Development of heptamethine carbocyanine based small molecule targeting platform for tumor specific drug delivery

Improved tumor-targeting strategies are needed to enhance a cytotoxic drug's therapeutic efficacy while minimizing systemic toxicity. Antibody-drug conjugates (ADCs) have shown this potential but are limited by a restricted tumor-epitope diversity; Each ADC is designed for a single tumor type, with development, manufacturing, and cold-chain requirements adding complexity and cost. A targeting platform that is inexpensive, modular, and applicable across tumor types could address these gaps. Dye-drug conjugates (DDC) have the potential to overcome these limitations. DDCs are composed of heptamethine carbocyanine (HCC) compounds that selectively interact with Organic Anion-Transporting Polypeptides (OATPs), chemically conjugated to diverse cytotoxic payloads. DDCs target OATPs which are overexpressed across human tumor types and represent a novel class of tumor-selective transporter targets. Additionally, they possess near-infrared (NIR) fluorescence enabling both imaging and therapy delivery making them attractive as a single small molecule delivery framework. Novel HCC analogs were synthesized and characterized as tumor-targeting scaffolds. These compounds exhibited minimal intrinsic cytotoxicity in normal and tumor cell lines. In vitro imaging of tumor cell lines confirmed HCC uptake. In vivo and ex vivo imaging of tumor xenografts following HCC systemic administration confirmed HCC tumor accumulation with minimal accumulation in other organs. Two of the novel HCCs were synthesized with cytotoxic payloads including doxorubicin, SN38 and MMAE as pro-drugs, linked via a cathepsin B cleavable linker. Upon linker cleavage, the released drugs become active. These DDCs were assessed for solubility, thermodynamic stability and plasma stability (mouse, human) identifying promising candidates. Studying candidates further, in tumor cell lines HCC-MMAE was internalized, and cleaved to release MMAE, resulting in cell death. At 72H, IC50 of HCC-MMAE in A549 cells was 92.7±9.6nM, in MCF7 cells 59.3±4.6nM, and SW620 79.6±37.6nM. The IC50 for MMAE alone was 0.49±0.2nM, while HCC alone >1,000nM across all cell lines. In vivo and ex vivo imaging of tumor xenografts following systemic administration confirmed DCC tumor accumulation with minimal accumulation in other organs. In conclusion, novel HCC-based DDCs have been developed as tumor-selective targeting agents. Conjugation of cytotoxic payloads to the HCC scaffold allowed effective delivery of payload, with the potential ability to use compounds previously limited by their systemic toxicity. The HCC platform allows conjugation of diverse payloads, with broad tumor applicability through the OATP target, and is synthetically simple and cost-effective to produce.