ABSTRACT
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
A critical component of innate immune response to infection and tissue damage is the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, and this pathway and its activation products have been implicated in the pathophysiology of a variety of diseases. NLRP3 inflammasome activation leads to the cleavage of pro-IL-1ß and pro-IL-18, as well as the subsequent release of biologically active IL-1ß, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome-selective, IL-1ß processing inhibitor CP-456,773 (also known as MCC950), and we demonstrate its efficacy in two in vivo models of inflammation. Specifically, we show that in human and mouse innate immune cells CP-456,773 is an inhibitor of the cellular release of IL-1ß, IL-1α, and IL-18, that CP-456,773 prevents inflammasome activation induced by disease-relevant soluble and crystalline NLRP3 stimuli, and that CP-456,773 inhibits R848- and imiquimod-induced IL-1ß release. In mice, CP-456,773 demonstrates potent inhibition of the release of proinflammatory cytokines following acute i.p. challenge with LPS plus ATP in a manner that is proportional to the free/unbound concentrations of the drug, thereby establishing an in vivo pharmacokinetic/pharmacodynamic model for CP-456,773. Furthermore, CP-456,773 reduces ear swelling in an imiquimod cream-induced mouse model of skin inflammation, and it reduces airway inflammation in mice following acute challenge with house dust mite extract. These data implicate the NLRP3 inflammasome in the pathogenesis of dermal and airway inflammation, and they highlight the utility of CP-456,773 for interrogating the contribution of the NLRP3 inflammasome and its outputs in preclinical models of inflammation and disease.
Subject(s)
Dermatitis/drug therapy , Heterocyclic Compounds, 4 or More Rings/pharmacology , Inflammasomes/antagonists & inhibitors , Inflammation/physiopathology , NLR Family, Pyrin Domain-Containing 3 Protein/antagonists & inhibitors , Pneumonia/drug therapy , Pneumonia/immunology , Sulfones/pharmacology , Animals , Cytokines/antagonists & inhibitors , Cytokines/immunology , Dermatitis/immunology , Dermatitis/physiopathology , Disease Models, Animal , Furans , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Humans , Immunity, Innate/drug effects , Indenes , Inflammation/drug therapy , Inflammation/immunology , Interleukin-18/antagonists & inhibitors , Interleukin-18/metabolism , Interleukin-1alpha/antagonists & inhibitors , Interleukin-1alpha/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/immunology , Mice , Pneumonia/physiopathology , Signal Transduction , Sulfonamides , Sulfones/administration & dosage , Sulfones/therapeutic useABSTRACT
The TLR pathway has been implicated in the pathogenesis of numerous diseases. IRAK-4 is integral to this pathway, making it a viable target for therapeutic intervention. This paper describes the application of a mechanistic pharmacodynamic model to assess the impact of IRAK-4 inhibition on the TLR-4 pathway. The model uses a minimal number of rate equations, molecular species, and parameters to characterize TLR signal transduction biology, including ligand-receptor interaction, protein complex formation, protein phosphorylation, negative regulation, and cytokine production. The model successfully reproduces the dynamic responses of TNFα to LPS stimulation, the tolerance to sequential LPS bolus dosing, the burst following a LPS bolus or infusion, and the modulation of pathway biomarkers following administration of an IRAK-4 inhibitor. Drug dosing schemes are evaluated for simulated disease states. The results emphasize the significance of LPS kinetics on response dynamics and the utility of a mechanistic model to help translate drug efficacy.
Subject(s)
Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Toll-Like Receptors/metabolism , Animals , Dose-Response Relationship, Drug , Humans , Lipopolysaccharides/pharmacology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BACKGROUND: In preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study. METHODS: Genes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey. RESULTS: Stimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration. CONCLUSIONS: A robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.
