Approaches to improve metabolic stability of a statine-based GRP receptor antagonist

The bombesin receptor family, in particular the gastrin-releasing peptide receptor (GRPr), is an attractive target in the field of nuclear oncology due to the high density of these receptors on the cell surface of several human tumors. The successful clinical implementation of ⁶⁴Cu-CB-TE2A-AR06, ⁶⁸Ga-RM2 and ⁶⁸Ga-NODAGA-MJ9, prompted us to continue the development of GRPr-antagonists. The aim of the present study was to assess if N-terminal modulations of the statine-based GRPr-antagonist influence the binding affinity, the pharmacokinetic performance and the in vivo metabolic stability.

The GRPr-antagonist (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH₂) was functionalized with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) via the spacer 4-amino-1-carboxymethyl-piperidine (Pip) and the amino acid N-Methyl-β-Ala, to obtain NMe-RM2 and labeled with ⁶⁸Ga and ¹⁷⁷Lu. The GRPr affinity of the corresponding metalloconjugates determined using [¹²⁵I-Tyr⁴]-BN as radioligand. In vitro evaluation included internalization studies using PC3 cells. The ⁶⁸Ga-conjugate was evaluated in PC3 xenografts by biodistribution and PET studies, while investigations on the metabolic stability and plasma protein binding were performed.

The half maximum inhibitory concentrations (IC₅₀) of the metalloconjugates, using [¹²⁵I-Tyr⁴]-BN, are in the low nanomolar range. PC3-cell culture binding studies of both metallated NMe-RM2 and RM2 show high GRPr-bound activity and low internalization. Metabolic studies showed that ⁶⁸Ga-NMe-RM2 and ⁶⁸Ga-RM2 are being cleaved in a similar fashion into three metabolites, with a good proportion of about 50% of the remaining blood activity at 15min post injection (p.i.) being represented by the intact radiotracer. ⁶⁸Ga-NMe-RM2 was shown to target specifically PC3 xenografts, with high and sustained tumor uptake of about 13% IA/g within a time frame of 3h. The PET images clearly visualized the tumor.

The relatively high percentage of the remaining intact radiotracer in blood 15min post injection sufficiently enables in vivo targeting of GRPr positive tumors, finding which has been also shown in clinical trials.