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.

ABSTRACT:The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.Clopidogrel is a thienopyridine antiplatelet agent that has been widely used in the management of cardiovascular diseases, including atherothrombosis, unstable angina, and myocardial infarction (Savi and Herbert, 2005). Clopidogrel is an inactive prodrug that needs to be converted to the pharmacologically active metabolite in vivo through the hepatic metabolism to exhibit the antiplatelet effect (Savi et al., 1992). Clopidogrel is first converted by the action of cytochrome P450 (P450) to 2-oxo-clopidogrel (a thiolactone) then in a second step converted to the pharmacologically active, thiol-containing metabolite as shown in Fig. 1 (Savi et al., 2000). The P450 isoforms involved in the bioactivation of clopidogrel have been suggested to be CYP1A2 in rats (Savi et al., 1994) and CYP3A in humans (Clarke and Waskell, 2003), although the contribution of these P450s to produce the active metabolite was still unclear. In addition, several recent clinical studies demonstrated that CYP3A4, CYP3A5, and CYP2C19 have a significant role in the formation of the active metabolite from clopidogrel (Hulot et al., 2006;Suh et al., 2006;Brandt et al., 2007;Farid et al., 2007Farid et al., , 2008. Furthermore, Brandt et al. (2007) reported that loss of function of CYP2C19 due to polymorphisms resulted in decreased exposure to...

Antibody–drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS‐8201a is a human epidermal growth factor receptor 2 (HER2)‐targeting antibody–drug conjugate prepared using a novel linker‐payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX‐8951 derivative, DXd). It was effective against trastuzumab emtansine (T‐DM1)‐insensitive patient‐derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS‐8201a was evaluated and compared with that of T‐DM1. We confirmed that the payload of DS‐8201a, DXd (1), was highly membrane‐permeable whereas that of T‐DM1, Lys‐SMCC‐DM1, had a low level of permeability. Under a coculture condition of HER2‐positive KPL‐4 cells and negative MDA‐MB‐468 cells in vitro, DS‐8201a killed both cells, whereas T‐DM1 and an antibody–drug conjugate with a low permeable payload, anti‐HER2‐DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2‐positive NCI‐N87 cells and HER2‐negative MDA‐MB‐468‐Luc cells by using an in vivo imaging system. In vivo, DS‐8201a reduced the luciferase signal of the mice, indicating suppression of the MDA‐MB‐468‐Luc population; however, T‐DM1 and anti‐HER2‐DXd (2) did not. Furthermore, it was confirmed that DS‐8201a was not effective against MDA‐MB‐468‐Luc tumors inoculated at the opposite side of the NCI‐N87 tumor, suggesting that the bystander killing effect of DS‐8201a is observed only in cells neighboring HER2‐positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS‐8201a has a potent bystander effect due to a highly membrane‐permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T‐DM1.

The greater in vivo antiplatelet effects of prasugrel as compared to clopidogrel reflect more efficient generation of its active metabolite with similar antiplatelet activity to that of clopidogrelÕs active metabolite. J Thromb Haemost 2007; 5: 1545-51.Summary. Background and methods: Prasugrel is a novel orally active thienopyridine prodrug with potent and longlasting antiplatelet effects. Platelet inhibition reflects inhibition of P2Y 12 receptors by its active metabolite (AM). Previous studies have shown that the antiplatelet potency of prasugrel is at least 10 times higher than that of clopidogrel in rats and humans, but the mechanism of its higher potency has not yet been fully elucidated. Results: Oral administration of prasugrel to rats resulted in dose-related and time-related inhibition of ex vivo platelet aggregation, and its effect was about 10 times more potent than that of clopidogrel. The plasma concentration of prasugrel AM was higher than that of clopidogrel AM despite tenfold higher doses of clopidogrel, indicating more efficient in vivo production of prasugrel AM than of clopidogrel AM. In rat platelets, prasugrel AM inhibited in vitro platelet aggregation induced by adenosine 5¢-diphosphate (ADP) (10 lM) with an IC 50 value of 1.8 lM. Clopidogrel AM similarly inhibited platelet aggregation with an IC 50 value of 2.4 lM. Similar results were also observed for ADP-induced (10 lM) decreases in prostaglandin E 1 -stimulated rat platelet cAMP levels. These results indicate that both AMs have similar in vitro antiplatelet activities. Conclusions: The greater in vivo antiplatelet potency of prasugrel as compared to clopidogrel reflects more efficient in vivo generation of its AM, which demonstrates similar in vitro activity to clopidogrel AM.

ABSTRACT:The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing active metabolites through corresponding thiolactone metabolites. The formation rate of clopidogrel active metabolite was much lower and more variable [0.164 ؎ 0.196 l/min/mg protein, coefficient of variation (CV) ‫؍‬ 120%] compared with the formation of prasugrel active metabolite (8.68 ؎ 6.64 l/min/mg protein, CV ‫؍‬ 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone metabolite (2.24 ؎ 1.00 l/min/mg protein, CV ‫؍‬ 45%) compared with the formation of prasugrel thiolactone metabolite (55.2 ؎ 15.4 l/min/mg protein, CV ‫؍‬ 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inactive acid metabolite (approximately 90% of total metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone metabolite formed was converted to an inactive thiolactone acid metabolite. The oxidation of clopidogrel to its thiolactone metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the active metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone metabolite to inactive acid metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone metabolite, which may contribute to the poorer and more variable active metabolite formation for clopidogrel than prasugrel.Prasugrel and clopidogrel are thienopyridine antiplatelet agents. Prasugrel is shown to reduce the rate of thrombotic cardiovascular events and stent thrombosis in patients with acute coronary syndrome and those to be managed with percutaneous coronary intervention (Wiviott et al., 2007). Likewise, clopidogrel is used for the management of patients after percutaneous coronary intervention and stent placement (Braunwald et al., 2002;Schulman, 2004). Thienopyridines are prodrugs that are converted in vivo to pharmacologically active metabolites through corresponding thiolactone intermediates. The thiol-containing active metabolites inhibit platelet function by irreversibly binding to the platelet P2Y 12 ADP receptor (Niitsu et al., 2005;Savi and Herbert, 2005;Algaier et al., 2008).