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 Fifty-four marketed and withdrawn drugs classified as DILI risks of "most concern", "less concern", and "no concern" were tested using a combination of four assays addressing mitochondrial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.

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.

Reactive oxygen species (ROS) assay-based photosafety screening for complex ingredients: Modification of the ROS assay protocol.

A reactive oxygen species (ROS) assay has been widely used for photosafety assessment; however, the phototoxic potential of complex materials, including plant extracts, essential oils, and functional polymers, is unevaluable because of their undefined molecular weights. The present study was undertaken to modify the ROS assay protocol for evaluating phototoxic potentials of those materials with use of their apparent molecular weight (aMw). On preparing sample solutions for the ROS assay, aMw ranging from 150 to 350 was tentatively employed for test substances. The modified ROS assays were applied to 45 phototoxic and 19 non-phototoxic substances, including 44 chemicals and 20 complex materials (plant extracts) for clarification of the predictive performance. Generation of ROS from photo-irradiated samples tended to increase as aMW grew, resulting in the largest number of false-positive predictions at aMW of 350. Some false-negative predictions were also observed when aMW was set at 200 or less. At aMw of 250, all tested phototoxic substances could be correctly identified as photoreactive with no false-negative predictions. Based on these observations, aMw of 250 was found to be suitable for the ROS assay on complex materials, and the sensitivity, specificity, and positive and negative predictivity for the proposed ROS assay were calculated to be 100, 52.6, 83.3, and 100%, respectively. Thus, the proposed approach may be efficacious for predicting phototoxic potentials of complex materials and contribute to the development of new products with a wide photosafety margin.

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.

Quantitative assessment of the effects of sulfamethoxazole on Toxoplasma gondii loads in susceptible WT C57BL/6 mice as an immunocompetent host model.

The abundance of Toxoplasma gondii with or without sulfamethoxazole (SMX) treatment was evaluated with quantitative competitive polymerase chain reaction in various organs of wild-type C57BL/6 mice, a susceptible immunocompetent host, after peroral infection with a cyst-forming Fukaya strain of T. gondii. SMX affected different organs in three ways: T. gondii was reduced independently of SMX (skin and kidney); T. gondii was not eradicated with continuous treatment (brain, heart, and lung); and T. gondii was eradicated with continuous treatment (tongue, skeletal muscle, and small intestine). The SMX concentrations in the brains, hearts, and lungs were higher in infected mice than in uninfected mice. These results indicate that even in an immunocompetent host, chemotherapy is necessary to reduce the parasite load and thus reduce the risk of recurrent disease.