Ko143

Preclinical Absorption, Distribution, Metabolism, Excretion, and Pharmacokinetics of A Novel Selective Inhibitor of Breast Cancer Resistance Protein (BCRP)

Mingxiang Liao, Bei-Ching Chuang, Qing Zhu, Yuexian Li, Emily Guan, Shaoxia Yu, Johnny Yang, Prakash Shimoga & Cindy Q Xia

KEYWORDS: BCRP inhibitor, Bcrp knockout, Ko143 analogue, ML753286, Preclinical ADME characterization

ABSTRACT

1. Breast cancer resistance protein (BCRP) plays an important role in drug absorption, distribution, and excretion. It is challenging to evaluate BCRP functions in preclinical models because commonly used BCRP inhibitors are nonspecific or unstable in animal plasma. 2. In the present work, in vitro absorption, distribution, metabolism and elimination (ADME) assays and pharmacokinetic (PK) experiments in Bcrp knockout (KO) (Abcg2-/-) and wild-type (WT) FVB mice and Wistar rats were conducted to characterize the preclinical properties of a novel selective BCRP inhibitor (ML753286, a Ko143 analog). 3. ML753286 is a potent inhibitor for BCRP, but not for P-glycoprotein (P-gp), organic anion-transporting polypeptide (OATP), or major cytochrome P450s (CYPs). It has high permeability, but is not an efflux transporter substrate. ML753286 has low to medium clearance in rodent and human liver S9 fractions, and is stable in plasma cross species. Bcrp inhibition affects oral absorption and clearance of sulfasalazine in rodents. A single dose of ML753286 at 50 to 300 mg/kg orally, and at 20 mg/kg intravenously or 25 mg/kg orally inhibits Bcrp functions in mice and rats, respectively. 4. These findings confirm that ML753286 is a useful selective inhibitor to evaluate BCRP/Bcrp activity in vitro and in rodent model systems.

INTRODUCTION

Breast cancer resistance protein (known as BCRP in humans and Bcrp in rodents), encoded by the ABCG2/Abcg2 gene, is a member of the ATP-binding cassette (ABC) family (Ejendal and Hrycyna 2002, Doyle and Ross 2003). The BCRP transporter was initially recognized to confer multidrug resistance in cancer cells (Doyle et al. 1998, Litman et al. 2000). It consists of 655 amino acids and 6 transmembrane domains and forms homodimers or homotetramers (Giacomini et al. 2010). BCRP is expressed broadly in human tissues such as mammary glands, intestine, liver, blood-brain barrier, testis, and placenta, is over-expressed in multiple tumor cells (Doyle et al. 1998, Litman et al. 2001, Maliepaard et al. 2001), and mediates the transport of various endogenous substrates (such as bile acids and estrones), dietary compounds (such as flavonoids and porphyrins), and drugs (such as chemotherapy and non-chemotherapy drugs) (Mao and Unadkat 2005, Ni et al. 2010, Robey et al. 2011). BCRP has influence on drug absorption, distribution, and elimination, and therefore, may impact drug toxicities, therapeutic efficacies, and drug-drug interactions (DDIs) (Giacomini et al. 2010). For example, clinical studies showed that the coadministration of curcumin resulted in a 3.2-fold increase in the AUC of sulfasalzine, possibly caused by BCRP inhibition (Shukla et al. 2009, Kusuhara et al. 2012). Additionally, the ABCG2 SNP, c.421C>A, p.Q141K, rs2231142 is associated with the altered pharmacokinetic (PK) of several drugs, such as rosuvastatin, sulphasalazine, and irinotecan, as well as the increased risk of gefitinib-induced diarrhea (Cusatis et al. 2006, Keskitalo et al. 2009, Giacomini et al. 2013). Based on these findings, regulatory guidelines have been issued by the United States Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommending the routine evaluation of BCRP in preclinical drug development to guide clinical DDI studies and drug labeling. However, there are still limited clinical cases of BCRP-mediated.

DDIs identified because of the overlap of substrates and inhibitors with other transporters and cytochrome P450s (CYPs). For example, the plasma AUC of rosuvastatin increased by 7-fold in subjects co-administered cyclosporine through the inhibition of organic anion-transporting polypeptide (OATP)1B1, 1B3, and BCRP (Poirier et al. 2014). At the early drug discovery stage, the in vivo PK profiling in preclinical animal models can provide critical information on optimizing ADME properties, understanding efficacy and safety, and reducing costs during drug development. BCRP inhibition can change drug PK properties in animals. For example, the plasma AUC of sulfasalazine after PO or IV administration in Bcrp1 (abcg2)-/- KO mice was 111- or 13-fold higher than that in WT mice (Zaher et al. 2006). Compared to genetic KO animals, which are relatively expensive, difficult to obtain, and potentially associated with the expression alterations of non-target transporters or drug metabolizing enzymes, chemical KO animals are more attractive test systems and have been widely used to evaluate the role of transporters in a drug’s ADME properties (Allen et al. 2002, Jonker et al. 2000, Shepard et al. 2003). However, the selection of specific transporter inhibitors is quite challenging. For example, elacridar is an extensively used inhibitor of efflux transporters P-gp and BCRP in rodents, and it inhibited P-gp, BCRP, and an unknown efflux transporter in liver (Hoffmaster et al. 2004). Therefore, potent and selective BCRP inhibitors are needed to prospectively define the contribution of BCRP to drug PK and DDIs in animals in drug discovery (Xia et al. 2007b). In recent years, efforts have been made to select or synthesize more potent and specific BCRP inhibitors through high-throughput screening of chemical compound libraries or synthesis of new classes of compounds (Marighetti et al. 2015, Wiese 2015, Li et al. 2016, Pires Ado et al. 2016, Song et al. 2017). However, the available PK and pharmacological data of those
BCRP inhibitors are insufficient, which limits their application to in vitro, in vivo, and clinical studies.

Based on the potency and specificity, the commonly used BCRP inhibitors can be categorized as those with relatively high potency and high specificity, such as fumitremorgin C and its analog Ko143, high potency and low specificity, including elacridar and tariquidar which are also P-gp inhibitors, and others, such as cyclosporine A (Allen et al. 2002, Kruijtzer et al. 2002, Xia et al. 2007a, Gardner et al. 2009). Ko143 (Figure 1A) is less toxic than fumitremorgin C, and has been widely used for in vitro ADME studies (Allen et al. 2002, Xia et al. 2007b, Muenster et al. 2008, Lee et al. 2015). However, high plasma clearance and low bioavailability impeded the application of Ko143 on BCRP studies in rats, a standard preclinical PK model (Weidner et al. 2015, Li et al. 2016). A recently described BCRP inhibitor (ML753286, known as Compound A in Li et al. 2016; Figure 1B), is a Ko143 analog showing an improved PK profile in rats, and is being considered as a promising selective BCRP inhibitor in animal models (Li et al. 2016). In the present work, a thorough characterization of preclinical in vitro and in vivo ADME properties of ML753286 was performed in order to support the application of this novel BCRP inhibitor on the in vivo evaluation of efflux transporter effects on PK profiles to optimize drug design and screening.

MATERIALS AND METHODS

Materials. The cell culture media and supplements were obtained from Invitrogen (Carlsbad, CA, USA). The common chemicals and reagents such as sulfasalazine, phenacetin, paclitaxel, diclofenac, NADPH, UDPGA, and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cryopreserved human hepatocytes were obtained from Life Techonologies Corp (Grand Island, NY, USA). Hepatic microsomes from male Sprague-Dawley (SD) rats were purchased from XenoTech, LLC (Lenexa, KS, USA). ML753286 was synthesized by Takeda Pharmaceuticals International Co. (Cambridge, MA, USA) (Li et al. 2016). Caco-2 Cellular Permeability. Caco-2 cell culture and bi-directional transport studies were conducted as previously described by Xia (2005). Briefly, Caco-2 cells (ATCC [Manassas, VA, USA]) were cultured on Costar 24-Transwell™ plates (0.33 cm2/well, 0.4-µm pore size; Corning Life Sciences [Corning, NY, USA]) for 21 to 25 days. The cell monolayers with transepithelial electrical resistance (TEER) values higher than 250 ohms × cm2 were used for the further assays. Prior to each experiment, the confluent cell monolayers on the Transwell inserts were washed and equilibrated for 30 minutes with transport media (Hank’s Balanced Salt Solution [HBSS] containing 25-mM 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES] and 10-mM glucose, pH 7.4). The assay was initiated by adding transport media containing Ko143 or ML753286 at 5 µM with 50 µM of lucifer yellow (LY) (used as a paracellular transport control to monitor cell monolayer integrity) to either the apical (for A-to-B transport) or basolateral (for B-to-A transport) compartment. The aliquots of the samples were withdrawn from the receiver side at 15, 30, 45, and 60 minutes. After LY fluorescence measurement, the samples were analyzed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) to quantify the amount of Ko143 and ML753286. The apparent permeability coefficient (Papp) of unidirectional fluxes for the test compounds was calculated according to Equation 1.

Where Papp = apparent permeability coefficient (expressed as cm/sec × 10-6), dQ/dt  total amount of drug present in the receiver chamber per unit time (nmol/sec), A = surface area (cm2, the present work used A = 0.33 cm2), and C0 = initial drug concentration in the donor chamber (nmol/mL). Efflux Transporter Inhibitor Study in Caco-2 Cells. The assay was initiated by adding transport media containing [3H]digoxin or [3H]estrone sulfate ([3H]E3S) in the absence or presence of various concentrations of Ko143 or ML753286 (testing from 30 µM with 3-fold serial dilution) or efflux inhibitor as positive control, to the basolateral side. At 30, 60, 90, and 120 minutes, an aliquot of the receiving solution was sampled from the receiver side. The radioactivity in each sample was measured using a 1450 MicroBeta® TriLux microplate scintillation and luminescence counter (PerkinElmer Life Sciences [Waltham, MA, USA]). The test compound IC50 of P-gp and BCRP was calculated using XLfit Microsoft® Excel (Microsoft Corp [Redmond, WA, USA]) Add-in, Version 5.3.1.3 (ID Business Solutions Ltd [Cambridge, MA, USA]). The transport rates of efflux pump substrates with positive inhibitor control treatment (LY335979 [5 µM] for P-gp and Ko143 [30 µM] for BCRP) were considered passive diffusion, and the transport rates with test compounds were expressed as passive diffusion and efflux transporter-mediated flux, which was not inhibited by test compound treatment. The remaining fraction of efflux transporter activity in the absence or presence of any treatment was calculated with Equation 2.

Where Papp,B-A = apparent permeability coefficient of efflux substrate in the basolateral-to-apical direction (cm/sec × 10-6) in the presence of tested inhibitor, Papp,B-A (PC)  Papp,B-A of test efflux pump substrates with positive inhibitor control treatment, and Papp,B-A (NC)  Papp,B-A of test efflux pump substrates without any inhibitor treatment.OATP Inhibitor Study in Human Hepatocytes. The uptake study in human hepatocytes was described by Zhu (2014). Briefly, the cryopreserved human hepatocytes were thawed and plated onto 48-well plates pre-coated with collagen IV (BD Biosciences [San Jose, CA, USA]) in William’s Medium E (WME) (Life Technologies [Carlsbad, CA, USA]) containing 0.1-mM nonessential amino acids, 2-mM L-glutamine, 4.5-g/L glucose, 10% fetal bovine serum, insulin- transferrin-selenium (1×) (BD Biosciences), 0.1-µM dexamethasone, and 100-unit/mL penicillin/streptomycin. The hepatocytes were incubated at 37°C for 3 hours before use in the uptake assays. The human hepatocytes were rinsed twice and incubated with HBSS containing [3H]rosuvastatin (50 nM) in the absence or presence of Ko143 or ML753286 (testing from 100 µM with 4-fold serial dilution) or an OATP inhibitor, rifampin, at 37C. After a 5-minute incubation, the assay was terminated by removing solution and washing the cells 3 times with ice-cold HBSS. Hepatocytes were lysed with 0.5% Triton X-100 for 30 minutes at room temperature. The radioactivity in cell lysates was determined using a scintillation and luminescence counter and the protein amounts were assessed by Pierce™ BCA Protein Assay (ThermoFisher Scientific Inc. [Rockford, IL, USA]).

The test compound IC50 of OATP was calculated using XLfit Microsoft® Excel Add-in, Version 5.3.1.3. The remaining fraction of OATP activity in the absence or presence of any treatment was calculated with Equation 3. Where Ruptake = uptake rate of OATP substrate in the presence of test inhibitor (total amount of drug accumulated in cells per unit time, pmol/min/mg of protein), Ruptake (PC)  Ruptake of test OATP substrate with positive inhibitor control (rifampin) treatment, and Ruptake (NC)  Papp,B-A of test OATP substrate without any inhibitor treatment. In Vitro Stability in Plasma. The in vitro stability of Ko143 and ML753286 was determined in CD-1 mouse, SD rat, beagle dog, cynomolgus monkey, and human plasma (BioreclamationIVT [Baltimore, MD, USA]). The plasma was incubated with 2 µM of test compound at 37°C. Aliquots of the samples were taken at 0, 10, 30, 60, and 120 minutes, and analyzed by LC/MS/MS to quantify the relative amount of Ko143 and ML753286. The half-life (t½) of the test compounds was calculated using Equation 4. In Vitro Plasma Protein Binding. The protein binding of ML753286 in mouse, rat, and human plasma was determined by a rapid equilbrium dialysis (RED) method. Briefly, the plasma was spiked with test compound at a concentration of 10 µM, and dialyzed against isotonic sodium phosphate buffer (120 mM, pH 7.5) using a RED device with a cutoff mass of 8000 Da (Linden Bioscience [Woburn, MA, USA]) at rotation rate of 750 rpm. After a 7-hour incubation at 37C, the buffer and plasma samples were collected and analyzed by a qualified LC/MS/MS method.

The test compound IC50 of the CYPs was calculated using XLfit Microsoft® Excel Add-in, Version 5.3.1.3. The remaining fraction of CYP activity in the absence or presence of any treatment was calculated with Equation 9. Where A = peak area ratio of known substrate with any treatment, and A (NC) = peak area ratio of known substrate without any treatment. PK Studies of Sulfasalazine in Male Bcrp KO (Abcg2−/−) and WT (Wistar) Rats in the Absence or Presence of ML753286. The doses of the Bcrp probe substrate, sulfasalazine in animal studies were selected according to published data (Maciej J. Zamek-Gliszczynski et al. 2012, Zaher et al. 2006) and the detection limit by LC/MS/MS. Eight male Bcrp KO and 24 WT rats were purchased from SAGE Labs (St. Louis, MI, US). The Bcrp KO rats were divided into 2 groups and the WT rats were divided into 6 groups; n = 4/group. For PO administration, 1 group of Bcrp KO rats were administered 0.5% HPMC/0.2% Tween 80 via oral gavage. Three groups of WT rats were administered a single PO dose of 25- or 50-mg/kg ML753286 or 0.5% HPMC/0.2% Tween 80. Twenty minutes later, 20-mg/kg sulfasalazine was administered to all 4 groups of rats via oral gavage. Blood was collected predose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours postdose and an aliquote was processed into plasma. In the IV study, 1 group of Bcrp KO rats were administered 10% ethanol/10% DMAC/40% PEG400/40% sterile water via IV injection. Similarly, 3 groups of WT rats were administered a single IV dose of ML753286 (10 or 20 mg/kg) or 10% ethanol/10% DMAC/40% PEG400/40% sterile water. Twenty minutes later, 2-mg/kg sulfasalazine was administered to all 4 groups of rats via IV bolus injection. Blood was collected predose and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours postdose and an aliquot was processed into plasma. The plasma concentrations of sulfasalazine and ML753286 were measured using appropriate liquid LC/MS/MS methods. The PK parameters were calculated by a noncompartmental model using Phoenix WinNonlin version 7.0 (Pharsight, a Certara Company, Princeton, NJ, USA).

PK Studies of Sulfasalazine in Female Bcrp (Abcg2−/−) KO and WT (FVB) Mice in the Absence or Presence of ML753286. Four female Bcrp KO and 20 WT mice were purchased from SAGE Labs. The WT mice were divided into 5 groups, n = 4/group. The Bcrp KO mice group was administered 0.5% HPMC/0.2% Tween 80 via IV injection, and the WT mice groups were administered a single PO dose of 50-, 100-, 200-, or 300-mg/kg ML753286 or 0.5% HPMC/0.2% Tween 80. Twenty minutes later, 4-mg/kg sulfasalazine was administered to all 6 groups of mice via oral gavage. Blood samples were collected predose and at 0.083, 0.5, 1, 4, 8, and 24 hours postdose. The blood concentrations of sulfasalazine and ML753286 were measured using appropriate liquid LC/MS/MS methods. The PK parameters were calculated by a noncompartmental model using Phoenix WinNonlin version 7.0. LC/MS/MS. The quantitative analysis of the test compounds was performed on a Shimadzu UFLC system coupled to a mass spectrometer (API4000 triple quadrupole equipped with a turbo ion spray source [Applied Biosystems (Foster City, CA, USA)]). The analytes were separated using a Phenomenex Synergi™ Hydro-RP C18 column (50 × 2.0 mm, 2.5-m PFP; Phenomenex [Torrance, CA, USA]) with initial solvent containing 90% mobile phase A (0.1% formic acid in water) and 10% mobile phase B (0.1% formic acid in ACN), with appropriate gradients and flow rates. MRM transitions were m/z 281 > m/z 193.1 for sulfasalazine, m/z 371 > m/z 126 for ML753286, and m/z 320.2 > m/z 247.2 for carbutamide.

DISCUSSION

Human BCRP can have an important impact on drug absorption, disposition, clearance, and DDI (Giacomini et al. 2010). Chemical KO animals are important tools to understand the role of transporters in drug ADME and DDI at the early discovery stage. A recently synthesized BCRP inhibitor, ML753286 (a Ko143 analogue), was demonstrated to inhibit BCRP function potently and with specificity, and has low clearance and high bioavailability in rats (Li et al. 2016). However, the optimal doses of ML753286 to completely inhibit Bcrp activity in commonly used preclinical animal models remain to be determined. In the present work, the in vitro ADME characteristics of ML753286 and Ko143 were compared. The in vivo PK studies were conducted in WT and Bcrp KO (Abcg2−/−) mice and rats to evaluate the in vivo potency to optimize the doses of ML753286 to inhibit Bcrp.

ML753286 showed high permeability and was not an efflux transporter substrate, while Ko143 had medium permeability and is an efflux pump substrate in Caco-2 cells (Table 1). The permeability of ML753286 was measured at 5 M, a concentration that is above the IC50 of ML753286 (0.6 M) for BCRP inhibition in Caco-2 cells, which suggested that the active efflux of ML753286 was possibly masked by its high permeability and high test concentration. When animals were treated with the recommended doses of ML753286, which are a single dose of ML753286 at 20 mg/kg IV or at 25 mg/kg PO in rats and at 50 to 300 mg/kg in mice to inhibit Bcrp functions, the gut concentrations of ML753286 were above its IC50 value and saturated the efflux pump. Therefore, the efflux would not affect the absorption of ML753286 in rodents. Ko143 was not stable in mouse and rat plasma with an estimated t1/2 of 20.3 and 2.1 minutes, respectively. In contrast, ML753286 was stable in mouse and rat plasma and was observed at 106.3% and 88.6%, respectively, after a 2-hour incubation (Table 2). The higher cellular permeability and plasma stability could be ascribed to the improved oral bioavailability of ML753286 compared to Ko143 (Li et al. 2016). Compared to Ko143, ML753286 had much lower in vitro hepatic clearance in mice and rats (Table 3), which further confirmed the application of ML753286 to investigate the effect Bcrp on its substrate’s PK in rodents.

The in vitro studies demonstrated that ML753286 showed lower in vitro hepatic clearance and no or weaker inhibitory effects on 5 major CYPs than Ko143 in humans (Table 3 and Table 4). These data demonstrated that ML753286 is suitable for clinical use; however, it is still unclear if ML753286 could be used to inhibit BCRP activities in humans. In addition, the differences in CYP isoforms among mice, rats, and humans may result in different inhibition profiles by ML753286. Sulfasalazine is a BCRP substrate, and has been widely used in animals and is also recommended in clinical trials to estimate BCRP-associated DDI and polymorphism effects (Zaher et al. 2006, Dahan and Amidon 2009, Zamek-Gliszczynski et al. 2012, Lee et al. 2015). In humans, the oral availability of sulfasalazine is low and highly variable.Sulfasalazine is extensively metabolized by a bacteria enzyme, azoreductase, to sulfapyridine (SP) and 5- aminosalicylic acid (5-ASA) in the colon, and then the absorbed sulfasalazine from intestine is metabolized to some extent to SP and 5-ASA in the liver. In the present work, after PO administration, the blood and plasma AUC of sulfasalazine in the Bcrp KO rats was 19.7- and 23.1-fold higher than in control WT rats, respectively, which was consistent with the observation of Zamek-Gliszczynski (2012). However, the administration of ML753286 at 25 and 50 mg/kg led to an unexpectedly higher sulfasalazine AUC in WT rats compared to Bcrp KO rats. The further analysis of sulfasalazine and ML753286 PK revealed that after PO administration, the plasma and blood Cmax and tmax of sulfasalazine were increased in Bcrp KO and 2 ML753286- predosed WT groups compared to the control group (Figure 2A and Table 5). Although the terminal phase of sulfasalazine was parallel in WT and Bcrp KO rats, it decreased in WT rats predosed ML753286 in a dose-dependent manner (Figure 2A).

In the present work, a single IV dose of 20 mg/kg of ML753286 increased the blood and plasma AUC of sulfasalazine after IV administration by 3.5- and 3.4-fold, respectively, compared to non-dosed animals, which were comparable to those in Bcrp (-/-) KO rats. However, the relatively longer t1/2 and higher AUC increase of sufasalazine in ML753286-PO dosed rats have been observed compared to these parameters in Bcrp KO rats. The different magnitude changes after PO and IV administration of ML753286 compared with the results from Bcrp KO rats suggested that there might be off-target inhibition in GI system by ML753286. In addition to BCRP inhibition, the inhibition of the intestinal bacterial metabolism may account for the elevated exposure of sulfasalzine in patients taking sulfasalazine concomitantly with certain BCRP inhibitory drugs, such as curcumin (Poirier et al. 2014). Sulfasalzine was described to interact with other transporter(s), such as the intestinal OATP2B1, to mediate the transport of sulfasalazine in vitro (Kusuhara et al. 2012, Tomaru et al. 2013). It will be interesting to further investigate if ML753286 has any inhibitory effect on the intestinal bacterial metabolism or OATP2B1 in the future. A ML753286 dose-dependent increase in blood and plasma AUC of sulfasalazine was observed in ML753286-predosed rats. The dose of 20 mg/kg IV was optimized for ML753286 to completely inhibit Bcrp-mediated sulfasalzine clearance in rats. The doses of ML753286 were optimized to be 25 mg/kg PO and 20 mg/kg IV to completely to inhibit Bcrp functions, and to reveal the Bcrp role in drug absorption and/or clearance in rats.

The PK studies were conducted in female FVB mice administered a single dose of 10 mg/kg (via oral gavage) or 1 mg/kg (via IV bolus injection) of ML753286. The plasma Cmax of ML753286 were determined to be 5570 nM (PO) and 2170 nM (IV), and the plasma AUC were 4980 nM·h (PO) and 947 nM·h (IV), in mice. The oral bioavailability of ML753286 was determined to be 52.6% in mice (Supplementary Figure 1). The additional PK studies were conducted on WT and Bcrp KO FVB mice administered sulfasalazine IV and ML753286 PO. To reach the equivalent Cmax observed in rats, ML753286 was administered to mice PO only due to its low solubility. Additionally, because the previous rat PK studies confirmed that Bcrp inhibition had the same affect on blood and plasma PK of sulfasalazine, only the blood samples was collected and analyzed in mouse PK studies. The AUC ratios of sulfasalazine in ML753286-dosed groups were less than that in the Bcrp KO mouse group, which was determined to be 12.4 and in agreement with the published data (13.3) (Zaher et al. 2006). The previous findings showed that the plasma AUC ratio of sulfasalazine in Bcrp KO mice compared to WT mice was 111-fold via PO administration and 13.3-fold via IV administration (Zaher et al. 2006), suggesting the important role of mouse Bcrp in the oral bioavailability and elimination of sulfasalazine. The PO administration of a Bcrp inhibitor, gefitinib, was reported to cause a 13-fold increase in the plasma AUC of sulfasalazine via PO administration in mice (Zaher et al. 2006). As discussed previously, in the genetic KO animals, the depletion of 1 transporter might lead to the alteration of the expression of other transporters and enzymes, as well as the physiological properties of animals. Although there is still a lack of evidence revealing effects on the expression of other ADME proteins in Bcrp KO mice, the difference in the blood AUC ratio of sulfasalazine in Bcrp KO and ML753286-dosed groups may be caused by the altered expression of other transporter(s) in Bcrp KO mice.

Considering the compelling evidence indicating BCRP as the major contributor to sulfasalazine disposition (Yamasaki et al. 2008, Zaher et al. 2006, Lee et al. 2015), ML753286 may cause a greater effect on absorption than clearance. ML753286 can be used as a useful potent and selective inhibitor to investigate the role of Bcrp in its substrate’s PK in mice. The rodent PK studies clearly showed that ML753286 can inhibit Bcrp in a manner similar to the KO animals. There were some unexpected results that might be due to effects of ML753286 on gut microflora, but the nature of the investigation (IV versus PO administration) suggests this was a related effect and was not the actual cause. In summary, in the present work, the in vitro ADME of novel selective BCRP inhibitor ML753286 was characterized and proved to be better than that of the more commonly used BCRP/Bcrp inhibitor Ko143. The mouse and rat in vivo PK studies confirmed the in vivo potency of ML753286 and identified the optimal doses of ML753286 to inhibit Bcrp in rodents. Those data suggested that ML753286 is a useful selective BCRP inhibitor to identify the role of this efflux transporter in its substrate’s PK in preclinical models.

ACKNOWLEDGMENTS

We thank Ms. Lili Yao, Ms. Bingli Ma, Mr. Lawrence Cohen, and Mr. Michael Johnson for valuable technical support. We also thank Ms. Ekta Kadakia for excellent assistance with calculating PK parameters. We would like to thank Ms. Rachelle Baker for expert proofreading.

DISCLOSURE STATEMENT

The authors declare that there is no conflict of interest regarding the publication of this manuscript.

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