of TDA compared with the baseline (0.61- and 0.76-fold, respectively) (Table 2) (P < 0.05). Unfortunately, the amount of HDA and TDA excreted in the urine were not analyzed in this study.

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Effect of 1000 mg PROB and 40 mg FSM doses on plasma concentration of HDA and TDA. The plasma concentration–time profiles of HDA (A) and TDA (B) are shown as the mean and S.D. values obtained from 14 healthy subjects before dose (open squares) and after a single oral dose of PROB (open circles), FSM (open triangles), and coadministration of PROB and FSM (open diamonds).

View this table:
  • View inline
  • View popup
TABLE 2

Comparison of pharmacokinetic parameters of HDA and TDA in healthy subjects after administration of PROB (1000 mg), FSM (40 mg), and coadministration of PROB and FSM

Data represent the means and S.D. from 14 subjects (n = 14).

Characterization of PROB as an In Vitro Inhibitor of OATP1B1 and OATP1B3.

To evaluate the inhibitory effect of PROB on OATP1B1 and OATP1B3, the uptake of 0.2 µM CPI (OATP1B1 and OATP1B3), 1 µM [3H]E17βG (OATP1B1), and 0.1 µM [3H]CCK-8 (OATP1B3) was measured in the presence of increasing concentrations of PROB in transporter-expressing cells. As shown in Fig. 3, PROB inhibited OATP1B1- and OATP1B3-mediated uptake of CPI in a concentration-dependent manner, with IC50 values of 167 ± 42.0 and 76.0 ± 17.2 µM, respectively (Table 4). PROB showed very similar inhibition on the uptake of [3H]E17βG and [3H]CCK-8 by OATP1B1 and OATP1B3, respectively (IC50 values for OATP1B1 and OATP1B3 were 121 ± 7.5 and 147 ± 37.5 µM, respectively).

Fig. 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 3.

Inhibitory effect of PROB on OATP1B1- (A) and OATP1B3-mediated transport of CPI and prototypical probe substrates. Uptake of 0.2 µM CPI, 1 µM E17βG (OATP1B1), and 1 M CCK-8 (OATP1B3) was assessed in stably transfected OATP1B1- and OATP1B3-HEK cells over 2 minutes in the absence (control) or presence of PROB at the indicated concentrations. Uptake changes are normalized to transporter-mediated uptake in the absence of PROB. Results represent the mean and S.D. from three independent determinations.

View this table:
  • View inline
  • View popup
TABLE 3

The hepatic uptake clearance of CP incubated in the presence and absence of PROB (100, 300, and 1000 µM)

The hepatic uptake clearance and percent uptake clearance inhibition were determined using the data in Fig. 4.

View this table:
  • View inline
  • View popup
TABLE 4

Prediction of OATP1B-mediated DDIs for PROB and FSM using R-value and endogenous biomarker methods

Effect of PROB on CP Uptake in Hepatocytes.

To further demonstrate the inhibition of PROB on the hepatic elimination of CP, the uptake of 1 µM CPI and CPIII in human hepatocytes was assessed. Figure 4 and Table 3 show the uptake of 1 µM CPI and CPIII into human hepatocytes in suspension in the presence of increasing concentrations of PROB. The CPI and CPIII influx was sensitive to the presence of PROB, as a PROB concentration of 100 µM reduced CPI and CPIII uptake by 12% and 33%, respectively, of the control. As the PROB concentration was raised, CPI and CPIII influx was further reduced so that at 1000 µM PROB, uptake has been inhibited by 56% (Table 3). The uptake of the reference substrate RSV (1 µM) also appeared to be inhibited to a similar extent by PROB in hepatocytes (Fig. 4; Table 3).

Fig. 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 4.

Inhibitory effect of PROB on the uptake of CPI (A), CPIII (B), and RSV (C) into human hepatocytes. Open circles, squares, triangles and diamonds, and closed circles represent the uptake of 1 μM substrate alone with 100 μM PROB, 300 μM PROB, 1000 μM PROB, and 200 μM RIF SV, respectively. Results represent the mean and S.D. from three independent determinations.

Discussion

The role and importance of OATP1B1 and OATP1B3 in the transport of drugs across the basolateral membranes of the hepatocytes are well recognized. It is within the last decade that the inhibition potential of a new molecular entity toward OATP1B transporter proteins has begun to be evaluated during drug discovery and development (Giacomini et al., 2010; Tweedie et al., 2013; Lee et al., 2017). For 34 small molecular drugs approved by the FDA in 2017, OATP1B together with CYP3A4 played a significant role in mediating more than half of the drug interactions with AUC changes ≥5-fold (Yu et al., 2019). Moreover, it has become increasingly evident that endogenous biomarkers of OATP1B, for example CPI, can be used to evaluate complex DDIs for better DDI risk assessment and management based on a mechanistic understanding in healthy subjects and patients (Suzuki et al., 2019; Jones et al., 2020; Mori et al., 2020). The described findings suggest that this is the case for PROB. This drug is regarded as a clinically efficient inhibitor of OAT1 and OAT3 since the benzoic acid derivative is known to compete for active renal tubular secretion primarily with acidic drugs (Maeda et al., 2014). Moreover, PROB has recently been identified as a weak inhibitor of the OATP1B-mediated transport of organic anions using a heterologous expression system (Hirano et al., 2006; Matsushima et al., 2008; Izumi et al., 2013, 2016). Accordingly, the in vivo and in vitro inhibition potential of PROB toward OATP1B1 and OATP1B3 was investigated using the endogenous probes CPI, CPIII, HDA, and TDA.

For the first time, the results of this study showed a significant increase in plasma concentration of CPI and CPIII [Cmax and AUC(0–24 h)], endogenous probes of OATP1B1 and OATP1B3, in humans during PROB treatments compared with baseline levels (1.34- to 1.62-fold) (Fig. 1; Table 1). These findings suggest that PROB inhibited the elimination of the endogenous substrates of OATP1B, and this might be explained by an inhibitory effect of PROB on renal and/or hepatic transporters, as CPI and CPIII are excreted not only in bile but also in the urine (Wolkoff et al., 1976; Lai et al., 2016; Shen et al., 2016). However, the renal clearance of CP was not significantly decreased by PROB (Table 1). When the plasma and urine findings are taken together, this suggests that the interaction occurs in the liver but not in the kidney. This is not unexpected, because CPI and CPIII are not substrates for OAT1 and OAT3 (Bednarczyk and Boiselle, 2016; Shen et al., 2017; Kunze et al., 2018), although OAT1 and OAT3 are capable of transporting small anionic endogenous substrates (e.g., p-aminohippurate and estrone-3-sulfate) and xenobiotics (e.g., adefovir, benzylpenicillin, and FSM) (Inui et al., 2000; Shen et al., 2019b). By contrast, OATP1B1 and OATP1B3 were found to effectively mediate the uptake of CPI and CPIII, which is consistent with the anionic nature of these endogenous compounds (Bednarczyk and Boiselle, 2016; Shen et al., 2016, 2017). Very recently, Wiebe et al. (2020) investigated the effects of four commonly employed drug transporter inhibitors on cocktail drug pharmacokinetics. PROB treatment increased Cmax and AUC of RSV by 328% and 123%, respectively. Although PROB decreased RSV CLR by 78%, its inhibition of renal clearance alone could not explain the pronounced AUC increase since approximately 28% of total body clearance of RSV was via the renal route. In line with the clinical data, a monkey transporter-mediated DDI study showed that PROB increased the AUC of RSV and pitavastatin by 2.6- and 2.1-fold, respectively (Kosa et al., 2018). In addition, PROB significantly inhibited the uptake of RSV and pitavastatin in monkey hepatocytes (Kosa et al., 2018). Therefore, the interaction between PROB and statin might be attributed to the decreased hepatic uptake–mediated clearance by monkey OATP1B. Likewise, PROB had an impact on the OATP1B-mediated uptake clearance of fexofenadine in humans since its inhibition of renal organic anion transporter alone could not explain the noted AUC change (Yasui-Furukori et al., 2005; Liu et al., 2008). However, we cannot rule out that the synthesis of CPI and CPIII may be altered by PROB treatment. Interestingly, the mean renal clearance of CPIII but not CPI was increased by PROB, although the increase was not statistically significant compared with the predose level (1.34- and 1.50-fold) (Table 1) (P > 0.05). Previously, we reported that CPIII but not CPI was subject to active tubular secretion in the kidney of monkeys and humans (Lai et al., 2016; Shen et al., 2016). One possible explanation for the unexpected increase of CPIII CLR is that PROB stimulated the function of transporter(s) responsible for the tubular secretion of CPIII. Such stimulation has been observed with many transporters such as multidrug resistance-associated protein (MRP) 2 (Gilibili et al., 2017). In addition, it is possible that CPIII undergoes both renal tubular secretion and reabsorption, and PROB inhibited the renal reabsorption of CPIII, resulting in the increased net renal excretion clearance of CPIII. Future work needs to be done to confirm the hypotheses. Servais et al. (2006) previously studied urinary coproporphyrin excretion in rats after inhibition of transporters by PROB and in mutant transport-deficient rats, in which MRP2 is lacking. Total urinary coproporphyrin excretion was similar in mutant transport-deficient rats and in normal rats with or without treatment by PROB, but relative urinary CPI excretion was increased in the mutant rats. Moreover, PROB is a weak inhibitor of MRP2 that is expressed on the apical surface of renal proximal tubule epithelial cells and hepatocytes. CPI and CPIII are substrates for MRP2 (Gilibili et al., 2017; Kunze et al., 2018), and the urinary CPI/(CPI + CPIII) ratio was proposed as a surrogate for MRP2 activity (Benz-de Bretagne et al., 2011, 2014). Unchanged CLR values of CPI in the subjects administered with PROB suggested that PROB did not affect MRP2 activity in the kidney in vivo (Table 1). The increased CLR of CPIII requires further study to identify the transporters responsible for the renal disposition of CPIII for better assessment of in vivo inhibition potential of an investigational drug using CPIII. Taken together, administration of PROB at a therapeutic dose can cause clinically relevant OATP1B inhibition.

In agreement with clinical findings, the IC50 of PROB in OATP1B1- and OATP1B3-HEK cells using CPI as a substrate is determined to be 167 and 76.0 µM, respectively, in this study (Fig. 3). This is consistent with recent reports employing dichlorofluorescein, bromosulfophthalein, E17βG, estrone-3-sulfate, pitavastatin, and fexofenadine as OATPIB1 and OATP1B3 substrates (Hirano et al., 2006; Matsushima et al., 2008; Izumi et al., 2013, 2016), in which the IC50 of PROB ranged from 39.8 to 227 µM. Furthermore, 100, 300, and 1000 µM PROB reduced the uptake of CPI, CPIII, and RSV in human hepatocytes in a concentration-dependent manner, with maximum inhibition by 56%, 56%, and 73%, respectively (Fig. 4; Table 3). The results suggested that PROB is a weak in vitro inhibitor of OATP1B1 and OATP1B3. However, because the free maximum concentration at the inlet to the liver (Iin,max,u) after 1000 mg PROB administration is large [34.6–41.5 µM, estimated by using the pharmacokinetic parameters reported previously (Shen et al., 2019a)], the predicted AUC ratio of a substrate drug of OATP1B in the presence and absence of PROB (R-value) is 1.46–1.55 using the IC50 value of 76.0 µM determined using CPI as a probe substrate (R-value = 1 + Iin,max,u/IC50), suggesting an in vivo inhibition potential of PROB with OATP1B (Table 4). These R-values, estimated using the method recommended in the guidance from the EMA and FDA (https://www.fda.gov/media/134582/download; https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-investigation-drug-interactions-revision-1_en.pdf), are consistent with the observed Cmax and AUC fold changes of endogenous probes CPI and CPIII. It is critical to include a sufficient number of subjects in a clinical study to detect weak drug transporter interaction. Barnett et al. (2018) demonstrated the sensitivity of CPI to identifying weak OATP1B inhibitors in an adequately powered clinical study using mode-based simulations and power calculations. The analysis showed the ability to identify a weak but clinically relevant OATP1B DDI using CPI as a probe (The ratio of victim drug area under the curve in the presence of the interacting drug relative to the control (AUCR) > 1.25 cutoff). Use of criterion of α = 0.01 and power of 0.8 required a sample size of 15 subjects (Barnett et al., 2018). This conclusion was further confirmed by CPI clinical data with several weak OATP1B inhibitors (Kunze et al., 2018; Liu et al., 2018). The clinical studies with 14 and 13 subjects indicated that CPI concentration changes were predictive for a weak clinically observed DDI in which CPI AUC increases of 1.6- and 1.4-fold were comparable with those observed for statins as victim drugs (Kunze et al., 2018; Liu et al., 2018). Therefore, we believe that our data generated with 14 subjects is sufficient to support the conclusions made in this study.

To quantify the response of HDA and TDA, two other endogenous probes of OATP1B (Yee et al., 2016; Shen et al., 2017; Yee et al., 2019), to PROB pretreatments in vivo, we measured the plasma HDA and TDA concentrations. The variations in plasma concentrations of HDA and TDA were larger than that of CPI and CPIII. The concentration-time profiles for HDA and TDA appear different between the PROB alone and PROB coadministration with FSM groups. Whereas the administration of PROB in combination with FSM significantly increased the AUC(0–24 h) of HDA and TDA by 1.71- and 1.62-fold, respectively, compared with FSM alone, the administration with PROB alone did not significantly alter the AUC(0–24 h) of HDA and TDA compared with prestudy levels (1.02- and 0.90-fold, respectively) (Fig. 2; Table 2). It is unclear why the PROB pretreatments show different effects on the plasma HDA and TDA levels. Unfortunately, the CLR of HDA and TDA could not be determined in the urine. In addition, the treatment with FSM alone significantly decreased the AUC(0–24 h) of TDA compared with the predose control (0.76-fold) (Table 2). We cannot rule out that the synthesis of the biomarkers may be affected by PROB and/or FSM. These results suggest that plasma HDA and TDA levels may not be good surrogate endogenous probes for weak OATP1B inhibition.

In conclusion, for the first time, our results demonstrate the weak inhibitory effect on OATP1B1 and OATP1B3 by PROB in vivo. Considering the fact that PROB is an index inhibitor for clinical OAT1/3 DDI study, these findings provide an explanation for the heretofore unknown mechanism responsible for the inhibition caused by PROB.

Acknowledgments

The authors acknowledge the following scientists for assistance with the clinical and preclinical studies: Susan Lubin and Erika Panfen.

Authorship Contributions

Participated in research design: Zhang, Holenarsipur, Zeng, Mariappan, Sinz, Shen.

Conducted experiments: Zhang, Kandoussi.

Contributed new reagents or analytic tools: Zhang, Kandoussi, Shen.

Performed data analysis: Zhang, Shen.

Wrote or contributed to the writing of the manuscript: Zhang, Sinz, Shen.

Footnotes

    • Received April 11, 2020.
    • Accepted July 13, 2020.
  • This study is supported by Bristol Myers Squibb Company.

  • https://doi.org/10.1124/dmd.120.000076.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

AUC
area under plasma concentration–time curve
AUC(0–24 h)
area under plasma concentration–time curve from time 0 to 24 hours
CCK-8
cholecystokinin octapeptide
CLR
renal clearance
CP
coproporphyrin
CPI
coproporphyrin I
CPIII
coproporphyrin III
DDI
drug-drug interaction
E17βG
estradiol-17β-d-glucuronide
FDA
Food and Drug Administration
FSM
furosemide
HBSS
Hanks’ balanced salt solution
HDA
hexadecanedioate
HEK
human embryonic kidney
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MRP
multidrug resistance-associated protein
OAT
organic anion transporter
OATP
organic anion–transporting polypeptide
PROB
probenecid
RIF SV
rifampicin SV
RSV
rosuvastatin
R-value
ratio of victim AUC in the presence and absence of perpetrators
TDA
tetradecanedioate
  • Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics

References

  1. ↵
    1. Barnett S,
    2. Ogungbenro K,
    3. Ménochet K,
    4. Shen H,
    5. Humphreys WG, and
    6. Galetin A
    (2019) Comprehensive evaluation of the utility of 20 endogenous molecules as biomarkers of OATP1B inhibition compared with rosuvastatin and coproporphyrin I. J Pharmacol Exp Ther 368:125–135.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Barnett S,
    2. Ogungbenro K,
    3. Menochet K,
    4. Shen H,
    5. Lai Y,
    6. Humphreys WG, and
    7. Galetin A
    (2018) Gaining mechanistic insight into coproporphyrin I as endogenous biomarker for OATP1B-mediated drug-drug interactions using population pharmacokinetic modeling and simulation. Clin Pharmacol Ther 104:564–574.
    OpenUrl
  3. ↵
    1. Bednarczyk D and
    2. Boiselle C
    (2016) Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III. Xenobiotica 46:457–466.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Benz-de Bretagne I,
    2. Respaud R,
    3. Vourc’h P,
    4. Halimi JM,
    5. Caille A,
    6. Hulot JS,
    7. Andres CR, and
    8. Le Guellec C
    (2011) Urinary elimination of coproporphyrins is dependent on ABCC2 polymorphisms and represents a potential biomarker of MRP2 activity in humans. J Biomed Biotechnol 2011:498757.
    OpenUrlPubMed
  5. ↵
    1. Benz-de Bretagne I,
    2. Zahr N,
    3. Le Gouge A,
    4. Hulot JS,
    5. Houillier C,
    6. Hoang-Xuan K,
    7. Gyan E,
    8. Lissandre S,
    9. Choquet S, and
    10. Le Guellec C
    (2014) Urinary coproporphyrin I/(I + III) ratio as a surrogate for MRP2 or other transporter activities involved in methotrexate clearance. Br J Clin Pharmacol 78:329–342.
    OpenUrl
  6. ↵
    1. Beringer PM,
    2. Kriengkauykiat J,
    3. Zhang X,
    4. Hidayat L,
    5. Liu S,
    6. Louie S,
    7. Synold T,
    8. Burckart GJ,
    9. Rao PA,
    10. Shapiro B, et al.
    (2008) Lack of effect of P-glycoprotein inhibition on renal clearance of dicloxacillin in patients with cystic fibrosis. Pharmacotherapy 28:883–894.
    OpenUrlPubMed
  7. ↵
    1. Chu X,
    2. Liao M,
    3. Shen H,
    4. Yoshida K,
    5. Zur AA,
    6. Arya V,
    7. Galetin A,
    8. Giacomini KM,
    9. Hanna I,
    10. Kusuhara H, et al., and International Transporter Consortium
    (2018) Clinical probes and endogenous biomarkers as substrates for transporter drug-drug interaction evaluation: perspectives from the international transporter consortium. Clin Pharmacol Ther 104:836–864.
    OpenUrl
  8. ↵
    1. Cunningham RF,
    2. Israili ZH, and
    3. Dayton PG
    (1981) Clinical pharmacokinetics of probenecid. Clin Pharmacokinet 6:135–151.
    OpenUrlCrossRefPubMed
    1. Ebner T,
    2. Ishiguro N, and
    3. Taub ME
    (2015) The use of transporter probe drug cocktails for the assessment of transporter-based drug-drug interactions in a clinical setting-proposal of a four component transporter cocktail. J Pharm Sci 104:3220–3228.
    OpenUrlCrossRef
  9. ↵
    1. Gewirtz DA,
    2. Plotkin JH, and
    3. Randolph JK
    (1984) Interaction of probenecid with methotrexate transport and release in the isolated rat hepatocyte in suspension. Cancer Res 44:3846–3850.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Giacomini KM,
    2. Huang SM,
    3. Tweedie DJ,
    4. Benet LZ,
    5. Brouwer KL,
    6. Chu X,
    7. Dahlin A,
    8. Evers R,
    9. Fischer V,
    10. Hillgren KM, et al., and International Transporter Consortium
    (2010) Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Gilibili RR,
    2. Chatterjee S,
    3. Bagul P,
    4. Mosure KW,
    5. Murali BV,
    6. Mariappan TT,
    7. Mandlekar S, and
    8. Lai Y
    (2017) Coproporphyrin-I: a fluorescent, endogenous optimal probe substrate for ABCC2 (MRP2) suitable for vesicle-based MRP2 inhibition assay. Drug Metab Dispos 45:604–611.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Han YH,
    2. Busler D,
    3. Hong Y,
    4. Tian Y,
    5. Chen C, and
    6. Rodrigues AD
    (2010) Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human liver drug transporters. Drug Metab Dispos 38:1072–1082.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Hirano M,
    2. Maeda K,
    3. Shitara Y, and
    4. Sugiyama Y
    (2006) Drug-drug interaction between pitavastatin and various drugs via OATP1B1. Drug Metab Dispos 34:1229–1236.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Inotsume N,
    2. Nishimura M,
    3. Nakano M,
    4. Fujiyama S, and
    5. Sato T
    (1990) The inhibitory effect of probenecid on renal excretion of famotidine in young, healthy volunteers. J Clin Pharmacol 30:50–56.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Inui KI,
    2. Masuda S, and
    3. Saito H
    (2000) Cellular and molecular aspects of drug transport in the kidney. Kidney Int 58:944–958.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Izumi S,
    2. Nozaki Y,
    3. Komori T,
    4. Maeda K,
    5. Takenaka O,
    6. Kusano K,
    7. Yoshimura T,
    8. Kusuhara H, and
    9. Sugiyama Y
    (2013) Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17β-glucuronide, estrone-3-sulfate, and sulfobromophthalein. Drug Metab Dispos 41:1859–1866.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Izumi S,
    2. Nozaki Y,
    3. Komori T,
    4. Takenaka O,
    5. Maeda K,
    6. Kusuhara H, and
    7. Sugiyama Y
    (2016) Investigation of fluorescein derivatives as substrates of organic anion transporting polypeptide (OATP) 1B1 to develop sensitive fluorescence-based OATP1B1 inhibition assays. Mol Pharm 13:438–448.
    OpenUrl
  18. ↵
    1. Jones NS,
    2. Yoshida K,
    3. Salphati L,
    4. Kenny JR,
    5. Durk MR, and
    6. Chinn LW
    (2020) Complex DDI by fenebrutinib and the use of transporter endogenous biomarkers to elucidate the mechanism of DDI. Clin Pharmacol Ther 107:269–277.
    OpenUrl
  19. ↵
    1. Kandoussi H,
    2. Zeng J,
    3. Shah K,
    4. Paterson P,
    5. Santockyte R,
    6. Kadiyala P,
    7. Shen H,
    8. Shipkova P,
    9. Langish R,
    10. Burrrell R, et al.
    (2018) UHPLC-MS/MS bioanalysis of human plasma coproporphyrins as potential biomarkers for organic anion-transporting polypeptide-mediated drug interactions. Bioanalysis 10:633–644.
    OpenUrl
  20. ↵
    1. Kosa RE,
    2. Lazzaro S,
    3. Bi YA,
    4. Tierney B,
    5. Gates D,
    6. Modi S,
    7. Costales C,
    8. Rodrigues AD,
    9. Tremaine LM, and
    10. Varma MV
    (2018) Simultaneous assessment of transporter-mediated drug-drug interactions using a probe drug cocktail in cynomolgus monkey. Drug Metab Dispos 46:1179–1189.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Kunze A,
    2. Ediage EN,
    3. Dillen L,
    4. Monshouwer M, and
    5. Snoeys J
    (2018) Clinical investigation of coproporphyrins as sensitive biomarkers to predict mild to strong OATP1B-mediated drug-drug interactions. Clin Pharmacokinet 57:1559–1570.
    OpenUrl
  22. ↵
    1. Lai Y,
    2. Mandlekar S,
    3. Shen H,
    4. Holenarsipur VK,
    5. Langish R,
    6. Rajanna P,
    7. Murugesan S,
    8. Gaud N,
    9. Selvam S,
    10. Date O, et al.
    (2016) Coproporphyrins in plasma and urine can Be appropriate clinical biomarkers to recapitulate drug-drug interactions mediated by organic anion transporting polypeptide inhibition. J Pharmacol Exp Ther 358:397–404.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Lappin G,
    2. Shishikura Y,
    3. Jochemsen R,
    4. Weaver RJ,
    5. Gesson C,
    6. Houston B,
    7. Oosterhuis B,
    8. Bjerrum OJ,
    9. Rowland M, and
    10. Garner C
    (2010) Pharmacokinetics of fexofenadine: evaluation of a microdose and assessment of absolute oral bioavailability. Eur J Pharm Sci 40:125–131.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Lee SC,
    2. Arya V,
    3. Yang X,
    4. Volpe DA, and
    5. Zhang L
    (2017) Evaluation of transporters in drug development: current status and contemporary issues. Adv Drug Deliv Rev 116:100–118.
    OpenUrl
  25. ↵
    1. Liu L,
    2. Cheeti S,
    3. Yoshida K,
    4. Choo E,
    5. Chen E,
    6. Chen B,
    7. Gates M,
    8. Singel S,
    9. Morley R,
    10. Ware J, et al.
    (2018) Effect of OATP1B1/1B3 inhibitor GDC-0810 on the pharmacokinetics of pravastatin and coproporphyrin I/III in healthy female subjects. J Clin Pharmacol 58:1427–1435.
    OpenUrl
  26. ↵
    1. Liu S,
    2. Beringer PM,
    3. Hidayat L,
    4. Rao AP,
    5. Louie S,
    6. Burckart GJ, and
    7. Shapiro B
    (2008) Probenecid, but not cystic fibrosis, alters the total and renal clearance of fexofenadine. J Clin Pharmacol 48:957–965.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Maeda K,
    2. Tian Y,
    3. Fujita T,
    4. Ikeda Y,
    5. Kumagai Y,
    6. Kondo T,
    7. Tanabe K,
    8. Nakayama H,
    9. Horita S,
    10. Kusuhara H, et al.
    (2014) Inhibitory effects of p-aminohippurate and probenecid on the renal clearance of adefovir and benzylpenicillin as probe drugs for organic anion transporter (OAT) 1 and OAT3 in humans. Eur J Pharm Sci 59:94–103.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Matsushima S,
    2. Maeda K,
    3. Ishiguro N,
    4. Igarashi T, and
    5. Sugiyama Y
    (2008) Investigation of the inhibitory effects of various drugs on the hepatic uptake of fexofenadine in humans. Drug Metab Dispos 36:663–669.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Molimard M,
    2. Diquet B, and
    3. Benedetti MS
    (2004) Comparison of pharmacokinetics and metabolism of desloratadine, fexofenadine, levocetirizine and mizolastine in humans. Fundam Clin Pharmacol 18:399–411.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Mori D,
    2. Ishida H,
    3. Mizuno T,
    4. Kusumoto S,
    5. Kondo Y,
    6. Izumi S,
    7. Nakata G,
    8. Nozaki Y,
    9. Maeda K,
    10. Sasaki Y, et al.
    (2020) Alteration in the plasma concentrations of endogenous OATP1B-biomarkers in non-small cell lung cancer patients treated with paclitaxel. Drug Metab Dispos 48:387–394.
    OpenUrlAbstract/FREE Full Text
    1. Müller F,
    2. Sharma A,
    3. König J, and
    4. Fromm MF
    (2018) Biomarkers for in vivo assessment of transporter function. Pharmacol Rev 70:246–277.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Panfen E,
    2. Chen W,
    3. Zhang Y,
    4. Sinz M,
    5. Marathe P,
    6. Gan J, and
    7. Shen H
    (2019) Enhanced and persistent inhibition of organic cation transporter 1 activity by preincubation of cyclosporine A. Drug Metab Dispos 47:1352–1360.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Pitkin D,
    2. Dubb J,
    3. Actor P,
    4. Alexander F,
    5. Ehrlich S,
    6. Familiar R, and
    7. Stote R
    (1981) Kinetics and renal handling of cefonicid. Clin Pharmacol Ther 30:587–593.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Rodrigues AD,
    2. Taskar KS,
    3. Kusuhara H, and
    4. Sugiyama Y
    (2018) Endogenous probes for drug transporters: balancing vision with reality. Clin Pharmacol Ther 103:434–448.
    OpenUrl
  34. ↵
    1. Santockyte R,
    2. Kandoussi H,
    3. Chen W,
    4. Zheng N,
    5. Venkatarangan L,
    6. Gan J,
    7. Shen H,
    8. Bonacorsi SJ,
    9. Easter J,
    10. Burrell R, et al.
    (2018) LC-MS/MS bioanalysis of plasma 1, 14-tetradecanedioic acid and 1, 16-hexadecanedioic acid as candidate biomarkers for organic anion-transporting polypeptide mediated drug-drug interactions. Bioanalysis 10:1473–1485.
    OpenUrl
  35. ↵
    1. Servais A,
    2. Lechat P,
    3. Zahr N,
    4. Urien S,
    5. Aymard G,
    6. Jaudon MC,
    7. Deray G, and
    8. Isnard Bagnis C
    (2006) Tubular transporters and clearance of adefovir. Eur J Pharmacol 540:168–174.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Shen H
    (2018) A pharmaceutical industry perspective on transporter and CYP-mediated drug-drug interactions: kidney transporter biomarkers. Bioanalysis 10:625–631.
    OpenUrl
  37. ↵
    1. Shen H,
    2. Chen W,
    3. Drexler DM,
    4. Mandlekar S,
    5. Holenarsipur VK,
    6. Shields EE,
    7. Langish R,
    8. Sidik K,
    9. Gan J,
    10. Humphreys WG, et al.
    (2017) Comparative evaluation of plasma bile acids, dehydroepiandrosterone sulfate, hexadecanedioate, and tetradecanedioate with coproporphyrins I and III as markers of OATP inhibition in healthy subjects. Drug Metab Dispos 45:908–919.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Shen H,
    2. Christopher L,
    3. Lai Y,
    4. Gong J,
    5. Kandoussi H,
    6. Garonzik S,
    7. Perera V,
    8. Garimella T, and
    9. Humphreys WG
    (2018) Further studies to support the use of coproporphyrin I and III as novel clinical biomarkers for evaluating the potential for organic anion transporting polypeptide 1B1 and OATP1B3 inhibition. Drug Metab Dispos 46:1075–1082.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Shen H,
    2. Dai J,
    3. Liu T,
    4. Cheng Y,
    5. Chen W,
    6. Freeden C,
    7. Zhang Y,
    8. Humphreys WG,
    9. Marathe P, and
    10. Lai Y
    (2016) Coproporphyrins I and III as functional markers of OATP1B activity: in vitro and in vivo evaluation in preclinical species. J Pharmacol Exp Ther 357:382–393.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Shen H,
    2. Holenarsipur VK,
    3. Mariappan TT,
    4. Drexler DM,
    5. Cantone JL,
    6. Rajanna P,
    7. Singh Gautam S,
    8. Zhang Y,
    9. Gan J,
    10. Shipkova PA, et al.
    (2019a) Evidence for the validity of pyridoxic acid (PDA) as a plasma-based endogenous probe for OAT1 and OAT3 function in healthy subjects. J Pharmacol Exp Ther 368:136–145.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Shen H,
    2. Scialis RJ, and
    3. Lehman-McKeeman L
    (2019b) Xenobiotic transporters in the kidney: function and role in toxicity. Semin Nephrol 39:159–175.
    OpenUrl
  42. ↵
    1. Shen H,
    2. Yang Z,
    3. Mintier G,
    4. Han YH,
    5. Chen C,
    6. Balimane P,
    7. Jemal M,
    8. Zhao W,
    9. Zhang R,
    10. Kallipatti S, et al.
    (2013) Cynomolgus monkey as a potential model to assess drug interactions involving hepatic organic anion transporting polypeptides: in vitro, in vivo, and in vitro-to-in vivo extrapolation. J Pharmacol Exp Ther 344:673–685.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Suzuki Y,
    2. Ono H,
    3. Tanaka R,
    4. Sato F,
    5. Sato Y,
    6. Ohno K,
    7. Mimata H, and
    8. Itoh H
    (2019) Recovery of OATP1B activity after living kidney transplantation in patients with end-stage renal disease. Pharm Res 36:59.
    OpenUrl
  44. ↵
    1. Takehara I,
    2. Yoshikado T,
    3. Ishigame K,
    4. Mori D,
    5. Furihata KI,
    6. Watanabe N,
    7. Ando O,
    8. Maeda K,
    9. Sugiyama Y, and
    10. Kusuhara H
    (2018) Comparative study of the dose-dependence of OATP1B inhibition by rifampicin using probe drugs and endogenous substrates in healthy volunteers. Pharm Res 35:138.
    OpenUrl
  45. ↵
    1. Tweedie D,
    2. Polli JW,
    3. Berglund EG,
    4. Huang SM,
    5. Zhang L,
    6. Poirier A,
    7. Chu X,
    8. Feng B, and International Transporter Consortium
    (2013) Transporter studies in drug development: experience to date and follow-up on decision trees from the International Transporter Consortium. Clin Pharmacol Ther 94:113–125.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Vlasses PH,
    2. Holbrook AM,
    3. Schrogie JJ,
    4. Rogers JD,
    5. Ferguson RK, and
    6. Abrams WB
    (1980) Effect of orally administered probenecid on the pharmacokinetics of cefoxitin. Antimicrob Agents Chemother 17:847–855.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Vree TB,
    2. van den Biggelaar-Martea M, and
    3. Verwey-van Wissen CP
    (1995) Probenecid inhibits the renal clearance of frusemide and its acyl glucuronide. Br J Clin Pharmacol 39:692–695.
    OpenUrlPubMed
  48. ↵
    1. Welling PG,
    2. Dean S,
    3. Selen A,
    4. Kendall MJ, and
    5. Wise R
    (1979) Probenecid: an unexplained effect on cephalosporin pharmacology. Br J Clin Pharmacol 8:491–495.
    OpenUrlPubMed
  49. ↵
    1. Wiebe ST,
    2. Giessmann T,
    3. Hohl K,
    4. Schmidt-Gerets S,
    5. Hauel E,
    6. Jambrecina A,
    7. Bader K,
    8. Ishiguro N,
    9. Taub ME,
    10. Sharma A, et al.
    (2020) Validation of a drug transporter probe cocktail using the prototypical inhibitors rifampin, probenecid, verapamil, and cimetidine. Clin Pharmacokinet DOI: 10.1007/s40262-020-00907-w [published ahead of print].
  50. ↵
    1. Wolkoff AW,
    2. Wolpert E,
    3. Pascasio FN, and
    4. Arias IM
    (1976) Rotor’s syndrome. A distinct inheritable pathophysiologic entity. Am J Med 60:173–179.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Yasui-Furukori N,
    2. Uno T,
    3. Sugawara K, and
    4. Tateishi T
    (2005) Different effects of three transporting inhibitors, verapamil, cimetidine, and probenecid, on fexofenadine pharmacokinetics. Clin Pharmacol Ther 77:17–23.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Yee SW,
    2. Giacomini MM,
    3. Hsueh CH,
    4. Weitz D,
    5. Liang X,
    6. Goswami S,
    7. Kinchen JM,
    8. Coelho A,
    9. Zur AA,
    10. Mertsch K, et al.
    (2016) Metabolomic and genome-wide association studies reveal potential endogenous biomarkers for OATP1B1. Clin Pharmacol Ther 100:524–536.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Yee SW,
    2. Giacomini MM,
    3. Shen H,
    4. Humphreys WG,
    5. Horng H,
    6. Brian W,
    7. Lai Y,
    8. Kroetz DL, and
    9. Giacomini KM
    (2019) Organic anion transporter polypeptide 1B1 polymorphism modulates the extent of drug-drug interaction and associated biomarker levels in healthy volunteers. Clin Transl Sci 12:388–399.
    OpenUrl
  54. ↵
    1. Yu J,
    2. Petrie ID,
    3. Levy RH, and
    4. Ragueneau-Majlessi I
    (2019) Mechanisms and clinical significance of pharmacokinetic-based drug-drug interactions with drugs approved by the U.S. Food and Drug Administration in 2017. Drug Metab Dispos 47:135–144.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Zhang Y,
    2. Panfen E,
    3. Fancher M,
    4. Sinz M,
    5. Marathe P, and
    6. Shen H
    (2019) Dissecting the contribution of OATP1B1 to hepatic uptake of statins using the OATP1B1 selective inhibitor estropipate. Mol Pharm 16:2342–2353.
    OpenUrl
PreviousNext
Back to top

In this issue

Drug Metabolism and Disposition: 48 (10)
Drug Metabolism and Disposition
Vol. 48, Issue 10
1 Oct 2020
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Index by author
  • Editorial Board (PDF)
  • Front Matter (PDF)
Download PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Drug Metabolism & Disposition article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Detection of Weak Organic Anion–Transporting Polypeptide 1B Inhibition by Probenecid with Plasma-Based Coproporphyrin in Humans
(Your Name) has forwarded a page to you from Drug Metabolism & Disposition
(Your Name) thought you would be interested in this article in Drug Metabolism & Disposition.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Research ArticleArticle

Use of Coproporphyrin to Detect Weak OATP1B Inhibition by PROB

Yueping Zhang, Vinay K. Holenarsipur, Hamza Kandoussi, Jianing Zeng, T. Thanga Mariappan, Michael Sinz and Hong Shen
Drug Metabolism and Disposition October 1, 2020, 48 (10) 841-848; DOI: https://doi.org/10.1124/dmd.120.000076

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero

Share
Research ArticleArticle

Use of Coproporphyrin to Detect Weak OATP1B Inhibition by PROB

Yueping Zhang, Vinay K. Holenarsipur, Hamza Kandoussi, Jianing Zeng, T. Thanga Mariappan, Michael Sinz and Hong Shen
Drug Metabolism and Disposition October 1, 2020, 48 (10) 841-848; DOI: https://doi.org/10.1124/dmd.120.000076
Reddit logo Twitter logo Facebook logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Acknowledgments
    • Authorship Contributions
    • Footnotes
    • Abbreviations
    • References
  • Figures & Data
  • Info & Metrics
  • eLetters
  • PDF + SI
  • PDF

Related Articles

Cited By...

More in this TOC Section

  • PK-PD studies of active ingredient in CH-I
  • IVIVE of aldehyde oxidase-mediated clearance
  • ALTBio Consortium developed for drug metabolism research.
Show more Articles

Similar Articles

Advertisement
  • Home
  • Alerts
Facebook   Twitter   LinkedIn   RSS

Navigate

  • Current Issue
  • Fast Forward by date
  • Fast Forward by section
  • Latest Articles
  • Archive
  • Search for Articles
  • Feedback
  • ASPET

More Information

  • About DMD
  • Editorial Board
  • Instructions to Authors
  • Submit a Manuscript
  • Customized Alerts
  • RSS Feeds
  • Subscriptions
  • Permissions
  • Terms & Conditions of Use

ASPET's Other Journals

  • Journal of Pharmacology and Experimental Therapeutics
  • Molecular Pharmacology
  • Pharmacological Reviews
  • Pharmacology Research & Perspectives
ISSN 1521-009X (Online)

Copyright © 2023 by the American Society for Pharmacology and Experimental Therapeutics