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Research ArticleMetabolism, Transport, and Pharmacogenomics

Coproporphyrins in Plasma and Urine Can Be Appropriate Clinical Biomarkers to Recapitulate Drug-Drug Interactions Mediated by Organic Anion Transporting Polypeptide Inhibition

Yurong Lai, Sandhya Mandlekar, Hong Shen, Vinay K. Holenarsipur, Robert Langish, Prabhakar Rajanna, Senthilkumar Murugesan, Nilesh Gaud, Sabariya Selvam, Onkar Date, Yaofeng Cheng, Petia Shipkova, Jun Dai, William G. Humphreys and Punit Marathe
Journal of Pharmacology and Experimental Therapeutics September 2016, 358 (3) 397-404; DOI: https://doi.org/10.1124/jpet.116.234914

Abstract

In the present study, an open-label, three-treatment, three-period clinical study of rosuvastatin (RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12 male healthy subjects to determine if coproporphyrin I (CP-I) and coproporphyrin III (CP-III) could serve as clinical biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 that belong to the solute carrier organic anion gene subfamily. Genotyping of the human OATP1B1 gene was performed in all 12 subjects and confirmed absence of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III prior to drug administration were 0.91 ± 0.21 and 0.15 ± 0.04 nM, respectively, with minimum fluctuation over the three periods. CP-I was passively eliminated, whereas CP-III was actively secreted from urine. Administration of RSV caused no significant changes in the plasma and urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration (Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as compared with the predose values. The area under the plasma concentration curves from time 0 to 24 h (AUC0–24h) of CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively, when compared with RSV alone. In agreement with this finding, Cmax and AUC0–24h of RSV increased by 13.2- and 5.0-fold, respectively, when RIF was coadministered. Collectively, we conclude that CP-I and CP-III in plasma and urine can be appropriate endogenous biomarkers specifically and reliably reflecting OATP inhibition, and thus the measurement of these molecules can serve as a useful tool to assess OATP drug-drug interaction liabilities in early clinical studies.

Introduction

Human hepatic organic anion-transporting polypeptides (OATPs) are expressed on the basolateral membrane of hepatocytes and are responsible for the hepatic uptake of numerous drugs and endogenous compounds. These transporters are the most important players in the disposition of a wide range of drugs with active hepatic uptake as the rate-determining step for drug clearance (Kalliokoski and Niemi, 2009; Niemi et al., 2011; Yoshida et al., 2012; Shitara et al., 2013a; Varma et al., 2015). OATP inhibition can cause drug-drug interactions (DDIs) leading to drug attrition or limitation of use in the clinic for both metabolically stable and unstable drugs. Understanding the potential pharmacokinetic (PK) interactions of a new chemical entity with commonly administered comedications is important for patient safety and is required as part of the regulatory approval process for drugs unless OATP inhibition can be definitively ruled out based on preclinical experiments.

Per the current regulatory guidance, the approach for detecting the potential for OATP-mediated drug interactions for a new chemical entity in the clinic is to conduct a drug-interaction study using a probe substrate of OATP, such as rosuvastatin (RSV). In general, whether to conduct these expensive clinical DDI trials is determined by a static mathematical approach through computing the ratio of unbound maximum portal vein inhibitor concentration in vivo for the new chemical entity against the in vitro OATP IC50. Although the current approaches in the regulatory guidance are likely sufficient to minimize the chance of a false-negative interaction, the potential for a high-rate of false-positive predictions has been a particular concern in the pharmaceutical industry (Prueksaritanont et al., 2013; Tweedie et al., 2013). The estimation is challenged or compromised by the uncertainty of protein binding values and the rate and fraction of oral absorption, as well as the mechanisms of transporter inhibition, e.g., long-lasting effects (Shitara et al., 2013b). Challenges also remain in our ability to overcome limitations of in vitro IC50 assessment using different probe substrates, incubation conditions, and variability when using different expression systems (Amundsen et al., 2010; Izumi et al., 2013, 2015; Shitara et al., 2013b).

From the industry perspective, the potential PK interactions should be addressed as early in drug development as possible, allowing important plans and decisions to be made about compound selection for clinical development prior to significant investment of late-phase clinical trials. In particular, the dose selection remains a dilemma in the design of a clinical DDI study, when the clinically efficacious dose is yet to be defined. Collectively, to avoid expensive false-positive clinical trials or the risk of late-stage failures, sensitive and specific endogenous biomarkers that can be measured during phase I dose-escalation trials would have substantial benefits for the pharmaceutical industry.

Previously, we conducted preclinical studies to investigate coproporphyrin I and III (CP-I and CP-III) in plasma and in urine as markers of OATP activity. CP-I and III are porphyrin metabolites arising from heme synthesis, and appear to be substrates for human and monkey OATP1B1 and 1B3 (Bednarczyk and Boiselle, 2015; Shen et al., 2016a). They are stable in the systemic circulation and in metabolically active tissues, (e.g. hepatocytes) (Bednarczyk and Boiselle, 2015; Shen et al., 2016a) and eliminated in bile and urine as intact forms (Aziz et al., 1964a, b; French and Thonger, 1966; Koskelo et al., 1966, 1967; Koskelo and Toivonen, 1966; Aziz and Watson, 1969; Ben-Ezzer et al., 1971; Kaplowitz et al., 1972). Changes in elimination of CPs in the urine are found to be related to hepatic transporter function. For example, Rotor’s syndrome, a genetic human disease with complete and simultaneous deficiencies of OATP1B1 and OATP1B3 (van de Steeg et al., 2012), is diagnosed by a marked preponderance of CP-I over CP-III in the urine (Ben-Ezzer et al., 1971; Wolkoff et al., 1976). In cynomolgus monkeys, the area under the plasma-concentration curves (AUCs) of CP-I and CP-III is markedly increased following the administration of OATP inhibitors, cyclosporine A, or rifampicin (RIF). The results suggest that both CP-I and CP-III in plasma and urine may be novel clinical endogenous biomarkers for assessing OATP-mediated DDIs.

In the present study, an open-label, three-treatment, three-period oral comparative bioavailability study was conducted to elucidate whether CP-I and CP-III in plasma and urine are sensitive, specific, and reliable probes reflecting hepatic OATP inhibition. RSV was administered in one arm as a sensitive probe for OATP function, and RIF was administered in two arms as a potent inhibitor of OATP function.

Materials and Methods

Chemicals and Drugs.

Coproporphyrin I dihydrochloride (97%) and coproporphyrin III dihydrochloride (97%) were purchased from Frontier Scientific Inc. (Logan, UT). Isotopically labeled CP-I sodium bisulfate salt (15N4-CP-I, 98%), used as an internal standard, was purchased from Toronto Research Chemicals (North York, Canada). Pooled, stock human plasma (3× charcoal stripped) was purchased from BioreclamationIVT (Westbury, NY). High-performance liquid chromatography (HPLC) grade acetonitrile, HPLC grade water, ethyl acetate, formic acid, and bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO). Artificial urine was purchased from Pickering Laboratories (Mountainview, CA). HPLC grade methanol, formic acid, and acetonitrile were purchased from Sigma-Aldrich. HPLC water was obtained from a Barnstead Nanopure deionizing system (Thermo Scientific, Waltham, MA).

RIF 600-mg capsules (RCIN) were obtained from Lupin Pharmaceuticals (Baltimore, MD). RSV (Crestor) 5-mg tablets were from AstraZeneca (Bangalore, India). Analytical reference standard for RIF was purchased from Angene International (London, UK) and for RSV from Apollo Scientific Limited (Stockport, UK). Control human plasma and human urine for the preparation of calibration standards and quality controls of RIF and RSV were procured from Syngene International Limited (Bangalore, India). Ritonavir was from USP (Rockville, MD). All other reagents and solvents were of HPLC grade, unless specified, and purchased from Sigma-Aldrich Corporation (Bangalore, India). All compounds were of analytical grade (≥95% purity).

Subject Selection.

Healthy male volunteers aged between 18 and 45 years with normal body mass index (18.50–24.99 kg/m2), minimum weight of 50 kg, and no clinically relevant conditions identified from the medical history, physical examination, electrocardiography, or chest X-ray were eligible for inclusion. Volunteers were excluded if any clinically relevant laboratory abnormality was identified in clinical chemistry tests (including tests of hepatic and renal biochemistry), hematology tests, urinalysis, or if values for total bilirubin, alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase were outside the normal reference ranges at the start of the trial.

Clinical Study Design.

This was an open-label, three-fixed-treatment, three-period, single-dose crossover study in 12 healthy, male, Indian, adult subjects under fasting conditions. The study protocol and the informed consent were reviewed and approved by the institutional independent ethics committee of Syngene International Ltd. The study was conducted in accordance with relevant Syngene clinical development standard operating procedures, International Council for Harmonisation “Guidance on Good Clinical Practice,” (http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R2__Addendum_Step2.pdf) Declaration of Helsinki (http://www.wma.net/en/30publications/10policies/b3/17c.pdf), Central Drugs Standard Control Organization guidelines (http://www.cdsco.nic.in/writereaddata/cdsco-guidanceforindustry.pdf), Indian Council of Medical Research guidelines (http://www.icmr.nic.in/ethical_guidelines.pdf), and other applicable regulatory requirements. The clinical part of the study was conducted at one clinical site (Human Pharmacology Unit, Syngene International Ltd. Clinical Development, Electronics City, Phase-II, Bangalore, India). Subjects agreed to refrain from use of any medicines for 14 days preceding the study. Each subject provided written informed consent prior to initiation of study procedures.

The trial consisted of three periods with a 7-day washout between periods. Volunteers received 600-mg RIF capsules, 5-mg RSV tablets, or 600-mg RIF capsules plus 5-mg RSV tablets in period 1, 2, and 3, respectively. In each period, investigational products were administered orally with 240 ml of water after an overnight fast of at least 10 hours. A standardized meal was provided 4 hours postdose. Water was provided ad libitum except for 1 hour before and 1 hour after dose administration. In each period of the study, 12 blood samples (∼3 ml) were collected predose and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 hours postdose. Samples were collected into tubes containing K2EDTA as anticoagulant and centrifuged (2600g at 4°C for 10 minutes). The plasma was separated into two aliquots. Fifty microliters of 1 M ammonium acetate buffer (pH 5.0) was added to one aliquot consisting of 500 μl of plasma and mixed for the measurements of RSV and RIF concentrations in the plasma. The other aliquot was used for CP-I and CP-III measurements. All plasma samples were stored at −70 ± 10°C until analysis.

At predose, about 120 µl of blood was collected from each subject on FTA elute cards (GE Life Sciences, Buckinghamshire, UK) as a dry blood spot, and the cards were dried for 2 hours under ambient conditions. These samples were used for genotyping SLCO1B1 polymorphism (A388 > G and T521 > C).

Urine samples were collected in each period during predose (−7 to 0 hours) and at about 0–7 and 7–24 hours postdose. Multiple urine samples in each interval were pooled, and the total volume of urine in each interval was recorded. A 20-ml aliquot of urine sample was transferred into two polypropylene tubes and stored at −70°C until analysis.

Safety and tolerability were assessed with clinical evaluations, which included a physical examination and laboratory assessments. Total and direct bilirubin in plasma were measured by an AU 400 automated clinical chemistry analyzer (Beckman Coulter, Brea, CA) according to the manufacturer’s instructions. Indirect bilirubin was calculated by subtracting direct bilirubin from total bilirubin. Adverse experiences were monitored throughout the study.

Quantification of CP-I and CP-III by Liquid Chromatography–Tandem Mass Spectrometry.

All samples were kept from light exposure as much as possible during sample preparation. Urine samples were diluted using artificial urine (Pickering Laboratories) with 0.1% bovine serum albumin to ensure that the mass spectrometry response was within the range of the calibration curve. Urine or plasma aliquots (100 µl) were transferred to a 1-ml 96-well plate and then mixed with 50 µl of internal standard solution (1.5 nM 15N4-CP-I in 12 M formic acid for plasma samples, 2.5 nM 15N4-CP-I in 6 M formic acid for urine samples) and 500 µl of ethyl acetate. After vortex mixing, the plate was centrifuged at 4000 rpm for 15 minutes. The supernatant (380 µl) was transferred to a 1-ml 96-well plate and dried using a nitrogen plate dryer with a heat setting of 55°C. Samples were then reconstituted with 60 µl of 1 M formic acid for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

The sample analysis was conducted on a SCIEX 5500 tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Toronto, Canada) coupled to a UPLC (Ultra High Performance Liquid Chromatography) FLUX pump and an HTS PAL autosampler (CTC Analytics, Zwingen, Switzerland). Samples (10 µl) were injected onto an Ace Excel 2 C18 2.1 × 150 mm UHPLC column (particle size 1.7 µm; Advanced Chromatography Technologies, Aberdeen, Scotland) and eluted by a gradient program of 20% B to 60% B in 4 minutes, 60% B to 100% B in 0.5 minute, and held 100% B for 0.5 minute. The mobile phase was a mixture of 0.1% formic acid in water (A) and 98% acetonitrile in water containing 0.1% formic acid (B). The column temperature was maintained at 65°C, and the flow rate was 0.5 ml/min. The mass spectrometer was operated in positive, multiple reaction monitoring (MRM) mode. The MRM precursor/product ion transitions were as follows: m/z 655.3 > 596.3 for CP-I and -III, and m/z 659.3 > 600.3 for the internal standard, 15N4-CP-I. The instrument settings on the mass spectrometer were as follows: declustering potential, 130 V; collision energy, 65 V; and dwell time, 25 ms. All peak integration and data processing were performed using SCIEX Analyst 1.6.2 (Applied Biosystems/MDS SCIEX). The LC-MS/MS method validation and quality controls are presented in the Supplemental Material.

Quantification of RSV and RIF by LC-MS/MS.

Sample extraction for RSV and RIF was conducted in 96-well plates using protein precipitation with acetonitrile. In brief, plasma and urine samples (30 µl) for RSV measurement were mixed with 125 µl of ice-cold acetonitrile containing 200 nM ritonavir as an internal standard in a hydrophilic solvinert plate (Millipore Corporation, Billerica, MA). Similarly, an aliquot of 20 µl of plasma and urine samples (50× diluted in control plasma) for RIF measurement was mixed with 180 µl of ice-cold acetonitrile containing 200 nM ritonavir in a hydrophilic solvinert plate. Samples were vortexed and centrifuged at 4°C and 4000 rpm for 5 minutes. The supernatant (5 µl for RSV measurement and 3 µl for RIF measurement) was injected onto LC-MS/MS.

The liquid chromatography system, ACQUITY UHPLC, consisted of ACQUITY binary solvent manager and ACQUITY sample manager with sample organizer (Waters Corporation, Milford, MA). Chromatographic separation was achieved by gradient elution on an ACQUITY C18 BEH, 1.7-µm, 2.1*50-mm column (Waters Corporation) maintained at 40°C. The mobile phase was a mixture of 0.1% formic acid in 10 mM ammonium formate (A) and 0.1% formic acid in acetonitrile (B). The gradient for RSV was set as follows: 10% B to 95% B in 2 minutes, held 95% B for 0.4 minute, 95% B to 10% B in 0.1 minute, and held 10% B for 0.5 minute. The flow rate was 0.6 ml/min. The gradient for RIF was set as follows: 2% B to 50% B in 0.5 minute, 50% B to 100% B in 0.01 minute, held 100% B for 0.09 minute, 100% B to 2% B in 0.05 minute, and held 2% B for 0.35 minute. The flow rate was 0.8 ml/min.

Mass spectrometric detection for RSV and RIF was performed on an AB Sciex 5500 QTRAP and 4000 QTRAP (Applied Biosystems, Foster City, CA), respectively, equipped with electrospray ionization source. The mass spectrometer was operated in positive ion mode, and MRM transitions were used for RSV (m/z 482.1 > 258.07) and RIF (m/z 823.4 > 791.3) detection. The instrument settings on the mass spectrometers were as follows: ion spray voltage, 5.5 kV; temperature, 550°C; declustering potential, 100 V; collision energy, 47 V and 25 V (for RSV and RIF, respectively); entrance potential, 10 V; and collision cell exit potential, 15 V. Peak integration and data processing were performed using Analyst version 1.6.2 (Applied Biosystems). The LC-MS/MS method validation and quality controls are presented in the Supplemental Material.

Identification of Variants in OATP1B1 Gene.

Blood from each subject was spotted on FTA Elute Micro Cards and dried. One 6-mm sample from each spot was punched out and placed in a 1.5-ml sterile centrifuge tube, to which 500 µl of nuclease-free water was added and vortexed for about 5 seconds to rinse the punch. Water was removed using a sterile pipette followed by the addition of 100 µl of nuclease-free water and heating at 95°C for 30 minutes. The samples were removed from the heat block and vortexed for 30 seconds. The samples were centrifuged for 30 seconds to separate the matrix and eluate containing purified DNA. The eluate was then transferred into a fresh tube.

Single nucleotide polymorphism (SNP) probe sets (rs2306283 for Asn130Asp and rs4149056 for Val174Ala for the OATP1B1 gene) were procured from Thermo Fisher Scientific (Grand Island, NY). Human PPIA (cyclophilin A) endogenous control probe mix (NM_021130.3) was used as a positive control for each sample. Taqman assay was performed using 5 µl of iQ multiplex powermix (Bio-Rad, Hercules, CA), 1 µl of DNA eluate from each sample, 0.5 µl of probe mix, and 3.5 µl of nuclease-free water. Polymerase chain reactions were conducted in triplicate for each probe set in a 384-well hard-shell plate using the CFX 384 real time polymerase chain reaction detection system from Bio-Rad. Reaction conditions were as follows: enzyme activation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 1 minute and for a total of 50 cycles. The probes contained a fluorescent reporter dye (VIC specific for allele 1, and FAM specific for allele 2) attached to its 5′ end and a quencher dye at its 3′ end. Allelic discrimination was analyzed based on relative fluorescence from the probe sets using CFX manager software from Bio-Rad. The presence of a VIC-only fluorescent signal represents homozygosity for allele 1, e.g., 388A, and an FAM-only fluorescent signal indicates homozygosity for allele 2, e.g., 388G. The presence of both signals suggests heterozygosity of alleles 1 and 2.

Pharmacokinetic Analysis.

The PK parameters were obtained by noncompartmental analysis of plasma concentration versus time data (KINETICA software, version 4.4.1; Thermo Fisher Scientific Corporation, Philadelphia, PA). The maximum plasma concentration (Cmax) and time for Cmax (Tmax) were recorded directly from experimental observations. The AUC was calculated using the mixed log-linear trapezoidal rule up to the last quantifiable concentration (Clast). Estimations of AUC and terminal elimination rate constant were made using a minimum of three time points with quantifiable concentrations. Concentrations below the limit of quantitation were considered as zero for calculations.

Renal clearance (CLr) was estimated by the following equations:Embedded Image(1)where X0-t is the cumulative amount excreted in the urine during the time interval (0∼t).

Statistical Analysis.

To test for statistically significant differences in CP concentrations in the plasma and urine excretion among multiple days, one-way analysis of variance followed by Dunnett’s comparisons was performed. Student’s t test was used to assess the statistical significance of differences between two sets of data. All statistical analyses were performed using Prism version 5.0 (GraphPad Software, Inc., San Diego, CA). A p value less than 0.05 was considered statistically significant.

Results

Study Enrollment.

Single doses of RSV administered either alone or coadministered with RIF were well tolerated in healthy subjects. No adverse events, serious adverse experiences, laboratory adverse experiences, or events of clinical interest were reported during the entire duration of the study. No subjects were discontinued by the study investigator. No clinically significant changes in the measured values of blood pressure, pulse rate, respiratory rate, or oral temperature which could be related to the study drug were observed during the entire duration of the study. Twelve subjects were enrolled at the beginning of the study. Subject 7 could not attend period 2, but visited the facility and completed period 3. Subject 8 completed period 1 and period 2 but dropped out of the study prior to period 3 due to personal reasons. All other subjects completed the entire study. Genotyping of two common SNPs of the human OATP1B1 gene, A388G (Asn130Asp) and T521A (Val174Ala), was performed in all 12 subjects. OATP1B1*5 and OATP1B1*15 mutations were absent in all 12 subjects. Nine subjects were heterozygous and three subjects were homozygous for OATP1B1*1b.

Pharmacokinetics of RIF.

The mean plasma concentration versus time profiles of RIF following a single oral dose of 600 mg of RIF were similar to those observed in combination with oral administration of 5 mg of RSV (Fig. 1; Table 1). RIF achieved a maximum plasma concentration (Cmax) of 26.7 and 30.6 µM at 2.5 and 2.2 hours postdose in period 1 and period 3, respectively. The average plasma concentrations of RIF from the combination treatment were about 6.8 and 0.7 µM at 12 and 24 hours postdose, respectively. At 12 hours postdose, RIF-free concentration (89% protein bound) was 0.75 µM, which is above the IC50 values of human OATP1B1 (0.55 ± 0.07 µM) or OATP1B3 (0.46 ± 0.13 µM) (Shen et al., 2013).

Fig. 1.
Fig. 1.

Plasma concentration–time profiles of RIF. Plasma concentrations of RIF were determined following administration of 600 mg of RIF alone (period 1) or a combination of 600 mg of RIF and 5 mg of RSV (period 3). The data are expressed as the mean ± S.D. of 12 (period 1) or 11 (period 3) subjects.

View this table:
TABLE 1

Pharmacokinetics parameters of RIF and RSV

Pharmacokinetics of RSV.

The mean plasma concentration versus time profiles and pharmacokinetic parameters of RSV following a single oral dose of 5 mg of RSV (period 2) or in combination with 600 mg of RIF (period 3) are depicted in Fig. 2 and Table 1. The Cmax and area under the concentration-time curve from time 0 to 24 h (AUC0–24h) of RSV following RSV dose alone were 9.17 ± 3.85 nM and 75.6 ± 26.5 nM*h, respectively. RIF markedly increased the Cmax and AUC0–24h of RSV by 13.2- and 5-fold, respectively. Tmax and terminal half-life (T1/2) of RSV decreased significantly by the coadministration of RIF (Table 1). As expected, urinary excretion of RSV was minimal, accounting for about 5% of the total dose. RIF increased the amount of RSV in urine to 24% of the total dose but had no impact on CLr of RSV (Table 1).

Fig. 2.
Fig. 2.

Effects of RIF on RSV exposure. RSV plasma concentration–time profiles were determined following oral administration of 5 mg of RSV alone (period 2) or a combination of 5 mg of RSV with 600 mg of RIF (period 3). The data are expressed as the mean ± S.D. of 11 subjects.

CP-I and CP-III Concentrations in Plasma and Urine.

Mean plasma concentration versus time profiles of CP-I and CP-III after RIF alone (period 1), RSV alone (period 2), or in combination (period 3) are shown in Fig. 3. Both CP-I and CP-III concentrations in plasma were not affected by RSV, as compared with the predose values (Table 2). Changes in CP-I and CP-III plasma concentrations in period 1 are considered to be independent of time (statistically not significant); therefore, Cmax and Tmax were not computed for CP-I and CP-III for this treatment (Table 2). There was no significant difference in the predose plasma concentrations and urine excretion [Xe(−7–0h)] of CP-I and CP-III in the three periods (Table 2), suggesting that CP-I and CP-III in the plasma and urine at baseline are constant. The ratio of CP-I versus CP-III at baseline was about 6:1 in the plasma, and about 1:1 in the urine.

Fig. 3.
Fig. 3.

Effects of RIF on plasma CP concentrations. CP plasma concentrations were determined following oral administration of 600 mg of RIF alone (period 1), 5 mg of RSV alone (period 2), and the combination of 600 mg of RIF and 5 mg of RSV (period 3). (A) CP-I. (B) CP-III. The data are expressed as the mean ± S.D. of 12 (period 1) or 11 (period 2 and period 3) subjects.

View this table:
TABLE 2

Comparison of pharmacokinetic parameters of CP-I and CP-III following oral administration of 600 mg of RIF alone, 5 mg of RSV alone, or the combination of 5 mg of RSV and 600 mg of RIF

Data are expressed as the mean ± SD.

Following administration of a single dose of 600 mg of RIF alone or in combination with 5 mg of RSV, a significant increase in CP-I Cmax (5.7- to 5.9-fold) was observed when compared with the predose concentration. Likewise, RIF, either alone or in combination with RSV, significantly increased CP-III Cmax 5.4- to 6.5-fold. Plasma AUC0–24h values for CP-I and CP-III were also significantly increased 4- and 3.4-fold, respectively, following RIF (Fig. 3; Table 2) compared with RSV alone. Plasma concentrations of both CP-I and CP-III returned to predose concentrations after 24 hours post RIF dose, the time at which free plasma concentration of RIF is lower than the OATP IC50 values (Shen et al., 2013).

The cumulative amount of CP-I excreted in urine during 24 hours [Xe(0–24h)] increased by 3.6-fold (RIF alone) or 3.4-fold (RIF+RSV), whereas the CP-III urinary amount increased by 1.6-fold (RIF alone) or 1.4-fold (RIF+RSV) (Table 2). As expected, the renal clearance of CP-I was not altered by RIF (Table 2). The administration of RIF alone or in combination with RSV reduced the CLr of CP-III; however, the reduction was not statistically different due to the large interindividual variation (Table 2).

Total, Direct, and Indirect Bilirubin in Human Plasma.

As indirect bilirubin is a substrate of OATP transporters and increases in plasma bilirubin in cynomolgus monkeys and rats after RIF administration have been reported (Chu et al., 2015), we further examined total, direct, and indirect bilirubin in plasma of human subjects receiving RSV alone (period 1) or the combination of RIF and RSV (period 3). As shown in Fig. 4, a slight (∼32%) but statistically significant increase in total bilirubin was detected in the combination treatment, in comparison with RSV alone. Indirect bilirubin, which was increased about 2-fold following the combination treatment, appeared to contribute to the increase in total bilirubin. In contrast, the changes in direct bilirubin in plasma were similar in the two treatments. No abnormal changes in other liver enzymes, e.g., alanine aminotransferase, were reported in any subject.

Fig. 4.
Fig. 4.

Effects of RIF on plasma bilirubin. Mean plasma concentrations of total (A), direct (B), and indirect bilirubin (C) were determined following RSV alone and RSV+RIF administration. The AUC0–24h values of total, direct, and indirect bilirubin are listed in the table. Data are expressed as the mean ± S.D. of 11 subjects. *P < 0.05, statistically significant difference between the subjects administered RIF alone (period 2) and RIF+RSV (period 3).

Discussion

CP-I and CP-III are actively taken up by human hepatocytes and human embryonic kidney 293 cells overexpressing human OATP1B1 or 1B3 protein (Bednarczyk and Boiselle, 2015; Shen et al., 2016a). The results suggested that CPs could be potential endogenous biomarkers of OATP activity in vivo (Benz-de Bretagne et al., 2011; van de Steeg et al., 2012). Indeed, the plasma concentrations of both CP-I and CP-III in cynomolgus monkeys were markedly increased following administration of OATP inhibitors cyclosporine A (100 mg/kg oral) or RIF (15 mg/kg oral) (Shen et al., 2015, 2016b). The observations in monkey could explain the clinical findings that cyclosporine A- or RIF-induced porphyria in patients is likely due to the inhibition of OATP transporters (Millar, 1980; Hivnor et al., 2003). In addition, changes in plasma CP-I and CP-III in cynomolgus monkey are in line with RSV exposure increase after coadministration with OATP inhibitors, suggesting CPs could be biomarkers for in vivo OATP-mediated DDIs (Shen et al., 2016a).

In the current clinical study, we found that plasma concentrations of both CP-I and CP-III were relatively stable at predose and after administration of RSV, and increased following administration of RIF. In line with the increase in RSV exposure in human subjects receiving a single oral dose of 600 mg of RIF, Cmax of CP-I and CP-III increased by 5.7- to 5.9-fold and 5.4- to 6.5-fold, whereas AUC0–24h increased by 4- and 3.4-fold, respectively. Although the Cmax increase of CP-I and CP-III was less than that of RSV (5.4- to 6.5-fold vs. 13.2-fold), the changes in AUC are comparable to AUC changes of RSV found in the current study (Table 1) and another report (4.4-fold) (Prueksaritanont et al., 2014). RSV is specifically distributed in the liver, where it is eliminated into the bile by canalicular efflux transporters (Nezasa et al., 2002). The inhibition of OATP function could decrease both systemic clearance and volume distribution of RSV. Indeed, plasma clearance over bioavailability of RSV was decreased 4.7-fold following the administration of RIF, whereas volume distribution over bioavailability (Vss/F) decreased by 8.7-fold, resulting in the decrease of T1/2 (0.7-fold). As AUC is independent of Vss, greater changes in the Cmax (13.2-fold) over AUC0–24h (5-fold) of RSV can be explained by the decrease in Vss.

Dubin-Johnson syndrome, an autosomal recessive genetic disorder leading to deficiency of human multidrug resistance–associated protein 2 (MRP2), causes an increase in ratio of urinary CP-I to CP-III. Urinary elimination of CPs is dependent on ABCC2 polymorphisms and represents a potential biomarker of MRP2 activity in humans, suggesting that CPs are substrates for MRP2. Furthermore, in vitro data show that rifampicin is a substrate for P-glycoprotein, MRP2, and breast cancer resistance protein (https://www.druginteractioninfo.org). Although clinical DDI results of pitavastatin suggested that inhibition of OATP1B-mediated hepatic uptake of pitavastatin is primarily responsible for the increased pitavastatin exposure in the presence of RIF, and the contribution of MRP2 inhibition to the rifampicin-pitavastatin interaction is considered small (Prueksaritanont et al., 2014), the potential impact of MRP2 inhibition on plasma CPs cannot be completely excluded, and further investigations are needed.

At predose, CP-I and CP-III amounts were about the same in human urine, and the ratio of CP-I over total CPs was close to that previously reported (0.29∼0.44) (Koskelo et al., 1966, 1967; Gebril et al., 1990) but different from the profiles in monkey, where CP-III was much higher than CP-I (Shen et al., 2016a). Urinary excretion of CP-I increased (3.6-fold) to a similar extent as that of CP-I AUC0–24h (4.0-fold) after RIF treatment, as compared with RSV alone. The increase in urinary excretion of CP-III (1.6-fold) was less compared with CP-I after RIF (3.3-fold), resulting in the preponderance of CP-I over CP-III in urine. The result is consistent with the abnormal distribution of CP isomers reported in the urine of patients with Rotor syndrome (Ben-Ezzer et al., 1971; Wolkoff et al., 1976). RIF treatment appeared to decrease CLr of CP-III, suggesting involvement of renal transporters (Koskelo et al., 1966, 1967; Gebril et al., 1990). Although CP-III, but not CP-I, appeared to be transported by organic anion transporter 1 (Bednarczyk and Boiselle, 2015), further characterization of the transporter(s) responsible for active renal secretion of CP-III and its association with genetic polymorphism is necessary. Overall, since CP-I is passively cleared in the urine, and the urinary output is proportional to the AUC increase, CP-I in the urine may have better value than CP-III as an in vivo biomarker for OATP function without complication by renal transporters.

Two SLCO1B1 SNPs, OATP1B1*5 and *15, could cause decreased cell surface expression and/or affect in vitro and in vivo transport activities, and are associated with increased plasma exposure (AUC) of substrates (Nishizato et al., 2003; Mwinyi et al., 2004; Pasanen et al., 2008; Seithel et al., 2008). For example, plasma exposure of repaglinide or statins (AUC) is 1.5- to 3-fold higher in subjects heterozygous or homozygous for the SLCO1B1 521CC genotype (Val174Ala) compared with subjects with the SLCO1B1 521TT genotype (Kivisto and Niemi, 2007; Kalliokoski et al., 2008; Niemi et al., 2011). The OATP1B1*1b haplotype is reported to be associated with increased activity of OATP1B1 (Katz et al., 2006) and lower AUC of pitavastatin in Japanese subjects (Maeda et al., 2006); controversially, the observed pharmacokinetic differences for RSV between Asian and Caucasian subjects is not due to the SLCO1B1*1b genotype (Lee et al., 2005; Choi et al., 2008). To our knowledge, there are no data describing the association between SLCO1B1 SNPs and plasma concentration of CP-I and CP-III. In the present study, three subjects were found to be OATP1B1*1b homozygous, and nine were OATP1B1*1b heterozygous. Although no significant differences in CP-I and CP-III baseline between subjects homozygous and heterozygous for OATP1B1*1b were found (Supplemental Fig. 1; Supplemental Table 1), the small study size did not allow us to draw any conclusions of the impact of OATP1B1 genotypes on the plasma CPs. Further investigation of the impact of OATP genetic polymorphisms on CP plasma concentration is warranted.

OATP1B1 and 1B3 have been shown to transport both unconjugated and conjugated bilirubin in vitro (Kalliokoski and Niemi, 2009). OATP inhibition can cause hyperbilirubinemia (Campbell et al., 2004). Indeed, bilirubin levels are markedly increased in the plasma of cynomolgus monkeys and rats after administration of RIF (Chu et al., 2015; Watanabe et al., 2015); thus, the authors proposed bilirubin as a biomarker for hepatic OATP inhibition. 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 changes in bilirubin plasma concentration. Furthermore, serum bilirubin, along with bile acids and other liver enzymes, are altered with drug-induced liver injuries (Ozer et al., 2008). In the present study, we showed that the increase in total and indirect bilirubin was marginal (<2-fold). The increase in total bilirubin could be attributed to the increase in indirect bilirubin in plasma, suggesting the inhibitory effect occurs on the sinusoidal side of hepatocytes (OATP uptake) rather than canalicular efflux (MRP2).

Collectively, the results presented herein showed that there is a strong mechanistic relationship between OATP inhibition and CP-I and CP-III plasma and urine concentrations. As previously mentioned, although the impact of age, gender, contributions of inhibition for hepatic efflux transporters and genetic polymorphisms, etc., on CP-I and CP-III remains to be further resolved by incoming clinical data, CP-I and CP-III in plasma and CP-I in urine could be surrogate endpoints for DDIs mediated by OATP inhibition in humans. Importantly, CP-I and CP-III measurements can be incorporated into phase I dose-escalation studies to provide evidence of OATP inhibition across all doses. We propose that CP-I and CP-III measurements should be incorporated in the decision tree for OATP DDI risk assessment and thereby inform planning and design of downstream clinical studies. Further investigations are needed to understand the influence of other intrinsic and extrinsic factors, such as age, gender, genetic polymorphism, and organ impairment, on CP-I and CP-III plasma and urine concentrations.

Acknowledgments

The authors thank Dr. Kurex Sidik for help with statistical analysis; Dr. Anil K., Dr. Siddangouda Patil, Jaya Patel (Syngene Clinical Development), Kamala Venkatesh, and Uday Kanni (Biocon Bristol-Myers Squibb R&D Center, Bangalore, India) for bilirubin analysis; Dr. Hemant Bhutani and Shishir Prasad (Biocon Bristol-Myers Squibb R&D Center) for assay of formulations; and Dr. Randy C. Dockens for reviewing the protocol of clinical studies. The authors also acknowledge Anne Rose and Tongtong Liu for handling clinical samples, and Anthony Marino for reviewing the manuscript.

Authorship Contributions

Participated in research design: Lai, Mandlekar, Shen, Cheng, Shipkova, Humphreys, Marathe.

Conducted experiments: Holenarsipur, Langish, Rajanna, Murugesan, Gaud, Selvam, Date, Dai.

Contributed new reagents or analytic tools: Langish, Rajanna, Murugesan, Gaud, Selvam, Date, Shipkova, Dai.

Performed data analysis: Lai, Mandlekar, Shen, Holenarsipur, Gaud, Cheng, Humphreys, Marathe.

Wrote or contributed to the writing of the manuscript: Lai, Mandlekar, Shen, Holenarsipur, Rajanna, Cheng, Shipkova, Humphreys, Marathe.

Footnotes

Abbreviations

AUC
area under the concentration-time curve
AUC0-24hr
area under the concentration-time curve from time 0 to 24 h
CLr
renal clearance
CP-I
coproporphyrin I
CP-III
coproporphyrin III
DDI
drug-drug interaction
HPLC
high-performance liquid chromatography
LC-MS/MS
liquid chromatography–tandem mass spectrometry
MRM
multiple reaction monitoring
MRP2
multidrug resistance–associated protein 2
OATP
organic anion transporting polypeptide
PK
pharmacokinetic
RIF
rifampicin
RSV
rosuvastatin
SNP
single nucleotide polymorphism
UHPLC
ultra high performance liquid chromatography

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Research ArticleMetabolism, Transport, and Pharmacogenomics

Coproporphyrins Are Clinical Biomarkers for OATP Inhibition

Yurong Lai, Sandhya Mandlekar, Hong Shen, Vinay K. Holenarsipur, Robert Langish, Prabhakar Rajanna, Senthilkumar Murugesan, Nilesh Gaud, Sabariya Selvam, Onkar Date, Yaofeng Cheng, Petia Shipkova, Jun Dai, William G. Humphreys and Punit Marathe
Journal of Pharmacology and Experimental Therapeutics September 1, 2016, 358 (3) 397-404; DOI: https://doi.org/10.1124/jpet.116.234914
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