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1.
J Pharm Biomed Anal ; 143: 130-140, 2017 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-28595106

RESUMO

CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel CD4+ T-cell epitope of human heat shock protein 60 (83-109, MW 2988.38g/mol) with a mutation (D100→L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical method, based on LC-MS/MS analysis, in the SRM mode was developed and fully validated to quantify this peptide in human plasma. An internal standard with the same amino acid sequence but labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear range from 1.5 to 48ng/mL (without matrix effect and carry over) and an accuracy and precision good enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The peptide was administered subcutaneously in three dose levels (1, 2.5 and 5mg) not normalized to the body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of 1.5ng/mL enabled the PK analysis. The Cmax, reached at 0.5h, showed a great variability, that was most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the variability between patients. A rapid clearance of this peptide (4-6h) is advantageous for an immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore peripheral tolerance.


Assuntos
Artrite Reumatoide , Linfócitos T CD4-Positivos , Cromatografia Líquida , Humanos , Peptídeos , Espectrometria de Massas em Tandem
2.
Cell Stress Chaperones ; 21(4): 735-44, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27241313

RESUMO

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by a chronic relapsing-remitting joint inflammation. Perturbations in the balance between CD4 + T cells producing IL-17 and CD4 + CD25(high)FoxP3 + Tregs correlate with irreversible bone and cartilage destruction in RA. APL1 is an altered peptide ligand derived from a CD4+ T-cell epitope of human HSP60, an autoantigen expressed in the inflamed synovium, which increases the frequency of CD4 + CD25(high)FoxP3+ Tregs in peripheral blood mononuclear cells from RA patients. The aim of this study was to evaluate the suppressive capacity of Tregs induced by APL1 on proliferation of effector CD4+ T cells using co-culture experiments. Enhanced Treg-mediated suppression was observed in APL1-treated cultures compared with cells cultured only with media. Subsequent analyses using autologous cross-over experiments showed that the enhanced Treg suppression in APL1-treated cultures could reflect increased suppressive function of Tregs against APL1-responsive T cells. On the other hand, APL1-treatment had a significant effect reducing IL-17 levels produced by effector CD4+ T cells. Hence, this peptide has the ability to increase the frequency of Tregs and their suppressive properties whereas effector T cells produce less IL-17. Thus, we propose that APL1 therapy could help to ameliorate the pathogenic Th17/Treg balance in RA patients.


Assuntos
Antígenos/imunologia , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Chaperonina 60/química , Peptídeos/farmacologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Artrite Reumatoide/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Separação Celular , Células Cultivadas , Humanos , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Pessoa de Meia-Idade , Fenótipo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Adulto Jovem
3.
Int Immunopharmacol ; 17(4): 1075-83, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24177275

RESUMO

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA.


Assuntos
Anti-Inflamatórios/farmacologia , Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Chaperonina 60/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Adulto , Idoso , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chaperonina 60/imunologia , Fragmentação do DNA/efeitos dos fármacos , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Ligantes , Masculino , Pessoa de Meia-Idade , Proteínas Mitocondriais/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
4.
Autoimmunity ; 44(6): 471-82, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21370936

RESUMO

Induction of immune tolerance as therapeutic approach for autoimmune diseases constitutes a current research focal point. In this sense, we aimed to evaluate an altered peptide ligand (APL) for induction of peripheral tolerance in patients with rheumatoid arthritis (RA). A novel T-cell epitope from human heat-shock protein 60 (Hsp60), an autoantigen involved in the pathogenesis of RA, was identified by bioinformatics tools and an APL was design starting from this epitope. We investigated the ability of this APL for inducing regulatory T cells (Treg cells) in mice and evaluated the therapeutic effect of this peptide in an adjuvant-induced arthritis (AA) rat model. Clinical score, TNFα levels and histopathology were monitored, as well as the capacity of this APL for inducing Treg cells. Finally, the potentialities of the APL for inducing Treg cells were evaluated in ex vivo assays using mononuclear cells isolated from peripheral blood (PBMC). The APL induced an increase of the proportions of Treg cells in the draining lymph nodes of the injected site in mice. The APL efficiently inhibited the course of AA, with significant reduction of the clinical and histopathology score. This effect was associated with an increase of the proportions of Treg cells and a decrease of TNFα levels in spleen. Finally, stimulation of PBMCs from RA patients by the APL increases the proportions of the CD4(+)CD25(high)FoxP3(+) Treg cells. These results indicate a therapeutic potentiality of APL and support further investigation of this candidate drug for treatment of RA.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Chaperonina 60/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Idoso , Animais , Chaperonina 60/uso terapêutico , Feminino , Humanos , Tolerância Imunológica , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Peptídeos/imunologia , Ratos , Ratos Endogâmicos Lew , Linfócitos T Reguladores/metabolismo
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