RESUMO
Molecules containing lysine-ureido-glutamate functional groups bind to the active site of prostate specific membrane antigen, which is overexpressed in prostate cancer. To prepare copper radiopharmaceuticals for the diagnosis and therapy of prostate cancer, macrobicyclic sarcophagine ligands tethered to either one or two lysine-ureido-glutamate functional groups through an appropriate linker have been prepared. Sarcophagine ligands can be readily radiolabeled with positron-emitting copper-64 at room temperature. The bivalent agent, in which two targeting groups are tethered to a single copper complex, dramatically outperforms the monomeric agent with respect to tumor uptake and retention. The high tumor uptake, low background, and prolonged tumor retention, even at 24â hours post injection, suggest the bivalent agent is a promising diagnostic for prostate cancer and could be used for prospective dosimetry for therapy with a copper-67 variant.
Assuntos
Radioisótopos de Cobre/química , Dipeptídeos/química , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamatos/química , Neoplasias da Próstata , Compostos Radiofarmacêuticos/química , Animais , Antígenos de Superfície , Sítios de Ligação , Linhagem Celular Tumoral , Radioisótopos de Cobre/metabolismo , Glutamatos/farmacocinética , Humanos , Lisina/análogos & derivados , Lisina/química , Masculino , Camundongos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/terapia , Ligação Proteica , Compostos Radiofarmacêuticos/farmacocinética , Nanomedicina Teranóstica , Distribuição Tecidual , Ureia/análogos & derivados , Ureia/químicaRESUMO
The intracellular distribution of fluorescently labeled copper and zinc bis(thiosemicarbazonato) complexes was investigated in M17 neuroblastoma cells and primary cortical neurons with a view to providing insights into the neuroprotective activity of a copper bis(thiosemicarbazonato) complex known as Cu(II)(atsm). Time-resolved fluorescence measurements allowed the identification of the Cu(II) and Zn(II) complexes as well as the free ligand inside the cells by virtue of the distinct fluorescence lifetime of each species. Confocal fluorescent microscopy of cells treated with the fluorescent copper(II)bis(thiosemicarbazonato) complex revealed significant fluorescence associated with cytoplasmic puncta that were identified to be lysosomes in primary cortical neurons and both lipid droplets and lysosomes in M17 neuroblastoma cells. Fluorescence lifetime imaging microscopy confirmed that the fluorescence signal emanating from the lipid droplets could be attributed to the copper(II) complex but also that some degree of loss of the metal ion led to diffuse cytosolic fluorescence that could be attributed to the metal-free ligand. The accumulation of the copper(II) complex in lipid droplets could be relevant to the neuroprotective activity of Cu(II)(atsm) in models of amyotrophic lateral sclerosis and Parkinson's disease.
Assuntos
Complexos de Coordenação/farmacocinética , Cobre/química , Fluorescência , Tiossemicarbazonas/química , Zinco/química , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Humanos , Modelos Moleculares , Estrutura Molecular , Espectrometria de Fluorescência , Fatores de Tempo , Distribuição TecidualRESUMO
Imaging of activated platelets using an activation specific anti-GPIIb/IIIa integrin single-chain antibody (scFvanti-LIBS) conjugated to a positron emitting copper-64 complex of a cage amine sarcophagine chelator (MeCOSar) is reported. This tracer was compared in vitro to a (64)Cu(II) complex of the scFv conjugated to another commonly used macrocycle, DOTA. The scFvanti-LIBS-MeCOSar conjugate was radiolabeled with (64)Cu(II) rapidly under mild conditions and with higher specific activity than scFvanti-LIBS-DOTA. The utility of scFvanti-LIBS-MeCOSar as a diagnostic agent was assessed in vivo in a mouse model of acute thrombosis. The uptake of scFvanti-LIBS-(64)CuMeCOSar in the injured vessel was significantly higher than the noninjured vessel. Positron emission tomography (PET) was used to show accumulation of scFvanti-LIBS-(64)CuMeCOSar with high and specific uptake in the injured vessel. ScFvanti-LIBS-(64)CuMeCOSar is an excellent tool for highly sensitive in vivo detection of activated platelets in PET and has the potential to be used for early diagnosis of acute thrombotic events.
Assuntos
Plaquetas/efeitos dos fármacos , Quelantes/química , Tomografia por Emissão de Pósitrons , Anticorpos de Cadeia Única/química , Animais , Plaquetas/metabolismo , Artérias Carótidas/fisiopatologia , Cobre/química , Radioisótopos de Cobre/química , Diagnóstico por Imagem , Modelos Animais de Doenças , Citometria de Fluxo , Compostos Heterocíclicos com 1 Anel/química , Inflamação , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB C , Ativação Plaquetária , Compostos Radiofarmacêuticos , Trombose/diagnóstico , Microtomografia por Raio-XRESUMO
Demyelination within the central nervous system (CNS) is a significant feature of debilitating neurological diseases such as multiple sclerosis and administering the copper-selective chelatorcuprizone to mice is widely used to model demyelination in vivo. Conspicuous demyelination within the corpus callosum is generally attributed to cuprizone's ability to restrict copper availability in this vulnerable brain region. However, the small number of studies that have assessed copper in brain tissue from cuprizone-treated mice have produced seemingly conflicting outcomes, leaving the role of CNS copper availability in demyelination unresolved. Herein we describe our assessment of copper concentrations in brain samples from mice treated with cuprizone for 40 d. Importantly, we applied an inductively coupled plasma mass spectrometry methodology that enabled assessment of copper partitioned into soluble and insoluble fractions within distinct brain regions, including the corpus callosum. Our results show that cuprizone-induced demyelination in the corpus callosum was associated with decreased soluble copper in this brain region. Insoluble copper in the corpus callosum was unaffected, as were pools of soluble and insoluble copper in other brain regions. Treatment with the blood-brain barrier permeant copper compound CuII(atsm) increased brain copper levels and this was most pronounced in the soluble fraction of the corpus callosum. This effect was associated with significant mitigation of cuprizone-induced demyelination. These results provide support for the involvement of decreased CNS copper availability in demyelination in the cuprizone model. Relevance to human demyelinating disease is discussed.
Assuntos
Cuprizona , Doenças Desmielinizantes , Humanos , Animais , Camundongos , Cuprizona/efeitos adversos , Corpo Caloso , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Cobre/farmacologia , Oligodendroglia , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Bainha de MielinaRESUMO
Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound CuII(atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with CuII(atsm) showing initial promise.
RESUMO
The first member of a water-soluble family of bis(thiosemicarbazone) ligands is reported. It forms a 1:1 complex with Zn(II) that absorbs intensely in the visible region (lambda(max) = 414 nm; epsilon = 1.8(4) x 10(4) M(-1) cm(-1); pH 7.3). Its affinity for Zn(II) (K(D) = 5.9(3) x 10(-9) M at pH 7.3) was determined by competition with ligand ethylene glycol O,O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid. Its potential application as a chromophoric probe was demonstrated by estimation of the Zn(II) binding affinities of the soluble metal-binding domains of two plant metal-transporting proteins.
Assuntos
Tiossemicarbazonas/química , Zinco/análise , Ligantes , Modelos Moleculares , Proteínas de Plantas/metabolismo , Ligação Proteica , Solubilidade , Água/química , Zinco/metabolismoRESUMO
Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.
Assuntos
Compostos Organometálicos/farmacologia , Medula Espinal/enzimologia , Medula Espinal/patologia , Superóxido Dismutase/metabolismo , Tiossemicarbazonas/farmacologia , Administração Oral , Animais , Complexos de Coordenação , Cobre/metabolismo , Citocromos c/metabolismo , Gliose/metabolismo , Gliose/patologia , Humanos , Fígado/enzimologia , Camundongos Transgênicos , Mitocôndrias/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação/genética , Compostos Organometálicos/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Fenótipo , Análise de Sobrevida , Tiossemicarbazonas/administração & dosagem , Extratos de TecidosRESUMO
New macrobicyclic cage amine or "sarcophagine" (sar) bifunctional chelators have been synthesised that form copper complexes of exceptional in vivo stability and incorporate isothiocyanate (-NCS) functional groups for conjugation to an antibody. The chelators were synthesised from the methyl-capped complex [Mg(II)(CH3)(NH2)sar](2+). Coordination of Mg(II) within the cavity of the cage amine ligand protects the secondary amine atoms from reacting with the -NCS functional groups. Two different [Mg(II)(NCS-sar)](2+) derivatives were conjugated to the HER2/neu-targeting antibody trastuzumab and the progress of the reaction monitored by electrospray mass spectrometry. The Mg(II) ion was removed from the immunoconjugates under mild conditions (0.1 M citrate buffer, pH 6). Labelling of the (CH3)(p-NCS-Ph)sar-trastuzumab conjugate with (64)Cu(II), a radioisotope suitable for positron emission tomography (PET), was fast (â¼5 min) and easily performed at room temperature with high radiochemical purity (>95%). Biodistribution and PET imaging studies in vivo showed that (64)Cu-labelled (CH3)(p-NCS-Ph)sar-trastuzumab maintained high stability under physiological conditions with high and selective uptake in a HER2-positive cancer cell line. The stability of the copper complex and the 12.7 h half-life of the radioisotope allows clear visualisation of tumours out to 48 h.
Assuntos
Aminas/química , Radioisótopos de Cobre , Imunoconjugados , Isotiocianatos/química , Compostos Macrocíclicos/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Linhagem Celular Tumoral , Quelantes/química , Estabilidade de Medicamentos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Marcação por Isótopo , Magnésio/química , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Receptor ErbB-2/metabolismo , Trastuzumab/químicaRESUMO
Quadridentate ligand H(2)L binds Cu(I) to form salt [Cu(I)(H(2)L)]BF(4) which undergoes aerial oxidation in solution with isolation of the diamagnetic salt [Cu(II)LË(-)]BF(4) where π-radical anion LË(-) is the (1e(-), 2H(+)) oxidation product of H(2)L.