RESUMO
We show that mouse embryonic endothelial progenitor cells (eEPCs) home preferentially to hypoxic lung metastases when administered intravenously. This specificity is inversely related to the degree of perfusion and vascular density in the metastasis and directly related to local levels of hypoxia and VEGF. Ex vivo expanded eEPCs that were genetically modified with a suicide gene specifically and efficiently eradicated lung metastases with scant patent blood vessels. eEPCs do not express MHC I proteins, are resistant to natural killer cell-mediated cytolysis, and can contribute to tumor vessel formation also in nonsyngeneic mice. These results indicate that eEPCs can be used in an allogeneic setting to treat hypoxic metastases that are known to be resistant to conventional therapeutic regimes.
Assuntos
Hipóxia Celular , Desenvolvimento Embrionário e Fetal/fisiologia , Endotélio Vascular/embriologia , Genes Transgênicos Suicidas , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Células-Tronco/fisiologia , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Efeito Espectador , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/terapia , Citosina Desaminase/genética , Citosina Desaminase/metabolismo , Endotélio Vascular/metabolismo , Fluoruracila/metabolismo , Marcação de Genes , Terapia Genética , Vetores Genéticos , Injeções Intravenosas , Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neovascularização Patológica/prevenção & controle , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/terapia , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Pró-Fármacos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Taxa de Sobrevida , Uracila/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In this study 'second generation' AnxV was specifically labeled with (99m)Tc in three different ways outside the binding region of the protein to obtain an improved target-to-background activity ratio. The compounds were tested in vitro and in vivo in normal mice and in a model of hepatic apoptosis (anti-Fas mAb). The apoptosis binding was most prominent for the HIS-tagged 'second generation' AnxV labeled with (99m)Tc(CO)(3) in comparison to (99m)Tc-HYNIC-cys-AnxV and (99m)Tc(CO)(3)-DTPA-cys-AnxV.
Assuntos
Anexina A5 , Apoptose , Tecnécio , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Anexina A5/química , Hepatócitos/citologia , Camundongos , Tecnécio/químicaRESUMO
AIMS: The integrin alpha v beta3 is highly expressed in atherosclerotic plaques by medial and intimal smooth muscle cells and by endothelial cells of angiogenic microvessels. In this study, we have assessed non-invasive molecular magnetic resonance imaging (MRI) of plaque-associated alpha v beta3 integrin expression on transgenic ApoE-/- mice with a low molecular weight peptidomimetic of Arg-Gly-Asp (mimRGD) grafted to gadolinium diethylenetriaminepentaacetate (Gd-DTPA-g-mimRGD). The analogous compound Eu-DTPA-g-mimRGD was employed for an in vivo competition experiment and to confirm the molecular targeting. The specific interaction of mimRGD conjugated to Gd-DTPA or to 99mTc-DTPA with alpha v beta3 integrin was furthermore confirmed on Jurkat T lymphocytes. METHODS AND RESULTS: The mimRGD was synthesized and conjugated to DTPA. DTPA-g-mimRGD was complexed with GdCl3.6H2O, EuCl3.6H2O, or with [99mTc(CO)3(H2O)3]+. MRI evaluation was performed on a 4.7 T Bruker imaging system. Blood pharmacokinetics of Gd-DTPA-g-mimRGD were assessed in Wistar rats and in c57bl/6j mice. The presence of angiogenic blood vessels and the expression of alpha v beta3 integrin were confirmed in aorta specimens by immunohistochemistry. Gd-DTPA-g-mimRGD produced a strong enhancement of the external structures of the aortic wall and of the more profound layers (possibly tunica media and intima). The aortic lumen seemed to be restrained and distorted. Pre-injection of Eu-DTPA-g-mimRGD diminished the Gd-DTPA-g-mimRGD binding to atherosclerotic plaque and confirmed the specific molecular targeting. A slower blood clearance was observed for Gd-DTPA-g-mimRGD, as indicated by a prolonged elimination half-life and a diminished total clearance. CONCLUSION: The new compound is potentially useful for the diagnosis of vulnerable atherosclerotic plaques and of other pathologies characterized by alpha v beta3 integrin expression, such as cancer and inflammation. The delayed blood clearance, the significant enhancement of the signal-to-noise ratio, and the low immunogenicity of the mimetic molecule highlight its potential for an industrial and clinical implementation.
Assuntos
Aorta/metabolismo , Aterosclerose/metabolismo , Meios de Contraste/metabolismo , Integrina alfaVbeta3/metabolismo , Angiografia por Ressonância Magnética/métodos , Compostos Organometálicos/metabolismo , Ácido Pentético/análogos & derivados , Animais , Aorta/patologia , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/patologia , Ligação Competitiva , Meios de Contraste/farmacocinética , Modelos Animais de Doenças , Feminino , Humanos , Imuno-Histoquímica , Células Jurkat , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Compostos Organometálicos/farmacocinética , Ácido Pentético/metabolismo , Ácido Pentético/farmacocinética , Compostos Radiofarmacêuticos/metabolismo , Ratos , Ratos Wistar , Processamento de Sinais Assistido por Computador , Pentetato de Tecnécio Tc 99m/metabolismoRESUMO
A novel cys-annexin A5 with a single cysteine-residue at its concave side has been developed by site-directed mutagenesis to allow conjugation through thiol-chemistry without affecting its apoptotic cell binding properties and was derivatized with HYNIC in a 1:1 stoichiometry. Similar to that of the 1st generation 99mTc-HYNIC-annexin A5, the novel 99mTc-HYNIC-cys-annexin A5 derivative shows in normal mice mainly renal and, to a lesser extent, hepatobiliary excretion. In murine models of hepatic apoptosis there was 257% increase in hepatic uptake of 99mTc-HYNIC-cys-annexin A5 as compared to normal mice. Using the novel tracer agent, acute reperfused myocardial infarction in rabbits was unequivocally delineated at 7h post-injection by muSPECT. The results indicate that the novel 99mTc-HYNIC-cys-annexin A5 shows similar apoptosis avidity as the 1st generation 99mTc-HYNIC-annexin A5.
Assuntos
Anexina A5/química , Apoptose , Cisteína/análogos & derivados , Compostos de Organotecnécio/farmacologia , Animais , Anexina A5/farmacologia , Química Farmacêutica/métodos , Cisteína/química , Cisteína/farmacologia , Desenho de Fármacos , Fígado/metabolismo , Camundongos , Modelos Químicos , Infarto do Miocárdio/metabolismo , Coelhos , Compostos de Sulfidrila/química , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
BACKGROUND: Stem cell transplantation (SCT) is potentially curative for high-risk leukemia patients. Conditioning regimens affect relapse rate and treatment-related mortality. We evaluated biodistribution, radiation absorbed organ doses and immediate toxicities of myeloablative radioimmunotherapy with marrow selective 188rhenium (188Re)-labeled anti-CD66 monoclonal antibody (mAb). METHODS: Fifty high-risk leukemia patients were treated 14 +/- 2 days prior to SCT. Dosimetric measurements were performed at 1.5, 3, 20, 26, and 44 hours after about 1 GBq of 188Re followed by radioimmunotherapy with about 10 GBq 188Re. Standard conditioning consisted of high-dose chemotherapy and 12 Gy total-body irradiation. Forty-six patients received allogenic, and four received autologous, stem cell grafts. RESULTS: The mean radiation absorbed doses (in Gy) were: marrow, 13.9 +/- 4.6; liver, 5.7 +/- 2.7; spleen, 22.6 +/- 25.5; kidneys, 6.8 +/- 2.6; lungs, 0.8 +/- 0.7; total body, 1.4 +/- 0.3. The tumor-to-organ-ratios were 2.4 for liver, 0.6 for the spleen, 2.0 for the kidneys and 17.8 for the lungs. Type of leukemia did not affect radiation absorbed doses of marrow, lungs, kidneys and liver. Mean marrow dose of transplanted patients in complete remission was 1.37 +/- 0.43 Gy/GBq, compared with 1.34 +/- 0.29 Gy/GBq for patients with leukemic blast marrow infiltration of 5-25%. Immediate side effects were moderate. All patients showed primary engraftment. After a median follow-up of 11.0 +/- 7.4 months 28/50 patients (56%) are in ongoing complete remission. Nine patients (5%) have relapsed, seven (4%) of them have died. Another 13 patients (7%) died of treatment-related causes. CONCLUSIONS: Due to its biodistribution, radiation absorbed organ doses, low toxicity and clinical data, myeloablative radioimmunotherapy with 188Re-labeled anti-CD66 mAb seems to be a promising method for improving standard conditioning of high-risk leukemia patients prior to SCT.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia/metabolismo , Leucemia/terapia , Radioimunoterapia , Rênio/farmacocinética , Condicionamento Pré-Transplante , Adolescente , Adulto , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/uso terapêutico , Medula Óssea/imunologia , Medula Óssea/efeitos da radiação , Moléculas de Adesão Celular , Feminino , Humanos , Leucemia/diagnóstico por imagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico por imagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Leucemia Mieloide/diagnóstico por imagem , Leucemia Mieloide/metabolismo , Leucemia Mieloide/terapia , Fígado/metabolismo , Fígado/efeitos da radiação , Pulmão/metabolismo , Pulmão/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico por imagem , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Doses de Radiação , Radioisótopos/efeitos adversos , Radioisótopos/farmacocinética , Radioisótopos/uso terapêutico , Cintilografia , Rênio/efeitos adversos , Rênio/uso terapêutico , Taxa de Sobrevida , Distribuição TecidualRESUMO
Triplex forming oligonucleotides (TFO) provide a promising tool for gene therapy. DNA damaging agents have been successfully coupled to TFOs and induce site-directed DNA damages. Here, we attempted to apply this antigen strategy using a TFO incorporated with a Conversion-electron-emitter, (99m)technetium, to target bcl-2 gene, the prototypical inhibitor of apoptosis. In the bcl-2 promoter region, we found two TFO binding sites which bind corresponding TFOs with very high specificity and affinity. Both partially and completely phosphorothioated TFOs form stable triplexes and significantly inhibit gene transcription in vitro. We also found that purine motif TFO with a thymidine opposite a thymidine interruption at the polypurine strand can form a stable triplex. In addition, (99m)technetium-conjugated TFOs were found to form a stable triplex and to inhibit bcl-2 gene transcription in vitro. Our results suggest a promising application of this triplex-forming oligonucleotide based Conversion-electron-emitter mediated gene radiotherapy in diseases related to bcl-2 overexpression.
Assuntos
Genes bcl-2 , Terapia Genética , Oligonucleotídeos/química , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Tecnécio/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Células HeLa , Humanos , Conformação de Ácido Nucleico , Regiões Promotoras GenéticasRESUMO
A new tropane derivative was synthesized by combining a tridentate ligand, N-(2-picolylamine)-N-acetic acid (2-PAA), and a phenyltropane derivative. It was labelled with a [(99m)Tc(CO)(3)](+) moiety, resulting in the formation of two stable and neutral lipophilic isomers. Their identity was confirmed using radio-LC-MS. In normal mice, no brain uptake was observed for any of the isomers and in vitro autoradiography using mouse brain sections showed no specific uptake in the striatal area.
Assuntos
Compostos de Organotecnécio/química , Tropanos/síntese química , Tropanos/farmacocinética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Avaliação Pré-Clínica de Medicamentos , Técnicas In Vitro , Marcação por Isótopo , Camundongos , Conformação Molecular , Relação Estrutura-Atividade , Distribuição Tecidual , Tropanos/químicaRESUMO
N-(2-Mercapto-propyl)-1,2-phenylenediamine (MPPDA) and N-beta-aminoethylglycine (AEG) were labelled with 99mTc(CO)3(+) to form the neutral complexes [99mTc(CO)3(MPPDA)] and [99mTc(CO)3(AEG)]. Both complexes were formed in excellent yields and their identity was confirmed by LC-MS. In mice, none of the new tracer agents showed brain uptake. [(99m)Tc(CO)3(MPPDA)] was trapped mainly in the liver and excreted via the hepatobiliary system, whereas [99mTc(CO)3(AEG)] was excreted rapidly via the kidneys to the urine.
Assuntos
Compostos de Organotecnécio/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Ligantes , Fígado/metabolismo , Espectrometria de Massas , Camundongos , Compostos de Organotecnécio/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
The anions [ReX3(CO)2(NO)]- (with X = Cl, 1; X = Br, 2) have been prepared with different counterions. Complex 1 was found to lose its chloride ligands in water within 24 h. The [Re(H2O)3(CO)2(NO)]2+ cation obtained after hydrolysis is a strong acid, which consequently undergoes a slow condensation reaction in water to form the very stable [Re(mu3-O)(CO)2(NO)]4 cluster 4 at pH > 2, that precipitates from the aqueous solution and is insoluble also in organic solvents. Fast deprotonation of [Re(H2O)3(CO)2(NO)]2+ did not lead to 4 but rather to the mononuclear species [Re(OH)(H2O)2(CO)2(NO)]+. Subsequent attack of OH- at a CO group resulted in the formation of a rhenacarboxylic acid and its carboxylate anion. For solutions of even higher pH, IR spectroscopy provided evidence for the formation of a Re(C(O)ON(O)) species. These processes were found to be reversible on lowering the pH. Starting from cluster 4 it was possible to obtain complexes of the types [ReX(CO)2(NO)L2] or [Re(CO)2(NO)L3](L2 = 2-picolinate, 2,2'-bipyridine, L-phenylalanate; L3 = tris(pyrazolyl)methane, 1,4,7-trithiacyclononane) in the presence of an acid in protic solvents, but only in low yields. In further synthetic studies, complexes 1 and 2 were found to be superior starting materials for substitution reactions to form [ReX(CO)2(NO)L2] or [Re(CO)2(NO)L3] complexes.
Assuntos
Compostos Organometálicos/química , Rênio/química , Prótons , SoluçõesRESUMO
Radioimmunotherapy (RIT) with radiolabelled monoclonal antibodies (mAbs) is an effective method of achieving myeloablation in leukaemia patients prior to stem cell transplantation (SCT). We wished to compare the approaches of specific binding to leukaemic blasts and non-specific binding to benign red marrow cells, which results in a myeloablative "cross-fire" effect. Therefore, we prospectively evaluated the biodistribution and biokinetics of the anti-CD45 mAb YTH 24.5 and the anti-CD66 mAb BW 250/183 with regard to their suitability for myeloablative RIT. The red marrow selective anti-CD66 mAb BW 250/183 (IgG1) binds to normal granulopoietic cells. In contrast, the anti-CD45 mAb YTH 24.5 (IgG2b) binds to 85-90% of acute leukaemic blasts and almost all haematopoietic white cells. Patients with leukaemic blast infiltration of the marrow <25% and assigned for RIT and SCT were included. Twelve patients (eight male, four female; median age 46+/-7 years) with AML (5), CML (5) or ALL (2) were examined. Both mAbs were labelled with technetium-99m. Within 48 h, 906+/-209 MBq (99m)Tc-anti-CD66 mAb and 760+/-331 MBq (99m)Tc-anti-CD45 mAb were injected consecutively. Scintigraphic and urinary measurements were performed 1, 2, 4 and 24 h after injection. Serum activities were evaluated 2, 5, 10, 15, 30 and 60 min and 2, 4 and 24 h after injection. Compared with the anti-CD45 mAb, the anti-CD66 mAb showed an approximately fourfold higher accumulation in the red marrow, a 2.5-fold lower accumulation in the liver and similar accumulation in the kidneys. The serum activity (% of the injected dose) initially decreased faster for the anti-CD45 mAb but was similar for the two mAbs 24 h after injection: 3.3%+/-1.2% (anti-CD66 mAb) and 2.4%+/-1.1% (anti-CD45 mAb). The cumulated urinary excretion was 17%+/-6.6% (anti-CD66 mAb) and 27.3%+/-7.9% (anti-CD45 mAb) 24 h after application. In these patients with low tumour load, the anti-CD66 mAb BW 250/183 showed more favourable properties in terms of biodistribution and pharmacokinetics. Thus, it appears superior to anti-CD45 mAb YTH 24.5 in selectively increasing the marrow dose and avoiding extramedullary organ toxicity.