(T1030-03-18) In Vivo Tissue Distribution of a Model PROTACs and Its Metabolites Following Subcutaneous Administration To Male Rats Via DESI - MS Imaging

Purpose: The aim of this study was to determine the tissue distribution of a model PROTACs and its metabolites following a single dose subcutaneous does to male rats.

Methods: PROTACs-3-gefitinib and  vehicle only control, were subcutaneously dosed to male Wistar rats (n=5) at 0 (vehicle) and 10 mg/kg (drug). Blood samples were collected via tail bleed microsampling at 0, 0.08, 0.25, 0.5, 1, 3, 6, 8 and 24 h post dose. Urine samples were collected at 0, 1-3, 1-8 and 8-24 h post dose. Animals were sacrificed and organs were harvested at 1, 3, 8 and 24 h post dose.   Drug metabolites were identified in plasma and urine using reversed - phase UHPLC connected to a QTof type HRMS  mass spectrometer operated in +ve and -ve ESI mode.  Rat liver samples were frozen at -20 C and sectioned for tissue imaging. The liver tissue slices were investigated for the presence of the PROTACs drug and identified metabolites using DESI-Tandem Quadrupole MS imaging operating +ve ESI mode.

Results: HRMS analysis suggested that the PROTACs molecule was subject to cleavage of the linker, via N-deamination or ester hydrolysis, to produce the VHL ligase and the gefitinib target binding moieties (TBM). Two VHL ligase-linker and two gefitinib-linker metabolites were identified, which differed by the length of the conserved linker chain. The gefitinib-linker cleavage biotransformation’s were further conjugated to two sulphate and two glucuronide metabolites. The urine provided only a minor route of excretion for the unchanged PROTACs molecule which was detected only at low concentrations (50 – 100 ng/mL). Much more significant amounts of the 2 major N-deamination cleavage products (400 ug.equiv over 24 h) as well as sulphate and glucuronide conjugates of the TBM cleavage product (300 µg.equiv. over 24 h) were excreted in urine.  DESI - MS/MS imaging showed the presence of the linker cleavage metabolite and glucuronide metabolite as well as the PROTACs drug 1, 3, 6 and 8 h samples.  The glucuronide metabolite showed the most intense signal, followed by the linker cleavage metabolite.  The PROTACs drug molecule was only visible in the 3 and 6 hr samples.  DESI MS imaging also revealed  that there was an increase in carnitine, and choline concentrations at the 6 and 8 h time period following PROTACs administration.

Conclusion: The drug metabolism and tissue distribution of a PROTACs drug molecule was determined following subcutaneous dosing to male rats.  The major route of metabolism was vis N-dealkylation of the aliphatic linker followed by conjugation via sulphation and glucuronidation of the gefitinib like moiety.  The drug metabolites were cleared from the plasma by 24 h still detectable in the urine samples at the 24 h time point.  DESI MS tissue imaging revealed the presence of the PROTACs drug, cleavage metabolite and glucuronide metabolite in the 1, 3 h liver samples.  There was also a significant increase in carnitine and choline concentration at the 3, 8 and 24 h time points.

Acknowledgements: Nikunj Tanna, Andrew Leightner and Robert Plumb are employees of Waters Corporation.


DESI MS imaging of Carnitine in Rat Liver Samples following dosing at 10 mg/kg SC


DESI MS Imaging of PROTACs metabolite in rat liver samples following dosing at 10 mg/kg SC