RESUMO
Immunogenicity against intravitreally administered brolucizumab has been previously described and associated with cases of severe intraocular inflammation, including retinal vasculitis/retinal vascular occlusion (RV/RO). The presence of antidrug antibodies (ADAs) in these patients led to the initial hypothesis that immune complexes could be key mediators. Although the formation of ADAs and immune complexes may be a prerequisite, other factors likely contribute to some patients having RV/RO, whereas the vast majority do not. To identify and characterize the mechanistic drivers underlying the immunogenicity of brolucizumab and the consequence of subsequent ADA-induced immune complex formation, a translational approach was performed to bridge physicochemical characterization, structural modeling, sequence analysis, immunological assays, and a quantitative systems pharmacology model that mimics physiological conditions within the eye. This approach revealed that multiple factors contributed to the increased immunogenic potential of brolucizumab, including a linear epitope shared with bacteria, non-natural surfaces due to the single-chain variable fragment format, and non-native drug species that may form over prolonged time in the eye. Consideration of intraocular drug pharmacology and disease state in a quantitative systems pharmacology model suggested that immune complexes could form at immunologically relevant concentrations modulated by dose intensity. Assays using circulating immune cells from treated patients or treatment-naïve healthy volunteers revealed the capacity of immune complexes to trigger cellular responses such as enhanced antigen presentation, platelet aggregation, endothelial cell activation, and cytokine release. Together, these studies informed a mechanistic understanding of the clinically observed immunogenicity of brolucizumab and associated cases of RV/RO.
Assuntos
Complexo Antígeno-Anticorpo , Análise de Causa Fundamental , Humanos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Inflamação , Inibidores da Angiogênese , Injeções IntravítreasRESUMO
Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases. Here, we describe how structure-based optimization of compound 2 resulted in the discovery of 34 (MHV370, (S)-N-(4-((5-(1,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methyl)bicyclo[2.2.2]octan-1-yl)morpholine-3-carboxamide). Its in vivo activity allows for further profiling toward clinical trials in patients with autoimmune disorders, and a Phase 2 proof of concept study of MHV370 has been initiated, testing its safety and efficacy in patients with Sjögren's syndrome and mixed connective tissue disease.
RESUMO
Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.
Assuntos
Doenças Autoimunes , Lúpus Eritematoso Sistêmico , Humanos , Camundongos , Animais , Receptor 7 Toll-Like/metabolismo , Receptor 7 Toll-Like/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/metabolismo , Hidroxicloroquina/farmacologia , Hidroxicloroquina/uso terapêutico , InterferonsRESUMO
Inappropriate activation of TLR7 and TLR8 is linked to several autoimmune diseases, such as lupus erythematosus. Here we report on the efficient structure-based optimization of the inhibition of TLR8, starting from a co-crystal structure of a small screening hit. Further optimization of the physicochemical properties for cellular potency and expansion of the structure-activity relationship for dual potency finally resulted in a highly potent TLR7/8 antagonist with demonstrated in vivo efficacy after oral dosing.
RESUMO
IL-1 receptor-activated kinase 1 (IRAK1) is involved in signal transduction downstream of many TLRs and the IL-1R. Its potential as a drug target for chronic inflammatory diseases is underappreciated. To study its functional role in joint inflammation, we generated a mouse model expressing a functionally inactive IRAK1 (IRAK1 kinase deficient, IRAK1KD), which also displayed reduced IRAK1 protein expression and cell type-specific deficiencies of TLR signaling. The serum transfer model of arthritis revealed a potentially novel role of IRAK1 for disease development and neutrophil chemoattraction exclusively via its activity in nonhematopoietic cells. Consistently, IRAK1KD synovial fibroblasts showed reduced secretion of neutrophil chemoattractant chemokines following stimulation with IL-1ß or human synovial fluids from patients with rheumatoid arthritis (RA) and gout. Together with patients with RA showing prominent IRAK1 expression in fibroblasts of the synovial lining, these data suggest that targeting IRAK1 may be therapeutically beneficial. As pharmacological inhibition of IRAK1 kinase activity had only mild effects on synovial fibroblasts from mice and patients with RA, targeted degradation of IRAK1 may be the preferred pharmacologic modality. Collectively, these data position IRAK1 as a central regulator of the IL-1ß-dependent local inflammatory milieu of the joints and a potential therapeutic target for inflammatory arthritis.
Assuntos
Artrite Reumatoide , Quinases Associadas a Receptores de Interleucina-1 , Neutrófilos , Membrana Sinovial , Animais , Artrite Reumatoide/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-8/metabolismo , Camundongos , Neutrófilos/metabolismo , Membrana Sinovial/metabolismoRESUMO
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Piridonas/química , Piridonas/farmacologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores , Animais , Células Cultivadas , Descoberta de Drogas , Humanos , Indazóis/química , Indazóis/farmacocinética , Indazóis/farmacologia , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Piridonas/farmacocinética , Ratos Sprague-Dawley , Receptor 7 Toll-Like/química , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
Trace amines (TAs) such as ß-phenylethylamine, p-tyramine, or tryptamine are biogenic amines found in the brain at low concentrations that have been implicated in various neuropsychiatric disorders like schizophrenia, depression, or attention deficit hyperactivity disorder. TAs are ligands for the recently identified trace amine-associated receptor 1 (TAAR1), an important modulator of monoamine neurotransmission. Here, we sought to investigate the consequences of TAAR1 hypersignaling by generating a transgenic mouse line overexpressing Taar1 specifically in neurons. Taar1 transgenic mice did not show overt behavioral abnormalities under baseline conditions, despite augmented extracellular levels of dopamine and noradrenaline in the accumbens nucleus (Acb) and of serotonin in the medial prefrontal cortex. In vitro, this was correlated with an elevated spontaneous firing rate of monoaminergic neurons in the ventral tegmental area, dorsal raphe nucleus, and locus coeruleus as the result of ectopic TAAR1 expression. Furthermore, Taar1 transgenic mice were hyposensitive to the psychostimulant effects of amphetamine, as it produced only a weak locomotor activation and failed to alter catecholamine release in the Acb. Attenuating TAAR1 activity with the selective partial agonist RO5073012 restored the stimulating effects of amphetamine on locomotion. Overall, these data show that Taar1 brain overexpression causes hyposensitivity to amphetamine and alterations of monoaminergic neurotransmission. These observations confirm the modulatory role of TAAR1 on monoamine activity and suggest that in vivo the receptor is either constitutively active and/or tonically activated by ambient levels of endogenous agonist(s).
Assuntos
Anfetamina/farmacologia , Monoaminas Biogênicas/fisiologia , Química Encefálica/fisiologia , Estimulantes do Sistema Nervoso Central/farmacologia , Receptores Acoplados a Proteínas G/biossíntese , Receptores Acoplados a Proteínas G/fisiologia , Transmissão Sináptica/fisiologia , Compostos de Anilina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos , Imidazóis/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Microdiálise , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Técnicas de Patch-Clamp , Fenótipo , Área Tegmentar Ventral/efeitos dos fármacos , Área Tegmentar Ventral/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Purified and hydrated elastin is studied by both thermal and dielectric techniques to have insight into the chain dynamics of this protein. By differential scanning calorimetry, the glassy behavior of elastin is highlighted; the glass transition temperature (T(g)) of elastin is found to be widely dependent on hydration, falling from 200 degrees C in the dehydrated state to 30 degrees C for 30% hydration. A limit of T(g) at around 0 degrees C is found when crystallizable water is present in the system, that is, when the formation of ice prevents motions of some 10 nm along the polypeptidic chains. The technique of thermally stimulated currents, carried out in the -180 to 0 degrees C temperature range, is useful to detect localized motions. In this case, too, the localized motions vary considerably according to hydration: a first relaxation mode is observed at -145 degrees C and it is associated with the reorientation of crystallizable water in ice I; a second relaxation mode, more complex and cooperative, occurs at around -80 degrees C and could be attributed to the complex constituted by the dipolar groups of the polypeptidic chain and noncrystallizable water, behaving as a glassy system.