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1.
Nat Commun ; 13(1): 3226, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35680875

RESUMO

Ultra-long-acting integrase strand transfer inhibitors were created by screening a library of monomeric and dimeric dolutegravir (DTG) prodrug nanoformulations. This led to an 18-carbon chain modified ester prodrug nanocrystal (coined NM2DTG) with the potential to sustain yearly dosing. Here, we show that the physiochemical and pharmacokinetic (PK) formulation properties facilitate slow drug release from tissue macrophage depot stores at the muscle injection site and adjacent lymphoid tissues following single parenteral injection. Significant plasma drug levels are recorded up to a year following injection. Tissue sites for prodrug hydrolysis are dependent on nanocrystal dissolution and prodrug release, drug-depot volume, perfusion, and cell-tissue pH. Each affect an extended NM2DTG apparent half-life recorded by PK parameters. The NM2DTG product can impact therapeutic adherence, tolerability, and access of a widely used integrase inhibitor in both resource limited and rich settings to reduce HIV-1 transmission and achieve optimal treatment outcomes.


Assuntos
Infecções por HIV , Inibidores de Integrase de HIV , Pró-Fármacos , Infecções por HIV/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis , Humanos , Oxazinas/uso terapêutico , Piperazinas , Pró-Fármacos/farmacologia , Piridonas/uso terapêutico
2.
Nat Commun ; 12(1): 3453, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103484

RESUMO

A once every eight-week cabotegravir (CAB) long-acting parenteral is more effective than daily oral emtricitabine and tenofovir disoproxil fumarate in preventing human immunodeficiency virus type one (HIV-1) transmission. Extending CAB dosing to a yearly injectable advances efforts for the elimination of viral transmission. Here we report rigor, reproducibility and mechanistic insights for a year-long CAB injectable. Pharmacokinetic (PK) profiles of this nanoformulated CAB prodrug (NM2CAB) are affirmed at three independent research laboratories. PK profiles in mice and rats show plasma CAB levels at or above the protein-adjusted 90% inhibitory concentration for a year after a single dose. Sustained native and prodrug concentrations are at the muscle injection site and in lymphoid tissues. The results parallel NM2CAB uptake and retention in human macrophages. NM2CAB nanocrystals are stable in blood and tissue homogenates. The long apparent drug half-life follows pH-dependent prodrug hydrolysis upon slow prodrug nanocrystal dissolution and absorption. In contrast, solubilized prodrug is hydrolyzed in hours in plasma and tissues from multiple mammalian species. No toxicities are observed in animals. These results affirm the pharmacological properties and extended apparent half-life for a nanoformulated CAB prodrug. The report serves to support the mechanistic design for drug formulation safety, rigor and reproducibility.


Assuntos
Liberação Controlada de Fármacos , Lipídeos/química , Nanopartículas/química , Pró-Fármacos/farmacologia , Piridonas/farmacocinética , Animais , Composição de Medicamentos , Endocitose , Humanos , Cinética , Masculino , Camundongos Endogâmicos BALB C , Piridonas/administração & dosagem , Piridonas/sangue , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Distribuição Tecidual
3.
Nat Mater ; 19(8): 910-920, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32341511

RESUMO

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.


Assuntos
Antirretrovirais/metabolismo , Nanoestruturas/química , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Piridonas/metabolismo , Animais , Antirretrovirais/farmacologia , Antirretrovirais/toxicidade , Transporte Biológico , Preparações de Ação Retardada , Composição de Medicamentos , Interações Medicamentosas , Estabilidade de Medicamentos , Camundongos , Piridonas/farmacologia , Piridonas/toxicidade
4.
J Control Release ; 311-312: 201-211, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31491432

RESUMO

Antiretroviral therapy requires lifelong daily dosing to attain viral suppression, restore immune function, and improve quality of life. As a treatment alternative, long-acting (LA) antiretrovirals can sustain therapeutic drug concentrations in blood for prolonged time periods. The success of recent clinical trials for LA parenteral cabotegravir and rilpivirine highlight the emergence of these new therapeutic options. Further optimization can improve dosing frequency, lower injection volumes, and facilitate drug-tissue distributions. To this end, we report the synthesis of a library of RPV prodrugs designed to sustain drug plasma concentrations and improved tissue biodistribution. The lead prodrug M3RPV was nanoformulated into the stable LA injectable NM3RPV. NM3RPV treatment led to RPV plasma concentrations above the protein-adjusted 90% inhibitory concentration for 25 weeks with substantial tissue depots after a single intramuscular injection in BALB/cJ mice. NM3RPV elicited 13- and 26-fold increases in the RPV apparent half-life and mean residence time compared to native drug formulation. Taken together, proof-of-concept is provided that nanoformulated RPV prodrugs can extend the apparent drug half-life and improve tissue biodistribution. These results warrant further development for human use.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Nanopartículas/administração & dosagem , Pró-Fármacos/administração & dosagem , Rilpivirina/administração & dosagem , Animais , Fármacos Anti-HIV/farmacocinética , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , HIV-1/efeitos dos fármacos , Humanos , Macaca mulatta , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Pró-Fármacos/farmacocinética , Rilpivirina/farmacocinética , Distribuição Tecidual
5.
Artigo em Inglês | MEDLINE | ID: mdl-31523362

RESUMO

Although the combination of highly active antiretroviral therapy (cART) can remarkably control human immunodeficiency virus type-1 (HIV-1) replication, it fails to cure HIV/AIDS disease. It is attributed to the incapability of cART to eliminate persistent HIV-1 contained in latent reservoirs in the central nervous system (CNS) and other tissue organs. Thus, withdrawal of cART causes rebound viral replication and resurgent of HIV/AIDS. The lack of success on non-ART approaches for elimination of HIV-1 include the targeted molecules not reaching the CNS, not adjusting well with drug-resistant mutants, or unable to eliminate all components of viral life cycle. Here, we show that our newly discovered Drug-S can effectively inhibit HIV-1 infection and persistence at the low concentration without causing any toxicity to neuroimmune cells. Our results suggest that Drug-S may have a direct effect on viral structure, prevent rebounding of HIV-1 infection, and arrest progression into acquired immunodeficiency syndrome. We also observed that Drug-S is capable of crossing the blood-brain barrier, suggesting a potential antiretroviral drug for elimination of CNS viral reservoirs and self-renewal of residual HIV-1. These results outlined the possible mechanism(s) of action of Drug-S as a novel antiretroviral drug for elimination of HIV-1 replication by interfering the virion structure.

6.
AIDS ; 33(3): 585-588, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30289818

RESUMO

: Long-acting antiretrovirals can improve therapy and prevention for HIV-1 infection. Current long-acting cabotegravir (CAB LAP) can be administered every other month. Previously, we demonstrated that a myristoylated CAB prodrug encased in poloxamer 407 provided extended plasma drug concentrations. We now demonstrate that this first-generation nanoformulated prodrug can sustain plasma CAB concentrations above the protein-adjusted 90% inhibitory concentration for 4 months in rhesus macaques. A 2.5-fold extension in CAB half-life and a 1.6-fold increase in area under the concentration-time curve were observed compared with CAB LAP.


Assuntos
Fármacos Anti-HIV/farmacocinética , Preparações de Ação Retardada/farmacocinética , Pró-Fármacos/farmacocinética , Piridonas/farmacocinética , Animais , Fármacos Anti-HIV/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Portadores de Fármacos/administração & dosagem , Meia-Vida , Macaca mulatta , Plasma/química , Poloxâmero/administração & dosagem , Pró-Fármacos/administração & dosagem , Piridonas/administração & dosagem
7.
Mol Neurobiol ; 55(7): 5937-5950, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29128903

RESUMO

Wernicke's encephalopathy, a common neurological disease, is caused by thiamine (vitamin B1) deficiency. Neuropathy resulting from thiamine deficiency is a hallmark of Wernicke-Korsakoff syndrome in chronic alcohol users. The underlying mechanisms of this deficiency and progression of neuropathy remain to be understood. To uncover the unknown mechanisms of thiamine deficiency in alcohol abuse, we used chronic alcohol consumption or thiamine deficiency diet ingestion in animal models. Observations from animal models were validated in primary human neuronal culture for neurodegenerative process. We employed radio-labeled bio-distribution of thiamine, qualitative and quantitative analyses of the various biomarkers and neurodegenerative process. In the present studies, we established that disruption of thiamine transport across the intestinal gut blood-brain barrier axis as the cause of thiamine deficiency in the brain for neurodegeneration. We found that reduction in thiamine transport across these interfaces was the cause of reduction in the synthesis of thiamine pyrophosphate (TPP), an active cofactor for pyruvate dehydrogenase E1α (PDHE1α). Our findings revealed that decrease in the levels of PDHE1α cofactors switched on the activation of PD kinase (PDK) in the brain, thereby triggering the neuronal phosphorylation of PDHE1α (p-PDHE1α). Dysfunctional phosphorylated PDHE1α causes the reduction of mitochondrial aerobic respiration that led to neurodegeneration. We concluded that impairment of thiamine transport across the gut-BBB-axis that led to insufficient TPP synthesis was critical to Wernicke-neuropathy, which could be effectively prevented by stabilizing the thiamine transporters.


Assuntos
Barreira Hematoencefálica/metabolismo , Trato Gastrointestinal/metabolismo , Tiamina/metabolismo , Encefalopatia de Wernicke/metabolismo , Encefalopatia de Wernicke/patologia , Animais , Transporte Biológico , Sobrevivência Celular , Dieta , Regulação para Baixo , Etanol , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Piruvato Desidrogenase (Lipoamida)/metabolismo , Distribuição Tecidual
8.
Biomaterials ; 151: 53-65, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29059541

RESUMO

Long-acting parenteral (LAP) antiretroviral drugs have generated considerable interest for treatment and prevention of HIV-1 infection. One new LAP is cabotegravir (CAB), a highly potent integrase inhibitor, with a half-life of up to 54 days, allowing for every other month parenteral administrations. Despite this excellent profile, high volume dosing, injection site reactions and low body fluid drug concentrations affect broad use for virus infected and susceptible people. To improve the drug delivery profile, we created a myristoylated CAB prodrug (MCAB). MCAB formed crystals that were formulated into nanoparticles (NMCAB) of stable size and shape facilitating avid monocyte-macrophage entry, retention and reticuloendothelial system depot formulation. Drug release kinetics paralleled sustained protection against HIV-1 challenge. After a single 45 mg/kg intramuscular injection to BALB/cJ mice, the NMCAB pharmacokinetic profiles was 4-times greater than that recorded for CAB LAP. These observations paralleled replicate measurements in rhesus macaques. The results coupled with improved viral restriction in human adult lymphocyte reconstituted NOD/SCID/IL2Rγc-/- mice led us to conclude that NMCAB can improve biodistribution and viral clearance profiles upon current CAB LAP formulations.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Nanopartículas/química , Pró-Fármacos/química , Piridonas/química , Adulto , Animais , Fármacos Anti-HIV/administração & dosagem , Portadores de Fármacos , Composição de Medicamentos , Liberação Controlada de Fármacos , Infecções por HIV/tratamento farmacológico , Meia-Vida , Humanos , Cinética , Macaca mulatta , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Tamanho da Partícula , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Piridonas/administração & dosagem , Piridonas/farmacologia , Solubilidade , Propriedades de Superfície
9.
Artigo em Inglês | MEDLINE | ID: mdl-29061742

RESUMO

A nanoformulated myristoylated dolutegravir prodrug (NMDTG) was prepared using good laboratory practice protocols. Intramuscular injection of NMDTG (118 ± 8 mg/ml, 25.5 mg of DTG equivalents/kg of body weight) to three rhesus macaques led to plasma DTG levels of 86 ± 12 and 28 ± 1 ng/ml on days 35 and 91, respectively. The NMDTG platform showed no significant adverse events. Further modification may further extend the drug's apparent half-life for human use.


Assuntos
Compostos Heterocíclicos com 3 Anéis/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Preparações de Ação Retardada , Inibidores de Integrase de HIV/administração & dosagem , Inibidores de Integrase de HIV/sangue , Inibidores de Integrase de HIV/farmacocinética , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/sangue , Injeções Intramusculares , Macaca mulatta , Masculino , Nanocompostos/administração & dosagem , Oxazinas , Piperazinas , Pró-Fármacos/administração & dosagem , Pró-Fármacos/síntese química , Piridonas
10.
Brain Behav Immun ; 62: 291-305, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28232172

RESUMO

Epidemiological studies showed a strong association between alcoholism and incidence of stroke, for which the underlying causative mechanisms remain to be understood. Here we found that infiltration of immune cells and deposition of cholesterol at the site of brain artery/capillary injury induced atherosclerosis in chronic alcohol (ethanol) consumption in the presence or absence of high-fat diet. Conversion of cholesterol into sharp edges of cholesterol crystals (CCs) in alcohol intake was key to activation of NLRP3 inflammasome, induction of cerebral atherosclerosis, and development of neuropathy around the atherosclerotic lesions. The presence of alcohol was critical for the formation of CCs and development of the neuropathology. Thus, we observed that alcohol consumption elevated the level of plasma cholesterol, deposition and crystallization of cholesterol, as well as activation of NLRP3 inflammasome. This led to arteriole or capillary walls thickening and increase intracranial blood pressure. Distinct neuropathy around the atherosclerotic lesions indicated vascular inflammation as an initial cause of neuronal degeneration. We demonstrated the molecular mechanisms of NLRP3 activation and downstream signaling cascade event in primary culture of human brain arterial/capillary endothelial cells in the setting of dose-/time-dependent effects of alcohol/CCs using NLRP3 gene silencing technique. We also detected CCs in blood samples from alcohol users, which validated the clinical importance of the findings. Finally, combined therapy of acetyl-l-carnitine and Lipitor® prevented deposition of cholesterol, formation of CCs, activation of NLRP3, thickening of vessel walls, and elevation of intracranial blood pressure. We conclude that alcohol-induced accumulation and crystallization of cholesterol activates NLRP3/caspase-1 in the cerebral vessel that leads to early development of atherosclerosis.


Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Aterosclerose/metabolismo , Encéfalo/metabolismo , Colesterol/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Pressão Sanguínea/fisiologia , Dieta Hiperlipídica , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
11.
Arterioscler Thromb Vasc Biol ; 32(5): 1167-77, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22402362

RESUMO

OBJECTIVE: Blood-brain barrier (BBB) dysfunction caused by activation of matrix metalloproteinases (MMPs) is a pathological feature in vascular/neurological disease. We describe the mechanisms of BBB dysfunction and neuroinflammation as a result of MMP-3/9 activation and disruption of vascular endothelial growth factor (VEGF)-A/VEGFR-2 interaction, impairing effective angiogenesis. METHODS AND RESULTS: We investigate the hypothesis in human brain endothelial cells and animal model of chronic alcohol ingestion. Proteome array analysis, zymography, immunofluorescence, and Western blotting techniques detected the activation, expression, and levels of MMP-3 and MMP-9. We found that degradation of VEGFR-2 and BBB proteins, for example, occludin, claudin-5, and ZO-1 by MMP-3/9, causes rupture of capillary endothelium and BBB leakiness. Impairment of BBB integrity was demonstrated by increased permeability of dye tracers and Fluo-3/calcein-AM-labeled monocyte adhesion or infiltration and decrease in transendothelial electric resistance. Alcohol-induced degradation of endothelial VEGFR-2 by MMP-3/9 led to a subsequent elevation of cellular/serum VEGF-A level. The decrease in VEGFR-2 with subsequent increase in VEGF-A level led to apoptosis and neuroinflammation via the activation of caspase-1 and IL-1ß release. The use of MMPs, VEGFR-2, and caspase-1 inhibitors helped to dissect the underlying mechanisms. CONCLUSIONS: Alcohol-induced MMPs activation is a key mechanism for dysfunction of BBB via degradation of VEGFR-2 protein and activation of caspase-1 or IL-1ß release. Targeting VEGF-induced MMP-3/9 activation can be a novel preventive approach to vascular inflammatory disease in alcohol abuse.


Assuntos
Consumo de Bebidas Alcoólicas/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Endotélio Vascular/metabolismo , Metaloproteinases da Matriz/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/fisiopatologia , Animais , Barreira Hematoencefálica/metabolismo , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/patologia , Humanos , Masculino , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Ratos
12.
PLoS One ; 6(4): e19258, 2011 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-21556365

RESUMO

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial ß-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.


Assuntos
Acetilcarnitina/metabolismo , Astrócitos/efeitos dos fármacos , Glucose/antagonistas & inibidores , Metanfetamina/farmacologia , Neurônios/efeitos dos fármacos , Astrócitos/metabolismo , Western Blotting , Metabolismo Energético , Glucose/metabolismo , Humanos , Imuno-Histoquímica , Neurônios/metabolismo
13.
Mol Neurodegener ; 6: 23, 2011 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-21426580

RESUMO

BACKGROUND: Methamphetamine (METH), an addictive psycho-stimulant drug with euphoric effect is known to cause neurotoxicity due to oxidative stress, dopamine accumulation and glial cell activation. Here we hypothesized that METH-induced interference of glucose uptake and transport at the endothelium can disrupt the energy requirement of the blood-brain barrier (BBB) function and integrity. We undertake this study because there is no report of METH effects on glucose uptake and transport across the blood-brain barrier (BBB) to date. RESULTS: In this study, we demonstrate that METH-induced disruption of glucose uptake by endothelium lead to BBB dysfunction. Our data indicate that a low concentration of METH (20 µM) increased the expression of glucose transporter protein-1 (GLUT1) in primary human brain endothelial cell (hBEC, main component of BBB) without affecting the glucose uptake. A high concentration of 200 µM of METH decreased both the glucose uptake and GLUT1 protein levels in hBEC culture. Transcription process appeared to regulate the changes in METH-induced GLUT1 expression. METH-induced decrease in GLUT1 protein level was associated with reduction in BBB tight junction protein occludin and zonula occludens-1. Functional assessment of the trans-endothelial electrical resistance of the cell monolayers and permeability of dye tracers in animal model validated the pharmacokinetics and molecular findings that inhibition of glucose uptake by GLUT1 inhibitor cytochalasin B (CB) aggravated the METH-induced disruption of the BBB integrity. Application of acetyl-L-carnitine suppressed the effects of METH on glucose uptake and BBB function. CONCLUSION: Our findings suggest that impairment of GLUT1 at the brain endothelium by METH may contribute to energy-associated disruption of tight junction assembly and loss of BBB integrity.

14.
Brain Behav Immun ; 25 Suppl 1: S129-36, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21262340

RESUMO

Microvessels, the main components of the blood-brain barrier (BBB) are vulnerable to oxidative damage during alcohol-induced stress. Alcohol produces oxidative damage within the vessels and in the brain. Using our animal model of catheter implant into the common carotid artery (CCA), we trace the footprints of alcohol-induced oxidative damage and inflammatory process at the BBB and into the brain. The uniqueness of the finding is that ethanol causes oxidative damage in all neurovascular components by activating NADPH oxidase and inducible nitric oxide synthase in the brain. It is not the oxidants but the ethanol that traverses through the BBB because we found that the highly reactive peroxynitrite does not cross the BBB. Thus, oxidative damage is caused at the site of oxidant production in the microvessels and in the brain. Our data indicate that acetaldehyde (the primary metabolite of ethanol) is the inducer/activator of these enzymes that generate oxidants in brain neurovascular cells. Evidence for alcohol-induced BBB damage is indicated by the alterations of the tight junction protein occludin in intact microvessels. Importantly, we demonstrate that the site of BBB oxidative damage is also the site of immune cells aggregation in the microvessels, which paves the path for inflammatory footprints. These findings reveal the underlying mechanisms that ethanol-elicited BBB oxidative damage initiates the brain vascular inflammatory process, which ultimately leads to neuroinflammation.


Assuntos
Barreira Hematoencefálica/patologia , Etanol/farmacologia , Microvasos/patologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Western Blotting , Imuno-Histoquímica , Inflamação/metabolismo , Inflamação/patologia , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Ratos , Ratos Sprague-Dawley , Junções Íntimas/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-21258656

RESUMO

Alcohol consumption causes neurocognitive deficits, neuronal injury, and neurodegeneration. At the cellular level, alcohol abuse causes oxidative damage to mitochondria and cellular proteins and interlink with the progression of neuroinflammation and neurological disorders. We previously reported that alcohol inhibits glucose transport across the blood-brain barrier (BBB), leading to BBB dysfunction and neurodegeneration. In this study, we hypothesized that ethanol (EtOH)-mediated disruption in glucose uptake would deprive energy for human astrocytes and neurons inducing neurotoxicity and neuronal degeneration. EtOH may also have a direct effect on glucose uptake in neurons and astrocytes, which has not been previously described. Our results indicate that ethanol exposure decreases the uptake of D-(2-(3)H)-glucose by human astrocytes and neurons. Inhibition of glucose uptake correlates with a reduction in glucose transporter protein expression (GLUT1 in astrocytes and GLUT3 in neurons). Acetyl-L-carnitine (ALC), a neuroprotective agent, suppresses the effects of alcohol on glucose uptake and GLUT levels, thus reducing neurotoxicity and neuronal degeneration. These findings suggest that deprivation of glucose in brain cells contributes to neurotoxicity in alcohol abusers, and highlights ALC as a potential therapeutic agent to prevent the deleterious health conditions caused by alcohol abuse.

16.
Psychopharmacology (Berl) ; 214(3): 707-18, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21079922

RESUMO

PURPOSE: Evidence shows that alcohol intake causes oxidative neuronal injury and neurocognitive deficits that are distinct from the classical Wernicke-Korsakoff neuropathy. Our previous findings indicated that alcohol-elicited blood-brain barrier (BBB) damage leads to neuroinflammation and neuronal loss. The dynamic function of the BBB requires a constant supply and utilization of glucose. Here we examined whether interference of glucose uptake and transport at the endothelium by alcohol leads to BBB dysfunction and neuronal degeneration. MATERIAL AND METHODS: We tested the hypothesis in cell culture of human brain endothelial cells, neurons and alcohol intake in animal by immunofluorescence, Western blotting and glucose uptake assay methods. RESULTS: We found that decrease in glucose uptake correlates the reduction of glucose transporter protein 1 (GLUT1) in cell culture after 50 mM ethanol exposure. Decrease in GLUT1 protein levels was regulated at the translation process. In animal, chronic alcohol intake suppresses the transport of glucose into the frontal and occipital regions of the brain. This finding is validated by a marked decrease in GLUT1 protein expression in brain microvessel (the BBB). In parallel, alcohol intake impairs the BBB tight junction proteins occludin, zonula occludens-1, and claudin-5 in the brain microvessel. Permeability of sodium fluorescein and Evans Blue confirms the leakiness of the BBB. Further, depletion of trans-endothelial electrical resistance of the cell monolayer supports the disruption of BBB integrity. Administration of acetyl-L: -carnitine (a neuroprotective agent) significantly prevents the adverse effects of alcohol on glucose uptake, BBB damage and neuronal degeneration. CONCLUSION: These findings suggest that alcohol-elicited inhibition of glucose transport at the blood-brain interface leads to BBB malfunction and neurological complications.


Assuntos
Acetilcarnitina/uso terapêutico , Álcoois/farmacologia , Transporte Biológico/efeitos dos fármacos , Barreira Hematoaquosa/metabolismo , Glucose/metabolismo , Doenças Neurodegenerativas , Nootrópicos/uso terapêutico , Animais , Barreira Hematoaquosa/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Colina O-Acetiltransferase/metabolismo , Modelos Animais de Doenças , Interações Medicamentosas , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Azul Evans , Feto , Proteína Glial Fibrilar Ácida/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Inibição Neural/efeitos dos fármacos , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Proteínas de Neurofilamentos/metabolismo , Neurônios/efeitos dos fármacos , Fosfoproteínas/metabolismo , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteína da Zônula de Oclusão-1 , Fator de von Willebrand/metabolismo
17.
Free Radic Biol Med ; 49(10): 1494-504, 2010 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-20708681

RESUMO

The studies presented here demonstrate the protective effect of acetyl-L-carnitine (ALC) against alcohol-induced oxidative neuroinflammation, neuronal degeneration, and impaired neurotransmission. Our findings reveal the cellular and biochemical mechanisms of alcohol-induced oxidative damage in various types of brain cells. Chronic ethanol administration to mice caused an increase in inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine adduct formation in frontal cortical neurons but not in astrocytes from brains of these animals. Interestingly, alcohol administration caused a rather selective activation of NADPH oxidase (NOX), which, in turn, enhanced levels of reactive oxygen species (ROS) and 4-hydroxynonenal, but these were predominantly localized in astrocytes and microglia. Oxidative damage in glial cells was accompanied by their pronounced activation (astrogliosis) and coincident neuronal loss, suggesting that inflammation in glial cells caused neuronal degeneration. Immunohistochemistry studies indicated that alcohol consumption induced different oxidative mediators in different brain cell types. Thus, nitric oxide was mostly detected in iNOS-expressing neurons, whereas ROS were predominantly generated in NOX-expressing glial cells after alcohol ingestion. Assessment of neuronal activity in ex vivo frontal cortical brain tissue slices from ethanol-fed mice showed a reduction in long-term potentiation synaptic transmission compared with slices from controls. Coadministration of ALC with alcohol showed a significant reduction in oxidative damage and neuronal loss and a restoration of synaptic neurotransmission in this brain region, suggesting that ALC protects brain cells from ethanol-induced oxidative injury. These findings suggest the potential clinical utility of ALC as a neuroprotective agent that prevents alcohol-induced brain damage and development of neurological disorders.


Assuntos
Acetilcarnitina/farmacologia , Encéfalo/efeitos dos fármacos , Etanol/toxicidade , Fármacos Neuroprotetores/farmacologia , Aldeídos , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/fisiologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/metabolismo
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