RESUMO
Rationale: Multiple myeloma (MM) is a multifocal malignancy of bone marrow plasma cells, characterized by vicious cycles of remission and relapse that eventually culminate in death. The disease remains mostly incurable largely due to the complex interactions between the bone microenvironment (BME) and MM cells (MMC). In the "vicious cycle" of bone disease, abnormal activation of osteoclasts (OCs) by MMC causes severe osteolysis, promotes immune evasion, and stimulates the growth of MMC. Disrupting these cancer-stroma interactions would enhance treatment response. Methods: To disrupt this cycle, we orthogonally targeted nanomicelles (NM) loaded with non-therapeutic doses of a photosensitizer, titanocene (TC), to VLA-4 (α4ß1, CD49d/CD29) expressing MMC (MM1.S) and αvß3 (CD51/CD61) expressing OC. Concurrently, a non-lethal dose of a radiopharmaceutical, 18F-fluorodeoxyglucose ([18F]FDG) administered systemically interacted with TC (radionuclide stimulated therapy, RaST) to generate cytotoxic reactive oxygen species (ROS). The in vitro and in vivo effects of RaST were characterized in MM1.S cell line, as well as in xenograft and isograft MM animal models. Results: Our data revealed that RaST induced non-enzymatic hydroperoxidation of cellular lipids culminating in mitochondrial dysfunction, DNA fragmentation, and caspase-dependent apoptosis of MMC using VLA-4 avid TC-NMs. RaST upregulated the expression of BAX, Bcl-2, and p53, highlighting the induction of apoptosis via the BAK-independent pathway. The enhancement of multicopper oxidase enzyme F5 expression, which inhibits lipid hydroperoxidation and Fenton reaction, was not sufficient to overcome RaST-induced increase in the accumulation of irreversible function-perturbing α,ß-aldehydes that exerted significant and long-lasting damage to both DNA and proteins. In vivo, either VLA-4-TC-NM or αvß3-TC-NMs RaST induced a significant therapeutic effect on immunocompromised but not immunocompetent MM-bearing mouse models. Combined treatment with both VLA-4-TC-NM and αvß3-TC-NMs synergistically inhibited osteolysis, reduced tumor burden, and prevented rapid relapse in both in vivo models of MM. Conclusions: By targeting MM and bone cells simultaneously, combination RaST suppressed MM disease progression through a multi-prong action on the vicious cycle of bone cancer. Instead of using the standard multidrug approach, our work reveals a unique photophysical treatment paradigm that uses nontoxic doses of a single light-sensitive drug directed orthogonally to cancer and bone cells, followed by radionuclide-stimulated generation of ROS to inhibit tumor progression and minimize osteolysis in both immunocompetent murine and immunocompromised human MM models.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Compostos Organometálicos/farmacologia , Osteoclastos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/metabolismo , Neoplasias Ósseas , Osso e Ossos/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fluordesoxiglucose F18/farmacologia , Humanos , Cadeias alfa de Integrinas/efeitos dos fármacos , Cadeias alfa de Integrinas/metabolismo , Camundongos , Mieloma Múltiplo/metabolismo , Compostos Organometálicos/metabolismo , Osteoclastos/efeitos dos fármacos , Osteólise/patologia , Radioisótopos/farmacologia , Compostos Radiofarmacêuticos/uso terapêutico , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Nanomedicina Teranóstica/métodos , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: In multiple myeloma, drug-resistant cells underlie relapse or progression following chemotherapy. Cell adhesion-mediated drug resistance (CAM-DR) is an established mechanism used by myeloma cells (MMC) to survive chemotherapy and its markers are upregulated in residual disease. The integrin very late antigen 4 (VLA4; α4ß1) is a key mediator of CAM-DR and its expression affects drug sensitivity of MMCs. Rather than trying to inhibit its function, here, we hypothesized that upregulation of VLA4 by resistant MMCs could be exploited for targeted delivery of drugs, which would improve safety and efficacy of treatments. EXPERIMENTAL DESIGN: We synthetized 20 nm VLA4-targeted micellar nanoparticles (V-NP) carrying DiI for tracing or a novel camptothecin prodrug (V-CP). Human or murine MMCs, alone or with stroma, and immunocompetent mice with orthotopic multiple myeloma were used to track delivery of NPs and response to treatments. RESULTS: V-NPs selectively delivered their payload to MMCs in vitro and in vivo, and chemotherapy increased their uptake by surviving MMCs. V-CP, alone or in combination with melphalan, was well tolerated and prolonged survival in myeloma-bearing mice. V-CP also reduced the dose requirement for melphalan, reducing tumor burden in association with suboptimal dosing without increasing overall toxicity. CONCLUSIONS: V-CP may be a safe and effective strategy to prevent or treat relapsing or refractory myeloma. V-NP targeting of resistant cells may suggest a new approach to environment-induced resistance in cancer.
Assuntos
Integrina alfa4beta1/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/metabolismo , Animais , Camptotecina/uso terapêutico , Adesão Celular , Linhagem Celular Tumoral , Dexametasona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Humanos , Melfalan/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/mortalidade , Inibidores da Topoisomerase I/uso terapêuticoRESUMO
Tumor-associated macrophages (TAMs) enhance tumor growth in mice and are correlated with a worse prognosis for breast cancer patients. While early therapies sought to deplete all macrophages, current therapeutics aim to reprogram pro-tumor macrophages (M2) and preserve those necessary for anti-tumor immune responses (M1). Recent studies have shown that c-MYC (MYC) is induced in M2 macrophages in vitro and in vivo where it regulates the expression of tumor-promoting genes. In a myeloid lineage MYC KO mouse model, MYC had important roles in macrophage maturation and function leading to reduced tumor growth. We therefore hypothesized that targeted delivery of a MYC inhibitor to established M2 TAMs could reduce polarization toward an M2 phenotype in breast cancer models. Methods: In this study, we developed a MYC inhibitor prodrug (MI3-PD) for encapsulation within perfluorocarbon nanoparticles, which can deliver drugs directly to the cytosol of the target cell through a phagocytosis independent mechanism. We have previously shown that M2-like TAMs express significant levels of the vitronectin receptor, integrin ß3, and in vivo targeting and therapeutic potential was evaluated using αvß3 integrin targeted rhodamine-labeled nanoparticles (NP) or integrin αvß3-MI3-PD nanoparticles. Results: We observed that rhodamine, delivered by αvß3-rhodamine NP, was incorporated into M2 tumor promoting macrophages through both phagocytosis-independent and dependent mechanisms, while NP uptake in tumor suppressing M1 macrophages was almost exclusively through phagocytosis. In a mouse model of breast cancer (4T1-GFP-FL), M2-like TAMs were significantly reduced with αvß3-MI3-PD NP treatment. To validate this effect was independent of drug delivery to tumor cells and was specific to the MYC inhibitor, mice with integrin ß3 knock out tumors (PyMT-Bo1 ß3KO) were treated with αvß3-NP or αvß3-MI3-PD NP. M2 macrophages were significantly reduced with αvß3-MI3-PD nanoparticle therapy but not αvß3-NP treatment. Conclusion: These data suggest αvß3-NP-mediated drug delivery of a c-MYC inhibitor can reduce protumor M2-like macrophages while preserving antitumor M1-like macrophages in breast cancer.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Nanopartículas/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Animais , Antineoplásicos/química , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/imunologia , Linhagem Celular Tumoral/transplante , Avaliação Pré-Clínica de Medicamentos , Feminino , Fluorocarbonos/administração & dosagem , Fluorocarbonos/química , Técnicas de Inativação de Genes , Humanos , Integrina alfaVbeta3 , Integrina beta3 , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia , Camundongos , Nanopartículas/química , Fagocitose , Cultura Primária de Células , Pró-Fármacos/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologiaRESUMO
Robust thrombus imaging is an unresolved clinical unmet need dating back to the mid 1970s. While early molecular imaging approaches began with nuclear SPECT imaging, contrast agents for virtually all biomedical imaging modalities have been demonstrated in vivo with unique strengths and common weaknesses. Two primary molecular imaging targets have been pursued for thrombus imaging: platelets and fibrin. Some common issues noted over 40 years ago persist today. Acute thrombus is readily imaged with all probes and modalities, but aged thrombus remains a challenge. Similarly, anti-coagulation continues to interfere with and often negate thrombus imaging efficacy, but heparin is clinically required in patients suspected of pulmonary embolism, deep venous thrombosis or coronary ruptured plaque prior to confirmatory diagnostic studies have been executed and interpreted. These fundamental issues can be overcome, but an innovative departure from the prior approaches will be needed.
Assuntos
Imagem Molecular/história , Trombose/diagnóstico por imagem , História do Século XX , História do Século XXI , HumanosRESUMO
Treatment of advanced heart failure with implantable LVADs is increasing, driven by profound unmet patient need despite potential serious complications: bleeding, infection, and thrombus. The experimental objective was to develop a sensitive imaging approach to assess early thrombus accumulation in LVADs under operational high flow and high shear rates. Methods: A monomeric bifunctional ligand with a fibrin-specific peptide, a short spacer, and 99mTc chelating amino acid sequence (F1A) was developed and compared to its tetrameric PEG analogue (F4A). Results:99mTc attenuation by LVAD titanium (1 mm) was 23%. 99mTc-F1A affinity to fibrin was Kd ~10 µM, whereas, the bound 99mTc-F4A probe was not displaced by F1A (120,000:1). Human plasma interfered with 99mTc-F1A binding to fibrin clot (p<0.05) in vitro, whereas, 99mTc-F4A targeting was unaffected. The pharmacokinetic half-life of 99mTc-F4A was 28% faster (124±41 min) than 99mTc-F1A (176±26 min) with both being bioeliminated through the urinary system with negligible liver or spleen biodistribution. In mice with carotid thrombus, 99mTc-F4A binding to the injured carotid was much greater (16.3±3.3 %ID/g, p=0.01) than that measured with an irrelevant negative control, 99mTc-I4A (3.4±1.6 %ID/g). In an LVAD mock flow-loop (1:1, PBS:human plasma:heparin) operating at maximal flow rate, 99mTc-F4A bound well to phantom clots in 2 min (p<0.05), whereas 99mTc-F1A had negligible targeting. Excised LVADs from patients undergoing pump exchange or heart transplant were rewired, studied in the mock flow loop, and found to have spatially variable fibrin accumulations in the inlet and outlet cannulas and bearings. Conclusions:99mTc-F4A is a high-avidity prototype probe for characterizing thrombus in LVADs that is anticipated to help optimize anticoagulation, reduce thromboembolic events, and minimize pump exchange.
Assuntos
Fibrina/metabolismo , Coração Auxiliar/efeitos adversos , Proteínas Recombinantes/metabolismo , Coloração e Rotulagem/métodos , Tecnécio/análise , Tecnécio/metabolismo , Trombose/diagnóstico , Animais , Meia-Vida , Insuficiência Cardíaca/terapia , Humanos , Camundongos , Ligação Proteica , Proteínas Recombinantes/farmacocinéticaRESUMO
Most cancer patients succumb to disseminated disease because conventional systemic therapies lack spatiotemporal control of their toxic effects in vivo, particularly in a complicated milieu such as bone marrow where progenitor stem cells reside. Here, we demonstrate the treatment of disseminated cancer by photoactivatable drugs using radiopharmaceuticals. An orthogonal-targeting strategy and a contact-facilitated nanomicelle technology enabled highly selective delivery and co-localization of titanocene and radiolabelled fluorodeoxyglucose in disseminated multiple myeloma cells. Selective ablation of the cancer cells was achieved without significant off-target toxicity to the resident stem cells. Genomic, proteomic and multimodal imaging analyses revealed that the downregulation of CD49d, one of the dimeric protein targets of the nanomicelles, caused therapy resistance in small clusters of cancer cells. Similar treatment of a highly metastatic breast cancer model using human serum albumin-titanocene formulation significantly inhibited cancer growth. This strategy expands the use of phototherapy for treating previously inaccessible metastatic disease.
Assuntos
Neoplasias Mamárias Experimentais/terapia , Mieloma Múltiplo/terapia , Compostos Organometálicos/administração & dosagem , Fotoquimioterapia , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Integrina alfa4beta1 , Camundongos Endogâmicos C57BL , Camundongos SCID , Micelas , Terapia de Alvo Molecular , Nanopartículas , Ratos , Albumina Sérica Humana , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
While the in vivo efficacy of Sn-2 phosphatidylcholine prodrugs incorporated into targeted, non-pegylated lipid-encapsulated nanoparticles was demonstrated in prior preclinical studies, the microscopic details of cell prodrug internalization and trafficking events are unknown. Classic fluorescence microscopy, fluorescence lifetime imaging microscopy, and single-molecule super-resolution microscopy were used to investigate the cellular handling of doxorubicin-prodrug and AlexaFluor™-488-prodrug. Sn-2 phosphatidylcholine prodrugs delivered by hemifusion of nanoparticle and cell phospholipid membranes functioned as phosphatidylcholine mimics, circumventing the challenges of endosome sequestration and release. Phosphatidylcholine prodrugs in the outer cell membrane leaflet translocated to the inner membrane leaflet by ATP-dependent and ATP-independent mechanisms and distributed broadly within the cytosolic membranes over the next 12 h. A portion of the phosphatidylcholine prodrug populated vesicle membranes trafficked to the perinuclear Golgi/ER region, where the drug was enzymatically liberated and activated. Native doxorubicin entered the cells, passed rapidly to the nucleus, and bound to dsDNA, whereas DOX was first enzymatically liberated from DOX-prodrug within the cytosol, particularly in the perinuclear region, before binding nuclear dsDNA. Much of DOX-prodrug was initially retained within intracellular membranes. In vitro anti-proliferation effectiveness of the two drug delivery approaches was equivalent at 48 h, suggesting that residual intracellular DOX-prodrug may constitute a slow-release drug reservoir that enhances effectiveness. We have demonstrated that Sn-2 phosphatidylcholine prodrugs function as phosphatidylcholine mimics following reported pathways of phosphatidylcholine distribution and metabolism. Drug complexed to the Sn-2 fatty acid is enzymatically liberated and reactivated over many hours, which may enhance efficacy overtime.
RESUMO
Although angiogenesis is a hallmark feature of asthmatic inflammatory responses, therapeutic anti-angiogenesis interventions have received little attention. Objective: Assess the effectiveness of anti-angiogenic Sn2 lipase-labile prodrugs delivered via αvß3-micellar nanotherapy to suppress microvascular expansion, bronchial remodeling, and airway hyper-responsiveness in Brown Norway rats exposed to serial house dust mite (HDM) inhalation challenges. Results: Anti-neovascular effectiveness of αvß3-mixed micelles incorporating docetaxel-prodrug (Dxtl-PD) or fumagillin-prodrug (Fum-PD) were shown to robustly suppress neovascular expansion (p<0.01) in the upper airways/bronchi of HDM rats using simultaneous 19F/1H MR neovascular imaging, which was corroborated by adjunctive fluorescent microscopy. Micelles without a drug payload (αvß3-No-Drug) served as a carrier-only control. Morphometric measurements of HDM rat airway size (perimeter) and vessel number at 21d revealed classic vascular expansion in control rats but less vascularity (p<0.001) after the anti-angiogenic nanotherapies. CD31 RNA expression independently corroborated the decrease in airway microvasculature. Methacholine (MCh) induced respiratory system resistance (Rrs) was high in the HDM rats receiving αvß3-No-Drug micelles while αvß3-Dxtl-PD or αvß3-Fum-PD micelles markedly and equivalently attenuated airway hyper-responsiveness and improved airway compliance. Total inflammatory BAL cells among HDM challenged rats did not differ with treatment, but αvß3+ macrophages/monocytes were significantly reduced by both nanotherapies (p<0.001), most notably by the αvß3-Dxtl-PD micelles. Additionally, αvß3-Dxtl-PD decreased BAL eosinophil and αvß3+ CD45+ leukocytes relative to αvß3-No-Drug micelles, whereas αvß3-Fum-PD micelles did not. Conclusion: These results demonstrate the potential of targeted anti-angiogenesis nanotherapy to ameliorate the inflammatory hallmarks of asthma in a clinically relevant rodent model.
Assuntos
Remodelação das Vias Aéreas , Inibidores da Angiogênese/administração & dosagem , Asma/tratamento farmacológico , Asma/patologia , Nanoestruturas/administração & dosagem , Animais , Asma/diagnóstico por imagem , Cicloexanos/administração & dosagem , Modelos Animais de Doenças , Docetaxel , Portadores de Fármacos/administração & dosagem , Ácidos Graxos Insaturados/administração & dosagem , Imageamento por Ressonância Magnética , Microscopia de Fluorescência , Pró-Fármacos/administração & dosagem , Pyroglyphidae/patogenicidade , Ratos , Sesquiterpenos/administração & dosagem , Taxoides/administração & dosagem , Resultado do TratamentoRESUMO
Fumagillin, an unstable anti-angiogenesis mycotoxin, was synthesized into a stable lipase-labile prodrug and incorporated into integrin-targeted lipid-encapsulated nanoparticles (αvß3-Fum-PD NP). Dual anti-angiogenic therapy combining αvß3-Fum-PD NP with zoledronic acid (ZA), a long-acting osteoclast inhibitor with proposed anti-angiogenic effects, was evaluated. In vitro, αvß3-Fum-PD NP reduced (P<0.05) endothelial cell viability without impacting macrophage viability. ZA suppressed (P<0.05) macrophage viability at high dosages but not endothelial cell proliferation. 3D MR neovascular imaging of rabbit Vx2 tumors showed no effect with ZA, whereas αvß3-Fum-PD NP alone and with ZA decreased angiogenesis (P<0.05). Immunohistochemistry revealed decreased (P<0.05) microvascularity with αvß3-Fum-PD NP and ZA and further microvascular reduction (P<0.05) with dual-therapy. In vivo, ZA did not decrease tumor macrophage numbers nor cancer cell proliferation, whereas αvß3-Fum-PD-NPs reduced both measures. Dual-therapy with ZA and αvß3-Fum-PD-NP may provide enhanced neo-adjuvant utility if macrophage ZA uptake is increased. From the Clinical Editor: Although anti-angiogenesis is one of the treatment modalities in the fight against cancer, many cancers become resistant to VEGF pathway inhibitors. In this article, the authors investigated the use of dual therapy using fumagillin, integrin-targeted lipid-encapsulated nanoparticles (αvß3- Fum-PD NP) and zoledronic acid (ZA), in both in-vitro and in-vivo experiments. This combination approach may provide an insight to the design of future drugs against cancers.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Difosfonatos/administração & dosagem , Imidazóis/administração & dosagem , Integrina alfaVbeta3/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Pró-Fármacos/administração & dosagem , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/química , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/química , Linhagem Celular Tumoral , Difusão , Difosfonatos/química , Imidazóis/química , Masculino , Terapia de Alvo Molecular/métodos , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Pró-Fármacos/química , Coelhos , Resultado do Tratamento , Ácido ZoledrônicoRESUMO
Multiple myeloma pathogenesis is driven by the MYC oncoprotein, its dimerization with MAX, and the binding of this heterodimer to E-Boxes in the vicinity of target genes. The systemic utility of potent small molecule inhibitors of MYC-MAX dimerization was limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. We hypothesized that new lipid-based MYC-MAX dimerization inhibitor prodrugs delivered via integrin-targeted nanoparticles (NP) would overcome prior shortcomings of MYC inhibitor approaches and prolong survival in a mouse model of cancer. An Sn 2 lipase-labile prodrug inhibitor of MYC-MAX dimerization (MI1-PD) was developed which decreased cell proliferation and induced apoptosis in cultured multiple myeloma cell lines alone (P < 0.05) and when incorporated into integrin-targeted lipid-encapsulated NPs (P < 0.05). Binding and efficacy of NPs closely correlated with integrin expression of the target multiple myeloma cells. Using a KaLwRij metastatic multiple myeloma mouse model, VLA-4-targeted NPs (20 nm and 200 nm) incorporating MI1-PD (D) NPs conferred significant survival benefits compared with respective NP controls, targeted (T) no-drug (ND), and untargeted (NT) control NPs (T/D 200: 46 days vs. NT/ND: 28 days, P < 0.05 and T/D 20: 52 days vs. NT/ND: 29 days, P = 0.001). The smaller particles performed better of the two sizes. Neither MI1 nor MI1-PD provided survival benefit when administered systemically as free compounds. These results demonstrate for the first time that a small molecule inhibitor of the MYC transcription factor can be an effective anticancer agent when delivered using a targeted nanotherapy approach.
Assuntos
Integrina alfa4beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas/química , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Integrina alfa4beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Camundongos , Mieloma Múltiplo/patologia , Pró-Fármacos/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacosRESUMO
In the extension phase of acute kidney injury, microvascular thrombosis, inflammation, vasoconstriction, and vascular endothelial cell dysfunction promote progressive damage to renal parenchyma after reperfusion. In this study, we hypothesized that direct targeting and pharmaceutical knockdown of activated thrombin at the sites of injury with a selective nanoparticle (NP)-based thrombin inhibitor, PPACK (phenylalanine-proline-arginine-chloromethylketone), would improve kidney reperfusion and protect renal function after transient warm ischemia in rodent models. Saline- or plain NP-treated animals were employed as controls. In vivo 19F magnetic resonance imaging revealed that kidney nonreperfusion was evident within 3 h after global kidney reperfusion at 34 ± 13% area in the saline group and 43 ± 12% area in the plain NP group and substantially reduced to 17 ± 4% (â¼50% decrease, P < 0.05) in the PPACK NP pretreatment group. PPACK NP pretreatment prevented an increase in serum creatinine concentration within 24 h after ischemia-reperfusion, reflecting preserved renal function. Histologic analysis illustrated substantially reduced intrarenal thrombin accumulation within 24 h after reperfusion for PPACK NP-treated kidneys (0.11% ± 0.06%) compared with saline-treated kidneys (0.58 ± 0.37%). These results suggest a direct role for thrombin in the pathophysiology of AKI and a nanomedicine-based preventative strategy for improving kidney reperfusion after transient warm ischemia.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Inibidores de Cisteína Proteinase/farmacologia , Células Endoteliais/efeitos dos fármacos , Nanopartículas de Magnetita/administração & dosagem , Traumatismo por Reperfusão/tratamento farmacológico , Trombina/antagonistas & inibidores , Injúria Renal Aguda/patologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Células Cultivadas , Creatinina/urina , Modelos Animais de Doenças , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/patologiaRESUMO
A high r1 relaxivity manganese-gadolinium nanocolloid (αvß3-MnOL-Gd NC) was developed and effectively detected atherosclerotic angiogenesis in rabbits fed cholesterol-rich diets for 12 months using a clinical MRI scanner (3T). 3D mapping of neovasculature signal intensity revealed the spatial coherence and intensity of plaque angiogenic expansion, which may, with other high risk MR bioindicators, help identify high-risk patients with moderate (40% to 60%) vascular stenosis. Microscopy confirmed the predominant media and plaque distribution of fluorescent αvß3-MnOL-Gd NC, mirroring the MR data. An expected close spatial association of αvß3-integrin neovasculature and macrophages was noted, particularly within plaque shoulder regions. Manganese oleate bioelimination occurred via the biliary system into feces. Gd-DOTA was eliminated through the bile-fecal and renal excretion routes. αvß3-MnOL-Gd NC offers an effective vehicle for T1w neovascular imaging in atherosclerosis. From the clinical editor: Cerebrovascular accidents are a leading cause of mortality and morbidity worldwide. The acute formation of thrombus following atherosclerotic plaque rupture has been well recognized as the etiology of stroke. The authors studied microanatomical features of vulnerable atherosclerotic plaque in this article, in an attempt to identify those with high risk of rupture. Gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC) was developed as a novel contrast agent for MRI. They show that this agent is effective in providing neovascular imaging.
Assuntos
Aterosclerose/diagnóstico por imagem , Meios de Contraste/farmacologia , Gadolínio/farmacologia , Hiperlipidemias/diagnóstico por imagem , Manganês/farmacologia , Neovascularização Patológica/diagnóstico por imagem , Placa Aterosclerótica/diagnóstico por imagem , Animais , Coloides , Meios de Contraste/química , Gadolínio/química , Manganês/química , Coelhos , RadiografiaRESUMO
High-relaxivity T1-weighted (T1w) MR molecular imaging nanoparticles typically present high surface gadolinium payloads that can elicit significant acute complement activation (CA). The objective of this research was to develop a high T1w contrast nanoparticle with improved safety. We report the development, optimization, and characterization of a gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC; 138±10 (Dav)/nm; PDI: 0.06; zeta: -27±2 mV). High r1 particulate relaxivity with minute additions of Gd-DOTA-lipid conjugate to the MnOL nanocolloid surface achieved an unexpected paramagnetic synergism. This hybrid MnOL-Gd NC provided optimal MR TSE signal intensity at 5 nM/voxel and lower levels consistent with the level expression anticipated for sparse biomarkers, such as neovascular integrins. MnOL NC produced optimal MR TSE signal intensity at 10 nM/voxel concentrations and above. Importantly, MnOL-Gd NC avoided acute CA in vitro and in vivo while retaining minimal transmetallation risk. From the clinical editor: The authors developed a gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC) in this study. These were used as a high-relaxivity paramagnetic MR molecular imaging agent in experimental models. It was shown that MnOL-Gd NC could provide high T1w MR contrast for targeted imaging. As the level of gadolinium used was reduced, there was also reduced risk of systemic side effects from complement activation.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Meios de Contraste , Gadolínio , Imageamento por Ressonância Magnética , Manganês , Nanopartículas , Animais , Biomarcadores/sangue , Coloides , Meios de Contraste/efeitos adversos , Meios de Contraste/química , Meios de Contraste/farmacologia , Avaliação Pré-Clínica de Medicamentos , Gadolínio/efeitos adversos , Gadolínio/química , Gadolínio/farmacologia , Manganês/efeitos adversos , Manganês/química , Manganês/farmacologia , Camundongos , Nanopartículas/efeitos adversos , Nanopartículas/químicaRESUMO
In nanomedicine, the hydrophobic nature of paclitaxel has favored its incorporation into many nanoparticle formulations for anti-cancer chemotherapy. At lower doses taxanes are reported to elicit anti-angiogenic responses. In the present study, the facile synthesis, development and characterization of a new lipase-labile docetaxel prodrug is reported and shown to be an effective anti-angiogenic agent in vitro and in vivo. The Sn 2 phosphatidylcholine prodrug was stably incorporated into the lipid membrane of α(v)ß3-integrin targeted perfluorocarbon (PFC) nanoparticles (α(v)ß3-Dxtl-PD NP) and did not appreciably release during dissolution against PBS buffer or plasma over three days. Overnight exposure of α(v)ß3-Dxtl-PD NP to plasma spiked with phospholipase enzyme failed to liberate the taxane from the membrane until the nanoparticle integrity was compromised with alcohol. The bioactivity and efficacy of α(v)ß3-Dxtl-PD NP in endothelial cell culture was as effective as Taxol(®) or free docetaxel in methanol at equimolar doses over 96 hours. The anti-angiogenesis effectiveness of α(v)ß3-Dxtl-PD NP was demonstrated in the Vx2 rabbit model using MR imaging of angiogenesis with the same α(v)ß3-PFC nanoparticle platform. Nontargeted Dxtl-PD NP had a similar MR anti-angiogenesis response as the integrin-targeted agent, but microscopically measured decreases in tumor cell proliferation and increased apoptosis were detected only for the targeted drug. Equivalent dosages of Abraxane(®) given over the same treatment schedule had no effect on angiogenesis when compared to control rabbits receiving saline only. These data demonstrate that α(v)ß3-Dxtl-PD NP can reduce MR detectable angiogenesis and slow tumor progression in the Vx2 model, whereas equivalent systemic treatment with free taxane had no benefit.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Nanopartículas/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Pró-Fármacos/uso terapêutico , Taxoides/uso terapêutico , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Apoptose , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Células Cultivadas , Docetaxel , Células Endoteliais/efeitos dos fármacos , Fluorocarbonos/química , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Nanopartículas/química , Fosfolipases/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Coelhos , Taxoides/química , Taxoides/farmacologiaRESUMO
IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48- and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.
Assuntos
Interleucina-1/farmacologia , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Linhagem Celular , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.
Assuntos
MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 3/metabolismo , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Receptor 8 Toll-Like/fisiologia , Animais , Fibroblastos/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1 , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de SinaisRESUMO
TNF receptor (TNFR) superfamily members, CD40, and BAFFR play critical roles in B cell survival and differentiation. Genetic deficiency in a novel adaptor molecule, Act1, for CD40 and BAFF results in a dramatic increase in peripheral B cells, which culminates in lymphadenopathy and splenomegaly, hypergammaglobulinemia, and autoantibodies. While the B cell-specific Act1 knockout mice displayed a similar phenotype with less severity, the pathology of the Act1-deficient mice was mostly blocked in CD40-Act1 and BAFF-Act1 double knockout mice. CD40- and BAFF-mediated survival is significantly increased in Act1-deficent B cells, with stronger IkappaB phosphorylation, processing of NF-kappaB2 (p100/p52), and activation of JNK, ERK, and p38 pathways, indicating that Act1 negatively regulates CD40- and BAFF-mediated signaling events. These findings demonstrate that Act1 plays an important role in the homeostasis of B cells by attenuating CD40 and BAFFR signaling.