The application of organ-on-chip models for the prediction of human pharmacokinetic profiles during drug development.

Organ-on-chip (OoC) technology has led to in vitro models with many new possibilities compared to conventional in vitro and in vivo models. In this review, the potential of OoC models to improve the prediction of human oral bioavailability and intrinsic clearance is discussed, with a focus on the functionality of the models and the application in current drug development practice. Multi-OoC models demonstrating the application for pharmacokinetic (PK) studies are summarized and existing challenges are identified. Physiological parameters for a minimal viable platform of a multi-OoC model to study PK are provided, together with PK specific read-outs and recommendations for relevant reference compounds to validate the model. Finally, the translation to in vivo PK profiles is discussed, which will be required to routinely apply OoC models during drug development.

Application of proteomics to understand maturation of drug metabolizing enzymes and transporters for the optimization of pediatric drug therapy.

Drug disposition in children is different compared to adults. Growth and developmental change the processes involved in drug disposition and efficacy, including membrane transporters and drug metabolizing enzymes, but for many of these proteins, the exact changes have not been fully elucidated to date. Quantitative proteomics offers a solution to analyze many DME and DT proteins at once and can be performed with very small tissue samples, overcoming many of the challenges previously limiting research in this pediatric field. Liquid chromatography tandem mass spectrometry (LC-MS/MS) based methods for quantification of (membrane) proteins has evolved as a golden standard for proteomic analysis. The last years, big steps have been made in maturation studies of hepatic and renal drug transporters and drug metabolizing enzymes using this method. Protein and organ specific maturation patterns have been identified for the human liver and kidney, which aids pharmacological modelling and predicting drug dosing in the pediatric population. Further research should focus on other organs, like intestine and brain, as well as on innovative methods in which proteomics can be used to further overcome the limited access to pediatric tissues, including liquid biopsies and organoids. In this review there is aimed to provide an overview of available human pediatric proteomics data, discuss its challenges and provide guidance for future research.

The Progress of Intestinal Epithelial Models from Cell Lines to Gut-On-Chip.

Over the past years, several preclinical in vitro and ex vivo models have been developed that helped to understand some of the critical aspects of intestinal functions in health and disease such as inflammatory bowel disease (IBD). However, the translation to the human in vivo situation remains problematic. The main reason for this is that these approaches fail to fully reflect the multifactorial and complex in vivo environment (e.g., including microbiota, nutrition, and immune response) in the gut system. Although conventional models such as cell lines, Ussing chamber, and the everted sac are still used, increasingly more sophisticated intestinal models have been developed over the past years including organoids, InTESTine™ and microfluidic gut-on-chip. In this review, we gathered the most recent insights on the setup, advantages, limitations, and future perspectives of most frequently used in vitro and ex vivo models to study intestinal physiology and functions in health and disease.

Towards human ex vivo organ perfusion models to elucidate drug pharmacokinetics in health and disease.

To predict the absorption, distribution, metabolism and excretion (ADME) profile of candidate drugs a variety of preclinical models can be applied. The ADME and toxicological behavior of newly developed drugs are often investigated prior to assessment in humans, which is associated with long time-lines and high costs. Therefore, good predictions of ADME profiles earlier in the drug development process are very valuable. Good prediction of intestinal absorption and renal and biliary excretion remain especially difficult, as there is an interplay of active transport and metabolism involved. To study these processes, including enterohepatic circulation, ex vivo tissue models are highly relevant and can be regarded as the bridge between in vitro and in vivo models. In this review the current in vitro, in vivo and in more detail ex vivo models for studying pharmacokinetics in health and disease are discussed. Additionally, we propose novel models, i.e., perfused whole-organs, which we envision will generate valuable pharmacokinetic information in the future due to improved translation to the in vivo situation. These machine-perfused organ models will be particularly interesting in combination with biomarkers for assessing the functionality of transporter and CYP450 proteins.

Regional Expression Levels of Drug Transporters and Metabolizing Enzymes along the Pig and Human Intestinal Tract and Comparison with Caco-2 Cells.

Intestinal transporter proteins and metabolizing enzymes play a crucial role in the oral absorption of a wide variety of drugs. The aim of the current study was to characterize better available intestinal in vitro models by comparing expression levels of these proteins and enzymes between porcine intestine, human intestine, and Caco-2 cells. We therefore determined the absolute protein expression of 19 drug transporters and the mRNA expression of 12 metabolic enzymes along the pig intestinal tract (duodenum, jejunum, ileum; N = 4), in human intestine (jejunum; N = 9), and Caco-2 cells. Expression of the included transporters and enzymes was in general well comparable between porcine and human intestinal tissue, although breast cancer resistance protein, monocarboxylate transporter 5, multidrug resistance protein (MRP) 1, MRP1, MRP3 (∼2-fold), and organic anion-transporting polypeptide (OATP) 4A1 (∼6-fold) was higher expressed in pig compared with human jejunum. Alternatively, expression level of relevant transporter proteins (glucose transporter 1, OATP4A1, MRP2, MRP1, and OATP2B1) was significantly higher (3- to 130-fold) in Caco-2 cells compared with human jejunum. Moreover, all examined CYPs showed at least a fivefold lower gene expression in Caco-2 cells compared with human jejunum, with the smallest differences for CYP1A1 and CYP3A5 and the largest difference for CYP3A4 (871-fold higher expression in human jejunum compared with Caco-2 cells). In conclusion, a comprehensive overview is provided of the expression levels of clinically relevant transporter proteins and metabolic enzymes in porcine and human intestinal tissue and Caco-2 cells, which may assist in deciding upon the most suitable model to further improve our understanding of processes that determine intestinal absorption of compounds.

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes.

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.

Proteomic Analysis of the Developmental Trajectory of Human Hepatic Membrane Transporter Proteins in the First Three Months of Life.

Human hepatic membrane-embedded transporter proteins are involved in trafficking endogenous and exogenous substrates. Even though impact of transporters on pharmacokinetics is recognized, little is known on maturation of transporter protein expression levels, especially during early life. We aimed to study the protein expression of 10 transporters in liver tissue from fetuses, infants, and adults. Transporter protein expression levels [ATP-binding cassette transporter (ABC)B1, ABCG2, ABCC2, ABCC3, bile salt efflux pump, glucose transporter 1, monocarboxylate transporter 1, organic anion transporter polypeptide (OATP)1B1, OATP2B1, and organic cation/carnitine transporter 2) were quantified using ultraperformance liquid chromatography tandem mass spectrometry in snap-frozen postmortem fetal, infant, and adult liver samples. Protein expression was quantified in isolated crude membrane fractions. The possible association between postnatal and postmenstrual age versus protein expression was studied. We studied 25 liver samples, as follows: 10 fetal [median gestational age 23.2 wk (range 16.4-37.9)], 12 infantile [gestational age at birth 35.1 wk (27.1-41.0), postnatal age 1 wk (0-11.4)], and 3 adult. The relationship of protein expression with age was explored by comparing age groups. Correlating age within the fetal/infant age group suggested four specific protein expression patterns, as follows: stable, low to high, high to low, and low-high-low. The impact of growth and development on human membrane transporter protein expression is transporter-dependent. The suggested age-related differences in transporter protein expression may aid our understanding of normal growth and development, and also may impact the disposition of substrate drugs in neonates and young infants.

Quantifying phenotypic flexibility as the response to a high-fat challenge test in different states of metabolic health.

Metabolism maintains homeostasis at chronic hypercaloric conditions, activating postprandial response mechanisms, which come at the cost of adaptation processes such as energy storage, eventually with negative health consequences. This study quantified the metabolic adaptation capacity by studying challenge response curves. After a high-fat challenge, the 8 h response curves of 61 biomarkers related to adipose tissue mass and function, systemic stress, metabolic flexibility, vascular health, and glucose metabolism was compared between 3 metabolic health stages: 10 healthy men, before and after 4 wk of high-fat, high-calorie diet (1300 kcal/d extra), and 9 men with metabolic syndrome (MetS). The MetS subjects had increased fasting concentrations of biomarkers representing the 3 core processes, glucose, TG, and inflammation control, and the challenge response curves of most biomarkers were altered. After the 4 wk hypercaloric dietary intervention, these 3 processes were not changed, as compared with the preintervention state in the healthy subjects, whereas the challenge response curves of almost all endocrine, metabolic, and inflammatory processes regulating these core processes were altered, demonstrating major molecular physiologic efforts to maintain homeostasis. This study thus demonstrates that change in challenge response is a more sensitive biomarker of metabolic resilience than are changes in fasting concentrations.

Human OATP1B1, OATP1B3 and OATP1A2 can mediate the in vivo uptake and clearance of docetaxel.

Organic anion transporting polypeptides (human: OATPs and mouse: Oatps) are uptake transporters with important roles in drug pharmacokinetics and toxicity. We aimed to study the in vivo impact of mouse and human OATP1A/1B transporters on docetaxel plasma clearance and liver and intestinal uptake. Docetaxel was administered to Oatp1a/1b knockout and liver-specific humanized OATP1B1, OATP1B3 and OATP1A2 transgenic mice. Experiments were conducted with a low polysorbate 80 (2.8%) formulation, as 8% polysorbate somewhat inhibited docetaxel plasma clearance after intravenous administration. After intravenous administration (10 mg/kg), Oatp1a/1b knockout mice had an approximately threefold higher plasma area under the curve (AUC). Impaired liver uptake was evident from the significantly reduced (approximately threefold) liver-to-plasma AUC ratios. Absence of mouse Oatp1a/1b transporters did not affect the intestinal absorption of orally administered docetaxel (10 mg/kg), while the systemic exposure of docetaxel was again substantially increased owing to impaired liver uptake. Most importantly, liver-specific expression of each of the human OATP1B1, OATP1B3 and OATP1A2 transporters provided a nearly complete rescue of the increased plasma levels of docetaxel in Oatp1a/1b-null mice after intravenous administration. Our data show that one or more of the mouse Oatp1a/1b transporters and each of the human OATP1A/1B transporters can mediate docetaxel uptake in vivo. This might be clinically relevant for OATP1A/1B-mediated tumor uptake of docetaxel and for docetaxel clearance in patients in whom the transport activity of OATP1A/1B transporters is reduced owing to genetic variation or pharmacological inhibition, leading to potentially altered toxicity and therapeutic efficacy of this drug.

Bcrp1;Mdr1a/b;Mrp2 combination knockout mice: altered disposition of the dietary carcinogen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and its genotoxic metabolites.

The multidrug transporters breast cancer resistance protein (BCRP), multidrug-resistance protein 1 (MDR1), and multidrug-resistance-associated protein (MRP) 2 and 3 eliminate toxic compounds from tissues and the body and affect the pharmacokinetics of many drugs and other potentially toxic compounds. The food-derived carcinogen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) is transported by BCRP, MDR1, and MRP2. To investigate the overlapping functions of Bcrp1, Mdr1a/b, and Mrp2 in vivo, we generated Bcrp1;Mdr1a/b;Mrp2(-/-) mice, which are viable and fertile. These mice, together with Bcrp1;Mrp2;Mrp3(-/-) mice, were used to study the effects of the multidrug transporters on the pharmacokinetics of PhIP and its metabolites. Thirty minutes after oral or intravenous administration of PhIP (1 mg/kg), the PhIP levels in the small intestine were reduced 4- to 6-fold in Bcrp1;Mdr1a/b;Mrp2(-/) (-) and Bcrp1;Mrp2;Mrp3(-/-) mice compared with wild-type mice. Fecal excretion of PhIP was reduced 8- to 20-fold in knockouts. Biliary PhIP excretion was reduced 41-fold in Bcrp1;Mdr1a/b;Mrp2(-/-) mice. Biliary and small intestine levels of PhIP metabolites were reduced in Bcrp1;Mrp2-deficient mice. Furthermore, in both knockout strains, kidney levels and urinary excretion of genotoxic PhIP-metabolites were significantly increased, suggesting that reduced biliary excretion of PhIP and PhIP metabolites leads to increased urinary excretion of these metabolites and increased systemic exposure. Bcrp1 and Mdr1a limited PhIP brain accumulation. In Bcrp1;Mrp2;Mrp3(-/-), but not Bcrp1;Mdr1a/b;Mrp(-/-) mice, the carcinogenic metabolites N2-OH-PhIP (2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and PhIP-5-sulfate (a genotoxicity marker) accumulated in liver tissue, indicating that Mrp3 is involved in the sinusoidal secretion of these compounds. We conclude that Bcrp1, Mdr1a/b, Mrp2, and Mrp3 significantly affect tissue disposition and biliary and fecal elimination of PhIP and its carcinogenic metabolites and may affect PhIP-induced carcinogenesis as a result.

A host-microbial metabolite interaction gut-on-a-chip model of the adult human intestine demonstrates beneficial effects upon inulin treatment of gut microbiome.

Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes, Bifidobacterium, Blautia, and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.

Murine Oatp1a/1b uptake transporters control rosuvastatin systemic exposure without affecting its apparent liver exposure.

Organic anion-transporting polypeptides (OATPs) mediate the liver uptake and hence plasma clearance of a broad range of drugs. For rosuvastatin, a cholesterol-lowering drug and OATP1A/1B substrate, the liver represents both its main therapeutic target and its primary clearance organ. Here we studied the impact of Oatp1a/1b uptake transporters on the pharmacokinetics of rosuvastatin using wild-type and Oatp1a/1b-null mice. After oral administration (15 mg/kg), intestinal absorption of rosuvastatin was not impaired in Oatp1a/1b-null mice, but systemic exposure (area under the curve) was 8-fold higher in these mice compared with wild-type. Although liver exposure was comparable between the two mouse strains (despite the increased blood exposure), the liver-to-blood ratios were markedly decreased (>10-fold) in the absence of Oatp1a/1b transporters. After intravenous administration (5 mg/kg), systemic exposure was 3-fold higher in Oatp1a/1b-null mice than in the wild-type mice. Liver, small intestinal, and kidney exposure were slightly, but not significantly, increased in Oatp1a/1b-null mice. The biliary excretion of rosuvastatin was very fast, with 60% of the dose eliminated within 15 minutes after intravenous administration, and also not significantly altered in Oatp1a/1b-null mice. Rosuvastatin renal clearance, although still minor, was increased ∼15-fold in Oatp1a/1b-null males, suggesting a role of Oatp1a1 in the renal reabsorption of rosuvastatin. Absence of Oatp1a/1b uptake transporters increases the systemic exposure of rosuvastatin by reducing its hepatic extraction ratio. However, liver concentrations are not significantly affected, most likely due to the compensatory activity of high-capacity, low-affinity alternative uptake transporters at higher systemic rosuvastatin levels and the absence of efficient alternative rosuvastatin clearance mechanisms.

Gut-on-a-Chip Research for Drug Development: Implications of Chip Design on Preclinical Oral Bioavailability or Intestinal Disease Studies.

The gut plays a key role in drug absorption and metabolism of orally ingested drugs. Additionally, the characterization of intestinal disease processes is increasingly gaining more attention, as gut health is an important contributor to our overall health. The most recent innovation to study intestinal processes in vitro is the development of gut-on-a-chip (GOC) systems. Compared to conventional in vitro models, they offer more translational value, and many different GOC models have been presented over the past years. Herein, we reflect on the almost unlimited choices in designing and selecting a GOC for preclinical drug (or food) development research. Four components that largely influence the GOC design are highlighted, namely (1) the biological research questions, (2) chip fabrication and materials, (3) tissue engineering, and (4) the environmental and biochemical cues to add or measure in the GOC. Examples of GOC studies in the two major areas of preclinical intestinal research are presented: (1) intestinal absorption and metabolism to study the oral bioavailability of compounds, and (2) treatment-orientated research for intestinal diseases. The last section of this review presents an outlook on the limitations to overcome in order to accelerate preclinical GOC research.

Novel Explanted Human Liver Model to Assess Hepatic Extraction, Biliary Excretion and Transporter Function.

Realistic models predicting hepatobiliary processes in health and disease are lacking. We therefore aimed to develop a physiologically relevant human liver model consisting of normothermic machine perfusion (NMP) of explanted diseased human livers that can assess hepatic extraction, clearance, biliary excretion, and drug-drug interaction (DDI). Eleven livers were included in the study, seven with a cirrhotic and four with a noncirrhotic disease background. After explantation of the diseased liver, NMP was initiated. After 120 minutes of perfusion, a drug cocktail (rosuvastatin, digoxin, metformin, and furosemide; OATP1B1/1B3, P-gp, BCRP, and OCT1 model compounds) was administered to the portal vein and 120 minutes later, a second bolus of the drug cocktail was co-administered with perpetrator drugs to study relevant DDIs. The explanted livers showed good viability and functionality during 360 minutes of NMP. Hepatic extraction ratios close to in vivo reported values were measured. Hepatic clearance of rosuvastatin and digoxin showed to be the most affected by cirrhosis with an increase in maximum plasma concentration (Cmax ) of 11.50 and 2.89 times, respectively, compared with noncirrhotic livers. No major differences were observed for metformin and furosemide. Interaction of rosuvastatin or digoxin with perpetrator drugs were more pronounced in noncirrhotic livers compared with cirrhotic livers. Our results demonstrated that NMP of human diseased explanted livers is an excellent model to assess hepatic extraction, clearance, biliary excretion, and DDI. Gaining insight into pharmacokinetic profiles of OATP1B1/1B3, P-gp, BCRP, and OCT1 model compounds is a first step toward studying transporter functions in diseased livers.

Organic anion-transporting polypeptides 1a/1b control the hepatic uptake of pravastatin in mice.

Organic anion-transporting polypeptides (OATPs) mediate the hepatic uptake of many drugs. Hepatic uptake is crucial for the therapeutic effect of pravastatin, a cholesterol-lowering drug and OATP1A/1B substrate. We aimed to gain empirical insight into the relationship between OATPs and pravastatin pharmacokinetics and toxicity. We therefore compared the distribution and toxicity of pravastatin in wild-type and Oatp1a/1b-null mice. Intestinal absorption of pravastatin was not affected by Oatp1a/1b absence, but systemic plasma exposure (AUC) increased up to 30-fold after oral bolus administration. This increased plasma exposure resulted from reduced hepatic uptake, as evident from 10 to 100-fold lower liver-to-plasma concentration ratios. However, the reductions in liver exposure were far smaller (<2-fold) than the increases in plasma exposure. Reduced pravastatin liver uptake in Oatp1a/1b-null mice was more obvious shortly after intravenous administration, with 8-fold lower biliary pravastatin excretion. Although mice chronically exposed to pravastatin for 60 days evinced little muscular toxicity, Oatp1a/1b-null mice displayed 10-fold higher plasma concentrations and 8-fold lower liver concentrations than wild-type mice. Thus, Oatp1a/1b transporters importantly control the hepatic uptake of pravastatin. Activity-reducing human OATP1B polymorphisms may therefore both reduce pravastatin therapeutic efficacy in the liver and increase systemic toxicity risks, thus compromising its therapeutic index in a two-edged way.

Intestinal explant barrier chip: long-term intestinal absorption screening in a novel microphysiological system using tissue explants.

The majority of intestinal in vitro screening models use cell lines that do not reflect the complexity of the human intestinal tract and hence often fail to accurately predict intestinal drug absorption. Tissue explants have intact intestinal architecture and cell type diversity, but show short viability in static conditions. Here, we present a medium throughput microphysiological system, Intestinal Explant Barrier Chip (IEBC), that creates a dynamic microfluidic microenvironment and prolongs tissue viability. Using a snap fit mechanism, we successfully incorporated human and porcine colon tissue explants and studied tissue functionality, integrity and viability for 24 hours. With a proper distinction of transcellular over paracellular transport (ratio >2), tissue functionality was good at early and late timepoints. Low leakage of FITC-dextran and preserved intracellular lactate dehydrogenase levels indicate maintained tissue integrity and viability, respectively. From a selection of low to high permeability drugs, 6 out of 7 properly ranked according to their fraction absorbed. In conclusion, the IEBC is a novel screening platform benefitting from the complexity of tissue explants and the flow in microfluidic chips.

A proof of concept using the Ussing chamber methodology to study pediatric intestinal drug transport and age-dependent differences in absorption.

Little is known about the impact of age on the processes governing human intestinal drug absorption. The Ussing chamber is a system to study drug transport across tissue barriers, but it has not been used to study drug absorption processes in children. This study aimed to explore the feasibility of the Ussing chamber methodology to assess pediatric intestinal drug absorption. Furthermore, differences between intestinal drug transport processes of children and adults were explored as well as the possible impact of age. Fresh terminal ileal leftover tissues from both children and adults were collected during surgery and prepared for Ussing chamber experiments. Paracellular (enalaprilat), transcellular (propranolol), and carrier-mediated drug transport by MDR1 (talinolol) and BCRP (rosuvastatin) were determined with the Ussing chamber methodology. We calculated apparent permeability coefficients and efflux ratios and explored their relationship with postnatal age. The success rate for the Ussing chamber experiments, as determined by electrophysiological measurements, was similar between children (58%, N = 15, median age: 44 weeks; range 8 weeks to 17 years) and adults (67%, N = 13). Mean serosal to mucosal transport of talinolol by MDR1 and rosuvastatin by BCRP was higher in adult than in pediatric tissues (p = 0.0005 and p = 0.0091). In contrast, within our pediatric cohort, there was no clear correlation for efflux transport across different ages. In conclusion, the Ussing chamber is a suitable model to explore pediatric intestinal drug absorption and can be used to further elucidate ontogeny of individual intestinal pharmacokinetic processes like drug metabolism and transport.

Impact of abcc2 [multidrug resistance-associated protein (MRP) 2], abcc3 (MRP3), and abcg2 (breast cancer resistance protein) on the oral pharmacokinetics of methotrexate and its main metabolite 7-hydroxymethotrexate.

The ATP-binding cassette (ABC) transporters ABCC2 [multidrug resistance-associated protein (MRP) 2], ABCC3 (MRP3), and ABCG2 (breast cancer resistance protein) are involved in the efflux of potentially toxic compounds from the body. We have shown before that ABCC2, ABCC3, and ABCG2 together influence the pharmacokinetics of the anticancer and antirheumatic drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) after intravenous MTX administration. We now have used Abcc2;Abcc3;Abcg2(-/-) and corresponding single and double knockout mice to investigate the relative impact of these transporters on MTX and 7OH-MTX pharmacokinetics after oral MTX administration (50 mg/kg). The plasma areas under the curve (AUC(plasma)) in Abcg2(-/-) and Abcc2;Abcg2(-/-) mice were 1.7- and 3.0-fold higher than those in wild-type mice, respectively, suggesting additive effects of Abcc2 and Abcg2 on oral MTX pharmacokinetics. However, the AUC(plasma) in Abcc2;Abcc3;Abcg2(-/-) mice was not different from that in wild-type mice, indicating that Abcc3 protein is necessary for increased MTX plasma concentrations in the absence of Abcc2 and/or Abcg2. Furthermore, 2 h after administration, MTX liver levels were increased in Abcg2-deficient strains and MTX kidney levels were 2.2-fold increased in Abcc2;Abcg2(-/-) mice compared with those in wild-type mice. The absence of Abcc2 and/or Abcg2 also led to significantly increased liver and kidney levels of 7OH-MTX. Our results suggest that inhibition of ABCG2 and/or ABCC2, genetic polymorphisms or mutations reducing expression or activity of these proteins may increase the oral availability of MTX. Such conditions may also present risk factors for increased MTX-related toxicity in patients treated with oral MTX.

Methotrexate pharmacokinetics in transgenic mice with liver-specific expression of human organic anion-transporting polypeptide 1B1 (SLCO1B1).

Human organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter that can transport a wide variety of drugs. In the present study, we have generated and characterized a transgenic mouse model with specific and functional expression of human OATP1B1 (SLCO1B1) in the liver. Immunohistochemical staining revealed basolateral localization of transgenic OATP1B1 in the liver, whereas no expression of OATP1B1 was found in the kidney and small intestine. Using this transgenic model, the in vivo role of human OATP1B1 in the disposition of the anticancer drug methotrexate (MTX) was studied. In mice on a semisynthetic diet, the area under the plasma concentration-time curve for intravenous methotrexate in SLCO1B1 transgenic mice was 1.5-fold decreased compared with wild-type mice. Furthermore, the amount of MTX in the liver was markedly higher ( approximately 2-fold) in the SLCO1B1 transgenic mice compared with wild-type mice, resulting in 2- to 4-fold higher liver-plasma ratios of MTX. Some murine liver Slco genes were markedly down-regulated on the semisynthetic diet compared with a standard diet, which probably reduced murine Oatp-mediated MTX uptake in the liver and therefore facilitated detection of the function of the transgenic OATP1B1. Taken together, these data demonstrate a marked and possibly rate-limiting role for human OATP1B1 in MTX elimination in vivo. Variation in OATP1B1 activity due to genetic polymorphisms, drug-drug interactions, and possibly dietary conditions may therefore play a role in the severity of MTX-related toxicity. SLCO1B1 transgenic mice could be a useful tool in studying the in vivo role of human OATP1B1 in drug pharmacokinetics.

Organic anion-transporting polypeptide 1B1 mediates transport of Gimatecan and BNP1350 and can be inhibited by several classic ATP-binding cassette (ABC) B1 and/or ABCG2 inhibitors.

Organic anion-transporting polypeptides (OATPs) are important uptake transporters that can have a profound impact on the systemic pharmacokinetics, tissue distribution, and elimination of several drugs. Previous in vivo studies of the pharmacokinetics of the lipophilic camptothecin (CPT) analog gimatecan suggested that the ATP-binding cassette (ABC) B1 (P-glycoprotein) and/or ABCG2 (breast cancer resistance protein) inhibitors elacridar and pantoprazole could inhibit transporters other than ABCB1 and ABCG2. In this study, we tested the possible role of OATP1B1 in this interaction by screening a number of CPT analogs for their transport affinity by human OATP1B1 in vitro. In addition, the impact of several widely used ABCB1 and/or ABCG2 modulators on this OATP1B1-mediated transport was assessed. We identified two novel CPT anticancer drugs, gimatecan and BNP1350, as OATP1B1 substrates, whereas irinotecan, topotecan, and lurtotecan were not transported by OATP1B1. It is interesting to note that transport of 17beta-estradiol 17beta-d-glucuronide (control), gimatecan, and BNP1350 by OATP1B1 could be completely inhibited by the classic ABCB1 and/or ABCG2 inhibitors elacridar, valspodar, pantoprazole, and, to a lesser extent, zosuquidar and verapamil. Therefore, the effect of these ABCB1 and ABCG2 modulators on the plasma pharmacokinetics of gimatecan and BNP1350 (and possibly also other OATP1B1 substrates) may be partly because of inhibition of OATP1B1 besides inhibition of ABCB1 and/or ABCG2. The findings of this study suggest that OATP1B1 polymorphisms or coadministration with one of the ABCB1/ABCG2 inhibitors could affect drug uptake, tissue distribution, and elimination of some CPT anticancer drugs, thereby modifying their efficacy and/or safety profile.