HLA-B*57:01-dependent intracellular stress in keratinocytes triggers dermal hypersensitivity reactions to abacavir.

Specific human leukocyte antigen (HLA) polymorphisms combined with certain drug administration strongly correlate with skin eruption. Abacavir hypersensitivity (AHS), which is strongly associated with HLA-B*57:01, is one of the most representative examples. Conventionally, HLA transmits immunological signals via interactions with T cell receptors on the cell surface. This study focused on HLA-mediated intracellular reactions in keratinocytes that might determine the onset of skin immunotoxicity by drug treatments. Abacavir exposure resulted in keratinocytes expressing HLA-B*57:01 exhibiting endoplasmic reticulum (ER) stress responses, such as immediate calcium release into the cytosol and enhanced HSP70 expression. In contrast, keratinocytes expressing HLA-B*57:03 (closely related to HLA-B*57:01) did not show these changes. This indicated that HLA-B*57:01 has a specific intracellular response to abacavir in keratinocytes in the absence of lymphocytes. Furthermore, abacavir exposure in HLA-B*57:01-expressing keratinocytes elevated the expression of cytokines/chemokines such as interferon-γ, interleukin-1ß, and CCL27, and induced T lymphoblast migration. These effects were suppressed by ER stress relief using 4-phenylbutyrate (4-PB). HLA-B*57:01-transgenic mice also exhibited ER stress in epidermal areas following abacavir administration, and abacavir-induced skin toxicity was attenuated by the administration of 4-PB. Moreover, abacavir bound to HLA-B*57:01 within cells and its exposure led to HLA-B*57:01 protein aggregation and interaction with molecular chaperones in the ER of keratinocytes. Our results underscore the importance of HLA-mediated intracellular stress responses in understanding the onset of HLA-B*57:01-mediated AHS. We provide the possibility that the intracellular behavior of HLA is crucial for determining the onset of drug eruptions.

Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells.

Although increased aerobic glycolysis is common in various cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive a state of glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, an inhibitor of lysine-specific histone demethylase 1A (LSD1), which dramatically decreased the growth of PDAC PANC-1 cells and showed an anti-tumoral effect in tumor-bearing mice. The growth of glycolysis-suppressed PANC-1 cells was also inhibited by another LSD1 inhibitor, OG-L002. Similarly, the other two PDAC cells (PK-1 and KLM-1) with suppressed glycolysis exhibited anticancer effects against SP-2509. However, the anticancer effects on PDAC cells were unrelated to LSD1. To investigate how PDAC cells survive in a glycolysis-suppressed condition, we conducted proteomic analyses. These results combined with our previous findings suggested that glucose-starvation causes PDAC cells to enhance mitochondrial oxidative phosphorylation. In particular, mitochondrial fatty acid metabolism was identified as a key factor contributing to the survival of PDAC cells under glycolysis suppression. We further demonstrated that SP-2509 and OG-L002 disturbed fatty acid metabolism and induced lipid droplet accumulation through the impairment of lipophagy, but not bulk autophagy. These findings indicate a significant potential association of lipophagy and anticancer effects in glycolysis-suppressed PDAC cells, offering ideas for new therapeutic strategies for PDAC by dual inhibition of glycolysis and fatty acids metabolism.

Pathological changes in various organs in HLA-B*57:01 transgenic mice with abacavir-induced skin eruption.

Several patients with cutaneous adverse drug reactions exhibit extracutaneous organ damages, and it becomes severe in a few patients resulting in death due to multiorgan failure. Understanding the sequential changes in various organs in patients with cutaneous eruption following drug administration will help understand disease onset and progression, aiding the development of prevention strategies and interventions. Therefore, we aimed to understand the effects of abacavir (ABC) on various organs in patients with ABC-induced eruptions by evaluating its effects in a mouse model. We found pathological changes in various organs of HLA-B*57:01 transgenic mice (B*57:01-Tg) following oral administration of ABC (20 mg/body/day). B*57:01-Tg exhibited a significant body weight decrease from day 1 of ABC administration, and reddening of the auricle was observed from day 5, and approximately 2/3 mice died by day 7. Histopathological examination revealed severe thymic atrophy after day 3, infiltration of inflammatory cells, predominantly lymphocytes with neutrophils, not only in the skin but also in the liver, kidney, and lung after day 5, and an increased number of lymphocytes with enlarged nuclei and granulocytic hematopoiesis were observed in the spleen after day 5. Blood chemistry revealed that albumin/globulin ratio was below 1.0 on day 5, reflecting a systemic inflammatory response, and the aspartate aminotransferase concentration rose to 193 ± 93.0 U/L on day 7, suggesting that cell damage may have occurred in various organs including liver accompanying inflammatory cell infiltration. These examinations of a mouse model of ABC-induced skin eruption show that disorders in various organs other than the skin should be considered and provide insights into the unexpected early systemic responses dependent on HLA-B*57:01. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00220-1.

New in vitro screening system to detect drug-induced liver injury using a culture plate with low drug sorption and high oxygen permeability.

Drug-induced liver injury (DILI) is a major factor underlying drug withdrawal from the market. Therefore, it is important to predict DILI during the early phase of drug discovery. Metabolic activation and mitochondrial toxicity are good indicators of the potential for DILI. However, hepatocyte function, including drug-metabolizing enzyme activity and mitochondrial function, reportedly decreases under conventional culture conditions; therefore, these conditions fail to precisely detect metabolic activation and mitochondrial toxicity-induced cell death. To resolve this issue, we employed a newly developed cell culture plate with high oxygen permeability and low drug sorption (4-polymethyl-1-pentene [PMP] plate). Under PMP plate conditions, cytochrome P450 (CYP) activity and mitochondrial function were increased in primary rat hepatocytes. Following l-buthionine-sulfoximine-induced glutathione depletion, acetaminophen-induced cell death significantly increased under PMP plate conditions. Additionally, 1-aminobenzotriazole reduced cell death. Moreover, mitochondrial toxicity due to mitochondrial complex inhibitors (ketoconazole, metformin, and phenformin) increased under PMP plate conditions. In summary, PMP plate conditions could improve CYP activity and mitochondrial function in primary rat hepatocytes and potentially detect metabolic activation and mitochondrial toxicity.

Drug-induced altered self-presentation increases tumor immunogenicity.

Anti-human immunodeficiency virus (HIV) drug abacavir (ABC) binds to the specific allele of human leukocyte antigen (HLA-B*57:01) and activates CD8+ T cells by presenting altered abnormal peptides. Here, we examined the effect of ABC-induced altered self-presentation by HLA-B*57:01 on immunogenicity of cancer cells and CD8+ T-cell-dependent anti-tumor immunity. We established human-mouse chimeric HLA-B*57:01-expressing tumor cell lines (B16F10 and 3LL) and tested the anti-tumor effect of ABC in vivo. ABC treatment inhibited the growth of HLA-B*57:01-expressing tumors by a CD8+ T-cell-dependent mechanism. ABC treatment induced CXCR3-dependent infiltration of CD8+ T cells into HLA-B*57:01-expressing tumors, and activated those tumor-infiltrating CD8+ T cells to proliferate and secrete IFN-γ. The activation of CD8+ T cells using drug-induced altered self-presentation may be a new strategy to increase tumor immunogenicity and improve the efficacy of immunotherapy.

Potential neonatal toxicity of new psychoactive substances.

Cannabis, cocaine, 3,4-methylenedioxymethamphetamine, and lysergic acid diethylamide are psychoactive substances with a significant increase in consumption during the 21st century due to their popularity in medicinal and recreational use. New psychoactive substances (NPSs) mimic established psychoactive substances. NPSs are known as being natural and safe to consumers; however, they are neither natural nor safe, causing severe adverse reactions, including seizures, nephrotoxicity, and sometimes death. Synthetic cannabinoids, synthetic cathinones, phenethylamines, and piperazines are all examples of NPSs. As of January 2020, nearly 1000 NPSs have become documented. Due to their low cost, ease of availability, and difficulty of detection, misuse of NPSs has become a familiar and growing problem, especially in adolescents and young adults in the past decade. The use of NPSs is associated with higher risks of unplanned sexual intercourse and pregnancy. As many as 4 in 100 women seeking treatment for substance abuse are pregnant or nursing. Animal studies and human clinical case reports have shown that exposure to certain NPSs during lactation periods has toxic effects on neonates, increasing various risks, including brain damage. Nevertheless, neonatal toxicity effects of NPSs are usually unrecognized and overlooked by healthcare professionals. In this review article, we introduce and discuss the potential neonatal toxicity of NPSs, emphasizing synthetic cannabinoids. Utilizing the established prediction models, we identify synthetic cannabinoids and their highly accumulative metabolites in breast milk.

Estimating drug-induced liver injury risk by in vitro molecular initiation response and pharmacokinetic parameters for during early drug development.

Drug-induced liver injury (DILI) is a major factor influencing new drug withdrawal; therefore, an appropriate toxicity assessment at the preclinical stage is required. Previous in silico models have been established using compound information listed in large data sources, thereby limiting the DILI risk prediction for new drugs. Herein, we first constructed a model to predict DILI risk based on a molecular initiating event (MIE) predicted by quantitative structure-activity relationships, admetSAR parameters (e.g. cytochrome P450 reactivity, plasma protein binding, and water-solubility), and clinical information (maximum daily dose [MDD] and reactive metabolite [RM]) for 186 compounds. The accuracy of the models using MIE, MDD, RM, and admetSAR alone were 43.2%, 47.3%, 77.0%, and 68.9%, while the "predicted MIE + admetSAR + MDD + RM" model's accuracy was 75.7%. The contribution of MIE to the overall prediction accuracy was little effect or rather worsening it. However, it was considered that MIE was a valuable parameter and that it contributed to detect high DILI risk compounds in the early development stage. We next examined the effect of stepwise changes in MDD on altering the DILI risk and estimating the maximum safety dose (MSD) for clinical use based on structural information, admetSAR, and MIE parameters because it is important to estimate the dose that could prevent the DILI onset in clinical conditions. Low-MSD compounds might increase the DILI risk, as these compounds were classified as "most-DILI concern" at low doses. In conclusion, MIE parameters were especially useful to check the DILI concern compounds and to prevent the underestimation of DILI risk in the early stage of drug development.

Lipopolysaccharide administration increases the susceptibility of mitochondrial permeability transition pore opening via altering adenine nucleotide translocase conformation in the mouse liver.

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, induces various biological reactions in vivo. Our previous study suggested that LPS administration disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in the liver mitochondria, and sensitizes mitochondrial permeability transition (MPT) pore opening in rats. However, it is unknown whether LPS-induced MPT pore opening in rats is similarly observed in mice and whether the mechanism is the same. LPS administration to mice increased not only cyclosporin A-sensitive swelling (MPT pore opening) susceptibility, but also induced cyclosporin A-insensitive basal swelling, unlike in rats. In addition, respiratory activity observed after adding ADP was significantly decreased. Based on these results, we further investigated the role of adenine nucleotide translocase (ANT). Carboxyatractyloside (CATR; an ANT inhibitor) treatment decreased respiratory activity after ADP was added in vehicle-treated mitochondria similarly to LPS administration. Additionally, CATR treatment increased MPT pore opening susceptibility in LPS-treated mitochondria compared to that of vehicle-treated mitochondria. Our study shows that ANT maintained a c-state conformation upon LPS administration, which increased MPT pore opening susceptibility in mice. These results suggest that LPS enhances MPT pore opening susceptibility across species, but the mechanism may differ between rat and mouse.

siRNA delivery to lymphatic endothelial cells via ApoE-mediated uptake by lipid nanoparticles.

Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.

TARC/CCL17 Expression Is Associated with CD8+ T Cell Recruitment in Abacavir-Induced Skin Hypersensitivity in HLA-Transgenic Mice.

Abacavir (ABC)-induced hypersensitivity (AHS) is strongly associated with human leukocyte antigen (HLA)-B*57 : 01 expression. Previous studies have demonstrated the feasibility of applying the HLA-transgenic mouse model in this context. ABC-induced adverse reactions were observed in HLA-B*57 : 01 transgenic (B*57 : 01-Tg) mice. Moreover, regulating immune tolerance could result in severe AHS that mimics symptoms observed in the clinical setting, which were modeled in CD4+ T cell-depleted programmed death-1 receptor (PD-1) knockout B*57 : 01-Tg (B*57 : 01-Tg/PD-1-/-) mice. Here, we aimed to examine whether thymus and activation-regulated chemokine (TARC)/CCL17 level can be used as a biomarker for AHS. Serum TARC levels increased in HLA-B*57 : 01-transgenic mice following oral administration of ABC; this increase was associated with the severity of skin toxicity. In ABC-fed CD4+ T cell-depleted B*57 : 01-Tg/PD-1-/- mice, TARC was detected in the epidermal keratinocytes of the ear. Skin toxicity was characterized by the infiltration of CD8+ T cells partially expressing C-C chemokine receptor type 4, which is the primary receptor for TARC. In vivo TARC neutralization effectively alleviated the symptoms of ear skin redness and blood vessel dilatation. Moreover, TARC neutralization suppressed the infiltration of CD8+ T cells to the ear skin but did not affect the ABC-induced adaptive immune response. Therefore, TARC was involved in ABC-induced skin toxicity and contributed to the recruitment of CD8+ T cells to skin. This evidence suggests that serum TARC level may be a functional biomarker for AHS.

In Vitro Assay System to Detect Drug-Induced Bile Acid-Dependent Cytotoxicity Using Hepatocytes.

Inhibition of bile acid excretion by drugs is a significant factor in the development of drug-induced cholestatic liver injury. We constructed a new in vitro assay system to detect bile acid-dependent cytotoxicity in hepatocytes. This cell-based system can assess the toxicity of the parent compound, as well as the contribution of metabolite(s). In addition, this system can utilize several types of hepatocytes (primary hepatocytes, hepatoma cell line, and induced pluripotent stem cell-induced hepatocytes). In this chapter, a method to detect drug-induced bile acid-dependent toxicity in hepatocytes is described.

PINK1-dependent and Parkin-independent mitophagy is involved in reprogramming of glycometabolism in pancreatic cancer cells.

Cancer cells rely on glycolysis to generate ATP for survival. However, inhibiting glycolysis is insufficient for the eradication of cancer cells because glycolysis-suppressed cells undergo metabolic reprogramming toward mitochondrial oxidative phosphorylation. We previously described that upon glycolytic suppression in pancreatic cancer cells, intracellular glycometabolism is shifted toward mitochondrial oxidative phosphorylation in an autophagy-dependent manner for cellular survival. Here, we hypothesized that mitophagy, which selectively degrades mitochondria via autophagy, is involved in mitochondrial activation under metabolic reprogramming. We revealed that glycolytic suppression notably increased mitochondrial membrane potential and mitophagy in a pancreatic cancer cell model (PANC-1). PTEN-induced kinase 1 (PINK1), a ubiquitin kinase that regulates mitophagy in healthy cells, regulated mitochondrial activation through mitophagy by glycolytic suppression. However, Parkin, a ubiquitin ligase regulated by PINK1 in healthy cells to induce mitophagy, was not involved in the PINK1-dependent mitophagy of the cancer glycometabolism. These results imply that cancer cells and healthy cells have different regulatory pieces of machinery for mitophagy, and inhibition of cancer-specific mechanisms may be a potential strategy for cancer therapy targeting metabolic reprogramming.

Conjugation of human serum albumin and flucloxacillin provokes specific immune response in HLA-B*57:01 transgenic mice.

Flucloxacillin (FLX) induces adverse liver reactions, which has been reported to be related to human leukocyte antigen (HLA)-B*57:01. In a previous study, abacavir-induced hypersensitivity was induced in HLA-B*57:01-transgenic mice (B*57:01-Tg), originally constructed by our group (Susukida et al., 2021). In this study, B*57:01-Tg mice were used to reproduce FLX-induced liver injury. However, treatment of B*57:01-Tg mice with FLX alone did not increase serum ALT levels. Immune-deficient B*57:01-Tg/PD-1-/-mice were produced by mating B*57:01-Tg with PD-1-/- mice. The immune response of B*57:01-Tg/PD-1-/- mice was further modulated by co-administration of CpG-oligodeoxynucleotides and anti-CD4 mAb. Nevertheless, immune regulation in B*57:01-Tg mice did not contribute to the onset of FLX-induced liver injury or immune activation. Moreover, we generated an FLX-human serum albumin (HSA) conjugate and showed that FLX covalently bound to HSA in a time-dependent manner. The FLX-HSA conjugate was administered to the B*57:01-Tg mice. The immune response was investigated using flow cytometry, revealing the phenotype of CD44highCD62Llow in CD8+ T cells (TEM cells). Administration of the FLX-HSA conjugate resulted in an HLA-B*57:01 restricted immune response as shown by the stimulation of TEM cells in the draining lymph nodes. In conclusion, administration of FLX alone to B*57:01-Tg mice did not induce liver injury or immune activation. Immune system sensitivity does not play a decisive role in this process. The conjugation of FLX and HSA results in specific TEM cell stimulation, which suggests that HLA-B*57:01 drives a stronger interaction with CD8+ T cells. These results suggest that patients carrying HLA-B*57:01 could be more susceptible to a conjugate of FLX and albumin and drive CD8+ T cell activation, which may be a vital risk factor for FLX-induced liver injury. In addition, the application of the FLX-HSA adduct may be an effective method for the construction of FLX-induced idiosyncratic liver injury in mice.

Weak complex formation of adverse drug reaction-associated HLAB57, B58, and B15 molecules.

The combination of certain human leukocyte antigen (HLA) polymorphisms with administration of certain drugs shows a strong correlation with developing drug hypersensitivity. Examples of typical combinations are HLA-B*57:01 with abacavir and HLA-B*15:02 with carbamazepine. However, despite belonging to the same serotype, HLA-B*57:03 and HLA-B*15:01 are not associated with drug hypersensitivity. Recent studies have shown that several HLA polymorphisms are associated with multiple drugs rather than a single drug, all resulting in drug hypersensitivity. In this study, we compared the molecular structures and intracellular localization of HLA-B*57:01, HLA-B*58:01, and HLA-B*15:02, which pose risks for developing drug hypersensitivity, as well as HLA-B*57:03 and HLA-B*15:01 that do not present such risks. We found that HLA molecules posing risks have a low affinity for the subunit ß2-microglobulin; notably, the weak hydrogen bond formed via Gln96 of the HLA molecule contributes to this behavior. We also clarified that these HLA molecules are easily accumulated in the endoplasmic reticulum, exhibiting a low expression on the cell surface. Considering that these hypersensitivity risk-associated HLA molecules form complexes with ß2-microglobulin and peptides in the endoplasmic reticulum, we assumed that their low complex formation ability in the endoplasmic reticulum facilitates the interaction with multiple drugs.

A novel perfusion culture system for screening mitochondrial toxicity in primary mouse hepatocytes.

The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.

The PD1 inhibitory pathway and mature dendritic cells contribute to abacavir hypersensitivity in human leukocyte antigen transgenic PD1 knockout mice.

Based on recent genome-wide association studies, abacavir-induced hypersensitivity is highly associated with human leukocyte antigen (HLA)-B*57:01 allele. However, the underlying mechanism of this occurrence is unclear. To investigate the underlying mechanism, we developed HLA-B*57:01 transgenic mice and found that application of abacavir could cause CD8 T cell activation with elevation in PD1 expression; however, severe skin hypersensitivity was not observed. To eliminate the immunosuppressive effect of PD1, HLA-B*57:01 transgenic/PD1 knockout (01Tg/PD1) mice were generated by mating HLA-B*57:01 transgenic mice and PD1 knockout mice. Thereafter, 01Tg/PD1 mice were treated with abacavir. Similar to the above results, severe skin hypersensitivity was not observed. Therefore, we treated 01Tg/PD1 mice with an anti-CD4 antibody to deplete CD4 T cells, followed by abacavir topically and orally. Severe abacavir-induced skin hypersensitivity was observed in 01Tg/PD1 mice after depletion of CD4 T cells, in addition to significant CD8 T cell activation and dendritic cell maturation. Taken together, we succeeded in reproducing severe skin hypersensitivity in a mouse model. And we found that through the combined depletion of PD1 and CD4 T cells, CD8 T cells could be activated and could proceed to clonal proliferation, which is promoted by mature dendritic cells, thereby eventually inducing severe skin hypersensitivity.

Regulation of the immune tolerance system determines the susceptibility to HLA-mediated abacavir-induced skin toxicity.

Idiosyncratic drug toxicity (IDT) associated with specific human leukocyte antigen (HLA) allotype is a rare and unpredictable life-threatening adverse drug reaction for which prospective mechanistic studies in humans are difficult. Here, we show the importance of immune tolerance for IDT onset and determine whether it is susceptible to a common IDT, HLA-B*57:01-mediated abacavir (ABC)-induced hypersensitivity (AHS), using CD4+ T cell-depleted programmed death-1 receptor (PD-1)-deficient HLA-B*57:01 transgenic mice (B*57:01-Tg/PD-1-/-). Although AHS is not observed in B*57:01-Tg mice, ABC treatment increases the proportion of cytokine- and cytolytic granule-secreting effector memory CD8+ T cells in CD4+ T cell-depleted B*57:01-Tg/PD-1-/- mice, thereby inducing skin toxicity with CD8+ T cell infiltration, mimicking AHS. Our results demonstrate that individual differences in the immune tolerance system, including PD-1highCD8+ T cells and regulatory CD4+ T cells, may affect the susceptibility of humans to HLA-mediated IDT in humans.

Role of respiratory uncoupling in drug-induced mitochondrial permeability transition.

Mitochondrial injury contributes to severe drug-induced liver injury. Particularly, mitochondrial permeability transition (MPT) is thought to be relevant to cytolytic hepatitis. However, the mechanism of drug-induced MPT is unclear and prediction of MPT is not adequately evaluated in the preclinical stage. In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling. Herein, we investigated whether other drugs that induce MPT share similar properties as troglitazone, using isolated mitochondria from rat liver. Of the 22 test drugs examined, six drugs, including troglitazone, induced MPT and showed an uncoupling effect. Additionally, receiver operating characteristic analysis was conducted to predict the MPT potential from the respiratory control ratio, an indicator of uncoupling intensity. Results showed that 2.5 was the best threshold that exhibited high sensitivity (1.00) and high specificity (0.81), indicating that uncoupling was correlated with MPT potential. Activation of calcium-independent phospholipase A2 appeared to be involved in uncoupling-induced MPT. Furthermore, a strong relationship between MPT intensity and the uncoupling effect among similar compounds was confirmed. These results may help in predicting MPT potential using cultured cells and modifying the chemical structures of the drugs to reduce MPT risk.

In Vitro Model for a Drug Assessment of Cytochrome P450 Family 3 Subfamily A Member 4 Substrates Using Human Induced Pluripotent Stem Cells and Genome Editing Technology.

In drug development, a system for predicting drug metabolism and drug-induced toxicity is necessary to ensure drug safety. Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) is an important drug-metabolizing enzyme expressed in the liver and small intestine, and predicting CYP3A4-mediated drug metabolism and drug-induced toxicity is essential. We previously developed procedures to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells (HLCs) or intestinal epithelial-like cells (IECs) with a fetal phenotype as well as a highly efficient genome editing technology that could enhance the homologous recombination efficiency at any locus, including CYP3A4. By using human iPS cells and our genome editing technology, we generated CYP3A4-knockout (KO) iPS cell-derived HLCs and IECs for the evaluation of CYP3A4-mediated drug metabolism and drug-induced toxicity. CYP3A4 deficiency did not affect pluripotency and hepatic and intestinal differentiation capacities, and CYP3A4 activity was entirely eradicated by CYP3A4 KO. Off-target effects (e.g., inhibition of bile acid excretion) were hardly observed in CYP3A4-KO cells but were observed in CYP3A4 inhibitor-treated (e.g., ketoconazole) cells. To evaluate whether drug-induced hepatotoxicity and enterotoxicity could be predicted using our model, we exposed CYP3A4-KO HLCs and IECs to acetaminophen, amiodarone, desipramine, leflunomide, tacrine, and tolcapone and confirmed that these cells could predict CYP3A4-mediated toxicity. Finally, we examined whether the therapeutic effects of an anti-hepatitis C virus (HCV) drug metabolized by CYP3A4 would be predicted using our model. CYP3A4-KO HLCs were treated with asunaprevir (antiviral drug metabolized by CYP3A4) after HCV infection, and the anti-viral effect was indeed strengthened by CYP3A4 KO. Conclusion: We succeeded in generating a novel evaluation system for prediction of CYP3A4-mediated drug metabolism and drug-induced toxicity.

Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments.