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Aim: Proteolysis targeting chimeras (PROTACs) exhibit a unique and promising pharmacology. However, this comes with molecular properties exceeding the 'drug-like' rule of five chemical space, which often limits gastrointestinal absorption. This in vivo study aimed to investigate the contribution of luminal and plasma stability, intestinal effective permeability, P-glycoprotein (P-gp) efflux, and bile excretion, on the rat intestinal absorption and systemic exposure of two PROTACs, ARV-110 (812 Da, LogD_7.4 4.8) and ARV-471 (724 Da, LogD_7.4 4.6).

Methods: Luminal stability and effective intestinal permeability were determined directly from luminal disappearance using single-pass intestinal perfusion, with and without a protease inhibitor, or a P-gp/Cytochrome P450 CYP3A inhibitor (ketoconazole) in rats. Plasma stability was tested by in vitro incubations. Intestinal absorption, systemic exposure, and biliary excretion were examined after intraduodenal and intravenous dosing with ketoconazole or the P-gp selective inhibitor (encequidar).

Results and discussion: Both PROTACs were degraded in the intestinal lumen and in plasma by peptidases. The intestinal effective permeability in rats was moderate for ARV-110 (0.62 x 10^-4 cm/s) and low for ARV-471 (0.23 x 10^-4 cm/s). P-gp inhibition increased the permeability 1.6-and 2.3-fold for ARV-110 and ARV-471, respectively. After intraduodenal dosing with the P-gp inhibitors a corresponding increase in systemic exposure was observed for both PROTACs. There was only a minor difference in the increased systemic exposure induced by the two inhibitors, suggesting that the mechanisms were primarily P-gp inhibition, rather than gut-wall and hepatic extraction. Biliary excretion was a minor pathway and did not affect the absorption and systemic exposure of the PROTACs to a large extent.

Conclusion: In the rat, ARV-110 and ARV-471 were enzymatically degraded in the intestinal lumen and in plasma, and their intestinal permeability and systemic exposure seem to be reduced due to P-gp efflux.