Synthesis and characterization of Bombesin derivatives with potential applications as nuclear medicine imaging/therapeutic agents [abstract]

Abstract only availableFaculty Mentor: Charles J. Smith, RadiologyThe use of tissue specific radiopharmaceuticals presents great promise for diagnostic and therapeutic applications in a number of human cancers. Gastrin-releasing peptide (GRP) receptors are known to be over-expressed on a variety of malignancies including breast, gastric, colon, pancreatic, prostate, and small-cell lung cancers. Experimental work with bombesin (BBN), an amphibian analogue to mammalian GRP, has demonstrated the ability of BBN to bind, with high affinity and specificity, to the GRP receptor. The use of a bifunctional chelating agent (BFCA) allows biologically active molecules to maintain receptor affinity while at the same time complexing a radionuclide. Spacer groups separating the BFCA and biological vector allow for fine tuning of the pharmacokinetics. Bombesin conjugates effectively complexing radioactive copper (i.e. Cu-64; t1/2=12.7h, Eγ=1345.8keV, Eβ-=578keV, Eβ+=651keV) posses potential as imaging agents for diagnostic positron emission tomography (PET) and therapeutic applications.Recently our research group has focused efforts on developing new BBN derivatives of Triaza (1,4,7-triazacyclononane) or functionalized derivatives of Triaza for complexing of specific radionuclides. Our laboratory has previously reported on a series of new Triaza-BBN conjugates. A derivative of this Triaza ligand system, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), has been synthesized by alkylation of the secondary amines of triaza by α-chloroacetic acid prior to conjugation to the biologically active BBN targeting vector. Bombesin conjugates were derived of the form, NOTA-X-BBN (X = ßAla, GGG, SSS). Synthesis of the unligated BBN [7-14] peptide with spacer group was conducted by Fmoc-protected solid-phase peptide synthesis (SPPS). The bombesin constructs were purified prior to conjugation of the ligand framework by means of reverse phase-high performance liquid chromatography (RP-HPLC). The NOTA ligand was conjugated to the N-terminus of the peptide by means of an activated ester derived from N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl in aqueous solution. The final NOTA-X-BBN derivatives were purified by RP-HPLC and confirmed via electrospray ionization-mass spectrometry (ESI-MS). Further studies to evaluate the ability of the derivatives to complex Cu-64 are underway. Subsequent studies will be conducted to evaluate the in vitro and in vivo characteristics of the radiopharmaceutical

Evaluation of a new series of copper-64-nota-bombesin targeted radiopharmecuticals with PET imaging potential [abstract]

Abstract only availableTissue-specific radiopharmaceuticals having promise as diagnostic and therapeutic targeting vectors for human cancers is of significant interest. In recent years, our group and many others have focused upon design and development of targeting vectors having high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRP receptors are known to be over-expressed on a variety of malignancies including breast, lung, pancreatic, and prostate cancers. Bombesin (BBN), an amphibian analogue of mammalian GRP, has demonstrated the ability to bind with high affinity and specificity to the GRP receptor. In this study, we report the synthesis of the bifunctional chelating agent (BFCA) 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and conjugation to the biologically active BBN targeting vector. Bombesin conjugates were derived of the form, NOTA-X-BBN (X = GGG, SSS, ßAla, 5Ava, and 8Aoc). The unligated BBN [7-14] peptides with spacer groups were synthesized by solid-phase peptide synthesis (SPPS) and purified by reverse phase-high performance liquid chromatography (RP-HPLC). NOTA chelator was conjugated to the N-terminal primary amine of BBN manually. The final NOTA-X-BBN derivatives were purified by RP-HPLC and their chemical constitution verified. [64Cu-NOTA-X-BBN]-conjugates were prepared by addition of 64CuCl2 to a buffered solution containing the conjugate followed by purification via RP-HPLC. In vivo studies of [64Cu-NOTA-8-Aoc-BBN]-conjugates in normal CF-1 mice showed receptor-specific uptake in normal pancreatic tissue, an organ known to express the GRPr in very high numbers. Furthermore, studies in human-prostate tumor-bearing mice have demonstrated the ability of [64Cu-NOTA-8-Aoc-BBN] to undergo receptor-specific tumor uptake. Diagnostic positron emission tomography (PET) imaging using this conjugate was successful in resolving xenografted tumors in a mouse model. Both normal and tumor-bearing mice exhibited low liver accumulation of Cu-64, a known complication of 64Cu-containing radiopharmaceuticals. Further studies are needed to verify the efficacy of [64Cu-NOTA-8-Aoc-BBN] to target other human malignancies.Life Sciences Undergraduate Research Opportunity Progra

In vitro/in vivo assessment of novel 99mTc-bombesin conjugates in human cancer tissue [abstract]

Abstract only availableReceptor-specific, radiolabeled peptides are becoming increasingly popular as targeting vectors for the development of new diagnostic radiopharmaceuticals. The over-expression of certain receptors such as the gastrin releasing peptide receptor (GRPr) on human cancer cells makes this method of drug development a viable tool for tumor targeting in vivo. Breast, pancreatic, prostate, gastric, colon, and small-cell lung cancer have demonstrated GRPr expression. In this project, we have conjugated a diaminoproionic acid (DPR) bifunctional chelator to bombesin (BBN) peptide targeting vector by solid phase peptide synthesis. BBN is an analogue of human gastrin releasing peptide (GRP) that binds to the GRPr with high affinity and specificity. Conjugates of the general structure [DPR-(X)-BBN(7-14)NH2] (X = a series of amino acid pharmacokinetic modifiers) were purified by reverse-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectrometry. Radiolabeling investigations of with fac-[99mTc(CO)3(H2O)3]+ (Isolink®) provided for metallated conjugates of the following general structure: [99mTc(CO)3-DPR-(X)-BBN(7-14)NH2]. These new conjugates demonstrated the ability to target specific human tumors in rodent models. Subsequent radiolabeling studies of [DPR-(X)-BBN(7-14)NH2] with fac-[188Re(CO)3(H2O)3]+, the therapeutic surrogate precursor of Tc-99m, have given us the potential to treat specific human tumors via these new targeting vectors. Detailed radiolabeling protocols, in vitro cell binding studies, and in vivo biodistribution assays will be reported.Harry S. Truman Memorial VA Hospita

99mTc-DPR-SSS-BBN for diagnosis of human cancers [abstract]

Abstract only availableReceptor-specific, radiolabeled peptides have become increasingly popular as targeting vectors for the development of new diagnostic radiopharmaceuticals. The over-expression of certain receptors such as the gastrin releasing peptide receptor (GRPr) on human cancer cells makes this method of drug development a viable tool for tumor targeting in vivo. Breast, pancreatic, prostate, gastric, colon, and small-cell lung cancer have demonstrated GRPr expression. In this project, we have conjugated a diaminoproionic acid (DPR) bifunctional chelator to bombesin (BBN) peptide targeting vector by solid phase peptide synthesis. BBN is an analogue of human gastrin releasing peptide (GRP) that binds to the GRPr with high affinity and specificity. A conjugate, [DPR-SSS-BBN(7-14)NH] was purified by reverse-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectrometry. Radiolabeling investigations of with fac-[99mTc(CO)3(H2O)3]+ (Isolink®) provided for the metallated conjugate [99mTc(CO)3-DPR-SSS-BBN(7-14)NH2]. This new conjugate demonstrated the ability to target specific human tumors in rodent models. In vitro cell binding studies, and in vivo biodistribution assays will be reported.Life Sciences Undergraduate Research Opportunity Progra