ABSTRACT
OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.
Subject(s)
Arthritis, Experimental/drug therapy , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Isoquinolines/therapeutic use , Lactams/therapeutic use , Macrophages/drug effects , Rheumatic Diseases/drug therapy , Synoviocytes/drug effects , Animals , Arthritis, Experimental/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Disease Models, Animal , Humans , Inflammation/drug therapy , Inflammation/immunology , Isoquinolines/pharmacology , Lactams/pharmacology , Leukocytes, Mononuclear/immunology , Macrophages/immunology , Mice , Rats , Rheumatic Diseases/immunology , Synoviocytes/immunologyABSTRACT
Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).
Subject(s)
Antitubercular Agents/pharmacology , Arthritis, Experimental/prevention & control , Janus Kinase 1/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , TYK2 Kinase/antagonists & inhibitors , Tuberculosis/complications , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/microbiology , Female , Molecular Structure , Rats , Rats, Inbred Lew , Tuberculosis/microbiologyABSTRACT
Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.
Subject(s)
Autoimmune Diseases/drug therapy , Cyclobutanes/pharmacology , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Sulfonamides/pharmacology , Animals , Arthritis, Experimental/drug therapy , Cyclobutanes/chemistry , Cyclobutanes/pharmacokinetics , Cyclobutanes/therapeutic use , Dogs , Drug Evaluation, Preclinical , Humans , Inhibitory Concentration 50 , Janus Kinase 1/chemistry , Janus Kinase 2/antagonists & inhibitors , Models, Molecular , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Pyrimidines/therapeutic use , Pyrroles/chemistry , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use , Rats , Substrate Specificity , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic use , Tissue DistributionABSTRACT
Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.
Subject(s)
Drug Discovery , Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Isoquinolines/pharmacology , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Dose-Response Relationship, Drug , Humans , Interleukin-1 Receptor-Associated Kinases/metabolism , Isoquinolines/administration & dosage , Isoquinolines/chemistry , Lactams , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.
Subject(s)
Arthritis, Experimental/drug therapy , Arthritis, Experimental/metabolism , Janus Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , RANK Ligand/metabolism , Animals , Arthritis, Experimental/immunology , Bone Resorption/drug therapy , Bone Resorption/immunology , Bone Resorption/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Disease Models, Animal , Female , Humans , Janus Kinases/metabolism , Macrophages/cytology , Macrophages/drug effects , Monocytes/cytology , Monocytes/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Osteoclasts/enzymology , Piperidines , Rats , Rats, Inbred Lew , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/enzymologyABSTRACT
Sensory irritants initiate respiratory reflexes by stimulating trigeminal sensory nerves. The vanilloid receptor (TRPV1) is expressed on sensory C fibers. The current experiments were aimed at examining the role of this receptor in mediating responses to several airborne irritants including an acidic (acetic acid), electrophilic (acrolein), and lipophilic solvent (styrene) vapor. Wild-type (C57Bl/6J) and VR1 knockout [B6.129S4-VR1(tm1jul)] mice were exposed to these irritants and breathing pattern responses were assessed by plethysmographic techniques; both wild-type and knockout animals responded similarly to the irritants. The TRPV1 antagonist iodoresiniferatoxin was also without effect on the responses to the irritants. Thus, in the C57Bl/6J mouse the TRPV1 receptor does not appear to play a major role in the stimulation of nasal trigeminal central reflex responses by these irritant air pollutants.
Subject(s)
Air Pollutants/pharmacology , Inhalation/drug effects , Receptors, Drug/physiology , Acetic Acid/pharmacology , Acrolein/pharmacology , Animals , Female , Inhalation/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Drug/deficiency , Receptors, Drug/genetics , Styrene/pharmacologyABSTRACT
The immediate responses of the upper respiratory tract (URT) to the irritants acrolein and acetic acid were examined in healthy and allergic airway-diseased C57Bl/6J mice. Acrolein (1.1 ppm) and acetic acid (330 ppm) vapors induced an immediate increase in flow resistance, as measured in the surgically isolated URT of urethane-anesthetized healthy animals. Acrolein, but not acetic acid, induced a small URT vasodilatory response. In awake spontaneously breathing mice, both vapors induced a prolonged pause at the start of expiration (a response mediated via stimulation of nasal trigeminal nerves) and an increase in total respiratory specific airway flow resistance, the magnitude of which was similar to that observed in the isolated URT. Both responses were significantly reduced in animals pretreated with large doses of capsaicin to defunctionalize sensory nerves, strongly suggesting a role for sensory nerves in development of these responses. The breathing pattern and/or obstructive responses were enhanced in mice with ovalbumin-induced allergic airway disease. These results suggest that the primary responses to acrolein and acetic acid vapors are altered breathing patterns and airway obstruction, that sensory nerves play an important role in these responses, and that these responses are enhanced in animals with allergic airway disease.
Subject(s)
Hypersensitivity/physiopathology , Irritants/pharmacology , Neurons, Afferent/physiology , Respiration/drug effects , Respiratory Physiological Phenomena , Respiratory Tract Diseases/physiopathology , Acetic Acid/administration & dosage , Acrolein/administration & dosage , Administration, Inhalation , Airway Obstruction/chemically induced , Airway Resistance/drug effects , Animals , Female , Hypersensitivity/immunology , Male , Mice , Mice, Inbred C57BL , Ovalbumin/immunology , Pulmonary Ventilation/drug effects , Respiratory Mechanics/drug effects , Respiratory System/blood supply , Respiratory System/drug effects , Respiratory Tract Diseases/immunology , VasodilationABSTRACT
The effect of exposure to irritant air pollutants on the development of allergic airway disease is poorly understood. This study examines the effects of the lower respiratory tract irritant, NO(2), on the outcome of ovalbumin (OVA)-induced allergic airway disease. Male and female C57Bl/6 mice were sensitized by weekly intraperitoneal (ip) OVA injections for 3 wk followed by daily 1-h OVA aerosol inhalation challenge for 3 or 10 d. Initially, mice were exposed daily for 3 d to air or 0.7 or 5 ppm NO(2) for 2 h following each OVA aerosol challenge. OVA exposure resulted in pronounced lower airway inflammation, as evidenced by a significant increase in bronchoalveolar lavage (BAL) total cellularity and eosinophil levels. BAL eosinophil levels were significantly lower in OVA-NO(2) compared to OVA-air animals. The reduction was similar at both NO(2) exposure concentrations. In a subsequent study, sensitized animals were exposed for 3 or 10 d to aerosolized OVA followed by air or 0.7 ppm NO(2). BAL eosinophils were again reduced at 3 d by OVA-NO(2) exposure compared to OVA-air mice. At 10 d the eosinophilia was virtually abolished. This reduction in OVA-induced cellular inflammation by NO(2) was confirmed by histopathological analysis. Contrary to expectations, exposure to NO(2) during the aerosol challenge to OVA dramatically diminished the outcome of allergic disease in lungs as measured by airway cellular inflammation.