The kidney is the main dose-limiting organ in radioligand therapy (RLT), and there is an urgent need for reducing renal radioactivity accumulation. According to the enzymolysis clearance strategy, the first objective of this study is to test whether enzymolysis efficiency can be improved by introducing a hydrophobic amino acid with a bulkier side chain to the second position of the cleavable sequence, and the second objective is to screen an optimal sequence to minimize the renal uptake. Four exendin 4 (Ex4) peptide analogues with different cleavable sequences were synthesized and labeled with ⁶⁸Ga. Both in vitro and in vivo metabolism studies were performed using either the model compounds or the complete probes. The in vitro stabilities of the tracers were evaluated in PBS and mouse serum. The microPET images were acquired in the INS-1 tumor model at different time points, and the radioactivity uptakes of the probes in tumors and kidneys were determined and compared. All the probes were stable in both PBS and mouse serum for at least 1 h. The in vitro cleavage study for both model compounds and intact probes showed enzymolysis efficiency in the following order: MWK > MFK > MVK > MGK. The in vivo metabolism study confirmed that a fragment of ⁶⁸Ga-NOTA-Met-OH appeared in both kidney and urine samples for all analogues with MVK, MFK, and MWK sequences. The microPET images showed that the tumor uptakes of all the modified probes were comparable to those of the control, while the kidney uptakes were significantly reduced by inserting the MWK, MFK, or MVK linker. The tumor-to-kidney ratios at 0.5, 1, and 2 h time points showed the following order: ⁶⁸Ga-NOTA-MWK-Ex4 > ⁶⁸Ga-NOTA-MFK-Ex4 > ⁶⁸Ga-NOTA-MVK-Ex4. In this study, based on the enzymolysis clearance strategy and the preference of the enzyme, different sequences were designed and compared both in vitro and in vivo. The results indicated that the larger the steric hindrance of the second hydrophobic amino acid side chain, the more effective the enzymatic hydrolysis, with enzymolysis efficiency in the following order: MWK > MFK > MVK > MGK. MWK appears to be the most effective sequence in reducing renal radioactivity accumulation of exendin 4 peptide derivatives.

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