Optimization of the pharmacokinetic profile of 99mTc-N4-Bombesin derivatives by modification of the pharmacophoric Gln-Trp sequence

1474 Objectives: In order to optimize the pharmacokinetic profile of 99mTc-labeled Gastrin-releasing peptide receptor (GRPR)-targeted antagonists, we substituted Gln7 or Trp8 within the pharmacophore of the N4 chelator-conjugated, RM2-like peptide MJ9 (N4-Pip5-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) by the unnatural amino acids homoserine (Hse7), citrulline (Cit7), β-(3-benzothienyl) alanine (Bta8) or α-methyl tryptophan (α-Me-Trp8). Results of the comparative preclinical evaluation of N4-[Hse7]MJ9 (1), N4-[Cit7]MJ9 (2), N4-asp-[Bta8]MJ9 (3) and N4-[α-Me-Trp8]MJ9 (4) were compared to the unmodified N4-MJ9 (R1) and N4-asp-MJ9 (R2). Methods: All compounds were synthesized via standard Fmoc-based SPPS. 99mTc-labeling (95 °C, 10 min) was carried out in a NaHPO4/citrate-buffered solution of SnCl2 and ascorbate. GRPR affinity (inverse IC50) was evaluated on PC-3 cells using [3-I-tyr6]MJ9 as cold competitor and the radiolabeled new ligands. Lipophilicity was measured as distribution coefficient in n-octanol/PBS (logD7.4). Metabolite studies in vitro were carried out on murine and human plasma (37 °C, 24 ± 2 h). Biodistribution and μSPECT/CT studies at 1 h p.i. were carried out on PC-3 tumor-bearing CB17-SCID mice. Results: Synthesis of all derivatives yielded 5-18% HPLC-purified labeling precursor. 99mTc-labeling resulted in RCYs of >97%. All peptides showed comparable GRPR affinity (IC50, inverse [nM](sd)): 99mTc-R1: 2.8(0.5), 99mTc-R2: 2.5(0.8), 99mTc-1: 2.6(0.1), 99mTc-2: 3.1(0.1), 99mTc-3: 3.0(1.3) and 99mTc-4: 3.7(0.5). LogD7.4 values (sd) of the 99mTc-N4-Bombesin analogs were distinctly higher than that of the well-known GRPR antagonist 68Ga/177Lu-RM2 (−2.35(0.04)/−2.51(0.02)): 99mTc-R1: −1.55(0.03), 99mTc-R2: −1.86(0.03), 99mTc-1: −1.26(0.02), 99mTc-2: −1.30(0.04), 99mTc-3: −1.33(0.06) and 99mTc-4: −1.29(0.08). Due to decreased metabolic stability, 99mTc-1 and 99mTc-3 exhibited, when compared to 99mTc-R2, lower pancreatic uptake [%ID/g(sd)] at 1 h p.i. (10.0(4.2) and 4.8(1.4) vs 16.0(1.6)). In contrast, due to an enhanced metabolic stability, 99mTc-2 and 99mTc-4 showed increased pancreatic uptake at 1 h p.i. (25.8(2.4) and 39.1(0.3)). Tumor accumulation [%ID/g(sd)] was comparable for 99mTc-2, 99mTc-3 and 99mTc-4 (9.9(0.1), 9.3(1.6) and 9.4(0.5)), but enhanced for 99mTc-R2 and 99mTc-1 at 1 h p.i. (11.7(1.6) and 14.6(2.3)). Due to its decreased metabolic stability and thus accelerated clearance kinetics, μSPECT/CT imaging with 99mTc-3 revealed the most favorable tumor-to-background contrast at 1 h p.i. Conclusion: We could demonstrate that the pharmacokinetic profile of Bombesin antagonists can be optimized via modification of the Gln or Trp moiety within the pharmacophore. Despite its slightly enhanced lipophilicity, the moderate metabolic instability of 99mTc-N4-asp-[Bta8]MJ9 (3) results in a favorable biodistribution pattern at 1 h p.i. and may be well suitable for diagnosis of GRPR-expressing malignancies in men.