Macrocyclic Chelator Research Articles

Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe

Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide.Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data.Results: The dual-labeled probe 64Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026).Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.

Nephrogenic Systemic Fibrosis (NSF): What It Really Means for Materials Scientists?

Noninvasive MR molecular imaging and targeted drug delivery systems, often referred to as theranostic agents, are being developed to enable improved detection, patient risk stratification, site-specific treatment and longitudinal monitoring [1]. Gadolinium (Gd), a paramagnetic lanthanide has been the dominant metal for T1 weighted MR contrast agents to produce bright signal in vivo. With seven unpaired electron (+3 oxidation state) in the outer d-orbital, it strongly influences the T1 relaxation times of the soft, biological tissues to which the metal has access. Gd exhibits a tendency to alter signal intensities by shortening the T1 time in its surroundings. The efficacy is measured by the relaxivity, which is dependent on the chemical properties of the metal, typically as a coordinated complex. Gadolinium is toxic in its free form [2]. The free ion (i.e. chloride, sulfate, or acetate form) has inadequate solubility in water in the neutral pH range, and is poorly tolerated in vivo. However in the clinics it is routinely administered in a chelated (linear or macrocylics) form such as DTPA or DOTA, still maintaining the strong relaxivity [3]. By far, in terms of their chemical properties, Gd is superior in comparison to the other paramagnetic metals e.g. manganese (Mn) and copper (Cu). Despite the long history of Gd blood pool contrast agents [4] and the preclinical success of nanotechnology platforms [5] based on this metal, the recent discovery of nephrogenic systemic fibrosis (NSF) has raised concern on currently approved MR contrast agents [6]. NSF is a serious and unexpected side effect of gadolinium blood pool agents observed in some patients with renal disease or following liver transplant [7]. It was first identified in 1997 in the US when a small group of renal transplant patients were found to have developed complications such as joint contractures, brawny in duration and hyper pigmentation of the skin in their extremities. The disease was initially referred to as ‘nephrogenic fibrosing dermopathy’; however, the close connection of visceral led to the name ‘nephrogenic systemic fibrosis’. Interestingly, all affected individuals were observed to have chronic kidney disease. Later, its association with Gd was clinically observed by Grobner et al. [8] and subsequently by others [9-10] (Figure 1). Patients with NSF develop thickening of the skin and connective tissues that can inhibit arm and leg movements and even lead to bone fractures. Approximately 5% of patients experience a rapidly progressive course, which may result in death due to widespread fibrosis [11]. The cause of NSF is unknown, and there is no effective treatment of this condition. All these findings prompted the FDA to issue a boxed warning of gadolinium blood pool agents specifying that gadolinium based contrast agents (GBCAs) increase the risk for NSF in patients with: 1) acute or chronic, severe renal insufficiency (glomerular filtration rate <30 mL/min/1.73m2), 2) acute renal insufficiency of any severity due to the hepato-renal syndrome, or 3) in the perioperative liver transplantation period [12]. Although dialysis of patients may be considered promptly following GBCA administration, there is no evidence that hemodialysis prevents NSF. Linear chelates (e.g. Omniscan®, Magnevist® and OptiMARK®) are the most commonly reported agents differentiating them from macrocyclic molecules. In terms of the kinetic, thermodynamic, conditional stability as well as their tendency for transmetallation with zinc and other cations, macrocyclic chelates are superior to the linear forms [13]. Myriad of advancement has been made to create targeted nanoagents carrying high payload of paramagnetic Gd to characterize tissues based on the presence of pathognomonic biosignatures [14]. Some of these examples include e.g. metal chelates bearing liposomal vesicles [15] and lipoprotein micelles; [16] ανβ3-targeted paramagnetic polymerized liposome [17] and perfluorocarbon (PFC) nanoparticles; [18] paramagnetic micelles for macrophage imaging within atherosclerotic plaque [19] etc.

Macrocyclic coordination chemistry

This chapter reviews the literature published on macrocyclic coordination chemistry during 2011. The aim is to describe the key advances, focusing predominantly on complexes formed with transition metal and lanthanide ions. Porphyrin ligands and supramolecular chemistry are not covered. Trends in the development of macrocyclic chelator design and the impact of the coordination chemistry of the chelators on both existing and emerging applications are discussed.

Cyclization of RGD peptide sequences via the macrocyclic chelator DOTA for integrin imaging

Two bicyclic compounds containing Arg-Gly-Asp (RGD) motifs (RGDf and RGD) were synthesized by cyclizing the peptide sequence across the macrocyclic ring of DOTA via two non-adjacent carboxylate pendent arms. The Lu(3+) or Cu(2+) complexes of these compounds, c(DOTA-RGDf) and c(DOTA-RGD), showed a metal dependent affinity towards integrin α(v)β(3)in vitro and the (177)Lu(3+) or (64)Cu(2+) labelled derivatives showed specific tumour uptake in MCF7 and U87MG tumour bearing mice.

Preliminary evaluation of a microwave-assisted metal-labeling strategy for quantification of peptides via RPLC–ICP-MS and the method of standard additions

NIST has performed preliminary research on applying a calibration methodology based on the method of standard additions to the quantification of peptides via reverse-phase liquid chromatography coupled to inductively coupled plasma mass spectrometry (RPLC–ICP-MS). A microwave-assisted lanthanide labeling procedure was developed and applied to derivatize peptides using the macrocyclic bifunctional chemical chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which significantly improved the lanthanide labeling yield and reduced reaction times compared to benchtop labeling procedures. Biomolecular MS technologies of matrix-assisted laser desorption ionization (MALDI)-MS and electrospray ionization (ESI)-MS/MS were used in concert with ICP-MS to confirm the results of microwave labeling, sample cleanup and standard additions experiments for several test peptides. The calibration scheme is outlined in detail and contextualized against complementary high accuracy calibration strategies currently employed for ICP-MS detection of biomolecules. Standard additions experiments using native, non-isotopic peptide calibrants confirm the simplicity of the scheme and the potential of applying a blending (recombined sample and spike) procedure, facilitating calibration via co-elution of lanthanide labeled peptides. Ways to improve and fully leverage the analytical methodology are highlighted.

Convenient Synthesis and Evaluation of Heptadentate Bifunctional Ligand for Radioimmunotherapy Applications.

An efficient synthetic route to a bifunctional chelating agent C-NE3TA-NCS for antibody-targeted radioimmunotherapy (RIT) applications was developed. Various synthetic methods centered on the key reaction steps including bimolecular cyclization, ring opening reactions of aziridine and aziridinium cations, and reductive aminiation were explored to optimize the preparation of a tetraaza-based chelate TANPA and C-NE3TA analogues. Heptadentate C-NE3TA-NCS was conjugated to a tumor targeting antibody and compared to hexadentate C-NOTA-NCS for radiolabeling reaction kinetics with lanthanides for RIT. C-NE3TA-antibody conjugate displayed significantly enhanced complexation kinetics with 90Y as compared to C-NOTA-antibody conjugate. The synthetic methods for TANPA and C-NE3TA-NCS reported herein have broad applications for preparation of bifunctioanl macrocyclic chelating agents.

Synthesis and characterization of scVEGF‐PEG‐[68Ga]NOTA and scVEGF‐PEG‐[68Ga]DOTA PET tracers

Vascular endothelial growth factor (VEGF) signaling via vascular endothelial growth factor receptor 2 (VEGFR‐2) on tumor endothelial cells is a critical driver of tumor angiogenesis. Novel anti‐angiogenic drugs target VEGF/VEGFR‐2 signaling and induce changes in VEGFR‐2 prevalence. To monitor VEGFR‐2 prevalence in the course of treatment, we are evaluating 68Ga positron emission tomography imaging agents based on macrocyclic chelators, site‐specifically conjugated via polyethylene glycol (PEG) linkers to engineered VEGFR‐2 ligand, single‐chain (sc) VEGF. The 68Ga‐labeling was performed at room temperature with NOTA (2,2′,2′′‐(1,4,7‐triazonane‐1,4,7‐triyl) triacetic acid) conjugates or at 90 °C by using either conventional or microwave heating with NOTA and DOTA (2,2′,2′′,2′′′‐(1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetrayl) tetraacetic acid) conjugates. The fastest (~2 min) and the highest incorporation (&gt;90%) of 68Ga into conjugate that resulted in the highest specific radioactivity (~400 MBq/nmol) was obtained with microwave heating of the conjugates. The bioactivity of the NOTA‐ and DOTA‐containing tracers was validated in 3‐D tissue culture model of 293/KDR cells engineered to express high levels of VEGFR‐2. The NOTA‐containing tracer also displayed a rapid accumulation (~ 20 s after intravenous injection) to steady‐state level in xenograft tumor models. A combination of high specific radioactivity and maintenance of functional activity suggests that scVEGF‐PEG‐[68 Ga]NOTA and scVEGF‐PEG‐[68 Ga]DOTA might be promising tracers for monitoring VEGFR‐2 prevalence and should be further explored. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Metallofullerene-based Nanoplatform for Brain Tumor Brachytherapy and Longitudinal Imaging in a Murine Orthotopic Xenograft Model

To demonstrate in an orthotopic xenograft brain tumor model that a functionalized metallofullerene (f-Gd₃N@C₈₀) can enable longitudinal tumor imaging and, when radiolabeled with lutetium 177 (¹⁷⁷Lu) and tetraazacyclododecane tetraacetic acid (DOTA) (¹⁷⁷Lu-DOTA-f-Gd₃N@C₈₀), provide an anchor to deliver effective brachytherapy. All experiments involving the use of mice were carried out in accordance with protocols approved by the institutional animal care and use committee. Human glioblastoma U87MG cells were implanted by using stereotactic procedures into the brains of 37 female athymic nude-Foxn1nu mice and allowed to develop into a tumor for 8 days. T1- and T2-weighted magnetic resonance (MR) imaging was performed in five mice. Biodistribution studies were performed in 12 mice at four time points over 7 days to evaluate gadolinium content. Survival studies involved 20 mice that received infusion of a nanoplatform by means of convection-enhanced delivery (CED) 8 days after tumor implantation. Mice in survival studies were divided into two groups: one comprised untreated mice that received f-Gd₃N@CC₈₀ alone and the other comprised mice treated with brachytherapy that received 1.11 MBq of ¹⁷⁷Lu-DOTA-f-Gd₃N@CC₈₀. Survival data were evaluated by using Kaplan-Meier statistical methods. MR imaging showed extended tumor retention (25.6% ± 1.2 of the infused dose at 52 days, confirmed with biodistribution studies) of the f-Gd₃N@CC₈₀ nanoplatform, which enabled longitudinal imaging. Successful coupling of ¹⁷⁷Lu to the f-Gd₃N@CC₈₀ surface was achieved by using a bifunctional macrocyclic chelator. The extended tumor retention allowed for effective brachytherapy, as indicated by extended survival time (> 2.5 times that of the untreated group) and histologic signs of radiation-induced tumor damage. The authors have developed a multimodal nanoplatform and have demonstrated longitudinal tumor imaging, prolonged intratumoral probe retention, biodistribution, and extended survival in an orthotopic xenograft brain tumor model.

Chemical and Radiochemical Considerations in Radiolabeling with α-Emitting Radionuclides

A review of chemical and radiochemical factors that must be considered when radiolabeling targeting agents with radionuclides is presented. The review discusses factors that are important in choice of radionuclide and choice of chelation or bonding reagents to use in the development of an α-emitting radiopharmaceutical. Chemical parameters, such as physical properties and pendant groups for radiolabeling, are reviewed. A major portion of the review outlines the development of chelates and labeling conditions for radiometals, and application of these reagents/conditions to radiometals. Acyclic and macrocyclic chelates containing amine and carboxylic acid coordination groups are highlighted, with examples of bifunctional chelates for biomolecule conjugation. Information is presented on over 60 radiometal-binding chelates. 211At radiolabeling is separated from that of radiometals, and the various reagents used for radiolabeling have been reviewed. Although not all 211At-labeling reagents are reviewed (due to another recent review), nearly 50 reagents studied in the development of pendant groups for labeling with 211At are described. The review also discusses how therapeutic doses of α-emitting radiopharmaceuticals can be affected by the radionuclide used and how radiation damage to the radiopharmaceutical can be minimized.

PET of Somatostatin Receptor–Positive Tumors Using 64Cu- and 68Ga-Somatostatin Antagonists: The Chelate Makes the Difference

Somatostatin-based radiolabeled peptides have been successfully introduced into the clinic for targeted imaging and radionuclide therapy of somatostatin receptor (sst)-positive tumors, especially of subtype 2 (sst2). The clinically used peptides are exclusively agonists. Recently, we showed that radiolabeled antagonists may be preferable to agonists because they showed better pharmacokinetics, including higher tumor uptake. Factors determining the performance of radioantagonists have only scarcely been studied. Here, we report on the development and evaluation of four (64)Cu or (68)Ga radioantagonists for PET of sst2-positive tumors. The novel antagonist p-Cl-Phe-cyclo(D-Cys-Tyr-D-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)D-Tyr-NH(2) (LM3) was coupled to 3 macrocyclic chelators, namely 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A), 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), and DOTA. (64/nat)Cu- and (68/nat)Ga-NODAGA-LM3 were prepared at room temperature, and (64/nat)Cu-CB-TE2A-LM3 and (68/nat)Ga-DOTA-LM3 were prepared at 95°C. Binding affinity and antagonistic properties were determined with receptor autoradiography and immunofluorescence microscopy using human embryonic kidney (HEK)-sst2 cells. In vitro internalization and dissociation was evaluated using the same cell line. Biodistribution and small-animal PET studies were performed with HEK-sst2 xenografts. All metallopeptides demonstrated antagonistic properties. The affinities depend on chelator and radiometal and vary about 10-fold; (68/nat)Ga-NODAGA-LM3 has the lowest half maximal inhibitory concentration (1.3 ± 0.3 nmol/L). The biodistribution studies show impressive tumor uptake at 1 h after injection, particularly of (64)Cu- and (68)Ga-NODAGA-LM3 (∼40 percentage injected dose per gram of tissue [%ID/g]), which were proven to be specific. Background clearance was fast and the tumor washout relatively slow for (64)Cu-NODAGA-LM3 (∼15 %ID/g, 24 h after injection) and almost negligible for (64)Cu-CB-TE2A-LM3 (26.9 ± 3.3 %ID/g and 21.6 ± 2.1 %ID/g, 4 and 24 h after injection, respectively). Tumor-to-normal-tissue ratios were significantly higher for (64)Cu-NODAGA-LM3 than for (64)Cu-CB-TE2A-LM3 (tumor-to-kidney, 12.8 ± 3.6 and 1.7 ± 0.3, respectively; tumor-to-muscle, 1,342 ± 115 and 75.2 ± 8.5, respectively, at 24 h, P < 0.001). Small-animal PET shows clear tumor localization and high image contrast, especially for (64)Cu- and (68)Ga-NODAGA-LM3. This article demonstrates the strong dependence of the affinity and pharmacokinetics of the somatostatin-based radioantagonists on the chelator and radiometal. (64)Cu- and (68)Ga-NODAGA-LM3 and (64)Cu-CB-TE2A-LM3 are promising candidates for clinical translation because of their favorable pharmacokinetics and the high image contrast on PET scans.

One-step radiosynthesis of 18F-AlF-NOTA-RGD2 for tumor angiogenesis PET imaging

One of the major obstacles of the clinical translation of (18)F-labeled arginine-glycine-aspartic acid (RGD) peptides has been the laborious multistep radiosynthesis. In order to facilitate the application of RGD-based positron emission tomography (PET) probes in the clinical setting we investigated in this study the feasibility of using the chelation reaction between Al(18)F and a macrocyclic chelator-conjugated dimeric RGD peptide as a simple one-step (18)F labeling strategy for development of a PET probe for tumor angiogenesis imaging. Dimeric cyclic peptide E[c(RGDyK)](2) (RGD(2)) was first conjugated with a macrocyclic chelator, 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and the resulting bioconjugate NOTA-RGD(2) was then radiofluorinated via Al(18)F intermediate to synthesize (18)F-AlF-NOTA-RGD(2). Integrin binding affinities of the peptides were assessed by a U87MG cell-based receptor binding assay using (125)I-echistatin as the radioligand. The tumor targeting efficacy and in vivo profile of (18)F-AlF-NOTA-RGD(2) were further evaluated in a subcutaneous U87MG glioblastoma xenograft model by microPET and biodistribution. NOTA-RGD(2) was successfully (18)F-fluorinated with good yield within 40 min using the Al(18)F intermediate. The IC(50) of (19)F-AlF-NOTA-RGD(2) was determined to be 46 ± 4.4 nM. Quantitative microPET studies demonstrated that (18)F-AlF-NOTA-RGD(2) showed high tumor uptake, fast clearance from the body, and good tumor to normal organ ratios. NOTA-RGD(2) bioconjugate has been successfully prepared and labeled with Al(18)F in one single step of radiosynthesis. The favorable in vivo performance and the short radiosynthetic route of (18)F-AlF-NOTA-RGD(2) warrant further optimization of the probe and the radiofluorination strategy to accelerate the clinical translation of (18)F-labeled RGD peptides.

Synthesis and radiolabeling of chelator–RNA aptamer bioconjugates with copper-64 for targeted molecular imaging

Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g., copper-64, 64Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of 64Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator–aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [ p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [ p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10-3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g., pH, temperature, and time) for each chelator–RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with 64Cu.

Macrocyclic chelator assembled RGD multimers for tumor targeting.

Macrocyclic chelators have been extensively used for complexation of metal ions. A widely used chelator, DOTA, has been explored as a molecular platform to assemble multiple bioactive peptides in this paper. The multivalent DOTA-peptide bioconjugates demonstrate promising tumor targeting ability.

Studies on Efficacy of a Novel 177Lu-Labeled Porphyrin Derivative in Regression of Tumors in Mouse Model

The aim of the present study was to develop a (177)Lu-labeled porphyrin derivative having favorable characteristics for use in targeted radiotherapy of cancer and to evaluate its biological behavior in mouse tumor models with respect to its effectiveness in tumor regression. Owing to the inherent affinity of porphyrins to accumulate in the tumors, suitably modified porphyrin derivative was chosen as the vehicle for the targeted delivery of the radionuclide. (177)Lu was preferred as the radionuclide of choice due to its suitable nuclear decay characteristics [E(β(max)) = 497 keV, Eγ = 208 keV (11%), 113 keV (6.4%)], comparatively longer half-life (6.73 d) and ease of production in adequate quantity and sufficiently high specific activity using medium flux research reactors. A novel porphyrin analogue, 5,10,15,20-tetrakis[4-carboxymethyleneoxyphenyl]porphyrin was synthesized inhouse and coupled with a macrocyclic bi-functional chelating agent, namely p-amino-benzyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid. The porphyrin-BFCA conjugate was labeled with (177)Lu and the biological behavior of the radiolabeled conjugate was studied by biodistribution and imaging in Swiss mice bearing either fibrosarcoma or thymic lymphoma tumors. Effectiveness of the agent in controlling the growth of tumor volumes was also studied by administering various doses of the radiolabeled preparation in the mouse tumor models. (177)Lu-labeled porphyrin-BFCA conjugate was prepared with high radiochemical purity ( > 99%) and adequate invitro stability. Biodistribution and imaging studies revealed good uptake and retention of the agent in the tumors with encouraging tumor to blood and tumor to muscle ratios at various post-administration time points. Tumor regression studies showed that the administration of the agent increased the average tumor doubling time and decreased the average specific growth rate substantially in both the types of tumors. However, thymic lymphoma was found to be more sensitive to the radiolabeled conjugate compared to fibrosarcoma. Preliminary biological evaluation and tumor regression studies carried out in two different tumor models in Swiss mice exhibited the promising nature of (177)Lu-labeled porphyrin-BFCA conjugate as an agent for targeted tumor therapy. However, further detailed investigations are warranted to evaluate the true potential of the developed agent.

Clinical, Biological, and Skin Histopathologic Effects of Ionic Macrocyclic and Nonionic Linear Gadolinium Chelates in a Rat Model of Nephrogenic Systemic Fibrosis

the purpose of this study was to compare the clinical, pathologic, and biochemical effects of repeated administrations of ionic macrocyclic or nonionic linear gadolinium chelates (GC) in rats with impaired renal function. rats submitted to subtotal nephrectomy were allocated to single injections of 2.5 mmol/kg of gadodiamide (nonionic linear chelate), nonformulated gadodiamide (ie, without the free ligand caldiamide), gadoterate (ionic macrocyclic chelate), or saline for 5 consecutive days. Blinded semi-quantitative histopathologic and immunohistochemical examinations of the skin were performed, as well as clinical, hematological, and biochemical follow-up. Rats were killed at day 11. Long-term (up to day 32) follow-up of rats was also performed in an auxiliary study. epidermal lesions (ulcerations and scabs) were found in 4 of the 10 rats treated with nonformulated gadodiamide. Two rats survived the study period. Inflammatory signs were observed in this group. No clinical, hematological, or biochemical signs were observed in the saline and gadoterate- or gadodiamide-treated groups. Plasma fibroblast growth factor-23 levels were significantly higher in the gadodiamide group than in the gadoterate group (day 11). Decreased plasma transferrin-bound iron levels were measured in the nonformulated gadodiamide group. Histologic lesions were in the range: nonformulated gadodiamide (superficial epidermal lesions, inflammation, necrosis, and increased cellularity in papillary dermis) > gadodiamide (small superficial epidermal lesions and signs of degradation of collagen fibers in the dermis) > gadoterate (very few pathologic lesions, similar to control rats). repeated administration of the nonionic linear GC gadodiamide to renally impaired rats is associated with more severe histologic lesions and higher FGF-23 plasma levels than the macrocyclic GC gadoterate.

Design, synthesis and biological evaluation of choline based SPECT imaging agent: Ga(iii)-DO3A-EA-Choline

The enhanced choline uptake and phosphorylation in tumor cells has motivated the development of radiolabeled choline derivatives as diagnostic markers for imaging cell membrane proliferation and noninvasive detection of prostate, brain and breast tumors. In the present work, we report a facile strategy for the synthesis of choline functionalized macrocyclic chelating agent (DO3A-EA-choline) and its radiocomplexation with (67)Ga for potential tumor imaging applications. The synthesis of the desired compound featured quaternization of N,N-dimethylaminoethanol with 1,2-dibromoethane followed by subsequent alkylation with trisubstituted cyclen (DO3A). All intermediates and final compounds have been fully characterized by spectroscopic techniques, namely, (1)H, (13)C NMR and mass spectroscopy. The compound has been successively labeled with (67)Ga-citrate in ammonium acetate buffer (pH 6.5) at 80 °C. MTT assays have been performed on the HEK cell line to determine the cytotoxicity of the compound. Cell uptake studies carried out on the U-87 MG cell line exhibited saturable binding of the radioconjugate in picomolar range with a K(d) value of 0.528 pM. The in vivo biodistribution and blood kinetics studies exhibited rapid clearance of the radiolabeled complex and excretion through the renal and hepatobiliary route. The present studies demonstrate the potential applications of (67)Ga-DO3A-EA-choline as a radiopharmaceutical for molecular imaging using ((67/68)Ga) SPECT and PET modalities.

Combination of Gene Therapy and Nanoparticle Imaging for Improving T-Cell Therapy.

AbstractAbstract 1479Despite a functioning immune system, malignant tumors develop strategies to avoid immune recognition and elimination by T cells. Gene therapy can be used to render T cells capable of targeting tumors in an antigen-specific manner. The ability to genetically manipulate a T cell ex vivo provides new opportunities to enhance their biological activity in vivo. To explore new treatment approaches we propose a novel paradigm in which T cells are developed as biological delivery vehicles for desired genes and nanoparticles. We have combined gene therapy with nanotechnology to generate T cells that express a chimeric antigen receptor (CAR) selectively expressed on T cells operating within the tumor microenvironment and to introduce radiolabeled gold nanoparticles for tracking T cells in vivo.T cells can home to tumor microenvironments to exert their immunoreceptor-dependent cytolytic activity. However, despite the ability of T-cell receptors (TCRs) and CAR to recognize tumor cells, the anti-tumor effect can be incomplete. The ability to transfer a wide range of materials into T cells via electroporation can be exploited to adapt T cells as biological delivery agents capable of targeting specific tissues. This method may also circumvent the limited biodistribution of “raw” nanomaterials due to rapid clearance by the liver and other reticuloendothelial system (RES) organs. Gold is an attractive biocompatible nanomaterial which can be synthesized in compliance with current good manufacturing practice (cGMP) guidelines required for clinical-grade production and can be chemically functionalized for specific imaging or therapeutic functions.We first tested whether commercially available nanoparticles can be electro-transferred into cultured and primary human T cells. 43 nm diameter latex nanoparticles and 7 nm gold nanoparticles (GNPs) were electro-transferred into T cells and visualized by TEM and confocal imaging (Figure 1). We have modified GNPs on the surface with the chelator, diethylenetriaminepentaacetic acid (DTPA), for stable coordination with 111In followed by GNP PEGylation. We have shown that these radiolabeled GNPs can be readily electro-transferred into T cells suitable for combined single-photon emission computed tomography (SPECT) and computed tomography (CT) (Figure 2). We used a clinical SPECT/CT scanner to detect 111In-GNP in T cells (∼2.1 × 104 nanoparticles/cell) at a sensitivity of ∼760 cells/mL. After developing electroporation protocols of nanoparticles and in vivo imaging of 111In-GNPs, additional sensitivity was achieved by modifying the chelating chemistry using the macrocyclic chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), to bind 64Cu to GNPs, increasing the number of gold particles/cell, and using 64Cu-labeled GNPs for imaging by positron emission tomography (PET). Before tail vein injection to a mouse, 11.4 mCi was detected from 10 million T cells (suspended in 300 μL PBS) electroporated using a BTX ECM830 device with the following settings: 1 kV/cm, 4 ms duration, single square pulse (Figure 3). The estimated concentration of nanoparticles transferred into T cells was ∼2.3 × 105 nanoparticles/cell as determined by a gamma counter (2470 Wizard, PerkinElmer) and nanoparticle titration. While 20 nm GNPs were used for 111In labeling, 7 nm GNPs were chosen for 64Cu labeling because of the improved (10-fold) electroporation efficiency.We are currently investigating whether multi-functional GNPs encapsulating chemotherapy drugs can add both in vivo T-cell imaging capability and enhanced cytotoxicity to CAR-redirected T cells. In aggregate, this may improve the potency of clinical-grade genetically modified T cells as a vehicle with the imaging capability for targeted delivery of drug-loaded GNPs to the tumor microenvironment. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Synthesis, Characterization, and Catalytic Reactivity of a Highly Basic Macrotricyclic Aminopyridine

The synthesis methods, physicochemical and structural characteristics, and catalytic reactivity of new macrocyclic proton chelators, N,N',N''-tris(p-tolyl)azacalix[3](2,6)(4-pyrrolidinopyridine) and N,N',N''-tris(p-tolyl)azacalix[3](2,6)(4-piperidinopyridine), are studied. The introduction of pyrrolidino and piperidino groups into the pyridine unit enables the enhancement of the synergistic proton affinity of the cavity of the macrotricycle giving a high basicity (pK(BH+) = 28.1 and 27.1 in CD(3)CN), resulting in a catalytic activity for the Michael addition of nitromethane with α,β-unsaturated carbonyl compounds.

Synthesis and evaluation of macrocyclic amino acid derivatives for tumor imaging by gallium-68 positron emission tomography

68Ga PET imaging in clinical oncology represents a notable development because the availability of 68Ga is not dependent on a cyclotron. Furthermore, labeled amino acid derivatives have been proven to be useful for the imaging many tumor types. In the present study, we synthesized β-aminoalanine, γ-aminohomoalanine, and lysine conjugates of macrocyclic bifunctional chelating agents, such as, NOTA ( 1a– c) and DOTA ( 2a– c). The compounds produced were found to be potential useful as 68Ga-PET imaging agents. In particular, they showed high tumor uptakes in vitro and in vivo, and had high labeling yields and excellent stabilities. The co-ordination chemistry of NOTA-monoamide compound 1a was studied by multinuclear NMR. In vitro studies showed that the synthesized compounds were taken up by cancer cells more than controls ( 68Ga-NOTA and 68Ga-DOTA). Furthermore, in vivo studies showed that they have high tumor to muscle and tumor to blood ratios, and small-animal PET imaging revealed high tumor uptakes as compared with other organs, and high bladder activities, indicating rapid renal excretion. These results might motivate the use of 68Ga amino acid PET for tumor diagnosis.

Biological evaluation of avidin-based tumor pretargeting with DOTA-Triazole-Biotin constructed via versatile Cu(I) catalyzed click chemistry

Background: The biotin-avidin interaction remains a gold standard for the two-step pretargeting approach to image tumor sites. We aim to develop two-step pretargeting systems utilizing 99mTc labeled biotin functionalized macrocyclic chelating agents synthesized using the highly efficient Cu(I) catalyzed azide–alkyne cycloaddition for potential radioimaging applications.Methods: A facile synthesis of DOTA-Triazole-Biotin, radiocomplexation with 99mTc and the pretargeting protocol is described. The synthesis features Cu(I) catalyzed click conjugation between biotinylated azide and propynyl functionalized DO3A. 99mTc radiolabeling was performed to detect the accumulation of avidin as the pretargeting agent. Cytotoxicity was determined using the trypan blue exclusion assay, macrocolony, and MTT assay. Cell uptake studies were performed using radiolabeled DOTA-Triazole-Biotin and compared with avidin treated cells for 2 h. Tumor imaging was performed in U-87MG cell line implanted tumor bearing nude mice and uptake of the radiotracer was estimated.Results: All compounds have been successfully characterized by NMR and MS spectroscopy. More than 96% radiolabeling efficiency was obtained and the radioconjugate exhibited sufficient stability under physiological conditions.Conclusion: To summarize, a new candidate for avidin based two-step pretargeting of tumors has been synthesized and evaluated for potential imaging and diagnostic applications. The chelate possesses high stability under physiological conditions, exhibits effective interaction with its avidin target, and low nonspecific retention in vivo.