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

Dynamic redox signaling during TGF-beta-induced epithelial-mesenchymal transition

Individual biological processes operate within larger contexts and can participate in the emergence of complex phenotypes. The morphogen transforming growth factor β (TGFβ) can initiate diverse cellular responses, including down-regulation of numerous antioxidant species. TGFβ signaling itself has been shown to exhibit redox sensitivity and in the context of TGFβ-mediated epithelial-mesenchymal transition (EMT), there exists a possibility of a positive feedback loop operating over multiple temporal and biological scales to stabilize a mesenchymal phenotype. Additionally, drug resistant side populations (SP) arise in populations that exhibit heterogeneity of ABCG2 transporter activity, which is regulated within the same cellular program as antioxidants. Therefore, it is possible that SPs reflect heterogeneity in redox regulation within a population; however, how single-cell ABCG2 activity heterogeneity manifests at the population level is not known. The overall objective of this research was to investigate how redox regulated processes contribute to complex phenotypes that arise in the context of TGFβ-mediated EMT using multivariate and systems approaches. We investigated the dynamics of redox regulation in the context of EMT, hypothesizing that decreased nucleophilic tone acquired during EMT strengthens TGFβ signaling enhancing acquisition and stabilization of the mesenchymal phenotype. We demonstrated the sensitivity of TGFβ signaling to antioxidants and the down-regulation of antioxidants within a singular model. We developed in-cell western assays to evaluate multivariate phenotype states as they developed during EMT. TGFβ treatment decreased H2O2 degradation rates and increased glutathione redox potential, indicating decreased nucleophilic tone. Epithelial/mesenchymal differentiation and redox time course data were paired using principal component analysis (PCA) to construct a multivariate representation of phenotype over the time course of EMT. We found that decreased nucleophilic tone during EMT coincides with acquisition of a mesenchymal phenotype over too long a time scale to enable enhancement of EMT. In the second portion of this research, we investigated the role of heterogeneity of ABCG2 activity at the single cell level in the emergence of SPs at the population level and the means by which TGFβ signaling modulates heterogeneity to affect SP size. TGFβ was found to decrease the size of SPs as well as the magnitude of response. A multiscale ensemble model consisting of a heterogeneous population of individual cells was used to interrogate multiple kinetic schemas and identified a highly active subpopulation juxtaposed by an inactive main population, suggesting the SP cells may exhibit a distinct redox profile from main cells, the frequency of which was decreased in response to TGFβ. In summary, we developed an approach to investigate the dynamics of redox regulation during TGFβ-mediated EMT from the perspective of a multivariate phenotype, simultaneously accounting for changes in epithelial/mesenchymal differentiation and to the intracellular redox environment. Additionally, we developed a multiscale ensemble modeling approach to investigate the kinetic mechanisms by which heterogeneity of ABCG2 regulation at the single-cell level leads to emergence of a SP at the population level.Ph.D

In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H 2 N-glycylglycylglycine-BBN[7–14]NH 2 peptide with the following general sequence: H 2 N-G-G-G-Q-W-A-V-G-H-L-M-(NH 2 ). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H 2 N-G-G-G-BBN[7–14]NH 2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125 I-Tyr 4 -BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity

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

In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH 2

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H 2 N-glycylglycylglycine-BBN[7–14]NH 2 peptide with the following general sequence: H 2 N-G-G-G-Q-W-A-V-G-H-L-M-(NH 2 ). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H 2 N-G-G-G-BBN[7–14]NH 2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125 I-Tyr 4 -BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH 2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity