Adam Prasanphanich

[64Cu-NOTA-8-Aoc-BBN(7-14)NH2] targeting vector for positron-emission tomography imaging of gastrin-releasing peptide receptor-expressing tissues

Radiolabeled peptides hold promise as diagnostic/therapeutic targeting vectors for specific human cancers. We report the design and development of a targeting vector, [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid, 8-Aoc = 8-aminooctanoic acid, and BBN = bombesin), having very high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). GRPrs are expressed on a variety of human cancers, including breast, lung, pancreatic, and prostate, making this a viable approach toward site-directed localization or therapy of these human diseases. In this study, [NOTA-X-BBN(7-14)NH2] conjugates were synthesized, where X = a specific pharmacokinetic modifier. The IC50 of [NOTA-8-Aoc-BBN(7-14)NH2] was determined by a competitive displacement cell-binding assay in PC-3 human prostate cancer cells using 125I-[Tyr4]-BBN as the displacement ligand. An IC50 of 3.1 ± 0.5 nM was obtained, demonstrating high binding affinity of [NOTA-8-Aoc-BBN] for the GRPr. [64Cu-NOTA-X-BBN] conjugates were prepared by the reaction of 64CuCl2 with peptides in buffered aqueous solution. In vivo studies of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] in tumor-bearing PC-3 mouse models indicated very high affinity of conjugate for the GRPr. Uptake of conjugate in tumor was 3.58 ± 0.70% injected dose (ID) per g at 1 h postintravenous injection (p.i.). Minimal accumulation of radioactivity in liver tissue (1.58 ± 0.40% ID per g, 1 h p.i.) is indicative of rapid renal-urinary excretion and suggests very high in vivo kinetic stability of [64Cu-NOTA-8-Aoc-BBN(7-14)NH2] with little or no in vivo dissociation of 64Cu2+ from the NOTA chelator. Kidney accumulation at 1 h p.i. was 3.79 ± 1.09% ID per g. Molecular imaging studies in GRPr-expressing tumor models produced high-contrast, high-quality micro-positron-emission tomography images.

In vitro and in vivo analysis of [64Cu-NO2A-8-Aoc-BBN(7–14)NH2]: a site-directed radiopharmaceutical for positron-emission tomography imaging of T-47D human breast cancer tumors

INTRODUCTION Human breast cancer, from which the T-47D cell line was derived, is known to overexpress the gastrin-releasing peptide receptor (GRPR) in some cases. Bombesin (BBN), an agonist for the GRPR, has been appended with a radionuclide capable of positron-emission tomography (PET) imaging and therapy. (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) (NO2A=1,4,7-triazacyclononane-1,4-diacetate) has produced high-quality microPET images of GRPR-positive breast cancer xenografted tumors in mice. METHODS The imaging probe was synthesized by solid-phase peptide synthesis followed by manual conjugation of the 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) bifunctional chelator and radiolabeling in aqueous solution. The radiolabeled conjugate was subjected to in vitro and in vivo studies to determine its specificity for the GRPR and its pharmacokinetic profile. A T-47D tumor-bearing mouse was imaged with microPET/CT and microMRI imaging. RESULTS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) targeting vector was determined to specifically localize in GRPR-positive tissue. Accumulation was observed in the tumor in sufficient quantities to allow for identification of tumors in microPET imaging procedures. For example, uptake and retention in T-47D xenografts at 1, 4 and 24 h were determined to be 2.27+/-0.08, 1.35+/-0.14 and 0.28+/-0.07 % ID/g, respectively. CONCLUSIONS The (64)Cu-NO2A-8-Aoc-BBN(7-14)NH(2) produced high-quality microPET images. The pharmacokinetic profile justifies investigation of this bioconjugate as a potentially useful diagnostic/therapeutic agent. Additionally, the bioconjugate would serve as a good starting point for modification and optimization of similar agents to maximize tumor uptake and minimize nontarget accumulation.

In Vitro and In Vivo Evaluation of Alexa Fluor 680- Bombesin(7-14)NH 2 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 H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2). 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]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF). In vitro competitive binding assays, using 125I-Tyr4-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]NH2 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.

Synthesis, Radiolabeling and In vitro GRP Receptor Targeting Studies of 99mTc‐Triaza‐X‐BBN[7‐14]NH2 (X=Serylserylserine, Glycylglycylglycine, Glycylserylglycine, or Beta Alanine)

Gastrin‐releasing peptide (GRP) receptors have been shown to be over‐expressed on several types of human cancer cells including prostate, breast, small cell lung and pancreatic cancers. Bombesin (BBN) is a 14 amino acid peptide that is an analogue of human gastrin‐releasing peptide, binding to GRP receptors (GRPr) with high affinity and specificity. The aim of these studies was to develop new 99mTc‐labeled BBN analogs having high tumor uptake and optimal pharmacokinetics for specific targeting of human prostate cancers. In this study, a new tridentate bifunctional chelating agent, 2‐(4,7‐bis(tert‐butoxycarbonyl)‐1,4,7‐triazonan‐1‐yl)‐acetic acid, was synthesized by first reacting 2 equivalents of BOC‐ON with 1,4,7‐triazacyclanone (Triaza) in CHCl3 at room temperature. The product, N, N′‐bis‐(tert‐butoxycarbonyl)‐1,4,7‐triazacyclanone, was alkylated using BrCH2COOH in acetonitrile. This new ligand framework was characterized by 1H and 13C NMR and electrospray ionization mass spectrometry (ESI‐MS). Solid‐phase peptide synthesis (SPPS) was used to produce Triaza‐X‐BBN[7‐14]NH2 conjugates with the following structure: Triaza‐X‐Q‐W‐A‐V‐G‐H‐L‐M‐(NH2), where the spacer group X=SSS, GGG, GSG and β‐Alanine (SSS=Serylserylserine, GGG=Glycylglycylglycine, and GSG=Glycylserylglycine). These conjugates were purified by reversed phase‐HPLC (RP‐HPLC) and characterized by ESI‐MS. In vitro competitive binding assays, using 125I‐Tyr4‐BBN as the radiolabelling gold standard, demonstrated IC50 values in the nanomolar range for all the new non‐metallated conjugates. For example, IC50s were 1.8±0.4, 3.9.±0.4, 1.9±0.3, and 1.3±0.2 nM for X=SSS, GGG, GSG and β‐Alanine, respectively. The new BBN conjugates were radiolabeled with Tc‐99m in moderate yield via the Isolink® radiolabeling kit available from Tyco Healthcare, St. Louis, MO. In vitro internalization and externalization analyses indicated receptor binding to be receptor specific in human, PC‐3, prostate cancer cells. These studies indicate that future in vivo studies in tumor‐bearing mouse models are justified.

Abstract 2504: Preferential cell cycle expression of the bombesin receptor (BB2r) in prostate cancer cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Aberrant expression of the bombesin receptor subtype 2 (BB2r) has been found in over 80% of prostate cancers. The functional role of the receptor in cancer has been identified as a promoter of growth, morphogenesis and mutagenesis in tumor cell lines. The BB2r binds with high affinity to the bombesin peptide (BN), stimulating agonist mediated receptor-peptide internalization. Accordingly, targeting the BB2r with radioisotopes conjugated to BN represents a promising method for therapeutic radiopharmaceutical development. We look to optimize ionizing radiation damage to targeted tumor cells by combining BB2r targeted radiotherapy with the radiosensitizing chemotherapeutic docetaxel. The hallmark mechanism of docetaxel is the promotion of G2/M phase arrest, which is also the most radiosensitive phase of the cell cycle. The objectives of this study are to characterizing general BB2r expression in the cell cycle and the receptors response to cell cycle perturbations caused by docetaxel. We have synthesized, purified, and characterized two BN analogs [BN(7-14) agonist and scrambled sequence] conjugated to a fluorophore to be used in combination with cell cycle analysis in multiparameter flow cytometry. For evaluation of cell surface receptor expression or agonist mediated internalization of the BB2r, PC-3 cell experiments were performed at either 4°C or 37°C, respectively. Our data demonstrates constitutive BB2r expressed in all phases of the cell cycle. However, there is a 60% increase in fluorophore conjugated agonist binding, representative of bombesin receptor expression, in the G2/M phase as compared to the G/G1 phase. To determine if G2/M phase preferential expression could be exploited during G2/M phase arrest, PC-3 cells were exposed to physiologically obtainable serum concentrations of docetaxel at 1, 5 or 10 nM for 48 hours. A dose dependent response in G2/M phase cell arrest was only observed at 5 and 10 nM. At docetaxel concentrations of 1, 5 and 10 nM we observed an increase of 60%, 200% and 300% in BB2r expression in the G2/M phase as compared to the G/G1 phase. However, with increasing concentrations of docetaxel, a net decrease in BB2r expression was observed in the entire cell population with a 13%, 34% and 48% decrease in receptor expression at 1, 5 and 10 nM docetaxel as compared to control. These findings suggest preferential expression of the BB2r in the G2/M phase of the cell cycle, ideal for optimizing targeted radiotherapy. We also report an unanticipated down-regulation of the BB2r with docetaxel treatment which may contribute to chemotherapeutic efficacy by reducing mitogen stimulation through the BB2r. Overall, the disproportionate BB2r expression in the G2/M phase coupled with reduced mitogen stimulation achieved with docetaxel therapy may contribute to chemotherapeutic synergy with BB2r targeted radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2504.

A new near-infrared absorption and fluorescent probe based on bombesin for molecular imaging

We have developed a series of new dye bombesin conjugates for site-specific absorption and fluorescence imaging of human prostate and breast cancers. Bombesin (BBN), an amphibian analog to the endogenous ligand, binds to the gastrin releasing peptide (GRP) receptors with high specificity and affinity. Previously, we developed an Alexa Fluor 680-GGG-BBN peptide conjugate which demonstrated high binding affinity and specificity for breast cancer cells in the in vitro and in vivo tests (Ref: Ma et al., Molecular Imaging, vol. 6, no. 3, 2007: 171-180). This probe can not be used as an absorption probe in near-infrared imaging because its absorption peak is in the visible wavelength range. In addition, site specific longer wavelength fluorescent probe is desired for in vivo molecular imaging because long wavelength photons penetrate deeper into tissue. The new absorption and fluorescent probe we developed is based on the last eight-residues of BBN, -Q-W-A-V-G-H-L-M-(NH2), and labeled with AlexaFluor750 through a chemical linker, beta-alanine. The new probe, Alexa Fluor 750-BetaAla-BBN(7-14)NH2, exhibits optimal pharmacokinetics for specific targeting and optical imaging of the GRP receptor over-expressing cancer cells. Absorption spectrum has been measured and showed absorption peaks at 690nm, 720nm and 735nm. Fluorescent band is located at 755nm. In vitro and in vivo investigations have demonstrated the effectiveness of the new conjugates to specifically target human cancer cells overexpressing GRP receptors and tumor xenografts in severely compromised immunodeficient mouse model.