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The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.

BACKGROUND AND PURPOSE: Chronic spontaneous urticaria presents as a heterogeneous syndrome characterised by wheals, angioedema, or both for greater than 6 weeks. Spleen tyrosine kinase mediates allergen‐induced mast cell degranulation via the IgE signalling pathway, a central component of wheal formation and inflammation. In this study, we investigated the effects of perfused or topically administered GSK2646264 on IgE‐mediated histamine release from mast cells in an ex vivo human skin model. EXPERIMENTAL APPROACH: Using a novel SkiP device, ex vivo human skin from mastectomy surgeries was challenged with anti‐IgE, complement 5a (C5a), and buffer to induce histamine release from skin mast cells. Histamine was collected via microdialysis fibres and measured fluorometrically. GSK2646264 was delivered via perfusion either using microdialysis fibres or topically in a cream. Drug concentrations in the skin were measured by LC–MS, and a pharmacokinetic/ pharmacodynamic (PK/PD) relationship developed. KEY RESULTS: Perfused GSK2646264 significantly inhibited anti‐IgE (but not C5a)‐induced histamine release in a concentration‐dependent manner. The 0.5, 1, and 3% cream delivered GSK2646264 to the dermis above the IC(90) and dose‐dependently attenuated anti‐IgE‐induced histamine release. CONCLUSIONS AND IMPLICATIONS: GSK2646264 administered topically or direct to the dermis blocked histamine release from in situ skin mast cells. A PK/PD relationship curve suggests that dermal concentrations above 6.8 μM should lead to approximately 90% inhibition of histamine release from skin mast cells following activation of the Fc fragment of IgE receptor 1a, implicating a potential use for the compound in skin mast cell diseases such as urticaria.

OBJECTIVE: Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. DESIGN: In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. RESULTS: In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4±0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2±0.7; GSK143 (1 mg/kg): 7.6±0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0-90 μM) and trinitrophenyl (0-4 μg/ml) induced a concentration-dependent release of β-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03-10 μM) concentration dependently blocked substance P and trinitrophenyl induced β-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. CONCLUSIONS: The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI. ispartof: Gut vol:62 issue:11 pages:1581-90 ispartof: location:England status: published

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.

The coupling of the techniques, high-performance liquid chromatography (HPLC), orthogonal acceleration time-of-flight mass spectrometry (OATOF-MS) and inductively coupled plasma mass spectrometry (ICP-MS) provides a very powerful method for identifying and quantifying the products of bradykinin metabolism. In this study, we were able to identify the major metabolites of bradykinin degradation reported in the literature. In addition, a new bradykinin metabolite corresponding to bradykinin 5,9 fragment (BK-(5,9)-fragment) was identified as a product of neutral endopeptidase (NEP) activity. This finding establishes that NEP cleaves bradykinin simultaneously at the positions 4–5 and 7–8. We also demonstrate the equivalent participation of NEP and angiotensin-converting enzyme (ACE) within the rat lung tissue membranes (RLTM) in bradykinin degradation, suggesting its suitability as a model for the assay of dual ACE/NEP inhibitors. On the contrary, in rat kidney brush border membranes (KBBM), ACE is not significantly involved in bradykinin metabolism, with NEP being the major enzyme. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

Replying to B. Heinemann . 10.1038/nature13688 (2014) We welcome the accompanying Comment1 by Heinemann , in which the authors use an extensive panel of sensitive KDM assays to independently confirm our results2 that GSK-J1 is a potent KDM6 inhibitor. Additionally, Heinemann et al.1 demonstrate that GSK-J1 has some, albeit weaker, activity towards KDM5B and KDM5C, for which we only had preliminary data available at the time of our original publication. As our jumonji assay portfolio expands, we have continued to update the GSK-J1 activity profile on the SGC website ( http://www.thesgc.org/chemical-probes/GSKJ1 ); this includes KDM5 inhibition activity by GSK-J1 similar to that reported by Heinemann. In conclusion, GSK-J1 remains the most selective KDM inhibitor yet disclosed and thus a valuable chemical tool.

Background: Recent advances underline intestinal inflammation, induced by handling of the gut during surgery, as crucial mechanism in the pathophysiology of postoperative ileus (POI). Macrophages, in the muscularis layer, and mast cells are the key players in the induction of this inflammatory process. Spleen tyrosine kinase (Syk) is an important kinase involved in macrophage activation as well as in mast cell degranulation and therefore inhibition of Syk pathway may represent an interesting therapeutic approach for POI. In the current study, we have evaluated the effect of the Syk-inhibitor GSK143 as potential treatment to shorten POI. Methods: The effect of an oral single dose (3mg/kg, 1.5 hours before surgery) was evaluated in a mouse model of POI, by analyzing gastrointestinal transit and intestinal muscularis inflammation. The in vitro effect of GSK143 (1 and 3 μM) was evaluated on cultured peritoneal mast cells (pMCs) and bone marrow derived macrophages (BMDMs) stimulated with immune complexes with IgE-anti-TNP (40 ng/mL) and LPS (100 ng/ml) respectively. Results: Administration of GSK143 (3 mg/kg) resulted in a serum level of 0.38±0.24 μM 1.5 hours after oral delivery. Treatment with a single dose of GSK143 (3 mg/kg) significantly improved gastrointestinal transit after surgical intestinal manipulated mice compared to placebo (Geometric Center (GC): Placebo, 4.2 ±0.4 vs GSK143, GC; 6.9±0.6). In addition, GSK143 was able to reduce the number of recruited myeloperoxidase (MPO) positive cells (placebo, 187±30 vs GSK143, 44±8, per 0.5mm2), neutrophils (placebo, 4.6x10-5±0.2 x10-5 vs GSK143, 1.7x10-5 ±0.7 x10-5) and monocytes (placebo, 1.6x106±0.4 x10-6 vs GSK143, 0.8x10-6 ±0.25 x10-6) in the muscularis externa compared to placebo. To define the possible target cells affected, GSK143 was tested in vitro on pMCs and BMDMs. Thirty minutes after GSK143 (1 and 3 μM) or vehicle treatment, pMCs degranulation, quantified by assessment of β-hexosaminidase, was induced by immune complexes with IgE-anti-TNP. Interestingly, GSK143 significantly reduced pMCs degranulation compared to vehicle in a dose dependent manner (GSK143 1μM, 62±7.5% and GSK143 3μM, 76±3.3% inhibition vs vehicle). In line, pre-treatment of BMDMs with GSK143 (1 and 3 μM) prior to LPS challenge significantly lowered the expression of IL-6 (21±4% to 31± 3 % inhibition vs vehicle ), TNFα (39±0.8% to 54±0.7% inhibition vs vehicle ), IL1 β (31±1.3% to 39±1.06% inhibition vs vehicle) and CCL2 (33±5.3% to 43±7% inhibition vs vehicle). Conclusion: Pre-treatment with the Syk-inhibitor GSK143 attenuates intestinal inflammation and consequently restores gastrointestinal transit in a model of POI. In vitro data suggests that both macrophages and mast cells are targeted by GSK143. These findings strongly suggest that Syk inhibition may be a new therapeutical tool to shorten POI.