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Research ArticleMetabolism, Transport, and Pharmacogenomics

Coproporphyrins I and III as Functional Markers of OATP1B Activity: In Vitro and In Vivo Evaluation in Preclinical Species

Hong Shen, Jun Dai, Tongtong Liu, Yaofeng Cheng, Weiqi Chen, Chris Freeden, Yingru Zhang, W. Griffith Humphreys, Punit Marathe and Yurong Lai
Journal of Pharmacology and Experimental Therapeutics May 2016, 357 (2) 382-393; DOI: https://doi.org/10.1124/jpet.116.232066

Abstract

Inhibition of organic anion-transporting polypeptide (OATP)1B function can lead to serious clinical drug-drug interactions, thus a thorough evaluation of the potential for this type of interaction must be completed during drug development. Therefore, sensitive and specific biomarkers for OATP function that could be used in conjunction with clinical studies are currently in demand. In the present study, preclinical evaluations were conducted to characterize the suitability of coproporphyrins (CPs) I and III as markers of hepatic OATP functional activity. Active uptake of CPs I and III was observed in human embryonic kidney (HEK) 293 cells singly expressing human OATP1B1 (hOATP1B1), hOATP1B3, cynomolgus monkey OATP1B1 (cOATP1B1), or cOATP1B3, as well as human and monkey hepatocytes. Cyclosporin A (100 mg/kg, oral) markedly increased the area under the curve (AUC) plasma concentrations of CPs I and III by 2.6- and 5.2-fold, while rifampicin (15 mg/kg, oral) increased the AUCs by 2.7- and 3.6-fold, respectively. As the systemic exposure increased, the excretion of both isomers in urine rose from 1.6- to 4.3-fold in monkeys. In agreement with this finding, the AUC of rosuvastatin (RSV) in cynomolgus monkeys increased when OATP1B inhibitors were coadministered. In Oatp1a/1b gene cluster knockout mice (Oatp1a/1b−/−), CPs in plasma and urine were significantly increased compared with wild-type animals (7.1- to 18.4-fold; P < 0.001), which were also in agreement with the changes in plasma RSV exposure (14.6-fold increase). We conclude that CPs I and III in plasma and urine are novel endogenous biomarkers reflecting hepatic OATP function, and the measurements have the potential to be incorporated into the design of early clinical evaluation.

Introduction

Human organic anion-transporting polypeptide (OATP)1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are expressed on the basolateral membrane of hepatocytes and are responsible for the hepatic uptake of numerous drugs and endogenous compounds. Transporter-mediated hepatic uptake could be a rate-determining process in elimination of both metabolically stable and unstable drugs (Kalliokoski and Niemi, 2009; Niemi et al., 2011; Yoshida et al., 2012; Shitara et al., 2013a). Hepatic uptake has clearly been demonstrated to be a potential source of pharmacokinetic variability for drugs such as rosuvastatin (RSV) and atorvastatin (ATV). For example, in 32 healthy volunteers with different SLCO1B1 genotypes dosed with 20 mg ATV, the mean area under the curve (AUC) of ATV was 144% higher in subjects with the 521CC genotype (n = 4) than in subjects with the 521TT genotype (reference genotype; n = 16) (Pasanen et al., 2007), although ATV is mainly eliminated via metabolism mediated by cytochrome P450 enzymes (Neuvonen et al., 2006).

Currently, both the Food and Drug Administration (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM292362.pdf) and the European Medicines Agency (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/07/WC500129606.pdf) require new chemical entities to be characterized for OATP1B inhibition and propose decision trees for assessing OATP1B inhibitors. The Food and Drug Administration and European Medicines Agency recommend using a static mathematical approach to estimate the risk of clinically relevant OATP1B-mediated drug-drug interactions (DDIs) by calculating the ratio of unbound maximum portal vein drug concentration in vivo against in vitro IC50. However, this approach is solely based on in vitro-to-in vivo extrapolation without any in vivo calibration. In addition, the use of this in vitro-to-in vivo extrapolation approach is challenged by the uncertain impact of protein binding–measured, drug absorption, and interlaboratory variability in IC50 assessment using different probe substrate and incubation conditions (Amundsen et al., 2010; Izumi et al., 2013, 2015; Shitara et al., 2013b). Therefore, the availability of endogenous biomarkers to assess transporter activities during early drug development would have substantial benefits for the pharmaceutical industry in order to avoid expensive clinical trials and also minimize the risk of late-stage failures and even drug withdrawal. In this regard, many attempts have been made to identify endogenous compounds that could reflect the activities of enzymes and transporters, such as CYP3A4 (Kanebratt et al., 2008; Diczfalusy et al., 2011; Shin et al., 2013; Kasichayanula et al., 2014), CYP2D6 (Tay-Sontheimer et al., 2014), MATEs (Ito et al., 2012; Kato et al., 2014; Müller et al., 2015), and OAT3 (Imamura et al., 2014) to improve prediction of DDI. However, as far as we know, no validated in vivo clinical endogenous probe of OATP1B has been identified.

Coproporphyrin (CP) is one of the by-products of heme synthesis, and its physiologic role is unclear. CPs I and III are not enzymatically altered in the liver (Kaplowitz et al., 1972), but instead are removed from the body via the bile and urine as intact molecules, albeit unequally. While CP III predominates over CP I in urine, the reverse is encountered in bile (Aziz et al., 1964a,b; French and Thonger, 1966; Koskelo et al., 1966; Koskelo and Toivonen, 1966; Koskelo et al., 1967; Aziz and Watson, 1969; Ben-Ezzer et al., 1971). There are relatively limited data available in the literature regarding CP plasma concentrations and how they change in response to physiologic alterations because of limited sensitivity of the common assays (Table 4). Abnormal distribution of CP isomers was reported in the urine of patients with Rotor syndrome, i.e., a marked preponderance of CP I over CP III (Ben-Ezzer et al., 1971; Wolkoff et al., 1976). In addition, administration of ethinylestradiol and phenodibromophthalein disulfonate was shown to result in a reduction in biliary CP excretion and an increase in urinary CP excretion in rats (Kaplowitz et al., 1972). Ethinylestradiol was later found to be a potent inhibitor of OATPs (De Bruyn et al., 2013) and phenodibromophthalein disulfonate also appears to be an OATP inhibitor (Milne et al., 2000). The genetic basis of Rotor’s syndrome was recently identified to be linked to the mutations that cause complete and simultaneous deficiencies of OATP1B1 and OATP1B3 (van de Steeg et al., 2012). Taken together, these observations suggest that CPs I and III are possible substrates for hepatic OATP transporters and can potentially be used as endogenous biomarkers to assess OATP1B1 and OATP1B3 activities. However, direct evidence of in vivo activity is still lacking. In the present study, we describe a combination of in vitro and in vivo investigations in cynomolgus monkeys and Oatp1a/1b−/− knockout (KO) mice to demonstrate that CPs I and III are suitable endogenous indicators of hepatic OATP activity in animals.

Materials and Methods

Chemicals and Supplies for In vitro and In vivo Experiments

Chemicals.

CP I dihydrochloride, CP III dihydrochloride, and deuteroporphyrin IX dihydrochloride were purchased from Frontier Scientific, Inc. (Logan, UT). Rifampicin (RIF), RSV, and RSV-d6 were purchased from Toronto Research Chemicals Inc. (North York, Ontario, Canada). Estradiol-17β-D-glucuronide (E17βG), cholecystokinin octapeptide (CCK-8), ammonium phosphate, and high-performance liquid chromatography (HPLC) grade methanol and acetonitrile were purchased from Sigma Aldrich (St. Louis, MO). HPLC water was obtained from a Barnstead Nanopure deionizing system (Thermo Scientific, Waltham, MA). Formic acid and concentrated hydrochloric acid (12 N) were obtained from EMD Chemicals Inc. (Gibbstown, NJ). [3H]E17βG (34.3 Ci/mmol) and [3H]CCK-8 (97.5 mCi/mmol) were purchased from Perkin Elmer Life and Analytical Sciences (Waltham, MA). All compounds were of analytical grade (≥95% purity). Stock solutions of CPs and RIF were prepared in dimethylsulfoxide and stored at −70°C.

Cell Culture and Reagents.

Hygromycin and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). Other cell culture media and reagents including Dulbecco’s modified Eagle’s growth medium, fetal bovine serum, nonessential amino acid, L-glutamine, N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid, phosphate-buffered saline, and Hanks’ balanced salt solution were purchased from Corning (Manassas, VA). Biocoat poly-d-lysine 24-well plates were purchased from BD Biosciences (San Jose, CA). Cryopreserved mouse, cynomolgus monkey, and human hepatocytes were purchased from BioreclamationIVT (Baltimore, MD). Pierce BCA protein assay kit was purchased from Thermo Scientific (Rockford, IL).

In vitro Studies

CP Uptake and Inhibition Studies in Cynomolgus Organic Anion-Transporting Polypeptide (cOATP)1B1, cOATP1B3, Human Organic Anion-Transporting Polypeptide (hOATP)1B1, and hOATP1B3 Transfected Human Embryonic Kidney (HEK) 293 Cells.

Recombinant cells expressing human and cynomolgus monkey OATP1B transporters were cultured as described previously (Shen et al., 2013). Uptake was initiated by the addition of transport buffer containing 3 µM CP isomer and 100 µM RIF or dimethylsulfoxide vehicle after washing cells with transport buffer. In control reactions, CP isomer was replaced with 1 μM [3H]E17βG and 0.1 μM [3H]CCK-8 for OATP1B1 and OATP1B3, respectively, to confirm activity of the transporter. The final concentration of dimethylsulfoxide was 0.5% (v/v) in all incubations. Each test condition was assessed in triplicate. After 5 minutes, uptake was terminated by washing cells 3 times with ice-cold transport buffer and the plate was air dried for at least 30 minutes in the fume hood. To measure levels of tritium-labeled probe substrates, cells in the dried plate were lysed with 300 µl 0.1% Triton-X 100. Aliquots of cell lysates (200 µl) were used to count radioactivity using a Tri-Carb 3100TR liquid scintillation counter (PerkinElmer Life Sciences, Boston). Protein concentrations in the cell lysate aliquots (20 µl) were measured using the Pierce BCA protein assay kit (Thermo Scientific). To measure levels of CP isomers, cells in the dried plate were lysed in a 300 µl 3:1 (v/v) ratio of acetonitrile and water at room temperature for 1 hour. The lysate was centrifuged through a 96-well filter plate (0.45 µm low-binding hydrophilic polytetrafluoroethylene) at 3700 rpm for 15 minutes. The filtrate was then diluted with 900 µl 4N hydrochloric acid containing deuteroporphyrin IX as an internal standard, and 50 µl injection was used for HPLC fluorescence detection (FLD) analysis. Quantification of CP by the HPLC-FLD method is described subsequently.

CP Uptake in Human and Monkey Hepatocytes.

Cryopreserved human (Lot NRJ) and cynomolgus monkey hepatocytes (Lot DQA) were processed in a stepwise manner, according to the instructions provided by the manufacturer (BioreclamationIVT). The uptake studies of CPs I and III were conducted as described previously (Morse et al., 2015). In brief, hepatocytes were incubated with the pre-prepared Krebs-Henseleit buffer containing CP isomers (1 µM) or RSV (1 μM, positive control) with or without RIF (as the OATP inhibitor, 100 µM, respectively). At designated time points, the cells were spun down through the oil layer and the hepatocyte pellet was then lysed in a 300 µl 2:1 (v/v) ratio of acetonitrile and 1N hydrochloric acid containing deuteroporphyrin IX as an internal standard, and a 1:1 (v/v) ratio of acetonitrile and water at room temperature, for CP and RSV measurements, respectively. The contents were filtered through a 96-well filter plate (0.45 µm of low-binding hydrophilic polytetrafluoroethylene) and the filtrate was dried under nitrogen. The CPs I and III samples were reconstituted in 10% formic acid (v/v) (100 µl) and a 10 µl sample was injected for HPLC-FLD analysis as described subsequently. The RSV samples were reconstituted in 0.1% formic acid in acetonitrile/water (20:80, v/v) (100 µl) and analyzed by liquid chromatography–tandem mass spectrometry. The stability of the CP isomers in human, monkey, and mouse hepatocytes over 4 hours and in fresh human plasma and urine over 24 hours was examined. A detailed description of cell stability assays is available in the Supplemental Material.

In Vivo Studies

Effect of Cyclosporin A (CsA) Administration on Disposition of CPs in Monkeys.

Plasma and urine samples were obtained from the pharmacokinetic interaction study between CsA (100 mg/kg) and RSV in cynomolgus monkeys conducted previously (Shen et al., 2015). Plasma and urine samples were stored at −70°C until analysis. All animal experiments were approved by the Bristol-Myers Squibb Institutional Animal Care and Use Committee and were performed under the standards recommended by the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).

Effect of RIF Administration on Disposition of CPs I and III in Monkeys.

To assess the effect of a single dose of RIF on plasma concentration and urinary excretion of CPs I and III, a 2-way crossover study was performed in three male cynomolgus monkeys. Male cynomolgus monkeys (4–8 kg) were purchased from Charles River Laboratories (Wilmington, MA), and placed in stainless steel metabolic cages prior to the experiments. The animals were administered with vehicle (i.e., saline) after an overnight fast on day 1, followed by a single oral dose of 15 mg/kg RIF dissolved in saline on day 8 via oral gavage. Serial blood samples (1 ml) were collected from the saphenous vein at 0 (predose), 0.25, 0.5, 0.75, 1, 2, 3, 5, 7, 24, and 48 hours. The plasma samples were obtained, followed by centrifugation at 3,000 g for 10 minutes, and stored at −70°C until analysis. Urine samples were collected 0–7, 7–24, and 24–48 hours after dosing.

Baseline levels in the plasma and urine were obtained from four normal male monkeys. On days 1, 4, and 7, the animals were placed in metabolic cages and serial blood samples (1 ml) were collected from the saphenous vein at 0, 4, and 7 hours following vehicle (i.e., saline) administration. On days 1, 4, and 7, urine samples were collected 0–7 and 7–24 hours after vehicle dosing. The total volumes of urine samples collected during different periods were measured and recorded. Aliquots (10 ml) of urine samples collected from each period were frozen on dry ice and stored at −70°C until analysis.

Disposition of CPs I and III in Oatp1a/1b Gene KO Mice.

Male Oatp1a/1b−/− KO and wild-type (WT) mice (30–40 g) were purchased from Taconic Biosciences (Hudson, NY). Animals were placed in metabolism cages and fasted overnight before dosing. Urine was collected in a 0–24 hour interval to evaluate baseline urinary excretion of CPs I and III.

Plasma concentrations and urine excretion of CPs I and III were characterized in comparison with the change in plasma concentration of RSV following oral administration of 15 mg/kg RSV in saline to three male WT and Oatp1a/1b−/− KO mice. Animals were euthanized at 3 hours postdose by terminal bleeding through cardiac puncture under isoflurane anesthesia, and urine and liver tissues were isolated. The urine samples collected from three animals in each group were pooled and preserved at −70°C until analysis. The liver was dissected and rinsed with phosphate-buffered saline, weighted, and stored at −70°C until analysis.

Analytical Assays

Quantification of CPs I and III by HPLC-FLD.

All samples were kept in the dark and sample processes were carried out by protecting them from light as much as possible. Samples from in vitro cell lysate were treated before HPLC-FLD analysis as described previously. Urine samples (100 µl) were vortex mixed with 10 µl concentrated hydrochloric acid and centrifuged at 12,000 g for 10 minutes. A total of 10 µl of the supernatant was injected for HPLC-FLD analysis. Plasma samples (100 µl) were first acidified with 10 µl concentrated hydrochloric acid, followed by vortex mixing with 250 µl acetonitrile containing deuteroporphyrin IX as an internal standard. After centrifuging at 12,000 g for 10 minutes, the supernatant was dried under nitrogen. Samples were then reconstituted in 50 µl 10% formic acid and 25 µl of the resulting solution was injected for HPLC-FLD analysis.

Chromatographic separations were carried out using an Agilent 1260 Infinity Quaternary LC system equipped with a quaternary pump, an autosampler, a column temperature controller, a diode array detector, and a 1260 Infinity fluorescence detector with an 8 µl biofluorescence flow cell (Agilent Technologies, Santa Clara, CA). The HPLC sample tray was kept at 4°C. The fluorescence signal was monitored at an excitation wavelength of 391 nm and emission wavelength of 621 nm. Data were collected and processed using Agilent Chemstation software. Chromatographic separation was conducted on a 100 mm × 3 mm (particle size 2.5 µm) Synergi Hydro-RP analytical column (Phenomenex, Torrance, CA) by gradient elution at a flow rate of 0.6 ml/min. The gradient HPLC mobile phase A contained 25 mM ammonium phosphate buffer at pH 6, while mobile phase B consisted of a 50/50 (v/v) mixture of methanol and acetonitrile. The elution program was adjusted for different matrices to optimize separation and reduce analysis time. For example, analyses of mouse plasma samples were conducted with an elution program of isocratic at 20% B from 0 to 6 minutes, linear increase from 20% to 35% B from 6 to 20 minutes, and holding at 95% B from 20 to 25 minutes. The column was equilibrated under initial conditions (20% B) for 5 minutes between injections. Column temperature was set at 25°C.

Quantification of RSV by Liquid Chromatography–Tandem Mass Spectrometry.

The cell lysate, plasma, and liver concentrations of RSV were determined by liquid chromatography–tandem mass spectrometry analysis as described previously (Shen et al., 2015).

Data Analysis

Hepatocyte Uptake Data Analysis.

The calculated amounts of compound taken into hepatocytes in the absence and presence of RIF were graphed with mean data (up to 5.0 minutes) (Fig. 2). The initial uptake rate (Vuptake) was calculated based on the rate of uptake during the linear phase (up to 1.0 or 1.5 minutes) (Fig. 2). The uptake clearance (CLu) and percent active uptake were calculated using the following equations:Embedded Image(1)Embedded Image(2)where C is the measured concentration of the test compound in the incubation buffer, and CLu,vehicle and CLu,100 µM RIF are the uptake clearance values in the absence and presence of inhibitor, respectively.

Pharmacokinetic Analysis.

The AUC from time zero to 24 or 48 hours (AUC(0–24 h) or AUC(0–48 h)) was calculated by mixed log-linear trapezoidal summations using validated software (Kinetica version 5.0; Thermo Electron, Philadelphia). Maximum plasma concentrations (Cmax) were directly obtained by observation. The renal clearance (CLR) and renal extraction ratio (ERR) were estimated by the following equations:Embedded Image(3)Embedded Image(4)where Xe is the cumulative amount of CP I or CP III excreted in urine; fu is the fraction of CP unbound in human plasma reported (i.e., 0.28) (Kaplowitz et al., 1972); and GFR is the glomerular filtration rate in monkeys (i.e., 2.1 ml/min/kg) (Davies and Morris, 1993).

Statistical Analysis.

The in vitro data represented the results of triplicates run in one experiment with a minimum of two experiments. The two-tailed unpaired Student’s t test was used to evaluate the statistical significance of differences between two sets of data generated from in vitro studies. The one-sided paired Student’s t test was used to assess the statistical significance of differences between two sets of data in monkey studies because of the crossover design, while the one-sided unpaired Student’s t test was used for mouse studies. To test for statistically significant differences among multiple time points in different days for CP plasma level in monkeys, two-way analysis of variance was performed. To test for statistically significant differences among multiple days for CP urinary excretion in monkeys, one-way analysis of variance was performed. When the F ratio showed that there were significant differences among days, the Dunnett method of multiple comparisons was used to determine which treatments differ. All statistical analyses were performed using Prism version 5.0 (GraphPad Software, Inc., San Diego, CA). Results were presented as the mean ± S.E.M. Differences were considered statistically significant when P < 0.05.

Results

Transport of CPs I and III in HEK 293 Cells Stably Transfected with Monkey and Human OATP1B1 and OATP1B3

A significant increase in the uptake rate of CPs I and III (3 µM) was observed in cOATP1B1-HEK, cOATP1B3-HEK, hOATP1B1-HEK, and hOATP1B3-HEK cells, relative to parental HEK 293 cells (Mock-HEK) (3.1- to 17.4-fold; P < 0.001) (Fig. 1, A and B). OATP1B involvement in the transport of CP was further confirmed by inhibition of the uptake by coincubation of the cells with 100 µM RIF, a potent inhibitor of each of these OATP transporters (Shen et al., 2013, Chu et al., 2015). The validity of the OATP assays was confirmed with commonly used positive controls for monkey and human OATP1B1 ([3H]E17βG) and OATP1B3 ([3H]CCK-8). Probe substrate uptake by the HEK cells expressing OATPs were significantly enhanced compared with MOCK-HEK cells and uptakes were greatly inhibited by 100 µM RIF, which confirmed the functional activity in cellular uptake of OATP substrates.

Fig. 1.
Fig. 1.

CPs I and III transport by HEK 293 cells expressing cOATP1B1, cOATP1B3, hOATP1B1, or hOATP1B3. Uptake of CP I (3 µM) (A), CP III (3 µM) (B), [3H]E17βG (1 μM) (C), and [3H]CCK-8 (0.1 μM) (D) was measured in HEK 293 cells stably transfected with control vector (Mock-HEK) and individual transporter in the absence and presence of RIF (100 μM). The data are expressed as the mean ± S.E.M. of three separate determinations (***P < 0.001, statistically significant difference compared with Mock-HEK cells).

Transport of CPs I and III in Human and Monkey Hepatocytes

To understand hepatic transport of CP, the uptake study with 1 µM CPs I and III was conducted in human and cynomolgus monkey hepatocytes at 37°C in the presence or absence of 100 µM RIF. As shown in Fig. 2, CPs I and III exhibited saturable and time-dependent uptake by human and monkey hepatocytes. The initial linear phase for uptake was maintained for at least 1 minute of incubation in both human and monkey hepatocytes. The initial uptake clearance was comparable for CPs I and III for each species (3.8 versus 5.6 µl/million cells and 8.2 versus 11.7 µl/million cells for human and monkey hepatocytes, respectively), whereas uptake clearance in monkey hepatocyte was about 2-fold higher than that in human. Approximately 75%–85% of CPs I and III uptake in human and monkey hepatocytes was inhibited by RIF, indicating that the entry of CPs into hepatocyte is mainly via active processes mediated by OATP.

Fig. 2.
Fig. 2.

CPs I and III transport by human and monkey hepatocytes. Time course of uptake of CPs (1 µM) and RSV (1 µM) was measured in human (A and B) and monkey hepatocytes (C and D) in the absence and presence of RIF (100 μM). The data are expressed as the mean ± S.E.M. of three separate determinations.

After a 4-hour incubation of CPs I and III (1 µM) with the hepatocytes of the three different species, greater than 97% of CPs I and III remained unchanged. The results suggested that CPs I and III are metabolically stable in human, monkey, and mouse hepatocytes (Supplemental Fig. 2, A–C). CPs I and III were also shown to be stable in human plasma and urine samples (Supplemental Fig. 2D).

CPs I and III Levels in Monkey Plasma and Urine Following the Administration of CsA.

Mean CPs I and III plasma concentrations over time with RSV (3 mg/kg) alone and in combination with CsA (100 mg/kg) in cynomolgus monkeys are shown in Fig. 3A. A significant 3.3-fold increase in CP I Cmax (P = 0.0045) and a 6.9-fold increase in CP III Cmax (P = 0.0003) (Table 1) were observed when codosed. Plasma CPs I and III AUC(0–24 h) values were significantly increased by 2.6-fold from 15.9 ± 2.5 to 41.5 ± 4.7 nM·h and by 5.2-fold from 4.1 ± 1.2 to 20.3 ± 2.8 nM·h (Fig. 3A; Table 1), respectively, whereas the amounts of CPs I and III excreted in urine Xe (0–24 h) were increased by 3.5-fold from 1.4 ± 0.61 to 4.8 ± 1.7 nmol and by 1.6-fold from 17.1 ± 6.1 to 26.6 ± 10.1 nmol (Fig. 2, B and C; Table 1). Consequently, renal clearance of CPs I and III (RSV alone versus the codosed) was not significantly different (1.5 ± 0.75 versus 1.9 ± 0.55 ml/min and 75.5 ± 40.0 versus 21.5 ± 5.2 ml/min, respectively). CP III was the dominant form in urine in both RSV alone and combination groups (92.4% ± 1.4% and 84.5% ± 4.4%, respectively), while CP I was the dominant form in monkey plasma for both groups (79.8% ± 4.7% and 67.7% ± 5.0%, respectively). Although plasma and urine samples were collected over 48 hours, there was no difference in plasma concentration and urinary excretion of CPs I and III beyond 24 hours between groups (Fig. 3, A–C). As a result, the plasma concentrations at 48 hours and urinary excretion during 24–48 hours were not included in the pharmacokinetic analysis.

Fig. 3.
Fig. 3.

Effects of CsA on CP disposition in cynomolgus monkeys. CP plasma concentration-time profiles (A), the 0–7-hour, 7–24-hour, and 24–48-hour urinary excretion rates of CP I (B), CP III (C), and total CP (D) were determined following oral administration of 3 mg/kg RSV alone and 3 mg/kg RSV with 100 mg/kg CsA. CsA was dosed 1 hour ahead of RSV. The data are expressed as the mean ± S.E.M. of three animals (*P < 0.05, statistically significant difference compared with the RSV alone group).

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TABLE 1

Comparison of pharmacokinetic parameters of CPs I and III following oral administration of 3 mg/kg RSV alone and 3 mg/kg RSV with 100 mg/kg CsA in cynomolgus monkeys

When CPs I and III renal clearance was normalized for fraction unbound and functional nephron mass (i.e., GFR), the ERR of CP I in monkeys treated with RSV alone was 0.68 ± 0.32 and was not significantly different from unity, suggesting no involvement of active renal secretion and reabsorption. In contrast, the ERR of CP III was substantially greater in monkeys treated with RSV alone (33.6 ± 17.0), indicating a net secretory mechanism for renal clearance. In the presence of CsA, the ERR of CP I did not change as expected. Although the ERR of CP III was reduced from 33.6 ± 17.0 to 9.7 ± 1.6, the reduction was not statistically different (Table 1).

CPs I and III Levels in Monkey Plasma and Urine Following Administration of RIF

Plasma samples for the determination of CPs I and III concentrations were collected from predose until 48 hours postdose during both vehicle and RIF (15 mg/kg) treatment periods. A significant 2.7 ± 0.75-fold increase in CP I AUC(0–48 h) (P < 0.05) and a 3.6 ± 0.74-fold increase in CP III AUC(0–48 h) (P < 0.01) (Table 2) were observed in RIF-treated monkeys compared with the control animals. There was no difference in CP plasma concentration between the two group animals before administration of RIF (i.e., at predose).

View this table:
TABLE 2

Comparison of pharmacokinetic parameters of CP I and CP III following oral administration of vehicle and 15 mg/kg RIF in cynomolgus monkeys

The amounts of CPs I and III excreted in urine over 48 hours were consistently higher in RIF-treated monkeys compared with the vehicle-treated animals (fold increases of 4.3 ± 1.6 and 3.1 ± 1.5, respectively), and the differences were statistically significant (P < 0.05) (Fig. 4, B–D; Table 2). However, the renal clearance of CPs I and III appeared to be unaffected by RIF. In addition, no significant differences were discerned between the RIF and vehicle groups with respect to the ERR values of CPs I and III (P > 0.05) (Table 2), indicating that the presence of RIF did not interfere with the tubular disposition of CPs. Furthermore, the ERR values of CP I in both treatments did not significantly differ from unity (0.68 ± 0.34 and 1.1 ± 0.40, respectively). On the other hand, the ERR values for CP III were significantly greater than unity, presenting further evidence that this isomer undergoes active tubular secretion in agreement with CsA experiments.

Fig. 4.
Fig. 4.

Effects of RIF on CP disposition in cynomolgus monkeys. CP plasma concentrations (A), the 0–7-hour, 7–24-hour, and 24–48-hour urinary excretion rates of CP I (B) and CP III (C), and total CP (D) were determined following oral administration of vehicle and 15 mg/kg RIF. The data are expressed as the mean ± S.E.M. of three animals (*P < 0.05 and **P < 0.01, statistically significant difference compared with the vehicle control group).

The basal plasma concentrations and urinary excretion of CPs I and III were studied over a 7-day period and the levels were found to be stable throughout the experiment in four untreated animals, as shown in Fig. 5. The mean plasma levels and 24-hour urine excretion of CPs I and III on day 1 did not statistically differ from those on days 4 and 7 (P > 0.05; within a 1.5-fold difference) (Fig. 5). In addition, the degree of intraindividual variability of CPs I and III basal plasma concentrations and urine excretion is low (within 1.9-fold; data not shown).

Fig. 5.
Fig. 5.

Mean plasma concentrations (A) and 24-hour urinary excretion rates of CPs I and III (B) on days 1, 4, and 7 in cynomolgus monkeys. The data are expressed as the mean ± S.E.M. of four animals. There are no significant differences in CP plasma level and urinary excretion among time points and days (P > 0.05).

CPs I and III in Plasma and Urine in the Oatp1a/1b Cluster Genes KO Mice

To further understand the effects of OATP transporters on the disposition of CPs, we compared urinary excretion and plasma concentrations of CPs I and III between WT and Oatp1a/1b−/− KO mice, which are functionally deficient in Oatp1a1, Oatp1a4, Oatp1a5, Oatp1a6, and Oatp1b2. Urinary excretion analyses were performed for both 0–24 and 0–3 hour periods with RSV administration. As shown in Figs. 6 and 7B, the 24- and 3-hour urinary excretion of CPs I and III were markedly increased in Oatp1a/1b−/− KO mice. In this regard, there were approximately 12.4- and 18.4-fold higher Xe (0–24 h) and 7.1- and 12.1-fold higher Xe (0–3 h) values for CPs I and III excretion, respectively (P < 0.001 for 24-hour analyses). In addition, Oatp1a/1b−/− KO mice demonstrated 7.4- and 15.2-fold higher CPs I and III plasma levels at 3 hours post-RSV dose, respectively, compared with WT mice (P < 0.001) (Fig. 7A). Total CP excretion in urine and concentration in plasma of Oatp1a/1b−/− KO mice were 10.8- to 16.7-fold higher than in WT mice (P < 0.001) (Fig. 7, A and B). CP III was the predominant form in both plasma and urine (73%–90%) (Figs. 6 and 7). Interestingly, a similar increase was observed in plasma RSV concentration at 3 hours postdose when comparing Oatp1a/1b−/− KO mice with WT mice (14.6-fold: 289 ± 62.2 versus 19.8 ± 1.3 nM; P < 0.001) (Fig. 7C). However, no significant difference was found in the liver concentrations of RSV at 3 hours postdose between KO and WT mice (Fig. 7D).

Fig. 6.
Fig. 6.

Effects of Oatp1a/1b gene deletion on urinary excretion of CP in mice. The 24-hour urinary excretion rates of CP I, CP III, and total CP were determined in WT and Oatp1a/1b−/− KO mice. The data are expressed as the mean ± S.E.M. of six animals (***P < 0.001, statistically significant difference compared with the WT group).

Fig. 7.
Fig. 7.

Effects of Oatp1a/1b gene deletion on CP and RSV disposition in mice. CP plasma concentrations (A), 3-hour CP urinary excretion (B), RSV plasma concentrations (C), and RSV liver concentrations (D) at 3 hours following oral administration of 15 mg/kg RSV to WT and Oatp1a/1b−/− KO mice were determined. The data are expressed as the mean ± S.E.M. of three animals (***P < 0.001, statistically significant difference compared with the WT group).

Discussion

Although CPs have been suggested as putative endogenous markers of OATP1B activity (Benz-de Bretagne et al., 2011; van de Steeg et al., 2012), no studies to date have reported their in vivo disposition, in comparison with known OATP1B probe substrates, after administration of a potent OATP inhibitor in animal models and humans. In vitro interaction of several porphyrins including CPs I and III with OATP1B1 has been reported (Campbell et al., 2009; Li et al., 2015). In addition, while this paper was in preparation, Bednarczyk and Boiselle (2016) assessed the in vitro transport of CP by OATP1B1 and OATP1B3 using transporter-overexpressing cells. These authors demonstrated that the in vitro transport of CPs I and III by OATP1B1 and OATP1B3 was time dependent, and can be saturated and inhibited. However, it is not entirely clear if there is temporal association between the potential biomarkers and OATP-based DDI in vivo. In addition, the driving force of OATP1B-mediated CP transport has not been investigated in the report and remains unclear in the literature (Hagenbuch and Stieger, 2013). In the present study, the uptake of CPs I and III by hOATP1B1-, hOATP1B3-, cOATP1B1-, and cOATP1B3-HEK cells was inhibited by coincubation with 100 µM RIF, a potent inhibitor of each of these OATP transporters (Shen et al., 2013) (Fig. 1, A and B). Additionally, OATP involvement was confirmed by demonstration of RIF-sensitive uptake in cryopreserved human and monkey hepatocytes. Incubation in the presence of 100 µM RIF, a condition known to deplete active transport by OATP1B1 and OATP1B3 but unlikely by sodium-taurocholate cotransporting polypeptide (IC50 = 277 µM) (Prueksaritanont et al., 2014), reduced the uptake of CP by 75%–85% (Fig. 2).

We have reported that the administration of CsA (100 mg/kg) and RIF (15 mg/kg) to cynomolgus monkeys significantly increased the AUC values of RSV, a well-established exogenous OATP1B probe, by 6.3- and 2.9-fold, respectively, due to OATP inhibition (Shen et al., 2013, 2015). The changes in AUC are comparable to those observed in humans (i.e., 7.1- and 4.4-fold, respectively) (Simonson et al., 2004; Prueksaritanont et al., 2014). In line with the increase in RSV exposure, as seen in Tables 1 and 2, CsA increased CPs I and III plasma exposures by 2.7- and 5.2-fold, and RIF increased those by 2.7- and 3.6-fold, respectively. These observations agree with clinical findings that CsA and RIF caused drug-induced porphyria, which refers to increased levels of circulating porphyrins including CP in patients (Millar, 1980; Hivnor et al., 2003), likely due to the inhibition of OATP1B1 and OATP1B3. Urinary excretion of CP I was increased to a similar extent compared with the changes in CP I AUC (3.5- versus 2.7-fold and 2.7- versus 4.3-fold by CsA and RIF, respectively). However, urinary excretion of CP III was increased to a smaller extent compared with the changes in CP III AUC by CsA but not RIF (1.6- versus 5.2-fold and 3.1- versus 3.6-fold by CsA and RIF, respectively). Apparently, transporter-mediated tubular secretion accounts for most of the renal clearance of CP III in monkeys since the ERR value is significantly greater than unity (approximately 10- to 30-fold) (Tables 1 and 2). CP III constitutes the major form of CP excreted in normal urine in monkeys (Table 3), which has also been observed in humans (Koskelo et al., 1966, 1967; Gebril et al., 1990). The active tubular secretion was unlikely inhibited by RIF and slightly reduced by CsA in this study. As a result, the CP III changes in urine in monkeys are small. These findings indicated that inhibition of OATP function could increase plasma CP concentrations and shunt the elimination pathway to renal excretion. The verification of CP as an OATP biomarker was then extended in Oatp1a/1b−/− KO mice, where similar observations were found for CP plasma levels and urinary excretion. While Oatp1a/1b−/− KO mice showed significant 7.4- and 15.2-fold increases in plasma concentrations of CPs I and III at 3 hours compared with WT animals, respectively, the plasma concentration of RSV at 3 hours following the single oral dose of 15 mg/kg RSV increased by 14.6-fold (Fig. 7, A and C). Urinary excretion values of CPs I and III over 3 and 24 hours are also increased by the disruption of Oatp1a and Oatp1b transporters. However, the effect of Oatp1a and 1b cluster gene deletion on urinary excretion of CP I was less than CP III (7.1- versus 12.1-fold and 12.4- versus 18.4-fold for Xe (0–3 h) and Xe (0–24 h), respectively). Taken together, these results demonstrate a validated assessment of CPs I and III activity using plasma CP concentrations and urinary CP excretion values either with administration of potent OATP1B inhibitor or genetic mutation of OATP genes relative to the well-established exogenous probe. Additionally, the time course of CP plasma concentration and urinary excretion in animal experiments displayed the temporal changes, allowing an assessment of the utility of these indices in studies of varying duration.

View this table:
TABLE 3

Comparison of urinary excretion of CPs I and III in cynomolgus monkeys, mice, and humans

View this table:
TABLE 4

Comparison of plasma profiles of CPs I and III in cynomolgus monkeys, mice, and humans

When the CP renal clearance was corrected by protein binding and renal function (i.e., GFR), the ERR value of CP I approximates unity, whereas that of CP III is substantially greater than unity in untreated monkeys (0.68 ± 0.32 versus 33.6 ± 17.0 and 0.68 ± 0.34 versus 24.1 ± 9.8 for the CsA and RIF studies, respectively) (Tables 1 and 2). These findings suggest that the renal elimination of CP I is likely mediated by glomerular filtration only; in contrast, active tubular secretion plays an important role in CP III renal clearance, although the isomers differ only in the position of the methyl and propionic acid groups in the porphyrin molecule (Supplemental Fig. 1). This observation agrees with other CP disposition studies in which CP III favors urinary excretion over biliary excretion in both humans and animals (Aziz et al., 1964a; Koskelo et al., 1967; Kaplowitz et al., 1972). In addition, CsA, an inhibitor of multiple transporters, reduced the mean CLR and ERR values of CP III in monkeys, while RIF, a relatively specific inhibitor for OATP1B1 and OATP1B3 (Prueksaritanont et al., 2014), did not decrease the mean CLR and ERR values of CP III compared with the vehicle control (62.9 ± 21.8 versus 49.4 ± 5.1 ml/min and 24.1 ± 9.8 versus 18.7 ± 1.7, respectively) (Table 2). Nevertheless, further studies on the characterization of CP excretion in the bile and elucidation of active CP III renal secretion are required. These results suggest that CP I may have better value in vivo as a biomarker of OATP1B compared with CP III because CP I is specific to hepatic uptake and is unlikely to be subject to active renal transport.

The urinary CP isomer ratio has been proposed as a biomarker of MRP2 activity, although there is no in vitro evidence indicating CPs I and III are substrates for MRP2 (Benz-de Bretagne et al., 2011). The proportion of each CP isomer in urine of patients with Dubin-Johnson syndrome (mutation in the ABCC2 gene) differed from normal subjects. CP III predominated in the urine of normal subjects, whereas the proportion of CP I was higher in the urine of patients with Dubin-Johnson syndrome, accounting for approximately 80% of the CP present. However, in terms of absolute amounts, urinary excretion of CP I was significantly increased compared with normal subjects, while the absolute amounts of CP III were decreased (Koskelo et al., 1967; Ben-Ezzer et al., 1971; Wolkoff et al., 1976; Frank et al., 1990; Toh et al., 1999). The mechanism of these changes has not yet been fully worked out. It is worth noting that the clinical relevance of MRP2 inhibition DDI is minimal in humans, with exposure levels reaching 121%–234% of those in normal subjects, depending on the drug considered (Chester et al., 2003; Suwannakul et al., 2008; Ieiri et al., 2009; Brennan et al., 2015; Davenport et al., 2015).

Recently, the specificity of CPs I and III transport was studied using cells transfected with various transporters (Bednarczyk and Boiselle, 2016). Besides OATP1B1- and OATP1B3-mediated transport of CPs I and III, OATP2B1 demonstrated significant transport of CP III, while sodium-taurocholate cotransporting polypeptide, OCT1, OCT2, OAT1, and OAT3 were negative for CP transport. However, CP III appeared to be transported by OAT1 with the ratio of uptake rate of transporter over vector transfected cells of 2.4, while CP I was not transported by OAT1 (Bednarczyk and Boiselle, 2016). Therefore, it is important to reexamine whether renal organic anion transporters OAT1, OAT3, and OATP4C1 contribute to the renal secretion of CP III if a clinical study confirms apparent renal tubular secretion of CP III. Such a study will provide comprehensive understanding of CP disposition and dissect OATP-mediated DDI from other mechanisms underlying CP excretion. Furthermore, the in vivo CP findings from animals need to be carefully translated to humans since CP disposition may not be specific to OATP. Indeed, species-dependent differences in disposition were observed for CP (Tables 3 and 4). While CP III predominated in the urine of monkeys with a mean CP I/CP III ratio of 0.07–0.08, mice and humans excreted relatively more CP I (0.29–0.44 CP I/CP III ratio); therefore, characterization of CP disposition in humans is critical for the use of CP biomarkers for OATP functional inhibition, and the report of clinical studies is forthcoming.

Bilirubin and bile acids are proposed to be biomarkers for hepatic OATP inhibition as suggested by their increased plasma levels in cynomolgus monkeys and rats after administration of RIF (Chu et al., 2015; Watanabe et al., 2015). However, in addition to the biochemical defect leading to reduced hepatic uptake of conjugated and unconjugated bilirubin, other factors such as impaired efflux transporters (MRP2 and MRP3) and enzyme activity (UGT1A1) may also result in the change in bilirubin plasma concentration. Bile acids are associated with other intestinal and hepatic transporters, including the sodium-taurocholate cotransporting polypeptide and bile salt export pump. Furthermore, serum bilirubin and bile acids levels, along with other liver enzymes, are complicated with drug-induced liver injuries (Ozer et al., 2008). In this regard, our results indicated that the CP levels in plasma and urine are independent of changes in liver function; therefore, they are suitable biomarkers for inhibition of hepatic OATP1B used in the clinical setting.

Based on the results presented herein, it is concluded that CP I and CP III can be used as endogenous probes to assess hepatic OATP activity in cynomolgus monkeys and mice. Furthermore, if our findings can indeed be extrapolated to humans, CPs I and III will provide a unique tool for predicting and explaining OATP-based DDIs.

Acknowledgments

The authors thank Dr. Elizabeth Dierks, Dr. Chunlin Chen, and Dr. Jinping Gan for scientific discussions and R. Marcus Fancher for help in conducting the monkey study.

Authorship Contributions

Participated in research design: Shen, Dai, Cheng, Zhang, Humphreys, Marathe, Lai.

Conducted experiments: Shen, Dai, Liu, Cheng, Chen, Freeden.

Contributed new reagents or analytic tools: Shen, Dai, Liu, Lai.

Performed data analysis: Shen, Dai, Lai.

Wrote or contributed to the writing of the manuscript: Shen, Dai, Cheng, Humphreys, Marathe, Lai.

Footnotes

Abbreviations

ATV
atorvastatin
AUC
area under the curve
CCK-8
cholecystokinin octapeptide
CLR
renal clearance
cOATP
cynomolgus organic anion-transporting polypeptide
CP
coproporphyrin
CsA
cyclosporin A
DDI
drug-drug interaction
ERR
renal extraction ratio
E17βG
estradiol-17β-D-glucuronide
FLD
fluorescence detection
GFR
glomerular filtration rate
HEK
human embryonic kidney
hOATP
human organic anion-transporting polypeptide
HPLC
high-performance liquid chromatography
KO
knockout
OATP
organic anion-transporting polypeptide
RIF
rifampicin
RSV
rosuvastatin
WT
wild type
Xe
amount of compound excreted into urine

References

  1. ↵
    1. Amundsen R,
    2. Christensen H,
    3. Zabihyan B, and
    4. Asberg A
    (2010) Cyclosporine A, but not tacrolimus, shows relevant inhibition of organic anion-transporting protein 1B1-mediated transport of atorvastatin. Drug Metab Dispos 38:1499–1504.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Aziz MA,
    2. Schwartz S, and
    3. Watson CJ
    (1964a) Studies of coproporphyrin. VII. Adaptation of the Eriksen paper chromatographic method to the quantitative analysis of the isomers in normal human urine. J Lab Clin Med 63:585–595.
    OpenUrlPubMed
  3. ↵
    1. Aziz MA,
    2. Schwartz S, and
    3. Watson CJ
    (1964b) Studies of coproporphyrin. VIII. Reinvestigation of the isomer distribution in jaundice and liver diseases. J Lab Clin Med 63:596–604.
    OpenUrlPubMed
  4. ↵
    1. Aziz MA and
    2. Watson CJ
    (1969) An analysis of the porphyrins of normal and cirrhotic human liver and normal bile. Clin Chim Acta 26:525–531.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Bednarczyk D and
    2. Boiselle C
    (2016) Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III. Xenobiotica 46:457–466.
    OpenUrlCrossRef
  6. ↵
    1. Ben-Ezzer J,
    2. Rimington C,
    3. Shani M,
    4. Seligsohn U,
    5. Sheba C, and
    6. Szeinberg A
    (1971) Abnormal excretion of the isomers of urinary coproporphyrin by patients with Dubin-Johnson syndrome in Israel. Clin Sci 40:17–30.
    OpenUrlCrossRef
  7. ↵
    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.
  8. ↵
    1. Brennan BJ,
    2. Poirier A,
    3. Moreira S,
    4. Morcos PN,
    5. Goelzer P,
    6. Portmann R,
    7. Asthappan J,
    8. Funk C, and
    9. Smith PF
    (2015) Characterization of the transmembrane transport and absolute bioavailability of the HCV protease inhibitor danoprevir. Clin Pharmacokinet 54:537–549.
    OpenUrlCrossRef
  9. ↵
    1. Campbell SD,
    2. Lau WF, and
    3. Xu JJ
    (2009) Interaction of porphyrins with human organic anion transporting polypeptide 1B1. Chem Biol Interact 182:45–51.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Chester JD,
    2. Joel SP,
    3. Cheeseman SL,
    4. Hall GD,
    5. Braun MS,
    6. Perry J,
    7. Davis T,
    8. Button CJ, and
    9. Seymour MT
    (2003) Phase I and pharmacokinetic study of intravenous irinotecan plus oral ciclosporin in patients with fuorouracil-refractory metastatic colon cancer. J Clin Oncol 21:1125–1132.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Chu X,
    2. Shih SJ,
    3. Shaw R,
    4. Hentze H,
    5. Chan GH,
    6. Owens K,
    7. Wang S,
    8. Cai X,
    9. Newton D,
    10. Castro-Perez J,
    11. et al.
    (2015) Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans. Drug Metab Dispos 43:851–863.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Davenport JM,
    2. Covington P,
    3. Bonifacio L,
    4. McIntyre G, and
    5. Venitz J
    (2015) Effect of uptake transporters OAT3 and OATP1B1 and efflux transporter MRP2 on the pharmacokinetics of eluxadoline. J Clin Pharmacol 55:534–542.
    OpenUrlCrossRef
  13. ↵
    1. Davies B and
    2. Morris T
    (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10:1093–1095.
    OpenUrlCrossRefPubMed
  14. ↵
    1. De Bruyn T,
    2. van Westen GJ,
    3. Ijzerman AP,
    4. Stieger B,
    5. de Witte P,
    6. Augustijns PF, and
    7. Annaert PP
    (2013) Structure-based identification of OATP1B1/3 inhibitors. Mol Pharmacol 83:1257–1267.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Diczfalusy U,
    2. Nylén H,
    3. Elander P, and
    4. Bertilsson L
    (2011) 4β-Hydroxycholesterol, an endogenous marker of CYP3A4/5 activity in humans. Br J Clin Pharmacol 71:183–189.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Frank M,
    2. Doss M, and
    3. de Carvalho DG
    (1990) Diagnostic and pathogenetic implications of urinary coproporphyrin excretion in the Dubin-Johnson syndrome. Hepatogastroenterology 37:147–151.
    OpenUrlPubMed
  17. ↵
    1. French JM and
    2. Thonger E
    (1966) Ether-soluble porphyrins in bile, meconium and urine: a new appraisal by microscale counter-current analysis. Clin Sci 31:337–351.
    OpenUrl
  18. ↵
    1. Gebril M,
    2. Weinkove C,
    3. Ead R,
    4. McDonald K, and
    5. Morton R
    (1990) Plasma porphyrins in chronic renal failure. Nephron 55:159–163.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Hagenbuch B and
    2. Stieger B
    (2013) The SLCO (former SLC21) superfamily of transporters. Mol Aspects Med 34:396–412.
    OpenUrlCrossRefPubMed
    1. Hindmarsh JT,
    2. Oliveras L, and
    3. Greenway DC
    (1999) Plasma porphyrins in the porphyrias. Clin Chem 45:1070–1076.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Hivnor C,
    2. Nosauri C,
    3. James W, and
    4. Poh-Fitzpatrick M
    (2003) Cyclosporine-induced pseudoporphyria. Arch Dermatol 139:1373–1374.
    OpenUrlPubMed
  22. ↵
    1. Ieiri I,
    2. Higuchi S, and
    3. Sugiyama Y
    (2009) Genetic polymorphisms of uptake (OATP1B1, 1B3) and efflux (MRP2, BCRP) transporters: implications for inter-individual differences in the pharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs. Expert Opin Drug Metab Toxicol 5:703–729.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Imamura Y,
    2. Tsuruya Y,
    3. Damme K,
    4. Heer D,
    5. Kumagai Y,
    6. Maeda K,
    7. Murayama N,
    8. Okudaira N,
    9. Kurihara A,
    10. Izumi T,
    11. et al.
    (2014) 6β-Hydroxycortisol is an endogenous probe for evaluation of drug-drug interactions involving a multispecific renal organic anion transporter, OAT3/SLC22A8, in healthy subjects. Drug Metab Dispos 42:685–694.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Ito S,
    2. Kusuhara H,
    3. Kumagai Y,
    4. Moriyama Y,
    5. Inoue K,
    6. Kondo T,
    7. Nakayama H,
    8. Horita S,
    9. Tanabe K,
    10. Yuasa H,
    11. et al.
    (2012) N-methylnicotinamide is an endogenous probe for evaluation of drug-drug interactions involving multidrug and toxin extrusions (MATE1 and MATE2-K). Clin Pharmacol Ther 92:635–641.
    OpenUrlCrossRefPubMed
  25. ↵
    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
  26. ↵
    1. Izumi S,
    2. Nozaki Y,
    3. Maeda K,
    4. Komori T,
    5. Takenaka O,
    6. Kusuhara H, and
    7. Sugiyama Y
    (2015) Investigation of the impact of substrate selection on in vitro organic anion transporting polypeptide 1B1 inhibition profiles for the prediction of drug-drug interactions. Drug Metab Dispos 43:235–247.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Kalliokoski A and
    2. Niemi M
    (2009) Impact of OATP transporters on pharmacokinetics. Br J Pharmacol 158:693–705.
    OpenUrlCrossRefPubMed
    1. Kanayama N,
    2. Yamazaki T,
    3. Naruse H,
    4. Sumimoto K,
    5. Horiuchi K, and
    6. Terao T
    (1992) Determining zinc coproporphyrin in maternal plasma—a new method for diagnosing amniotic fluid embolism. Clin Chem 38:526–529.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Kanebratt KP,
    2. Diczfalusy U,
    3. Bäckström T,
    4. Sparve E,
    5. Bredberg E,
    6. Böttiger Y,
    7. Andersson TB, and
    8. Bertilsson L
    (2008) Cytochrome P450 induction by rifampicin in healthy subjects: determination using the Karolinska cocktail and the endogenous CYP3A4 marker 4β-hydroxycholesterol. Clin Pharmacol Ther 84:589–594.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Kaplowitz N,
    2. Javitt N, and
    3. Kappas A
    (1972) Coproporphyrin I and 3 excretion in bile and urine. J Clin Invest 51:2895–2899.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Kasichayanula S,
    2. Boulton DW,
    3. Luo WL,
    4. Rodrigues AD,
    5. Yang Z,
    6. Goodenough A,
    7. Lee M,
    8. Jemal M, and
    9. LaCreta F
    (2014) Validation of 4β-hydroxycholesterol and evaluation of other endogenous biomarkers for the assessment of CYP3A activity in healthy subjects. Br J Clin Pharmacol 78:1122–1134.
    OpenUrlCrossRef
  32. ↵
    1. Kato K,
    2. Mori H,
    3. Kito T,
    4. Yokochi M,
    5. Ito S,
    6. Inoue K,
    7. Yonezawa A,
    8. Katsura T,
    9. Kumagai Y,
    10. Yuasa H,
    11. et al.
    (2014) Investigation of endogenous compounds for assessing the drug interactions in the urinary excretion involving multidrug and toxin extrusion proteins. Pharm Res 31:136–147.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Koskelo P,
    2. Eisalo A, and
    3. Toivonen I
    (1966) Urinary excretion of porphyrin precursors and coproporphyrin in healthy females on oral contraceptives. BMJ 1:652–654.
    OpenUrlFREE Full Text
  34. ↵
    1. Koskelo P and
    2. Toivonen I
    (1966) Separation of urinary coproporphyrin isomers 1 and 3 by thin-layer chromatography. Studies in healthy subjects and patients with myocardial infarction. Scand J Clin Lab Invest 18:543–549.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Koskelo P,
    2. Toivonen I, and
    3. Adlercreutz H
    (1967) Urinary coproporphyrin isomer distribution in the Dubin-Johnson syndrome. Clin Chem 13:1006–1009.
    OpenUrlAbstract
  36. ↵
    1. Li XL,
    2. Guo ZT,
    3. Wang YD,
    4. Chen XY,
    5. Liu J, and
    6. Zhong DF
    (2015) Potential role of organic anion transporting polypeptide 1B1 (OATP1B1) in the selective hepatic uptake of hematoporphyrin monomethyl ether isomers. Acta Pharmacol Sin 36:268–280.
    OpenUrlCrossRef
  37. ↵
    1. Millar JW
    (1980) Rifampicin-induced porphyria cutanea tarda. Br J Dis Chest 74:405–408.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Milne RW,
    2. Larsen LA,
    3. Jørgensen KL,
    4. Bastlund J,
    5. Stretch GR, and
    6. Evans AM
    (2000) Hepatic disposition of fexofenadine: influence of the transport inhibitors erythromycin and dibromosulphothalein. Pharm Res 17:1511–1515.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Morse BL,
    2. Cai H,
    3. MacGuire JG,
    4. Fox M,
    5. Zhang L,
    6. Zhang Y,
    7. Gu X,
    8. Shen H,
    9. Dierks EA,
    10. Su H,
    11. et al.
    (2015) Rosuvastatin liver partitioning in cynomolgus monkeys: measurement in vivo and prediction using in vitro monkey hepatocyte uptake. Drug Metab Dispos 43:1788–1794.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Müller F,
    2. Pontones CA,
    3. Renner B,
    4. Mieth M,
    5. Hoier E,
    6. Auge D,
    7. Maas R,
    8. Zolk O, and
    9. Fromm MF
    (2015) N1-methylnicotinamide as an endogenous probe for drug interactions by renal cation transporters: studies on the metformin-trimethoprim interaction. Eur J Clin Pharmacol 71:85–94.
    OpenUrlCrossRefPubMed
  41. ↵
    National Research Council (2011) Guide for The Care and Use of Laboratory Animals, 8th ed, The National Academies Press, Washington, DC.
  42. ↵
    1. Neuvonen PJ,
    2. Niemi M, and
    3. Backman JT
    (2006) Drug interactions with lipid-lowering drugs: mechanisms and clinical relevance. Clin Pharmacol Ther 80:565–581.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Niemi M,
    2. Pasanen MK, and
    3. Neuvonen PJ
    (2011) Organic anion transporting polypeptide 1B1: a genetically polymorphic transporter of major importance for hepatic drug uptake. Pharmacol Rev 63:157–181.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Ozer J,
    2. Ratner M,
    3. Shaw M,
    4. Bailey W, and
    5. Schomaker S
    (2008) The current state of serum biomarkers of hepatotoxicity. Toxicology 245:194–205.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Pasanen MK,
    2. Fredrikson H,
    3. Neuvonen PJ, and
    4. Niemi M
    (2007) Different effects of SLCO1B1 polymorphism on the pharmacokinetics of atorvastatin and rosuvastatin. Clin Pharmacol Ther 82:726–733.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Prueksaritanont T,
    2. Chu X,
    3. Evers R,
    4. Klopfer SO,
    5. Caro L,
    6. Kothare PA,
    7. Dempsey C,
    8. Rasmussen S,
    9. Houle R,
    10. Chan G,
    11. et al.
    (2014) Pitavastatin is a more sensitive and selective organic anion-transporting polypeptide 1B clinical probe than rosuvastatin. Br J Clin Pharmacol 78:587–598.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Shen H,
    2. Su H,
    3. Liu T,
    4. Yao M,
    5. Mintier G,
    6. Li L,
    7. Fancher RM,
    8. Iyer R,
    9. Marathe P,
    10. Lai Y,
    11. et al.
    (2015) Evaluation of rosuvastatin as an organic anion transporting polypeptide (OATP) probe substrate: in vitro transport and in vivo disposition in cynomolgus monkeys. J Pharmacol Exp Ther 353:380–391.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    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,
    11. 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
  49. ↵
    1. Shin KH,
    2. Choi MH,
    3. Lim KS,
    4. Yu KS,
    5. Jang IJ, and
    6. Cho JY
    (2013) Evaluation of endogenous metabolic markers of hepatic CYP3A activity using metabolic profiling and midazolam clearance. Clin Pharmacol Ther 94:601–609.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Shitara Y,
    2. Maeda K,
    3. Ikejiri K,
    4. Yoshida K,
    5. Horie T, and
    6. Sugiyama Y
    (2013a) Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption. Biopharm Drug Dispos 34:45–78.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Shitara Y,
    2. Takeuchi K, and
    3. Horie T
    (2013b) Long-lasting inhibitory effects of saquinavir and ritonavir on OATP1B1-mediated uptake. J Pharm Sci 102:3427–3435.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Simonson SG,
    2. Raza A,
    3. Martin PD,
    4. Mitchell PD,
    5. Jarcho JA,
    6. Brown CD,
    7. Windass AS, and
    8. Schneck DW
    (2004) Rosuvastatin pharmacokinetics in heart transplant recipients administered an antirejection regimen including cyclosporine. Clin Pharmacol Ther 76:167–177.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Suwannakul S,
    2. Ieiri I,
    3. Kimura M,
    4. Kawabata K,
    5. Kusuhara H,
    6. Hirota T,
    7. Irie S,
    8. Sugiyama Y, and
    9. Higuchi S
    (2008) Pharmacokinetic interaction between pravastatin and olmesartan in relation to SLCO1B1 polymorphism. J Hum Genet 53:899–904.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Tay-Sontheimer J,
    2. Shireman LM,
    3. Beyer RP,
    4. Senn T,
    5. Witten D,
    6. Pearce RE,
    7. Gaedigk A,
    8. Gana Fomban CL,
    9. Lutz JD,
    10. Isoherranen N,
    11. et al.
    (2014) Detection of an endogenous urinary biomarker associated with CYP2D6 activity using global metabolomics. Pharmacogenomics 15:1947–1962.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Toh S,
    2. Wada M,
    3. Uchiumi T,
    4. Inokuchi A,
    5. Makino Y,
    6. Horie Y,
    7. Adachi Y,
    8. Sakisaka S, and
    9. Kuwano M
    (1999) Genomic structure of the canalicular multispecific organic anion–transporter gene (MRP2/cMOAT) and mutations in the ATP-binding–cassette region in Dubin-Johnson syndrome. Am J Hum Genet 64:739–746.
    OpenUrlCrossRefPubMed
  56. ↵
    1. van de Steeg E,
    2. Stránecký V,
    3. Hartmannová H,
    4. Nosková L,
    5. Hřebíček M,
    6. Wagenaar E,
    7. van Esch A,
    8. de Waart DR,
    9. Oude Elferink RP,
    10. Kenworthy KE,
    11. et al.
    (2012) Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J Clin Invest 122:519–528.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Watanabe T,
    2. Miyake M,
    3. Shimizu T,
    4. Kamezawa M,
    5. Masutomi N,
    6. Shimura T, and
    7. Ohashi R
    (2015b) Utility of bilirubins and bile acids as endogenous biomarkers for the inhibition of hepatic transporters. Drug Metab Dispos 43:459–466.
    OpenUrlAbstract/FREE Full Text
  58. ↵
    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
  59. ↵
    1. Yoshida K,
    2. Maeda K, and
    3. Sugiyama Y
    (2012) Transporter-mediated drug–drug interactions involving OATP substrates: predictions based on in vitro inhibition studies. Clin Pharmacol Ther 91:1053–1064.
    OpenUrlCrossRefPubMed
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Research ArticleMetabolism, Transport, and Pharmacogenomics

Coproporphyrin as Endogenous In Vivo Probe for OATP1B

Hong Shen, Jun Dai, Tongtong Liu, Yaofeng Cheng, Weiqi Chen, Chris Freeden, Yingru Zhang, W. Griffith Humphreys, Punit Marathe and Yurong Lai
Journal of Pharmacology and Experimental Therapeutics May 1, 2016, 357 (2) 382-393; DOI: https://doi.org/10.1124/jpet.116.232066
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