In the drug development in pharmaceuticals, development of drugs may be discontinued due to the toxicity and clinical side effect, therefore, safety assessment is one of the important factors in drug development. Consortium for Safety Assessment using Human Cells (CSAHi) has been launched for developing and standardizing a toxicity evaluation system for development of drug using human iPS cell differentiated cells. CSAHi focuses on hepato-, cardio-, and neuro-toxicities as important toxicity organs which are attributed to the causes of discontinuation of drug development. In neurotoxicity, seizure is an important finding because of high frequency expression in nonclinical. Multi-electrode array (MEA) systems have recently attracted attention as useful for evaluating seizure risk because they can non-invasively measure the electrophysiological activities of neural networks. We are evaluating the electrophysiological responses to several seizure compounds using MEA in cultured hiPSC-derived neurons. It is important to establish an analytical method to detecting seizure-like activities. We have focused the establish of the effective analysis parameters for detecting seizure risk. We identify to be separate the responses between seizure-positive and seizure-negative compounds using principal component analysis of 10 analysis parameters. In addition, we could separate the mechanism of action of the seizure-positive compounds by principal component analysis and cluster analysis using 10 parameters. It is considered that principal component analysis or cluster analysis could not only assess the seizure risk but also classify mechanism of action by in vitro MEA system using human iPS cell-derived neurons.

Development of an in vitro drug efficacy and safety assessment based on the function of the neural network is required in preclinical studies. A microelectrode array (MEA), which can simultaneously measure the electrical activity of a human induced pluripotent stem cell-derived neural network at multiple points, is an effective assay system. In this study, we focused on seizure liability and clarified the responsiveness to seizure-positive compounds depending on the excitatory and inhibitory balance (E/I balance) of each evaluation sample. In addition, it has been shown that multivariate analysis and AI analysis methods are effective for detecting toxicity and predicting drug mechanisms of action. The future challenge is to approach in vitro-to-in vivo extrapolation (IVIVE) for in vitro assessment. An assessment using brain organoids and low-frequency component analysis, in which enable comparison with in vivo ECoG are effective approaches to IVIVE. MEA can be applied to the central nervous system and the peripheral nervous system; therefore, MEA is also expected to become a highly useful assessment tool for peripheral neuropathy.

Human induced pluripotent stem cell-derived neurons (hiPSC-neurons) have potentials to improve the predictability of the adverse effects (AEs) in the human central nervous system (CNS). We have succeeded in reproducing the human neural networks stably on dish. This is important because most of the CNS AEs were caused not by the neuronal death but by the abnormal neural network activities. Our group have been attempting to establish the in vitro assay system to detect abnormal neural network activities by microelectrode array system (MEA). Furthermore, the algorithm that can determine the synchronized burst firing (SBF) objectively was developed in the collaboration of our iPS Non-clinical Experiments for Nervous System (iNCENS) project and Consortium for Safety Assessment using Human iPS Cells (CSAHi). iNCENS and CSAHi are also involved in the international Life Sciences Institute (ILSI): Health and Environmental Sciences Institute (HESI) HESI NeuTox MEA sub team, and are participating in the pilot study using 12 compounds. In the big flow to develop physiological CNS safety assessment system, we have to show Japan initiative based on our innovative iPS technology we have developed so far.

Disease-specific iPS cells have been considered and used as platforms for disease modeling and drug discovery for intractable diseases. In the field of cardiovascular medicine, iPS cells have been generated from patients with heart diseases including inherited cardiomyopathy. The disease-specific iPS cells showed the certain parts of phenotype of the disease on culture dishes in in vitro systems, but the cells do not necessarily recapitulate patients’ clinical properties, particularly those of physiological-/pathophysiological aspects. The point should be solved to establish disease reliable platforms. The discrepancy may be attributed to the lack of developmental process during culture procedure. To settle the problems, various techniques have been attempted such as culture dishes with specific structures. This review describes issues to be solved to recapitulate “heart diseases on culture dishes”, introducing the phenotype of disease specific iPS-cells from patients with cardiomyopathy.

It is reported that the incidence of atrial arrhythmias has been increasing year by year and it might increase from now on. Although not only aging but pharmaceutical drug treatments might relate to atrial arrhythmias, experimental method to detect drug-induced atrial arrhythmias has not been established so far. Therefore, we induced differentiation of atrial-like cardiomyocytes from human induced pluripotent stem (iPS) cell, and clarified their characteristics and verified their dug responsiveness. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their character was compared to RA-untreated cardiomyocytes. In gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have the characters of atrial or ventricular like cardiomyocytes, respectively. In addition, it was also confirmed that atrial-like cardiomyocytes induced reentry-like conduction disorder, which is atrial arrhythmias. Furthermore, as a result of examining the responsiveness of various ion channel inhibitors using these cells, the inhibition of ultra-rapid delayed rectifier potassium current (I_Kur) specifically existed in atrial muscle induced prolongation of PWD30cF (membrane potential duration at 30% depolarization corrected by Fridericia formula) only in atrial-like cardiomyocytes. In addition, ventricular-like cardiomyocytes alone exhibited an early after depolarization by treatment of rapid rectifier potassium current (I_Kr) inhibitor which induced ventricular arrhythmia in clinical situation. Based on above evidences, current evaluation systems using human iPS cell-derived atrial-like cardiomyocytes might be a valuable tool for drug-induced atrial arrhythmias.

Deep learning technology is rapidly advancing, and is now used to solve complex problems. induced pluripotent stem cells (iPSCs) can be used for several purposes such as regenerative medicine, disease modeling study and drug screening. It is inevitable to identify iPSC-derived differentiated cells in microscopy for any use. Here, we used deep learning to establish an automated method to identify endothelial cells derived from iPSCs, without the need for immunostaining or lineage tracing.

Various artificial cells and artificial tissues can be generated from induced pluripotent stem cells (iPS cells). There is now an urgent need to standardize the quality evaluation and management of iPS cells. Recently, artificial intelligence (AI) technology such as machine learning is providing evaluation method for the quality of iPS cells and iPS cell-derived somatic cells based on optical microscopy. Light, which is the principle of optical microscopy, has an interesting and important feature. There are various kinds of interaction between light and molecule, and the scattered light includes internal information of the molecule. Raman scattering inheres all the vibration mode of molecular bonds composing a molecule, and second harmonic generation (SHG) light, which is one of second-order non-linear scattering light, is derived from electric polarizations in the molecule, in other words, carries structural information within the protein. While states of a cell are usually defined by protein/gene expression patterns, we have proposed to apply Raman spectra for cellular fingerprinting as an alternative for identifying the cell state, and now succeeded in predicting gene-expression of antibiotic resistant bacteria in combination with machine learning technology. Meanwhile, SHG microscopy has been used to visualize fiber structures in living specimens, such as collagen, and microtubules as a label-free modality. By utilizing the feature that SHG senses protein structure change, we developed a new method to measure actomyosin activity in cardiac cells. The most important advantage of the use of the scattering light is their non-labeling and non-invasive capability.

In Japan, hypertensive patients are speculated to be 40 million, and 30 million among them are poor controlled. Moreover, 20% of them are estimated as “resistant hypertension” those are above their target blood pressure even in the simultaneous use of three different classes anti-hypertensive drugs. Recently, aldosterone is recognized as one of the main causes of the etiology of “resistant hypertension.” Therefore, it is important to discover novel drugs that inhibit the synthesis and secretion of aldosterone. We recently generated a stable H295R cell line expressing aldosterone synthase gene (CYP11B2) promoter (−1521~+2)/luciferase cDNA chimeric reporter construct. We thereafter established a high-throughput screening (HTS) system for the discovery of novel anti-hypertensive drugs that inhibit angiotensin II-induced CYP11B2 expression using the cell line. After confirmation of its validation (Z’ score > 0.5), we performed HTS using Core Library (9,600 chemical compounds) and Validated Compound Library (1,979 chemical compounds) obtained from Drug Discovery Initiative (The University of Tokyo), and Tohoku University Chemical Library (5,562 chemical compounds), respectively. We obtained several hit compounds from each library, and focused on one compound (bortezomib) obtained from Validated Compound Library. The compound did not affect cell viability by WST-1 assay, and was demonstrated to lower blood pressure of Tsukuba Hypertensive Mice, significantly. The compound may therefore be a potential candidate of novel anti-hypertensive drugs in the future.

With the recent progress in drug metabolism and pharmacokinetics studies, the attrition due to pharmacokinetics in clinical trials and post-marketing was reduced to less than 1%. On the other hand, attrition of clinical trials due to adverse effects and toxicity has remained high. In particular, drug-induced liver injury (DILI) is a major cause of discontinuation of clinical trials and withdrawal of drug candidates after marketing. DILI is roughly divided into intrinsic and idiosyncratic. The former is relatively easy to predict its onset in preclinical drug development, but the latter’s onset mechanism is still unknown and its onset prediction is difficult. We are investigating to develop an experimental animal model of idiosyncratic DILI (iDILI), clarify the pathogenic mechanism, and apply the obtained biomarker information to the establishment of an in vitro cell-based prediction test system. In this paper, we will introduce various animal models of iDILI, present status of pathogenic mechanism study, and classification of iDILI drugs, and introduce the recent progress of in vitro cell-based prediction test system and new causative factors of iDILI.

Tedizolid, a novel oxazolidinone antibacterial agent, is a protein synthesis inhibitor that acts on bacterial ribosomes to inhibit initiation of translation. Tedizolid phosphate, a prodrug of tedizolid, is rapidly converted to the active form of tedizolid by phosphatase after administration. Tedizolid has antimicrobial activity mainly against gram-positive pathogens, and generally shows 4–8 times stronger in vitro activity than linezolid, an oxazolidinone antibacterial agent. Tedizolid has antimicrobial activity against Staphylococcus aureus (S. aureus) regardless of being methicillin-resistant or susceptible, with 90% minimum inhibitory concentrations (MIC₉₀) ranging from 0.25–0.5 μg/mL. Although antimicrobial activity of tedizolid against linezolid-resistant S. aureus (LRSA) is generally reduced, tedizolid is still active to LRSA whose linezolid resistance is caused by cfr gene. Structure-activity relationship analysis suggests that the C-5 hydroxymethyl group, the C-ring pyridine, and the D-ring tetrazole group of tedizolid are associated with enhanced antimicrobial activity of tedizolid and its antimicrobial activity against linezolid-resistant bacteria by the cfr gene. Frequency of spontaneous resistance mutation to tedizolid is low, and about 16-fold lower than that to linezolid. Pharmacokinetic/pharmacodynamic (PK/PD) parameter most related to the efficacy of tedizolid is the area under free drug concentration-time curve/minimum inhibitory concentration (fAUC/MIC), and fAUC/MIC value required for bacteriostasis under immunocompetent conditions was calculated to be three. Phase III studies of tedizolid phosphate were conducted in Japan and overseas countries and demonstrated its efficacy and safety in patients with skin and soft tissue infections caused by gram positive organisms including methicillin-resistant S. aureus (MRSA).

Esaxerenone is a novel non-steroidal mineralocorticoid receptor antagonisit (MR blocker), whose unique binding to the MR-ligand domain yields a stronger MR antagonistic effect and higher selectivity than existing MR antagonisits. Esaxerenone was approved for the treatment of hypertension in Japan in January 2019. Esaxerenone suppresses the reduction of urinary Na+/K+ ratio in adrenalectomized rats and blood pressure increase, proteinuria, and renal tissue lesions in salt-sensitive hypertensive rats—all in a dose-dependent manner. Esaxerenone is rapidly absorbed and reaches intracellular targets because of its high membrane permeability, exhibits high bioavailability with small interindividual exposure variation, and is metabolized via several pathways (e.g., oxidation, glucuronidation, and hydrolysis), which is associated with low drug–drug interaction risk. As esaxerenone is slightly excreted into urine, its exposure is similar between elderly and non-elderly patients, and between patients with normal and moderately deteriorated renal function. Given its 19-hour half-life, once-daily administration would have a sustainable antihypertensive effect. The ESAX-HTN phase 3 study demonstrated the non-inferiority of esaxerenone’s antihypertensive effect versus that of eplerenone in essential hypertension. Another study showed a stable antihypertensive effect for 52 weeks as monotherapy or combination therapy. In hypertensive patients with moderate impairment or both type 2 diabetes and albuminuria treated with a renin–angiotensin system inhibitor, esaxerenone elicited a stable antihypertensive effect and manageable hyperkalemia incidence with titration from a low dose and monitoring including serum potassium. Thus, with careful monitoring of serum potassium, esaxerenone can be administered to patients with moderate renal impairment or both diabetes and albuminuria.