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.
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-tetrahydrobenzobthiophene-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.
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.
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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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Phosphatidylinositol 3-kinase (PI 3-kinase) is activated by the cytokine interleukin-2 (IL-2). We have used a constitutively active PI 3-kinase to identify IL-2-mediated signal transduction pathways ...directly regulated by PI 3-kinase in lymphoid cells. The serine/threonine protein kinase B (PKB)/Akt can act as a powerful oncogene in T cells, but its positioning in normal T cell responses has not been explored. Herein, we demonstrate that PKB is activated by IL-2 in a PI 3-kinase-dependent fashion. Importantly, PI 3-kinase signals are sufficient for PKB activation in IL-2-dependent T cells, and PKB is a target for PI 3-kinase signals in IL-2 activation pathways. The present study establishes also that PI 3-kinase signals or PKB signals are sufficient for activation of p70 S6 kinase in T cells. PI 3-kinase can contribute to, but is not sufficient for, activation of extracellular signal-regulated kinases (Erks) and Erk effector pathways. Therefore, PI 3-kinase is a selective regulator of serine/threonine kinase signal transduction pathways in T lymphocytes, and this enzyme provides a crucial link between the interleukin-2 receptor, the protooncogene PKB, and p70 S6 kinase.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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-1β 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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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Despite the established role for PI3Ks in cell migration, the PI3Ks involved in lymphocyte chemotaxis are poorly defined. In this study, we report that p110γ-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 p110δ, a major PI3K isoform in lymphocytes. B cells deficient in p110δ showed diminished chemotactic responses, especially to CXCL13. Adoptive transfer experiments with WMN-treated wild-type B cells and with p110δ-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 p110γ and p110δ function in lymphocyte chemotaxis, and show differential roles for PI3K family members in B and T cell migration.
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.
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 retainedand in some cases improveda 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.
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