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1.
J Pharmacol Exp Ther ; 360(1): 226-238, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27821712

RESUMO

Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinases involved in B-cell and myeloid cell signaling. Small molecule inhibitors of BTK are being investigated for treatment of several hematologic cancers and autoimmune diseases. GDC-0853 ((S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one) is a selective and reversible oral small-molecule BTK inhibitor in development for the treatment of rheumatoid arthritis and systemic lupus erythematosus. In Sprague-Dawley (SD) rats, administration of GDC-0853 and other structurally diverse BTK inhibitors for 7 days or longer caused pancreatic lesions consisting of multifocal islet-centered hemorrhage, inflammation, fibrosis, and pigment-laden macrophages with adjacent lobular exocrine acinar cell atrophy, degeneration, and inflammation. Similar findings were not observed in mice or dogs at much higher exposures. Hemorrhage in the peri-islet vasculature emerged between four and seven daily doses of GDC-0853 and was histologically similar to spontaneously occurring changes in aging SD rats. This suggests that GDC-0853 could exacerbate a background finding in younger animals. Glucose homeostasis was dysregulated following a glucose challenge; however, this occurred only after 28 days of administration and was not directly associated with onset or severity of pancreatic lesions. There were no changes in other common serum biomarkers assessing endocrine and exocrine pancreatic function. Additionally, these lesions were not readily detectable via Doppler ultrasound, computed tomography, or magnetic resonance imaging. Our results indicate that pancreatic lesions in rats are likely a class effect of BTK inhibitors, which may exacerbate an islet-centered pathology that is unlikely to be relevant to humans.


Assuntos
Pâncreas/efeitos dos fármacos , Piperazinas/toxicidade , Inibidores de Proteínas Quinases/toxicidade , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridonas/toxicidade , Pirróis/toxicidade , Tirosina Quinase da Agamaglobulinemia , Animais , Cães , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Humanos , Masculino , Camundongos , Pâncreas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Ratos , Especificidade da Espécie
2.
Anal Chem ; 88(23): 11521-11526, 2016 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-27797494

RESUMO

Deamidation of therapeutic antibodies may result in decreased drug activity and undesirable changes in pharmacokinetics and immunogenicity. Therefore, it is necessary to monitor the deamidation levels [during storage] and after in vivo administration. Because of the complexity of in vivo samples, immuno-affinity capture is widely used for specific enrichment of the target antibody prior to LC-MS. However, the conventional use of bead-based methods requires large sample volumes and extensive processing steps. Furthermore, with automation difficulties and extended sample preparation time, bead-based approaches may increase artificial deamidation. To overcome these challenges, we developed an automated platform to perform tip-based affinity capture of antibodies from complex matrixes with rapid digestion and peptide elution into 96-well microtiter plates followed by LC-MS analysis. Detailed analyses showed that the new method presents high repeatability and reproducibility with both intra and inter assay CVs < 8%. Using the automated platform, we successfully quantified the levels of deamidation of a humanized monoclonal antibody in cynomolgus monkeys over a time period of 12 weeks after administration. Moreover, we found that deamidation kinetics between in vivo samples and samples stressed in vitro at neutral pH were consistent, suggesting that the in vitro stress test may be used as a method to predict the liability to deamidation of therapeutic antibodies in vivo.


Assuntos
Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Desaminação , Animais , Anticorpos/sangue , Anticorpos/uso terapêutico , Automação , Células CHO , Cromatografia Líquida , Cricetulus , Eritrócitos , Feminino , Citometria de Fluxo , Humanos , Macaca fascicularis , Espectrometria de Massas
3.
Bioorg Med Chem Lett ; 26(2): 575-579, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26675441

RESUMO

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridazinas/química , Piridazinas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Tirosina Quinase da Agamaglobulinemia , Animais , Cães , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Tirosina Quinases/metabolismo , Piridazinas/metabolismo , Piridazinas/farmacocinética , Pirimidinonas/metabolismo , Pirimidinonas/farmacocinética , Ratos , Tiofenos/metabolismo , Tiofenos/farmacocinética
4.
Bioorg Med Chem Lett ; 25(6): 1333-7, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25701252

RESUMO

SAR studies focused on improving the pharmacokinetic (PK) properties of the previously reported potent and selective Btk inhibitor CGI-1746 (1) resulted in the clinical candidate GDC-0834 (2), which retained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models. Structure based design efforts drove this work as modifications to 1 were investigated at both the solvent exposed region as well as 'H3 binding pocket'. However, in vitro metabolic evaluation of 2 revealed a non CYP-mediated metabolic process that was more prevalent in human than preclinical species (mouse, rat, dog, cyno), leading to a high-level of uncertainly in predicting human pharmacokinetics. Due to its promising potency, selectivity, and preclinical efficacy, a single dose IND was filed and 2 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, 2 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure. This information informed the back-up program and discovery of improved inhibitors.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinonas/química , Tiofenos/química , Tirosina Quinase da Agamaglobulinemia , Animais , Benzamidas/química , Benzamidas/metabolismo , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cristalografia por Raios X , Cães , Meia-Vida , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Pirimidinonas/síntese química , Pirimidinonas/farmacocinética , Ratos , Relação Estrutura-Atividade , Tiofenos/síntese química , Tiofenos/farmacocinética
5.
Nat Chem Biol ; 7(1): 41-50, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21113169

RESUMO

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Linfócitos B/efeitos dos fármacos , Benzamidas/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Células Mieloides/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase da Agamaglobulinemia , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Benzamidas/química , Benzamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/farmacologia , Proteínas Tirosina Quinases/uso terapêutico , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
6.
Bioorg Med Chem Lett ; 23(17): 4953-9, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23867164

RESUMO

In an effort to identify potent and isoform selective inhibitors of PI3Kδ, GNE-293 (34) was identified. Inhibitor 2 was found to induce micronuclei formation in both the MNT and HCA in vitro assays. Compounds testing negative for genotoxicity were successfully identified through modifications of the 2-benzimidazole substituent and the methylene moiety to disrupt planarity. A variety of heteroatom linkers were explored to examine their effect on potency and isoform selectivity by restricting torsional angles to favor ligand interactions with PI3Kδ's Trp760. These modifications also resulted in an improved in vivo pharmacokinetic profile.


Assuntos
Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Purinas/química , Purinas/farmacologia , Animais , Linhagem Celular , Cães , Humanos , Simulação de Acoplamento Molecular , Testes de Mutagenicidade , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Ratos , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 22(13): 4296-302, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22672799

RESUMO

A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.


Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Triptofano/química , Animais , Sítios de Ligação , Simulação por Computador , Feminino , Injeções Intravenosas , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
8.
J Neurosci ; 30(6): 2025-38, 2010 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-20147531

RESUMO

To assess the effects and mechanisms of a CD200R1 agonist administered during the progressive stage of a multiple sclerosis model, we administered CD200R1 agonist (CD200Fc) or control IgG2a during the chronic phase of disease (days 10-30) in mice with experimental autoimmune encephalomyelitis (EAE), induced using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) peptide. We found that administration of CD200Fc during the chronic stages of EAE reduced disease severity, demyelination, and axonal damage, through the modulation of several key disease mechanisms. CD200Fc treatment suppressed macrophage and microglial accumulation within the CNS, in part through downregulation of adhesion molecules VLA-4 and LFA-1, which are necessary for macrophage migration. Additionally, expression of activation markers MHC-II and CD80 and production of proinflammatory cytokines IL-6, tumor necrosis factor-alpha, and nitric oxide by CD11b(+) cells were decreased in both the spleen and CNS in CD200Fc-treated animals. Antigen-presenting cell function in the spleen and CNS was suppressed in CD200Fc-treated mice, but there were no significant alterations on T cell activation or phenotype. CD200Fc increased apoptosis of CD11b(+) cells but not astrocytes. In contrast, addition of CD200Fc treatment protected oligodendrocytes from apoptosis in vitro and in vivo. Our results demonstrate that CD200R1 agonists modulate both myeloid- and non-myeloid-related mechanisms of chronic disease in the EAE model and may be effective in the treatment of progressive multiple sclerosis and other neurodegenerative diseases.


Assuntos
Antígenos de Superfície/genética , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Esclerose Múltipla/tratamento farmacológico , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Antígenos de Superfície/biossíntese , Apoptose/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/fisiologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Receptores de Orexina , Receptores de Superfície Celular/biossíntese , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Baço/imunologia , Baço/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia
9.
J Pharmacol Exp Ther ; 338(1): 154-63, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21521773

RESUMO

Bruton's tyrosine kinase (BTK) plays a critical role in the development, differentiation, and proliferation of B-lineage cells, making it an attractive target for the treatment of rheumatoid arthritis. The objective of this study was to evaluate the antiarthritis effect of GDC-0834 [R-N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide], a potent and selective BTK inhibitor, and characterize the relationship between inhibition of BTK phosphorylation (pBTK) and efficacy. GDC-0834 inhibited BTK with an in vitro IC(50) of 5.9 and 6.4 nM in biochemical and cellular assays, respectively, and in vivo IC(50) of 1.1 and 5.6 µM in mouse and rat, respectively. Administration of GDC-0834 (30-100 mg/kg) in a rat collagen-induced arthritis (CIA) model resulted in a dose-dependent decrease of ankle swelling and reduction of morphologic pathology. An integrated disease progression pharmacokinetic/pharmacodynamic model where efficacy is driven by pBTK inhibition was fit to ankle-diameter time-course data. This model incorporated a transit model to characterize nondrug-related decreases in ankle swelling occurring at later stages of disease progression in CIA rats. The time course of ankle swelling in vehicle animals was described well by the base model. Simultaneous fitting of data from vehicle- and GDC-0834-treated groups showed that overall 73% inhibition of pBTK was needed to decrease the rate constant describing the ankle swelling increase (k(in)) by half. These findings suggest a high degree of pBTK inhibition is required for maximal activity of the pathway on inflammatory arthritis in rats.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Modelos Químicos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Tirosina Quinase da Agamaglobulinemia , Animais , Antirreumáticos/química , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Bovinos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/química , Pirimidinonas/química , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Tiofenos/química , Resultado do Tratamento
10.
ACS Med Chem Lett ; 11(8): 1588-1597, 2020 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-32832028

RESUMO

Bruton's tyrosine kinase (Btk) is thought to play a pathogenic role in chronic immune diseases such as rheumatoid arthritis and lupus. While covalent, irreversible Btk inhibitors are approved for treatment of hematologic malignancies, they are not approved for autoimmune indications. In efforts to develop additional series of reversible Btk inhibitors for chronic immune diseases, we sought to differentiate from our clinical stage inhibitor fenebrutinib using cyclopropyl amide isosteres of the 2-aminopyridyl group to occupy the flat, lipophilic H2 pocket. While drug-like properties were retained-and in some cases improved-a safety liability in the form of hERG inhibition was observed. When a fluorocyclopropyl amide was incorporated, Btk and off-target activity was found to be stereodependent and a lead compound was identified in the form of the (R,R)- stereoisomer.

11.
Trends Cell Biol ; 12(8): 368-73, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12191913

RESUMO

T and B lymphocytes migrate hundreds of micrometers each day to survey the body's lymphoid tissues for antigens. No other mammalian cell type undergoes such extensive and continual movement, raising the question of whether lymphocytes have specializations to support their migratory behavior. This possibility has recently gained support from studies of mice deficient in DOCK2, a member of the Caenorhabditis elegans Ced-5, mammalian DOCK180 and Drosophila melanogaster myoblast city (CDM) family of scaffolding proteins. Migration of lymphocytes, but not other cell types, is severely disrupted in DOCK2-deficient mice. Despite the conserved role of CDM molecules in regulating Rac activation and actin assembly, relatively little is known about how these molecules function. Here, we review the role of DOCK2 in lymphocyte homing to lymphoid tissues and discuss recent findings for other CDM family molecules that provide a basis for understanding how DOCK2 might function in lymphocytes.


Assuntos
Linfócitos B/imunologia , Proteínas de Transporte/fisiologia , Movimento Celular , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina , Linfócitos T/imunologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Proteínas de Transporte/química , Quimiocinas CXC/classificação , Quimiocinas CXC/fisiologia , Quimiotaxia de Leucócito , Proteínas de Membrana/classificação , Proteínas de Membrana/fisiologia , Camundongos , Modelos Biológicos , Transdução de Sinais , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/química
12.
J Med Chem ; 61(6): 2227-2245, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29457982

RESUMO

Bruton's tyrosine kinase (Btk) is a nonreceptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Preclinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery and preclinical characterization of a potent, selective, and noncovalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in preclinical and Phase 2 studies ongoing in patients with rheumatoid arthritis, lupus, and chronic spontaneous urticaria. On the basis of its potency, selectivity, long target residence time, and noncovalent mode of inhibition, 29 has the potential to be a best-in-class Btk inhibitor for a wide range of immunological indications.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Tirosina Quinase da Agamaglobulinemia/efeitos dos fármacos , Tirosina Quinase da Agamaglobulinemia/genética , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/toxicidade , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Cães , Descoberta de Drogas , Humanos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Células Madin Darby de Rim Canino , Modelos Moleculares , Estrutura Molecular , Piperazinas/farmacocinética , Piperazinas/toxicidade , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Piridonas/farmacocinética , Piridonas/toxicidade , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley
13.
ACS Med Chem Lett ; 8(6): 608-613, 2017 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-28626519

RESUMO

In our continued effort to discover and develop best-in-class Bruton's tyrosine kinase (Btk) inhibitors for the treatment of B-cell lymphomas, rheumatoid arthritis, and systemic lupus erythematosus, we devised a series of novel tricyclic compounds that improved upon the druglike properties of our previous chemical matter. Compounds exemplified by G-744 are highly potent, selective for Btk, metabolically stable, well tolerated, and efficacious in an animal model of arthritis.

14.
JCI Insight ; 2(7): e90111, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28405610

RESUMO

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Linfócitos B/imunologia , Nefrite Lúpica/imunologia , Células Mieloides/metabolismo , Plasmócitos/patologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Autoanticorpos/imunologia , Linfócitos B/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon-alfa/imunologia , Rim/imunologia , Rim/patologia , Nefrite Lúpica/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NZB , Plasmócitos/efeitos dos fármacos
15.
Toxicol Sci ; 152(1): 72-84, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27103662

RESUMO

CRTh2 is expressed on immune cells that drive asthma pathophysiology. Current treatment options for severe asthma are inadequate and therapeutic antibody-mediated depletion of CRTh2-expressing cells represents a promising new therapeutic strategy. Here we report for the first time that CRTh2 is not only expressed on immune cells, but also on microvasculature in the central nervous system (CNS) and gastric mucosa in humans. Microvascular expression of CRTh2 raises a safety concern because a therapeutic antiCRTh2 antibody with enhanced depletion capacity could lead to vascular damage. To evaluate this safety risk, we characterized microvascular expression in human and in transgenic mice expressing human CRTh2 protein (hCRTh2.BAC.Tg) and found that CRTh2 is not localized to microvascular endothelium that is directly exposed to circulating therapeutic antibody, but rather, to pericytes that in the CNS are shielded from direct circulatory exposure by the blood-brain barrier. Immunohistochemical visualization of an intravenously administered antiCRTh2 antibody in transgenic mice revealed localization to microvascular pericytes in the gastric mucosa but not in the CNS, suggesting the blood-brain barrier effectively limits pericyte exposure to circulating therapeutic antibody in the CNS. Repeated dosing with a depleting antiCRTh2 antibody in hCRTh2.BAC.Tg mice revealed linear pharmacokinetics and no drug-related adverse findings in any tissues, including the CNS and gastric mucosa, despite complete depletion of CRTh2 expressing circulating eosinophils and basophils. Collectively, these studies demonstrate that the likelihood of drug-related CNS or gastrointestinal toxicity in humans treated with a therapeutic depleting antiCRTh2 antibody is low despite pericyte expression of CRTh2 in these tissues.


Assuntos
Antiasmáticos/farmacologia , Anticorpos Monoclonais/farmacologia , Asma/tratamento farmacológico , Sistema Nervoso Central/efeitos dos fármacos , Mucosa Gástrica/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Antiasmáticos/administração & dosagem , Antiasmáticos/farmacocinética , Antiasmáticos/toxicidade , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/toxicidade , Asma/imunologia , Asma/metabolismo , Barreira Hematoencefálica/metabolismo , Permeabilidade Capilar , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Humanos , Injeções Intravenosas , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pericitos/imunologia , Pericitos/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/imunologia , Receptores de Prostaglandina/metabolismo , Medição de Risco , Distribuição Tecidual
16.
JCI Insight ; 1(7): e86689, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27699264

RESUMO

Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.


Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Asma/terapia , Células Th2/citologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Basófilos/citologia , Citocinas , Modelos Animais de Doenças , Eosinófilos/citologia , Humanos , Imunidade Inata , Pulmão/citologia , Pulmão/imunologia , Linfócitos/citologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos SCID , Camundongos Transgênicos
17.
J Med Chem ; 55(12): 5887-900, 2012 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-22626259

RESUMO

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, ß, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, ß, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Inibidores do Citocromo P-450 CYP3A , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Artrite Reumatoide/enzimologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular , Citocromo P-450 CYP3A , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Conformação Proteica , Especificidade por Substrato , Fatores de Tempo
18.
J Immunol ; 173(4): 2236-40, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15294934

RESUMO

Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110gamma-deficient T cells, but not B cells, show reduced chemotactic responses to the lymphoid chemokines, CCL19, CCL21, and CXCL12. As B cell and T cell chemotactic responses were both sensitive to the general PI3K inhibitors, wortmannin (WMN) and LY294002, we explored whether B cell responses were affected in mice lacking p110delta, a major PI3K isoform in lymphocytes. B cells deficient in p110delta showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110delta-deficient B cells revealed diminished homing to Peyer's patches and splenic white pulp cords. WMN selectively inhibited CXCR5-dependent B cell homing to Peyer's patches. These observations establish that p110gamma and p110delta function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.


Assuntos
Linfócitos B/imunologia , Quimiotaxia de Leucócito/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Quimiocinas/imunologia , Quimiocinas/farmacologia , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/deficiência , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologia
19.
Nature ; 416(6876): 94-9, 2002 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-11882900

RESUMO

B lymphocytes re-circulate between B-cell-rich compartments (follicles or B zones) in secondary lymphoid organs, surveying for antigen. After antigen binding, B cells move to the boundary of B and T zones to interact with T-helper cells. Despite the importance of B--T-cell interactions for the induction of antibody responses, the mechanism causing B-cell movement to the T zone has not been defined. Here we show that antigen-engaged B cells have increased expression of CCR7, the receptor for the T-zone chemokines CCL19 and CCL21, and that they exhibit increased responsiveness to both chemoattractants. In mice lacking lymphoid CCL19 and CCL21 chemokines, or with B cells that lack CCR7, antigen engagement fails to cause movement to the T zone. Using retroviral-mediated gene transfer we demonstrate that increased expression of CCR7 is sufficient to direct B cells to the T zone. Reciprocally, overexpression of CXCR5, the receptor for the B-zone chemokine CXCL13, is sufficient to overcome antigen-induced B-cell movement to the T zone. These findings define the mechanism of B-cell relocalization in response to antigen, and establish that cell position in vivo can be determined by the balance of responsiveness to chemoattractants made in separate but adjacent zones.


Assuntos
Linfócitos B/fisiologia , Quimiocinas CC/fisiologia , Receptores de Quimiocinas/fisiologia , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Movimento Celular/fisiologia , Quimiocina CCL19 , Quimiocina CCL21 , Clonagem Molecular , Citometria de Fluxo , Humanos , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muramidase/imunologia , Receptores CCR7 , Receptores CXCR5 , Receptores de Citocinas/fisiologia
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