Jered C. Garrison

[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 Vivo Evaluation and Small-Animal PET/CT of a Prostate Cancer Mouse Model Using 64Cu Bombesin Analogs: Side-by-Side Comparison of the CB-TE2A and DOTA Chelation Systems

The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, β+: 18%, Eβ+max = 653 keV; β−: 37%, Eβ−max = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N′,N″,N‴-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. Methods: The CB-TE2A-8-AOC-BBN(7–14)NH2 and DOTA-8-AOC-BBN(7–14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. Results: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 ± 2.27 and 4.95 ± 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 ± 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 ± 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7–14)NH2 radioconjugate demonstrated significant clearance, 98.60 ± 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 ± 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7–14)NH2 radioconjugate because of nontarget retention. Conclusion: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7–14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.

Synthesis and structural characterization of a [Ag4]4+ cluster stabilized by a mixed-donor N-heterocyclic carbene linked cyclophane and the first reported synthesis of a N-heterocyclic carbene complex in water

Abstract The synthesis of the tetranuclear Ag44+ cluster stabilized by a N-heterocyclic carbene macrocycle (3)[PF6]4. Decomposition of the tetranuclear cluster (3)[PF6]4 in light forms the dimeric species (2)[PF6]2. In situ synthesis of the dimeric silver N-hetrocyclic carbene complex (2)[Br]2 in water.

Evaluation of the pharmacokinetic effects of various linking group using the 111In-DOTA-X-BBN(7-14)NH2 structural paradigm in a prostate cancer model.

The high incidence of BB2 receptor (BB2r) expression in various cancers has prompted investigators to pursue the development of BB2r-targeted agents for diagnostic imaging, chemotherapy, and radiotherapy. Development of BB2r-targeted agents, based on the bombesin (BBN) peptide, has largely involved the use of the bifunctional chelate approach in which the linking group serves several key roles including pharmacokinetic modification. Understanding the in vivo properties of the various pharmacokinetic modifying linking groups is crucial for developing BB2r-targeted agents with improved targeting and clearance characteristics. The goal of this study was to systematically evaluate the pharmacokinetic profile of aliphatic hydrocarbon, aromatic, and poly(ethylene glycol) (ether) functional groups in order to obtain a better understanding of the in vivo properties of these pharmacokinetic modifiers. Specifically, we synthesized six radioconjugates with the structure 111In-DOTA- X-BBN(7-14)NH2, where X = 8-aminooctanoic acid (8-AOC), 5-amino-3-oxapentyl-succinamic acid (5-ADS), 8-amino-3,6-dioxaoctyl-succinamic acid (8-AOS), p-aminobenzoic acid (AMBA), Gly-AMBA, and Gly- p-aminomethylbenzoic acid (Gly-AM2BA). All of the (nat)In-conjugates demonstrated nanomolar binding affinities to the BB2r. In CF-1 mice, the BB2r uptake in the pancreas of radioconjugates containing aromatic linking groups was found to be significantly higher at 1 h postinjection than the radioconjugates with ether linker moieties. For PC-3 tumor-bearing SCID mice, the tumor uptake was found to be 6.66 +/- 2.00, 6.21 +/- 1.57, 6.36 +/- 1.60, 4.46 +/- 0.81, and 7.76 +/- 1.19 %ID/g for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively, at 15 min postinjection. By 24 h postinjection, the radioconjugates containing aromatic groups exhibited the highest percentage tumor retention with 11.4%, 19.8%, 26.6%, 25.8%, and 25.5% relative to the 15 min values remaining in the tumor tissue for the 8-AOC, 8-ADS, AMBA, Gly-AMBA, and Gly-AM2BA radioconjugates, respectively. Fused Micro-SPECT/CT imaging studies performed at 24 h postinjection revealed substantial accumulation of radioactivity in the tumor tissue for all radioconjugates. In both biodistribution and Micro-SPECT/CT imaging studies, the radioconjugates containing aromatic linking groups typically exhibited significantly higher G.I. tract retention than the hydrocarbon or ether linking moieties. In conclusion, our studies indicate that radioconjugates incorporating aromatic linking groups, of the type investigated, generally demonstrated enhanced retention in BB2r expressing tissues in comparison to either the hydrocarbon or ether linking moieties. Furthermore, this investigation clearly demonstrates the significance of the linking group upon not only the in vivo clearance of the radiopharmaceutical, but also on the in vivo uptake and retention of the BB2r-targeted agent in tumor tissue. Future designs of BB2r-targeted agents should include a careful consideration of the effect linking group functionality has upon tumor targeting and retention.

Synthesis and structural characterization of two bis(imidazol-2-ylidene) complexes of Pt(II)

The air stable syntheses of two new chelating N-heterocyclic carbene complexes of Pt(II), bis(1,1′- n -butylimidazolium)-3,3′-methylenePtI 2 ( 2 ) and bis[bis(1,1′- n -butylimidazolium)-3,3′-methylene]PtI 2 ( 3 ) are described.

Synthesis and structural characterization of two bis-imidazolium-linked cyclophanes: precursors toward ‘carbeneporphyrinoid’ ligands

Abstract The synthesis and spectroscopic characterization of the imidazolium-linked cyclophanes [ 1 ][PF6]2 and [ 2 ][PF6]2 are described. Cyclophane [ 1 ][PF6]2 contains two imidazolium rings bridged by a 2,6-bis(methyl)-pyridine unit and a 2,5-bis(methyl)-pyrrole unit. Cyclophane [ 2 ][PF6]2 contains two imidazolium rings bridged by two 2,5-bis(methyl)-pyrrole units. Cyclophanes [ 1 ][PF6]2 and [ 2 ][PF6]2 have been structurally characterized by X-ray crystallography.

Synthesis and In Vitro and In Vivo Evaluation of Hypoxia-Enhanced 111In-Bombesin Conjugates for Prostate Cancer Imaging

Receptor-targeted agents, such as gastrin-releasing peptide receptor (BB2r)–targeted peptides, have been investigated extensively in preclinical and clinical studies. In an attempt to increase the effectiveness of diagnostic or radiotherapeutic agents, we have begun to explore the incorporation of the hypoxia-selective prodrug 2-nitroimidazole into receptor-targeted peptides. Hypoxia is a well-known characteristic of many solid tumors, including breast, prostate, and pancreatic cancers. The aim of this approach is to use the hypoxia-trapping capability of 2-nitroimidazoles to increase the retention of the agent in hypoxic, BB2r-positive tumors. We have demonstrated that incorporation of one or more 2-nitroimidazoles into the BB2r-targeted peptide significantly increases the in vitro retention of the agent in hypoxic prostate cancer cells. The study described herein represents our first investigation of the in vivo properties of these hypoxia-enhanced BB2r-targeted agents in a PC-3 xenograft mouse model. Methods: Four 111In-labeled BB2r-targeted conjugates—111In-1, 111In-2, 111In-3, and 111In-4, composed of 2-nitroimidazole moieties of 0, 1, 2, and 3, respectively—were synthesized, labeled, and purified. The BB2r binding affinities, externalization, and protein-association properties of these radioconjugates were assessed using the BB2r-positive PC-3 human prostate cancer cell line under hypoxic and normoxic environments. The in vivo biodistribution and micro-SPECT/CT imaging of the 111In-1, 111In-2, and 111In-4 radioconjugates were investigated in PC-3 tumor–bearing severely combined immunodeficient mice. Results: All conjugates and natIn-conjugates demonstrated nanomolar binding affinities. 111In-1, 111In-2, 111In-3, and 111In-4 demonstrated 41.4%, 60.7%, 69.1%, and 69.4% retention, correspondingly, of internalized radioactivity under hypoxic conditions relative to 34.8%, 35.3%, 33.2%, and 29.7% retention, respectively, under normoxic conditions. Protein-association studies showed significantly higher levels of association under hypoxic conditions for 2-nitroimidazole–containing BB2r-targeted radioconjugates than for controls. On the basis of the initial 1-h uptake in the PC-3 tumors, 111In-1, 111In-2, and 111In-4 demonstrated tumor retentions of 1.5%, 6.7%, and 21.0%, respectively, by 72 h after injection. Micro-SPECT/CT imaging studies of 111In-1, 111In-2, and 111In-4 radioconjugates resulted in clear delineation of the tumors. Conclusion: On the basis of the in vitro and in vivo studies, the BB2r-targeted agents that incorporated 2-nitroimidazole moieties demonstrated improved retention. These results indicate that further exploration into the potential of hypoxia-selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.

Synthesis and structural characterization of a silver complex of a mixed-donor N-heterocyclic carbene linked cyclophane

The synthesis of a dicationic imidazolium-linked cyclophane and a dimeric silver-N-heterocyclic carbene complex, that is the first silver complex with a N-heterocyclic carbene ligand involved in a pi-bonding interaction, is reported.

177Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model

INTRODUCTION A major barrier to the advancement of therapeutic nanomedicines has been the non-target toxicity caused by the accumulation of the drug delivery systems in organs associated with the reticuloendothelial system, particularly the liver and spleen. Herein, we report the development of peptide based metabolically active linkers (MALs) that are enzymatically cleaved by cysteine cathepsin B and S, two proteases highly expressed in the liver and spleen. The overall goal of this approach is to utilize the MALs to lower the non-target retention and toxicity of radiolabeled drug delivery systems, thus resulting in higher diagnostic and radiotherapeutic efficacy. METHODS In this study three MALs (MAL0, MAL1 and MAL2) were investigated. MAL1 and MAL2 are composed of known substrates of cathepsin B and S, respectively, while MAL0 is a non-cleavable control. Both MAL1 and MAL2 were shown to undergo enzymatic cleavage with the appropriate cathepsin protease. Subsequent to conjugation to the HPMA copolymer and radiolabeling with (177)Lu, the peptide-polymer conjugates were renamed (177)Lu-metabolically active copolymers ((177)Lu-MACs) with the corresponding designations: (177)Lu-MAC0, (177)Lu-MAC1 and (177)Lu-MAC2. RESULTS In vivo evaluation of the (177)Lu-MACs was performed in an HPAC human pancreatic cancer xenograft mouse model. (177)Lu-MAC1 and (177)Lu-MAC2 demonstrated 3.1 and 2.1 fold lower liver retention, respectively, compared to control ((177)Lu-MAC0) at 72h post-injection. With regard to spleen retention, (177)Lu-MAC1 and (177)Lu-MAC2 each exhibited a nearly fourfold lower retention, relative to control, at the 72h time point. However, the tumor accumulation of the (177)Lu-MAC0 was two to three times greater than (177)Lu-MAC1 and (177)Lu-MAC2 at the same time point. The MAL approach demonstrated the capability of substantially reducing the non-target retention of the (177)Lu-labeled HPMA copolymers. CONCLUSIONS While further studies are needed to optimize the pharmacokinetics of the (177)Lu-MACs design, the ability of the MAL to significantly decrease non-target retention establishes the potential this avenue of research may have for the improvement of diagnostic and radiotherapeutic drug delivery systems.

Development of hypoxia enhanced 111In-labeled Bombesin conjugates: design, synthesis, and in vitro evaluation in PC-3 human prostate cancer.

The gastrin-releasing peptide receptor (BB2r) has shown great promise for tumor targeting due to the increase of the receptor expression in a variety of human cancers including prostate, breast, small-cell lung, and pancreatic cancer. From clinical investigations, prostate cancer has been shown to be among the most hypoxic of the cancers investigated. Many solid tumors contain regions of hypoxia due to poor organization and efficiency of the vasculature. However, hypoxia is typically not present in normal tissue. Nitroimidazoles, a thoroughly investigated class of hypoxia selective drugs, have been shown to be highly retained in hypoxic tissues. The purpose of this study is to determine if the incorporation of hypoxia trapping moieties into the structural paradigm of BB2r-targeted peptides will increase the retention time of the agents in prostate cancer tumors. The present work involves the design, syntheses, purification, and in vitro investigation of hypoxia enhanced (111)In-BB2r-targeted radioconjugates. A total of four BB2r-targeted conjugates (1-4) were synthesized and coupled with increasing numbers of 2-nitroimidazoles, a hypoxia trapping moiety. Conjugates were radiolabeled with (111)In and purified by HPLC prior to in vitro studies. Receptor saturation assays under both normoxic and hypoxic conditions showed that the BB2r receptor expression on the PC-3 human prostate cancer cell line was not significantly affected by oxygen levels. Competitive binding assays revealed that incorporation of 2-nitroimidazoles had a detrimental effect to BB2r binding when adequate spacer groups, between the hypoxia trapping agent and the pharmacophore, were not employed. All of the 2-nitroimidazole containing BB2r-targeted agents exhibited significantly higher longitudinal retention in PC-3 cells under hypoxic conditions compared to the analogous normoxic studies. Protein association analysis revealed a 3-fold increase in binding of a 2-nitroimidazole containing BB2r-targeted agent under hypoxic relative to normoxic conditions. The positive nature of these results indicate that further exploration into the potential of hypoxia selective trapping agents for BB2r-targeted agents, as well as other targeted compounds, is warranted.