Targeting the Gastrin-Releasing Peptide Receptor (GRP-R) in Cancer Therapy: Development of Bombesin-Based Peptide-Drug Conjugates

Abstract

Targeted tumour therapy has proved to be an efficient alternative to overcome the limitations of conventional chemotherapy. Among several receptors upregulated in cancer cells, the gastrin-releasing peptide receptor (GRP-R) has recently emerged as a promising target for cancer imaging, diagnosing and treatment due to its overexpression on cancerous tissues such as breast, prostate, pancreatic and small-cell lung cancer. Herein, we report on the in vitro and in vivo selective delivery of the cytotoxic drug daunorubicin to prostate and breast cancer, by targeting GRP-R. Exploiting many bombesin analogues as homing peptides, including a newly developed peptide, we produced eleven daunorubicin-containing peptide-drug conjugates (PDCs), acting as drug delivery systems to safely reach the tumour environment. Two of our bioconjugates revealed remarkable anti-proliferative activity, an efficient uptake by all three tested human breast and prostate cancer cell lines, high stability in plasma and a prompt release of the drug-containing metabolite by lysosomal enzymes. Moreover, they revealed a safe profile and a consistent reduction of the tumour volume in vivo. In conclusion, we highlight the importance of GRP-R binding PDCs in targeted cancer therapy, with the possibility of further tailoring and optimisation.

Keywords: bombesin; breast cancer; drug delivery systems; gastrin-releasing peptide receptor; peptide–drug conjugates; prostate cancer; targeted tumour therapy.

Figure 1

Synthesis of Dau-BBN (7-14) conjugates and BBN (7-14) analogues as free peptides. (a) Ac₂O, DIPEA, DMF (1:1:3 v/v/v%), 30 min, RT. (b) (1) SPPS; (2) 3 eq >=Aoa-OH, 3 eq HOBt, 9 eq DIC in DMF, 1 h, RT. (c) TFA (95%), 2.5% TIS, 2.5% H₂O, 2 h (G1–G5, FP1, FP2) or 10 mL TFA, 750 mg crystal phenol, 250 µL EDT, 500 µL thioanisole, 250 µL H₂O (L1–L6). (d) Methoxyamine (1 M) in 0.1 M NH₄OAc buffer (pH 5), 1-2 h, RT. (e) Dau (1.3 eq) in 0.1 M NH₄OAc buffer (pH 5), o/n, RT. SPPS: Solid Phase Peptide Synthesis; Ac: acetyl; >=Aoa: isopropylidene aminooxyacetyl; Aoa: aminooxyacetyl; Aib: 2-aminoisobutyric acid; Sta: statine.

Figure 2

Localisation of Dau–BBN (7-14) conjugates in PC-3 cells, visualised by confocal laser scanning microscopy (CLSM). Conjugates were detected by the fluorescence of daunorubicin (red), nuclei were stained with Hoechst 33342 (blue). Scale bar: 10 μm.

Figure 3

Stability profile of the conjugates in lysosomes and mouse plasma. (A) Degradation of L1, L3, L5 and L6 and (B) release of the active metabolite Dau=Aoa-Leu-OH in rat liver lysosomal homogenate (peak area, mean/10¹⁰ ± SD, n = 3). (C) Stability of compounds L1, L5 and L6 in mouse plasma over 24 h (%, mean ± SD, n = 3).

Figure 4

In vivo toxicity and antitumour effect of conjugates L5 and L6. (A) Mouse body weight in chronic toxicity studies (%, mean ± SD, n = 3) after administration of either L5 or L6, in three different concentrations, calculated on Dau content: 5 mg/kg, 10 mg/kg and 20 mg/kg. Four treatments (red arrows), three mice per group. (B) Tumour volume (mm³, mean ± SEM, n = 8). Administration regime: 0.9% saline (control group, black arrows), Dau (1 mg/kg, orange arrows) and PDCs (10 mg/kg calculated on Dau content, black arrows), every 5th day starting from day 9. (C) Calculated tumour doubling time for control, Dau- and PDC-treated groups (days, mean ± SD, n = 8). ns: non-significant difference. **: significant difference at p < 0.01.