Development of High Affinity Camptothecin-Bombesin Conjugates That Have Targeted Cytotoxicity for Bombesin Receptor-containing Tumor Cells

Terry W Moody,Samuel A Mantey,Tapas K Pradhan,Michael Schumann,Tomoo Nakagawa,Alfredo Martinez,Joseph Fuselier,David H Coy,Robert T Jensen

doi:10.1074/jbc.m401938200

Abstract

Mammalian bombesin (BN) receptors are among those most frequently overexpressed by a number of common tumors including prostate, breast, lung, and colon cancers. The aim of this study was to develop a camptothecin-bombesin (CPT-BN) conjugate that interacts with all classes of BN receptors and possibly functions as a prodrug via a labile linker with site-specific cytotoxicity for cancer cells bearing these receptors. CPT was coupled to analogs of [D-Tyr6,beta-Ala11,Phe13,Nle14]BN-(6-14) (BA0) using carbamate linkers (L1 and L2) with built-in nucleophile-assisted releasing groups for intracellular cleavage of free cytotoxic agents. One conjugate, CPT-L2-BA3, bound to all three BN receptor classes with high affinity and functioned as a full agonist at each. 125I-CPT-L2-BA3 was rapidly internalized by cells expressing each BN receptor class and, using fluorescent imaging, was found to co-localize with BN receptors initially and later to be internalized in cytoplasmic compartments. HPLC analysis of internalized ligand showed that 40% was intact, 25% was metabolized by releasing free CPT, and 35% was metabolized to other breakdown products. CPT-L2-BA3 inhibited the growth of NCI-H1299 non-small cell lung cancer cells in 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) and clonal growth assays. CPT-L2-BA3 was cytotoxic in an MTT assay for cells transfected with each class of BN receptor; however, it had significantly less effect in cells lacking BN receptors. These results indicate that CTP-L2-BA3 is a potent agonist that is cytotoxic for cells overexpressing any of the three BN receptor classes and functions as a prodrug for receptor-mediated cytoxicity. It therefore should be a useful prototype to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism.

Highlights

In order to enhance tumor cytotoxicity and decrease side effects, there has been increased interest in the development of prodrugs that improve site-specific delivery of cytotoxic anticancer agents [1, 2]
CPT-L2-BA3 Binds with High Affinity to hGRPR, hNMBR, and hBRS3 and Has Biological Activity—In developing an agonist that could be coupled to cytotoxic compounds and still bind with high affinity to human BN receptors, we started with the standard BN analog, BA0 (Fig. 1, top)
A new carbamate linker system was utilized, which consisted of a series of built-in nucleophileassisted releasing (BINAR) groups, which allows more facile intracellular cleavage of free cytotoxic agents containing reactive OH groups [3]

Summary

Introduction

In order to enhance tumor cytotoxicity and decrease side effects, there has been increased interest in the development of prodrugs that improve site-specific delivery of cytotoxic anticancer agents [1, 2]. A synthetic analog of BN has been described, [D-Tyr6,␤-Ala11,Phe13,Nle14]BN-(6 –14) (BA0), which functions as a universal ligand for all three mammalian BN receptors [11, 12] This analog binds with high affinity to each of the three BN receptor classes and is rapidly internalized by each class of BN receptors [11, 12]. Recently, a new novel carbamate linker system [3] has been described that allows the conjugation of a peptide to either the topoisomerase I inhibitor, camptothecin (CPT), or the tubulin-binding agent, combretastatin This novel linker system [3] contains a built-in nucleophileassisted releasing (BINAR) group that enables fine timing of intracellular cleavage rates of free cytotoxic agents containing reactive hydroxyl groups such as CPT or combretastatin [3]

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Results

Conclusion