Enteroids to Study Pediatric Intestinal Drug Transport.

Intestinal maturational changes after birth affect the pharmacokinetics (PK) of drugs, having major implications for drug safety and efficacy. However, little is known about ontogeny-related PK patterns in the intestine. To explore the accuracy of human enteroid monolayers for studying drug transport in the pediatric intestine, we compared the drug transporter functionality and expression in enteroid monolayers and tissue from pediatrics and adults. Enteroid monolayers were cultured of 14 pediatric [median (range) age: 44 weeks (2 days-13 years)] and 5 adult donors, in which bidirectional drug transport experiments were performed. In parallel, we performed similar experiments with tissue explants in Ussing chamber using 11 pediatric [median (range) age: 54 weeks (15 weeks-10 years)] and 6 adult tissues. Enalaprilat, propranolol, talinolol, and rosuvastatin were used to test paracellular, transcellular, and transporter-mediated efflux by P-gp and breast cancer resistance protein (BCRP), respectively. In addition, we compared the expression patterns of ADME-related genes in pediatric and adult enteroid monolayers with tissues using RNA sequencing. Efflux transport by P-gp and BCRP was comparable between the enteroids and tissue. Efflux ratios (ERs) of talinolol and rosuvastatin by P-gp and BCRP, respectively, were higher in enteroid monolayers compared to Ussing chamber, likely caused by experimental differences in model setup and cellular layers present. Explorative statistics on the correlation with age showed trends of increasing ER with age for P-gp in enteroid monolayers; however, it was not significant. In the Ussing chamber setup, lower enalaprilat and propranolol transport was observed with age. Importantly, the RNA sequencing pathway analysis revealed that age-related variation in drug metabolism between neonates and adults was present in both enteroids and intestinal tissue. Age-related differences between 0 and 6 months old and adults were observed in tissue as well as in enteroid monolayers, although to a lesser extent. This study provides the first data for the further development of pediatric enteroids as an in vitro model to study age-related variation in drug transport. Overall, drug transport in enteroids was in line with data obtained from ex vivo tissue (using chamber) experiments. Additionally, pathway analysis showed similar PK-related differences between neonates and adults in both tissue and enteroid monolayers. Given the challenge to elucidate the effect of developmental changes in the pediatric age range in human tissue, intestinal enteroids derived from pediatric patients could provide a versatile experimental platform to study pediatric phenotypes.

Elevated risk of adverse effects from foodborne contaminants and drugs in inflammatory bowel disease: a review.

The global burden of Inflammatory bowel disease (IBD) has been rising over the last decades. IBD is an intestinal disorder with a complex and largely unknown etiology. The disease is characterized by a chronically inflamed gastrointestinal tract, with intermittent phases of exacerbation and remission. This compromised intestinal barrier can contribute to, enhance, or even enable the toxicity of drugs, food-borne chemicals and particulate matter. This review discusses whether the rising prevalence of IBD in our society warrants the consideration of IBD patients as a specific population group in toxicological safety assessment. Various in vivo, ex vivo and in vitro models are discussed that can simulate hallmarks of IBD and may be used to study the effects of prevalent intestinal inflammation on the hazards of these various toxicants. In conclusion, risk assessments based on healthy individuals may not sufficiently cover IBD patient safety and it is suggested to consider this susceptible subgroup of the population in future toxicological assessments.

Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

Age-Specific ADME Gene Expression in Infant Intestinal Enteroids.

In childhood, developmental changes and environmental interactions highly affect orally dosed drug disposition across the age range. To optimize dosing regimens and ensure safe use of drugs in pediatric patients, understanding this age-dependent biology is necessary. In this proof-of-concept study, we aimed to culture age-specific enteroids from infant tissue which represent its original donor material, specifically for drug transport and metabolism. Enteroid lines from fresh infant tissues (n = 8, age range: 0.3-45 postnatal weeks) and adult tissues (n = 3) were established and expanded to 3D self-organizing enteroids. The gene expression of drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), and PEPT1 (SLC15A1) and drug metabolizing enzymes CYP3A4, CYP2C18, and UGT1A1 was determined with RT-qPCR in fresh tissue and its derivative differentiated enteroids. Expression levels of P-gp, BCRP, MRP2, and CYP3A4 were similar between tissues and enteroids. PEPT1 and CYP2C18 expression was lower in enteroids compared to that in the tissue. The expression of UGT1A1 in the tissue was lower than that in enteroids. The gene expression did not change with the enteroid passage number for all genes studied. Similar maturational patterns in tissues and enteroids were visually observed for P-gp, PEPT1, MRP2, CYP3A4, CYP2C18, and VIL1. In this explorative study, interpatient variability was high, likely due to the diverse patient characteristics of the sampled population (e.g., disease, age, and treatment). To summarize, maturational patterns of clinically relevant ADME genes in tissue were maintained in enteroids. These findings are an important step toward the potential use of pediatric enteroids in pediatric drug development, which in the future may lead to improved pediatric safety predictions during drug development. We reason that such an approach can contribute to a potential age-specific platform to study and predict drug exposure and intestinal safety in pediatrics.

The potential of enteroids derived from children and adults to study age-dependent differences in intestinal CYP3A4/5 metabolism.

Drug metabolism in the intestinal wall affects bioavailability of orally administered drugs and is influenced by age. Hence, it is important to fully understand the drug metabolizing capacity of the gut to predict systemic exposure. The aim of this study was to investigate the potential of enteroids as a tool to study CYP3A4/5 -mediated metabolism in both children and adults. Bioconversion of midazolam, a CYP3A4/5 model substrate, was studied using enteroid monolayers as well as tissue explants in the Ussing chamber, both derived from pediatric [median (range age): 54 weeks (2 days - 13 years), n = 21] and adult (n = 5) tissue. Caco-2 cellular monolayers were employed as controls. In addition, mRNA expression of CYP3A4 was determined in enteroid monolayers (n = 11), tissue (n = 23) and Caco-2 using RT-qPCR. Midazolam metabolism was successfully detected in all enteroid monolayers, as well as in all tissue explants studied in the Ussing chamber, whereas Caco-2 showed no significant metabolite formation. The extracted fraction of midazolam was similar between enteroid monolayers and tissue. The fraction of midazolam extracted increased with age in enteroid monolayers derived from 0 to 70 week old donors. No statistically significant correlation was observed in tissue likely due to high variability observed and the smaller donor numbers included in the study. At the level of gene expression, CYP3A4 increased with age in tissues (n = 32), while this was not reflected in enteroid monolayers (n = 16). Notably, asymmetric metabolite formation was observed in enteroids and tissue, with higher metabolite formation on the luminal side of the barrier. In summary, we demonstrated that enteroids can be used to measure CYP3A4/5 midazolam metabolism, which we show is similar as observed in fresh isolated tissue. This was the case both in children and adults, indicating the potential of enteroids to predict intestinal metabolism. This study provides promising data to further develop enteroids to study drug metabolism in vitro and potentially predict oral absorption for special populations as an alternative to using fresh tissue.

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.

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.

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.

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.

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.

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.

A higher throughput and physiologically relevant two-compartmental human ex vivo intestinal tissue system for studying gastrointestinal processes.

A majority of the preclinical intestinal screening models do not properly reflect the complex physiology of the human intestinal tract, resulting in low translational value to the clinical situation. The often used cell lines such as Caco-2 or HT-29 are not well suited to investigate the different processes that predict oral bioavailability in real life, or processes involved in general gut health aspects. Therefore, highly realistic models resembling the human in vivo situation are needed; application of ex vivo intestinal tissue is an interesting and feasible alternative. After previously using porcine intestinal tissue as a predictive model for human intestinal absorption, we now have successfully applied human intestinal tissue into a newly developed InTESTine™ two-compartmental disposable device suitable for standard 6- or 24-well plate format. With this set-up we demonstrated (regional differences in) drug absorption, by using a subset of compounds with known varying Fa (fraction absorbed) values. A rank-order relationship of R2 = 0.85 could be established between the Fa and Papp of these commercially available drugs. Additionally, comparison between the InTESTine system and the established Ussing chamber technology showed a correlation of R2 = 0.94 (10 drugs) with respect to Papp values, indicating good comparison of both models. Besides absorption, intestinal wall metabolism of testosterone (CYP3A4) was determined by showing a linear formation (R2 = 0.99; up to 165 min) of the main metabolites androstenedione and 6Beta-hydroxytestosterone, indicating no loss of metabolic capacity of the intestinal tissue within the system. Enteroendocrine responses were assessed of the satiety hormones GLP-1 and PYY after stimulation with rebaudioside A and casein, resulting in significantly increased secretion to the luminal side as well as to the basolateral side. Incubation with the probiotic strain LGG showed to enhance the viability of the tissue by showing to decrease the LDH secretion compared to blank intestinal tissue. In conclusion, we show that human ex vivo intestinal tissue mounted in the higher throughput InTESTine 6- 24-transwell plate system is easy to handle and a suitable system to study diverse functional GI processes.

Proteomics of human liver membrane transporters: a focus on fetuses and newborn infants.

BACKGROUND: Hepatic membrane transporters are involved in the transport of many endogenous and exogenous compounds, including drugs. We aimed to study the relation of age with absolute transporter protein expression in a cohort of 62 mainly fetus and newborn samples. METHODS: Protein expressions of BCRP, BSEP, GLUT1, MCT1, MDR1, MRP1, MRP2, MRP3, NTCP, OCT1, OATP1B1, OATP1B3, OATP2B1 and ATP1A1 were quantified with LC-MS/MS in isolated crude membrane fractions of snap-frozen post-mortem fetal and pediatric, and surgical adult liver samples. mRNA expression was quantified using RNA sequencing, and genetic variants with TaqMan assays. We explored relationships between protein expression and age (gestational age [GA], postnatal age [PNA], and postmenstrual age); between protein and mRNA expression; and between protein expression and genotype. RESULTS: We analyzed 36 fetal (median GA 23.4 weeks [range 15.3-41.3]), 12 premature newborn (GA 30.2 weeks [24.9-36.7], PNA 1.0 weeks [0.14-11.4]), 10 term newborn (GA 40.0 weeks [39.7-41.3], PNA 3.9 weeks [0.3-18.1]), 4 pediatric (PNA 4.1 years [1.1-7.4]) and 8 adult liver samples. A relationship with age was found for BCRP, BSEP, GLUT1, MDR1, MRP1, MRP2, MRP3, NTCP, OATP1B1 and OCT1, with the strongest relationship for postmenstrual age. For most transporters mRNA and protein expression were not correlated. No genotype-protein expression relationship was detected. DISCUSSION AND CONCLUSION: Various developmental patterns of protein expression of hepatic transporters emerged in fetuses and newborns up to four months of age. Postmenstrual age was the most robust factor predicting transporter expression in this cohort. Our data fill an important gap in current pediatric transporter ontogeny knowledge.

Development of a mechanistic biokinetic model for hepatic bile acid handling to predict possible cholestatic effects of drugs.

Drug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80µM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20µM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP.

Comparison of In Vitro Assays in Selecting Radiotracers for In Vivo P-Glycoprotein PET Imaging.

Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands.

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.