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 In a previous study, we found that troglitazone, a drug withdrawn due to liver injury, induced MPT via mild depolarization probably resulting from uncoupling.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.

Mechanism-based integrated assay systems for the prediction of drug-induced liver injury.

Drug-induced liver injury (DILI) can cause hepatic failure and result in drug withdrawal from the market. It has host-related and compound-dependent mechanisms. Preclinical prediction of DILI risk is very challenging and safety assessments based on animals inadequately forecast human DILI risk. In contrast, human-derived in vitro cell culture-based models could improve DILI risk prediction accuracy. Here, we developed and validated an innovative method to assess DILI risk associated with various compounds. Fifty-four marketed and withdrawn drugs classified as DILI risks of "most concern", "less concern", and "no concern" were tested using a combination of four assays addressing mitochondrial injury, intrahepatic lipid accumulation, inhibition of bile canalicular network formation, and bile acid accumulation. Using the inhibitory potencies of the drugs evaluated in these in vitro tests, an algorithm with the highest available DILI risk prediction power was built by artificial neural network (ANN) analysis. It had an overall forecasting accuracy of 73%. We excluded the intrahepatic lipid accumulation assay to avoid overfitting. The accuracy of the algorithm in terms of predicting DILI risks was 62% when it was constructed by ANN but only 49% when it was built by the point-added scoring method. The final algorithm based on three assays made no DILI risk prediction errors such as "most concern " instead of "no concern" and vice-versa. Our mechanistic approach may accurately predict DILI risks associated with numerous candidate drugs.

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.

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.

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.

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.

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.

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.

Evaluation of Parent- and Metabolite-Induced Mitochondrial Toxicities Using CYP-Introduced HepG2 cells.

Mitochondrial toxicity is an important factor to predict drug-induced liver injury (DILI). Previous studies have focused predominantly on mitochondrial toxicities due to parent forms, and no study has adequately evaluated metabolite-induced mitochondrial toxicity. Moreover, previous studies have used HepG2 cells, which lack many cytochrome P450 (CYP) genes. To overcome this problem, CYP-introduced HepG2 cells were constructed using several gene transfer technologies, including adenoviruses and plasmids. However, these methods only led to a transient expression of CYP genes. In the present study, usefulness of four CYPs introduced-HepG2 (TC-Hep) cells previously constructed through mammalian artificial chromosome technology were examined, especially from the perspective of mitochondrial toxicity. First, we evaluated the effects of known compounds, such as rotenone and flutamide, on mitochondrial toxicity and cell death in TC-Hep cells cultured in galactose conditions. Expectedly, rotenone-induced cell death ameliorated because rotenone was metabolized by CYPs into inactive form(s) and flutamide-induced cell death increased in TC-Hep cells. Second, we evaluated five compounds that caused liver injury in clinical phase and were discontinued during pharmaceutical development. The present in vitro tool suggested that three of the five compounds caused metabolite-induced mitochondrial toxicities. In conclusion, the present in vitro tool could easily and inexpensively detect metabolite-induced mitochondrial toxicity; hence, it can be useful for predicting DILI in preclinical phase.

[The trends in predicting drug-induced liver injury].

Drug-induced liver injury (DILI) is the major reason for the discontinuation of new drug development and the withdrawal of drugs from the market. Hence, the evaluation systems which predict the onset of DILI in the pre-clinical stage are needed. To date, many researchers have conducted the mechanism of DILI, but the DILI prediction is poor because of the complexity of DILI. In this regard, based on the information obtained from basic research and clinical case, several pharmaceutical companies have been developed DILI prediction methods with high sensitivity and specificity by combining multiple targets. Another reason for low predictability is derived from the conventional culture method which causes a rapid decrease in hepatocyte function. To overcome these problems, the construction of a high-level in vitro evaluation system has been developed and applied to DILI evaluation. On the other hand, these in vitro evaluation methods require a lot of labor and cost so, in silico prediction methods have also been constructed in recent years. Based on this point, this article reviews the trends in DILI prediction systems in the non-clinical stage.

Hypoxia/reoxygenation exacerbates drug-induced cytotoxicity by opening mitochondrial permeability transition pore: Possible application for toxicity screening.

Recently, mitochondrial dysfunction is thought of as an important factor leading to a drug-induced liver injury. Our previous reports show that mitochondria-related toxicity, including respiratory chain inhibition (RCI) and reactive oxygen species (ROS) induction, can be detected by the modification of sugar resource substitution and high oxygen condition. However, this in vitro model does not detect mitochondrial permeability transition (MPT)-induced toxicity. Another study with a lipopolysaccharide-pre-administered rodent model showed that ischemia/reperfusion induced ROS, sensitized the susceptibility of MPT pore opening and, finally developed drug-induced liver toxicity. Based on this result, the present study investigated the effect of hypoxia/reoxygenation (H/R) treatment mimicking the ischemia/reperfusion on MPT-dependent toxicity, aiming to construct a system that can evaluate MPT by drugs in hepatocytes. Mitochondrial ROS were enhanced by H/R treatment only in the galactose culture condition. Amiodarone, benzbromarone, flutamide and troglitazone which induced MPT pore opening led to hepatocyte death only in combination with H/R and galactose. Moreover, this alteration was significantly suppressed in hepatocytes lacking cyclophilin D. In conclusion, MPT-induced cytotoxicity can be detected by activating mitochondrial function and H/R. This cell-based assay system could evaluate MPT induced-cytotoxicity by drugs, besides RCI and ROS induction.

Successful energy shift from glycolysis to mitochondrial oxidative phosphorylation in freshly isolated hepatocytes from humanized mice liver.

Mitochondrial toxicity is a factor of drug-induced liver injury. Previously, we reported an in vitro rat hepatocyte assay where mitochondrial toxicity was more sensitively evaluated, using sugar resource substitution and increased oxygen supply. Although this method could be applicable to human cell-based assay, cryopreserved human hepatocyte (CHH) has some disadvantages/uncertainty, including unstable same donor supply and potential organelle damage due to cryopreservation. Herein, we compared the mitochondrial functions of freshly-isolated hepatocytes from humanized chimeric mice liver (PXB-cells) and three CHH lots to determine the better cell source for mitochondrial toxicity assay. Two CHH lots declined after replacing glucose with galactose. To confirm the shift in energy production from glycolysis to oxidative phosphorylation, lactate and oxygen consumption rate (indicators of glycolytic activity and mitochondrial oxidative phosphorylation, respectively) were measured. In PXB-cells, lactate amount decreased, while oxygen consumption in 100 min increased. These effects were less evident in CHH. The cytotoxicity of the select respiratory chain inhibitors was enhanced in PXB-cells upon sugar replacement, but no change occurred with negative control drugs (bicalutamide and metformin). Altogether, PXB-cells was less vulnerable to sugar resource substitution than CHH. The substitution activated mitochondrial function and enhanced cytotoxicity of respiratory chain inhibitors in PXB-cells.

Inhibition of biliary network reconstruction by benzbromarone delays recovery from pre-existing liver injury.

The liver performs a variety of essential functions; hence drug-induced liver injury (DILI) is a serious concern that can ultimately lead to the withdrawal of a drug from the market or discontinuation of drug development. However, the mechanisms of drug-induced liver injury are not always clear. We hypothesized that drugs may inhibit the liver recovery process, especially bile canalicular (BC) network reformation, leading to persistent liver injury and deterioration, and tested this hypothesis in the present work. The BC structure disappeared in mice following treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or thioacetamide (TAA) for 4 weeks, then reappeared after 4 weeks of receiving a normal diet. By contrast, reconstruction of the BC structure was suppressed in mice fed a diet containing 0.3% benzbromarone (BBR; which can induce fatal liver injury in clinical settings) after liver injury. Plasma ALT levels were increased significantly in mice treated with BBR after DDC or TAA treatment, compared with BBR alone. To confirm whether BBR has a direct inhibitory effect on hepatocytes, we also examined BC reformation in primary cultured mouse hepatocytes with a sandwich configuration. Under these culture conditions, the BC network rapidly reformed from days 2 and 3 after seeding. During the reformation period, BBR inhibited BC reformation significantly. These results suggest that BBR inhibits BC reconstruction and delays recovery from pre-existing liver injury.

In vitro bile acid-dependent hepatocyte toxicity assay system using human induced pluripotent stem cell-derived hepatocytes: Current status and disadvantages to overcome.

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5ß-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.

Inhibition of bile canalicular network formation in rat sandwich cultured hepatocytes by drugs associated with risk of severe liver injury.

Idiosyncratic drug-induced liver injury is a clinical concern with serious consequences. Although many preclinical screening methods have been proposed, it remains difficult to identify compounds associated with this rare but potentially fatal liver condition. Here, we propose a novel assay system to assess the risk of liver injury. Rat primary hepatocytes were cultured in a sandwich configuration, which enables the formation of a typical bile canalicular network. From day 2 to 3, test drugs, mostly selected from a list of cholestatic drugs, were administered, and the length of the network was semi-quantitatively measured by immunofluorescence. Liver injury risk information was collected from drug labels and was compared with in vitro measurements. Of 23 test drugs examined, 15 exhibited potent inhibition of bile canalicular network formation (<60% of control). Effects on cell viability were negligible or minimal as confirmed by lactate dehydrogenase leakage and cellular ATP content assays. For the potent 15 drugs, IC50 values were determined. Finally, maximum daily dose divided by the inhibition constant gave good separation of the highest risk of severe liver toxicity drugs such as troglitazone, benzbromarone, flutamide, and amiodarone from lower risk drugs. In conclusion, inhibitory effect on the bile canalicular network formation observed in in vitro sandwich cultured hepatocytes evaluates a new aspect of drug toxicity, particularly associated with aggravation of liver injury.