Cu(I)-assisted click chemistry strategy for conjugation of non-protected cross-bridged macrocyclic chelators to tumour-targeting peptides.

Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry has inherent challenges for copper-labeled radiopharmaceuticals. An azide-modified phosphonate-based cross-bridged macrocyclic chelator was synthesized for click chemistry conjugation with azide-modified Y3-TATE (a somatostatin analogue) on resin, without the need for protecting the chelator. The (64)Cu-labeled bioconjugate shows favourable in vitro and in vivo behaviour.

Angiographic features, collaterals, and infarct topography of symptomatic occlusive radiation vasculopathy: a case-referent study.

BACKGROUND AND PURPOSE: Occlusive radiation vasculopathy (ORV) predisposes head-and-neck cancer survivors to ischemic strokes. METHODS: We analyzed the digital subtraction angiography acquired in 96 patients who had first-ever transient ischemic attack or ischemic strokes attributed to ORV. Another age-matched 115 patients who had no radiotherapy but symptomatic high-grade (>70%) carotid stenoses were enrolled as referent subjects. Digital subtraction angiography was performed within 2 months from stroke onset and delineated carotid and vertebrobasilar circulations from aortic arch up to intracranial branches. Two reviewers blinded to group assignment recorded all vascular lesions, collateral status, and infarct pattern. RESULTS: ORV patients had less atherosclerotic risk factors at presentation. In referent patients, high-grade stenoses were mostly focal at the proximal internal carotid artery. In contrast, high-grade ORV lesions diffusely involved the common carotid artery and internal carotid artery and were more frequently bilateral (54% versus 22%), tandem (23% versus 10%), associated with complete occlusion in one or both carotid arteries (30% versus 9%), vertebral artery (VA) steno-occlusions (28% versus 16%), and external carotid artery stenosis (19% versus 5%) (all P<0.05). With comparable rates of vascular anomaly, ORV patients showed more established collateral circulations through leptomeningeal arteries, anterior communicating artery, posterior communicating artery, suboccipital/costocervical artery, and retrograde flow in ophthalmic artery. In terms of infarct topography, the frequencies of cortical or subcortical watershed infarcts were similar in both groups. CONCLUSIONS: ORV angiographic features and corresponding collaterals are distinct from atherosclerotic patterns at initial stroke presentation. Clinical decompensation, despite more extensive collateralization, may precipitate stroke in ORV.

Young ischaemic stroke in South Auckland: a hospital-based study.

BACKGROUND AND PURPOSE: To analyse the risk factors, trends in incidence and aetiology of stroke in young adults in a hospital-based population in South Auckland, New Zealand. METHOD: A retrospective review of patients aged 15 years to 45 years with a discharge diagnosis of ischaemic stroke (ICD 10 Codes I63 - I65, G46, I672, I675-I679, I694- I694) from June 1 2004 to December 31, 2009. Vascular risk factors, demographic factors, stroke severity, stroke subtype, results of investigations and stroke outcome were determined from review of the hospital record (paper copy and electronic record). RESULTS: A total of 131 patients were identified, representing 4.6% of all stroke discharges. Over one-half of the patients were of "underdetermined cause" (Trial of Org 10172 in Acute Stroke Treatment [TOAST] criteria), (1) mainly due to incomplete investigation. Cardioembolism (16%) was the second most common cause of stroke, followed by small vessel disease and stroke of other determined aetiology (both 12.2%). Confirmed large vessel atherosclerosis (6.1%) was the least common cause of stroke in this study population. The most common risk factors were hyperlipidaemia (45.8%), hypertension (42.7%), current tobacco smoking (42.7%) and obesity (36.6%). The indigenous Maori and Pacific Island people had a higher rate of stroke, at least double of other ethnicities. The in-hospital fatality rate was 3.1%. All surviving patients were discharged home. Eighty-six percent of the survivors were independent. CONCLUSIONS: Our study demonstrates strokes of undetermined aetiology and cardioembolism were the most common cause of stroke in young people in South Auckland, and that Maori and Pacific Island people have a higher rate of stroke.

Isomeric trimethylene and ethylene pendant-armed cross-bridged tetraazamacrocycles and in vitro/in vivo comparisions of their copper(II) complexes.

Ethylene cross-bridged tetraamine macrocycles are useful chelators in coordination, catalytic, medicinal, and radiopharmaceutical chemistry. Springborg and co-workers developed trimethylene cross-bridged analogues, although their pendant-armed derivatives received little attention. We report here the synthesis of a bis-carboxymethyl pendant-armed cyclen with a trimethylene cross-bridge (C3B-DO2A) and its isomeric ethylene-cross-bridged homocyclen ligand (CB-TR2A) as well as their copper(II) complexes. The in vitro and in vivo properties of these complexes are compared with respect to their potential application as (64)Cu-radiopharmaceuticals in positron emission tomography (PET imaging). The inertness of Cu-C3B-DO2A to decomplexation is remarkable, exceeding that of Cu-CB-TE2A. Electrochemical reduction of Cu-CB-TR2A is quasi-reversible, whereas that of Cu-C3B-DO2A is irreversible. The reaction conditions for preparing (64)Cu-C3B-DO2A (microwaving at high temperature) are relatively harsh compared to (64)Cu-CB-TR2A (basic ethanol). The in vivo behavior of the (64)Cu complexes was evaluated in normal rats. Rapid and continual clearance of (64)Cu-CB-TR2A through the blood, liver, and kidneys suggests relatively good in vivo stability, albeit inferior to (64)Cu-CB-TE2A. Although (64)Cu-C3B-DO2A clears continually, the initial uptake is high and only about half is excreted within 22 h, suggesting poor stability and transchelation of (64)Cu to proteins in the blood and/or liver. These data suggest that in vitro inertness of a chelator complex may not always be a good indicator of in vivo stability.

Stroke caused by small vessel occlusion in a patient taking etoricoxib and thalidomide.

We report a 33-year-old man with seronegative arthritis who had an acute infarct at the left lentiform nucleus while taking etoricoxib and thalidomide regularly. Extensive investigations did not find any evidence of large artery atherosclerosis, vasculitis, cardioembolic source or anti-phospholipid antibodies. While it is possible that a short smoking history, hyperlipidemia, and the use of thalidomide could have contributed to the thrombosis of a small penetrator vessel, we postulated that the prolonged use of etoricoxib is another possible contributing factor.

Tracheobronchial injuries from blunt trauma.

Tracheobronchial injuries from trauma can be life threatening. We present a case report of a 23-year-old man who suffered a left main bronchus transection after a motorbike accident. The diagnostic and management issues surrounding tracheobronchial injuries are reviewed. Early diagnosis and treatment lead to the best outcome, with almost complete return of pulmonary function.

Optimization of labeling and metabolite analysis of copper-64-labeled azamacrocyclic chelators by radio-LC-MS.

The cross-bridged tetraamine ligand 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (H2CB-TE2A) allows formation of a radio-copper complex with higher in vivo stability than that of the corresponding non-cross-bridged analog 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). The structure of the natCu(II) complex of CB-TE2A has been previously determined by X-ray crystallography; however, direct high-pressure liquid chromatography (HPLC) characterization of the corresponding 64Cu complex was inaccessible due to the inability to detect the complex by ultraviolet absorbance at the radiotracer level. A reverse-phase HPLC separation of a series of natCu(II)-tetraazamacrocyclic complexes, both traditional and cross-bridged, was developed and applied toward characterization and assessment of the purity of the corresponding no-carrier-added 64Cu-labeled complexes. Verification of the identity of copper-64-labeled compounds was also achieved by coupling this HPLC method with mass spectrometry. The radio-liquid chromatography/mass spectrometry methodology was further extended to study the in vivo metabolic fates of 64Cu-azamacrocyclic complexes.

Preparation and biological evaluation of copper-64-labeled tyr3-octreotate using a cross-bridged macrocyclic chelator.

PURPOSE: Somatostatin receptors (SSTr) are expressed on many neuroendocrine tumors, and several radiotracers have been developed for imaging these types of tumors. For this reason, peptide analogues of somatostatin have been well characterized. Copper-64 (t(1/2) = 12.7 hours), a positron emitter suitable for positron emission tomography (PET) imaging, was shown recently to have improved in vivo clearance properties when chelated by the cross-bridged tetraazamacrocycle 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A) compared with 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). EXPERIMENTAL DESIGN: CB-TE2A and TETA were conjugated to the somatostatin analogue tyrosine-3-octreotate (Y3-TATE) for evaluation of CB-TE2A as a bifunctional chelator of 64Cu. The in vitro affinity of each compound for SSTr was determined using a homologous competitive binding assay. In vivo characteristics of both radiolabeled compounds were examined in biodistribution and microPET studies of AR42J tumor-bearing rats. RESULTS: Cu-CB-TE2A-Y3-TATE (Kd = 1.7 nmol/L) and Cu-TETA-Y3-TATE (Kd = 0.7 nmol/L) showed similar affinities for AR42J derived SSTr. In biodistribution studies, nonspecific uptake in blood and liver was lower for 64Cu-CB-TE2A-Y3-TATE. Differences increased with time such that, at 4 hours, blood uptake was 4.3-fold higher and liver uptake was 2.4-fold higher for 64Cu-TETA-Y3-TATE than for 64Cu-CB-TE2A-Y3-TATE. In addition, 4.4-times greater tumor uptake was detected with 64Cu-CB-TE2A-Y3-TATE than with 64Cu-TETA-Y3-TATE at 4 hours postinjection. MicroPET imaging yielded similar results. CONCLUSIONS: CB-TE2A appears to be a superior in vivo bifunctional chelator of 64Cu for use in molecular imaging by PET or targeted radiotherapy due to both improved nontarget organ clearance and higher target organ uptake of 64Cu-CB-TE2A-Y3-TATE compared with 64Cu-TETA-Y3-TATE.

Inside or outside a ligand cleft? Synthetic, structural, and kinetic inertness studies of zinc, cadmium, and mercury complexes of cross-bridged cyclam and cyclen.

Ethylene cross-bridging of the popular tetraazamacrocyclic ligand cyclam has led to metal complexes with enhanced kinetic inertness. The synthesis and spectral characterization of zinc(II), cadmium(II), and mercury(II) complexes of cross-bridged cyclam (L1) as well as cross-bridged cyclen (L2) are reported along with the details of our synthetic route to L2. X-ray structural studies revealed that all Zn(II) and Cd(II) cations are fully kappa(4)N-coordinated inside the respective ligand's molecular cleft with L1 providing the better fit for Zn(II). While Hg(II) is similarly coordinated to L2, it has been found to complex L1 outside the ligand cleft in a novel exo-kappa(2)N-mode. Solution NMR data of the kappa(4)N complexes are consistent with the presence of only a single cis-folded isomer in each case. Ligand (1)H and (13)C coupling to both (111,113)Cd and (199)Hg in their complexes can be clearly discerned. The relative kinetic inertness of representative cross-bridged complexes in acidic aqueous solution has been assessed and found to be in the following order: Zn(II) > Cd(II)[dbl greater-than] Hg(II). The data also reaffirm that cross-bridged cyclam ligand L1 forms a substantially more inert complex with zinc(II) than either the smaller cyclen analogue L2 or the unbridged 1,4,8,11-tetramethyl-cyclam L3.

Comparative in vivo stability of copper-64-labeled cross-bridged and conventional tetraazamacrocyclic complexes.

The increased use of copper radioisotopes in radiopharmaceutical applications has created a need for bifunctional chelators (BFCs) that form stable radiocopper complexes and allow covalent attachment to biological molecules. The chelators most commonly utilized for labeling copper radionuclides to biomolecules are analogues of 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); however, recent reports have communicated the instability of the radio-Cu(II)-TETA complexes in vivo. A class of bicyclic tetraazamacrocycles, the ethylene "cross-bridged" cyclam (CB-cyclam) derivatives, form highly kinetically stable complexes with Cu(II) and therefore may be less susceptible to transchelation than their nonbridged analogues in vivo. Herein we report results on the relative biological stabilities and identification of the resulting radiolabeled metabolites of a series of (64)Cu-labeled macrocyclic complexes. Metabolism studies in normal rat liver have revealed that the (64)Cu complex of 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane ((64)Cu-CB-TE2A) resulted in significantly lower values of protein-associated (64)Cu than (64)Cu-TETA [13 +/- 6% vs 75 +/- 9% at 4 h]. A similar trend was observed for the corresponding cyclen derivatives, with the (64)Cu complex of 4,10-bis(carboxymethyl)-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane ((64)Cu-CB-DO2A) undergoing less transchelation than the (64)Cu complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ((64)Cu-DOTA) [61 +/- 14% vs 90.3 +/- 0.5% protein associated (64)Cu at 4 h]. These data indicate that the structurally reinforcing cross-bridge enhances in vivo stability by reducing metal loss to protein in both the cyclam and cyclen cross-bridged (64)Cu complexes and that (64)Cu-CB-TE2A is superior to (64)Cu-CB-DO2A in that regard. These findings further suggest that a bifunctional chelator derivative of CB-TE2A is a highly desirable alternative for labeling copper radionuclides to biological molecules for diagnostic imaging and targeted radiotherapy.

Radiolabeling and in vivo behavior of copper-64-labeled cross-bridged cyclam ligands.

Macrocyclic chelators and their metal complexes have widespread applications in the biomedical sciences, including radiopharmaceutical chemistry. The use of copper radionuclides in radiopharmaceuticals is increasing. Macrocyclic chelators have been found to have enhanced in vivo stability over acyclic chelators such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). The currently used chelators of choice for labeling copper radionuclides to biological molecules are analogues of TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid); however, recent reports have demonstrated evidence of in vivo instability of the radio-Cu(II)-TETA complexes. A new class of structurally reinforced macrocycles, the "cross-bridged" cyclam derivatives, form highly stable complexes with Cu(II) that are resistant to dissociation in strong acid. Here, we evaluate a series of (64)Cu(II) cross-bridged macrocyclic complexes for biological stability and in vivo behavior. The ligands evaluated include the parent ligand, 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (1), and three 4,11-di-pendant arm derivatives: 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (2); 4,11-bis(N,N-diethyl-amidomethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (3); and 4,11-bis(amidoethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (4). Copper-64 formed complexes with ligands 1-4 in high radiochemical yields. The (64)Cu-2 complex was neutral, while (64)Cu complexes of 1, 3, and 4 were positively charged. All complexes showed no decomposition in rat serum out to 24 h. Biodistribution experiments in Sprague-Dawley rats indicated that (64)Cu-1, -3, and -4 were taken up by the liver and kidney and cleared slowly over 24 h, whereas (64)Cu-2 cleared rapidly from all tissues. The rapid clearance of the (64)Cu-2 complex from the blood and liver, as well as liver metabolism experiments in rats, suggests that it is highly stable in vivo. A bifunctional chelator of 2 is a significant candidate for labeling copper radionuclides to biological molecules for diagnostic imaging and targeted radiotherapy.