Technetium Nitrido Complexes of Tetradentate Thiosemicarbazones: Kit-Based Radiolabeling, Characterization, and In Vivo Evaluation.

Bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators have demonstrated utility in nuclear medicine. In particular, the 64Cu2+ complexes have been extensively developed for hypoxia imaging and molecular imaging of peptide and protein markers of disease. However, the chemistry and application of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators in combination with 99mTc, the most widely used radionuclide in nuclear medicine, is underexplored. Herein, a series of bis(thiosemicarbazone) and pyridylhydrazone-thiosemicarbazone chelators were radiolabeled with nitrido-technetium-99m in an optimized one-pot synthesis from [99mTc]TcO4-. Optimization of the radiochemical syntheses allowed for production of the complexes in >90% radiochemical conversion with apparent molar activities of 3.3-5 GBq/µmol. Competition experiments demonstrated the excellent stability of the complexes. The nitrido-technetium-99 complexes were synthesized, and the chemical identities were investigated using mass spectrometry, spectroscopy, and density functional theory calculations. Complexation of nitrido-rhenium(V) was achieved with the N4-dialkylated bis(thiosemicarbazones). Planar imaging and ex vivo biodistribution studies of the five 99mTc complexes were conducted on healthy BALB/c mice to determine in vivo behavior. The lipophilic nature of the complexes resulted in uptake of 1.6-5.7% ID g-1 in the brain at 2 min postinjection and retention of 0.4-1.7% ID g-1 at 15 min postinjection. The stability of the complexes and the biodistribution data demonstrate that these chelators are ideal platforms for future production of radiopharmaceutical candidates.

Pre-targeting amyloid-ß with antibodies for potential molecular imaging of Alzheimer's disease.

With the aim of developing the concept of pretargeted click chemistry for the diagnosis of Alzheimer's disease two antibodies specific for amyloid-ß were modified to incorporate trans-cyclooctene functional groups. Two bis(thiosemicarbazone) compounds with pendant 1,2,4,5-tetrazine functional groups were prepared and radiolabelled with positron emitting copper-64. The new copper-64 complexes rapidly react with the trans-cyclooctene functionalized antibodies in a bioorthogonal click reaction and cross the blood-brain barrier in mice.

A Biodistribution Study of the Radiolabeled Kv1.3-Blocking Peptide DOTA-HsTX1[R14A] Demonstrates Brain Uptake in a Mouse Model of Neuroinflammation.

The voltage-gated potassium channel Kv1.3 regulates the pro-inflammatory function of microglia and is highly expressed in the post-mortem brains of individuals with Alzheimer's and Parkinson's diseases. HsTX1[R14A] is a selective and potent peptide inhibitor of the Kv1.3 channel (IC50 ∼ 45 pM) that has been shown to decrease cytokine levels in a lipopolysaccharide (LPS)-induced mouse model of inflammation. Central nervous system exposure to HsTX1[R14A] was previously detected in this mouse model using liquid chromatography with tandem mass spectrometry, but this technique does not report on the spatial distribution of the peptide in the different brain regions or peripheral organs. Herein, the in vivo distribution of a [64Cu]Cu-labeled DOTA conjugate of HsTX1[R14A] was observed for up to 48 h by positron emission tomography (PET) in mice. After subcutaneous administration to untreated C57BL/6J mice, considerable uptake of the radiolabeled peptide was observed in the kidney, but it was undetectable in the brain. Biodistribution of a [68Ga]Ga-DOTA conjugate of HsTX1[R14A] was then investigated in the LPS-induced mouse model of neuroinflammation to assess the effects of inflammation on uptake of the peptide in the brain. A control peptide with very weak Kv1.3 binding, [68Ga]Ga-DOTA-HsTX1[R14A,Y21A,K23A] (IC50 ∼ 6 µM), was also tested. Significantly increased uptake of [68Ga]Ga-DOTA-HsTX1[R14A] was observed in the brains of LPS-treated mice compared to mice treated with control peptide, implying that the enhanced uptake was due to increased Kv1.3 expression rather than simply increased blood-brain barrier disruption. PET imaging also showed accumulation of [68Ga]Ga-DOTA-HsTX1[R14A] in inflamed joints and decreased clearance from the kidneys in LPS-treated mice. These biodistribution data highlight the potential of HsTX1[R14A] as a therapeutic for the treatment of neuroinflammatory diseases mediated by overexpression of Kv1.3.

Hexadentate technetium-99m bis(thiosemicarbazonato) complexes: synthesis, characterisation and biodistribution.

The syntheses of non-oxido/non-nitrido bis(thiosemicarbazonato)technetium(V) complexes featuring a series of alkyl and ether substituents is presented. The bis(thiosemicarbazones) were radiolabelled with technetium-99m using an optimised one-pot synthesis from [99mTc][TcO4]-. Mass spectrometry and computational chemistry data suggested a distorted trigonal prismatic coordination environment for the bis(thiosemicarbazonato)technetium(V) complexes by way of a bis(thiosemicarbazone)technetium(V)-oxido intermediate complex. The lipophilicities of the complexes were estimated using distribution ratios and three of the new complexes were investigated in mice using kinetic planar imaging and ex vivo biodistribution experiments and were compared to [99mTc][TcO4]-. Modification of the technetium complexes with various lipophilic functional groups altered the biodistributions of the complexes in mice despite evidence suggesting limited stability of the complexes to biologically relevant conditions. The most hydrophilic complex had higher uptake in the kidneys compared to the most lipophilic, which had higher liver uptake, suggesting modification of the excretion pathways.

A General Design Strategy Enabling the Synthesis of Hydrolysis-Resistant, Water-Stable Titanium(IV) Complexes.

Despite its prevalence in the environment, the chemistry of the Ti4+ ion has long been relegated to organic solutions or hydrolyzed TiO2 polymorphs. A knowledge gap in stabilizing molecular Ti4+ species in aqueous environments has prevented the use of this ion for various applications such as radioimaging, design of water-compatible metal-organic frameworks (MOFs), and aqueous-phase catalysis applications. Herein, we show a thorough thermodynamic screening of bidentate chelators with Ti4+ in aqueous solution, as well as computational and structural analyses of key compounds. In addition, the hexadentate analogues of catechol (benzene-1,2-diol) and deferiprone (3-hydroxy-1,2-dimethyl-4(1H)-pyridone), TREN-CAM and THPMe respectively, were assessed for chelation of the 45 Ti isotope (t1/2 =3.08 h, ß+ =85 %, Eß+ =439 keV) towards positron emission tomography (PET) imaging applications. Both were found to have excellent capacity for kit-formulation, and [45 Ti]Ti-TREN-CAM was found to have remarkable stability in vivo.

Modern Developments in Bifunctional Chelator Design for Gallium Radiopharmaceuticals.

The positron-emitting radionuclide gallium-68 has become increasingly utilised in both preclinical and clinical settings with positron emission tomography (PET). The synthesis of radiochemically pure gallium-68 radiopharmaceuticals relies on careful consideration of the coordination chemistry. The short half-life of 68 min necessitates rapid quantitative radiolabelling (≤10 min). Desirable radiolabelling conditions include near-neutral pH, ambient temperatures, and low chelator concentrations to achieve the desired apparent molar activity. This review presents a broad overview of the requirements of an efficient bifunctional chelator in relation to the aqueous coordination chemistry of gallium. Developments in bifunctional chelator design and application are then presented and grouped according to eight categories of bifunctional chelator: the macrocyclic chelators DOTA and TACN; the acyclic HBED, pyridinecarboxylates, siderophores, tris(hydroxypyridinones), and DTPA; and the mesocyclic diazepines.

The Synthesis of a Bis(thiosemicarbazone) Macrocyclic Ligand and the Mn(II), Co(II), Zn(II) and 68Ga(III) Complexes.

A 1,4,7,10-tetraazacyclododecane (cyclen) variant bearing two thiosemicarbazone pendant groups has been prepared. The ligand forms complexes with Mn2+, Co2+ and Zn2+. X-ray crystallography of the Mn2+, Co2+ and Zn2+ complexes showed that the ligand provides a six-coordinate environment for the metal ions. The Mn2+ and Zn2+ complexes exist in the solid state as racemic mixtures of the Δ(δ,δ,δ,δ)/Λ(λ,λ,λ,λ) and Δ(λ,λ,λ,λ)/Λ(δ,δ,δ,δ) diastereomers, and the Co2+ complex exists as the Δ(δ,δ,δ,δ)/Λ(λ,λ,λ,λ) and Δ(λ,λ,λ,δ)/Λ(δ,δ,δ,λ) diastereomers. Density functional theory calculations indicated that the relative energies of the diastereomers are within 10 kJ mol-1. Magnetic susceptibility of the complexes indicated that both the Mn2+ and Co2+ ions are high spin. The ligand was radiolabelled with gallium-68, in the interest of developing new positron emission tomography imaging agents, which produced a single species in high radiochemical purity (>95%) at 90 °C for 10 min.

An octadentate bis(semicarbazone) macrocycle: a potential chelator for lead and bismuth radiopharmaceuticals.

A variant of 1,4,7,10-tetraazacyclododecane (cyclen) bearing two semicarbazone pendant groups has been prepared. The octadentate ligand forms complexes with Bi3+ and Pb2+. X-ray crystallography showed that the neutral ligand provides an eight-coordinate environment for both metal ions and intermolecular hydrogen bond interactions have influenced the coordination environments of both complexes in the solid state. NMR spectroscopy revealed a fluxional environment for both complexes. The ligand was radiolabeled with the α-emitting radioactive isotope 213Bi3+, which is used in systemic targeted radiotherapy. The resulting complex was stable in serum for at least 90 min (two decay half-lives). The Pb2+ complex has reasonably fast kinetics of formation (t1/2 = 20 min) at 25 °C and pH 7.4. The Bi3+ and Pb2+ complexes show kinetic stability in 1.2 M HCl (half-lives of 214 min and 47 min, respectively). This is the first description of a macrocycle bearing semicarbazone pendant groups and its utility in coordinating main group metals, specifically those with radiotherapeutic potential.

Modulating Targeting of Poly(ethylene glycol) Particles to Tumor Cells Using Bispecific Antibodies.

Low-fouling or "stealth" particles composed of poly(ethylene glycol) (PEG) display a striking ability to evade phagocytic cell uptake. However, functionalizing them for specific targeting is challenging. To address this challenge, stealth PEG particles prepared by a mesoporous silica templating method are functionalized with bispecific antibodies (BsAbs) to obtain PEG-BsAb particles via a one-step binding strategy for cell and tumor targeting. The dual specificity of the BsAbs-one arm binds to the PEG particles while the other targets a cell antigen (epidermal growth factor receptor, EGFR)-is exploited to modulate the number of targeting ligands per particle. Increasing the BsAb incubation concentration increases the amount of BsAb tethered to the PEG particles and enhances targeting and internalization into breast cancer cells overexpressing EGFR. The degree of BsAb functionalization does not significantly reduce the stealth properties of the PEG particles ex vivo, as assessed by their interactions with primary human blood granulocytes and monocytes. Although increasing the BsAb amount on PEG particles does not lead to the expected improvement in tumor accumulation in vivo, BsAb functionalization facilitates tumor cell uptake of PEG particles. This work highlights strategies to balance evading nonspecific clearance pathways, while improving tumor targeting and accumulation.

Modification of Biodistribution and Brain Uptake of Copper Bis(thiosemicarbazonato) Complexes by the Incorporation of Amine and Polyamine Functional Groups.

The synthesis of new bis(thiosemicarbazonato)copper(II) complexes featuring polyamine substituents via selective transamination reactions is presented. Polyamines of different lengths, with different ionizable substituent groups, were used to modify and adjust the hydrophilic/lipophilic balance of the copper complexes. The new analogues were radiolabeled with copper-64 and their lipophilicities estimated using distribution coefficients. The cell uptake of the new polyamine complexes was investigated with preliminary in vitro biological studies using a neuroblastoma cancer cell line. The in vivo biodistribution of three of the new analogues was investigated in vivo in mice using positron-emission tomography imaging, and one of the new complexes was compared to [64Cu]Cu(atsm) in an A431 squamous cell carcinoma xenograft model. Modification of the copper complexes with various amine-containing functional groups alters the biodistribution of the complexes in mice. One complex, with a pendent ( N, N-dimethylamino)ethane functional group, displayed tumor uptake similar to that of [64Cu]Cu(atsm) but higher brain uptake, suggesting that this compound has the potential to be of use in the diagnostic brain imaging of tumors and neurodegenerative diseases.

Tuning the properties of tris(hydroxypyridinone) ligands: efficient 68Ga chelators for PET imaging.

The prototype tris(1,6-dimethyl-3-hydroxypyridin-4-one) chelator for gallium-68, THPMe, has shown great promise for rapid and efficient kit-based 68Ga labelling of PET radiopharmaceuticals. Peptide derivatives of THPMe have been used to image expression of their target receptors in vivo in preclinical and clinical studies. Herein we describe new synthetic routes to the THP platform including replacing the 1,6-dimethyl-3-hydroxypyridin-4-one N1-CH3 group of THPMe with O (tris(6-methyl-3-hydroxypyran-4-one, THPO) and N1-H (tris(6-methyl-3-hydroxypyridin-4-one), THPH) groups. The effect of these structural modifications on lipophilicity, gallium binding and metal ion selectivity was investigated. THPH was able to bind 68Ga in extremely mild conditions (5 min, room temperature, pH 6, 1 µM ligand concentration) and, notably, in vivo, when administered to a mouse previously injected with 68Ga acetate. The 67Ga radiolabelled complex was stable in serum for more than 7 days. [68Ga(THPH)] displayed a log P value of -2.40 ± 0.02, less negative than the log P = -3.33 ± 0.02 measured for [68Ga(THPMe)], potentially due to an increase in intramolecular hydrogen bonding attributable to the N1-H pyridinone units. Spectrophotometric determination of the Ga3+/Fe3+ complex formation constants for both THPMe and THPH revealed their preference for binding Ga3+ over Fe3+, which enabled selective labelling with 68Ga3+ in the presence of a large excess of Fe3+ in both cases. Compared to THPMe, THPH showed significantly reduced affinity for Fe3+, increased affinity for Ga3+ and improved radiolabelling efficiency. THPO was inferior to both THPH and THPMe in terms of labelling efficiency, but its benzylated precursor Bn-THPO (tris(6-methyl-3-benzyloxypyran-4-one)) provides a potential platform for the synthesis of a library of THP compounds with tunable chemical properties and metal preferences.

64Cu-SARTATE PET Imaging of Patients with Neuroendocrine Tumors Demonstrates High Tumor Uptake and Retention, Potentially Allowing Prospective Dosimetry for Peptide Receptor Radionuclide Therapy.

Imaging of somatostatin receptor expression is an established technique for staging of neuroendocrine neoplasia and determining the suitability of patients for peptide receptor radionuclide therapy. PET/CT using 68Ga-labeled somatostatin analogs is superior to earlier agents, but the rapid physical decay of the radionuclide poses logistic and regulatory challenges. 64Cu has attractive physical characteristics for imaging and provides a diagnostic partner for the therapeutic radionuclide 67Cu. Based on promising preclinical studies, we have performed a first-time-in-humans trial of 64Cu-MeCOSar-Tyr3-octreotate (64Cu-SARTATE) to assess its safety and ability to localize disease at early and late imaging time-points. Methods: In a prospective trial, 10 patients with known neuroendocrine neoplasia and positive for uptake on 68Ga-DOTA-octreotate (68Ga-DOTATATE) PET/CT underwent serial PET/CT imaging at 30 min, 1 h, 4 h, and 24 h after injection of 64Cu-SARTATE. Adverse reactions were recorded, and laboratory testing was performed during infusion and at 1 and 7 d after imaging. Images were analyzed for lesion and normal-organ uptake and clearance to assess lesion contrast and perform dosimetry estimates. Results:64Cu-SARTATE was well tolerated during infusion and throughout the study, with 3 patients experiencing mild infusion-related events. High lesion uptake and retention were observed at all imaging time-points. There was progressive hepatic clearance over time, providing the highest lesion-to-liver contrast at 24 h. Image quality remained high at this time. Comparison of 64Cu-SARTATE PET/CT obtained at 4 h to 68Ga-DOTATATE PET/CT obtained at 1 h indicated comparable or superior lesion detection in all patients, especially in the liver. As expected, the highest early physiologic organ uptake was in the kidneys, liver, and spleen. Conclusion:64Cu-SARTATE is safe and has excellent imaging characteristics. High late-retention in tumor and clearance from the liver suggest suitability for diagnostic studies and for prospective dosimetry for 67Cu-SARTATE peptide receptor radionuclide therapy, and the half-life of 64Cu would also facilitate good-manufacturing-practice production and distribution to sites without access to 68Ga.

Guest-induced Assembly of Bis(thiosemicarbazonato) Zinc(II) Coordination Nanotubes.

A ZnII complex of the dianionic tetradentate ligand formed by deprotonation of glyoxal-bis(4-phenyl-3-thiosemicarbazone) (H2 gtsp) is a [3+3] trinuclear triangular prism. Recrystallization of this complex in the presence of either CO2 , CS2 , or CH3 CN leads to the formation of [4+4] open-ended charge-neutral tetranuclear coordination nanotubes, approximately 2 nm in length and with internal dimensions large enough to accommodate linear guest molecules, which serve to template their formation. Upon removal of the templating molecules the nanotubes demonstrated reversible sorption of CO2 with an isosteric enthalpy of sorption of 28 kJ mol-1 at low loading.

Comparison of macrocyclic and acyclic chelators for gallium-68 radiolabelling.

Gallium-68 (68Ga) is a positron-emitting isotope used for clinical PET imaging of peptide receptor expression. 68Ga radiopharmaceuticals used in molecular PET imaging consist of disease-targeting biomolecules tethered to chelators that complex 68Ga3+. Ideally, the chelator will rapidly, quantitatively and stably coordinate 68Ga3+ at room temperature, near neutral pH and low chelator concentration, allowing for simple routine radiopharmaceutical formulation. Identification of chelators that fulfil these requirements will facilitate development of kit-based 68Ga radiopharmaceuticals. Herein the reaction of a range of widely used macrocyclic and acyclic chelators with 68Ga3+ is reported. Radiochemical yields have been measured under conditions of varying chelator concentrations, pH (3.5 and 6.5) and temperature (25 and 90 °C). These chelators are: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane macrocycles substituted with phosphonic (NOTP) and phosphinic (TRAP) groups at the amine, bis(2-hydroxybenzyl)ethylenediaminediacetic acid (HBED), a tris(hydroxypyridinone) containing three 1,6-dimethyl-3-hydroxypyridin-4-one groups (THP) and the hexadentate tris(hydroxamate) siderophore desferrioxamine-B (DFO). Competition studies have also been undertaken to assess relative complexation efficiencies of each chelator for 68Ga3+ under different pH and temperature conditions. Performing radiolabelling reactions at pH 6.5, 25 °C and 5-50 µM chelator concentration resulted in near quantitative radiochemical yields for all chelators, except DOTA. Radiochemical yields either decreased or were not substantially improved when the reactions were undertaken at lower pH or at higher temperature, except in the case of DOTA. THP and DFO were the most effective 68Ga3+ chelators at near-neutral pH and 25 °C, rapidly providing near-quantitative radiochemical yields at very low chelator concentrations. NOTP and HBED were only slightly less effective under these conditions. In competition studies with all other chelators, THP demonstrated highest reactivity for 68Ga3+ complexation under all conditions. These data point to THP possessing ideal properties for rapid, one-step kit-based syntheses of 68Ga-biomolecules for molecular PET imaging. LC-MS and 1H, 13C{1H} and 71Ga NMR studies of HBED complexes of Ga3+ showed that under the analytical conditions employed in this study, multiple HBED-bound Ga complexes exist. X-ray diffraction data indicated that crystals isolated from these solutions contained octahedral [Ga(HBED)(H2O)], with HBED coordinated in a pentadentate N2O3 mode, with only one phenolic group coordinated to Ga3+, and the remaining coordination site occupied by a water molecule.

Highly Sensitive Detection of Minimal Cardiac Ischemia using Positron Emission Tomography Imaging of Activated Platelets.

A reliable method for the diagnosis of minimal cardiac ischemia would meet a strong demand for the sensitive diagnosis of coronary artery disease in cardiac stress testing and risk stratification in patients with chest pain but unremarkable ECGs and biomarkers. We hypothesized that platelets accumulate early on in ischemic myocardium and a newly developed technology of non-invasive molecular PET imaging of activated platelets can thus detect minimal degrees of myocardial ischemia. To induce different degrees of minimal cardiac ischemia, the left anterior descending artery (LAD) was ligated for 10, 20 or 60 min. Mice were injected with a newly generated scFvanti-GPIIb/IIIa-64CuMeCOSar radiotracer, composed of a single-chain antibody that only binds to activated integrin GPIIb/IIIa (αIIbßIII) and thus to activated platelets, and a sarcophagine cage MeCOSar complexing the long half-life PET tracer copper-64. A single PET/CT scan was performed. Evans Blue/TTC staining to detect necrosis as well as classical serological biomarkers like Troponin I and heart-type fatty acid-binding protein (H-FABP) were negative, whereas PET imaging of activated platelets was able to detect small degrees of ischemia. Taken together, molecular PET imaging of activated platelets represents a unique and highly sensitive method to detect minimal cardiac ischemia.

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer.

The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.

The ionic charge of copper-64 complexes conjugated to an engineered antibody affects biodistribution.

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

Engineering poly(ethylene glycol) particles for improved biodistribution.

We report the engineering of poly(ethylene glycol) (PEG) hydrogel particles using a mesoporous silica (MS) templating method via tuning the PEG molecular weight, particle size, and the presence or absence of the template and investigate the cell association and biodistribution of these particles. An ex vivo assay based on human whole blood that is more sensitive and relevant than traditional cell-line based assays for predicting in vivo circulation behavior is introduced. The association of MS@PEG particles (template present) with granulocytes and monocytes is higher compared with PEG particles (template absent). Increasing the PEG molecular weight (from 10 to 40 kDa) or decreasing the PEG particle size (from 1400 to 150 nm) reduces phagocytic blood cell association of the PEG particles. Mice biodistribution studies show that the PEG particles exhibit extended circulation times (>12 h) compared with the MS@PEG particles and that the retention of smaller PEG particles (150 nm) in blood, when compared with larger PEG particles (>400 nm), is increased at least 4-fold at 12 h after injection. Our findings highlight the influence of unique aspects of polymer hydrogel particles on biological interactions. The reported PEG hydrogel particles represent a new class of polymer carriers with potential biomedical applications.

Tripodal tris(hydroxypyridinone) ligands for immunoconjugate PET imaging with (89)Zr(4+): comparison with desferrioxamine-B.

Due to its long half-life (78 h) and decay properties (77% electron capture, 23% ß(+), Emax = 897 keV, Eav = 397 keV, Eγ = 909 keV, Iγ = 100%) (89)Zr is an appealing radionuclide for immunoPET imaging with whole IgG antibodies. Derivatives of the siderophore desferrioxamine-B (H3DFO) are the most widely used bifunctional chelators for coordination of (89)Zr(4+) because the radiolabeling of the resulting immunoconjugates is rapid under mild conditions. (89)Zr-DFO complexes are reportedly stable in vitro but there is evidence that (89)Zr(4+) is released in vivo, and subsequently taken up by the skeleton. We have evaluated a novel tripodal tris(hydroxypyridinone) chelator, H3CP256 and its bifunctional maleimide derivative, H3YM103, for coordination of Zr(4+) and compared the NMR spectra, and the (89)Zr(4+) radiolabeling, antibody conjugation, serum stability and in vivo distribution of radiolabelled immunoconjugates with those of H3DFO and its analogues. H3CP256 coordinates (89)Zr(4+) at carrier-free concentrations forming [(89)Zr(CP256)](+). Both H3DFO and H3CP256 were efficiently radiolabelled using [(89)Zr(C2O4)4](4-) at ambient temperature in quantitative yield at pH 6-7 at millimolar concentrations of chelator. Competition experiments demonstrate that (89)Zr(4+) dissociates from [(89)Zr(DFO)](+) in the presence of one equivalent of H3CP256 (relative to H3DFO) at pH 6-7, resulting largely in [(89)Zr(CP256)](+). To assess the stability of H3DFO and H3YM103 immunoconjugates radiolabelled with (89)Zr, maleimide derivatives of the chelators were conjugated to the monoclonal antibody trastuzumab via reduced cysteine side chains. Both immunoconjugates were labelled with (89)Zr(4+) in >98% yield at high specific activities and the labeled immunoconjugates were stable in serum with respect to dissociation of the radiometal. In vivo studies in mice indicate that (89)Zr(4+) dissociates from YM103-trastuzumab with significant amounts of activity becoming associated with bones and joints (25.88 ± 0.58% ID g(-1) 7 days post-injection). In contrast, <8% ID g(-1) of (89)Zr activity becomes associated with bone in animals administered (89)Zr-DFO-trastuzumab over the course of 7 days. The tris(hydroxypyridinone) chelator, H3CP256, coordinates (89)Zr(4+) rapidly under mild conditions, but the (89)Zr-labelled immunoconjugate, (89)Zr-YM103-trastuzumab was observed to release appreciable amounts of (89)Zr(4+)in vivo, demonstrating inferior stability when compared with (89)Zr-DFO-trastuzumab. The significantly lower in vivo stability is likely to be a result of lower kinetic stability of the Zr(4+) tris(hydroxypyridinone complex) relative to that of DFO and its derivatives.