Category: Preclinical Development
Purpose: Chemotherapeutic agents are generally hydrophobic agents which results into nonselective distribution to healthy cells and cause toxicities. Ligand-targeted drugs (LTDs) are a promising class of targeted anticancer agents. The targeting ligands in the LTDs are mostly hydrophilic, which reduces its nonselective passive distribution and toxicities to normal healthy cells. Furthermore, the smaller size of these LTDs results in better tumor penetration in solid tumors. However, because of their hydrophilicity and smaller size, the short circulation half-life presents a significant challenge for utilizing their full therapeutic potential. The purpose of our current research is to extend the half-life of the targeted chemotherapeutic agents while maintaining their hydrophilicity and smaller size which will enhance the safety and efficacy profiles.
Methods: We conjugated hydrophobic chemotherapeutic agent MMAE with a targeting moiety (PSMA ligand) and a hydrophilic small molecule (Transthyretin ligand for half-life extension, TLHE) that binds reversibly to the serum protein transthyretin (TTR). The synthesis of both ligands was carried out following conventional synthetic procedures as reported earlier.1 Upon conjugation of both ligands to MMAE, we generated several Tri-Functional Molecules (TFMs). We also synthesized typical Bi-Functional molecules (BFMs) as reference molecules where MMAE was conjugated with the targeting moiety. After synthesis, we tested the binding and selectivity of TFMs to serum TTR using Fluorescence Polarization (FP) and Fluorescence Probe Exclusion (FPE) assays. We also tested the preferential binding of TFMs to PSMA in the presence of TTR. After confirming the preferential binding and selectivity, we tested and compared the cytotoxicity of BFMs and TFMs in prostate cancer cell lines, LNCaP (PSMA+), DU145 (PSMA-) and HeLa (non-PSMA) cell lines. Depending on the results of these in vitro assays, we selected one TFM molecule (TFM3) for in vivo studies. We investigated the pharmacokinetic profile of TFM3 and BFM2 in rats to find out their half-life. We used CD-1 male mice to determine the in vivo toxicity of TFM3 and compared with BMF2 and MMAE. Based on the preliminary toxicity study result, we treated tumor bearing (subcutaneous) male nude mice (nu/nu) with TFM3, BFM2 and MMAE for two weeks and compared their efficacy.
Results: From the in vitro binding and selectivity assays, we found that TFM3 maintained a very good binding affinity and selectivity towards TTR over other serum proteins which resulted into improved half-life in vivo which was evident from the pharmacokinetic profile in rats.1 TFM3 also showed selective cytotoxicity towards PSMA positive cell line, LNCaP. TFM3 had better toxicity profile in CD-1 male mice compared to both BFM2 and MMAE. TFM3 also showed superior in vivo efficacy compared to BFM2 and MMAE in nude mice model of prostate cancer.
Conclusion: We have developed a new platform approach that combines the advantages from both the small hydrophilic LTDs and large macromolecules, which has higher tumor penetration and extended circulation t1/2 respectively. By attaching small and hydrophilic TTR ligands to targeted chemotherapeutic agents, we generated hydrophilic small TFMs (< 3 kDa) which, compared to typical ligand targeted drugs, have enhanced pharmacokinetic and efficacy profiles. The same approach can potentially be useful for enhancing the pharmacokinetic properties and hydrophilicity of various biomolecules without significantly increasing their size.