Abstract 1305: Metabolism studies of SH7139, a small molecule drug targeting B-cell malignancies overexpressing HLA-DR10, confirm its prodrug function

Abstract

Abstract SH7139 is a small molecule drug developed to target B-cell malignancies overexpressing HLA-DR10. It contains a DOTA metal chelating ring and three recognition ligands (Ct, Cb, and Dv) that are linked together using a scaffold composed of lysines and mini-polyethylene glycols. The amide bonds within the scaffold have D-configurations to make them resistant to hydrolysis by native proteolytic enzymes. However, two of the ligands contain bonds and chemical groups that can be cleaved or modified by metabolic enzymes to produce known cytotoxic compounds. LCMS was used to identify the metabolites of SH7139 produced in cryopreserved hepatocytes, Burkitt's (Raji) lymphoma cells and the liver microsomes of several mammalian species. Following uptake of SH7139 by human, beagle and rat hepatocytes, which occurs slowly, the cleavage of an amide bond within the Ct ligand produced 2-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]oxyaniline (M8) and a large fragment containing the remainder of SH7139 (M2). Phase II metabolism led to rapid glucuronidation of M8 to facilitate its excretion. This result may explain the lack of SH7139 liver toxicity in animals studied to date. Raji lymphoma cells produce the same two Phase I metabolites, but glucuronidation of M8 is not observed. Thus, M8 is free to inhibit multiple activities required for tumor cell survival, including the processing of misfolded proteins, lipogenesis and sumoylation. Experiments performed with microsomes isolated from Raji cells and human, Cynomologus monkey, beagle, rat and mouse livers identified eight additional metabolites. Removal of a single methyl group from intact SH7139 produced M7. Hydrolysis of the azo group within the SH7139 ligand Dv in both M2 and M7 led to the production of small and large fragments in each case: dimethylphenylenediamine (M9) and M1 and methylphenylenediamine (M10) and M5, respectively. M9 and the phenylenediamine that is usually produced by the further metabolism of M9 and M10 have been shown by others to inhibit ATP production in mitochondria. A final set of demethylation, amide cleavage, oxidation, reduction and glucuronidation reactions involving M7 and M2 produced the remaining metabolite M6. While metabolic processing of the Cb ligand did not produce a biologically reactive metabolite, other experiments have found that the Cb ligand in intact SH7139 inhibits the hydrolysis of GTP by MgcRacGAP-Rac1 and the completion of cytokinesis. Collectively, these results confirm that SH7139 functions similar to both an antibody drug conjugate (ADC) and a pro-drug. In contrast to an ADC, however, the same SH7139 ligand that participates in the targeting function is also involved, either directly or following metabolic processing, in tumor cell killing. This research was supported by the National Cancer Institute Phase II SBIR Award R44CA159843. Citation Format: Monique Cosman Balhorn, Rod Balhorn. Metabolism studies of SH7139, a small molecule drug targeting B-cell malignancies overexpressing HLA-DR10, confirm its prodrug function. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1305.