In Vitro Metabolism of 2-Methoxy-N-[3-[4-[3-methyl-4-[(6-methyl- 3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) by Aldehyde Oxidase and Predicting Its Percent Contribution Relative to CYP-Mediated Metabolism.

2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) is an anticancer drug that was discontinued due to hepatotoxicity found in clinical studies. Metabolite analysis of CP-724,714 was conducted using human hepatocytes, in which twelve oxidative metabolites and one hydrolyzed metabolite were formed. Among the three mono-oxidative metabolites, the formation of two was inhibited by adding 1-aminobenzotriazole, a pan-CYP inhibitor. In contrast, the remaining one was not affected by this inhibitor but partially inhibited by hydralazine, indicating that aldehyde oxidase (AO) was involved in metabolizing CP-724,714, which contains a quinazoline substructure, a heterocyclic aromatic quinazoline ring, known to be preferably metabolized by AO. One of the oxidative metabolites of CP-724,714 observed in human hepatocytes was also generated in recombinant human AO. Although CP-724,714 is metabolized by both CYPs and AO in human hepatocytes, the contribution level of AO could not be evaluated using its specific inhibitors because of low AO activity in in vitro human materials. Here, we present a metabolic pathway for CP-724,714 in human hepatocytes and the involvement of AO in CP-724,714 metabolism. We showed here a plausible workflow for predicting AO contribution to the metabolism of CP-724,714 based on DMPK screening data. SIGNIFICANCE STATEMENT: 2-methoxy-N-[3-[4-[3-methyl-4-[(6-methyl-3-pyridinyl)oxy]anilino]-6-quinazolinyl]prop-2-enyl]acetamide (CP-724,714) was identified as a substrate of aldehyde oxidase (AO) rather than xanthine oxidase. Since CP-724,714 is also metabolized by cytochrome P450s (CYPs), the contribution levels of AO and CYPs in the metabolism of CP-724,714 were estimated simultaneously based on in vitro drug metabolism screening data.

Eribulin penetrates brain tumor tissue and prolongs survival of mice harboring intracerebral glioblastoma xenografts.

Glioblastoma is one of the most devastating human malignancies for which a novel efficient treatment is urgently required. This pre-clinical study shows that eribulin, a specific inhibitor of telomerase reverse transcriptase (TERT)-RNA-dependent RNA polymerase, is an effective anticancer agent against glioblastoma. Eribulin inhibited the growth of 4 TERT promoter mutation-harboring glioblastoma cell lines in vitro at subnanomolar concentrations. In addition, it suppressed the growth of glioblastoma cells transplanted subcutaneously or intracerebrally into mice, and significantly prolonged the survival of mice harboring brain tumors at a clinically equivalent dose. A pharmacokinetics study showed that eribulin quickly penetrated brain tumors and remained at a high concentration even when it was washed away from plasma, kidney or liver 24 hours after intravenous injection. Moreover, a matrix-assisted laser desorption/ionization mass spectrometry imaging analysis revealed that intraperitoneally injected eribulin penetrated the brain tumor and was distributed evenly within the tumor mass at 1 hour after the injection whereas only very low levels of eribulin were detected in surrounding normal brain. Eribulin is an FDA-approved drug for refractory breast cancer and can be safely repositioned for treatment of glioblastoma patients. Thus, our results suggest that eribulin may serve as a novel therapeutic option for glioblastoma. Based on these data, an investigator-initiated registration-directed clinical trial to evaluate the safety and efficacy of eribulin in patients with recurrent GBM (UMIN000030359) has been initiated.

Development of the Complement C5 Assay by LC-MS/MS in Monkey Serum and Comparison with Enzyme-Linked Immunosorbent Assay.

Complement C5 (C5) is the key component for the complement activation pathway, which is important for innate immunity, and inhibition of C5 is considered to be effective in antibody-mediated rejection in organ transplantation. Thus determination of C5 levels in systemic circulation is a simple way to understand efficacy of drugs that aim to inhibit C5 production. We have developed a simple liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for C5 in cynomolgus monkey serum. C5 in monkey serum was subjected to tryptic digestion, and two signature peptides, DSSVPNTGTAR and LQGTLPVEAR, were assayed by LC-MS/MS with electrospray ionization in the positive ion mode. Assay reproducibility in serum samples was evaluated, and the assay was applied to the C5 assay in monkey serum after administration of C5 siRNA encapsulated in lipid nanoparticles to monkeys. The time profiles of C5 after administration of C5 siRNA were comparable between the two signature peptides by LC-MS/MS and were also similar to those by an enzyme-linked immunosorbent assay using an assay kit. These findings suggest that the established LC-MS/MS assay of C5 is reliable to determine C5 levels in monkey serum.

Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction.

BACKGROUND & AIMS: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening. METHODS: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development. RESULTS: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity. CONCLUSIONS: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity.

A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.