Simultaneous Evaluation of Membrane Permeability and UDP-Glucuronosyltransferase-Mediated Metabolism of Food-Derived Compounds Using Human Induced Pluripotent Stem Cell-Derived Small Intestinal Epithelial Cells.

Pharmacokinetic prediction after oral ingestion is important for quantitative risk assessment of food-derived compounds. To evaluate the utility of human intestinal absorption prediction, we compared the membrane permeability and metabolic activities of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs) with Caco-2 cells or human primary enterocytes (hPECs). We found that membrane permeability in hiPSC-SIECs had better predictivity than that in Caco-2 cells against 21 drugs with known human intestinal availability (r = 0.830 and 0.401, respectively). Membrane permeability in hiPSC-SIECs was only 0.019-0.25-fold as compared with that in Caco-2 cells for 7 in 15 food-derived compounds, primarily those that were reported to undergo glucuronidation metabolism. The metabolic rates of the glucuronide conjugate were similar or higher in hiPSC-SIECs as compared with hPECs but lower in Caco-2 cells. Expression levels of UDP-glucuronosyltransferase (UGT) isoform mRNA in hiPSC-SIECs were similar or higher as compared with hPECs. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism. SIGNIFICANCE STATEMENT: Gastrointestinal absorption is an important step for predicting the internal exposure of food-derived compounds. This research revealed that human induced pluripotent stem cell-derived small intestinal cells (hiPSC-SIECs) had better predictivity of intestinal availability than Caco-2 cells; furthermore, the metabolic rates of UDP-glucuronosyltransferase (UGT) substrates of hiPSC-SIECs were closer to those of human primary enterocytes than those of Caco-2 cells. Therefore, hiPSC-SIECs could be a useful tool for predicting human intestinal absorption to simultaneously evaluate membrane permeability and UGT-mediated metabolism.

Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E-4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction.

Simultaneous detection of eight species of tree nut in foods using two tetraplex polymerase chain reaction assays.

Tree nuts comprise a category of food allergens that must be included in the food labels in several countries. We developed a polymerase chain reaction (PCR) method using eight specific primer pairs to detect eight representative tree nuts (almond, Brazil nut, cashew, hazelnut, macadamia nut, pecan, pistachio, and walnut) under the same experimental conditions. The specificity of the eight primer pairs was confirmed by PCR testing against a variety of plant and animal samples. The detection limit of the method ranged from 1 fg to 1 pg DNA of individual tree nuts. The method detected tree nut DNA in processed and unprocessed food. In addition, the primer pairs could be combined into two sets of tetraplex PCR system. The developed method is specific, sensitive, and efficient, making it useful for detecting trace amounts of eight species of tree nut in foods.

Construction of extended and functional bile canaliculi using long-term sandwich-cultured cryopreserved human hepatocytes and the application of hepatocytes for predicting the biliary excretion of pharmaceutical and food-related compounds.

The biliary excretion of pharmaceutical and food-related compounds is an important factor for assessing pharmacokinetics and toxicities in humans, and a highly predictive in vitro method for human biliary excretion is required. We have developed a simple in vitro culture method for generating extended and functional bile canaliculi using cryopreserved human hepatocytes. We evaluated the uptake of compounds by hepatocytes and bile canaliculi, and the biliary excretion index (BEI) was calculated. After 21 days of culture, the presence of extended and functional bile canaliculi was confirmed by the uptake of two fluorescent substrates. Positive BEIs were observed for taurocholic acid-d4, rosuvastatin, pitavastatin, pravastatin, valsartan, olmesartan, and topotecan (reported biliary-excreted compounds in humans), but no difference in BEI was observed for salicylic acid (a nonbiliary-excreted compound). Furthermore, 8 of 21 food-related compounds with specific structures and reported biliary transporter involvement exhibited positive BEIs. The developed in vitro system was characterized by functional bile canaliculus-like structures, and it could be applied to the prediction of the biliary excretion of pharmaceutical and food-related compounds.

In vitro-based prediction of human plasma concentrations of food-related compounds.

Efforts have been made to replace animal experiments in safety evaluations, including in vitro-based predictions of human internal exposures, such as predicting peak plasma concentration (Cmax) values for xenobiotics and comparing these values with in vitro-based toxicity endpoints. Herein, the authors predicted the Cmax values of food-related compounds in humans based on existing and novel in vitro techniques. In this study, 20 food-related compounds, which have been previously reported in human pharmacokinetic or toxicokinetic studies, were evaluated. Human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIEC) and Caco-2 cells, HepaRG cells, equilibrium dialysis of human plasma, and LLC-PK1 cell monolayer were used to assess intestinal absorption and availability, hepatic metabolism, unbound plasma fraction, and secretion and reabsorption in renal tubular cells, respectively. After conversion of these parameters into human kinetic parameters, the plasma concentration profiles of these compounds were predicted using in silico methods, and the obtained Cmax values were found to be between 0.017 and 183 times the reported Cmax values. When the in silico-predicted parameters were modified with in vitro data, the predicted Cmax values came within 0.1-10 times the reported values because the metabolic activities of hiPSC-SIECs, such as uridine 5'-diphospho-glucuronosyl transferase, are more similar to those of human primary enterocytes. Thus, combining in vitro test results with the plasma concentration simulations resulted in more accurate and transparent predictions of Cmax values of food-related compounds than those obtained using in silico-derived predictions alone. This method facilitates accurate safety evaluation without the need for animal experiments.

In vitro translation with [34S]-labeled methionine, selenomethionine, and telluromethionine.

Heteroisotope and heteroatom tagging with [(34)S]-enriched methionine (Met), selenomethionine (SeMet), and telluromethionine (TeMet) was applied to in vitro translation. Green fluorescent protein (GFP) and JNK stimulatory phosphatase-1 (JSP-1) genes were translated with wheat germ extract (WGE) in the presence of Met derivatives. GFPs containing Met derivatives were subjected to HPLC coupled with treble detection, i.e., a photodiode array detector, a fluorescence detector, and an inductively coupled plasma mass spectrometer (ICP-MS). The activities of JSP-1-containing Met derivatives were also measured. GFP and JSP-1 containing [(34)S]-Met and SeMet showed comparable fluorescence intensities and enzyme activities to those containing naturally occurring Met. TeMet was unstable and decomposed in WGE, whereas SeMet was stable throughout the experimental period. Thus, although Te was the most sensitive to ICP-MS detection among S, Se, and Te, TeMet was less incorporated into the proteins than Met and SeMet. Finally, the potential of heteroisotope and heteroatom tagging of desired proteins in in vitro translation followed by ICP-MS detection was discussed. [figure: see text] TeMet was less incorporated into GFP than Met and SeMet due to its instability in WGE.

Comprehensive in vitro cardiac safety assessment using human stem cell technology: Overview of CSAHi HEART initiative.

Recent increasing evidence suggests that the currently-used platforms in vitro IKr and APD, and/or in vivo QT assays are not fully predictive for TdP, and do not address potential arrhythmia (VT and/or VF) induced by diverse mechanisms of action. In addition, other cardiac safety liabilities such as functional dysfunction of excitation-contraction coupling (contractility) and structural damage (morphological damage to cardiomyocytes) are also major causes of drug attrition, but current in vitro assays do not cover all these liabilities. We organized the Consortium for Safety Assessment using Human iPS cells (CSAHi; http://csahi.org/en/), based on the Japan Pharmaceutical Manufacturers Association (JPMA), to verify the application of human iPS/ES cell-derived cardiomyocytes in drug safety evaluation. The main goal of the CSAHi HEART team has been to propose comprehensive screening strategies to predict a diverse range of cardiotoxicities by using recently introduced platforms (multi-electrode array (MEA), patch clamp, cellular impedance, motion field imaging [MFI], and Ca transient systems) while identifying the strengths and weaknesses of each. Our study shows that hiPS-CMs used in these platforms have pharmacological responses more relevant to humans in comparison with existent hERG, APD or Langendorff (MAPD/contraction) assays, and not only MEA but also other methods such as impedance, MFI, and Ca transient systems would offer paradigm changes of platforms for predicting drug-induced QT risk and/or arrhythmia or contractile dysfunctions. Furthermore, we propose a potential multi-parametric platform in which field potential (MEA)-Ca transient-contraction (MFI) could be evaluated simultaneously as an ideal novel platform for predicting a diversity of cardiac toxicities, namely whole effects on the excitation-contraction cascade.

CSAHi study: Detection of drug-induced ion channel/receptor responses, QT prolongation, and arrhythmia using multi-electrode arrays in combination with human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: The use of multi-electrode arrays (MEA) in combination with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provides a promising method to predict comprehensive cardiotoxicity, including drug-induced QT prolongation and arrhythmia. We previously demonstrated that MEA in combination with hiPSC-CMs could provide a generalizable platform by using 7 reference drugs at 10 testing facilities. Using this approach, we evaluated responses to reference drugs that modulate a range of cardiac ion currents and have a range of arrhythmogenic effects. METHODS: We used the MEA system (MED64) and commercially available hiPSC-CMs (iCell cardiomyocytes) to evaluate drug effects on the beat rate, field potential duration (FPD), FPD corrected by Fridericia's formula (FPDc), and the incidence of arrhythmia-like waveforms. RESULTS: This assay detected the repolarization effects of Bay K8644, mibefradil, NS1643, levcromakalim, and ouabain; and the chronotropic effects of isoproterenol, ZD7288, and BaCl2. Chronotropy was also affected by K+ and Ca2+ current modulation. This system detected repolarization delays and the arrhythmogenic effects of quinidine, cisapride, thioridazine, astemizole, bepridil, and pimozide more sensitively than the established guinea pig papillary muscle action potential assay. It also predicted clinical QT prolongation by drugs with multiple ion channel effects (fluoxetine, amiodarone, tolterodine, vanoxerine, alfuzosin, and ranolazine). DISCUSSION: MEA in combination with hiPSC-CMs may provide a powerful method to detect various cardiac electrophysiological effects, QT prolongation, and arrhythmia during drug discovery. However, the data require careful interpretation to predict chronotropic effects and arrhythmogenic effects of candidate drugs with multiple ion channel effects.

CSAHi study: Evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia--effects of 7 reference compounds at 10 facilities.

INTRODUCTION: Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia. METHODS: Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericia's formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated. RESULTS: The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms. DISCUSSION: MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans.