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Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation. Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.
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Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 µg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.
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Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Compuestos de Bifenilo/farmacología , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Procolágeno N-Endopeptidasa/metabolismo , Inhibidores de Proteasas/farmacología , Sulfonamidas/farmacología , Proteína ADAMTS4 , Animales , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacocinética , Diseño de Fármacos , Humanos , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Modelos Moleculares , Osteoartritis/tratamiento farmacológico , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.
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Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Química Farmacéutica/métodos , Osteoartritis/tratamiento farmacológico , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Procolágeno N-Endopeptidasa/metabolismo , Sulfonamidas/síntesis química , Proteína ADAMTS4 , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Modelos Químicos , Conformación Molecular , Proteoglicanos/química , Sulfonamidas/farmacologíaRESUMEN
Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM-Sv/HP and MM-Sv/HP-2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q-PCR) were performed on cells in monolayer culture and cells embedded in a three-dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP-2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro-inflammatory IL-1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP-13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP-2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL-1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase-cleaved aggrecan fragments. In summary, MM-Sv/HP and MM-Sv/HP-2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology.
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Proteínas Morfogenéticas Óseas/farmacología , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Mediadores de Inflamación/farmacología , Interleucina-1/farmacología , Factor de Crecimiento Transformador beta/farmacología , Agrecanos/metabolismo , Alginatos/farmacología , Animales , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2 , Cartílago Articular/enzimología , Línea Celular Transformada , Condrocitos/enzimología , Endopeptidasas/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fémur/citología , Fémur/efectos de los fármacos , Humanos , Ratones , FenotipoRESUMEN
Genome-wide association studies of asthma have identified genetic variants in the IL1RL1 gene, but the molecular mechanisms conferring risk are unknown. IL1RL1 encodes the ST2 receptor (ST2L) for IL-33 and an inhibitory decoy receptor (sST2). IL-33 promotes type 2 inflammation, which is present in some but not all asthmatics. We find that two single nucleotide polymorphisms (SNPs) in IL1RL1 - rs1420101 and rs11685480 - are strongly associated with plasma sST2 levels, though neither is an expression quantitative trait locus (eQTL) in whole blood. Rather, rs1420101 and rs11685480 mark eQTLs in airway epithelial cells and distal lung parenchyma, respectively. We find that the genetically determined plasma sST2 reservoir, derived from the lung, neutralizes IL-33 activity, and these eQTL SNPs additively increase the risk of airway type 2 inflammation among asthmatics. These risk variants define a population of asthmatics at risk of IL-33-driven type 2 inflammation.
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Asma/genética , Proteína 1 Similar al Receptor de Interleucina-1/genética , Sitios de Carácter Cuantitativo , Células Cultivadas , Predisposición Genética a la Enfermedad , Humanos , Inflamación , Interleucina-33 , Polimorfismo de Nucleótido SimpleRESUMEN
PURPOSE: In the treatment of osteoarthritis (OA), some of the therapeutic approaches require delivery of drug(s) to the diseased cartilage. Presence of adequate drug levels in the cartilage is one of the important criteria in selection and ranking of lead compounds. The purpose of this study was to investigate the correlation in cartilage compound levels between in vitro experiments and in vivo animal studies. MATERIALS AND METHODS: Bovine cartilage samples were incubated with test compounds of various concentrations in a culture medium, in the absence or presence of 25 mg/ml of serum albumin which served as an artificial synovial fluid (SF). The test compounds were also dosed to rabbits, the animal model used for efficacy studies, over a six-week treatment period. Test article concentrations in plasma, SF, and cartilage were determined by LC/MS/MS analysis. RESULTS AND CONCLUSIONS: A correlation in cartilage drug concentration was observed between in vitro and in vivo studies. Plasma protein binding and the test article's affinity to cartilage were the major determining factors for drug delivery to cartilage in vivo.
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Cartílago Articular , Inyecciones Intraarteriales , Animales , Área Bajo la Curva , Proteínas Sanguíneas/metabolismo , Cartílago Articular/química , Bovinos , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Excipientes , Articulaciones/química , Masculino , Espectrometría de Masas , Inhibidores de la Metaloproteinasa de la Matriz , Unión Proteica , Conejos , Albúmina Sérica/farmacología , Líquido Sinovial/metabolismoRESUMEN
OBJECTIVE: Protein kinase Czeta (PKCzeta), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKCzeta in interleukin-1beta (IL-1beta)-induced NF-kappaB signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. METHODS: Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKCzeta. Western blot analysis was used to evaluate phosphorylation of PKCzeta and NF-kappaB. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. RESULTS: Phosphorylation of PKCzeta and NF-kappaB was induced by IL-1beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCzeta suppressed IL-1beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCzeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKCzeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. CONCLUSION: Our results show that PKCzeta is involved in the regulation of IL-1beta-induced NF-kappaB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCzeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.
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Proteínas ADAM/metabolismo , Condrocitos/metabolismo , Interleucina-1beta/fisiología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Proteína Quinasa C/metabolismo , Proteína ADAMTS4 , Células Cultivadas , Condrocitos/patología , Endopeptidasas/metabolismo , Humanos , Óxido Nítrico/metabolismo , Osteoartritis/patología , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA.