Abstract
Most human tumors overexpress or ectopically express peptide hormone/neurotransmitter receptors, which are being increasingly studied as a means to selectively deliver cytotoxic agents. Although a number of peptide ligand-constructs demonstrate tumor cytotoxicity, the role of specific tumoral receptor interaction in its mediation is unclear. To address this question, we synthesized camptothecin (CPT) bombesin (Bn) analogs, in which CPT was coupled via a novel carbamate linker, L2 [N-(N-methyl-amino-ethyl)-glycine carbamate], that were chemically similar but differed markedly in their potency/affinity for human Bn receptors. We then examined their ability to interact with Bn receptors and cause in vitro and in vivo tumor cytotoxicity. CPT-L2-[d-Tyr6,β-Ala11,d-Phe13,Nle14] Bn (6-14) (BA3) bound with high affinity and had high potency for all three human Bn receptor subtypes, whereas CPT-L2-[d-Tyr6,β-Ala11, d-Phe13,Nle14] Bn (6-14) [d-Phe-CPT-L2-BA3] had >1400-fold lower affinity/potency. 125I-CPT-L2-BA3 but not 125I-d-Phe-CPT-L2-BA3 was internalized by Bn receptor subtype-containing cells. CPT-L2-BA3 displayed significantly more cytotoxicity than d-Phe-CPT-L2-BA3 toward NCI-H1299 lung cancer cells in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide and clonogenic assays and more potently inhibited H1299 xenograft growth in nude mice. CPT-L2-BA3 was also metabolically more stable than its parent peptide and inhibited growth of a number of other tumor cell lines in vitro and in vivo. These results demonstrate that specific tumoral receptor interaction is important in mediating the ability of peptide ligand-cytotoxic constructs to cause cytotoxicity. Because many tumors overexpress Bn receptors, these results also demonstrate that CPT-L2-BA3 will be a useful agent for delivering receptor-mediated cytotoxicity to many different human tumors.
Footnotes
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This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute Center for Cancer Research, National Institute of Diabetes and Digestive and Kidney Diseases, and by the Tulane University Peptide Research Fund.
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doi:10.1124/jpet.106.104141.
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ABBREVIATIONS: Bn, bombesin; GRPR, gastrin-releasing peptide receptor; NMBR, neuromedin B receptor; BRS3, bombesin receptor subtype 3; L2, N-(N-methyl-amino-ethyl)-glycine carbamate; CPT, camptothecin; BA3, [d-Tyr6,β-Ala11,Phe13,Nle14] Bn (6-14); DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; hBRS3, human BRS3; hNMBR, human NMBR; GRP, gastrin-releasing peptide; hGRPR, human GRP receptor; IP, inositol phosphate; SIT medium, RPMI 1640 containing 3 × 10-8 M sodium selenite, 5 μg/ml bovine insulin and 10 μg/ml transferrin; RIA, radioimmunoassay; s.c., subcutaneous; i.p., intraperitoneal.
- Received March 10, 2006.
- Accepted June 8, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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