(T1430-06-33) Comparison of the Pharmacokinetics and Metabolic Fate of Gefitinib – 3 – PROTACs and Gefitinib in the Rat Following Subcutaneous Dosing

Purpose: 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 via the cell machinery, and thus represent a new and attractive for of disease treatment. Gefitinib is a tyrosine kinase inhibitor for the treatment of non-small cell lung cancer, it is a potent epidermal growth factor receptor (EGFR) inhibitor which operates by interrupting cell signalling. The pharmacokinetics and in vivo metabolism of gefitinib in the rat and mouse have been previously reported [3,4] and showed that the compound was well absorbed with peak concentrations occurring at 1 h, with an estimated oral bioavailability of 50 – 60%, and a half-life of 3.8 h (2.6 h for the IV route). The aim of this study was to understand the pharmacokinetics and metabolism of the targeted protein degrader gefitinib – 3 – PROTACs in the rat and compare the resulting data with that of the POI alone (gefitinib).

Methods: Gefitinib – 3 – PROTACs and gefitinib were subcutaneously dosed separately at 0 or 8 mg/kg to male Wistar rats (n = 6), the animal study was carried out at Evotec UK under full management and ethical review in accordance with local regulations. The rats were housed in metabowls and blood samples (50 µL) were taken at regular intervals over a 24 h period, urine and faeces were collected over 0 – 24 h. Plasma derived from the blood samples were subject to protein precipitation using organic solvent containing gefitinib d6 as an internal standard (IS). The resulting solutions were analysed using a 2- min gradient reversed – phase +ESI UPLC-MS/MS (Waters Corporation, Milford, MA) using specific MRM transitions for gefitinib – 3 – PROTACs, gefitinib and IS. Metabolite identification was performed using reversed – phase UPLC-HRMS operated in both +ve and -ve ESI mode. The resulting concentration data was interrogated using PKSolver to determine the pharmacokinetics of both molecules. Metabolite identification was performed using MassMetasite (Mass Analytica, Barcelona, Spain).

Results: PROTACs consist of three main components i) target binding moiety, ii) linker and iii) ubiquitin E3 ligase binding moiety.  These heterobifunctional molecules function via the binding of the target moiety to the protein of interest (POI) while E3 ubiquitin ligase is simultaneously bound by the other end of the PROTACs molecule, Figure 1.  Binding of the POI and the ligase causes ubiquitination of the POI which is then degraded by the cellular ubiquitin-proteosome system, during which the PROTACs molecule is regenerated.  These PROTACs can be considered “large small molecules” and as such share many of the attributes of small molecules, such as synthesis scale up, cost of manufacture, shelf life, stability and route of administration. Plasma and urine calibration lines were prepared by the dilution of authentic standard of gefitinib and gefitinib – 3 – PROTACs, in control rat plasma over the range of 10 pg/mL to 1,000 ng/mL.  A bioanalytical LC-MS/MS method was developed for the simultaneous quantification of the PROTACs and POI drug.  LC-MS/MS was performed using a simple, 2- min reversed phase gradient, PROTACs and gefitinib were monitored using the transitions m/z 934.17 à 617.34 & m/z 447.25 à 128.20 respectively.  Under these conditions gefitinib and gefitinib based PROTACs-3 eluted with retention times of t_R = 1.40 and 1.86 min respectively.  The limit of detection in rat plasma was determined to be 20 pg/mL (2.5 fg on column) for both analytes.  The calibration line was determined to be linear over approximately 5 orders of magnitude (20 pg/mL – 1,000 ng/mL) using internal standard quantification and 1/x weighting. The methodology was developed and validated in accordance with the ICH M10 guideline.  Analysis of the LC-MS/MS data showed that a peak plasma concentration of 106 ng/mL was observed for gefitinib with T_max = 8 h, PROTACS – 3- gefitinib achieved a peak plasma concentration of 62 ng/mL with T_max = 6 h, AUCs = 818, 824ng.hr/mL for gefitinib and PROTACs respectively Figure 2.  HRMS analysis showed that gefitinib was extensively metabolized including O-demethylation, oxidative metabolism of the morpholine ring, and oxidative defluorination mediated via CYP3A4, 2D6 and 3A5 some of these oxidative metabolites were further conjugated to form sulfate and glucuronide metabolites

Conclusion: The pharmacokinetics and metabolism of gefitinib and gefitinib – 3 – PROTACs were determined following subcutaneous dosing to male rats.  The PROTACs drug showed a similar pharmacokinetics profile to gefitinib, with peak plasma concentration of 62 ng/mL with occurring 6 h post dose.

Acknowledgements: This study was funded by Waters Corporation and carried out by Evotec UK. A full management and ethical review of the study was performed to ensure that the design conformed to both National and EU guidelines prior to study commencement. Robert Plumb and Andrew Leightner are employees of Waters Corporation, Ian Wilson is a consultant for Waters Corporation.


PROTACs method of action


Concentration over time