Novel Inhaled Pan-JAK Inhibitor, LAS194046, Reduces Allergen-Induced Airway Inflammation, Late Asthmatic Response, and pSTAT Activation in Brown Norway Rats.

The Janus-activated kinase (JAK) family together with signal transducer and activator of transcription (STAT) signaling pathway has a key role in regulating the expression and function of many inflammatory cytokines. This has led to the discovery of JAK inhibitors for the treatment of inflammatory diseases, some of them already in the market. Considering the adverse effects associated with JAK inhibition by oral route, we wanted to explore whether JAK inhibition by inhaled route is enough to inhibit airway inflammation. The aim of this study was to characterize the enzymatic and cellular potency and the selectivity of LAS194046, a novel JAK inhibitor, compared with the reference compounds ruxolitinib and tofacitinib. The efficacy of this new JAK inhibitor is described in a model of ovalbumin (OVA)-induced airway inflammation in Brown Norway rats by inhaled administration. As potential markers of target engagement, we assessed the effect of LAS194046 on the STAT activation state. LAS194046 is a selective inhaled pan-JAK inhibitor that reduces allergen-induced airway inflammation, late asthmatic response, and phosphor-STAT activation in the rat OVA model. Our results show that topical inhibition of JAK in the lung, without relevant systemic exposure, is sufficient to reduce lung inflammation and improve lung function in a rat asthma model. In summary, JAK-STAT pathway inhibition by inhaled route constitutes a promising therapeutic option for lung inflammatory diseases.

Tofacitinib ameliorates inflammation in a rat model of airway neutrophilia induced by inhaled LPS.

BACKGROUND AND PURPOSE: The Janus Kinase (JAK) family mediates the cytokine receptor-induced signalling pathways involved in inflammatory processes. The activation of the signal transducers and activators of transcription (STATs) by JAK kinases is a key point in these pathways. Four JAK proteins, JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2) associate with the intracellular domains of surface cytokine receptors are phosphorylating STATs and modulating gene expression. The aim of this study was to explore the role of JAK inhibition in an acute model of inhaled lipopolysaccharide (LPS)-induced airway inflammation in rats through evaluating the effects of tofacitinib, a marketed pan-JAK inhibitor. Specifically, some pulmonary inflammation parameters were studied and the lung STAT3 phosphorylation was assessed as a target engagement marker of JAK inhibition in the model. EXPERIMENTAL APPROACH: Rats were exposed to an aerosol of LPS (0.1 mg/ml) or phosphate-buffered saline (PBS) during 40 min. Bronchoalveolar lavage fluid (BALF) and lung samples were collected 4 h after PBS or LPS exposure. Neutrophils in BALF were counted and a panel of cytokines were measured in BALF. Phosphorylation of STAT3 was studied in lung homogenates by ELISA and localization of phospho-STAT3 (pSTAT3) in lung tissue was also evaluated by immunohistochemistry. In order to assess the effect of JAK inhibition, tofacitinib was administered 1 h before challenge at doses of 3, 10 and 30 mg/kg p.o. KEY RESULTS: Inhaled LPS challenge induced an augment of neutrophils and cytokines in the BALF as well as an increase in pSTAT3 expression in the lungs. Tofacitinib by oral route inhibited the LPS-induced airway neutrophilia, the levels of some cytokines in the BALF and the phosphorylation of STAT3 in the lung tissue. CONCLUSIONS AND IMPLICATIONS: In summary, this study shows that JAK inhibition ameliorates inhaled LPS-induced airway inflammation in rats, suggesting that at least JAK/STAT3 signalling is involved in the establishment of the pulmonary neutrophilia induced by LPS. JAKs inhibitors should be further investigated as a potential therapy for respiratory inflammatory diseases.

Anti-inflammatory potential of PI3Kδ and JAK inhibitors in asthma patients.

BACKGROUND: Phosphatidylinositol 3-kinase delta (PI3Kδ) and Janus-activated kinases (JAK) are both novel anti-inflammatory targets in asthma that affect lymphocyte activation. We have investigated the anti-inflammatory effects of PI3Kδ and JAK inhibition on cytokine release from asthma bronchoalveolar lavage (BAL) cells and T-cell activation, and measured lung PI3Kδ and JAK signalling pathway expression. METHOD: Cells isolated from asthma patients and healthy subjects were treated with PI3Kδ or JAK inhibitors, and/or dexamethasone, before T-cell receptor stimulation. Levels of IFNγ, IL-13 and IL-17 were measured by ELISA and flow cytometry was used to assess T-cell activation. PI3Kδ, PI3Kγ, phosphorylated protein kinase B (pAKT) and Signal Transducer and Activator of Transcription (STAT) protein expression were assessed by immunohistochemistry in bronchial biopsy tissue from asthma patients and healthy subjects. PI3Kδ expression in BAL CD3 cells was measured by flow cytometry. RESULTS: JAK and PI3Kδ inhibitors reduced cytokine levels from both asthma and healthy BAL cells. Combining dexamethasone with either a JAK or PI3Kδ inhibitor showed an additive anti-inflammatory effect. JAK and PI3Kδ inhibitors were shown to have direct effects on T-cell activation. Immunohistochemistry showed increased numbers of PI3Kδ expressing cells in asthma bronchial tissue compared to controls. Asthma CD3 cells in BAL expressed higher levels of PI3Kδ protein compared to healthy cells. CONCLUSIONS: Targeting PI3Kδ or JAK may prove effective in reducing T-cell activation and the resulting cytokine production in asthma.

A novel inhaled Syk inhibitor blocks mast cell degranulation and early asthmatic response.

Spleen tyrosine kinase (Syk) is essential for signal transduction of immunoreceptors. Inhibition of Syk abrogates mast cell degranulation and B cell responses. We hypothesized that Syk inhibition in the lung by inhaled route could block airway mast cells degranulation and the early asthmatic response without the need of systemic exposure. We discovered LAS189386, a novel Syk inhibitor with suitable properties for inhaled administration. The aim of this study was to characterize the in vitro and in vivo profile of LAS189386. The compound was profiled in Syk enzymatic assay, against a panel of selected kinases and in Syk-dependent cellular assays in mast cells and B cells. Pharmacokinetics and in vivo efficacy was assessed by intratracheal route. Airway resistance and mast cell degranulation after OVA challenge was evaluated in an ovalbumin-sensitized Brown Norway rat model. LAS189386 potently inhibits Syk enzymatic activity (IC50 7.2 nM), Syk phosphorylation (IC50 41 nM), LAD2 cells degranulation (IC50 56 nM), and B cell activation (IC50 22 nM). LAS189386 inhibits early asthmatic response and airway mast cell degranulation without affecting systemic mast cells. The present results support the hypothesis that topical inhibition of Syk in the lung, without systemic exposure, is sufficient to inhibit EAR in rats. Syk inhibition by inhaled route constitutes a promising therapeutic option for asthma.

Characterization of a model of tracheal plasma extravasation in passively sensitized rats using anti-allergic and anti-inflammatory drugs by oral and intratracheal route.

The aim of the following study was to characterize a passive systemic anaphylaxis rat model of dinitrophenyl (DNP)-induced plasma extravasation in the trachea to determine if the model is appropriate for the evaluation of new drugs targeting airway mast cells by oral and intratracheal (i.t.) route. To this purpose we have used fluticasone and a range of anti-allergic drugs including compounds either active on mast cell activation, such as cromoglycate and the Syk inhibitor R406, or active on mast cell mediators, such as cetirizine and montelukast. To further characterize the model, the effect of fluticasone, cromoglycate and R406 on rat tracheal mast cell degranulation was also assessed histologically. DNP-induced tracheal plasma extravasation was inhibited by cromoglycate (i.v. and i.t.) and R406 (p.o.), but not by fluticasone (i.t.), cetirizine or montelukast (p.o.). Cromoglycate and R406 also showed inhibition of tracheal mast cell degranulation, whereas fluticasone was inactive. These results suggest that the DNP-induced tracheal plasma extravasation model constitutes a useful animal model for the evaluation, by oral and i.t. route, of new anti-allergic drugs intended to target airway mast cells.

Pyrazine-based Syk kinase inhibitors.

A series of aminopyrazines as inhibitors of Syk kinase activity and showing inhibition of LAD2 cells degranulation is described. Optimization of the carboxamide motif with aminomethylpiperidines provided high potency inhibiting Syk but low cellular activity. Amides of cis and trans adamantanol showed good inhibitory activity against Syk as well as remarkable activity in LAD2 cells degranulation assay.

Highly potent aminopyridines as Syk kinase inhibitors.

A novel class of potent Syk inhibitors has been developed from rational design. Highly potent aminopyridine derivatives bearing a 4-trifluoromethyl-2-pyridyl motif and represented by compound 13b IC(50): 0.6 nM were identified. Substitution by a 2-pyrazinyl motif and SAR expansion in position 4 of the central core provided diverse potent non-cytotoxic Syk inhibitors showing nanomolar activity inhibiting human mast cell line LAD2 degranulation.

The pan-JAK inhibitor LAS194046 reduces neutrophil activation from severe asthma and COPD patients in vitro.

Non-T2 severe asthma and chronic obstructive pulmonary disease (COPD) are airway chronic inflammatory disorders with a poor response to corticosteroids. LAS194046, a novel pan-Janus kinase (JAK) inhibitor, shows inhibitory effects on T2 allergic lung inflammation in rats. In this work we analyze the effects of LAS194046, fluticasone propionate and their combination in neutrophils from non-T2 severe asthma and COPD patients in vitro. Neutrophils from 23 healthy subjects, 23 COPD and 21 non-T2 severe asthma patients were incubated with LAS194046 (0.01 nM-1 µM), fluticasone propionate (0.1 nM-1 µM) or their combination and stimulated with lipopolysaccharide (LPS 1 µM). LAS194046 shows similar maximal % inhibition and potency inhibiting IL-8, MMP-9 and superoxide anion release in neutrophils from healthy, COPD and asthma. Fluticasone propionate suppresses mediator release only in neutrophils from healthy patients. The combination of LAS194046 with fluticasone propionate shows synergistic anti-inflammatory and anti-oxidant effects. The mechanisms involved in the synergistic effects of this combination include the increase of MKP1 expression, decrease of PI3Kδ, the induction of glucocorticoid response element and the decrease of ERK1/2, P38 and JAK2/STAT3 phosphorylation compared with monotherapies. In summary, LAS194046 shows anti-inflammatory effects in neutrophils from COPD and severe non-T2 asthma patients and induces synergistic anti-inflammatory effects when combined with fluticasone propionate.

Identification of 2-Imidazopyridine and 2-Aminopyridone Purinones as Potent Pan-Janus Kinase (JAK) Inhibitors for the Inhaled Treatment of Respiratory Diseases.

Janus kinases (JAKs) have a key role in regulating the expression and function of relevant inflammatory cytokines involved in asthma and chronic obstructive pulmonary disease. Herein are described the design, synthesis, and pharmacological evaluation of a series of novel purinone JAK inhibitors with profiles suitable for inhaled administration. Replacement of the imidazopyridine hinge binding motif present in the initial compounds of this series with a pyridone ring resulted in the mitigation of cell cytotoxicity. Further systematic structure-activity relationship (SAR) efforts driven by structural biology studies led to the discovery of pyridone 34, a potent pan-JAK inhibitor with good selectivity, long lung retention time, low oral bioavailability, and proven efficacy in the lipopolysaccharide-induced rat model of airway inflammation by the inhaled route.

PI3K, p38 and JAK/STAT signalling in bronchial tissue from patients with asthma following allergen challenge.

BACKGROUND: Inhaled allergen challenges are often used to evaluate novel asthma treatments in early phase clinical trials. Current novel therapeutic targets in asthma include phosphoinositide 3-kinases (PI3K) delta and gamma, p38 mitogen-activated protein kinase (p38) and Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathways. The activation of these pathways following allergen exposure in atopic asthma patients it is not known. METHODS: We collected bronchial biopsies from 11 atopic asthma patients at baseline and after allergen challenge to investigate biomarkers of PI3K, p38 MAPK and JAK/STAT activation by immunohistochemistry. Cell counts and levels of eosinophil cationic protein and interleukin-5 were also assessed in sputum and bronchoalvelar lavage. RESULTS: Biopsies collected post-allergen had an increased percentage of epithelial cells expressing phospho-p38 (17.5 vs 25.6%, p = 0.04), and increased numbers of sub-epithelial cells expressing phospho-STAT5 (122.2 vs 540.6 cells/mm2, p = 0.01) and the PI3K marker phospho-ribosomal protein S6 (180.7 vs 777.3 cells/mm2,p = 0.005). Type 2 inflammation was increased in the airways post allergen, with elevated levels of eosinophils, interleukin-5 and eosinophil cationic protein. CONCLUSIONS: Future clinical trials of novel kinase inhibitors could use the allergen challenge model in proof of concept studies, while employing these biomarkers to investigate pharmacological inhibition in the lungs.

The intestinal anti-inflammatory effects of the novel agent UR-1505 in the TNBS model of rat colitis are mediated by T-lymphocyte inhibition.

UR-1505 is a novel pentafluoropropoxy derivative of salicylic acid, selected from a series of salicylate derivatives, according to their activity as inhibitors of T-lymphocyte activation. This study describes the anti-inflammatory activity of UR-1505 on trinitrobenzenesulphonic acid-induced colitis in rat, an experimental model that resembles to Crohn's disease (CD), as well as its in vitro effects on T-cells and bone marrow-derived macrophages (BMDM) activation. UR-1505 showed intestinal anti-inflammatory effect, associated with reduced colonic levels of TNFalpha and LTB(4), inhibition of the expression of IFNgamma and iNOS, and lower colonic leukocyte infiltration. The in vitro assays revealed that UR-1505 also inhibited T-lymphocyte proliferation and IL-12/IFNgamma production, two of the main pro-inflammatory cytokines involved in the pathogenesis of CD. However, UR-1505 did not modify LPS- nor IFNgamma-induced activation in BMDM. Thus, UR-1505 specifically affects T-cells without modifying the activation of BMDM. In conclusion, the intestinal anti-inflammatory activity of UR-1505 seems to be mediated by a reduction in the recruitment of immune cells to the inflammatory foci, together with the inhibition of T-cell activation. These results suggest that UR-1505 may be an interesting candidate to be explored for the treatment of CD.

New water-soluble sulfonylphosphoramidic acid derivatives of the COX-2 selective inhibitor cimicoxib. A novel approach to sulfonamide prodrugs.

The synthesis and pharmacological evaluation of new water-soluble phosphoramidate derivatives of the COX-2 selective inhibitor cimicoxib (4) are described. The sulfonylphosphoramidic acid derivative 10 was converted to 4 in human plasma and showed excellent in vivo activity in the rat carrageenan-edema test. Pharmacokinetic evaluation in dogs indicated that 10 behaved as a prodrug, immediately converting to 4 and giving an identical profile to that of the parent compound. These results represent the first description of phosphoramidic acids as prodrugs for the sulfonamido group. Compound 10 also exhibited an important and sustained analgesic effect in the hyperalgesia test in rats and a high aqueous solubility at pH higher than 7. This profile led to the selection of 10 (UR-14048) for further development in the parenteral treatment of acute pain.

The intestinal anti-inflammatory activity of UR-12746S on reactivated experimental colitis is mediated through downregulation of cytokine production.

BACKGROUND: UR-12746S (dersalazine sodium) is cleaved by colonic bacteria delivering the PAF antagonist UR-12715 and 5-ASA. This study describes the anti-inflammatory activity of UR-12746S in an experimental model of reactivated colitis and its effects on cytokine production. METHODS: Rats were initially rendered colitic by a colonic instillation of 10 mg of trinitrobenzenesulphonic acid (TNBS) dissolved in 0.25 ml of 50 % ethanol, and colitis was reactivated two weeks after by a second administration of the same dose of TNBS. Two groups of colitic rats received UR-12746S (25 and 50 mg/kg daily, p.o.) and colonic damage was evaluated every week for 4 weeks. Different biochemical markers of colonic inflammation were assayed: MPO activity and cytokine (IL-1beta and TNFalpha) levels. Also, the in vitro effects of UR-12715 and 5-ASA on cytokine production were assayed. RESULTS: UR-12746S showed anti-inflammatory effect in reactivated colitis in rats, as evidenced by a significant reduction in MPO activity. Both doses of UR-12746S decreased IL-1beta production, while only the highest dose assayed inhibited TNFalpha production. In vitro studies revealed that UR-12715 or 5-ASA (from 10(-6) to 10(-4) M) inhibited IL-8 production (30-40%) in HT-29 cells when incubated with LPS. This inhibitory effect was enhanced when both compounds were administered simultaneously at 10(-4) M. In addition, UR-12715 inhibited IL-1beta or TNFalpha production in THP-1 or U937 cells, respectively, when these cells were stimulated by PMA and LPS; whereas 5-ASA only showed a weak effect in inhibiting IL-1beta production. CONCLUSION: UR-12746S was able to prevent relapse in experimental colitis and inhibition of proinflammatory cytokine production participates in the intestinal anti-inflammatory activity exerted by this compound.

The new salicylate derivative UR-1505 modulates the Th2/humoral response in a dextran sodium sulphate model of colitis that resembles ulcerative colitis.

The aim of the present study was to evaluate the inmunomodulatory effects of UR-1505, a new salicylate derivative, on the T helper (Th)2/humoral response produced during dextran sodium sulfate (DSS)-induced rat colitis. In the in vitro studies, UR-1505 (300 microM) inhibited both the production of interleukin (IL)-10 and IL-5 in concanavalin A (Con A)-activated splenocytes and the production of immunoglobulin (Ig) G and IgA by B-lymphocytes. However, in contrast to the in vitro results, the administration of UR-1505 (10 and 30 mg/kg per day) to rats with established DSS-colitis enhanced both IL-10 and IgA production, whereas it inhibited IgG production, thus ameliorating the intestinal inflammation.

UR-1505, a salicylate able to selectively block T-cell activation, shows intestinal anti-inflammatory activity in the chronic phase of the DSS model of rat colitis.

BACKGROUND: UR-1505 is a novel salicylate derivative compound that has been demonstrated to selectively down-regulate T-cell activation. The aim of the present study was to elucidate the mechanisms involved in the intestinal anti-inflammatory effects of UR-1505 in 2 protocols of a dextran sodium sulfate (DSS) model of rat colitis: acute and established colitis. METHODS: The first protocol consisted of incorporating DSS into the drinking water at a concentration of 5% (w/v) for 5 days (acute initial colitis). In the second protocol, once the acute colitis had been induced, the concentration of DSS was reduced to 2% (w/v) and maintained for 10 days (established colitis). RESULTS: The results obtained demonstrated that although UR-1505 did not exert a significant intestinal anti-inflammatory effect in ameliorating the initial steps of the intestinal inflammation induced by DSS, it had a beneficial effect on ongoing inflammation, most probably through inhibiting activation of T lymphocytes, thus avoiding perpetuation of the inflammatory process. CONCLUSIONS: These results suggest that this compound is a good candidate for inducing remission or maintaining therapies in human inflammatory bowel disease (IBD). Moreover, the different results obtained by UR-1505 in these 2 protocols of colitis induction (acute initial colitis versus established colitis) confirm the importance of selection and optimization of the experimental model to evaluate the drugs to be used in IBD therapy.

UR-1505, a new salicylate, blocks T cell activation through nuclear factor of activated T cells.

2-Hydroxy-4(-2,2,3,3,3-pentafluoropropoxy)-benzoic acid (UR-1505), a new molecule chemically related to salicylic acid, has immunomodulator properties and is currently under clinical development for treatment of atopic dermatitis. The present work describes the immunomodulatory profile of UR-1505. UR-1505 targets T cells, inhibiting their proliferation and cytokine production by blocking nuclear factor of activated T cells (NF-AT) DNA-binding activity. The effects of UR-1505 (100-300 microM) on T cell proliferation seems to be dependent on the stimulus, because UR-1505 inhibited CD3/CD28-induced T-cell proliferation, increased p27(KIP) levels, and induced G1/S cell arrest but, interestingly, did not inhibit the Janus tyrosine kinase/signal transducer and activator of transcription-induced T-cell proliferation. These data suggest that UR-1505 acts by means of a specific mechanism inhibiting T cell activation depending on T cell receptor signaling pathway. Furthermore, the antiproliferative effects of UR-1505 are not a consequence of decreased cell viability. In addition to the inhibition of T-cell proliferation, UR-1505 decreased, in a dose-dependent manner, the production of interleukin (IL)-5 and interferon (IFN)-gamma in activated T cells, and this effect was produced at the transcriptional level. Because T-cell proliferation and cytokine production were regulated through NF-AT, we examined the effect of UR-1505 on this transcription factor. According to its effect on IL-5 and IFN-gamma mRNA expression, UR-1505 specifically inhibited NF-AT DNA binding without effect on nuclear factor-kappaB and activator protein-1 activities. The effect of UR-1505 on NF-AT is not attributable to a blockade of nuclear import. In conclusion, UR-1505 is a new immunomodulator agent that specifically inhibits NF-AT activation. Because NF-AT regulates the transcription of most genes involved in lymphocyte activation, its selective inactivation results in both decreased T-cell proliferation and cytokine production.