COMBINATORIAL MOLECULAR PHARMACOLOGICAL AND PRECLINICAL STUDIES OF AN EPIGENETIC ANTICANCER DRUG, 3-DEAZANEPLANOCIN A, IN RATS

Pharmacologic reversal of epigenetic events including histone modification and DNA methylation has a therapeutic potential in cancer treatment through inducing cancer cell apoptosis. 3-deazaneplanocin A (DZNep) appears to be a unique chromatin remodelling (epigenetic) compound that can deplete the cellular enhancer of zeste homolog 2 (EZH2) protein, an enzyme responsible for modulation of histone methylation and in turn specifically induce breast cancer apoptosis. Hence, DZNep, alone or together with the other two epigenetic agents trichostatin A (TSA) and 5-Aza-2¿-deoxycytidine (AZA), were used to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells and to identify a comprehensive set of genes functionally enriched in inflammation network and abnormally regulated by EZH2 in breast cancer cells. From this set of genes, TNF, a cell death gene, was found to be associated with the DZNep plus TSA induced cell death in breast cancer cells. As the only antitumor agent through inhibition of EZH2 protein to date, the preclinical study of DZNep was thus performed. No observable adverse effect dose (NOAEL) of DZNep determined was 10 mg/kg in Sprague Dawley (SD) rats. DZNep caused nephrotoxicity and renal atrophy in rats in a dose-dependent manner but had no apparent effect on the hematological system of rats. A sensitive and reliable liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of DZNep in rat biosamples (rat plasma, urine, feces, and tissues). Following an intravenous bolus administration, DZNep had a dose-independent tri-exponential disposition profile with a short plasma elimination half-life (1.1 h) in SD rats. DZNep also showed a low protein binding in plasma (18.5 %) and a low partitioning to erythrocyte (0.78), but had extensive tissue distribution, especially in kidney (21.25 ± 2.12 µg/g at 5 min), and predominant renal excretion (80.3 % recovered as the unchanged drug by 72 h). DZNep was not extensively metabolized in vivo (86.80 % recovery of DZNep unchanged from urine and feces by 72 h). A unilamellar pegylated liposomal formulation containing DZNep (L-DZNep) was developed using a remote-loading method in the presence of phenylboronic acid and the encapsulation efficiency of DZNep in L-DZNep reached 47.5 %. Importantly, the entrapment of DZNep within the pegylated liposomal carrier noticeably ameliorated the pharmacokinetic characteristics of DZNep in vivo (e.g. elimination half-life, 8.0 h). The overall findings of these studies support that DZNep not only can serve as a molecular pharmacological tool to probe the complex interaction between epigenetic events but also hopefully become a potential chemotherapeutic agent for the clinical application.

Author dc:creator

• SUN FENG

• 3-deazaneplanocin A, epigenetics, molecular pharmacology, preclinical study, pharmacokinetics, liposomes