A comprehensive mechanistic investigation of factors affecting intestinal absorption and bioavailability of two PROTACs in rats

Abstract

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).

Highlights

The intestinal effective permeability was moderate for ARV-110 and low for ARV-471 in rats.

Intestinal P-gp contributes to limited intestinal permeability of ARV-110 and ARV-471.

CYP3A seems to play minor role in first-pass metabolism of ARV-110 and ARV-471.

The CRBN-based PROTACs undergo luminal enzyme-mediated degradation pre-absorption.

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 × 10^-4 cm/s) and low for ARV-471 (0.23 × 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.

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2.1. Model compounds and other chemicals

ARV-110 (purity > 98 %) and ARV-471 (purity > 95.4 %) were purchased from Excenen PharmaTech Co., Ltd. (Shanghai, China). Ketoconazole and 4-(2-aminoethyl)-benzene-sulfonyl fluoride (Pefabloc® SC) as well as HPLC grade dimethyl sulfoxide (DMSO), Cremophor EL, D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), carboxymethyl cellulose (CMC), sodium chloride (NaCl) and potassium dihydrogen phosphate (KH₂PO₄) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Polyethylene glycol 400 (PEG400) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Encequidar mesylate was purchased from TargetMol Chemicals Inc. (Boston, MA, USA).

Janis Niessen, Nathalie Arendt, Markus Sjöblom, Ilse R. Dubbelboer, Thomas Borchardt, Mirko Koziolek, Mikael Hedeland, Hans Lennernäs, Anura Indulkar, David Dahlgren, A comprehensive mechanistic investigation of factors affecting intestinal absorption and bioavailability of two PROTACs in rats, European Journal of Pharmaceutics and Biopharmaceutics, Volume 211, 2025, 114719, ISSN 0939-6411, https://doi.org/10.1016/j.ejpb.2025.114719.