RESUMO
INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.
Assuntos
Anticoagulantes/farmacologia , Antídotos/farmacologia , Avidina/farmacologia , Biotina/análogos & derivados , Oligossacarídeos/farmacologia , Adulto , Anticoagulantes/efeitos adversos , Anticoagulantes/sangue , Anticoagulantes/farmacocinética , Antídotos/efeitos adversos , Antídotos/farmacocinética , Avidina/efeitos adversos , Avidina/sangue , Avidina/farmacocinética , Biotina/efeitos adversos , Biotina/sangue , Biotina/farmacocinética , Biotina/farmacologia , Testes de Coagulação Sanguínea , Venenos de Crotalídeos/antagonistas & inibidores , Fator Xa , Humanos , Masculino , Metaloendopeptidases/antagonistas & inibidores , Oligossacarídeos/efeitos adversos , Oligossacarídeos/sangue , Oligossacarídeos/farmacocinética , Adulto JovemRESUMO
Alterations of cell monolayer integrity and increased vascular permeability are key to many pathologies, including atherosclerosis, stroke, lung injury, cancer, digestive disorders and others. Current approaches to probe cell permeability require specific culture conditions and provide an average estimation of trans-monolayer permeability, while analysis of regional monolayer permeability in static and mechanically challenged monolayer at a single-cell scale resolution remains unavailable. We describe a novel method for visualization and rapid quantification of trans-monolayer permeability based on high-affinity interactions between ligand (FITC-conjugated avidin) added in the culture medium, which permeates cell monolayer to reach substrate-bound acceptor (biotinylated gelatin or collagen). This approach was used to simultaneously evaluate general and local permeability responses by endothelial cell (EC) monolayer to a spectrum of barrier protective and barrier disruptive agonists and their combinations. The results revealed the paracellular pathway as the predominant mechanism of agonist-induced mass transport by pulmonary EC. We also detected for the first time, in a direct assay, a synergistic effect of pathologically relevant levels of cyclic stretch (CS) and edemagenic agent thrombin in the development of pulmonary EC hyper-permeability response observed in ventilator-induced lung injury. The reported novel assay provides unique information about local monolayer permeability changes induced by agonists, mechanical factors or molecular perturbations in single cells. However, the spectrum of substrates, assay formats and experimental conditions compatible with this assay suggest its broad application in the areas of endothelial and epithelial biology, cancer research and other fields.
Assuntos
Avidina/farmacocinética , Permeabilidade Capilar/fisiologia , Células Endoteliais/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Artéria Pulmonar/citologia , Análise Serial de Tecidos/métodos , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Avidina/metabolismo , Transporte Biológico/fisiologia , Biotinilação , Técnicas de Cultura de Células , Colágeno/metabolismo , Meios de Cultura/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluoresceína-5-Isotiocianato/farmacocinética , Imunofluorescência , Gelatina/metabolismo , HumanosRESUMO
As an alternative to directly targeting of necrotic tissue using hypericin, we synthesized a conjugate of hypericin to biotin for use in a pretargeting approach. With this conjugate, we explored the possibility of a two-step pretargeting strategy using (123)I-labeled avidin as effector molecule directed against necrotic RIF-1 tumors. Hypericin was conjugated to biotin-ethylenediamine in a straightforward coupling method using n-hydroxysuccinimide and dicyclohexylcarbodiimide. The necrosis avidity of the conjugate was first confirmed in necrotic liver tissue by means of fluorescence microscopy. Using autoradiography imaging and whole body-biodistribution, the accumulation of (123)I-avidin in necrotic tumor tissue was evaluated 24 h after administration and 48 h after pretargeting with hypericin-biotin. Analysis of autoradiography images show a higher accumulation of (123)I-avidin in pretargeted compared to nontargeted tissue. However, absolute accumulation of (123)I-avidin in necrotic tumors was low as shown by biodistribution experiments. Direct injection of hypericin-biotin or biotin-fluorescein did not substantially improve (123)I-avidin accumulation after pretargeting, pointing towards a poor penetration of avidin in necrotic tissue. Our results show the feasibility of a pretargeting technique using a small molecule as targeting agent. However, for a more efficient accumulation of the effector molecule in necrotic tissue, other pretargeting strategies need to be investigated.
Assuntos
Antineoplásicos/administração & dosagem , Avidina/administração & dosagem , Biotina/administração & dosagem , Necrose/metabolismo , Neoplasias/metabolismo , Perileno/análogos & derivados , Animais , Antracenos , Antineoplásicos/química , Avidina/farmacocinética , Biotina/química , Biotinilação , Linhagem Celular Tumoral , Meios de Contraste/administração & dosagem , Meios de Contraste/farmacocinética , Etanol , Feminino , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos C3H , Necrose/induzido quimicamente , Necrose/patologia , Neoplasias/patologia , Perileno/administração & dosagem , Perileno/química , Distribuição TecidualRESUMO
PURPOSE: Avidin-coupled monoclonal antibody MX35 (avidin-MX35) and astatine-211-labeled, biotinylated, succinylated poly-l-lysine ((211)At-B-PL(suc)) were administered in mice to assess potential efficacy as an intraperitoneal (i.p.) therapy for microscopic tumors. We aimed to establish a timeline for pretargeted radioimmunotherapy using these substances, and estimate the maximum tolerable activity. METHODS: (125)I-avidin-MX35 and (211)At-B-PL(suc) were administered i.p. in nude mice. Tissue distributions were studied at various time points and mean absorbed doses were estimated from organ uptake of (211)At-B-PL(suc). Studies of myelotoxicity were performed after administration of different activities of (211)At-B-PL(suc). RESULTS: We observed low blood content of both (125)I-avidin-MX35 and (211)At-B-PL(suc), indicating fast clearance. After sodium perchlorate blocking, the highest (211)At uptake was found in kidneys. Red bone marrow (RBM) accumulated some (211)At activity. Mean absorbed doses of special interest were 2.3 Gy/MBq for kidneys, 0.4 Gy/MBq for blood, and 0.9 Gy/MBq for RBM. An absorbed dose of 0.9 Gy to the RBM was found to be safe. These values suggested that RBM would be the key dose-limiting organ in the proposed pretargeting scheme, and that blood data alone was not sufficient for predicting its absorbed dose. CONCLUSIONS: To attain a favorable distribution of activity and avoid major toxicities, at least 1.0 MBq of (211)At-B-PL(suc) can be administered 24 hours after an i.p. injection of avidin-MX35. These results provide a basis for future i.p. therapy studies in mice of microscopic ovarian cancer.
Assuntos
Anticorpos Monoclonais/farmacocinética , Astato/farmacocinética , Avidina/farmacocinética , Lisina/farmacocinética , Radioimunoterapia/métodos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/química , Astato/administração & dosagem , Astato/química , Avidina/administração & dosagem , Avidina/química , Biotinilação/métodos , Medula Óssea/efeitos dos fármacos , Feminino , Isótopos de Iodo/administração & dosagem , Isótopos de Iodo/química , Isótopos de Iodo/farmacocinética , Marcação por Isótopo/métodos , Rim/efeitos dos fármacos , Lisina/administração & dosagem , Lisina/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/radioterapia , Polímeros/administração & dosagem , Polímeros/química , Polímeros/farmacocinética , Distribuição TecidualRESUMO
The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biological functions into cells has gained much attention in recent years. However, the internalization mechanisms and delivery properties of CPP-cargo remains controversial. In this study, low- and high-molecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluorescein isothiocyanate-labeled nona-arginine (CPP-FITC), and the latter was the fluorescently labeled nona-arginine-avidin complex (CPP-avidin). We measured the intracellular trafficking of CPP-FITC and CPP-avidin in four cancer cell lines in a series of microenvironments altered by the presence or absence of serum, different temperatures and different incubation times. The results revealed that CPP-cargo delivery exhibited no specificity toward any cell line, but the levels were found to be related to cell type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with related endocytic inhibitors. Two different types of CPP-cargo were required to cross the plasma membrane to bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs and small cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocytic route. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in an energy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependent pathway is not essential to the translocation of either type of CPP-cargo.
Assuntos
Arginina/química , Avidina/administração & dosagem , Peptídeos Penetradores de Células/química , Fluoresceína-5-Isotiocianato/administração & dosagem , Animais , Avidina/farmacocinética , Transporte Biológico , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sistemas de Liberação de Medicamentos , Endocitose , Fluoresceína-5-Isotiocianato/farmacocinética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Camundongos , Peso Molecular , Neoplasias/metabolismo , Ratos , Temperatura , Fatores de TempoRESUMO
We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 µg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 µL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions.
Assuntos
Avidina/farmacologia , Avidina/toxicidade , Biotina/administração & dosagem , Radioisótopos de Índio , Compostos Radiofarmacêuticos/farmacologia , Compostos Radiofarmacêuticos/toxicidade , Células 3T3 , Animais , Apoptose/efeitos dos fármacos , Avidina/imunologia , Avidina/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Macaca fascicularis , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Testes de Mutagenicidade/métodos , Compostos Radiofarmacêuticos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Testes de Toxicidade/métodosRESUMO
UNLABELLED: With the emergence of multimodal imaging strategies, genetically encoded reporters that can be flexibly combined with any imaging modality become highly attractive. Here we describe the use of glycosylphosphatidylinositol (GPI)-anchored avidin, an avidin moiety targeted to the extracellular side of cell membranes via a GPI anchor, as a reporter for in vivo imaging. Being present on the outside of cells, avidin can be visualized with any type of biotinylated imaging agent, without the requirement that the probe be membrane-permeable. We used the avidin-GPI system to monitor the activity of hypoxia-inducible factors (HIFs)-oxygen-sensing transcription factors, which play a major role in regulating cancer progression-in a mouse tumor allograft model. METHODS: Mouse C51 cells were stably transfected with pH3SVG, a reporter construct driving the expression of avidin-GPI from an HIF-sensitive promoter. The transfected cells were subcutaneously implanted into BALB/c nude mice. At 10 d after tumor inoculation, mice received an intravenous injection of either alexa-594-biocytin or (67)Ga-DOTA-biotin, and tumor HIF activity was imaged using fluorescence reflectance imaging or SPECT. RESULTS: In vitro cell experiments demonstrated the functionality and HIF-dependent regulation of the avidin-GPI reporter construct. In vivo, avidin-GPI was targeted specifically in allograft tumors with biotinylated imaging probes using both fluorescence imaging and SPECT. Analysis of the reporter expression pattern on ex vivo tumor tissue sections indicated a good overlap, with areas of hypoxia. CONCLUSION: We have demonstrated the utility of avidin-GPI as a reporter for multimodal in vivo imaging using both a fluorescence and a SPECT approach to assess intracellular oxygen signaling in a mouse tumor model.
Assuntos
Avidina/farmacocinética , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Glicosilfosfatidilinositóis/farmacocinética , Animais , Avidina/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Genes Reporter , Glicosilfosfatidilinositóis/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters - cell-penetrating peptides (CPPs) - are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP-cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs - transportan, oligoarginine and Tat - complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.
Assuntos
Avidina/farmacocinética , Endocitose , Peptídeos/farmacocinética , Sequência de Aminoácidos , Animais , Avidina/química , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Transporte ProteicoRESUMO
Avidin is a glycoprotein from hen egg white that binds biotin with very high affinity. Here we describe OXavidin, a product containing aldehyde groups, obtained by ligand-assisted sugar oxidation of avidin by sodium periodate. OXavidin chemically reacts with cellular and tissue proteins through Schiff's base formation thus residing in tissues for weeks while preserving the biotin binding capacity. The long tissue residence of OXavidin as well as that of OXavidin/biotinylated agent complex occurs in normal and neoplastic tissues and immunohistochemistry shows a strong and homogenous stromal localization. Once localized in tissue/tumor, OXavidin becomes an "artificial receptor" for intravenous injected biotin allowing tumor targeting with biotinylated therapeutics like radioisotopes or toxins. Moreover, present data also suggest that OXavidin might be useful for the homing of biotinylated cells. Overall, OXavidin exhibits a remarkable potential for many different therapeutic applications.
Assuntos
Avidina/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Imuno-Histoquímica/métodos , Animais , Avidina/química , Avidina/metabolismo , Avidina/uso terapêutico , Biotina/metabolismo , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Camundongos , Transplante de Neoplasias , Ácido Periódico , Estreptavidina/farmacocinéticaRESUMO
PURPOSE: In a continuous effort to seek for anticancer treatments with minimal side effects, we aim at proving the feasibility of the Intraoperative Avidination for Radionuclide Therapy, a new procedure for partial breast irradiation. EXPERIMENTAL DESIGN: To assess doses of 90Y-DOTA-biotin to target (i.e., breast tumor bed) and nontarget organs, we did simulation studies with 111In-DOTA-biotin in 10 candidates for conservative breast surgery. Immediately after quadrantectomy, patients were injected with 100-mg avidin in the tumor bed. On the following day, patients were given 111In-DOTA-biotin (approximately 111 MBq) i.v. after appropriate chase of biotinylated albumin (20 mg) to remove circulating avidin. Biokinetic studies were done by measuring radioactivity in scheduled blood samples, 48-h urine collection, and through scintigraphic images. The medical internal radiation dose formalism (OLINDA code) enabled dosimetry assessment in target and nontarget organs. RESULTS: Images showed early and long-lasting radioactive biotin uptake in the operated breast. Rapid blood clearance (<1% at 12 h) and urine excretion (>75% at 24 h) were observed. Absorbed doses, expressed as mean+/-SD in Gy/GBq, were as low as 0.15+/-0.05 in lungs, 0.10+/-0.02 in heart, 0.06+/-0.02 in red marrow, 1.30+/-0.50 in kidneys, 1.50+/-0.30 in urinary bladder, and 0.06+/-0.02 in total body, whereas in the targeted area, they increased to 5.5+/-1.1 Gy/GBq (50% ISOROI) and 4.8+/-1.0 Gy/GBq (30% ISOROI). CONCLUSION: Our preliminary results suggest that Intraoperative Avidination for Radionuclide Therapy is a simple and feasible procedure that may improve breast cancer patients' postsurgical management by shortening radiotherapy duration.
Assuntos
Avidina/administração & dosagem , Biotina/análogos & derivados , Braquiterapia/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/radioterapia , Compostos Organometálicos/administração & dosagem , Adolescente , Adulto , Idoso , Avidina/farmacocinética , Biotina/administração & dosagem , Biotina/farmacocinética , Neoplasias da Mama/cirurgia , Terapia Combinada , Feminino , Humanos , Radioisótopos de Índio , Injeções Intralesionais , Injeções Intravenosas , Período Intraoperatório , Mastectomia Segmentar , Pessoa de Meia-Idade , Compostos Organometálicos/farmacocinética , Projetos Piloto , Estudos Prospectivos , Cintilografia , Compostos Radiofarmacêuticos , Radioterapia Adjuvante , Biópsia de Linfonodo Sentinela , Distribuição Tecidual , Radioisótopos de ÍtrioRESUMO
A target cell-specific activation strategy for improved molecular imaging of peritoneal implants has been proposed, in which fluorophores are activated only in living targeted cells. A current example of an activatable fluorophore is one that is normally self-quenched by attachment to a peptide backbone but which can be activated by specific proteases that degrade the peptide resulting in "dequenching." In this study, an alternate fluorescence activation strategy is proposed whereby self-quenching avidin-rhodamine X, which has affinity for lectin on cancer cells, is activated after endocytosis and degradation within the lysosome. Using this approach in a mouse model of peritoneal ovarian metastases, we document target-specific molecular imaging of submillimeter cancer nodules with minimal contamination by background signal. Cellular internalization of receptor-ligand pairs with subsequent activation of fluorescence via dequenching provides a generalizable and highly sensitive method of detecting cancer microfoci in vivo and has practical implications for assisting surgical and endoscopic procedures.
Assuntos
Avidina/farmacocinética , Corantes Fluorescentes/farmacocinética , Neoplasias Ovarianas/metabolismo , Rodaminas/farmacocinética , Animais , Avidina/química , Linhagem Celular Tumoral , Detergentes/química , Feminino , Fluorescência , Corantes Fluorescentes/química , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Neoplasias Ovarianas/patologia , Rodaminas/química , Espectrometria de FluorescênciaRESUMO
A new procedure, known as Intraoperative Avidination for Radionuclide Therapy (IART), is described in breast cancer patients. In this paper, we provide proof of the principle that intraoperative injection of avidin in the tumour bed after quadrantectomy allows homing in of intravenously (IV) administered radioactive biotin to the target site. This approach of targeted therapy consists of two steps: (i) "avidination" of the anatomical area of the tumour with avidin injected by the surgeon, into and around the tumour bed; (ii) targeting the anatomical area of the tumour by IV injection of radiolabelled biotin. The scintigraphic images demonstrated fast and stable uptake of labelled biotin at the site of operated breast. The radiation dose released to the index quadrant was more than 5 Gy/GBq, consistent with a boost of 20 Gy for an activity of 3.7 GBq 90Y-biotin (100mCi). A further large clinical trial facing IART in combination with reduced external-beam radiotherapy is, in our opinion, fully justified.
Assuntos
Avidina/administração & dosagem , Biotina/administração & dosagem , Braquiterapia/métodos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/radioterapia , Adulto , Avidina/farmacocinética , Biotina/farmacocinética , Neoplasias da Mama/cirurgia , Terapia Combinada , Feminino , Humanos , Injeções Intralesionais , Injeções Intravenosas , Período Intraoperatório , Mastectomia Segmentar , Pessoa de Meia-Idade , Radiometria , Cintilografia , Dosagem Radioterapêutica , Radioterapia Adjuvante , Radioisótopos de ÍtrioRESUMO
A novel drug delivery vehicle that specifically targets using ultrasound radiation force (USRF) and biotin-avidin interactions is presented. Model vehicles consist of avidinated fluorescent nanobeads bound directly to the biotinylated lipid shells of preformed microbubbles. USRF was used to deflect the vehicle from the center of flow to a tube surface in order to facilitate receptor-ligand mediated adhesion. At wall shear stress levels commensurate with venous and arterial flow, USRF was used to direct the vehicles to a biotinylated tube surface. Subsequent high-pressure pulses fragmented the carrier, and molecular interactions induced deposition of the nanobeads on the wall. Targeting of nanobeads to the tube was molecularly specific and dependent on, in order of importance, vehicle concentration, wall shear stress, nanobead size, and insonation time. The observation that portions of the microbubble lipid monolayer shell remain attached to adherent nanobeads is important for future consideration of drug transport mechanisms. This versatile method of delivery is shown to enable targeted deposition of nanoparticles in shear flow and could be modified to carry therapeutic agents for controlled release in targeted delivery applications.
Assuntos
Portadores de Fármacos/farmacocinética , Microbolhas , Avidina/química , Avidina/farmacocinética , Portadores de Fármacos/química , Citometria de Fluxo/métodos , Modelos Químicos , Nanoestruturas/química , Tecnologia Farmacêutica/instrumentação , Tecnologia Farmacêutica/métodos , Transdutores , UltrassomRESUMO
A method for delivering drugs to sites of disease extension in mediastinal nodes is described. Mediastinal node and lymphatic distributions were determined after intracavitary injection of the avidin/biotin-liposome system in normal rats. The effect of the injected dose on lymphatic targeting of liposomes after intraperitoneal injection of (99m)Tc-blue-biotin-liposomes and intrapleural injection of avidin, and vice versa, is presented. Scintigraphic imaging was used to follow the movement of (99m)Tc-blue-biotin-liposomes to determine the pharmacokinetics and organ uptake. Tissue biodistribution studies were performed 22 h after injection of the (99m)Tc-blue-biotin-liposomes. Results indicated that independent of the cavity in which each agent was injected, a dose of 5.0 mg of each agent results in higher mediastinal node targeting (8%-10% ID/Organ) as compared with the injection of a 0.5 mg dose (2%-5% ID/Organ, p < 0.05). Targeting of diaphragm and associated lymphatics was observed when (99m)Tc-blue-biotin-liposomes were injected in peritoneum and avidin in pleural space. In contrast, pleural, and pericardial lymphatic targeting was observed when (99m)Tc-blue-biotin-liposomes were injected in pleural space and avidin in peritoneum. Intracavitary injection of the avidin/biotin-liposome system could potentially be used for the delivery of prophylactic drugs that could reduce tumor metastasis and infection spread to mediastinal nodes.
Assuntos
Avidina/farmacocinética , Biotina/farmacocinética , Lipossomos/farmacocinética , Animais , Avidina/administração & dosagem , Biotina/administração & dosagem , Diafragma/metabolismo , Injeções , Injeções Intraperitoneais , Lipossomos/administração & dosagem , Linfonodos/metabolismo , Vasos Linfáticos/metabolismo , Masculino , Mediastino , Cavidade Pleural , Ratos , Ratos Sprague-Dawley , TecnécioRESUMO
Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin-PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin-PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin-PEG 10 and 20 kDa prepared in the presence of imminobiotin-PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79-85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin-PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.
Assuntos
Avidina/química , Biotina/química , Polietilenoglicóis/química , Animais , Anticorpos/metabolismo , Avidina/farmacocinética , Sítios de Ligação/genética , Biotina/genética , Dicroísmo Circular , Imunoprecipitação , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/farmacocinética , Ligação Proteica , Conformação Proteica , TrítioRESUMO
The objective of this study was to develop a more effective liposome-based method for delivering drugs to mediastinal nodes. Nodal uptake was determined after intrapleural injection of the avidin/biotin-liposome system in normal rats. The effect of injection sequence (avidin injected 2 h before biotin-liposomes and vice versa), volume injected, and administered dose of the agents is described. Pharmacokinetics of the avidin/biotin-liposome system was monitored with scintigraphic imaging by labeling the biotin-liposomes with technetium-99m ((99m)Tc). To identify the nodes during the biodistribution studies, patent blue dye was encapsulated in the biotin-liposomes. Tissue biodistribution studies were performed 22 h after injection of the (99m)Tc-blue-biotin-liposomes. When avidin was injected before (99m)Tc-blue-biotin-liposomes, better mediastinal node targeting (15.7%; p < 0.05) was achieved than when biotin-liposomes were injected first (8.3%) or when only biotin-liposomes were injected (1.0%). Injection of a small dose of liposomes (0.5 mg phospholipid) and avidin (0.5 mg) resulted in the most favorable drug delivery to mediastinal nodes and other organs. Intrapleural injection of the avidin/biotin-liposome system could potentially be used for drug delivery to disease processes such as lung cancer, anthrax, and tuberculosis that invade mediastinal nodes and use them as centers of incubation and dissemination.
Assuntos
Avidina/farmacocinética , Biotina/farmacocinética , Linfonodos/metabolismo , Cavidade Pleural , Animais , Avidina/administração & dosagem , Biotina/administração & dosagem , Sistemas de Liberação de Medicamentos , Injeções , Lipossomos , Masculino , Mediastino , Ratos , Ratos Sprague-Dawley , Corantes de Rosanilina , Tecnécio , Fatores de Tempo , Distribuição TecidualRESUMO
Here we review an approach to the design and production of antibody/ligand pairs for use in cell targeting procedures, to achieve functional affinity far greater than avidin/biotin. Using fundamental chemical principles, we have developed antibody/ligand pairs that retain the binding specificity of the antibody, but do not dissociate. By eliminating the dissociation of the ligand from the antibody, we have made the affinity functionally infinite. This methodology is applicable to other biological binding pairs.
Assuntos
Anticorpos/efeitos dos fármacos , Metais Terras Raras , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Radioisótopos , Animais , Anticorpos/imunologia , Avidina/farmacocinética , Biotina/farmacocinética , Humanos , Radiografia , CintilografiaRESUMO
Avidin-poly(ethylene glycol) (PEG) conjugates were obtained by derivatization of about 10% of the protein amino groups (four amino groups per protein molecule) with linear 5 kDa PEG or branched 10 or 20 kDa PEGs. Circular dichroism analysis showed that the polymer conjugation neither altered the protein structure nor the environment of the aromatic amino acids which are present at the level of the biotin binding site. Spectroscopic studies were carried out to evaluate the biotin recognition activity of the conjugates either in terms of number of biotin binding sites or avidin/biotin affinity. Avidin-PEG 5 kDa and avidin-PEG 10 kDa displayed over 90% of the native protein biological activity while a reduction in the recognition of biotinylated antibodies of about 25% was found with PEG 20 kDa. In vivo studies demonstrated that the protein immunogenicity was in the order: wild type avidin>avidin-PEG 5 kDa>avidin-PEG 10 kDa>avidin-PEG 20 kDa. By intravenous injection into mice bearing a solid tumor, all conjugates displayed prolonged permanence in the circulation with respect to the native protein. The area under the curve values of avidin-PEG 5 kDa, avidin-PEG 10 kDa and avidin-PEG 20 kDa were about 3-, 7- and 30-times higher than the wild type avidin with reduced accumulation in kidneys and liver. Interestingly, all conjugates accumulated significantly in the tumor mass. In particular, in the case of avidin-PEG 20 kDa, 8% of the injected dose (ID)/g of tissue accumulated in the tumor after 5 h from the administration and over 6% of the ID/g was maintained throughout 72 h.
Assuntos
Avidina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Polietilenoglicóis/administração & dosagem , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Avidina/sangue , Avidina/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/farmacocinéticaRESUMO
To establish an effective nonviral gene delivery and a corresponding imaging method for i.p.-disseminated tumors, various oligonucleotide-carrier complexes were synthesized, and their in vitro and in vivo properties were examined. The 20-mer multiamino-linked oligonucleotide (oligo), synthesized as antisense against the c-erbB-2 sequence, and the 3'-biotinylated form of the same oligonucleotide (oligo-Bt) were (111)In labeled through a diethylenetriaminepentaacetic acid chelate. (111)In-oligo was mixed with generation 4 polyamidoamine dendrimer (G4) or with biotinylated G4 (G4-Bt), which are positively charged to form electrostatic complexes. (111)In-oligo/G4-Bt and (111)In-oligo-Bt were conjugated to avidin ((111)In-oligo/G4-Av and (111)In-oligo-Av, respectively). (111)In-oligo/G4, (111)In-oligo/G4-Av, (111)In-oligo-Av, and carrier-free (111)In-oligo (2.96 kBq/22.4-45.9 ng of oligo) were examined for internalization in vitro in human ovarian cancer cells (SHIN3). Biodistribution of (111)In-oligo-carrier complexes or (111)In-oligo was examined in normal (n = 4-7) or i.p. SHIN3 tumor-bearing (n = 6-10) mice 2-24 h after i.p. injection (74 kBq/125-300 ng). Scintigraphy of i.p. tumor-bearing and normal mice was performed at various times postinjection of (111)In-oligo-carrier complex or (111)In-oligo (1.85 MBq/2.2 ng). (111)In-oligo-carrier complexes bound to the tumor cells were internalized at a rate of 34-56% at 24 h. In vivo, G4, G4-Av, and Av significantly enhanced tumor delivery of (111)In-oligo [9.1, 14.5, and 24.4% of injected dose per g of tissue (ID/g) at 24 h; P < 0.05, < 0.01, and < 0.0001, respectively] compared with delivery without carrier (0.8% ID/g). Scintigrams of (111)In-oligo delivered to the i.p.-disseminated tumors by the carriers were successfully obtained. In conclusion, G4, G4-Av, and Av can effectively deliver (111)In-oligo to i.p.-disseminated tumors. (111)In-oligo-carrier complexes also have potential as tracers for imaging and monitoring of gene delivery.