A novel cholic acid-based contrast enhancement agent for targeted MRI

The novel Gd(III) complexes of heptadentate ligands NE3TA and NE3TA-Bn were prepared, and their relaxivities were measured and favorably compared to the commercially available MRI contrast enhancement agent Gd(DOTA). NE3TA was conjugated with cholic acid (CA) to produce CA-NE3TA. TEM images of Gd(CA-NE3TA) indicate that the complex self-assembles forming nano-sized micelles and displays an over threefold increased relaxivity compared to Gd(DOTA). The new cholic acid-conjugated nanoparticle MR contrast enhancement agent, Gd(CA-NE3TA) possesses great promise for use in targeted MRI.     

Novel bimodal bifunctional ligands for radioimmunotherapy and targeted MRI

The structurally novel bifunctional ligands C-NETA and C-NE3TA, each possessing both acyclic and macrocyclic moieties, were prepared and evaluated as potential chelates for radioimmunotherapy (RIT) and targeted magnetic resonance imaging (MRI). Heptadentate C-NE3TA was fortuitously discovered during the preparation of C-NETA. An optimized synthetic method to C-NETA and C-NE3TA including purification of the polar and tailing reaction intermediates, tert-butyl C-NETA (2) and tert-butyl C-NE3TA (3) using semiprep HPLC was developed. The new Gd(III) complexes of C-NETA and C-NE3TA were prepared as contrast enhancement agents for use in targeted MRI. The T 1 relaxivity data indicate that Gd(C-NETA) and Gd(C-NE3TA) possess higher relaxivity than Gd(C-DOTA), a bifunctional version of a commercially available MRI contrast agent; Gd(DOTA). C-NETA and C-NE3TA were radiolabeled with (177)Lu, (90)Y, (203)Pb, (205/6)Bi, and (153)Gd; and in vitro stability of the radiolabeled corresponding complexes was assessed in human serum. The in vitro studies indicate that the evaluated radiolabeled complexes were stable in serum for 11 days with the exception being the (203)Pb complexes of C-NETA and C-NE3TA, which dissociated in serum. C-NETA and C-NE3TA radiolabeled (177)Lu, (90)Y, or (153)Gd complexes were further evaluated for in vivo stability in athymic mice and possess excellent or acceptable in vivo biodistribution profile. (205/6)Bi- C-NE3TA exhibited extremely rapid blood clearance and low radioactivity level at the normal organs, while (205/6)Bi- C-NETA displayed low radioactivity level in the blood and all of the organs except for the kidney where relatively high renal uptake of radioactivity is observed. C-NETA and C-NE3TA were further modified for conjugation to the monoclonal antibody Trastuzumab.     

In vivo behavior of copper-64-labeled methanephosphonate tetraaza macrocyclic ligands

Copper-64 ( T(1/2)=12.7 h; beta(+): 0.653 MeV, 17.4%; beta(-): 0.578 MeV, 39%) is produced in a biomedical cyclotron and has applications in both imaging and therapy. Macrocyclic chelators are widely used as bifunctional chelators to bind copper radionuclides to antibodies and peptides owing to their relatively high kinetic stability. In this paper, we evaluated three tetraaza macrocyclic ligands with two, three, and four pendant methanephosphonate functional groups. DO2P [1,4,7,10-tetraazacyclododecane-1,7-di(methanephosphonic acid)], DO3P [1,4,7,10-tetraazacyclododecane-1,4,7-tri(methanephosphonic acid)], and DOTP [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid)] were all radiolabeled with (64)Cu in high radiochemical yields. Copper-64-labeled DO2P and DOTP were highly stable in rat serum out to 24 h, while (64)Cu-DO3P remained 73% intact, with the remainder possibly forming a (64)Cu(.)2DO3P dimer by 24 h. The biodistribution experiments were performed in normal Sprague-Dawley rats. Of the three complexes, (64)Cu-DO2P demonstrated the most optimal clearance through the blood and liver. Copper-64-DO3P and (64)Cu-DOTP exhibited higher liver uptake and longer retention of liver activity, possibly because of the large negative charge of the complexes under physiological conditions. All three (64)Cu-labeled complexes showed high accumulation in bone, likely due to the binding of the methanephosphonate groups to hydroxyapatite. These results suggest that this series of methanephosphonate macrocyclic ligands may be useful as potential bone-imaging agents. The thermodynamic stability constants of the Cu(II) complexes with these three ligands were determined, and were found to be significantly higher than those of their acetate analogues. The Cu(II)-DO2P complex exhibited the highest stability constant among divalent transition metal ion DO2P complexes. Metabolism studies of (64)Cu-DO2P in rat liver suggest that the DO2P ligand may be used as a bifunctional chelator for copper radionuclides in radiodiagnostic or radiotherapeutic studies.     

Binding of dexamethasone to rat liver nuclei in vivo and in vitro: evidence for two distinct binding sites

The binding of [3H]dexamethasone (DEX) to rat liver nuclei in vitro and in vivo have been compared. In vitro, purified nuclei displayed a single class of specific glucocorticoid binding sites with a dissociation constant (Kd) of approximately 10(-7) M for [3H]DEX at 4 degrees C. The glucocorticoid agonists prednisolone, cortisol, and corticosterone and the antagonists progesterone and cortexolone competed avidly for this site, but the potent glucocorticoid triamcinolone acetonide (TA) competed poorly in vitro. Nuclei isolated from the livers of intact rats contained 1-2 X 10(4) [3H]DEX binding sites/nucleus. Up to 85% of the binding sites were recovered in the nuclear envelope (NE) fraction when NE were prepared either before or after labeling with [3H]DEX in vitro. After adrenalectomy, the specific [3H]DEX binding capacity of both nuclei and NE decreased to 15-20% of control values, indicating sensitivity of the binding sites to hormonal status of the animals. Efforts to restore the binding capacity by administration of exogenous glucocorticoids, however, were unsuccessful. After labeling of rat liver nuclei in vivo by intraperitoneal injection of [3H]DEX or [3H]TA into living animals, the steroid specificity and subnuclear localization of radiolabel were different. Both [3H]TA (which did not bind in vitro) and [3H]DEX became localized to nuclei in a saturable fashion in vivo. With either of these ligands, approximately 20% of the total nuclear radiolabel was recovered in the NE fraction. These results suggest the presence of two separate and distinct binding sites in rat liver nuclei, one which is localized to the NE and binds [3H]DEX (but not [3H]TA) in vitro, and another which is not localized to the NE but binds [3H]DEX and [3H]TA in vivo.     

Complexation of the fungal metabolite tenuazonic acid with copper (II), iron (III), nickel (II), and magnesium (II) ions

Tenuazonic acid (TA) is a phytotoxin produced by a fungal pathogen of rice, Pyricularia oryzae. We have synthesized and characterized the metal complexes of TA with copper (II), iron (III), nickel (II), and magnesium (II). The stoichiometry of the complexes determined by microanalysis and mass spectroscopy (D/CI) are Cu(II)TA2, Fe(III)TA3, Ni(II)TA2, and Mg(TA)2. Voltammograms of Fe(III)TA3, and Cu(II)TA2 in methanolic solutions confirmed this stoichiometry. Ni(II)TA2 paramagnetism and visible absorption data suggest an octahedral geometry. Fe(III)TA3 showed a characteristic visible absorption at 450 nm. Addition of Fe(III)Cl3 and Mg(II)Cl2 did not reverse the toxicity of NaTA to rice and bacterial cells, showing that this toxicity is not due to the privation of the cells of these metals essential for cell growth.     

Novel Radiolabeled Peptides for Breast and Prostate Tumor PET Imaging: (64)Cu/and (68)Ga/NOTA-PEG-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]BBN(6-14)

Bombesin (BBN)-based radiolabeled peptides exhibit promising properties for targeted imaging of gastrin-releasing peptide receptors (GRPR)-positive tumors. The aim of this study was to evaluate with positron emission tomography (PET) the pharmacokinetic and imaging properties of two novel BBN-based radiolabeled peptides, (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14), for diagnosis of breast and prostate cancers using small animal models. Competitive binding assays on T47D breast and PC3 prostate cancer cells showed that the affinity for GRPR depends on the complexed metal and can vary up to a factor of about 3; (64)Cu/NOTA-PEG-BBN(6-14) was found to have the lowest inhibition constant (1.60 ± 0.59 nM). (64)Cu/and (68)Ga/NOTA-PEG-BBN(6-14) presented similar cell uptake on T47D and PC3 cells and were stable in vivo. Biodistribution studies of radiolabeled peptides carried out in Balb/c and tumor-bearing Balb/c nude mice showed that (64)Cu/NOTA-PEG-BBN(6-14) presented higher GRPR-mediated uptake in pancreas and adrenal glands, but comparable PC3 tumor uptake as (68)Ga/NOTA-PEG-BBN(6-14). Finally, receptor-dependent responses were observed during blocking studies with unlabeled peptide in both biodistribution and small-animal PET imaging studies. Our results confirmed the dependence of the affinity and pharmacokinetics of BBN-based radiopeptides on the complexed radiometal. Interspecies differences between mouse and human GRPR binding properties were also noted in these preclinical studies. Considering their good imaging characteristics, both (64)Cu/NOTA-PEG-BBN(6-14) and (68)Ga/NOTA-PEG-BBN(6-14) are promising candidates for GRPR-targeted PET imaging of breast and prostate cancers.     

Re-evaluating the effects of organic ligands on copper toxicity to barley root elongation in culture solution

Abstract

In the presence of weak ligands, both free ion activity and organic complexes of Cu should b considered when predicting Cu toxicity in aquatic and soil-plant systems. However, there is littl information about the quantitative contribution of Cu that is organically complexed to Cu toxicity. In thi study, a bioassay using barley root elongation in culture solution was used to investigate the effects o organic ligands with different conditional stability constants on Cu toxicity and the quantitativ contribution of the organically complexed Cu to the Cu toxicity. The results indicated that a significan decrease (p<0.05) in Cu toxicity, assessed by barley root elongation, was observed in response to th addition of organic ligands. The decrease differed, to some extent, with different organic ligands o disodium ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), oxalate and malate at low and constant free Cu²⁺ activity. Addition of EDTA or NTA resulted in strong reduction of Cu toxicity while modest reduction of Cu toxicity was observed for the addition of malate as the relatively wea ligand. Furthermore, the results of the present study revealed that the CuNTA^? and CuEDTA^2? complexes were not toxic, while the Cu–malate complexes were mildly toxic to barley root elongation More importantly, it was found that the toxicity of Cu–malate complexes were nearly 0.5-fold less than that of free Cu²⁺ ions.     

In vivo targeting of HER2-positive tumor using 2-helix affibody molecules

Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2<2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA-MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA-MUT-DS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA-MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA-MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA-MUT-DS was 4.66±0.38 or 2.17±0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n=3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA-MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA-MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA-MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA-MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.     

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, (64)Cu) 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 (64)Cu-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 (64)Cu.     

New macrobicyclic chelator for the development of ultrastable 64Cu-radiolabeled bioconjugate

Ethylene cross-bridged cyclam with two acetate pendant arms, ECB-TE2A, is known to form the most kinetically stable (64)Cu complexes. However, its usefulness as a bifunctional chelator is limited because of its harsh radiolabeling conditions. Herein, we report new cross-bridged cyclam chelator for the development of ultrastable (64)Cu-radiolabeled bioconjugates. Propylene cross-bridged TE2A (PCB-TE2A) was successfully synthesized in an efficient way. The Cu(II) complex of PCB-TE2A exhibited much higher kinetic stability than ECB-TE2A in acid decomplexation studies, and also showed high resistance to reduction-mediated demetalation. Furthermore, the quantitative radiolabeling of PCB-TE2A with (64)Cu was achieved under milder conditions compared to ECB-TE2A. Biodistribution studies strongly indicate that the (64)Cu complexes of PCB-TE2A cleared out rapidly from the body with minimum decomplexation.     

Antifungal potential of binary and mixed-ligand complexes of N,2'-diphenyl acetohydroxamic acid.

Chelating potential of N,2'-DPAHA with 3d metal ions such as Cu(II), Ni(II), Zn(II), and Cd(II) in the presence of Gly and Phen has been investigated. These experiments were designed to study the role of the stability of mixed-ligand complexes in the modulation of its fungicidal potential. The mixed-ligand complexes were found to be more stable than binary complexes. Enhanced stability of mixed-ligand complexes of Ni(II), Co(II), Zn(II), and Cd(II) is presumably due to pi-bonding effects. In the stabilization of the Cu(II) mixed-ligand complex system, the Jahn-Tellar effect may play a vital role, in addition to pi-bonding effects. Fungicidal activity of N,2'-DPAHA and its binary complexes with Cu(II), Ni(II), and Co(II) was examined against Fusarium oxysporum using the inhibition zone technique. Binary complexes of Zn(II) and Cd(II) with N,2'-DPAHA and mixed-ligand complexes M(II)-Gly or Phen-N,2'-DPAHA, where M(II) = Cu(II), Ni(II), Zn(II), Co(II), and Cd(II) were screened against Alternaria alternata by slide germination technique. All mixed-ligand complexes exhibited fungicidal activity but did not improve significantly compared to binary complexes. Synergistic action of primary and secondary ligands has increased the stability of the mixed-ligand complex compared to the binary complex (1:1) of the secondary ligand (N,2'-DPAHA), and the fungicidal potential of the mixed-ligand complex involving N,2'-DPAHA as secondary ligand was not increased.     

Mn2+, Co2+, Cu2+ and Zn2+ complexes with two macrocyclic ligands bearing L-lactate-like functions: potentiometric studies and evaluation of superoxide-scavenging properties of the Mn2+ complex

Some aerobic organisms devoid of SOD use Mn2+ chelates to scavenge the O2- radical. Since the Mn2+-bis(lactato)diaquo complex is known as having a high SOD-like activity, we prepared manganese(II) complexes with triazamacrocyclic ligands bearing L-lactate-like functions in order to obtain model compounds able to disproportionate the superoxide radical. Thus, two macrocyclic ligands, N,N',N"-tris[2(S)-hydroxybutyric acid]-1,4,7-triazacyclononane, L1, and N,N',N"-tris[2(S)-hydroxybutyric acid]-1,5,9-triazacyclododecane, L2, were prepared and their capacity to retain the Mn2+ ion in aqueous solution was determined from potentiometric experiments. The chelating properties in aqueous solution of each ligand towards Co2+, Cu2+ and Zn2+ ions were also determined. L1 forms complexes with Mn2+, Co2+, Cu2+ and Zn2+ ions with stability constants of 8.33(5), 15.78(5), 17.65(3) and 14.32(1), respectively. L2 forms complexes with Cu2+ and Zn2+ ions with stability constants of 10.67(1) and 6.98(3), respectively. But the constants related to the Mn2+ and Co2+ complexes were too low to be determined by the method used. The stability constants values calculated for L2 complexes are significantly lower than those for the corresponding complexes of L1. Additional spectroscopic measurements were carried out on the Mn2+-L1 system. The electronic spectrum of this system showed a pH-dependence that may be consistent with the formation of hydroxo-species as the ESR spectra recorded at 120 K did not show oxidation of the Mn2+ ion in the pH range studied. The superoxide-scavenging activity of the manganese(II)-L1 complex was investigated using the cytochrome c assay. The Mn2+-L1 system showed an IC50 value of 1.7 microM which indicates that it appears as a potent SOD mimic.     

Copper(II) complexes of a hydroxamic acid from maize

The thermodynamics of formation for DIMBOA-Cu(II) complexes (where DIMBOA = 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-4H-one, a hydroxamic acid from maize) has been investigated in aqueous solutions by a potentiometric method. DIMBOA forms 1:1 and 1:2 chelates with Cu(II) at ionic strength 0.05 M (NaCl04). The stability constants measured were about 10⁵ and 10⁴ for the 1:1 and 1:2 complexes respectively, determined at 10, 20 and 30°. The contribution of ΔH and ΔS to the stability of complexes is examined and the pK values are compared with other ligands found in maize. Although DIMBOA has similar or higher constants to form copper complexes than other plant ligands, its possible role as a transport agent in maize remains to be established.     

Synthesis,characterization and in vitro cytotoxic,mutagenic and antimicrobial activity of platinum(II) complexes with substituted benzimidazole ligands

In this study, six Pt(II) complexes bearing 5(6)-H or -CH(3)-2-phenyl or -(2'-pyridyl) or -mercaptomethylbenzimidazole ligands as 'carrier groups' were synthesized and characterized by elemental analysis, IR and (1)H-NMR spectra and evaluated for their preliminary in vitro cytotoxic activity to the human RD Rhabdomyosarcoma cell line and mutagenic properties in Salmonella typhimurium strains TA 98 and TA 100 in the absence of the S9 rat liver fraction. The preliminary test results showed that the complexes had slightly greater cytotoxic activity on the RD cell line at 1 microM concentration than cisplatin. Among the compounds tested for their mutagenicity, Pt(II) complexes of 2-(2'-pyridyl)- and 5(6)-methyl-2-(2'-pyridyl)benzimidazoles were found to be mutagenic. A comparative study of the MIC (minimum inhibitory concentration) values indicated that, in general, there were no differences between the poor antimicrobial activity values of the ligands and their Pt(II) complexes with respect to the tested microorganisms. These results suggest that the synthesized Pt(II) complexes should be considered for further antitumor activity studies.     

Antibacterial dimeric copper(II) complexes with chromone-derived compounds

A new series of six chromone-derived compounds and their Cu(II) complexes have been synthesized and characterized by their physical, spectral and analytical data. The ligands and their Cu(II) complexes were screened for their in vitro antibacterial activity against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri) and two Gram-positive (Bacillus subtilis, Staphylococcus aureus) bacterial strains by agar-well diffusion method. The ligands were found to exhibit either no or low-to-moderate activities against one or more bacterial species whereas, the Cu(II) complexes exhibited moderate-to-high activity. The ligands which were inactive before complexation became active upon complexation with the Cu(II) metal ion and less active became more active.     

Synthesis of novel chiral metal complexes derived from chiral thiosemicarbazide ligands as potential antioxidant agents

Two new chiral thiosemicarbazide ligands and their Cu (II), Ni (II), Pd (II), and Zn (II) complexes were synthesized and characterized by nuclear magnetic resonance (NMR) (only for ligand), Fourier transform infrared (FT‐IR), ultraviolet visible (UV‐Vis), mass, and elemental analysis. The antioxidant activity of ligands and their metal complexes was examined. It was found that the antioxidant activity of metal complexes was better than their ligands. In addition, the antioxidant activity, as reflected by free radical scavenging, was evaluated. Besides, Pd (II) complexes exhibited better antioxidant activity than Ni (II), Cu (II), and Zn (II) complexes. Therefore, complexes (3a‐Pd and 3b‐Pd) can be used as an antioxidant agent or antioxidant test standard.     

Evaluation of 64Cu-labeled bifunctional chelate-bombesin conjugates

Several bifunctional chelates (BFCs) were investigated as carriers of (64)Cu for PET imaging. The most widely used chelator for (64)Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N,N',N″,N'-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with (64)Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H(2)N-Aoc-[d-Tyr(6),βAla(11),Thi(13),Nle(14)]bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H(2)N-Bn-PCTA(Ot-Bu)(3) or p-H(2)N-Bn-DO3A(Ot-Bu)(3)) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with (64)Cu using [(64)Cu]Cu(OAc)(2) in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN (64)Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the (64)Cu/Oxo-DO3A complex remained intact after 20 h while the (64)Cu/DOTA-BBN complex was completely demetalated. In contrast, both (64)Cu/NOTA- and (64)Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that (64)Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor.     

Metal complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid and benzohydroxamic acid. Crystal and molecular structure of [Cu(phen)2(Cl)]Cl x H2Sha, a model for a peroxidase-inhibitor complex

Stability constants of iron(III), copper(II), nickel(II) and zinc(II) complexes of salicylhydroxamic acid (H2Sha), anthranilic hydroxamic acid (HAha) and benzohydroxamic acid (HBha) have been determined at 25.0 degrees C, I=0.2 mol dm(-3) KCl in aqueous solution. The complex stability order, iron(III) > copper(II) > nickel(II) approximately = zinc(II) was observed whilst complexes of H2Sha were found to be more stable than those of the other two ligands. In the preparation of ternary metal ion complexes of these ligands and 1,10-phenanthroline (phen) the crystalline complex [Cu(phen)2(Cl)]Cl x H2Sha was obtained and its crystal structure determined. This complex is a model for hydroxamate-peroxidase inhibitor interactions.     

Copper(II) Complexes Derived from Schiff Bases Containing 4-Methylbenzylamine as a Core Unit: Cytotoxicity,pBR322-DNA Studies,Biological Assays,and Quantum Chemical Parameters

Bivalent copper complexes, [Cu(SB¹)₂] 1 (SB¹=(2-(4-methylbenzylimino)methyl)-5-methylphenol, [Cu(SB²)₂] 2 (SB²=(2-(4-methylbenzylimino)methyl)-4-bromolphenol), and [Cu(SB³)₂] 3 (SB³=(2-(4-methylbenzylimino)methyl)-4,6-dibromophenol) were synthesized using the Schiff bases prepared from 4-methylbenzylamine (p-tolylmethanamine). These were characterized using a variety of spectro-analytical methods. For all copper complexes, a square planar geometry was determined through spectral analyses. Utilizing molecular orbital energies, the stability of the copper complexes was calculated from quantum chemical characteristics. The kinetic and thermal degradation parameters were calculated from the thermograms. Studies on DNA binding interactions, such as UV absorption and emission, have shown that the manner of DNA binding is intercalative, and the binding constant (K_b) order is 3 > 2 > 1 . Under oxidative and photolytic techniques, the copper complexes outperform the parent Schiff bases in their ability to cleave double-stranded pBR322 DNA. When tested for cytotoxicity on the KB3 and MCF7 cell lines, complexes displayed greater activity than their parent ligands. Studies on the complexes′ in-vitro antibacterial and antioxidant activity showed that they are significantly more powerful than the parent ligands.     

Biological stability evaluation of the α2β1 receptor imaging agents: diamsar and DOTA conjugated DGEA peptide

Robust chelating stability under biological condi-tions is critical for the design of copper-based radiopharmaceuticals. In this study, the stabilities of (64)Cu-DOTA and diamsar (two bifunctional Cu-64 chelators (BFCs)) conjugated DGEA peptides were evaluated. The in vitro stabilities of (64)Cu-DOTA-DGEA, (64)Cu-DOTA-Ahx-DGEA, and (64)Cu-Z-E(diamsar)-Ahx-DGEA were evaluated in PBS. A carboxyl-protected DOTA-DGEA was also synthesized to study the potential inter- and intramolecular interactions between DOTA and the carboxylate groups of DGEA peptide. microPET imaging of (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA were performed in PC-3 prostate tumor model to further investigate the in vivo behavior of the tracers. DOTA-DGEA, DOTA-Ahx-DGEA, Z-E(diamsar)-Ahx-DGEA, and protected DOTA-DGEA peptides were readily obtained, and their identities were confirmed by MS. (64)Cu(2+) labeling was performed with high radiochemical yields (>98%) for all tracers after 1 h incubation. Stability experiments revealed that (64)Cu-DOTA-DGEA had unexpectedly high (64)Cu(2+) dissociation when incubated in PBS (>55% free (64)Cu(2+) was observed at 48 h time point). The (64)Cu(2+) dissociation was significantly reduced in the carboxyl-protected (64)Cu-DOTA-DGEA complex but not in the (64)Cu-DOTA-Ahx-DGEA complex, which suggests the presence of competitive binding for (64)Cu(2+) between DOTA and the carboxyl groups of the DGEA peptide. In contrast, no significant (64)Cu(2+) dissociation was observed for (64)Cu-Z-E(diamsar)-Ahx-DGEA in PBS. For microPET imaging, the PC-3 tumors were clearly visualized with both (64)Cu-DOTA-DGEA and (64)Cu-Z-E(diamsar)-Ahx-DGEA tracers. However, (64)Cu-DOTA-DGEA demonstrated 5× higher liver uptake than (64)Cu-Z-E(diamsar)-Ahx-DGEA. This biodistribution variance could be attributed to the chelating stability difference between these two tracers, which correlated well with the PBS stability experiments. In summary, the in vitro and in vivo evaluations of (64)Cu-Z-E(diamsar)-Ahx-DGEA and (64)Cu-DOTA-DGEA have demonstrated the significantly superior Cu-chelation stability for the diamsar derivative compared with the established DOTA chelator. The results also suggest that diamsar may be preferred for Cu chelation especially when multiple carboxylic acid groups are present. Free carboxyl groups may naturally compete with DOTA for (64)Cu(2+) binding and therefore reduce the complex stability.