In vivo Activity of Rhodium Metalloinsertors and Exploration of Drug Delivery Systems

Abstract

Rhodium metalloinsertors are octahedral complexes developed to selectively target the mismatches and insertions/deletions (indels) that result from mismatch repair (MMR) deficient cancers. By incorporating particularly wide, aromatic, inserting ligands, these complexes are able to detect thermodynamically destabilized mismatch sites via a binding mode known as metalloinsertion, in which the inserting ligand binds DNA via the minor groove and results in ejection of the destabilized mismatched base pair. In vitro analyses of metalloinsertors have found that these complexes are selectively cytotoxic towards MMR-deficient cancer cells compared to MMR-proficient cells. Furthermore, the newest family of Rh-O metalloinsertors, which includes [Rh(phen)(chrysi)(PPO)]²⁺ (Rh-PPO), displays preferential cytotoxicities in the nanomolar range, which is significantly more potent than first generation metalloinsertors and many standard of care chemotherapeutics. Given the high level of potency and selectivity of Rh-O metalloinsertors, further clinical development of these complexes has been pursued. Here, we present the first preclinical mouse evaluation of a rhodium metalloinsertor as an anticancer agent. The Rh-O metalloinsertor Rh-PPO was evaluated in the HCT116 colorectal cancer xenograft tumor model alongside saline and oxaliplatin controls. Intraperitoneal studies with Rh-PPO showed significant decreases in tumor volumes over time and final tumor weights, indicating Rh-PPO has notable anticancer activity. Additionally, Rh-PPO treatment resulted in a noteworthy increase in the length of mouse survival that was on par with the FDA approved chemotherapeutic oxaliplatin. Pharmacokinetic analyses revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Importantly, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, which points to a need for more selective delivery methods in order to further metalloinsertor development. In order to target cancerous cells with still higher selectivity, routes to metalloinsertor antibody drug conjugate (ADC) designs were explored. By attaching Rh-O metalloinsertors to an antibody specific to cancer-associated antigens, our complexes may become even more specifically directed to induce selective cytotoxicity in diseased cells. Three ADC drug linkers that incorporate maleimide groups into the N^O coordinating ligand of a Rh-O metalloinsertor were designed, synthesized, and characterized. These complexes were evaluated for their cellular potency and selectivity toward MMR-deficient cancer cells. Studies revealed that functionalization of the hydroxyl-containing ancillary ligand resulted in decreased potency and abolished preferential cytotoxicity, contrary to previous studies that assessed modifications of this ligand. Liposomal formulations of Rh-PPO were also explored to further target metalloinsertors to malignant cells. Liposomal drug encapsulations have a demonstrated ability to decrease systemic toxicity and increase tumor drug uptake; therefore, the biological activity of Rh-PPO liposomal formulations was explored. Four distinct Rh-PPO liposome formation methods were developed and the resulting liposomes were assessed for their encapsulation efficiency, cellular toxicity, and stability. Remote loaded Rh-PPO liposomes were found to display the most promising chemical and biological characteristics, although additional optimization of encapsulation procedures is necessary for further preclinical evaluation of this metalloinsertor drug delivery approach. As metalloinsertors continue preclinical assessment and development, a greater understanding of their mechanism of action is imperative. Biological studies with Rh-PPO and the fluorescent analogue RhPPO-Cy3 have shown that DNA damage from metalloinsertor treatment involves the formation of DNA double strand breaks near metalloinsertor-mismatch binding sites. Furthermore, the DNA damage response, including recruitment of pH2AX and Rad51 proteins, becomes activated in response to Rh-PPO treatment. In order to further elucidate the unique mechanism of action of Rh-O metalloinsertors, which involves both metalloinsertor enantiomers binding to DNA mismatches and displaying biological activity, structural studies are ongoing. X-ray crystallography and microelectron diffraction (microED) techniques have been used in attempts to obtain a high resolution structure of Rh-O metalloinsertors bound to DNA mismatch sites. Gaining these structural insights will be critical to understanding the increased cytotoxic selectivity and uniquely high potency of these second generation metalloinsertor complexes. The experiments detailed in this thesis have advanced the preclinical development of rhodium metalloinsertors. The ability of Rh-O metalloinsertors to decrease tumor growth in vivo has been established. Additionally, liposomal and ADC metalloinsertor drug formulations have been pursued as drug delivery systems, and the biological mechanisms relevant to metalloinsertor activity have been analyzed. Additional efforts to study rhodium metalloinsertors will continue to advance these promising chemotherapeutics as novel, targeted treatments for MMR-deficient cancers.