Cytochrome P450 (P450 or CYP) is a family of drug-metabolizing enzymes that contribute to the metabolism and disposition of approximately half of the pharmaceutical drugs marketed for human use (Guengerich, 2015). In particular, members of the CYP1, CYP2, and CYP3 families, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, play crucial roles in drug metabolism in humans (Guengerich, 2015). It is well known that P450-dependent metabolism is involved not only in the detoxification and inactivation of xenobiotics but also in the formation of reactive metabolites (Park et al., 2011; Rendic and Guengerich, 2024), which may lead to chemical carcinogenesis and toxicity, including drug-induced liver injury (DILI). Additionally, the expression and activity of P450 may vary due to various endogenous and exogenous factors, such as liver diseases and co-administered drugs or foods (Guengerich, 2015). Since alterations in P450 activity by co-administered drugs may impact the pharmacological effects and toxicity of those drugs, referred to as drug-drug interactions, the potential for drug candidates to inhibit or induce P450s is assessed during drug development, with regulatory agencies requiring this data (U.S. Food and Drug Administration, 2020).

DILI is a major factor in the discontinuation of drug development and market withdrawal (Wilke et al., 2007; Cook et al., 2014). The mechanisms underlying DILI are diverse and not fully understood; however, various factors related to drug metabolism, such as the formation of reactive metabolites and the production of reactive oxygen species (ROS), are well recognized (Lammert et al., 2010; Park et al., 2011; Feng and He, 2013; Yu et al., 2014; Guengerich, 2015; Villanueva-Paz et al., 2021). These reactions can lead to mitochondrial dysfunction, cellular stress, immune responses, and inflammatory reactions (Park et al., 2011; Feng and He, 2013; Real et al., 2019; Garcia-Cortes et al., 2020; Villanueva-Paz et al., 2021). To mitigate DILI caused by these reactive metabolites, the U.S. Food and Drug Administration states in the guidance document “Drug-Induced Liver Injury: Premarketing Clinical Evaluation” that radiochemical in vitro assays are useful for evaluating the covalent binding of a drug or its metabolites to liver proteins (U.S. Food and Drug Administration, 2009).

In addition to DILI, adverse drug reactions (ADRs) in other organs and tissues, such as cardiotoxicity, neurotoxicity, renal toxicity, and myotoxicity, often lead to the discontinuation of drug development and withdrawal from the market (Wilke et al., 2007; Cook et al., 2014). For example, a number of noncardiovascular drugs, including anticancer drugs (e.g., cisplatin and gemcitabine), antimicrobial drugs (e.g., erythromycin and clarithromycin), and antipsychotic drugs (e.g., thioridazine, chlorpromazine, and haloperidol), can cause atrial fibrillation or Torsades de Pointes, a potentially fatal ventricular arrhythmia (Raj et al., 2009). Moreover, some drugs, including anticancer agents such as anthracyclines, cyclophosphamide, and 5-fluorouracil, are associated with cardiomyopathy and heart failure (Raj et al., 2009). Since extensive QT interval prolongation can ultimately lead to Torsades de Pointes, and QT interval prolongation mainly results from the reduction of rectifier potassium current (IKr) caused by the blockage of the potassium channel encoded by the human ether-a-go-go related gene (hERG), the inhibition of the hERG channel by drug candidates is routinely assessed during drug development, as suggested by the International Council on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) S7B guideline (Vicente et al., 2018).

Neurotoxicity is another type of toxicity closely linked to the discontinuation of drug development, particularly during clinical stages (Cook et al., 2014; Walker et al., 2018). This underscores the need for effective methods of neurotoxicity evaluation to be established at preclinical stages. While drugs indicated for the central nervous system (CNS) primarily cause CNS toxicity, those for the cardiovascular and gastrointestinal systems can also induce it (Cook et al., 2014; Walker et al., 2018). Other classes of drugs can lead to acute kidney injury, particularly tubular injury. These include aminoglycosides, acyclovir, cisplatin, ifosfamide, and proton pump inhibitors (Awdishu and Mehta, 2017). These drugs cause kidney injuries through various mechanisms, including reducing blood supply and pressure, direct or indirect damage to kidney cells (such as cell membranes and mitochondria), and blocking the ureter due to drug crystallization (Džidić-Krivić et al., 2024; Arakawa et al., 2025). Although novel biomarkers for early detection of drug-induced kidney injury, such as KIM-1, lipocalin associated with neutrophil gelatinase, and various microRNAs, have been identified, the mechanistic understanding of drug-induced kidney injury remains limited.

P450 inhibition assays are commonly performed during the early stages of drug development, particularly concerning drug-drug interactions (U.S. Food and Drug Administration, 2020), as they align well with high-throughput analysis. Based on this, we previously investigated whether P450 inhibition data could be utilized for evaluating DILI risk and found that most drugs demonstrating strong inhibition of human CYP1A1 or CYP1B1 were DILI-positive, indicating that the P450 inhibition data are valuable for predicting DILI (Shimizu et al., 2021). More recently, we demonstrated that P450 inhibition assay data are beneficial for DILI risk assessment through decision tree-based analyses (Kaito et al., 2024). Furthermore, through mechanistic analyses exploring the relationship between CYP1A1 inhibition and DILI, we demonstrated that in rats, CYP1A1 inhibition by a drug candidate activated the aryl hydrocarbon receptor (AHR) in the liver, resulting in hepatotoxicity, potentially due to the decreased clearance of endogenous AHR agonists, such as 6-formylindolo[3,2-b]carbazole (Yoda et al., 2022). These findings imply that P450 inhibition represents a novel DILI risk factor and that P450 inhibition assay could function as a potential tool for screening DILI risk.

Although ADRs other than DILI have been reported in clinical practice, the association between P450 inhibition and ADRs apart from DILI remains unclear. Meanwhile, P450-dependent metabolism plays a crucial role in the formation of reactive metabolites and ROS, which lead to tissue injury and, consequently, ADRs in extrahepatic tissues (Park et al., 2011; Feng and He, 2013; Guengerich, 2015; McMillan and Tyndale, 2018; Lash, 2021). Additionally, AHR is expressed in nearly all tissues and organs, and its activation can induce various types of toxicity beyond hepatotoxicity (Fernandez-Salguero et al., 1996; Wilson and Safe, 1998; Nguyen and Bradfield, 2008). These observations, along with our previous findings, suggest that data from P450 inhibition assays may be useful in predicting organ toxicities in addition to DILI. Therefore, this study aimed to test this possibility. To this end, we compiled a dataset containing toxicity information for various organs, utilizing the Side Effect Resource (SIDER) (Kuhn et al., 2016) and the Medical Dictionary for Regulatory Activities (MedDRA), an internationally harmonized medical glossary (Lindstrom-Gommers and Mullin, 2019), and investigated the relationship between organ toxicity and human P450 inhibition.