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1.
Neurol Ther ; 12(4): 1187-1203, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37195409

RESUMO

INTRODUCTION: Siponimod, a potent and selective sphingosine-1-phosphate (S1P1,5) agonist, is the only therapeutic agent that has shown efficacy against disability progression, decline in cognitive processing speed, total brain volume loss, gray matter atrophy and signs of demyelination in patients with secondary progressive multiple sclerosis (SPMS). Although the pathophysiology of progression in SPMS and primary progressive MS (PPMS) is thought to be similar, fingolimod, the prototype S1P1,3,45 agonist, failed to show efficacy against disability progression in PPMS. Differentiating siponimod from fingolimod at the level of their central effects is believed to be the key to a better understanding of the underlying characteristics that could make siponimod uniquely efficacious in progressive MS (PMS). METHODS: Here, we compared the central vs. peripheral dose-dependent drug exposures for siponimod and fingolimod in healthy mice and mice with experimental autoimmune encephalomyelitis (EAE). RESULTS: Siponimod treatment achieved dose-dependent efficacy and dose-proportional increases in steady-state drug blood levels, with a central nervous system (CNS)/blood drug-exposure ratio (CNS/bloodDER) of ~ 6 in both healthy and EAE mice. In contrast, fingolimod treatments achieved dose-proportional increases in fingolimod and fingolimod-phosphate blood levels, with respective CNS/bloodDER that were markedly increased (≥ threefold) in EAE vs. healthy mice. CONCLUSION: If proven to have translational value, these observations would suggest that CNS/bloodDER may be a key differentiator for siponimod over fingolimod for clinical efficacy in PMS.

2.
J Interferon Cytokine Res ; 43(6): 246-256, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36454249

RESUMO

Maladjusted immune responses to the coronavirus disease 2019 (COVID-19), for example, cytokine release syndrome, may result in immunopathology and acute respiratory distress syndrome. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, and its S1P receptor (S1PR) are crucial in maintaining endothelial cell chemotaxis and barrier integrity. Apart from the S1P1 receptor-mediated mechanisms of sequestration of cytotoxic lymphocytes, including Th-17 and S1P1/2/3-mediated endothelial barrier functions, S1PR modulators may also attenuate cytokine release via activation of serine/threonine protein phosphatase 2A and enhance the pulmonary endothelial barrier via the c-Abl tyrosine kinase pathway. Chronic treatment with fingolimod (S1PR1,3,4,5 modulator) and siponimod (S1PR1,5 modulator) has demonstrated efficacy in reducing inflammatory disease activity and slowing down disease progression in multiple sclerosis. The decision to selectively suppress the immunity of a critically ill patient with COVID-19 remains a difficult choice. It has been suggested that treatment with fingolimod or siponimod may be appropriate to attenuate severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-induced hyperinflammation in patients with COVID-19 since these patients are already monitored in an intensive care setting. Here, we review the use of S1PR modulators, fingolimod and siponimod, in regulating the inflammatory response to SARS-CoV-2 with the aim of understanding their potential rationale use in patients with COVID-19.


Assuntos
COVID-19 , Esclerose Múltipla , Moduladores do Receptor de Esfingosina 1 Fosfato , Humanos , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Receptores de Esfingosina-1-Fosfato , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico , SARS-CoV-2/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia , Esclerose Múltipla/metabolismo
3.
Int J Mol Sci ; 23(22)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36430692

RESUMO

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Although immune modulation and suppression are effective during relapsing-remitting MS, secondary progressive MS (SPMS) requires neuroregenerative therapeutic options that act on the CNS. The sphingosine-1-phosphate receptor modulator siponimod is the only approved drug for SPMS. In the pivotal trial, siponimod reduced disease progression and brain atrophy compared with placebo. The enteric nervous system (ENS) was recently identified as an additional autoimmune target in MS. We investigated the effects of siponimod on the ENS and CNS in the experimental autoimmune encephalomyelitis model of MS. Mice with late-stage disease were treated with siponimod, fingolimod, or sham. The clinical disease was monitored daily, and treatment success was verified using mass spectrometry and flow cytometry, which revealed peripheral lymphopenia in siponimod- and fingolimod-treated mice. We evaluated the mRNA expression, ultrastructure, and histopathology of the ENS and CNS. Single-cell RNA sequencing revealed an upregulation of proinflammatory genes in spinal cord astrocytes and ependymal cells in siponimod-treated mice. However, differences in CNS and ENS histopathology and ultrastructural pathology between the treatment groups were absent. Thus, our data suggest that siponimod and fingolimod act on the peripheral immune system and do not have pronounced direct neuroprotective effects.


Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Cloridrato de Fingolimode/farmacologia , Cloridrato de Fingolimode/uso terapêutico , Compostos de Benzil/farmacologia , Sistema Nervoso Central/patologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia
4.
Front Immunol ; 13: 814064, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967378

RESUMO

Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain.


Assuntos
Esclerose Múltipla , Estruturas Linfoides Terciárias , Administração Intravenosa , Animais , Anticorpos Monoclonais , Anticorpos Monoclonais Humanizados/farmacologia , Antígenos CD20 , Camundongos
5.
Artigo em Inglês | MEDLINE | ID: mdl-35354603

RESUMO

BACKGROUND AND OBJECTIVES: Siponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis. METHODS: Mouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis-optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing. RESULTS: An increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype. DISCUSSION: These results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations.


Assuntos
Remielinização , Animais , Azetidinas , Compostos de Benzil/farmacologia , Cuprizona/farmacologia , Camundongos , Microglia , Remielinização/fisiologia
6.
Mult Scler J Exp Transl Clin ; 7(4): 20552173211049168, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34777855

RESUMO

BACKGROUND: Siponimod (BAF312), a selective S1P1/S1P5 agonist, reduces disability progression in secondary progressive MS. Recent observations suggest it could act via S1P1/S1P5-dependent anti-inflammatory and pro-myelination effects on CNS-resident cells. OBJECTIVE: Generate preclinical evidence confirming siponimod's CNS penetration and activity. METHODS: Siponimod's CNS penetration and distribution was explored in rodents and non-human primates (NHPs) using: Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS), quantitative whole-body autoradiography (QWBA) using 14C-radiolabeled siponimod or non-invasive single-photon emission CT (SPECT) with a validated 123I-radiolabeled siponimod analog. Functional CNS activity was investigated by S1P1 receptor quantification in brain homogenates. RESULTS: In mice/rats, siponimod treatments achieved dose-dependent efficacy and dose-proportional increase in drug blood levels, with mean brain/blood drug-exposure ratio (Brain/BloodDER) of 6-7. Efficacy in rat brain tissues was revealed by a dose-dependent reduction in brain S1P1 levels. QWBA distribution analysis in rats indicated that [14C]siponimod related radioactivity could readily penetrate CNS, with particularly high uptakes in white matter of cerebellum, corpus callosum, and medulla oblongata versus lower exposures in other areas such as olfactory bulb. SPECT monitoring in NHPs revealed CNS distribution with a brain/bloodDER of ∼6, as in rodents. CONCLUSION: Findings demonstrate siponimod's CNS penetration and distribution across species, with high translational potential to human.

7.
J Med Chem ; 63(23): 14594-14608, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33216547

RESUMO

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.


Assuntos
Sangue/metabolismo , Inibidores de Caspase/uso terapêutico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Ureia/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Inibidores de Caspase/síntese química , Inibidores de Caspase/metabolismo , Inibidores de Caspase/farmacocinética , Linhagem Celular Tumoral , Feminino , Meia-Vida , Humanos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/metabolismo , Neoplasias/tratamento farmacológico , Pirazóis/síntese química , Pirazóis/metabolismo , Pirazóis/farmacocinética , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Ovinos , Ureia/síntese química , Ureia/metabolismo , Ureia/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Med Chem ; 63(23): 14576-14593, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33252239

RESUMO

MALT1 plays a central role in immune cell activation by transducing NF-κB signaling, and its proteolytic activity represents a key node for therapeutic intervention. Two cycles of scaffold morphing of a high-throughput biochemical screening hit resulted in the discovery of MLT-231, which enabled the successful pharmacological validation of MALT1 allosteric inhibition in preclinical models of humoral immune responses and B-cell lymphomas. Herein, we report the structural activity relationships (SARs) and analysis of the physicochemical properties of a pyrazolopyrimidine-derived compound series. In human T-cells and B-cell lymphoma lines, MLT-231 potently and selectively inhibits the proteolytic activity of MALT1 in NF-κB-dependent assays. Both in vitro and in vivo profiling of MLT-231 support further optimization of this in vivo tool compound toward preclinical characterization.


Assuntos
Inibidores de Caspase/uso terapêutico , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ureia/análogos & derivados , Ureia/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Caspase/síntese química , Inibidores de Caspase/farmacologia , Descoberta de Drogas , Feminino , Humanos , Imunidade Humoral/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Ureia/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Front Immunol ; 11: 745, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425939

RESUMO

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.


Assuntos
Diabetes Mellitus Tipo 1/congênito , Diarreia/etiologia , Doenças Genéticas Ligadas ao Cromossomo X/etiologia , Doenças do Sistema Imunitário/congênito , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/antagonistas & inibidores , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 1/etiologia , Cães , Feminino , Humanos , Doenças do Sistema Imunitário/etiologia , Inflamação/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Linfócitos T Reguladores/imunologia
10.
Arthritis Rheumatol ; 72(6): 919-930, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31943941

RESUMO

OBJECTIVE: Fcγ receptors (FcγR) play important roles in both protective and pathogenic immune responses. The assembly of the CBM signalosome encompassing caspase recruitment domain-containing protein 9, B cell CLL/lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) is required for optimal FcγR-induced canonical NF-κB activation and proinflammatory cytokine release. This study was undertaken to clarify the relevance of MALT-1 protease activity in FcγR-driven events and evaluate the therapeutic potential of selective MALT-1 protease inhibitors in FcγR-mediated diseases. METHODS: Using genetic and pharmacologic disruption of MALT-1 scaffolding and enzymatic activity, we assessed the relevance of MALT-1 function in murine and human primary myeloid cells upon stimulation with immune complexes (ICs) and in murine models of autoantibody-driven arthritis and immune thrombocytopenic purpura (ITP). RESULTS: MALT-1 protease function is essential for optimal FcγR-induced production of proinflammatory cytokines by various murine and human myeloid cells stimulated with ICs. In contrast, MALT-1 protease inhibition did not affect the Syk-dependent, FcγR-mediated production of reactive oxygen species or leukotriene B4 . Notably, pharmacologic MALT-1 protease inhibition in vivo reduced joint inflammation in the murine K/BxN serum-induced arthritis model (mean area under the curve for paw swelling of 45.42% versus 100% in control mice; P = 0.0007) but did not affect platelet depletion in a passive model of ITP. CONCLUSION: Our findings indicate a specific contribution of MALT-1 protease activity to FcγR-mediated events and suggest that MALT-1 protease inhibitors have therapeutic potential in a subset of FcγR-driven inflammatory disorders.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Receptores de IgG/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Plaquetas/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Humanos , Camundongos , Células Mieloides/metabolismo
11.
J Immunol ; 203(11): 2791-2806, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31659015

RESUMO

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.


Assuntos
Autoimunidade/imunologia , Homeostase/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/deficiência , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética
12.
Ann Rheum Dis ; 78(7): 974-978, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30902822

RESUMO

OBJECTIVE: To examine the role of CD40-CD154 costimulation and effects of therapeutic pathway blockade in the non-obese diabetic (NOD/ShiLtJ) model of Sjögren's syndrome (SS). METHODS: We assessed leucocyte infiltration in salivary glands (SGs) from NOD/ShiLtJ mice by immunohistochemistry and examined transcriptomics data of SG tissue from these animals for evidence of a CD40 pathway gene signature. Additionally, we dosed MR1 (anti-CD154 antibody) in NOD mice after the onset of SS-like disease and examined the effects of MR1 treatment on sialadenitis, autoantibody production, SG leucocyte infiltration, gene expression downstream of CD40 and acquaporin 5 (AQP5) expression. RESULTS: We could detect evidence of CD40 expression and pathway activation in SG tissue from NOD mice. Additionally, therapeutic treatment with MR1 suppressed CD40 pathway genes and sialadenitis, inhibited ectopic lymphoid structure formation and autoantibody production, as well as decreased the frequency of antibody-secreting cells in SGs but had minimal effects on AQP5 expression in NOD/ShiLtJ SGs. CONCLUSION: CD40-CD154 interactions play an important role in key pathological processes in a mouse model of SS, suggesting that blockade of this costimulatory pathway in the clinic may have beneficial therapeutic effects in patients suffering from this autoimmune exocrinopathy.


Assuntos
Ligante de CD40/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/administração & dosagem , Antígenos de Histocompatibilidade Menor/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Síndrome de Sjogren/tratamento farmacológico , Síndrome de Sjogren/imunologia , Animais , Aquaporina 5/metabolismo , Autoanticorpos/metabolismo , Ligante de CD40/imunologia , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/imunologia , Imuno-Histoquímica , Leucócitos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Antígenos de Histocompatibilidade Menor/imunologia , Glândulas Salivares/patologia , Sialadenite/tratamento farmacológico , Sialadenite/imunologia , Sialadenite/patologia , Transdução de Sinais/imunologia , Síndrome de Sjogren/patologia
13.
Xenobiotica ; 49(1): 13-21, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29299977

RESUMO

1. The utility of 1-aminobenzotriazole (ABT), incorporated in food, has been investigated as an approach for longer term inhibition of cytochrome P450 (P450) enzymes in mice. 2. In rats, ABT inhibits gastric emptying, to investigate this potential limitation in mice we examined the effect of ABT administration on the oral absorption of NVS-CRF38. Two hour prior oral treatment with 100 mg/kg ABT inhibited the oral absorption of NVS-CRF38, Tmax was 4 hours for ABT-treated mice compared to 0.5 hours in the control group. 3. A marked inhibition of hepatic P450 activity was observed in mice fed with ABT containing food pellets for 1 month. P450 activity, as measured by the oral clearance of antipyrine, was inhibited on day 3 (88% of control), week 2 (83% of control) and week 4 (80% of control). 4. Tmax values for antipyrine were comparable between ABT-treated mice and the control group, alleviating concerns about impaired gastric function. 5. Inclusion of ABT in food provides a minimally invasive and convenient approach to achieve longer term inhibition of P450 activity in mice. This model has the potential to enable pharmacological proof-of-concept studies for research compounds which are extensively metabolised by P450 enzymes.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Triazóis/farmacologia , Administração Oral , Animais , Camundongos , Oxazóis/metabolismo , Pirazóis/metabolismo
14.
PLoS One ; 11(12): e0168252, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28005953

RESUMO

RATIONAL: Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. RESULTS: NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3-4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences. CONCLUSIONS: Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates.


Assuntos
Compostos de Anilina/farmacologia , Artrite Experimental/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Dipeptídeos/farmacologia , Inflamação/fisiopatologia , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/análogos & derivados , Adjuvantes Imunológicos/toxicidade , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Homeostase/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismo
15.
J Immunol ; 194(8): 3723-34, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25762782

RESUMO

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.


Assuntos
Linfócitos B Reguladores/imunologia , Caspases/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Encefalomielite Autoimune Experimental/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B Reguladores/patologia , Caspases/genética , Diferenciação Celular/genética , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologia
16.
Biochim Biophys Acta ; 1841(5): 745-58, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24239768

RESUMO

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.


Assuntos
Medicina Clínica , Imunossupressores/farmacologia , Lisofosfolipídeos/metabolismo , Propilenoglicóis/farmacologia , Projetos de Pesquisa , Transdução de Sinais/efeitos dos fármacos , Esfingosina/análogos & derivados , Pesquisa Biomédica , Cloridrato de Fingolimode , Humanos , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologia
17.
Clin Transl Med ; 3: 36, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25984272

RESUMO

BACKGROUND: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood. METHODS: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based). RESULTS: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS. CONCLUSIONS: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.

18.
PLoS One ; 8(3): e59630, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23544080

RESUMO

BACKGROUND: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.


Assuntos
Aldeído Liases/deficiência , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/prevenção & controle , Aldeído Liases/metabolismo , Animais , Encéfalo/metabolismo , Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/sangue , Encefalomielite Autoimune Experimental/complicações , Fatores de Transcrição Forkhead/metabolismo , Hipersensibilidade Tardia/sangue , Hipersensibilidade Tardia/complicações , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/patologia , Memória Imunológica/imunologia , Integrases/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Ovinos , Esfingolipídeos/metabolismo , Baço/imunologia , Baço/patologia , Análise de Sobrevida , Timo/imunologia , Timo/patologia
19.
Chem Biol ; 19(9): 1142-51, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22999882

RESUMO

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.


Assuntos
Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/química , Animais , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Receptores de Esfingosina-1-Fosfato , Relação Estrutura-Atividade , Especificidade por Substrato
20.
Transplantation ; 93(2): 156-64, 2012 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-22179400

RESUMO

BACKGROUND: Sotrastaurin (STN), a novel oral protein kinase C inhibitor that inhibits early T-cell activation, was assessed in non-human primate recipients of life-supporting kidney allografts. METHODS: Cynomolgus monkey recipients of life-supporting kidney allografts were treated orally with STN alone or in combination with cyclosporine A (CsA). RESULTS: STN monotherapy at 50 mg/kg once daily prolonged recipient survival times to the predefined endpoint of 29 days (n=2); when given at 25 mg/kg twice daily, the median survival time (MST) was 27 days (n=4). Neither once-daily monotherapy of STN 20 mg/kg nor CsA 20 mg/kg was effective (MST 6 days [n=2] and 7 days [n=5], respectively). In combination, however, STN 20 mg/kg and CsA 20 mg/kg prolonged MST to more than 100 days (n=5). By combining lower once-daily doses of STN (7 or 2 mg/kg) with CsA (20 mg/kg), MST was more than 100 (n=3) and 22 days (n=2), respectively. Neither in single-dose pharmacokinetic studies nor the transplant recipients were STN or CsA blood levels for combined treatment greater than when either drug was administered alone. STN blood levels in transplant recipients during combination therapy were dose related (20 mg/kg, 30-182 ng/mL; 7 mg/kg, 7-41 ng/mL; and 2 mg/kg, 3-5 ng/mL). STN at a daily dose of up to 20 mg/kg was relatively well tolerated. CONCLUSIONS: STN prolonged survival times of non-human primate kidney allograft recipients both as monotherapy and most effectively in combination with CsA. Pharmacokinetic interactions were not responsible for the potentiation of immunosuppressive efficacy by coadministering STN and CsA.


Assuntos
Ciclosporina/administração & dosagem , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Rim/imunologia , Pirróis/administração & dosagem , Quinazolinas/administração & dosagem , Animais , Ciclosporina/efeitos adversos , Ciclosporina/farmacocinética , Sinergismo Farmacológico , Sobrevivência de Enxerto/imunologia , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Imunossupressores/farmacocinética , Transplante de Rim/patologia , Transplante de Rim/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Macaca fascicularis , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Pirróis/efeitos adversos , Pirróis/farmacocinética , Quinazolinas/efeitos adversos , Quinazolinas/farmacocinética , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Transplante Homólogo
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