Ryo Atsumi

A Zone Classification System for Risk Assessment of Idiosyncratic Drug Toxicity Using Daily Dose and Covalent Binding

The risk of idiosyncratic drug toxicity (IDT) is of great concern to the pharmaceutical industry. Current hypotheses based on retrospective studies suggest that the occurrence of IDT is related to covalent binding and daily dose. We determined the covalent binding of 42 radiolabeled drugs in three test systems (human liver microsomes and hepatocytes in vitro and rat liver in vivo) to assess the risk of IDT. On the basis of safety profiles given in official documentation, tested drugs were classified into the safety categories of safe, warning, black box warning, and withdrawn. The covalent binding in each of the three test systems did not distinguish the safety categories clearly. However, when the log-normalized covalent binding was plotted against the log-normalized daily dose, the distribution of the plot in the safety categories became clear. An ordinal logistic regression analysis indicated that both covalent binding and daily dose were significantly correlated with safety category and that covalent binding in hepatocytes was the best predictor among the three systems. When two separation lines were drawn on the correlation graph between covalent binding in human hepatocytes and daily dose by a regression analysis to create three zones, 30 of 37 tested drugs were located in zones corresponding to their respective classified safety categories. In conclusion, we established a zone classification system using covalent binding in human hepatocytes and daily dose for the risk assessment of IDTs.

Metabolism-dependent hepatotoxicity of amodiaquine in glutathione-depleted mice

We investigated the hepatotoxicity induced by AQ using a glutathione (GSH)-depleted mice model. Although sole administration of either AQ or l-buthionine-S,R-sulfoxinine (BSO), a well-known GSH synthesis inhibitor, produced no significant hepatotoxicity, combined administration of AQ with BSO induced hepatotoxicity characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Pretreatment of aminobenzotriazole, a nonspecific inhibitor for P450s, completely suppressed the above hepatotoxicity caused by AQ co-treatment with BSO. Administration of radiolabeled AQ in combination with BSO exhibited significantly higher covalent binding to mice liver proteins than that observed after sole dosing of radiolabeled AQ. The results obtained in this GSH-depleted animal model suggest that the reactive metabolite of AQ formed by hepatic P450 binds to liver proteins, and then finally leads to hepatotoxicity. These observations may help to understand the risk factors and the mechanism for idiosyncratic hepatotoxicity of AQ in humans.

DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1

Purpose: An anti-HER2 antibody–drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. Experimental Design: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. Results: DS-8201a exhibited a HER2 expression-dependent cell growth–inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1–insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. Conclusions: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1–insensitive HER2-positive cancers and low HER2–expressing cancers. Clin Cancer Res; 22(20); 5097–108. ©2016 AACR.

A Predictive Model of Human Myelotoxicity Using Five Camptothecin Derivatives and the In vitro Colony-Forming Unit Granulocyte/Macrophage Assay

Purpose: Many promising anticancer drugs are limited by myelosuppression. It is difficult to evaluate human myelotoxicity before a Phase I study because of the susceptibility of humans and animals to hematotoxicity. The purpose of this study was to establish a reliable method to predict the human maximum tolerated dose (MTD) of five camptothecin derivatives: SN-38, DX-8951f, topotecan, 9-aminocamptothecin, and camptothecin. Experimental Design: The myelotoxicity of SN-38 and DX-8951f were evaluated on bone marrow from mice, dogs, and humans using a 14-day colony-forming unit, granulocyte-macrophage (CFU-GM) assay to determine the 50%, 75%, and 90% inhibitory concentration values (IC50, IC75, and IC90, respectively). Results: Species differences in myelotoxicity were observed for SN-38 and DX-8951f. Using human and murine IC90s for myelotoxicity of these compounds and other camptothecin compounds (topotecan, 9-aminocamptothecin, and camptothecin), in vivo toxicological data, and pharmacokinetic parameters (data referred to in the literature), human MTDs were predicted retrospectively. The mechanism-based prediction model that is proposed uses the in vitro camptothecin assay and in vivo parameters on the basis of free fraction of area under the concentration-curve at the MTD (r2 = 0.887) and suggests that the human MTDs were well predicted for the five camptothecin derivatives by this model rather than by other models. Conclusion: The human MTDs of the camptothecin drugs were successfully predicted using the mechanism-based prediction model. The application of this model for in vitro hematotoxicology could play an important role for the development of new anticancer agents.

Ticlopidine-induced hepatotoxicity in a GSH-depleted rat model

We investigated hepatotoxicity induced by ticlopidine (TIC) in glutathione (GSH)-depleted rats by pre-treatment of a well-known GSH synthesis inhibitor, l-buthionine-S,R-sulfoxinine (BSO). Although sole administration of either TIC or BSO showed no signs of hepatotoxicity, combined administration of TIC with BSO induced hepatotoxicity, which was characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Administration of radio-labeled TIC in combination with BSO resulted in significantly higher covalent binding to rat liver proteins than that observed after sole dosing of radio-labeled TIC. Pre-treatment of 1-Aminobenzotriazole, a non-specific inhibitor of P450s, completely suppressed both hepatotoxicity and the increased hepatic covalent binding caused by TIC co-treatment with BSO. The results obtained in this animal model suggest that GSH depletion and covalent binding may be involved in hepatotoxicity induced by TIC. These observations may help to understand the risk factors and the mechanism of hepatotoxicity of TIC in humans.

New highly active taxoids from 9β-Dihydrobaccatin-9,10-acetals. Part 4

It was shown that a new taxane analogue 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both i.v. and p.o. administration, was prone to be metabolized by human liver microsomes. We identified a major metabolite, M-1, generated by human liver microsomes as 20a, a hydroxylated compound at the pyridine ring of 3. To improve the metabolic stability of 3, we designed and synthesized new taxane analogues based on the structure of M-1, and obtained some compounds that maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes.

Metabolism of Ticlopidine in Rats: Identification of the Main Biliary Metabolite as a Glutathione Conjugate of Ticlopidine S-Oxide

We have identified several novel metabolites of ticlopidine, a well known antiplatelet agent and have revealed its metabolic route in rats. The main biliary metabolite of ticlopidine was characterized as a glutathione (GSH) conjugate of ticlopidine S-oxide, in which conjugation had occurred at carbon 7a in the thienopyridine moiety. Quantitative analysis revealed that 29% of the dose was subjected to the formation of reactive intermediates followed by conjugation with GSH after oral administration of ticlopidine (22 mg/kg) to rats. In vitro incubation of ticlopidine with rat liver 9000g supernatant fraction (S9) fractions led to the formation of multiple metabolites, including 2-oxo-ticlopidine, the precursor for the pharmacologically active ticlopidine metabolite, [1-(2-chlorobenzyl)-4-mercaptopiperidin-(3Z)-ylidene] acetic acid. A novel thiophene ring-opened metabolite with a thioketone group and a carboxylic acid moiety has also been detected after incubation of 2-oxo-ticlopidine with rat liver microsomes or upon incubation of ticlopidine with rat liver S9 fractions.

Abstract A145: DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent anti-tumor efficacy with differentiation from T-DM1 in preclinical studies

Antibody-drug conjugates (ADCs) represent a promising drug class which expresses a wider therapeutic window than conventional chemotherapeutic agents by effecting efficient and specific drug delivery to antigen-expressing tumor cells. DS-8201a is a HER2-targeting ADC structurally composed of a humanized anti-HER2 antibody, enzymatically cleavable peptide-linker, and a novel topoisomerase I inhibitor (DXd), which is cell-membrane permeable and more potent than SN-38 the active metabolite of irinotecan. This ADC achieves a high drug-to-antibody-ratio (DAR, 7 to 8) with homogeneous conjugation with DXd. DS-8201a is cleaved by lysosomal enzymes and releases DXd in the cytoplasm after it binds to HER2 receptors and is internalized in tumor cells. In order to evaluate the pharmacological potential of DS-8201a, in vitro and in vivo studies were performed in comparison with T-DM1. T-DM1 is a commercialized HER2-targeting ADC with a tubulin polymerization inhibitor that is approved for treatment for HER2-positive breast cancer. In vitro studies indicated that DS-8201a exhibited a HER2 expression-dependent cell growth inhibitory activity. In vivo studies using a HER2-positive gastric cancer NCI-N87 cell line-derived xenograft (CDX) model suggested that DS-8201a induced a dose-dependent tumor growth inhibition and tumor regression with a single dosing at more than 1 mg/kg. In comparison with T-DM1, 1) DS-8201a was effective against T-DM1-insensitive breast and gastric patient-derived xenograft (PDX) models with high HER2 expression. 2) DS-8201a, but not T-DM1, demonstrated anti-tumor efficacy against several breast cancer PDX models with low HER2 expression. 3) DS-8201a showed a complete response in mice inoculated with a mixture of HER2-positive and -negative cells while T-DM1 did not. These differentiations may be due to the different mechanisms of action of each conjugated drug (topoisomerase I inhibition vs. tubulin polymerization inhibition), higher DAR of DS-8201a (7 to 8 vs. 3.5), and DS-8201a9s more potent bystander killing due to higher cell membrane permeability of conjugated toxin. When taken together with over 30 CDX and PDX models from 8 tumor types (breast, gastric, cholangiocarcinoma, esophagus, colon, NSCLC, pancreas, and ovary) conducted so far, DS-8201a induced tumor regression in over 20 models with various HER2 expression levels though T-DM1 induced regression in only 8 models with high HER2 expression. In conclusion, these studies suggest that DS-8201a, a novel HER2-targeting ADC, may be more efficacious in a broader patient population than T-DM1, and in tumors which current anti-HER2 therapies are ineffective, such as T-DM1 insensitive tumors and low HER2-expressing tumors, and in tumors with high HER2 heterogeneity. The first in human study will be initiated in 2015 3Q. Citation Format: Yusuke Ogitani, Junko Yamaguchi, Chiaki Ishii, Takehiro Hirai, Ryo Atsumi, Koji Morita, Ichiro Hayakawa, Hiroyuki Naito, Takeshi Masuda, Takashi Nakada, Takahiro Jikoh. DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent anti-tumor efficacy with differentiation from T-DM1 in preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A145.