CZ48 is a novel potent anticancer agent currently in a phase I clinical trial. The absorption of CZ48 in humans is approximately less than 10% based on the excretion ratio of the unchanged form in the feces following oral administration. It was reported that CZ48 acts as a pro-drug and exerts its anticancer activity through the active metabolite CPT in vivo. The specific enzymes involved in its biotransformation have not been identified. To date, it has been very difficult to predict drug-drug interactions with CZ48 in the clinical setting due to a lack of information. Cytochrome P450 enzymes are primarily found in liver cells but are also located in other organ cells throughout the body. Therefore, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite CPT. In this study, we conducted in vitro experiments by using in vitro human liver models which have been developed in the past few decades, including isoenzymes, microsomes, cytosol, S9 fraction, statins and inhibitors/inducers to allow us to evaluate the potential for drug-drug interactions.


Using an integrated approach CZ48 biotransformation was studied for CYP-mediated metabolic reaction with human liver microsomes (HLM), cytosol and S9 fraction. Seven major isoenzymes were used to investigate, through relative activity factor approach, the contribution of selective statins or of several inhibitors/inducers in the phase-I metabolism of CZ48.


Experiments revealed that 97.9% of CZ48 was metabolized in HLM in 2 hours, and 64.8% of CZ48 was biotransformed in the S9 fraction, while cytosol was less responsible (13.4%) for CZ48 biotransformation. The majority (68.5%) of CZ48 was metabolized into CPT when co-incubated with CYP3A4. The statins, mevastatin (lovastatin), atorvastatin and simvastatin, are specific CYP3A4 substrates and inhibited CZ48 biotransformation. In contrast, rosuvastatin showed no inhibitory effect. At CZ48 concentrations of 0.25, 0.5 and 1 uM, verapamil, erythromycin, clarithromycin and CYP3A4 showed the strong inhibitory effects on CZ48 biotransformation. However, based on results with human liver microsomes in the presence of the CYP3A4 inducers phenytoin, rifampicin and carbamazepine, it was suggested that cz48 was partly metabolized by other enzymes in the microsomes.


Our results establish the metabolic pathways of CZ48, revealing the significant role of CYPs in its metabolism. Of the major human CYPs, CYP3A4 is responsible for catalyzing the biotransformation of CZ48. Indeed, CYP3A4 has extremely broad substrate specificity and is responsible for the metabolism of 50% of drugs in current use. Our results provide insight into the underlying biochemical mechanisms of CZ48, which would help to predict clinical outcomes.

Note: This abstract was not presented at the meeting.

Citation Format: Xing Liu, Albert DeJesus, Dana Vardeman, Zhisong Cao, Beppino Giovanella. In vitro biotransformation of and inhibitory effects of CZ48 in human liver microsomes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2014-4337