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Research ArticleArticle

Evaluation of the Utility of PXB Chimeric Mice for Predicting Human Liver Partitioning of Hepatic Organic Anion-Transporting Polypeptide Transporter Substrates

Bo Feng, Rachel Pemberton, Wojciech Dworakowski, Zhengqi Ye, Craig Zetterberg, Guanyu Wang, Yoshio Morikawa and Sanjeev Kumar
Drug Metabolism and Disposition March 2021, 49 (3) 254-264; DOI: https://doi.org/10.1124/dmd.120.000276

Abstract

The ability to predict human liver-to-plasma unbound partition coefficient (Kpuu) is important to estimate unbound liver concentration for drugs that are substrates of hepatic organic anion-transporting peptide (OATP) transporters with asymmetric distribution into the liver relative to plasma. Herein, we explored the utility of PXB chimeric mice with humanized liver that are highly repopulated with human hepatocytes to predict human hepatic disposition of OATP substrates, including rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide. In vitro total uptake clearance and transporter-mediated active uptake clearance in C57 mouse hepatocytes were greater than in PXB chimeric mouse hepatocytes for rosuvastatin, pravastatin, pitavastatin, and valsartan. Consistent with in vitro uptake data, enhanced hepatic uptake and resulting total systemic clearance were observed with the above four compounds in severely compromised immune-deficient (SCID) control mice compared with the PXB chimeric mice, which suggest that mouse has a stronger transporter-mediated hepatic uptake than human. In vivo liver-to-plasma Kpuu from PXB chimeric and SCID control mice were also compared, and rosuvastatin and pravastatin Kpuu in SCID mice were more than 10-fold higher than that in PXB chimeric mice, whereas pitavastatin, valsartan, and repaglinide Kpuu in SCID mice were comparable with Kpuu in PXB chimeric mice. Finally, PXB chimeric mouse liver-to-plasma Kpuu values were compared with the reported human Kpuu, and a good correlation was observed as the PXB Kpuu vales were within 3-fold of human Kpuu. Our results indicate that PXB mice could be a useful tool to delineate hepatic uptake and enable prediction of human liver-to-plasma Kpuu of hepatic uptake transporter substrates.

SIGNIFICANCE STATEMENT We evaluated PXB mouse with humanized liver for its ability to predict human liver disposition of five organic anion-transporting polypeptide transporter substrates. Both in vitro and in vivo data suggest that mouse liver has a stronger transporter-mediated hepatic uptake than the humanized liver in PXB mouse. More importantly, PXB liver-to-plasma unbound partition coefficient (Kpuu) values were compared with the reported human Kpuu, and a good correlation was observed. PXB mice could be a useful tool to project human liver-to-plasma Kpuu of hepatic uptake transporter substrates.

Introduction

Assessment of unbound liver drug concentration is critical for understanding the pharmacological activity and developing pharmacokinetic (PK)/pharmacodynamic (PD) relationships for disease targets residing in the liver, predicting metabolic and biliary clearance as well as drug-drug interaction (DDI) potential, and anticipating the risk of liver adverse events. According to pharmacokinetic principles, it is often assumed that, for permeable compounds that distribute into noneliminating tissues via passive diffusion, unbound tissue concentration equals unbound plasma concentration at equilibrium. However, this assumption may not hold true in the case of liver, which expresses a variety of uptake and efflux drug transporters as well as drug-metabolizing enzymes. Based on the extended clearance concept, the unbound partition coefficient (Kpuu) between the liver and plasma in vivo is governed by a balance of passive diffusion, intrinsic clearance of active hepatic uptake, sinusoidal and biliary efflux, and metabolism (Shitara and Sugiyama, 2006; Watanabe et al., 2010; Yabe et al., 2011). Hence, unbound plasma concentrations cannot always serve as a surrogate for unbound liver concentrations. When drugs are actively taken up into liver through hepatic uptake transporters, such as organic anion-transporting proteins (OATPs), and/or are metabolized in the liver, Kpuu can be significantly different than unity. During drug discovery efforts focused at disease targets located in the liver, it is of great interest to accurately predict in vivo liver-to-plasma Kpuu in humans, since it enables direct estimation of the unbound liver concentrations, which are challenging to measure directly in humans, from the easily measurable unbound plasma concentrations. Accurate assessment of human liver-to-plasma Kpuu thus can help facilitate the design of drugs with targeted distribution to the liver, modeling of DDIs due to inhibition/induction of liver enzymes and transporters, and development of PK/PD relationships when transporters are involved in the asymmetric hepatic drug distribution process.

The ability to predict in vivo liver-to-plasma Kpuu from in vitro assays is highly desirable, but a direct extrapolation of in vitro results to in vivo Kpuu in humans is challenging because transporter protein levels and function in the in vitro systems could be quite different than those in vivo (Bow et al., 2008; Kunze et al., 2014; Morse et al., 2015; Vildhede et al., 2015). Meanwhile, the translational value of traditional preclinical animal models to humans is limited by the substantial species differences in hepatic transporter homology, sequence identity, expression pattern, substrate specificity, and affinity (Chu et al., 2013; Grime and Paine, 2013). OATPs are expressed on the sinusoidal membrane of hepatocytes and have been identified as main contributors to the disposition of statins and a number of other drug classes (Ieiri et al., 2009). The impact of OATPs on hepatic clearance of their substrates has been demonstrated via OATP1B1 polymorphisms and DDIs with OATP inhibitors (Neuvonen et al., 2006; Ieiri et al., 2009; Kalliokoski and Niemi, 2009; Niemi et al., 2011; Yoshida et al., 2012). The significant species differences in amino acid sequences between human OATPs and rodent Oatps make it difficult to translate PK or PD data from preclinical species to humans. In an attempt to overcome these limitations, different humanized animal models have been developed to gain insight into the determinants of liver drug disposition and bridge the gap between preclinical to clinical extrapolation. For example, knockout models lacking murine Oatp1a/1b isoforms and OATP1B1 or OATP1B3 humanized transgenic mice have been developed to estimate the pharmacokinetics of human OATP substrate drugs (van de Steeg et al., 2013; Zimmerman et al., 2013; Higgins et al., 2014; Salphati et al., 2014). However, the presence of three major hepatic OATPs in the human liver with broad overlap in substrate specificity could result in a different drug disposition profile relative to these transgenic mice that express each OATP individually. Additionally, mouse liver metabolism could be much different from human.

Another well studied humanized model is PXB mice, which are chimeric mice with humanized liver that express the urokinase plasminogen activator gene on a severely compromised immune-deficient (SCID) background (Tateno et al., 2015). These PXB mice have constitutive expression of a protease in mouse hepatocytes causing hepatic injury and thus permit the selective expansion of fully functional human hepatocytes upon transplantation. The livers of these mice can be highly (up to >90%–95%) repopulated with human hepatocytes, and based on mRNA and protein measurements, the expression levels of drug-metabolizing enzymes and drug transporters in hepatocytes of PXB mice were similar to those in human livers (Tateno et al., 2004; Nishimura et al., 2005; Okumura et al., 2007; Ohtsuki et al., 2014). PXB chimeric mice with humanized liver have been used for human PK prediction (Sanoh et al., 2012; Miyamoto et al., 2017; Naritomi et al., 2019), human-specific metabolite identification (Samuelsson et al., 2012; Bateman et al., 2014), and DDI studies (Katoh et al., 2007; Uchida et al., 2018).

The purpose of our studies was to evaluate the utility of PXB chimeric mouse model to predict liver disposition of OATP substrates in humans. To this end, systemic PK and liver distribution of five OATP substrates, including rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide, were studied in PXB versus SCID mice and compared with the available clinical data on their hepatic disposition. In addition, uptake kinetics of these five OATP substrates were characterized in plated hepatocytes from PXB and C57 mouse to compare with the findings from in vivo studies. These drugs have been reported to interact with the hepatic OATPs in either in vitro systems and/or clinical studies involving patients with polymorphic variants of OATP1B1 to support the contributing role of active uptake in their liver disposition (Ieiri et al., 2009; Niemi et al., 2011). Moreover, these drugs have also been associated with clinical DDIs due to inhibition of active transport, either in isolation or in conjunction with a reduction in metabolism (Hirano et al., 2006; Ieiri et al., 2009; Prueksaritanont et al., 2014). Given that the in vivo disposition of many OATP substrates is complex—involving multiple elimination pathways via various drug transporters and metabolizing enzymes—PXB chimeric mice could be a promising model for preclinical to clinical translation of hepatic drug disposition. However, understanding the PK and liver disposition of different OATP substrates in these mice relative to humans is essential before they can be used for mechanistic understanding and translational studies in drug discovery and development.

Materials and Methods

Materials.

Plateable mouse hepatocytes isolated from C57 mice were purchased from BioIVT (Hicksville, NY). PXB hepatocytes were isolated from chimeric PXB mice and freshly plated by PhoenixBio (Hiroshima, Japan). Hepatocyte plating and maintenance media were composed of Williams E medium supplemented with either Primary Hepatocyte Thawing and Plating or Primary Hepatocyte Maintenance Supplements (ThermoFisher Scientific, Waltham, MA). Universal cryopreservation recovery medium was obtained from In Vitro ADMET Laboratories (Columbia, MD). Twenty-four–well collagen I–coated plates were purchased from BD Biosciences (Oxford, UK). Bicinchoninic acid protein assay was purchased from Sigma-Aldrich (St. Louis, MO). Test compounds were obtained from Sigma-Aldrich or from the internal Vertex archive (Boston, MA).

Animal Husbandry.

Mice were kept in Techniplast Green Line individually ventilated mouse cages. Autoclaved food and water were available ad libitum. Light was on a 12:12 light/dark cycle with the lighting phase starting at 6:30 AM. Temperature and relative humidity were maintained between 21°C and 23°C and 30%–70%, respectively. Animals were acclimatized for at least 72 hours before starting an experimental procedure.

Animals and Treatments.

All animal studies were approved by the Institution for Animal Care and Use within an Association for Assessment and Accreditation of Laboratory Animal Care–accredited vivarium. PXB male mice were obtained from PhoenixBio at ages 16–22 weeks, and SCID male mice were purchased from Charles River Laboratories (Wilmington, MA; model #236) at an age range of 10–12 weeks and were further aged in house to be 12–16 weeks at time of study. Mice were dosed orally with one of the following OATP substrates: rosuvastatin calcium, pravastatin sodium, valsartan, repaglinide, or pitavastatin calcium. Initially, each substrate was administered at varying single doses according to Table 1 in PXB and SCID mice (n = 3 per substrate) with whole blood samples collected out to 24 hours to assess dose versus exposure relationship. Subsequently, these data were used to select doses for our primary goal of measuring liver-to-plasma Kpuu at clinically relevant systemic exposure. During the Kpuu studies, substrates were dosed in PXB and SCID mice (four to five mice per compound per strain) via oral administration using varying regimens according to Table 2. Terminal plasma and liver were collected at Cmax (Tmax) (formally determined from single dose PK studies) for each substrate according to Table 2. The vehicles used to formulate the OATP substrates were the same for single dose PK and Kpuu studies.

View this table:
TABLE 1

Pharmacokinetic parameters after a single oral dose of OATP substrates with whole blood samples taken out to 24 h in PXB and SCID mice

Data are means ± S.D.

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TABLE 2

Dose selections for liver-to-plasma Kpuu studies in PXB and SCID mice

Tissue and Blood/Plasma Sampling.

Whole blood samples (10 µl blood: 40 µl water) were collected via the tail vein at 30 minutes and 1, 2, 4, 7, and 24 hours for pharmacokinetic analyses. Blood was mixed with 40 µl of deionized water immediately after collection and then placed on dry ice. Whole blood samples were stored at −80°C until analysis.

For Kpuu evaluation, plasma was collected via the vena cava at Tmax, followed by cardiac perfusion with heparinized saline (50 µl of 1000 USP U/ml heparin per ml saline). Prior to perfusion, animals were aestheticized with ketamine (40 mg/ml) and xylazine (2 mg/ml) dosed i.p. 0.1 ml per 10 g body weight. Liver was resected, weighed, and placed on dry ice. Liver samples were homogenized in phosphate buffer (typically 3-fold dilution) for concentration determination, and the samples were further diluted as needed for the liver homogenate binding assay.

Plasma Protein Binding and Liver Binding Assays.

Rapid equilibrium dialysis (RED) plates and inserts (molecular weight cutoff: 8K) were purchased from Thermo Fisher Scientific. Dialysis phosphate buffer (100 mM sodium phosphate in 150 mM NaCl, pH 7.4) was prepared in house. The internal standard (IS) was a proprietary compound synthesized in house. Plasma dilution method was used as needed for the plasma protein binding assay. Aliquots (200 µl) of plasma, diluted plasma, or liver homogenate samples were loaded into the donor chamber of the RED device for dialysis against 350 µl of the phosphate buffer. The loaded RED device was sealed with an adhesive membrane and agitated at ∼450 rpm for 18 (plasma) or 6 hours (liver homogenate) in the 37°C incubator with 85% humidity. The assay was performed in triplicate. Time zero samples were prepared by adding an aliquot of the plasma, diluted plasma, or liver homogenate sample prior to dialysis to an equal volume of buffer and extracted with the IS solution in acetonitrile. After incubation, an aliquot of the sample was taken from the donor side of the RED device and added to an equal volume of buffer and extracted with the IS solution in acetonitrile. An aliquot of the dialysate sample was taken from the receiver side of the RED device and added to an equal volume of blank matrix (plasma, diluted plasma, or liver homogenate) and extracted with the IS solution. Samples were vortexed and centrifuged at 2950g for 20 minutes, and the supernatants were transferred to 96-well plates for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis.

Fraction Unbound Data Analysis.

Embedded ImageCfree indicates the peak area ratio of the compound in the dialysate (receiver side) after incubation; Ctotal indicates the peak area ratio of the compound in plasma or liver homogenate (donor side) after incubation.

The formula for calculating fraction unbound Fu (corrected) from Fu (uncorrected) (fraction unbound in diluted matrix) is as follows:Embedded Image

Quantification of Rosuvastatin, Pravastatin, Pitavastatin, Valsartan, Repaglinide in Whole Blood, Plasma, and Liver Samples.

The whole blood samples were lysed with four parts of water and extracted with the IS solution in acetonitrile at an aqueous-to-organic ratio of 1:10. The plasma and liver homogenate samples were extracted with the IS solution in acetonitrile at an aqueous-to-organic ratio of 1:10. Samples were vortexed and centrifuged at 2254g for 20 minutes. The extract was injected onto LC-MS/MS for concentration determination.

LC-MS/MS Analysis.

The LC-MS/MS analysis was performed on an API 5500 QTrap or 6500+ triple quadrupole mass spectrometer (AB Sciex, Framingham, MA) equipped with Agilent 1200 or 1290 series binary pumps from Agilent Technologies (Santa Clara, CA) and a CTC Analytics PAL autosampler (CTC Analytics AG, Zwingen, Switzerland). Mobile phase A was 10 mM ammonium acetate (pH 4), and mobile phase B was methanol/acetonitrile (1:1, v/v). The liquid chromatographic analysis was performed on a Unisol C18 column (2.1 × 30 mm, 5 μm) from Agela Technologies (Wilmington, DE). The analyte was eluted using a gradient method with an initial mobile phase composition of 100% A at a flow rate of 800 µl/min. The mobile phase B increased from 0% to 70% in 0.3 minutes and was held at 70% for 0.7 minutes before being ramped up to 99% in 0.2 minutes. The mobile phase was kept at 99% B for 1 minute at an increased flow rate of 1800 µl/min. It was then brought back to the initial condition in 0.3 minutes.

Pitavastatin, valsartan, repaglinide, rosuvastatin, and the IS were monitored in positive electrospray ionization mode using multiple reaction monitoring transitions of m/z 422 → 290, m/z 436 → 235, m/z 453 → 230, m/z 482 → 258, and m/z 406 → 346, respectively. Pravastatin and the IS were monitored in negative electrospray ionization mode using multiple reaction monitoring transitions of m/z 423 → 321 and m/z 404 → 102, respectively.

For plasma protein binding and tissue binding assays, quantitation of the compounds was based on peak area ratio of the compound versus the IS. For the PK studies, quantitation of the compounds was based on calibration curves prepared in blank matrix (plasma, liver homogenate, or a mixture of whole blood/water at 1:4). A fit-for-purpose method qualification was performed for all analytes. The lower limit of quantitation (LLOQ) in whole blood analysis was 5.0 ng/ml for rosuvastatin. The LLOQ in plasma was 1.0 ng/ml for pravastatin, repaglinide, rosuvastatin, and valsartan and 2.5 ng/ml for pitavastatin. The LLOQ in liver sample was 1.0 ng/g for pravastatin, repaglinide, and valsartan; 2.5 ng/g for pitavastatin; and 10 ng/g for rosuvastatin. The accuracy of the methods met the acceptance criteria of ±20% of nominal concentrations. The precision (CV) met the acceptance criteria of ≤20%. A summary of quality control sample data is presented in the Supplemental Table.

Pharmacokinetic Analysis.

Descriptive statistics of whole blood concentrations and pharmacokinetic parameter estimates were calculated using the Watson LIMS Version 7.4 software. The area under the plasma concentration-time curve (AUC) value from time zero to time of last measurable concentration was calculated using the linear trapezoidal rule. Pharmacokinetic parameters for all substrates were obtained by noncompartmental analysis. The slope of the elimination phase (λz) was estimated by linear regression. Cmax and Tmax were obtained directly from the observed values. Log-transformed plasma concentrations were plotted against log-transformed Kpuu values.

In Vitro Hepatocyte Uptake Studies.

Cryopreserved hepatocytes were rapidly thawed in a 37°C bath and poured into prewarmed universal cryopreservation recovery medium tubes. Mouse hepatocytes were centrifuged at 100g, then resuspended in plating media. Cell number and viability were determined using trypan blue exclusion. All hepatocyte suspensions seeded for uptake experiments contained >80% viable cells. Cells were diluted to a density of 0.4 × 106 cells/ml, and 0.5 ml was added to each well of a 24-well collagen I–coated plate. Plates were incubated for 4 hours in a humidified chamber at 37°C/5% CO2 to allow cell attachment. Plating media was replaced with maintenance media and incubated overnight prior to initiating experiments. Hepatocyte uptake of OATP substrates was measured at a single concentration of 0.1 µM, with exceptions for pravastatin (5 µM) and valsartan (1 µM) due to analytical and permeability characteristics. Total and passive clearance was determined from the initial rate of uptake at 37°C in the absence and presence of the OATPs inhibitor, 1 mM rifamycin SV. Cell monolayers were rinsed twice and preincubated with 250 µl Hanks’ balanced salt solution containing 1 mM rifamycin SV or DMSO solvent for at least 15 minutes. Incubations were initiated by adding 250 µl prewarmed volumes of 2× substrate drug ± 1 mM rifamycin SV to duplicate wells. Substrate was added at 30-second intervals for a total incubation time of 2 minutes, at which time aliquots were collected for analysis. The incubation was terminated by removal of remaining substrate solutions and washing the cell monolayers three times on ice with cold Hanks’ balanced salt solution. Cells were frozen at −20°C and subsequently lysed for 10 minutes in acetonitrile solution containing an analytical internal standard (tolbutamide). Lysates were mixed with an equal volume of water and transferred into tubes for centrifugation at 8385g. Supernatants were submitted for LC-MS/MS analysis to determine intracellular concentration of intracellular fraction, in addition to the unbound concentration of incubation solutions. Representative samples in each assay were treated as above but lysed in radioimmunoprecipitation assay buffer (RIPA buffer) to measure total protein by the bicinchoninic acid assay.

LC-MS/MS Analysis of Hepatocyte Uptake.

Samples were analyzed using an Agilent 1100 series high-performance liquid chromatography system (Agilent) paired with a SCIEX triple quadrupole 6500+ mass spectrometer (SCIEX, Framingham, MA). Aliquots were injected onto a Unisol C18 column (5 μm, 2.1 × 30 mm; Agela Technologies, Newark, DE) using a CTC Analytics PAL (LEAP) autosampler (CTC Analytics AG). The analytes and internal standard were eluted using a gradient method with a flow rate from 0.8 to 1.8 ml/min. Mobile phase A was 10 mM ammonium acetate in water (pH 4.0), and mobile phase B was acetonitrile/methanol (50/50, v/v). The peak area ratio of analyte/internal standard was quantified by standard regression, including only standards with response within 30% of nominal concentration. A fit-for-purpose method qualification was performed as well for all the analytes. The LLOQ was 50 pM. The same analysis acceptance criteria as described earlier were applied for each analytical run. A summary of quality control sample data is presented in the Supplemental Table.

Determination of Hepatocyte Kinetic Uptake Parameters.

Total uptake clearance (CLuptake) and passive clearance (CLpassive) values were measured by the rate of hepatocyte uptake in the absence and presence of an OATP inhibitor, 1 mM rifamycin SV, respectively. The initial rate of uptake was determined over 2 minutes including four time points, 0.5, 1, 1.5 and 2 minutes, as the slope of the linear regression of the intracellular concentration versus time plot. CLuptake and CLpassive were calculated by dividing slope by the measured average unbound concentration of the incubation solutions and normalizing to protein content. Active uptake clearance (CLactive) was calculated by subtracting CLpassive from CLuptake. The differences of in vitro hepatocyte uptake clearance between C57 and PXB mice were evaluated by unpaired t test with P value of 0.05.

Results

In Vitro Uptake of Five OATP Substrates in C57 Mouse and PXB Chimeric Mouse Hepatocytes.

Five OATP substrates, including rosuvastatin, pitavastatin, pravastatin, valsartan, and repaglinide, were investigated in the plated C57 mouse and PXB chimeric mouse hepatocytes. Unbound uptake clearance and passive diffusion were measured from initial rates at 0.5, 1.0, 1.5, and 2 minutes in the absence and presence of the OATP inhibitor, 1 mM rifamycin SV, respectively. The unbound uptake clearance (CLuptake) and transporter-mediated unbound CLactive are presented in Fig. 1. For rosuvastatin, pravastatin, and valsartan, CLuptake and CLactive in C57 mouse hepatocytes were substantially higher (close to 10-fold) than in PXB mouse hepatocytes. In the case of pitavastatin, CLuptake and CLactive in C57 mouse hepatocytes were moderately higher (2–4-fold) than in PXB mouse hepatocytes, whereas CLuptake and CLactive in C57 mouse and PXB chimeric mouse hepatocytes were comparable for repaglinide. Additionally, active uptake contributed to more than 90% of total uptake of rosuvastatin, pravastatin, pitavastatin, and valsartan in mouse hepatocytes, whereas the relative contribution of active transport to total uptake of repaglinide was much smaller (∼65%) in mouse hepatocytes (Fig. 1). Interestingly, the relative contribution of active transport to the total uptake was more profound in C57 mouse than PXB chimeric mouse hepatocytes. Particularly for pravastatin, active uptake contributed to more than 95% of total uptake in C57 mouse hepatocytes but only ∼60% of total uptake in PXB hepatocytes. It is worth pointing out that we were unable to acquire hepatocytes from control SCID mouse and tested the five OATP substrate in hepatocytes from C57 mouse instead. Hence, it is possible that the hepatocytes from C57 mouse function somewhat differently from SCID mouse.

Fig. 1.
Fig. 1.

Histograms comparing uptake clearance in PXB and C57 mouse hepatocytes for five OATP substrates, including rosuvastatin, pitavastatin, pravastatin, valsartan, and repaglinide. Total uptake clearance (A), active uptake clearance (B), and percentage of active uptake (C) are presented. Hepatocyte uptake of OATP substrates was measured at a single concentration of 0.1 µM, with exceptions for pravastatin (5 µM) and valsartan (1 µM) at four time points, including 0.5, 1, 1.5, and 2 minutes. Total and passive clearance was determined from the initial rate of uptake at 37°C in the absence and presence of the OATP inhibitor, 1 mM rifamycin SV, respectively. Data are means ± S.D. *P < 0.05, C57 mouse hepatocyte uptake vs. PXB mouse hepatocyte uptake.

PK of Rosuvastatin, Pitavastatin, Pravastatin, Valsartan, and Repaglinide in PXB and SCID Mice.

Systemic PK of the five OATP substrates, including rosuvastatin, pitavastatin, pravastatin, repaglinide, and valsartan, were assessed in PXB and SCID mice. After single oral administration of each compound, whole blood samples were collected for full PK profile up to 24 hours in PXB and the control SCID mice to assess dose-exposure relationship. The results from dose-proportionality PK studies (Table 1) were used to determine the doses necessary to reach the reported clinical Cmax for liver-to-plasma Kpuu studies. The reported clinical Cmax, measured human Fu of plasma (Fu,plasma), and calculated free Cmax, as well as the doses chosen for Kpuu studies of five compounds, are summarized in Table 2. Unbound fractions of five compounds in plasma and liver were determined by rapid equilibrium dialysis (Table 3) to calculate unbound plasma and unbound liver concentrations (Table 4) for liver-to-plasma Kpuu estimations. Statistical significance of liver-to-plasma Kpuu between PXB and SCID mouse was determined by unpaired Student’s t test with a P value of 0.05. Rosuvastatin (P < 0.01) showed the most significant difference.

View this table:
TABLE 3

Plasma protein binding and liver binding of rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide in PXB and SCID mice from ex vivo binding assay with tissues collected at Tmax (h)

Data are means ± S.D.

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TABLE 4

Unbound plasma and liver drug concentrations of rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide in PXB and SCID mice in liver-to-plasma Kpuu studies with tissues collected at Tmax (h)

Data are means ± S.D.

In Vivo Kpuu Measurement of Rosuvastatin.

The reported rosuvastatin clinical Cmax is 44 ng/ml (Luvai et al., 2012), which equates to ∼7.5 ng/ml unbound rosuvastatin in plasma with a human Fu,plasma of 17.1% (internal data). Based on dose-proportionality studies, single doses of 15 and 30 mg/kg for SCID mice and 1.5 and 3 mg/kg for PXB mice were selected for liver-to-plasma rosuvastatin Kpuu measurements (Table 2). It is worth mentioning that we have evaluated and compared the Kpuu values from multiple dose studies where rosuvastatin was dosed twice a day for 3 days to reach steady state or after a single dose with samples at Tmax, and the Kpuu values from the two methods were comparable. Thus, plasma and liver concentrations of rosuvastatin were measured at Tmax for each single dose. The plasma and liver binding of rosuvastatin in PXB and SCID mice were determined by rapid equilibrium dialysis (Table 3) to calculate unbound plasma and unbound liver concentrations (Table 4). Figure 2 shows rosuvastatin liver-to-plasma Kpuu values in both PXB and SCID mice at unbound plasma concentration matching the free Cmax in human, and the data clearly indicated that rosuvastatin Kpuu was dramatically increased in SCID mice relative to PXB mice. The average rosuvastatin liver-to-plasma Kpuu in PXB mice was ∼23, which was more than 10-fold lower than the average rosuvastatin Kpuu of ∼268 in SCID mice (Fig. 2). The data in Fig. 2 also demonstrated that although the dose in SCID mice was 10-fold higher than the dose in PXB mice, the unbound plasma concentrations in PXB and SCID mice were similar, suggesting more profound hepatic uptake in SCID mice compared with PXB mice. This is consistent with the observation that hepatic uptake of rosuvastatin (and resulting systemic clearance) in SCID mouse was much greater than in PXB mouse in the dose-proportionality study of rosuvastatin (Table 1).

Fig. 2.
Fig. 2.

Rosuvastatin liver-to-plasma Kpuu in PXB and SCID mouse. Rosuvastatin was dosed to SCID mice at 15 and 30 mg/kg and PXB mice at 1.5 and 3 mg/kg. Plasma and liver concentrations of rosuvastatin were measured at 1 hour after dose. The data for each point was from one mouse, and average Kpuu values are presented with S.D. as error bars. **P < 0.01, rosuvastatin liver-to-plasma Kpuu in PXB vs. SCID mouse.

In Vivo Kpuu Measurement of Pravastatin.

Based on the reported clinical Cmax (25 ng/ml) of pravastatin (Hatanaka, 2000), 0.6 and 3 mg/kg for PXB mice and multiple dose levels (1.8, 3, 10, 20, and 40 mg/kg) for SCID mice were selected for pravastatin liver-to-plasma Kpuu evaluation, as well as understanding the relationship between the unbound plasma concentrations in PXB and SCID mice and Kpuu (Table 2). Unbound fractions of pravastatin in plasma and liver were determined (Table 3) to calculate unbound plasma and unbound liver concentrations (Table 4). Figure 3 depicts pravastatin liver-to-plasma Kpuu in PXB and SCID mice, and the data revealed that greater systemic exposures in SCID mice correlated with lower Kpuu values, which is likely due to the saturation of hepatic uptake transporters. At approximate human pravastatin unbound Cmax (∼13 ng/ml), the liver-to-plasma Kpuu in PXB mice was ∼4.4, whereas the Kpuu in SCID mice was ∼121, which is in agreement with the previous reports (Higgins et al., 2014; Salphati et al., 2014), remarkably larger than Kpuu in PXB mice. Additionally, more than 20-fold higher dose of pravastatin was needed in SCID mice as compared with PXB mice to reach the unbound clinical Cmax, indicating that the hepatic uptake (and as a result, systemic clearance) of pravastatin in SCID mice was considerably higher than that in PXB mice, which is also consistent with the observation in pravastatin dose-proportionality study (Table 1).

Fig. 3.
Fig. 3.

Pravastatin liver-to-plasma Kpuu in PXB and SCID mouse. Pravastatin was dosed to SCID mice at 1.8, 3, 10, 20 and 40 mg/kg and PXB mice at 0.6 and 3 mg/kg. Plasma and liver concentrations of pravastatin were measured at 0.5 hours after dose. The average Kpuu for SCID mice was calculated from the doses of 10, 20, and 40 mg/kg, and the average Kpuu for PXB mice was calculated from the dose of 0.6 mg/kg. Each point was from one mouse, and average Kpuu values are presented with S.D. as error bars. BID, twice a day. *P < 0.05, pravastatin liver-to-plasma Kpuu in PXB vs. SCID mouse.

In Vivo Kpuu Measurement of Pitavastatin.

Based on the reported clinical Cmax of pitavastatin (38 ng/ml) (Luo et al., 2015), 0.1/0.2 and 0.2/0.5 mg/kg of pitavastatin were dosed in PXB and SCID mice, respectively, for liver-to-plasma Kpuu measurements (Table 2). Pitavastatin unbound fractions in plasma and liver were measured (Table 3) to calculate unbound plasma and unbound liver concentrations of pitavastatin (Table 4), as well as liver-to-plasma Kpuu (Fig. 4). In contrast to rosuvastatin and pravastatin, pitavastatin liver-to-plasma Kpuu of ∼16 in PXB mice at the unbound pitavastatin plasma concentration, matching the approximate clinical unbound Cmax, was similar to the Kpuu of ∼18 in the control SCID mice, which is consistent with the previously reported liver-to-plasma partition coefficient (Kp) of ∼20 in FVB mice (Salphati et al., 2014). As expected, hepatic uptake of pitavastatin in PXB mice was weaker than in SCID mice but to a much lesser extent than rosuvastatin and pravastatin. These data suggest that the hepatic uptake (and as a result, systemic clearance) of pitavastatin in SCID mice was slightly higher than that in PXB mice.

Fig. 4.
Fig. 4.

Pitavastatin liver-to-plasma Kpuu in PXB and SCID mouse. Pitavastatin was dosed to SCID mice at 0.2 and 0.5 mg/kg and PXB mice at 0.1 and 0.2 mg/kg. Plasma and liver concentrations of pitavastatin were measured at 0.25 hours after dose. The data for each point was from one mouse, and average Kpuu values are presented with S.D. as error bars.

In Vivo Kpuu Measurement of Valsartan.

Based on the reported clinical Cmax (3250 ng/ml) of valsartan (Siddiqui et al., 2011), 8 mg/kg valsartan was dosed in SCID mice, and 5 and 8 mg/kg valsartan were dosed in PXB mice to generate valsartan liver-to-plasma Kpuu (Table 2). Interestingly, valsartan liver-to-plasma Kpuu in PXB mice was about 2-fold higher than Kpuu in SCID mice, with an average valsartan Kpuu of ∼82 in PXB mice and ∼40 in SCID mice at the approximate human unbound Cmax of valsartan, as shown in Fig. 5. Aligning with the single dose PK studies (Table 1), the valsartan total Cmax values in PXB mice were strikingly higher than those in SCID mice in Kpuu studies. However, since SCID mouse plasma Fu of valsartan is ∼10-fold higher than PXB mouse plasma Fu (Table 3), the free plasma concentrations of valsartan in SCID mice were only ∼2-fold lower than those in PXB mice at the same dose, suggesting that the hepatic uptake (and as a result, unbound systemic clearance) of valsartan in SCID mice was only modestly higher (∼2-fold) than that in PXB mice.

Fig. 5.
Fig. 5.

Valsartan liver-to-plasma Kpuu in PXB and SCID mouse. Valsartan was dosed to SCID mice at 8 mg/kg and PXB mice at 5 and 8 mg/kg. Plasma and liver concentrations of valsartan were measured at 0.5 hours after dose. The data for each point were from one mouse, and average Kpuu values are presented with S.D. as error bars. BID, twice a day. *P < 0.05, valsartan liver-to-plasma Kpuu in PXB vs. SCID mouse.

In Vivo Kpuu Measurement of Repaglinide.

Similar to the previous studies, based on the reported clinical Cmax (65 ng/ml) of repaglinide (Hatorp, 2002), 1.5/1, and 0.8 mg/kg of repaglinide were dosed into SCID and PXB mice, respectively, for liver-to-plasma Kpuu studies. Repaglinide liver and plasma protein binding in PXB and SCID mice were determined (Table 3) to calculate unbound plasma and unbound liver concentrations (Table 4), as well as Kpuu (Fig. 6). Repaglinide liver-to-plasma Kpuu values were indistinguishable between PXB and SCID mice, with values slightly higher than 1 in both mouse strains. Additionally, the systemic clearance of repaglinide in SCID and PXB mice was also similar in the Kpuu study, which is consistent with the observation in dose proportionality study of repaglinide. These data imply that hepatic uptake of repaglinide (and resulting systemic clearance) in SCID and PXB mice is similar.

Fig. 6.
Fig. 6.

Repaglinide liver-to-plasma Kpuu in PXB and SCID mouse. Repaglinide was dosed to SCID mice at 1 and 1.5 mg/kg, and to PXB mice at 0.8 mg/kg. Plasma and liver concentrations of repaglinide were measured at 0.5 hours after dose. The data for each point were from one mouse, and average Kpuu values are presented with S.D. as error bars. BID, twice a day.

Discussion

The utility of animal models in understanding drug disposition and its extrapolation to human is challenging due to differences in enzyme and transporter substrate specificity as well as expression levels across species (Hagenbuch and Meier, 2003; Chu et al., 2013; Grime and Paine, 2013). On the other hand, certain clinical pharmacology or mechanistic studies cannot be conducted in humans for ethical and practical reasons. Therefore, humanized animal models could be very valuable to bridge the gap between preclinical and clinical studies to understand the determinants of human drug disposition. PXB chimeric mice with humanized liver have been successfully used for metabolite profiling, PK prediction, and DDI studies. However, to the best of our knowledge, no studies using PXB mice to predict human liver disposition of hepatic uptake transporter substrates have been reported. Hence, a comprehensive understanding of the PK and liver disposition of different hepatic OATP substrates in these mice is needed before they can be used for translational prediction in drug discovery and development. Given that the in vivo disposition of many OATP substrates is complex, involving multiple elimination pathways via various drug transporters and metabolizing enzymes, we have studied hepatic uptake of five different OATP transporter substrates, including rosuvastatin, pravastatin, pitavastatin, valsartan, and repaglinide, in PXB chimeric and the control SCID mice. Liver-to-plasma Kpuu were measured in both PXB and SCID mice and compared with the published human Kpuu data from the clinical imaging studies, PK/PD studies, or in vitro studies. The goal of these studies was to assess PXB mice as a potential tool for studying the role of hepatic uptake transporters in drug disposition in translational research.

The hypothesis was that if the liver of PXB chimeric mouse truly functions like a human liver in terms of hepatic drug uptake, metabolism, and biliary clearance, then the liver uptake and liver-to-plasma Kpuu of OATP substrates in PXB mice will be comparable to humans and different from that in mice. It was reported previously that the active uptake clearance in rat hepatocytes on average was 7.1-fold higher than in human hepatocytes based on a comparison of uptake clearance of seven OATP substrates in rat and human hepatocytes (Ménochet et al., 2012a,b). Additionally, human OATP–transgenic mice had a weaker uptake efficiency for statins than control mice (Higgins et al., 2014; Salphati et al., 2014), which could be explained by differences in transporter expression and/or specificity between the two mouse strains. In line with this observation, mouse Oatp expression levels in liver and hepatocytes were reported to be much greater than that in human liver and hepatocytes (Wang et al., 2015). Given that the sequence homology of Oatps between mice and rats is quite high (Meier-Abt et al., 2005), it is expected that there are minimal species differences between rat and mouse Oatps.

First the uptake of the five well characterized OATP substrates, including rosuvastatin, pitavastatin, pravastatin, valsartan, and repaglinide, in hepatocytes from C57 mouse and PXB chimeric mouse were characterized (Fig. 1). In the case of rosuvastatin, pravastatin, and valsartan, the overall and transporter-mediated active uptake clearances in mouse hepatocytes were markedly higher than in PXB chimeric mouse hepatocytes. For pitavastatin, the uptake in mouse hepatocytes was moderately higher than PXB hepatocytes, whereas repaglinide had a similar uptake in mouse and PXB mouse hepatocytes. As described above, it has been reported previously that rat hepatocytes exhibit greater uptake activities for OATP substrates, and the intrinsic active uptake of rosuvastatin, pravastatin, and valsartan in rat hepatocytes was much greater than that in human hepatocytes (Ménochet et al., 2012a; Grime and Paine, 2013); in contrast, repaglinide and pitavastatin active uptake clearance values were within 3-fold of each other in the two species. Thus, the uptake differences between C57 mouse and PXB mouse hepatocytes observed in our studies are aligned with the reported uptake differences between rat and human hepatocytes. These data substantiate that PXB mouse hepatocytes function more like human hepatocytes. Although in general the expression levels of Oatps are higher in rodents than humans, the substrate affinity toward human transporters could be greater than rodent hepatocytes for drugs such as pitavastatin (Ménochet et al., 2012b). Consequently, the species difference in hepatocyte uptake of pitavastatin is less pronounced than rosuvastatin, pravastatin, and valsartan. Despite the species difference in hepatic uptake clearance being compound dependent, in general higher hepatic uptake is observed in rodents than humans. It is not surprising that repaglinide displayed similar uptake in mouse and PXB chimeric mouse hepatocytes because the transporter contribution to hepatic uptake of highly permeable repaglinide is much less than the other four compounds.

PK studies of the five OATP substrates demonstrated that the total blood exposures to rosuvastatin, pravastatin, and valsartan in PXB chimeric mice were ∼10-fold or more higher than that in control SCID mice at the same dose, and the blood exposure to pitavastatin in PXB mice was ∼8-fold higher than that in control SCID mice (Table 1). In contrast to these four OATP substrates, the blood exposure to repaglinide in PXB mice was indistinguishable from that in SCID mice at the same dose (Table 1). Considering the plasma binding of rosuvastatin and pravastatin is similar in PXB and SCID mouse, whereas the plasma binding in PXB mouse is higher than SCID mouse for valsartan (∼10-fold) and pitavastatin (∼3-fold) (Table 3), the unbound plasma concentrations in SCID mice were much lower than PXB mice for rosuvastatin and pravastatin and slightly lower (∼2-fold) for pitavastatin and valsartan. These data suggest that the hepatic uptake of these four compounds in SCID mice is greater than that in PXB mice, whereas the hepatic uptake of repaglinide in SCID and PXB mice is comparable. These PK data are reasonably in line with in vitro hepatocyte uptake data (Fig. 1), which also demonstrated equal uptake of repaglinide in PXB and C57 mouse hepatocytes but greater uptake of the other four compounds in mouse hepatocytes than PXB mouse hepatocytes. Quantitatively, the uptake difference of valsartan in mouse and PXB hepatocytes in vitro (∼10-fold) was considerably higher than the uptake difference in SCID and PXB mouse in vivo (∼2-fold), and the reason for this discrepancy remains to be understood. On the other hand, because of a higher passive permeability, the contribution of active uptake to the total hepatic uptake of repaglinide is much less than the aforementioned four OATP substrates, which has been confirmed in DDI studies with OATP inhibitors as well as by the effect of OATP1B1 polymorphisms on repaglinide pharmacokinetics (Kalliokoski et al., 2008; Yoshikado et al., 2017). As a result, the impact of species difference in OATPs/Oatps on hepatic uptake and pharmacokinetics of repaglinide is likely to be smaller. Consistent with the similar hepatic uptake of repaglinide in SCID and PXB mice, the blood exposures to repaglinide in PXB chimeric and SCID mice were comparable.

Since liver-to-plasma Kpuu can be concentration dependent, it is important to measure Kpuu at the plasma concentrations that approximate the unbound Cmax in the clinic. Using data from dose-ranging PK studies, the doses of each compound were selected for Kpuu study to ensure that the unbound plasma concentrations in PXB mouse were close to the human unbound Cmax. The unbound systemic clearance of rosuvastatin in SCID mouse was considerably greater than in PXB mouse, and rosuvastatin liver-to-plasma Kpuu in SCID mice was observed to be remarkably higher (>10-fold) than that in PXB mice (Fig. 2). Consistent with our observation, Uchida et al. (2018) also reported that the total systemic clearance of rosuvastatin was substantially higher in SCID mice than in PXB mice after intravenous administration, and its blood concentrations were ∼5-fold lower in SCID than in PXB mice. Thus, rosuvastatin liver-to-blood Kp was markedly higher (close to 10-fold) in SCID mice than in PXB mice, which indicates a higher uptake in SCID mice than in PXB mice. Moreover, Iusuf et al. (2013) reported that normal mice had lower systemic concentrations of rosuvastatin compared with human OATP–transgenic mice, mainly due to a higher liver distribution. Similar to rosuvastatin, pravastatin liver-to-plasma Kpuu (Fig. 3) in PXB mice was dramatically lower than that in SCID mice, whereas pitavastatin (Fig. 4) and repaglinide Kpuu (Fig. 6) in PXB and SCID mice were similar. Interestingly, valsartan liver-to-plasma Kpuu (Fig. 5) in PXB mice were ∼2-fold of Kpuu in SCID mice.

According to the extended clearance concept, liver-to-plasma Kpuu is governed by multiple clearance pathways, including the intrinsic clearance of passive diffusion, active hepatic uptake, sinusoidal efflux, biliary excretion, and metabolism. Differences in the metabolism of these OATP substrates in mice and humans can potentially obscure the in vivo impact of the OATP transporters on drug disposition in these two mouse models. Rosuvastatin and pravastatin are hydrophilic with a lower passive permeability than other statins, and their systemic clearance is mostly mediated by hepatic uptake and biliary clearance as well as renal clearance of the unchanged drug, with negligible metabolism in mice and humans (Martin et al., 2003; Kitamura et al., 2008; Iusuf et al., 2013). Furthermore, hepatic uptake of rosuvastatin and pravastatin via OATPs significantly influences their PK rather than biliary efflux via other transporters, because the uptake process is the rate-determining step in their disposition (Shitara and Sugiyama, 2006; Elsby et al., 2012; Shitara et al., 2013). Accordingly, rosuvastatin and pravastatin liver-to-plasma Kpuu is mainly determined by active hepatic uptake. Thus, Kpuu in PXB mice is expected to be much smaller than that in SCID mice because of species difference in OATP/Oatp-mediated hepatic uptake as discussed previously. Additionally, the contribution of active uptake to total uptake of pravastatin in PXB hepatocytes was much smaller (∼60%) compared with C57 mouse hepatocytes (>95%) (Fig. 1). Consistent with this, the liver-to-plasma Kpuu of pravastatin in PXB mouse was much lower than that in SCID mice and only modestly greater than unity.

Compared with rosuvastatin and pravastatin, pitavastatin has a higher passive permeability. The unbound plasma concentrations of pitavastatin in PXB mice were slightly higher than SCID mice at the same dose in liver-to-plasma Kpuu studies, suggesting that the unbound hepatic uptake of pitavastatin in SCID mice is marginally higher than PXB. These observations are consistent with the fact that pitavastatin AUC increases upon coadministration with rifampin (an OATP/Oatp inhibitor) were similar in both humans (Prueksaritanont et al., 2014) and mice (internal data), suggesting that hepatic uptake of pitavastatin in human and mouse are similar. Additionally, the major clearance pathway for pitavastatin is breast cancer resistance protein (Bcrp)-mediated biliary clearance, and mouse liver Bcrp expression level is substantial higher (3–8-fold) than human (Chu et al., 2013). Both of these factors could contribute to a similar liver-to-plasma Kpuu for pitavastatin in SCID and PXB mice.

Although the unbound hepatic uptake and systemic clearance of valsartan in SCID mice were moderately higher than PXB mice (∼2-fold), valsartan liver-to-plasma Kpuu in SCID was ∼2-fold lower than PXB mice (Fig. 5). It has been reported that multidrug resistance-associated protein 2 (MRP2)–mediated biliary clearance plays a prominent role in valsartan liver disposition, and the unbound valsartan biliary clearance in rats was ∼35-fold higher than that in humans (Grime and Paine, 2013), which is consistent with the report that the amount of Mrp2 in rat liver was approximately 10-fold greater than that in human (Li et al., 2009; Wang et al., 2015; Fallon et al., 2016). It is possible that a much greater biliary clearance of valsartan in SCID mouse partially masked the difference in liver-to-plasma Kpuu values of valsartan between PXB and SCID mice. In the case of repaglinide, both hepatic active uptake and metabolism are important determinants of its hepatic clearance (Patilea-Vrana and Unadkat, 2016). As discussed previously, the contribution of active uptake to the total hepatic uptake of repaglinide was much smaller than other compounds. As such, repaglinide liver-to-plasma Kpuu in PXB and SCID mice are anticipated to be comparable and close to unity. Additionally, although the uptake clearance of valsartan was much lower than repaglinide, since the contributions of active uptake to total uptake were substantially higher for valsartan than repaglinide in PXB and SCID mouse hepatocytes (Fig. 1), it is not surprising that valsartan liver-to-plasma Kpuu were much higher than repaglinide in both SCID and PXB mice.

The ultimate objective of these studies was to compare liver-to-plasma Kpuu in PXB mice with human Kpuu to validate whether PXB mice would be an acceptable translational model to predict human liver disposition of hepatic uptake transporter substrates. It should be noted that Kpuu values are concentration dependent, and higher plasma concentrations can saturate hepatic uptake transporters, leading to lower Kpuu. Pravastatin Kpuu data have clearly demonstrated that Kpuu decreased with higher plasma concentrations of pravastatin (Fig. 3). Therefore, the dosing duration and regimen in PXB mice were appropriately selected to ensure that Kpuu values were determined at clinically relevant in vivo exposure. A comparison of the measured liver-to-plasma Kpuu from PXB mice, SCID mice and human Kpuu from positron emission tomography (PET), PK/PD modeling, and in vitro human hepatocytes is shown in Table 5. The measured Kpuu from PXB mice are in good agreement with the reported human liver Kpuu (within 2-fold), except for pitavastatin. High-quality in vivo human Kpuu data are relatively very scarce since liver biopsy is invasive and PET imaging has some limitations (e.g., interference from metabolites, nonspecific binding to tissues). Thus, very few in vivo human Kpuu values are available for comparison with PXB mice. Human liver PET data has been reported for [11C] rosuvastatin (Billington et al., 2019) and its liver-to-plasma Kp was estimated to be ∼38 using the terminal phase (after 15 minutes) of rosuvastatin liver and plasma data. Then Kp was corrected with our in-house–measured rosuvastatin human Fu,plasma of 17.1% and PXB mouse liver Fu of 19.1% to estimate liver-to-plasma Kpuu of ∼43 in human, which is within 2-fold of rosuvastatin Kpuu of ∼23 observed in PXB mouse study. Besides rosuvastatin, human liver PET data has also been reported for [11C]dehydropravastatin (DHP) (Kaneko et al., 2018), an analog of pravastatin. With the similar transporter interactions between DHP and pravastatin, the Kpuu value of DHP could be used as a surrogate for the Kpuu of pravastatin. The Kpuu was estimated to be ∼7 using terminal phase (after 15 minutes) of DHP data with pravastatin human Fu,plasma of 52.0% and human liver Fu of 49.0% (internal data), which is within 2-fold of pravastatin liver-to-plasma Kpuu of ∼4.4 measured in PXB mice. Moreover, human Kpuu values of rosuvastatin and pravastatin have been calculated using PK/PD modeling. These estimates carry some uncertainty because these are indirectly derived from a number of in vitro and in vivo parameters. Nevertheless, rosuvastatin human Kpuu of ∼10 was estimated from PK/PD (Riccardi et al., 2017), which is ∼2-fold of rosuvastatin Kpuu of ∼23 in PXB mice. In addition, pravastatin liver-to-plasma Kpuu of ∼5.3 was estimated based on PK/PD (Riccardi et al., 2017), which is also close to the pravastatin Kpuu of ∼4.4 measured in PXB mice. Due to lack of human PET and PK/PD modeling data, human liver-to-plasma Kpuu of pitavastatin, valsartan, and repaglinide were generated from in vitro human hepatocytes (Table 5) (De Bruyn et al., 2018). The data indicate that the Kpuu values of valsartan and repaglinide from PXB mice are similar to those generated in human hepatocytes. However, pitavastatin PXB mouse Kpuu of ∼14 is almost 10-fold higher than the Kpuu of 1.7 generated using in vitro hepatocytes. Interestingly, another group reported pitavastatin liver-to-plasma Kpuu values from 2.10 to 14.58 using different methods in human hepatocytes (Riede et al., 2017). The reason for the discrepancy is not yet clear, and further studies are warranted to understand this disconnect. It is possible that in vitro hepatocytes data could misrepresent in vivo human Kpuu due to the different hepatic uptake transporter expressions and functions between in vitro and in vivo settings. Overall, given the uncertainty of the human Kpuu values from PET, PK/PD modeling and human hepatocytes, the Kpuu for the five OATP substrates from PXB mice appear to suggest a reasonable translational accuracy for the model. To further evaluate the utility of PXB mice for predicting human liver partitioning of hepatic OATP substrates, high quality human liver PET or biopsy data are greatly desired.

View this table:
Table 5

Comparison of liver-to-plasma Kpuu of OATP substrates in PXB, SCID mouse and human

In summary, our data suggest that PXB chimeric mice with humanized liver offer promise for gaining an understanding of human hepatic uptake and the anticipated human liver-to-plasma Kpuu in drug discovery and development, which can help develop the confidence for preclinical to clinical translation of liver drug disposition. This model can be a potentially useful tool for quantitative absorption, distribution, metabolism, and excretion characterization and mechanistic studies, particularly for drugs where multiple enzymes/transporters play a role in their disposition.

Acknowledgments

The authors thank the Chimeric Mouse with Humanized Liver (CMHL) Consortium for the support of the study.

Authorship Contributions

Participated in research design: Feng, Pemberton, Dworakowski, Kumar.

Conducted experiments: Pemberton, Ye, Zetterberg.

Contributed new reagents or analytic tools: Ye, Zetterberg, Morikawa.

Performed data analysis: Feng, Pemberton, Ye, Zetterberg, Wang.

Wrote or contributed to the writing of the manuscript: Feng, Pemberton, Ye, Zetterberg, Kumar.

Footnotes

    • Received October 6, 2020.
    • Accepted December 8, 2020.

  • ↵1 Current affiliation: Syros Pharmaceuticals, Cambridge, Massachusetts.

  • The work was supported by Vertex Pharmaceutical Company Limited and Chimeric Mouse with Humanized Liver (CMHL) Consortium. Y.M. is an employee of PhoenixBio Co., Ltd., Hiroshima, Japan. He provided PXB mice but did not have any additional role in conducting the experiment, performing the data analysis, or preparing the manuscript. All other authors (B.F., R.P., W.D., Z.Y., C.Z., G.W., and S.K.) declare no competing interest.

  • https://doi.org/10.1124/dmd.120.000276.

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

Abbreviations

AUC
area under the plasma concentration-time curve
CLactive
active uptake clearance
CLpassive
passive clearance
CLuptake
total uptake clearance
DDI
drug-drug interaction
DHP
dehydropravastatin
Fu
fraction unbound
Fu,plasma
fraction unbound of plasma
IS
internal standard
Kpuu
unbound partition coefficient
LC-MS/MS
liquid chromatography–tandem mass spectrometry
LLOQ
lower limit of quantitation
MRP2
multidrug resistance-associated protein 2
OATP
organic anion-transporting polypeptide
PD
pharmacodynamic
PET
positron emission tomography
PK
pharmacokinetic
PXB
urokinase plasminogen activator/SCID repopulated with over 90% human hepatocytes
RED
rapid equilibrium dialysis
SCID
severely compromised immune-deficient
Tmax
time to Cmax

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Human Liver Partitioning of OATP Substrates

Bo Feng, Rachel Pemberton, Wojciech Dworakowski, Zhengqi Ye, Craig Zetterberg, Guanyu Wang, Yoshio Morikawa and Sanjeev Kumar
Drug Metabolism and Disposition March 1, 2021, 49 (3) 254-264; DOI: https://doi.org/10.1124/dmd.120.000276
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