Abstract 3170: DMSO 2.0 - An advanced DMSO product for 2D and 3D cell culture of PROTACs and hydrophobic drugs

Abstract Dimethyl sulfoxide (DMSO) is the most commonly used aprotic solvent for solubilizing poorly water-soluble molecules in cell culture assays. It is an inert, biocompatible solvent with high solubilization capacity. In the last decade, there has been a big parade shift in cancer drug discovery with the development of next-generation cancer therapeutics, especially oncogenic protein-specific small molecules and targeted protein degraders, also known as Proteolysis Targeting Chimera (PROTACs). Such novel drugs are efficiently degrading/inhibiting the key oncoproteins that remained “undruggable” for many years before 2010 e.g., KRAS, PARP, BRAF, etc. Though very efficient, the majority of them fall in beyond Lipinski rule 5 space (bRo5) due to extreme hydrophobicity. Due to poor solubility and rapid precipitation tendency, such molecules pose great challenges during in vitro cell culture and preclinical testing. In our lab, we have observed that stock solution of anticancer molecules (BRD4 PROTAC, EGFR PROTAC, BRD4/MDM2 PROTAC, Sorafenib, Adagrasib, Trametinib, Vemurafenib, Lenvatinib) prepared in DMSO showed immediate to sustained precipitation of drug in DMEM/RPMI media with 5-10% serum. It resulted in significant variation and inaccuracy in cytotoxicity assays. Precipitation of the molecule within the tip (while mixing it from stock solution to media) led to inaccuracy in concentration. Thus, designing and developing an advanced DMSO formulation for testing such novel anticancer molecules is imperative. After screening over 15 hydrophilic adjuvants and their combinations, DMSO 2.0 was developed and tested using the above anticancer molecules. 50 mM stock solution of molecules was prepared in DMSO and DMSO 2.0, followed by addition into cell culture media. Precipitation was monitored by microscopy and high-performance liquid chromatography (HPLC) method. DMSO 2.0 led to the easy mixing of stock solutions with the media by preventing the precipitation of molecules within the tip. Most importantly, DMSO 2.0 led to marked inhibition/slowing down the precipitation of molecules in media compared to DMSO. Precipitation was inhibited at as high as 50 µM and all the subsequent dilutions. DMSO 2.0 showed no apparent cytotoxicity at a concentration below 0.5% v/v in lung cancer cells. Overall, DMSO 2.0 could be a promising solvent for cancer drug discovery, and its drug-specific composition will provide crucial insight into early formulation development strategy for preclinical testing. Citation Format: Ketan Patel. DMSO 2.0 - An advanced DMSO product for 2D and 3D cell culture of PROTACs and hydrophobic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3170.