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1.
Yakugaku Zasshi ; 140(10): 1235-1242, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999202

RESUMO

The central nervous system (CNS) is segregated from the circulating blood and peripheral tissues by endothelial and epithelial barriers. To overcome refractory CNS diseases, it is important to understand the membrane transport systems of drugs and the endogenous compounds that relate to the pathogenesis of CNS diseases at these barriers. The endothelial barrier in the brain is the blood-brain barrier (BBB). Our studies clarified the efflux transport of prostaglandin E2 (PGE2), a modulator of neural excitation and inflammatory responses, across the BBB via plasma membrane transporters such as organic anion transporter 3 (Oat3) and multidrug resistance-associated protein 4 (Mrp4). This efflux transport was attenuated by peripheral inflammation or cerebral treatment with neuroexcitatory l-glutamate, suggesting that BBB-mediated PGE2 elimination was altered under several pathological conditions. We also examined excitatory amino acid transporter (EAAT) 1 and 3 as l-glutamate efflux transporters of the inner blood-retinal barrier (BRB) and blood-cerebrospinal barrier. It was considered that these efflux membrane transporters participated in the homeostasis of neuroexcitatory and neuroinflammatory responses in the brain and retina. Moreover, we identified connexin 43 (Cx43) hemichannels as a new membrane transport system that is activated under pathological conditions and recognizes several monocarboxylate drugs, such as valproate. As it is expected that the action of these membrane transporters across the CNS barriers is of great importance in understanding the pathology of various neuroexcitatory diseases, our studies should contribute to the establishment of therapeutic strategies for refractory CNS diseases.


Assuntos
Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/metabolismo , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/metabolismo , Desenvolvimento de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Retina/metabolismo , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Conexina 43/metabolismo , Dinoprostona/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo
2.
Nat Commun ; 11(1): 4929, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004789

RESUMO

Non-invasive, molecularly-specific, focal modulation of brain circuits with low off-target effects can lead to breakthroughs in treatments of brain disorders. We systemically inject engineered ultrasound-controllable drug carriers and subsequently apply a novel two-component Aggregation and Uncaging Focused Ultrasound Sequence (AU-FUS) at the desired targets inside the brain. The first sequence aggregates drug carriers with millimeter-precision by orders of magnitude. The second sequence uncages the carrier's cargo locally to achieve high target specificity without compromising the blood-brain barrier (BBB). Upon release from the carriers, drugs locally cross the intact BBB. We show circuit-specific manipulation of sensory signaling in motor cortex in rats by locally concentrating and releasing a GABAA receptor agonist from ultrasound-controlled carriers. Our approach uses orders of magnitude (1300x) less drug than is otherwise required by systemic injection and requires very low ultrasound pressures (20-fold below FDA safety limits for diagnostic imaging). We show that the BBB remains intact using passive cavitation detection (PCD), MRI-contrast agents and, importantly, also by sensitive fluorescent dye extravasation and immunohistochemistry.


Assuntos
Barreira Hematoencefálica/metabolismo , Encefalopatias/tratamento farmacológico , Portadores de Fármacos/efeitos da radiação , Agonistas de Receptores de GABA-A/administração & dosagem , Ultrassonografia de Intervenção/métodos , Animais , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/efeitos da radiação , Relação Dose-Resposta à Radiação , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Feminino , Agonistas de Receptores de GABA-A/farmacocinética , Humanos , Imagem por Ressonância Magnética , Modelos Animais , Muscimol/administração & dosagem , Muscimol/farmacocinética , Ratos , Técnicas Estereotáxicas , Ondas Ultrassônicas
3.
Nat Commun ; 11(1): 4790, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963242

RESUMO

Preventing aggregation of amyloid beta (Aß) peptides is a promising strategy for the treatment of Alzheimer's disease (AD), and gold nanoparticles have previously been explored as a potential anti-Aß therapeutics. Here we design and prepare 3.3 nm L- and D-glutathione stabilized gold nanoparticles (denoted as L3.3 and D3.3, respectively). Both chiral nanoparticles are able to inhibit aggregation of Aß42 and cross the blood-brain barrier (BBB) following intravenous administration without noticeable toxicity. D3.3 possesses a larger binding affinity to Aß42 and higher brain biodistribution compared with its enantiomer L3.3, giving rise to stronger inhibition of Aß42 fibrillation and better rescue of behavioral impairments in AD model mice. This conjugation of a small nanoparticle with chiral recognition moiety provides a potential therapeutic approach for AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/efeitos dos fármacos , Ouro/farmacologia , Transtornos da Memória/tratamento farmacológico , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Masculino , Transtornos da Memória/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/metabolismo , Estereoisomerismo
4.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
5.
PLoS One ; 15(9): e0236611, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941446

RESUMO

Treatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium. DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with single- or double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA. Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts. Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS.


Assuntos
Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Nanopartículas Metálicas/química , Oligodesoxirribonucleotídeos/metabolismo , Astrócitos/metabolismo , Linhagem Celular , Células Cultivadas , Galactose/química , Ouro/química , Humanos , Oligodesoxirribonucleotídeos/administração & dosagem
6.
Artigo em Inglês | MEDLINE | ID: mdl-32978291

RESUMO

OBJECTIVE: To investigate the pathophysiologic mechanism of encephalopathy and prolonged comatose or stuporous state in severally ill patients with coronavirus disease 2019 (COVID-19). METHODS: Eight COVID-19 patients with signs of encephalopathy were tested for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the serum and CSF using a Food and Drug Administration-approved and independently validated ELISA. Blood-brain barrier (BBB) integrity and immunoglobulin G (IgG) intrathecal synthesis were further tested using albumin and IgG indices. The CSF was also tested for autoimmune encephalitis antibodies and 14-3-3, a marker of ongoing neurodegeneration. RESULTS: All patients had anti-SARS-CoV-2 antibodies in their CSF, and 4 of 8 patients had high titers, comparable to high serum values. One patient had anti-SARS-CoV-2 IgG intrathecal synthesis, and 3 others had disruption of the blood-brain barrier. The CSF in 4 patients was positive for 14-3-3-protein suggesting ongoing neurodegeneration. In all patients, the CSF was negative for autoimmune encephalitis antibodies and SARS-CoV-2 by PCR. None of the patients, apart from persistent encephalopathic signs, had any focal neurologic signs or history or specific neurologic disease. CONCLUSIONS: High-titer anti-SARS-CoV-2 antibodies were detected in the CSF of comatose or encephalopathic patients demonstrating intrathecal IgG synthesis or BBB disruption. A disrupted BBB may facilitate the entry of cytokines and inflammatory mediators into the CNS enhancing neuroinflammation and neurodegeneration. The observations highlight the need for prospective CSF studies to determine the pathogenic role of anti-SARS-CoV-2 antibodies and identify early therapeutic interventions.


Assuntos
Autoanticorpos/líquido cefalorraquidiano , Betacoronavirus/isolamento & purificação , Barreira Hematoencefálica/metabolismo , Coma/líquido cefalorraquidiano , Infecções por Coronavirus/líquido cefalorraquidiano , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Pneumonia Viral/líquido cefalorraquidiano , Estupor/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/líquido cefalorraquidiano , Coma/diagnóstico , Infecções por Coronavirus/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doenças do Sistema Nervoso/diagnóstico , Pandemias , Pneumonia Viral/diagnóstico , Estupor/diagnóstico , Resultado do Tratamento
7.
PLoS One ; 15(9): e0238238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881880

RESUMO

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.


Assuntos
Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Radiossensibilizantes/uso terapêutico , Temozolomida/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dacarbazina/análise , Dacarbazina/metabolismo , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Cromatografia Gasosa-Espectrometria de Massas , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Radiossensibilizantes/análise , Radiossensibilizantes/metabolismo , Radiossensibilizantes/farmacologia , Temozolomida/análise , Temozolomida/metabolismo , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
PLoS One ; 15(9): e0238301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881954

RESUMO

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) is a primary astrocytopathy driven by antibodies directed against the aquaporin-4 water channel located at the end-feet of the astrocyte. Although blood-brain barrier (BBB) breakdown is considered one of the key steps for the development and lesion formation, little is known about the molecular mechanisms involved. The aim of the study was to evaluate the effect of human immunoglobulins from NMOSD patients (NMO-IgG) on BBB properties. METHODS: Freshly isolated brain microvessels (IBMs) from rat brains were used as a study model. At first, analysis of the secretome profile from IBMs exposed to purified NMO-IgG, to healthy donor IgG (Control-IgG), or non-treated, was performed. Second, tight junction (TJ) proteins expression in fresh IBMs and primary cultures of brain microvascular endothelial cells (BMEC) was analysed by Western blotting (Wb) after exposition to NMO-IgG and Control-IgG. Finally, functional BBB properties were investigated evaluating the presence of rat-IgG in tissue lysate from brain using Wb in the rat-model, and the passage of NMO-IgG and sucrose in a bicameral model. RESULTS: We found that NMO-IgG induces functional and morphological BBB changes, including: 1) increase of pro-inflammatory cytokines production (CXCL-10 [IP-10], IL-6, IL-1RA, IL-1ß and CXCL-3) in IBMs when exposed to NMO-IgG; 2) decrease of Claudin-5 levels by 25.6% after treatment of fresh IBMs by NMO-IgG compared to Control-IgG (p = 0.002), and similarly, decrease of Claudin-5 by at least 20% when BMEC were cultured with NMO-IgG from five different patients; 3) a higher level of rat-IgG accumulated in periventricular regions of NMO-rats compared to Control-rats and an increase in the permeability of BBB after NMO-IgG treatment in the bicameral model. CONCLUSION: Human NMO-IgG induces both structural and functional alterations of BBB properties, suggesting a direct role of NMO-IgG on modulation of BBB permeability in NMOSD.


Assuntos
Aquaporina 4/imunologia , Barreira Hematoencefálica/metabolismo , Imunoglobulina G/farmacologia , Neuromielite Óptica/patologia , Permeabilidade/efeitos dos fármacos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Claudina-5/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Imunoglobulina G/isolamento & purificação , Microvasos/citologia , Microvasos/metabolismo , Neuromielite Óptica/metabolismo , Ratos
9.
Int J Mol Sci ; 21(15)2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32751841

RESUMO

Increasing evidence suggests that Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-CoV-2) can also invade the central nervous system (CNS). However, findings available on its neurological manifestations and their pathogenic mechanisms have not yet been systematically addressed. A literature search on neurological complications reported in patients with COVID-19 until June 2020 produced a total of 23 studies. Overall, these papers report that patients may exhibit a wide range of neurological manifestations, including encephalopathy, encephalitis, seizures, cerebrovascular events, acute polyneuropathy, headache, hypogeusia, and hyposmia, as well as some non-specific symptoms. Whether these features can be an indirect and unspecific consequence of the pulmonary disease or a generalized inflammatory state on the CNS remains to be determined; also, they may rather reflect direct SARS-CoV-2-related neuronal damage. Hematogenous versus transsynaptic propagation, the role of the angiotensin II converting enzyme receptor-2, the spread across the blood-brain barrier, the impact of the hyperimmune response (the so-called "cytokine storm"), and the possibility of virus persistence within some CNS resident cells are still debated. The different levels and severity of neurotropism and neurovirulence in patients with COVID-19 might be explained by a combination of viral and host factors and by their interaction.


Assuntos
Betacoronavirus/fisiologia , Sistema Nervoso Central/virologia , Infecções por Coronavirus/patologia , Pneumonia Viral/patologia , Animais , Betacoronavirus/isolamento & purificação , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/virologia , Encefalopatias/complicações , Encefalopatias/patologia , Sistema Nervoso Central/metabolismo , Infecções por Coronavirus/virologia , Encefalite/complicações , Encefalite/patologia , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia
10.
Yakugaku Zasshi ; 140(8): 1007-1012, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741858

RESUMO

We previously showed that increased permeability of the blood-brain barrier (BBB) after ischemic stroke enables extravasation of nano-sized liposomes and accumulation in the ischemic region, and that delivery of neuroprotective agents using liposomal drug delivery systems (DDS) is applicable for treating cerebral ischemia/reperfusion (I/R) injury. However, entry of liposomes into the brain parenchyma was limited in the early stages after I/R possibly due to microvascular dysfunction induced by pathological progression. As such, new approaches to overcome the BBB are needed. Leukocytes can pass through inflamed BBB in I/R region due to membrane proteins displayed on their surface. We thus hypothesized that incorporation of leukocyte membrane proteins onto liposomal membranes would impart leukocyte-mimicking functions to liposomes and that leukocyte-mimetic liposomes (LM-Lipo) may pass through inflamed endothelial cells and BBB, similar to leukocytes. LM-Lipo prepared using intermembrane protein transfer from human leukemia cells showed significantly increased association to inflamed human umbilical vein endothelial cells relative to plain liposomes. Moreover, LM-Lipo passed through inflamed endothelial cell layer by regulating intercellular junctions. These results suggest that imparting leukocyte-like properties to liposomes via intermembrane protein transfer would be an effective strategy to overcome inflamed endothelial barriers. In this review, we describe our findings on ischemic stroke treatment using liposomal DDS and the potential of LM-Lipo to overcome inflamed endothelial barriers.


Assuntos
Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Leucócitos , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Humanos , Permeabilidade , Transporte Proteico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo
11.
Int J Nanomedicine ; 15: 5113-5129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764940

RESUMO

Background: Low bioavailability and poor permeability of the blood-brain barrier are problematic when delivering therapeutic agents and particularly anti-human immunodeficiency virus therapy to the central nervous system. The intranasal route offers an alternative for central nervous system delivery. Cubosomes have been reported as helpful vehicles for intranasal delivery of therapeutics to enable brain targeting. Objective: In this study, we aimed to develop the intranasal cubosomal thermogelling dispersion of saquinavir mesylate for central nervous system delivery. Methods: The Box-Behnken design was applied to study the effect of monoolein, Poloxamer 407, and polyvinyl alcohol as independent factors and the particle size, entrapment efficiency, gelation temperature, and stability index as responses. The optimized cubosomes were evaluated using transmission electron microscopy, ex vivo permeation, and in vivo pharmacokinetics. Results: The optimized formula consisting of monoolein (8.96%), Poloxamer 407 (17.45%), and polyvinyl alcohol (7.5%) was prepared and evaluated. Higher values for the steady-state flux, permeability coefficient, and enhancement factor were observed for the cubosomal thermogelling dispersion of saquinavir during ex vivo permeation in comparison with an aqueous suspension of saquinavir. From the pharmacokinetic profile, the relative bioavailability for the intranasal optimized formula was approximately 12-fold higher when compared with oral aqueous suspension and 2.5-fold greater when compared to the intranasal aqueous suspension of saquinavir. Conclusion: Overall, the saquinavir-loaded cubosomal thermogelling formulation is promising for central nervous system delivery by intranasal administration.


Assuntos
Portadores de Fármacos/química , Cristais Líquidos/química , Nanoestruturas/química , Saquinavir/farmacologia , Temperatura , Administração Intranasal , Animais , Disponibilidade Biológica , Barreira Hematoencefálica/metabolismo , Géis , Glicerídeos/química , Masculino , Tamanho da Partícula , Permeabilidade , Poloxâmero/química , Álcool de Polivinil/química , Saquinavir/administração & dosagem , Saquinavir/metabolismo
12.
Proc Natl Acad Sci U S A ; 117(33): 19854-19865, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759214

RESUMO

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.


Assuntos
Células Endoteliais/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Claudina-5/genética , Claudina-5/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Edição de Genes , Genoma , Humanos , Camundongos , Camundongos Knockout , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Pirazóis/farmacologia , Piridinas/farmacologia , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
Nature ; 583(7816): 425-430, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612231

RESUMO

The vascular interface of the brain, known as the blood-brain barrier (BBB), is understood to maintain brain function in part via its low transcellular permeability1-3. Yet, recent studies have demonstrated that brain ageing is sensitive to circulatory proteins4,5. Thus, it is unclear whether permeability to individually injected exogenous tracers-as is standard in BBB studies-fully represents blood-to-brain transport. Here we label hundreds of proteins constituting the mouse blood plasma proteome, and upon their systemic administration, study the BBB with its physiological ligand. We find that plasma proteins readily permeate the healthy brain parenchyma, with transport maintained by BBB-specific transcriptional programmes. Unlike IgG antibody, plasma protein uptake diminishes in the aged brain, driven by an age-related shift in transport from ligand-specific receptor-mediated to non-specific caveolar transcytosis. This age-related shift occurs alongside a specific loss of pericyte coverage. Pharmacological inhibition of the age-upregulated phosphatase ALPL, a predicted negative regulator of transport, enhances brain uptake of therapeutically relevant transferrin, transferrin receptor antibody and plasma. These findings reveal the extent of physiological protein transcytosis to the healthy brain, a mechanism of widespread BBB dysfunction with age and a strategy for enhanced drug delivery.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Barreira Hematoencefálica/metabolismo , Transcitose , Fosfatase Alcalina/metabolismo , Animais , Anticorpos/metabolismo , Transporte Biológico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/metabolismo , Proteínas Sanguíneas/farmacocinética , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Saúde , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasma/metabolismo , Proteoma/administração & dosagem , Proteoma/metabolismo , Proteoma/farmacocinética , Receptores da Transferrina/imunologia , Transcrição Genética , Transferrina/metabolismo
14.
ACS Chem Neurosci ; 11(15): 2137-2144, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32639711

RESUMO

Now, it has been evidenced that Covid19 (SARS-CoV-2) infects the brain tissues. Along with this, a challenge has been raised for research professionals to find effective drugs for its treatment since the recent spread of this virus from Wuhan, China. Targeting the treatment of brain infection, it has also been a challenge that the clinical drug should have good CNS penetration ability to cross the blood-brain barrier.


Assuntos
Betacoronavirus , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/metabolismo , Antivirais/administração & dosagem , Antivirais/metabolismo , Betacoronavirus/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/virologia , Encéfalo/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/administração & dosagem , Fármacos do Sistema Nervoso Central/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Metilprednisolona/administração & dosagem , Metilprednisolona/metabolismo , Pandemias , Resultado do Tratamento
15.
Proc Natl Acad Sci U S A ; 117(32): 19141-19150, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703811

RESUMO

Current strategies to direct therapy-loaded nanoparticles to the brain rely on functionalizing nanoparticles with ligands which bind target proteins associated with the blood-brain barrier (BBB). However, such strategies have significant brain-specificity limitations, as target proteins are not exclusively expressed at the brain microvasculature. Therefore, novel strategies which exploit alternative characteristics of the BBB are required to overcome nonspecific nanoparticle targeting to the periphery, thereby increasing drug efficacy and reducing detrimental peripheral side effects. Here, we present a simple, yet counterintuitive, brain-targeting strategy which exploits the higher impermeability of the BBB to selectively label the brain endothelium. This is achieved by harnessing the lower endocytic rate of brain endothelial cells (a key feature of the high BBB impermeability) to promote selective retention of free, unconjugated protein-binding ligands on the surface of brain endothelial cells compared to peripheral endothelial cells. Nanoparticles capable of efficiently binding to the displayed ligands (i.e., labeled endothelium) are consequently targeted specifically to the brain microvasculature with minimal "off-target" accumulation in peripheral organs. This approach therefore revolutionizes brain-targeting strategies by implementing a two-step targeting method which exploits the physiology of the BBB to generate the required brain specificity for nanoparticle delivery, paving the way to overcome targeting limitations and achieve clinical translation of neurological therapies. In addition, this work demonstrates that protein targets for brain delivery may be identified based not on differential tissue expression, but on differential endocytic rates between the brain and periphery.


Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos/instrumentação , Células Endoteliais/metabolismo , Nanopartículas/metabolismo , Animais , Transporte Biológico , Encéfalo/irrigação sanguínea , Endotélio/metabolismo , Humanos , Ratos , Ratos Sprague-Dawley
16.
J Stroke Cerebrovasc Dis ; 29(8): 104977, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32689608

RESUMO

BACKGROUND: Ischemic stroke is a severe neurological disorder that affected millions of people worldwide. Neuro-inflammation and apoptosis play an essential role in the pathogenesis of neuronal death during ischemic stroke. Alpha-pinene is a bicyclic terpenoid with anti-inflammatory and anti-apoptotic activities. Accordingly, the main purpose of this study was to assess the protective effect of α-pinene in ischemic stroke. MATERIALS AND METHODS: To induce ischemic stroke in male Wistar rats, the middle cerebral artery was occluded for 60 min followed by 24 h reperfusion. Alpha-pinene was injected intraperitoneally at the beginning of reperfusion. A day after reperfusion, the neurological deficits, volume of infarct area, and blood-brain barrier (BBB) permeability were evaluated. The mRNA expression of inflammatory cytokines as well as pro- and anti-apoptotic genes was assessed by using reverse transcription-polymerase chain reaction. The protein levels of inflammatory cytokines were also measured by ELISA method. RESULTS: The results showed that α-pinene (50 and 100 mg/kg) significantly improved sensorimotor function and decreased the volume of infarct area in the brain. The high permeability of BBB was also alleviated by α-pinene (50 and 100 mg/kg) in ischemic areas. Besides, α-pinene (100 mg/kg) attenuated neuro-inflammation through decreasing both the gene and protein expression of TNF-α and IL-1ß in the hippocampus, cortex, and striatum. Besides, α-pinene (100 mg/kg) suppressed apoptosis via downregulation of the pro-apoptotic Bax mRNA expression with a concomitant upregulation of anti-apoptotic Bcl-2 gene expression. CONCLUSIONS: Overall, it was concluded that α-pinene exerts neuroprotective effect during ischemic stroke through attenuating neuroinflammation and inhibition of apoptosis.


Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Monoterpenos Bicíclicos/farmacologia , Encéfalo/efeitos dos fármacos , Citocinas/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Citocinas/genética , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
J Stroke Cerebrovasc Dis ; 29(8): 104997, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32689627

RESUMO

OBJECTIVE: To study association between blood pressure (BP) and development of early neurological deterioration (END) in acute ischemic stroke patients with intravenous rt-PA thrombolysis and its possible mechanism. METHODS: We prospectively collected data of acute ischemic stroke patients with intravenous rt-PA thrombolysis from March 2015 to June 2019. The collected data include general, clinical data and laboratory test. Moreover, serum levels or activity of malondialdehyde (MDA), superoxide dismutase (SOD), matrix metalloproteinase-9 (MMP-9), occludin(OCLN), ZO-1 and aquaporin 4(AQP-4) were determined. RESULTS: A total of 357 acute ischemic stroke patients received intravenous rt-PA thrombolysis and 16 cases were eventually excluded. Finally, 341 patients were eligible in this study and 65 patients (19.06%) experienced END. Mean systolic blood pressure (SBP) within 24 h, serum levels or activity of MDA, SOD, MMP-9, ZO-1, OCLN, AQP-4 at 24 h after thrombolysis were the independent predictors for END in the total and hypertension population using multivariate logistic regression analysis, and mean SBP within 24 h was the best predictor for END. Receiver operating characteristic (ROC) analysis found that cutoff mean SBP for END was 148.16 mmHg, and sensitivity was 85.6%. The best target SBP level is 140ཞ149 mmHg. Further, spearman correlation tests indicated that BP levels were directly proportional to serum levels or activity of MDA, MMP-9, ZO-1, OCLN, AQP-4 at 24 h after thrombolysis and neurological deterioration severity. CONCLUSIONS: END frequently occurs after thrombolysis, and the best predictor of END is SBP which may be mediated through oxidative stress induced blood-brain barrier disruption and AQP4 upregulation.


Assuntos
Aquaporina 4/sangue , Pressão Sanguínea , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Fibrinolíticos/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica/efeitos adversos , Ativador de Plasminogênio Tecidual/efeitos adversos , Idoso , Biomarcadores/sangue , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/sangue , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/fisiopatologia , Progressão da Doença , Feminino , Fibrinolíticos/administração & dosagem , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo , Ativador de Plasminogênio Tecidual/administração & dosagem , Resultado do Tratamento , Regulação para Cima
18.
ACS Chem Neurosci ; 11(12): 1704-1705, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: covidwho-505550

RESUMO

The coronavirus disease 19 (COVID-19) pandemic has brought a great threat to global public health. Currently, mounting evidence has shown the occurrence of neurological symptoms in patients with COVID-19. However, the detailed mechanism by which the SARS-CoV-2 attacks the brain is not well characterized. Recent investigations have revealed that a cytokine storm contributes to brain inflammation and subsequently triggers neurological manifestations during the COVID-19 outbreak. Targeting brain inflammation may provide significant clues to the treatment of neurologic complications caused by SARS-CoV-2. Vascular growth factor (VEGF), which is widely distributed in the brain, probably plays a crucial role in brain inflammation via facilitating the recruitment of inflammatory cells and regulating the level of angiopoietins II (Ang II). Also, Ang II is considered as the products of SARS-CoV-2-attacking target, angiotensin-converting enzyme 2 (ACE2). Further investigation of the therapeutic potential and the underlying mechanisms of VEGF-targeted drugs on the neurological signs of COVID-19 are warranted. In any case, VEGF is deemed a promising therapeutic target in suppressing inflammation during SARS-CoV-2 infection with neurological symptoms.


Assuntos
Encéfalo/metabolismo , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Pneumonia Viral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Betacoronavirus/metabolismo , Barreira Hematoencefálica/metabolismo , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo
19.
ACS Chem Neurosci ; 11(12): 1704-1705, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32485101

RESUMO

The coronavirus disease 19 (COVID-19) pandemic has brought a great threat to global public health. Currently, mounting evidence has shown the occurrence of neurological symptoms in patients with COVID-19. However, the detailed mechanism by which the SARS-CoV-2 attacks the brain is not well characterized. Recent investigations have revealed that a cytokine storm contributes to brain inflammation and subsequently triggers neurological manifestations during the COVID-19 outbreak. Targeting brain inflammation may provide significant clues to the treatment of neurologic complications caused by SARS-CoV-2. Vascular growth factor (VEGF), which is widely distributed in the brain, probably plays a crucial role in brain inflammation via facilitating the recruitment of inflammatory cells and regulating the level of angiopoietins II (Ang II). Also, Ang II is considered as the products of SARS-CoV-2-attacking target, angiotensin-converting enzyme 2 (ACE2). Further investigation of the therapeutic potential and the underlying mechanisms of VEGF-targeted drugs on the neurological signs of COVID-19 are warranted. In any case, VEGF is deemed a promising therapeutic target in suppressing inflammation during SARS-CoV-2 infection with neurological symptoms.


Assuntos
Encéfalo/metabolismo , Infecções por Coronavirus/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Pneumonia Viral/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Betacoronavirus/metabolismo , Barreira Hematoencefálica/metabolismo , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo
20.
PLoS Pathog ; 16(6): e1008381, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32525948

RESUMO

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.


Assuntos
Astrócitos , Barreira Hematoencefálica , Infecções por HIV , HIV-1/metabolismo , Hipocampo , Liberação de Vírus , Animais , Antirretrovirais/farmacologia , Astrócitos/metabolismo , Astrócitos/patologia , Astrócitos/virologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/virologia , Linhagem Celular Tumoral , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/patologia , HIV-1/genética , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID
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