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

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,β-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. 125ICPT-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. 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,β-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. 125ICPT-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. 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 anti-cancer agents (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 2Dubowchik G.M. Walker M.A. Pharmacol. Ther. 1999; 83: 67-123Crossref PubMed Scopus (257) Google Scholar). Prodrug conjugates have been described utilizing antibodies directed against specific tumor-associated antigens, hydrophilic polymers, and peptide or steroid hormones that interact with receptors overexpressed or ectopically expressed on the tumor (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 2Dubowchik G.M. Walker M.A. Pharmacol. Ther. 1999; 83: 67-123Crossref PubMed Scopus (257) Google Scholar). The goal of the prodrug is to deliver the therapeutic agent to the target cell, at which time a tumor-specific process (enzyme activity, specific cellular degradation, etc.) will site-specifically release the active drug (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar). For peptide hormone receptor prodrugs, this requires coupling of the cytotoxic agent to a peptide hormone through a coupling mechanism that retains high affinity for the peptide hormone receptor and allows the cytotoxic drug to be released after receptor specific internalization and degradation (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar, 3Fuselier J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar, 4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar). Peptide receptor ligands as vehicles to deliver cytotoxic agents are receiving considerable attention, because numerous studies using imaging methods or autoradiographic methods have shown many common malignant tumors either ectopically expressing large numbers of peptide receptors (4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar, 5Reubi J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar, 6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, 9Hoffman T.J. Quinn T.P. Volkert W.A. Nucl. Med. Biol. 2001; 28: 527-539Crossref PubMed Scopus (56) Google Scholar) or overexpressing them, thus allowing enhanced uptake of selective ligands for these receptors (6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar). The mammalian bombesin (BN) 1The abbreviations used are: BN, bombesin; CPT, camptothecin; GRP, gastrin-releasing peptide; BRS3, bombesin receptor subtype 3; hBRS3, human BRS3; GRPR, gastrin-releasing peptide receptor; hGRPR, human GRPR; NMBR, neuromedin B receptor; human neuromedin non-small cell lung built-in nucleophile-assisted high abbreviations used are: BN, bombesin; CPT, camptothecin; GRP, gastrin-releasing peptide; BRS3, bombesin receptor subtype 3; hBRS3, human BRS3; GRPR, gastrin-releasing peptide receptor; hGRPR, human GRPR; NMBR, neuromedin B receptor; human neuromedin non-small cell lung built-in nucleophile-assisted high of receptors peptide receptor neuromedin B receptor and the bombesin receptor subtype are for possibly cytotoxic agents to malignant many common tumors frequently and these the BN of receptors is of the receptor most frequently expressed by and the for these receptors as growth J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, D. J. Curr. 2003; PubMed Scopus Google Scholar). BN receptors have been on of lung malignant colon and cancer J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar). a of BN has been [d-Tyr6,β-Ala11,Phe13,Nle14]BN-(6-14) which functions as a ligand for all three mammalian BN receptors M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar). with high affinity to each of the three BN receptor classes and is rapidly internalized by each class of BN receptors M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar). is an because tumors BN receptor classes J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google and this ligand interact with high affinity with each. a carbamate linker J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar) has been described that allows the of a peptide to either the or the linker J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar) a built-in nucleophile-assisted releasing that of intracellular cleavage of free cytotoxic agents groups as CPT or J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). In the have a number of CPT analogs coupled to bombesin through carbamate linkers (L1 and L2) to a BN receptor cytotoxic have CPT-L2-BA3, which high affinity for all three mammalian BN is active at each and is rapidly internalized by each receptor CPT-L2-BA3 is cytotoxic for NCI-H1299 non-small cell lung cancer which receptors M. J. 24: PubMed Scopus Google Scholar) and for cells receptors those lacking these cells and the cell lung NCI-H1299 and cells and and and BN, GRP, and antibodies and and The mammalian and and The was and NCI-H1299 cells in NCI-H1299 cells with cells cells expressing human receptors human receptors or human receptors as described M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Pharmacol. Google Scholar, Coy D.H. Pharmacol. Google Scholar) in cell with cells in with and The cells NCI-H1299 and cells and used in the growth after at in of peptide of the conjugates by methods using the J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). on and was for the of for the of and for the groups of and was coupled to the of the which was with a of CPT was at by a in with and a of in by of and in of the was to the in and to and of the the conjugate was using the by of the and and of the peptide with to by HPLC and by and For the shown in had a of and was found to have a of had a of and was found to have a of had a of and was found to have a of of had an of and CPT-L2-BA3 had a of and was found to have a of of and with specific of as described M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar) or as described of in was to a and with of for and which this of of the of peptide in of and of and for at and The was with of For and which have of was and for at The using a and by high on a The with the and with and with at of to was as described M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar, Pharmacol. Google Scholar). The and cells expressing or NCI-H1299 cells with ligand at for and through of in at for using a The with and for in a The was the of with cells in and was of in all the using a and the Pharmacol. PubMed Scopus Google Scholar). cells transfected with hGRPR, or with as as described Pharmacol. Google Scholar, Coy D.H. Pharmacol. Google Scholar). after the to a with of and to the the cells the was and the cells with In all in the of ligand to in are expressed as the of that is or internalized The internalization of conjugates was in human NCI-H1299 cells that M. J. 24: PubMed Scopus Google Scholar). was with NCI-H1299 cells for at of cells three in receptor to free One was with and for at to peptide bound to the cell The was in a of cells was with to at of cells at for and three in receptor to free One was with and for at to peptide bound to the cell The was in a of cells was with to at of cells with transfected bombesin receptors and cells transfected with at for with in and of with with at and to ligand for at with and as described cells in of with and the was to the of the was on an HPLC with a and with a at in at and at and in each was by in a of in as described Pharmacol. Google Scholar, Coy D.H. Pharmacol. Google Scholar). or cells in and for at in a The cells with in growth with for an the by for at with of The was and with of assay and with or any of the of at the by the of of in was by as described Pharmacol. Google Scholar, Coy D.H. Pharmacol. Google Scholar). with of to free and with of to of to the to was with and for in a of the conjugates to was as described S. J. Life Sci. PubMed Scopus Google Scholar). NCI-H1299 cells and cells and with at for The which at for and at the in and with the cells in a with a mechanism and The was at of and using an of to and after the of studies using the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) studies in using MTT assays. NCI-H1299 or cells in and of CPT-L2-BA3 of MTT was and after of was the at was using an The the with of CPT-L2-BA3 using the cells as of on the growth of NCI-H1299 and cells using a assay S. J. J. A. Coy D. Cancer Res. Google Scholar). The of of in in The of of in and NCI-H1299 after of was and after at the for The number of in using an analysis of of the used was to that described Pharmacol. Google Scholar, Pharmacol. Google Scholar). the was the and of the using the the analysis of the was using an of cells on at a of the cells transfected using of and of the One after the cells and on in at a of cells with and with or the CPT-L2-BA3 in for at The cells three in to the The cells bound CPT-L2-BA3 and in with a as and a as The was using a as and a as the after using and on with was using a fluorescent CPT-L2-BA3 with to hGRPR, and and an agonist that be coupled to cytotoxic and with high affinity to human BN with the BN studies by M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar) and J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar) have that this BN ligand is a high affinity agonist for all human BN receptor as as the BN a carbamate linker was which of a of which allows intracellular cleavage of free cytotoxic agents groups J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). linker coupled to CPT or allows release of the free cytotoxic agent J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). In the coupled analogs of the BN receptor to the which found to have a useful and the cytotoxicity with coupled to analogs J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar) These of an linker and linker of these linkers was coupled to the in of CPT and the of analogs of the BN receptor agonist in which in bound with high affinity to cells transfected and with of and of the was for the to interact with the three human BN receptor hGRPR, and For each of these three human BN receptor CTP-L2-BA3 the high affinity with the for all three human BN receptor In CPT-L2-BA3 had a affinity for human of a affinity for the human of and an affinity for the human cells of In and had for and for the and and for The high affinity to for the and showed a affinity for of high affinity for all three BN receptor CPT-L2-BA3 as the conjugate for full in this and of BN analogs and conjugates for human BN receptor for for in a In the effect on receptor affinity of the of CPT with the effect of the of the linker to the of studies the of the and CPT-L2-BA3 to to cells hGRPR, and was For all three BN receptor CPT-L2-BA3 was the most potent in the of the analogs of each of these because with of a the the J. Google Scholar) of the three and was on the and affinity for the was as as for and of the specific to the and the specific of and all inhibited in a of CPT-L2-BA3 and to that with the results with and to and cells each of these human BN receptor of CPT-L2-BA3 and These studies that each of these to each of the human BN receptors and with the affinity for each human BN receptor The of and CTP-L2-BA3 was using cells transfected with BN receptors and with of the three ligands and in in a BN had a and in at the hGRPR, and hBRS3, BN in the for each of the BN receptor with the hGRPR, of CPT-L2-BA3, and at and with the cells and cells the to These results that CPT-L2-BA3 had and for each of the three human BN receptor to either the or the BN linker CPT-L2-BA3 of each of the three BN analogs to be internalized by each of the three human BN receptor was each of the three rapidly internalized was internalized rapidly by and internalization internalization using cells and rapidly internalized with of the ligand internalized after For internalization at with of the bound ligand internalized and internalization by cells or These results indicate that is internalized by cells BN receptors at of the and the internalization of or by human BN with with with with with with in a for the internalization of CPT-L2-BA3 by used cells transfected with an the receptor was to the with agonist after CPT-L2-BA3 was the receptors and to the and frequently of the receptors however, had internalized after B and a with CPT-L2-BA3, most of the receptors internalized the had internalized intracellular however, there was with These results that after the receptor and conjugate have These results that the BN conjugate CPT-L2-BA3 is internalized by a process and rapidly in intracellular after the internalized the of the internalized cells and cells transfected with with for and the ligand was by The internalized was and and by HPLC the 40% of the internalized in the as 25% as and 35% as In with the cells internalization or of the ligand These results that CPT-L2-BA3 is internalized and metabolized by releasing CPT and as as other the of is for internalization and of CPT-L2-BA3 of in NCI-H1299 bound with high affinity to NCI-H1299 cells and was internalized CPT-L2-BA3 had a affinity for receptors on these cells CPT-L2-BA3 a in in NCI-H1299 cells a specific receptor S. J. Life Sci. PubMed Scopus Google had effect on inhibited the in by or CPT-L2-BA3 The of CPT-L2-BA3, however, the by the receptor results using or cells this with high affinity to or M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, S. J. Life Sci. PubMed Scopus Google this that CPT-L2-BA3 is an in by of on NCI-H1299 CPT-L2-BA3 had effect on the of to NCI-H1299 cells increased CPT-L2-BA3 an in of that with of CPT-L2-BA3 a in and the of had effect CPT-L2-BA3 of CPT-L2-BA3 to cellular was in the MTT CPT-L2-BA3 a of by NCI-H1299 cells and cells with to CPT-L2-BA3 had a growth effect in NCI-H1299 cells of the was and for and cells human BN the was and for cells transfected with or The results indicate that CPT-L2-BA3 was potent at the of cells BN receptors cells lacking BN receptors. a CPT-L2-BA3 inhibited cellular of NCI-H1299 cells in a NCI-H1299 in the of conjugates The in number and using or CPT-L2-BA3 or and using CPT-L2-BA3 For the NCI-H1299 the for CPT-L2-BA3 to was In CPT-L2-BA3 the of NCI-H1299 cells on and as as These results indicate that CPT-L2-BA3 was cytotoxic for NCI-H1299 using analogs (6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google that the or of peptide hormone receptors by tumors (4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar) be to cytotoxicity (6Kwekkeboom D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar). is using peptide receptor ligands coupled to agents for a number of peptide hormone receptors frequently on common malignant tumors prostate, breast, lung and (4Langer M. Beck-Sickinger A.G. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 71-93Crossref PubMed Scopus (109) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, A.V. Nagy A. J. 1999; PubMed Scopus Google Scholar, N. S. PubMed Scopus Google Scholar). cytotoxic agent using bombesin receptors as a target be useful for a number of BN receptor on all J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar, J. Clin. PubMed Scopus Google of J. Clin. PubMed Scopus Google Scholar, A.V. Nagy A. M. A. 2001; PubMed Scopus Google of cell or non-small cell lung J. Clin. PubMed Scopus Google Scholar, N. A.G. Pharmacol. Ther. 1999; PubMed Scopus Google of S. Cancer Res. Google of of malignant A.V. Nagy A. M. A. 2001; PubMed Scopus Google of cancer A.V. 2000; PubMed Scopus Google of cell of the and J. Cancer 2002; PubMed Scopus Google and of colon cancer J. Clin. PubMed Scopus Google Scholar, S. J. PubMed Scopus Google Scholar). which as for each of the three BN receptor are rapidly internalized Coy D.H. Pharmacol. Google Scholar, S. Coy D.H. Pharmacol. Scholar, J. 28: PubMed Scopus Google Scholar, Google Scholar). studies using analogs of that these be used to target BN and these analogs are internalized in high by these tumors T.J. Quinn T.P. Volkert W.A. Nucl. Med. Biol. 2001; 28: 527-539Crossref PubMed Scopus (56) Google Scholar). develop a bombesin that be cytotoxic to tumor cells overexpressing mammalian BN to be the BN cytotoxic conjugate should with high affinity to hGRPR, and is because tumors classes of BN receptors in J.C. Wenger S. Schumuckli-Maurer J. Schaer J.C. Gugger M. Clin. Cancer Res. 2002; 8: 1139-1146PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar). was by using the bombesin D. Krenning E.P. deJong M. J. Nucl. Med. 2000; 41: 1704-1713PubMed Google Scholar, 7Reubi J.C. Endocr. Rev. 2003; 24: 389-427Crossref PubMed Scopus (988) Google Scholar, 8Schally A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, 9Hoffman T.J. Quinn T.P. Volkert W.A. Nucl. Med. Biol. 2001; 28: 527-539Crossref PubMed Scopus (56) Google Scholar, D. J. Curr. 2003; PubMed Scopus Google Scholar, M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar, M. J. 24: PubMed Scopus Google Scholar, Pharmacol. Google which with high affinity for each of the three classes of mammalian bombesin receptors M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google Scholar). a coupling to be used that be by intracellular to release the cytotoxic agent the tumor cell (1deGroot F.M. Damen E.W. Scheeren H.W. Curr. Med. Chem. 2001; 8: 1093-1122Crossref PubMed Scopus (116) Google Scholar). carbamate linkers J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google which groups that coupling to agents that a used to analogs to or CPT, was found to high affinity for be in and have tumor cytotoxicity at the of cytotoxic agent J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). In a study J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google groups had on receptor affinity or peptide carbamate an linker and an CPT conjugate, in with BN linkers and the at the of had a effect on affinity to BN receptors. of linker and peptide conjugate coupled to high affinity for all three BN and this was in the number of results the that the bombesin has the to be an for site-specific delivery of agents to cells mammalian bombesin receptors. These the The bombesin functions as a high affinity agonist at each class of mammalian bombesin receptor and is rapidly internalized by each receptor and the of has on any of these bombesin had high affinity to the M. Coy D.H. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. J. Pharmacol. PubMed Scopus Google for each of the three classes of mammalian bombesin receptors. of each of the mammalian bombesin receptors results in Coy D.H. Pharmacol. Google Scholar, Pharmacol. Google Scholar, S. Pharmacol. Google Scholar, Med. Res. Rev. PubMed Scopus Google and this was this had an or for each receptor and of as as to the analogs of the bombesin and the to that was internalized by cells each of the three mammalian bombesin and the internalization and of internalization for the bombesin the of to the linker bombesin decrease the receptor in in a in affinity for a for the and an in affinity for the the for this increased affinity is and of the found to be for high affinity by M. J. Biol. Chem. PubMed Scopus Google Scholar, Coy D.H. Pharmacol. 2002; PubMed Scopus Google and possibly CPT-L2-BA3 interacts with affinity with these or to the increased by the of CPT to the the had and to the for and, functioned as a full The coupling of to the bombesin with internalization by the three mammalian bombesin in significantly increased the internalized by for the receptor and for the studies that are rapidly internalized and that the BN is initially in and to in the it is Pharmacol. Google Scholar, Google Scholar, J. Biol. Chem. PubMed Scopus Google Scholar, J. Biol. Chem. PubMed Google Scholar). results using fluorescent are with the that CPT-L2-BA3 is in a by the found that the CPT-L2-BA3 on the cell with the was internalized in the These results indicate that CPT-L2-BA3 is internalized as a of receptor ligands are by and A. Pharmacol. PubMed Scopus Google Scholar, J. PubMed Scopus (29) Google Scholar). in released the and metabolized by to release intracellular HPLC results of internalized by cells after with and of ligand this These results that CPT-L2-BA3 was and it was metabolized to by releasing free internalization was on the of on the cells and thus that CPT-L2-BA3 was as a prodrug by the cytotoxic conjugate to the cytotoxic agent be released by intracellular to in analogs of activity, and, study the of the intracellular of free are to be metabolized by which is in cancer cells Lett. 2000; PubMed Scopus Google Scholar). is to to cytotoxicity J.A. Sun L. Woltering S.N. Murphy W.A. Vasilevich N. Coy D.H. Bioorg. Med. Chem. Lett. 2003; 13: 799-803Crossref PubMed Scopus (29) Google Scholar). is to be in numerous human tumors including breast, lung, and as as to be by human tumors S. 1999; Google Scholar). number of results and those in the the that the CPT-L2-BA3, will be a useful prototype agent for the of site-specific delivery of this agent to cytotoxicity in tumors overexpressing mammalian bombesin receptors. CPT-L2-BA3 bound with high affinity and was rapidly internalized by on NCI-H1299 the CPT-L2-BA3 was internalized and metabolized releasing free CPT-L2-BA3 was active in NCI-H1299 cells in that it and was by specific using the MTT and growth CPT-L2-BA3 inhibited the growth of NCI-H1299 In a bombesin coupled to CPT had effect on in cells that transfected with any of the three classes of mammalian bombesin the CPT-L2-BA3 cytotoxicity in the cells bombesin the of the bombesin receptor to enhanced a number of of CPT-L2-BA3 and results of in the in that it should have an CPT-L2-BA3 is hydrophilic allowing and should be to on bombesin for overexpressing bombesin receptors should target the allowing with is by results with analogs coupled to peptide delivery A.V. Nagy A. Life Sci. 2003; 72: 2305-2320Crossref PubMed Scopus (122) Google Scholar, A.V. Nagy A. J. 1999; PubMed Scopus Google Scholar). In results that CPT-L2-BA3 with high affinity to all mammalian BN receptor functions as a potent and receptor agonist at each receptor is is metabolized to and is cytotoxic for tumor cells and cells transfected with human BN receptors. should be useful to explore the effectiveness of tumor-specific cytotoxicity delivery using a receptor-mediated mechanism. for