Radiation causes tissue damage by dysregulating inflammasome-gasdermin D signaling in both host and transplanted cells.

Radiotherapy is a commonly used conditioning regimen for bone marrow transplantation (BMT). Cytotoxicity limits the use of this life-saving therapy, but the underlying mechanisms remain poorly defined. Here, we use the syngeneic mouse BMT model to test the hypothesis that lethal radiation damages tissues, thereby unleashing signals that indiscriminately activate the inflammasome pathways in host and transplanted cells. We find that a clinically relevant high dose of radiation causes severe damage to bones and the spleen through mechanisms involving the NLRP3 and AIM2 inflammasomes but not the NLRC4 inflammasome. Downstream, we demonstrate that gasdermin D (GSDMD), the common effector of the inflammasomes, is also activated by radiation. Remarkably, protection against the injury induced by deadly ionizing radiation occurs only when NLRP3, AIM2, or GSDMD is lost simultaneously in both the donor and host cell compartments. Thus, this study reveals a continuum of the actions of lethal radiation relayed by the inflammasome-GSDMD axis, initially affecting recipient cells and ultimately harming transplanted cells as they grow in the severely injured and toxic environment. This study also suggests that therapeutic targeting of inflammasome-GSDMD signaling has the potential to prevent the collateral effects of intense radiation regimens.

ATI-1777, a Topical Jak1/3 Inhibitor, May Benefit Atopic Dermatitis without Systemic Drug Exposure: Results from Preclinical Development and Phase 2a Randomized Control Study ATI-1777-AD-201.

Introduction: Atopic dermatitis, a chronic, pruritic skin disease, affects 10-30% of children and up to 14% of adults in developed countries. ATI-1777, a potent and selective Jak1/3 inhibitor, was designed with multiple sites of metabolism to deliver local efficacy in the skin and limit systemic exposure. In preclinical studies, ATI-1777 selectively inhibited Jak1/3 with limited systemic exposure and without any adverse effects. Primary objective: The primary goal of this study was to assess the preliminary clinical efficacy of ATI-1777 topical solution in adults with moderate or severe atopic dermatitis. Design: ATI-1777-AD-201, a phase 2a, first-in-human, randomized, double-blind, vehicle-controlled, parallel-group study, evaluated the efficacy, safety, tolerability, and pharmacokinetics of ATI-1777 topical solution in 48 participants with atopic dermatitis over 4 weeks. Primary endpoint: The primary endpoint was a reduction of a modified Eczema Area and Severity Index score from baseline. Results: Reduction was significantly greater in the ATI-1777-treated group on day 28 than in vehicle-treated group (percentage reduction from baseline = 74.45% [standard error = 6.455] and 41.43% [standard error = 6.189], respectively [P < .001]). Average plasma concentrations of ATI-1777 were <5% of the half-maximal inhibitory concentration of ATI-1777 for inhibiting Jak1/3. No deaths or serious adverse events were reported. Conclusion: Topical ATI-1777 does not lead to pharmacologically relevant systemic drug exposure and may reduce clinical signs of atopic dermatitis. Trial Registration: The study was registered at ClinicalTrials.gov with the number NCT04598269.

Selective Inhibition of the MK2 Pathway: Data From a Phase IIa Randomized Clinical Trial in Rheumatoid Arthritis.

OBJECTIVE: The study objective was to evaluate the safety, tolerability, pharmacodynamics, and preliminary efficacy of ATI-450 with methotrexate in patients with rheumatoid arthritis (RA). METHODS: A parallel-assignment, placebo-controlled, investigator-blinded/patient-blinded multicenter study evaluated patients with moderate-to-severe RA aged 18 to 70 years. Eligible patients were randomized (1:1) to ATI-450 50-mg oral tablets twice daily or placebo with a stable weekly dose of methotrexate for 12 weeks. The primary objective was to assess ATI-450 safety and tolerability. The secondary objectives were to assess the median percentage change from baseline high-sensitivity C-reactive protein (hs-CRP) levels, the mean change from baseline in Disease Activity Score in 28 joints based on CRP level (DAS28-CRP) and Rheumatoid Arthritis Magnetic Resonance Imaging Score hand-wrist assessments of synovitis or bone erosion at week 12, and the proportion of patients with American College of Rheumatology 20/50/70 (ACR 20/50/70) and with DAS28-CRP scores of less than 2.6. The exploratory outcomes were change from baseline in endogenous and ex vivo-stimulated cytokine levels. RESULTS: ATI-450 was well tolerated with no severe adverse events reported. ATI-450 reduced median hs-CRP levels by 42% or more at all posttreatment timepoints. In the ATI-450 group, a mean (median) decrease in DAS28-CRP score of 2.0 (2.1) was observed at week 12; proportions of patients with an ACR 20/50/70 response in the per-protocol population were 60%, 33%, and 20%, respectively, at week 12. Endogenous plasma levels of key inflammatory cytokines (tumor necrosis factor α, macrophage inflammatory protein 1ß, interleukin 6, interleukin 8) were reduced across the 12 treatment weeks. CONCLUSION: This is the first clinical study demonstrating that selective mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) pathway blockade leads to a sustained antiinflammatory effect. This suggests that targeting the MK2 pathway mitigates the tachyphylaxis observed with p38 MAPK inhibitors in RA and supports further exploration.

p38MAPKα Stromal Reprogramming Sensitizes Metastatic Breast Cancer to Immunotherapy.

Metastatic breast cancer is an intractable disease that responds poorly to immunotherapy. We show that p38MAPKα inhibition (p38i) limits tumor growth by reprogramming the metastatic tumor microenvironment in a CD4+ T cell-, IFNγ-, and macrophage-dependent manner. To identify targets that further increased p38i efficacy, we utilized a stromal labeling approach and single-cell RNA sequencing. Thus, we combined p38i and an OX40 agonist that synergistically reduced metastatic growth and increased overall survival. Intriguingly, patients with a p38i metastatic stromal signature had better overall survival that was further improved by the presence of an increased mutational load, leading us to ask if our approach would be effective in antigenic breast cancer. The combination of p38i, anti-OX40, and cytotoxic T-cell engagement cured mice of metastatic disease and produced long-term immunologic memory. Our findings demonstrate that a detailed understanding of the stromal compartment can be used to design effective antimetastatic therapies. SIGNIFICANCE: Immunotherapy is rarely effective in breast cancer. We dissected the metastatic tumor stroma, which revealed a novel therapeutic approach that targets the stromal p38MAPK pathway and creates an opportunity to unleash an immunologic response. Our work underscores the importance of understanding the tumor stromal compartment in therapeutic design. This article is highlighted in the In This Issue feature, p. 1275.

Interleukin-1ß-induced interleukin-6 production in A549 cells is mediated by both phosphatidylinositol 3-kinase and interleukin-1 receptor-associated kinase-4.

The aim of this study is to investigate whether PI3K (phosphatidylinositol-3-kinase) is involved in IL-1ß (interleukin-1ß)-induced IL-6 production in A549 (human lung adenocarcinoma epithelial cell) and human RASF (rheumatoid arthritis synovial fibroblast). PI3K inhibitor, LY294002 significantly reduced IL-1ß-induced IL-6 production in A549 cells but not in RASF, indicating that IL-1ß-induced IL-6 production was partially mediated by PI3Kin A549 cells but not in RASF. siRNA (small interfering RNA) of IRAK4 (IL-1 receptor-associated kinase 4) treatment decreased IRAK4 mRNA level by up to 90% in A549 cells. In this condition, IL-1ß-induced increase of IL-6 mRNA and protein level was decreased by up to 93% and 70%, respectively. Furthermore, the combination of IRAK4 siRNA and LY294002 treatment decreased protein induction level of IL-6 in A549 cells compared with that of IRAK4 siRNA or LY294002 alone. These results indicate that IL-1ß-induced IL-6 production in A549 cells is mediated by both PI3K and IRAK4 and suggest that involvement of PI3K in the IL-1-induced IL-6 production is cell type specific.

Substituted N-aryl-6-pyrimidinones: a new class of potent, selective, and orally active p38 MAP kinase inhibitors.

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase.

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase.

The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.

Proinflammatory cytokines, IL-1ß and TNF-α, induce expression of interleukin-34 mRNA via JNK- and p44/42 MAPK-NF-κB pathway but not p38 pathway in osteoblasts.

The aim of this study is to investigate the induction of interleukin-34 (IL-34) and macrophage colony-stimulating factor (M-CSF) mRNA by inflammatory cytokines and the involvement of mitogen-activated protein kinases (MAPKs) in this signaling pathway in human osteoblasts as both IL-34 and M-CSF bind to the same receptor c-FMS. Among four inflammatory cytokines [(IL-1ß, IL-6, IL-17, and tumor necrosis factor-α (TNF-α)], IL-34 mRNA expression level was dramatically induced by IL-1ß (17-fold) and TNF-α (74-fold). IL-1ß and TNF-α activated the intracellular mitogen-activated protein kinases (MAPKs): p44/42 MAPK, p38, and c-Jun N-terminal kinase (JNK) as well as nuclear factor-κB (NF-κB) in osteoblasts. IL-1ß- and TNF-α-mediated induction of IL-34 mRNA expression was decreased by JNK inhibitor. Interestingly, although treatment of MEK-1/2 inhibitor showed no reduction in the increase of IL-34 mRNA expression by cytokines, combination of MEK-1/2 inhibitor and JNK inhibitor significantly inhibited IL-1ß- and TNF-α-mediated IL-34 mRNA expression level compared to those by each inhibitor alone. On the other hand, M-CSF mRNA expression level was significantly induced by both IL-1ß and TNF-α by up to 7- and 11-fold, respectively. IL-1ß- and TNF-α-mediated induction of M-CSF mRNA was not affected by p38, JNK, and MEK-1/2 inhibitors. However, NF-κB inhibitor completely inhibited the elevation of M-CSF mRNA expression by these cytokines. These results showed that proinflammatory cytokines, IL-1ß and TNF-α, induced the expression of IL-34 mRNA via JNK and p44/42 MAPK but not p38 in human osteoblasts while p38, JNK, and p44/42 MAPK were not involved in the induction of M-CSF mRNA expression by these cytokines.

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the MK2 Inhibitor ATI-450 in Healthy Subjects: A Placebo-Controlled, Randomized Phase 1 Study.

PURPOSE: ATI-450 is an oral, small-molecule inhibitor of the p38α mitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2 (MK2) inflammatory signaling pathway. This phase 1, single and multiple ascending dose (SAD, MAD) study evaluated ATI-450 safety, tolerability, pharmacokinetics, and pharmacodynamics. PATIENTS AND METHODS: Healthy adults were randomly assigned to SAD (10, 30, 50, 100 mg; n=24) and MAD (10, 30, 50 mg twice daily [BID] for 7 days; n=24) cohorts of ATI-450 or placebo (n=14). Safety and tolerability were evaluated through clinical and laboratory assessments. Pharmacokinetic parameters were evaluated in plasma samples; pharmacodynamic assessments included quantification of cytokine levels (tumor necrosis factor α [TNF-α], interleukin [IL]-1ß, IL-6, IL-8) and phosphorylation of the MK2 downstream substrate, heat shock protein 27 (p-HSP27). RESULTS: The most common adverse events were headache (10/48, 20.8%), dizziness (6/48, 12.5%), upper respiratory tract infection (3/48, 6.3%), and constipation (3/48, 6.3%). Pharmacokinetics were dose-proportional, with a terminal half-life of 9‒12 hours in the MAD cohorts on day 7. Dose- and concentration-dependent inhibition of ex vivo stimulated cytokines and target biomarker was observed. On day 7, patients in the 50 mg BID dose cohort recorded mean trough drug levels that were 1.4, 2.2, 2.3, and 2.4 times greater than the IC80 for TNF-α, IL-1ß, IL-8, and p-HSP27, respectively. Mean Cmax was 3.5, 5.4, 5.6, and 6.0 times greater than the IC80 for TNF-α, IL-1ß, IL-8, and p-HSP27, respectively. IL-6 inhibition >50% was noted for part of the dosing interval. CONCLUSION: ATI-450 was well tolerated at the doses investigated, exhibited dose- and time-independent (ie, linear) pharmacokinetics, and dose-related pharmacodynamic effects. These results support further study of ATI-450 in immunoinflammatory diseases in phase 2 trials.

The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress.

Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factor­α (TNFα), causing autocrine phosphorylation and activation of transforming growth factor­ß­activated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38­induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress­induced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.

NLRP3 inflammasome activation triggers gasdermin D-independent inflammation.

NOD-like receptor (NLR), family pyrin domain containing 3 (NLRP3) assembles a protein complex known as the NLRP3 inflammasome upon sensing certain pathogen products or sterile danger signals. Gain-of-function mutations such as the D301N substitution in NLRP3, which cause its constitutive activation (NLRP3CA) also results in inflammasome assembly. This inflammasome processes pro­interleukin-1 ß (pro­IL-1ß) and pro­IL-18 into bioactive IL-1ß and IL-18, respectively, and cleaves gasdermin D (GSDMD). GSDMD amino-terminal fragments form plasma membrane pores that facilitate the secretion of IL-1ß and IL-18 and lead to the inflammatory cell death pyroptosis. Accordingly, GSDMD inactivation results in negligible spontaneous inflammation in various experimental models such as in Nlrp3CA/+ mice lacking GSDMD (Nlrp3CA/+;Gsdmd−/− mice). Here, we found that Nlrp3CA/+;Gsdmd−/− mice, when challenged with LPS or TNF-α, still secreted IL-1ß and IL-18, indicating inflammasome activation independent of GSDMD. Accordingly, Gsdmd−/− macrophages failed to secrete IL-1ß and undergo pyroptosis when briefly exposed to NLRP3 inflammasome activators but released these cytokines when persistently activated. Sustained NLRP3 inflammasome induced caspase-8/-3 and GSDME cleavage and IL-1ß maturation in vitro in Gsdmd−/− macrophages. Thus, a salvage inflammatory pathway involving caspase-8/-3­GSDME was activated after NLRP3 activation when the canonical NLRP3-GSDMD signaling was blocked. Consistent with genetic data, the active metabolite of FDA-approved disulfiram CuET, which inhibited GSDMD and GSDME cleavage in macrophages, reduced the severe inflammation and tissue damage that occurred in the Nlrp3CA/+ mice. Thus, NLRP3 inflammasome activation overwhelms the protection afforded by GSDMD deficiency, rewiring signaling cascades through mechanisms that include GSDME to propagate inflammation.

Macrophage-colony stimulating factor and interleukin-34 induce chemokines in human whole blood.

The aim of this study is to investigate if macrophage-colony stimulating factor (M-CSF) or interleukin-34 (IL-34) induces cytokines or chemokines using human whole blood (HWB) and if an M-CSF- or IL-34-induced cytokine or chemokine production from HWB is inhibited by soluble M-CSF receptor or c-FMS kinase inhibitors. Among eight cytokines or growth factors tested, only IL-6 level was increased by up to 6-fold by M-CSF or IL-34 in HWB. In contrast, chemokine levels (IP-10/CXCL10, IL-8/CXCL8, and MCP-1/CCL2) were dramatically increased by M-CSF or IL-34 in HWB while exhibiting a large variation among donors. Variability of the MCP-1 signal induced by M-CSF or IL-34 was relatively less among donors compared to the IP-10 and IL-8 signals. The elevation of these chemokine levels was significantly decreased by soluble M-CSF receptor, indicating the elevation of these chemokines was mediated by M-CSF or IL-34. Furthermore, GW2580, a c-FMS kinase inhibitor, inhibited the induction of MCP-1 by M-CSF or IL-34 in a concentration dependent manner. These indicate MCP-1 is the most appropriate chemokine target for a chemokine release assay to evaluate the potency of c-FMS kinase inhibitors and MCP-1 release assay using HWB would be useful, relevant tool for translational pharmacology of c-FMS kinase inhibitors.

Identification of SD-0006, a potent diaryl pyrazole inhibitor of p38 MAP kinase.

Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38alpha kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Therapy-Induced Senescence Drives Bone Loss.

Chemotherapy is important for cancer treatment, however, toxicities limit its use. While great strides have been made to ameliorate the acute toxicities induced by chemotherapy, long-term comorbidities including bone loss remain a significant problem. Chemotherapy-driven estrogen loss is postulated to drive bone loss, but significant data suggests the existence of an estrogen-independent mechanism of bone loss. Using clinically relevant mouse models, we showed that senescence and its senescence-associated secretory phenotype (SASP) contribute to chemotherapy-induced bone loss that can be rescued by depleting senescent cells. Chemotherapy-induced SASP could be limited by targeting the p38MAPK-MK2 pathway, which resulted in preservation of bone integrity in chemotherapy-treated mice. These results transform our understanding of chemotherapy-induced bone loss by identifying senescent cells as major drivers of bone loss and the p38MAPK-MK2 axis as a putative therapeutic target that can preserve bone and improve a cancer survivor's quality of life. SIGNIFICANCE: Senescence drives chemotherapy-induced bone loss that is rescued by p38MAPK or MK2 inhibitors. These findings may lead to treatments for therapy-induced bone loss, significantly increasing quality of life for cancer survivors.

Structural bioinformatics-based prediction of exceptional selectivity of p38 MAP kinase inhibitor PH-797804.

PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.

Discovery of N-substituted pyridinones as potent and selective inhibitors of p38 kinase.

The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38alpha. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.

SD0006: a potent, selective and orally available inhibitor of p38 kinase.

SD0006 is a diarylpyrazole that was prepared as an inhibitor of p38 kinase-alpha (p38alpha). In vitro, SD0006 was selective for p38alpha kinase over 50 other kinases screened (including p38gamma and p38delta with modest selectivity over p38beta). Crystal structures with p38alpha show binding at the ATP site with additional residue interactions outside the ATP pocket unique to p38alpha that can confer advantages over other ATP competitive inhibitors. Direct correlation between inhibition of p38alpha activity and that of lipopolysaccharide-stimulated TNFalpha release was established in cellular models and in vivo, including a phase 1 clinical trial. Potency (IC(50)) for inhibiting tumor necrosis factor-alpha (TNFalpha) release, in vitro and in vivo, was <200 nmol/l. In vivo, SD0006 was effective in the rat streptococcal-cell-wall-induced arthritis model, with dramatic protective effects on paw joint integrity and bone density as shown by radiographic analysis. In the murine collagen-induced arthritis model, equivalence was demonstrated to anti-TNFalpha treatment. SD0006 also demonstrated good oral anti-inflammatory efficacy with excellent cross-species correlation between the rat, cynomolgus monkey, and human. SD0006 suppressed expression of multiple proinflammatory proteins at both the transcriptional and translational levels. These properties suggest SD0006 could provide broader therapeutic efficacy than cytokine-targeted monotherapeutics.