Reducing kidney uptake of radiolabelled exendin-4 using variants of the renally cleavable linker MVK

Background

Peptidic radiotracers are preferentially excreted through the kidneys, which often results in high persistent renal retention of radioactivity, limiting or even preventing therapeutic clinical translation of these radiotracers. Exendin-4, which targets the glucagon-like-peptide 1 receptor (GLP-1R) overexpressed in insulinomas and in congenital hyperinsulinism, is an example thereof. The use of the tripeptide MVK, which is readily cleaved between methionine and valine by neprilysin at the renal brush border membrane, already showed promising results in reducing kidney uptake as reported in the literature. Based on our previous findings we were interested how linker variants with multiple copies of the MV-motive influence renal washout of radiolabelled exendin-4.

Results

Three exendin-4 derivatives, carrying either one MVK, a MV-MVK or a MVK-MVK linker were synthesized and compared to a reference compound lacking a cleavable linker. In vivo results of a biodistribution in GLP-1R overexpressing tumour bearing mice at 24 h post-injection demonstrated a significant reduction (at least 57%) of renal retention of all ¹¹¹In-labeled exendin-4 compounds equipped with a cleavable linker compared to the reference compound. While the insertion of the single linker MVK led to a reduction in kidney uptake of 70%, the dual approach with the linker MV-MVK slightly, but not significantly enhanced this effect, with 77% reduction in kidney uptake compared to the reference. In vitro IC₅₀ and cell uptake studies were conducted and demonstrated that though the cleavable linkers negatively influenced the affinity towards the GLP-1R, cell uptake remained largely unaffected, except for the MV-MVK cleavable linker conjugate, which displayed lower cell uptake than the other compounds. Importantly, the tumour uptake in the biodistribution study was not significantly affected with 2.9, 2.5, 3.2 and 1.5% iA/g for radiolabelled Ex4, MVK-Ex4, MV-MVK-Ex4 and MVK-MVK-Ex4, respectively.

Conclusion

Cleavable linkers are highly efficient in reducing the radioactivity burden in the kidney. Though the dual linker approach using the instillation of MV-MVK or MVK-MVK between exendin-4 and the radiometal chelator did not significantly outperform the single cleavable linker MVK, further structural optimization or the combination of different cleavable linkers could be a stepping stone in reducing radiation-induced nephrotoxicity.