Peptide–Drug Conjugate GnRH–Sunitinib Targets Angiogenesis Selectively at the Site of Action to Inhibit Tumor Growth

Abstract

The potential to heighten the efficacy of antiangiogenic agents was explored in this study based on active targeting of tumor cells overexpressing the gonadotropin-releasing hormone receptor (GnRH-R). The rational design pursued focused on five analogues of a clinically established antiangiogenic compound (sunitinib), from which a lead candidate (SAN1) was conjugated to the targeting peptide [D-Lys 6 ]-GnRH, generating SAN1GSC. Conjugation of SAN1 did not disrupt any of its antiangiogenic or cytotoxic properties in GnRH-R-expressing prostate and breast tumor cells. Daily SAN1GSC treatments in mouse xenograft models of castration-resistant prostate cancer resulted in significant tumor growth delay compared with equimolar SAN1 or sunitinib alone. This efficacy correlated with inhibited phosphorylation of AKT and S6, together with reduced Ki-67 and CD31 expression. The superior efficacy of the peptide-drug conjugate was also attributed to the finding that higher amounts of SAN1 were delivered to the tumor site ($4-fold) following dosing of SAN1GSC compared with equimolar amounts of nonconjugated SAN1. Importantly, treatment with SAN1GSC was associated with minimal hematotoxicity and cardiotoxicity based on measurements of the left ventricular systolic function in treated mice. Our results offer preclinical proof-of-concept for SAN1GSC as a novel molecule that selectively reaches the tumor site and downregulates angiogenesis with negligible cardiotoxicity, thus encouraging its further clinical development and evaluation.

Targeting of the breast cancer microenvironment with a potent and linkable oxindole based antiangiogenic small molecule

“…Results and comparative IC 50 values are presented in Figure 2D. SAP was equipotent to sunitinib, but also to our previously generated SAN1 molecule [18] in all cell lines with IC 50 values ranging from 6.5 ± 2.4 μmol/L to 15.8 ± 1.7 μmol/L.…”

“…No signs of discomfort or overt toxicity were observed in any of the treated mice. Interestingly these pharmacokinetic measurements were superior to sunitinib when directly compared with previous results using the same administration route (IP) and an equimolar dose (Cmax for sunitinib was 6.5 ± 0.9 μmol/L at 0.25 h with an AUC 0−24 h of 21.4 ± 3.6 h x μmol/L) [18]. According to the biochemical assays SAP concentrations of approximately 0.1 μmol/L were needed for the inhibition of the target kinases (Figure 2A–2C).…”

“…We had previously encountered a related cardiotoxic incident with another analogue of sunitinib (SAN1) [18] but this effect was alleviated when SAN1 was conjugated to a peptide moiety. It seems reasonable to assume that the same outcome could be achieved with SAP in future conjugation studies of SAP with a targeting moiety.…”

“…Cardiotoxicity was assessed as described previously [18, 50]. For the hematotoxicity experiments approximately 300 μL of blood were collected from each animal in a vacutainer blood collection tube containing EDTA (BD Biosciences).…”

“…We have recently demonstrated that conjugation of a rationally designed analogue of sunitinib (called SAN1) to a decapeptide moiety (generating SAN1GSC) alleviated such toxicity possibly due to the ability of SAN1GSC to selectively deliver the pharmacophore to the tumor microenvironment, as shown in a castration resistant prostate cancer animal model [18]. SAN1 was also active in BrCa cell lines but its development encountered various hurdles, namely a low synthetic yield (71%), and the limited conjugation options that the free hydroxyl group of SAN1 allowed for.…”