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1.
J Infect Dis ; 225(10): 1765-1772, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-33507266

RESUMO

BACKGROUND: REGN3048 and REGN3051 are human monoclonal antibodies (mAb) targeting the spike glycoprotein on the Middle East respiratory syndrome coronavirus (MERS-CoV), which binds to the receptor dipeptidyl peptidase-4 (DPP4) and is necessary for infection of susceptible cells. METHODS: Preclinical study: REGN3048, REGN3051 and isotype immunoglobulin G (IgG) were administered to humanized DPP4 (huDPP4) mice 1 day prior to and 1 day after infection with MERS-CoV (Jordan strain). Virus titers and lung pathology were assessed. Phase 1 study: healthy adults received the combined mAb (n = 36) or placebo (n = 12) and followed for 121 days. Six dose levels were studied. Strict safety criteria were met prior to dose escalation. RESULTS: Preclinical study: REGN3048 plus REGN3051, prophylactically or therapeutically, was substantially more effective for reducing viral titer, lung inflammation, and pathology in huDPP4 mice compared with control antibodies and to each antibody monotherapy. Phase 1 study: REGN3048 plus REGN3051 was well tolerated with no dose-limiting adverse events, deaths, serious adverse events, or infusion reactions. Each mAb displayed pharmacokinetics expected of human IgG1 antibodies; it was not immunogenic. CONCLUSIONS: REGN3048 and REGN3051 in combination were well tolerated. The clinical and preclinical data support further development for the treatment or prophylaxis of MERS-CoV infection.


Assuntos
Infecções por Coronavirus , Coronavírus da Síndrome Respiratória do Oriente Médio , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Dipeptidil Peptidase 4/metabolismo , Humanos , Imunoglobulina G , Camundongos , Glicoproteína da Espícula de Coronavírus
2.
Bioanalysis ; 13(24): 1827-1836, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34743612

RESUMO

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.


Assuntos
Anticorpos Monoclonais/análise , COVID-19/terapia , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , SARS-CoV-2/imunologia , Anticorpos Monoclonais/uso terapêutico , COVID-19/virologia , Técnicas Eletroquímicas , Humanos , Luminescência
3.
Anal Chem ; 93(38): 12889-12898, 2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34463470

RESUMO

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.


Assuntos
COVID-19 , Anticorpos Monoclonais , Cromatografia Líquida , Humanos , SARS-CoV-2 , Espectrometria de Massas em Tandem
4.
J Immunol Res ; 2016: 6262383, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27556048

RESUMO

Anti-drug antibodies induced by biologic therapeutics often impact drug pharmacokinetics, pharmacodynamics response, clinical efficacy, and patient safety. It is critical to assess the immunogenicity risk of potential biotherapeutics in producing neutralizing and nonneutralizing anti-drug antibodies, especially in clinical phases of drug development. Different assay methodologies have been used to detect all anti-drug antibodies, including ELISA, radioimmunoassay, surface plasmon resonance, and electrochemiluminescence-based technologies. The most commonly used method is a bridging assay, performed in an ELISA or on the Meso Scale Discovery platform. In this report, we aim to review the emerging new assay technologies that can complement or address challenges associated with the bridging assay format in screening and confirmation of ADAs. We also summarize generic anti-drug antibody assays that do not require drug-specific reagents for nonclinical studies. These generic assays significantly reduce assay development efforts and, therefore, shorten the assay readiness timeline.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos/análise , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Anticorpos Monoclonais/uso terapêutico , Terapia Biológica , Tolerância a Medicamentos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Imunoensaio/instrumentação , Imunoensaio/métodos , Imunoensaio/normas , Reação em Cadeia da Polimerase/métodos
5.
J Neuroinflammation ; 7: 97, 2010 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-21192826

RESUMO

BACKGROUND: Recently, we reported a neuroprotective effect for Hawthorn (Crataegus oxyacantha) ethanolic extract in middle cerebral artery occlusion-(MCAO) induced stroke in rats. The present study sheds more light on the extract's mechanism of neuroprotection, especially its immunomodulatory effect. METHODS: After 15 days of treatment with Hawthorn extract [100 mg/kg, pretreatment (oral)], male Sprague Dawley rats underwent transient MCAO for 75 mins followed by reperfusion (either 3 or 24 hrs). We measured pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6), ICAM-1, IL-10 and pSTAT-3 expression in the brain by appropriate methods. We also looked at the cytotoxic T cell sub-population among leukocytes (FACS) and inflammatory cell activation and recruitment in brain (using a myeloperoxidase activity assay) after ischemia and reperfusion (I/R). Apoptosis (TUNEL), and Bcl-xL- and Foxp3- (T(reg) marker) positive cells in the ipsilateral hemisphere of the brain were analyzed separately using immunofluorescence. RESULTS: Our results indicate that occlusion followed by 3 hrs of reperfusion increased pro-inflammatory cytokine and ICAM-1 gene expressions in the ipsilateral hemisphere, and that Hawthorn pre-treatment significantly (p ≤ 0.01) lowered these levels. Furthermore, such pre-treatment was able to increase IL-10 levels and Foxp3-positive cells in brain after 24 hrs of reperfusion. The increase in cytotoxic T cell population in vehicle rats after 24 hrs of reperfusion was decreased by at least 40% with Hawthorn pretreatment. In addition, there was a decrease in inflammatory cell activation and infiltration in pretreated brain. Hawthorn pretreatment elevated pSTAT-3 levels in brain after I/R. We also observed an increase in Bcl-xL-positive cells, which in turn may have influenced the reduction in TUNEL-positive cells compared to vehicle-treated brain. CONCLUSIONS: In summary, Hawthorn extract helped alleviate pro-inflammatory immune responses associated with I/R-induced injury, boosted IL-10 levels, and increased Foxp3-positive T(regs) in the brain, which may have aided in suppression of activated inflammatory cells. Such treatment also minimizes apoptotic cell death by influencing STAT-3 phosphorylation and Bcl-xL expression in the brain. Taken together, the immunomodulatory effect of Hawthorn extract may play a critical role in the neuroprotection observed in this MCAO-induced stroke model.


Assuntos
Crataegus/química , Fatores Imunológicos/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/imunologia , Adulto , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/patologia , Citocinas/imunologia , Modelos Animais de Doenças , Encefalite/tratamento farmacológico , Encefalite/imunologia , Encefalite/patologia , Humanos , Infarto da Artéria Cerebral Média , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-10/imunologia , Masculino , Peroxidase/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/prevenção & controle , Fator de Transcrição STAT3/metabolismo , Acidente Vascular Cerebral/patologia , Proteína bcl-X/metabolismo
6.
Int J Dev Neurosci ; 27(8): 799-803, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19712738

RESUMO

UNLABELLED: In our present investigation the neuroprotective effect of alcoholic extract of Hawthorn (Crataegus oxycantha) was evaluated against middle cerebral artery occlusion induced ischemia/reperfusion injury in rats. Male Sprague-Dawley rats were pretreated with 100 mg/kg body weight of the extract by oral gavage for 15 days. The middle cerebral artery was then occluded for 75 min followed by 24 h of reperfusion. The pretreated rats showed significantly improved neurological behavior with reduced brain infarct when compared to vehicle control rats. The glutathione level in brain was found to be significantly (p<0.05) low in vehicle control rats after 24 h of reperfusion when compared to sham operated animals. However, in Hawthorn extract pretreated rats the levels were found to be close to that of sham. Malondialdehyde levels in brain of sham and pretreated group were found to be significantly lower than the non-treated vehicle group (p<0.05). The nitric oxide levels in brain were measured and found to be significantly (p<0.05) higher in vehicle than in sham or extract treated rats. CONCLUSION: Our results suggest that Hawthorn extract which is a well known prophylactic for cardiac conditions may very well protect the brain against ischemia-reperfusion. The reduced brain damage and improved neurological behavior after 24 h of reperfusion in Hawthorn extract pretreated group may be attributed to its antioxidant property which restores glutathione levels, circumvents the increase in lipid peroxidation and nitric oxide levels thereby reducing peroxynitrite formation and free radical induced brain damage.


Assuntos
Infarto Encefálico , Crataegus/química , Infarto da Artéria Cerebral Média/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/patologia , Infarto Encefálico/fisiopatologia , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Testes Neuropsicológicos , Óxido Nítrico/metabolismo , Oxirredução , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Ratos , Ratos Sprague-Dawley
7.
J Neurochem ; 102(2): 365-77, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17437550

RESUMO

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti-inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke's secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose-dependent manner (1-10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down-regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule-1 and E-selectin), cytokines (tumor necrosis factor-alpha and interleukin-1beta) and inducible nitric oxide synthase. Anti-inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down-regulating caspase 3 and up-regulating anti-apoptotic protein Bcl-xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.


Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Encefalite/tratamento farmacológico , Ataque Isquêmico Transitório/tratamento farmacológico , Álcool Feniletílico/análogos & derivados , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Ácidos Cafeicos/uso terapêutico , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalite/metabolismo , Encefalite/fisiopatologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/fisiopatologia , Masculino , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Óxido Nítrico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Álcool Feniletílico/farmacologia , Álcool Feniletílico/uso terapêutico , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologia , Resultado do Tratamento
8.
J Neuroinflammation ; 3: 12, 2006 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-16689995

RESUMO

BACKGROUND: Stroke is one of the leading causes of death worldwide and a major cause of morbidity and mortality in the United States of America. Brain ischemia-reperfusion (IR) triggers a complex series of biochemical events including inflammation. Leukotrienes derived from 5-lipoxygenase (5-LOX) cause inflammation and are thus involved in the pathobiology of stroke injury. METHODS: To test the neuroprotective efficacy of 5-LOX inhibition in a rat model of focal cerebral IR, ischemic animals were either pre- or post-treated with a potent selective 5-LOX inhibitor, (N- [3-[3-(-fluorophenoxy) phenyl]-1-methyl-2-propenyl]-N-hydroxyurea (BW-B 70C). They were evaluated at 24 h after reperfusion for brain infarction, neurological deficit score, and the expression of 5-LOX. Furthermore, the mechanism and the anti-inflammatory potential of BW-B 70C in the regulation of nuclear factor kappa B (NF-kappaB) and inflammatory inducible nitric oxide synthase (iNOS) were investigated both in vivo and in vitro. RESULTS AND DISCUSSION: Both pre- and post-treatment with BW-B 70C reduced infarctions and improved neurological deficit scores. Immunohistochemical study of brain sections showed IR-mediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-kappaB activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2,3,5-trimethyl-6-[12-hydroxy-5,10-dodecadiynyl]-1,4-benzoquinone (AA-861). As with BW-B 70C, they provided remarkable neuroprotection. Furthermore, in vitro, BW-B 70C inhibited lipopolysaccharide (LPS) mediated nitric oxide production, iNOS induction and NF-kappaB activation in the BV2 microglial cell line. Treating rat primary microglia with BW-B70C confirmed blockage of LPS-mediated translocation of the p65 subunit of NF-kappaB from cytosol to nucleus. CONCLUSION: The study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-kappaB in a rat model of experimental stroke.

9.
Nitric Oxide ; 15(2): 114-24, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16524750

RESUMO

The efficacy of nitric oxide (NO) treatment in ischemic stroke, though well recognized, is yet to be tested in clinic. NO donors used to treat ischemic injury are structurally diverse compounds. We have shown that treatment of S-nitrosoglutathione (GSNO) protects the brain against injury and inflammation in rats after experimental stroke [M. Khan, B. Sekhon, S. Giri, M. Jatana, A. G. Gilg, K. Ayasolla, C. Elango, A. K. Singh, I. Singh, S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke, J. Cereb. Blood Flow Metab. 25 (2005) 177-192.]. In this study, we tested structurally different NO donors including GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), methylamine hexamethylene methylamine NONOate (MAHMA), propylamine propylamine NONOate (PAPA), 3-morpholinosydnonimine (SIN-1) and compared their neuroprotective efficacy and antioxidant property in rats after ischemia/reperfusion (I/R). GSNO, in addition to neuroprotection, decreased nitrotyrosine formation and lipid peroxidation in blood and increased the ratio of reduced versus oxidized glutathione (GSH/GSSG) in brain as compared to untreated animals. GSNO also prevented the I/R-induced increase in mRNA expression of ICAM-1 and E-Selectin. SNAP and SNP extended limited neuroprotection, reduced nitrotyrosine formation in blood and blocked increase in mRNA expression of ICAM-1 and E-Selectin in brain tissue. PAPA, MAHMA, and SIN-1 neither protected the brain nor reduced oxidative stress. We conclude that neuroprotective action of NO donors in experimental stroke depends on their ability to reduce oxidative stress both in brain and blood.


Assuntos
Isquemia Encefálica/prevenção & controle , Doadores de Óxido Nítrico/uso terapêutico , Substâncias Protetoras/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Química Encefálica , Glutationa/análise , Dissulfeto de Glutationa/análise , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Óxido Nítrico/análise , Doadores de Óxido Nítrico/química , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tirosina/análogos & derivados , Tirosina/sangue , Tirosina/metabolismo
10.
J Cereb Blood Flow Metab ; 25(2): 177-92, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15647746

RESUMO

Preservation of endothelial functions with low-dose nitric oxide (NO) and inhibition of excessive production of NO from inducible NO synthase (iNOS) is a potential therapeutic approach for acute stroke. Based on this hypothesis, an NO modulator, S-nitrosoglutathione (GSNO) was used, which provided neuroprotection in a rat model of focal cerebral ischemia. Administration of GSNO after the onset of ischemia reduced infarction and improved cerebral blood flow. To understand the mechanism of protection, the involvement of inflammation in ischemic brain injury was examined. Treatment with GSNO reduced the expression of tumor necrosis factor-alpha, interleukin-1beta, and iNOS; inhibited the activation of microglia/macrophage (ED1, CD11-b); and downregulated the expression of leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 in the ischemic brain. The number of apoptotic cells (including neurons) and the activity of caspase-3 were also decreased after GSNO treatment. Further, the antiinflammatory effect of GSNO on expression of iNOS and activation of NF-kappaB machinery in rat primary astrocytes and in the murine microglial cell line BV2 was tested. Cytokine-mediated expression of iNOS and activation of NF-kappaB were inhibited by GSNO treatment. That GSNO protects the brain against ischemia/reperfusion injury by modulating NO systems, resulting in a reduction in inflammation and neuronal cell death was documented by the results.


Assuntos
Isquemia Encefálica/prevenção & controle , Inflamação/prevenção & controle , Fármacos Neuroprotetores/farmacologia , S-Nitrosoglutationa/farmacologia , Acidente Vascular Cerebral/complicações , Animais , Apoptose/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Isquemia Encefálica/etiologia , Isquemia Encefálica/patologia , Linhagem Celular , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/etiologia , Inflamação/patologia , Masculino , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Permeabilidade/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/prevenção & controle , Acidente Vascular Cerebral/patologia
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