DMPK of Gefitinib & Gefitinib-3-PROTAC Following Subcutaneous Administration to the Rat Using Micro Sampling and UPLC-MS/MS

Proteolysis Targeting Chimeras (PROTACs) are a new class of drug molecules which work by mobilizing the ubiquitin–proteasome system to achieve proteasome-mediated degradation of the target protein. PROTACs consist of three components i) target binding moiety, ii) linker and iii) ubiquitin E3 ligase binding moiety. PROTACs eliminate all functions of the protein, providing differentiated pharmacology and do not require binding moieties that inhibit protein function. They also significantly increase the number of “druggable” proteins opening up the possibility for new safer medicines. PROTACs are “large small molecules” >800g/mol and understanding their DMPK properties presents a new challenge in. Here we report UHPLC-MS/MS to determine the pharmacokinetics and metabolic biotransformation of gefitinib and gefitinib-PROTACs 3 in male Wistar rats. Male Wistar rats were dosed subcutaneously with either gefitinib, gefitinib-PROTACs 3 or drug free vehicle at 10 mg/kg. Blood was collected via at regular intervals the following 24 hours. Derived plasma (10 µL) was subjected to protein precipitation with acetonitrile (containing d6 gefitinib as internal standard, 50ng/mL), vortex mixed and centrifuged prior to analysis. Gefitinib and gefitinib-PROTACs-3 pharmacokinetics was obtained via quantitative analysis using a 2 min reversed - phase UHPLC-MS/MS method coupled to a tandem quadrupole mass spectrometer operating in positive ion MRM mode. Metabolite identification was performed on a 10 min reversed – phase UHPLC coupled to a Multi Reflecting Time-of-Flight mass spectrometer operating in positive mode.