Methods and compounds for the treatment or prevention of hypercytokinemia and severe influenza

Abstract

The invention provides a p38 MAPK inhibitor of Formula I, or a pharmaceutically acceptable salt or solvate thereof: Formula I for use in the treatment or prevention of hypercytokinemia in a human patient; wherein R is C.sub.1-3alkyl, optionally substituted by one or more halo, NR.sup.1R.sup.2 or hydroxy, and R.sup.1 and R.sup.2 are independently H, halo or C.sub.1-3alkyl, optionally substituted by one or more F. Also provided are compositions for use in the treatment or prevention of hypercytokinemia comprising the p38 MAPK inhibitor of Formula I; and methods for treating or preventing hypercytokinemia in a human patient in need thereof comprising administering to the patient a therapeutically or prophylactically effective amount of a p38 MAPK inhibitor of Formula I. The invention also provides a p38 MAPK inhibitor and an antimicrobial agent, such as an antiviral agent, for use in the treatment or prevention of hypercytokinemia. ##STR00001##

Claims

1. A method of treating hypercytokinemia in a human or animal patient in need thereof comprising administering to the patient a therapeutically effective amount of a p38 MAP kinase inhibitor of Formula I, or a pharmaceutically acceptable salt or solvate thereof: ##STR00015## wherein R is C.sub.1-3 alkyl, optionally substituted by one or more halo, NR.sup.1R.sup.2 or hydroxy, and R.sup.1 and R.sup.2 are independently H, halo or C.sub.1-3alkyl, optionally substituted by one or more F.

2. The method of claim 1, wherein the p38 MAP kinase inhibitor is of Formula II, or a pharmaceutically acceptable salt or solvate thereof: ##STR00016##

3. The method of claim 1, wherein the p38 MAP kinase inhibitor is of Formula III, or a pharmaceutically acceptable salt or solvate thereof: ##STR00017##

4. The method of claim 1, wherein the hypercytokinemia occurs due to one or more of severe influenza virus infection; graft-versus-host disease (GVHD); acute respiratory distress syndrome (ARDS); sepsis; Ebola; smallpox; systemic inflammatory response syndrome (SIRS); bacterial infection; and cancer.

5. The method of claim 1, wherein the hypercytokinemia occurs due to severe influenza virus infection.

6. The method of claim 1, further comprising administering an antimicrobial agent.

7. The method of claim 6, wherein the antimicrobial agent is an antiviral agent.

8. The method of claim 7, wherein the antiviral agent is oseltamivir or a pharmaceutically acceptable salt thereof.

9. The method of claim 1, further comprising administering an anticancer agent.

10. The method of claim 1, wherein the p38 MAP kinase inhibitor is administered orally.

11. The method of claim 1, wherein the hypercytokinemia occurs due to exposure to a pathogen.

12. The method of claim 11, wherein the hypercytokinemia occurs due to a viral infection.

13. The method of claim 12, wherein the immune response in the patient is triggered by a respiratory viral infection.

14. The method of claim 13, wherein the hypercytokinemia occurs due to an influenza virus infection.

15. The method of claim 1, wherein the hypercytokinemia is triggered by cancer.

16. The method of claim 1, wherein the hypercytokinemia is triggered by an autoimmune response.

17. The method of claim 14, wherein the hypercytokinemia occurs due to a viral infection.

18. The method of claim 17, wherein the immune response in the patient is triggered by a respiratory viral infection.

19. The method of claim 18, wherein the hypercytokinemia occurs due to an influenza virus infection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates the identification of signalling pathways.

(2) FIG. 2 illustrates the mapping the activity of genes in IL-1, TNFα and IL-6 stimulated pathways. These cytokines are produced by influenza virus infected cells and found to be increased in the blood of people infected with influenza, particularly those hospitalised with severe influenza. The hatched lines indicate gene expression levels with lines sloping from top right to bottom left indicating upregulation (e.g. TNF-α), and lines sloping from top left to bottom right (e.g. LBP) indicating down-regulation; genes that are both upregulated and down-regulated are indicated by cross-hatching. The line spacing represents the intensity of up- or down-regulation, with more densely packed lines indicating greater activity. The maps were generated using IPA. A “route” through a pathway is defined as a single contiguous connection of proteins that extend from the plasma membrane through to the nucleus.

(3) FIG. 3 shows an example of scoring a route through the IL-6 pathway of FIG. 2.

(4) FIG. 4 shows three key cell types involved in the pathology of severe influenza.

(5) FIG. 5 is a schematic of influenza ‘soup’ production and electrogenerated chemiluminescence analysis in human epithelial cells. Infected cell “soups” contain key cytokines found in clinical samples.

(6) FIG. 6 shows Western blotting of phosphorylated P38 and HSP27 in response to viral soup application and effects of P38 inhibition in epithelial cells using the p38 MAPK inhibitor PH 797804. Electrogenerated chemiluminescence analysis of inflammatory cytokine production in response to viral soup application, and effects of P38 inhibition in epithelial cells.

(7) FIG. 7 shows Western blotting of phosphorylated HSP27 in response to viral soup application and effects of P38 inhibition in endothelial cells. Electrogenerated chemiluminescence analysis of inflammatory cytokine production in response to viral soup application, and effects of P38 inhibition in endothelial cells.

(8) FIG. 8 shows Western blotting of phosphorylated HSP27 in response to viral soup application and effects of P38 inhibition in immune cells. Electrogenerated chemiluminescence analysis of inflammatory cytokine production in response to viral soup application, and effects of P38 inhibition in immune cells. Cell viability of immune cells in response to increasing concentrations of P38 MAPK inhibitor.

(9) FIG. 9 shows electrogenerated chemiluminescence analysis of inflammatory cytokine production in response to LPS, CD-3 and viral soup application. Effects of P38 inhibition on viral soup induced inflammatory cytokine in immune cells.

(10) FIG. 10 shows the effects of p38 and MEK inhibitors on IP10 production by HUVECs stimulated with 70% HBEC viral soup. Secreted cytokine levels were assayed by electrogenerated chemiluminescence. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison post hoc test.

(11) FIG. 11 shows compound effects on IL-1b production from immune cells. Each point on the dot plot represents an individual experiment. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison post hoc test.

(12) FIG. 12. shows compound effects on TNFa production from immune cells. Each point on the dot plot represents an individual experiment. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison post hoc test.

(13) FIG. 13 shows compound effects on IP10 production from endothelial cells. Each point on the dot plot represents an individual experiment. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison post hoc test.

(14) FIG. 14 shows compound effects on IL8 production from endothelial cells. Each point on the dot plot represents an individual experiment. Statistical significance was calculated using one-way ANOVA with Dunnett's multiple comparison post hoc test.

(15) FIG. 15 shows the cytokine levels in 28 healthy volunteers infected with influenza virus (samples collected at Day −1 to Day 28, hollow dots) and 30 individuals hospitalised with severe influenza (stippled dots). Blood samples from the severe patients were collected between 24 and 72 hours after admission to hospital. The statistical significance of differences in cytokine levels between the infected healthy volunteers (D2 and D3) and the hospitalised patients (influenza A or B) was calculated using the Mann-Whitney U test.

(16) FIG. 16 shows the effect of HBEC viral soup on inflammatory mediator production in HBEC cells measured by MSD analysis and the inhibitory effect of the compound of Formula III.

(17) FIG. 17 shows inflammatory cytokine production by HUVEC cells treated with either TNFa plus IL-6, or HBEC viral soup as measured by MSD analysis in endothelial cells and the inhibitory effect of the compound of Formula III.

(18) FIG. 18 shows inflammatory cytokine production in immune cells treated with either TNFa plus IL-6, or A549 viral soup as measured by MSD analysis in immune cells and the inhibitory effect of the compound of Formula III.

(19) FIG. 19 shows the effect of combining p38 MAPK inhibitors with oseltamivir.

(20) FIG. 20 shows inflammatory cytokine production by HUVEC cells treated with either TNFa plus IL-6, or HBEC viral soup as measured by MSD analysis in endothelial cells and the inhibitory effect of the compound of Formula II.

(21) FIG. 21 shows inflammatory cytokine production in immune cells treated with either TNFa plus IL-6, or A549 viral soup as measured by MSD analysis in immune cells and the inhibitory effect of the compound of Formula II.

(22) FIG. 22 shows the effect of combining the compound of Formula II with oseltamivir on the anti-viral properties of oseltamivir.

(23) FIG. 23 shows the effect of combining oseltamivir the compound of Formula II on the anti-inflammatory properties of the compound of Formula II.

EXAMPLES

Example 1

Identification of p38 MAPK by Transcriptomic Analysis

(24) Bioinformatics analysis of transcriptomic data from blood samples collected from human volunteers and patients infected with influenza was used to map the signalling pathways activated in the human host response to influenza infection in both uncomplicated (mild and moderate) and severe influenza (see PHE Guidance on Use of Anti-Viral Agents for the Treatment and Prophylaxis of Seasonal Influenza (2015-16), version 6.0, September 2015). Human viral challenge studies were carried out and transcriptomic data from those studies were used for mapping the former, while transcriptomic data from a field-based sampling study (Hoang, L. T. et al., 2014) were used for mapping the latter. Comparison of signalling pathways identified by comparing both datasets enabled the identification of signalling pathways that are very active in severe influenza versus mild and moderate influenza. Further analysis of individual pathway components identified p38 MAPK as a key “node” in a number of these active pathways.

(25) Healthy human volunteers were intranasally challenged with influenza A/Wisconsin/67/2005 (H3N2) (Zaas, A. K. et al., Gene expression signatures diagnose influenza and other symptomatic respiratory viral infection in humans, Cell Host Microbe, 2009; 17: 207-217 and Davenport, E. E. et al., Transcriptomic profiling facilitates classification of response to influenza challenge, J. Mol. Med., 2015; 93: 105-114) or with influenza A/Perth/16/2009 (H3N2) (internal study, not published). PAXgene™ samples of whole blood were collected from the volunteers at various time points for subsequent transcriptome analysis. Methods for influenza A/Wisconsin/67/2005 (H3N2) viral challenge, case definitions, sample collection, RNA purification and microarray analysis are as detailed in Zaas et al., 2009 and Davenport et al., 2015. Methods for influenza A/Perth/16/2009 (H3N2) viral challenge, case definitions and sample collection were as described for the Wisconsin strain except RNA purification and microarray analysis using Affymetrix HGU133 Plus 2.0 arrays were performed by Almac (https://web.archive.org/web/20160317153848/http://www.almacgroup.com/). Methods for recruiting patients with severe influenza, blood sample collection, RNA purification and microarray analysis are as detailed in Hoang et al., 2014.

(26) Microarray data files for the Zaas et al., 2009, Davenport et al., 2015 and Hoang et al., 2014 studies were downloaded from the Gene Expression Omnibus (GEO) database (https://web.archive.org/web/20160622040853/http://www.ncbi.nlm.nih.gov/geo/) using the accession numbers GSE52428, GSE61754 and GSE61821, respectively. Microarray data (.CEL) files for the unpublished study were downloaded from Almac and stored locally for bioinformatics analysis. All four transcriptomic datasets were processed and analysed using the R (version 3.0.2.) integrated suite of software facilities for data manipulation, calculation and graphical display (https://web.archive.org/web/20160623011408/http://www.R-project.org). Quality assessment of raw microarray data was performed using statistical methods standard in the art (e.g. Heber, S. and Sick, B., Quality assessment of affymetrix genechip data, Omics, 2006; 10: 358-368). Affymetrix datasets were normalised using the Robust Multi-array Average (RMA) method [https://www.bioconductor.org/packages/3.3/bioc/manuals/affy/man/affy.pdf] and Illumina datasets were normalised using the Lumi package [https://www.bioconductor.org/packages/3.3/bioc/manuals/lumi/man/lumi.pdf]. Both packages were executed in the R environment. To facilitate annotation of probe-sets and gene names, Affymetrix chip definition files (version 17.1.0) were downloaded from the BrainArray website (https://web.archive.org/web/2016062311275 820160623112758/http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/17.1.0/ensg.asp) and Illumina chip definition files (illuminaHumanv4.db) were downloaded from the Bioconductor website (https://web.archive.org/web/20151209032754/http://bioconductor.org/packages/release/dat a/annotation/html/illuminaHumanv4.db.html).

(27) The latter files were used with microarray data from Davenport et al., 2015 and Hoang et al, 2014.

(28) Normalised Zaas et al., 2009, Davenport et al., 2015 and Perth datasets were individually merged with the Hoang et al., 2014 dataset using the COMBAT module in the InSilicoMerging package in Bioconductor (https://web.archive.org/web/20150905151657/http://www.bioconductor.org/packages/relea se/bioc/html/inSilicoMerging.html). Differential gene expression analysis on merged data sets was carried out using the limma package in R (https://www.bioconductor.org/packages/3.3/bioc/vignettes/limma/inst/doc/usersguide.pdf). For pairwise comparisons only data from infected volunteers in the Zaas et al., 2009, Davenport et al., 2015 and Perth datasets were used, equating to 11, 14 and 5 subjects, respectively. From the Hoang et al., 2014 dataset only data for the three H3N2-infected severe influenza patients in the data set were used. For each merged data set two pairwise comparisons were performed to identify genes that were upregulated relative to baseline levels after infection with virus and then further upregulated in the severe patient samples:

(29) Perth: day −1 vs day 3 and; day 3 vs Hoang et al., 2014 severe.

(30) Zaas et al., 2009: day −1 vs 60 hours and; 60 hours vs Hoang et al., 2014 severe.

(31) Davenport et al., 2015: day 0 vs 48 hours and; 48 hours vs Hoang et al., 2014 severe.

(32) To maximise the number of upregulated genes that could be mapped to pathways, all genes showing fold-changes>0 were identified. Each of the 6 resulting gene lists were analysed through the use of QIAGEN's Ingenuity® Pathway Analysis (IPA®, QIAGEN Redwood City, https://web.archive.org/web/20131021061639/http://www.ingenuity.com/). This resulted in the identification of 650 signalling pathways which were reduced to 353 after the removal of 297 metabolic pathways. FIG. 1 summarises this process.

(33) In order to interrogate the relevance of each of these signalling pathways to the pathogenesis of severe influenza, a manual scoring approach was devised to identify very active “routes” within these pathways in the complicated versus the “uncomplicated” influenza datasets. In this context “routes” are defined as contiguous connections of proteins in a canonical pathway that extend from the plasma membrane through to the nucleus. As a result, a canonical pathway may have a number of different routes through it. Using this scoring approach, routes within IPA canonical pathways were mapped directionally from the plasma membrane to the nucleus and the ‘overlay’ function in IPA was used to show gene activity. To illustrate this process an example of three pathways identified using this method is shown in FIG. 2.

(34) Individual routes in the identified 353 pathways were manually scored for gene activity as exemplified in FIG. 3 for a route through the IL-1 canonical pathway in which:

(35) TABLE-US-00001 TABLE 1 Nodes Nodes not Pathway Route Up-regulated up-regulated IL-6 IL6R + GP130- 81% SHC-Ras signalling SHC-GRB2-SOS- Ras-cRAF-MEK- ERK-ELK + SRF

(36) In all, 491 routes showing >75% up-regulated nodes were identified (exemplified in Table 2 below). Of these, 95 routes containing >3 nodes were identified in which 100% of all the nodes in the route were upregulated in the Hoang et al., 2014 severe influenza dataset versus baseline (D-1 or D0) in the Zaas et al., 2009, Davenport et al., 2015 and Perth datasets (Table 3). Twenty-four of these 95 routes were shown to be >75% upregulated compared with pathways derived from the mild and moderate H3N2 and H1N1 influenza datasets from Hoang et al., 2014 (H3N2 and H1N1—mild and moderate) and Zaas et al., 2009 (H1N1-D-1 and 60 h; Table 4).

(37) Inspection of the 95 routes highlighted a number of potentially targetable nodes from which p38MAPK was chosen because of its well characterised role in inflammation and the availability of high quality clinically tested small molecule inhibitors for use in in vitro and ex vivo studies.

(38) TABLE-US-00002 TABLE 2 An example of route scoring analysis. Nodes↑ Pathway Route Nodes (%) ↓Nodes NFKB growth factor receptors-RAS- 10 92 IKKB RAF-MEKK1-IKKa-NFKB2- RELB-lymphogenesis NFKB IL-1R/TLR-MYD88-TYRAP- 8 93 IKKB IRAP-IRAK-TRAF6-TAK1- IKKa-IKBP65-P65NFKB- P65NFKB-inflammation Role of IL21Ralpha/IL2Rgamma-JAK3- 4 100 JAK1 and STAT1/3/5 JAK3 in cytokine signalling PI3K-AKT Integrin-PINCH-ILK-PI3K-PP2A- 9 89 signalling AKT-CRAF-MEK1/2-ERK1/2- RTK P70S6K-cell growth PI3K-AKT Integrin-PINCH-ILK-PI3K-PP2A- 9 89 signalling AKT-CRAF-MEK1/2-ERK1/2- RTK P70S6K-cell growth PI3K-AKT Integrin-PINCH-ILK-PI3K-PP2A- 9 78 signalling AKT-CRAF-MEK1/2-ERK1/2- integrin P70S6K-cell growth NFKB TNF-TANK-TRAF-FADD-RIP- 7 87.5 TNF- MAP3K3-IKKa-IKBP65- R/ P65NFKB-P65NFKB- IKKB inflammation CNTF (CNTFR-LIFR-GP130)-JAK1/2- 10 90 SHP2 signalling SHP2-GRB2-SOS-RAS-CRAF- MEK1/2-ERK1/2-P( )RSK-gene expression CNTF (CNTFR-LIFR-GP130)-TYK2- 3 100 signalling STAT1/3-gene expression role of JAK (GP130-OSMR)-intermediate 4 100 in IL-6 type signalling-ERK1/2-p38MAPK- cytokine JNK-signalling signalling role of JAK (GP130-OSMR)-JAK2- 3 100 in IL-6 type STAT1/3/5-gene expression cytokine signalling role of JAK (GP130-OSMR)-STAT1/3-gene 2 100 in IL-6 type expression cytokine signalling HER-2 (HER1/HER2)-GRB2-SOS-RAS- 5 80 HER1- signalling (CYCLIND1-CDK6-CYCLINE- HER2 in breast p27KIP1)-cell cycle progression cancer and proliferation HER-2 (HER1/HER2)-PI3K-AKT- 4 75 HER1- signalling CYCLIND1-cell cycle progression HER2 in breast cancer role of (IFNAR1-IFNAR2)-TYK2- 4 100 JAk1, STAT2-STAT1-gene expression JAK2 and TYK2 in interferon signalling IL-9 (IL-9R-IL2R)-JAK3-IRS1/2-PI3K- 5 100 signalling PI3ksignalling

(39) TABLE-US-00003 TABLE 3 Ninety-five routes containing 100% upregulated nodes in the Hoang et al., 2014 severe influenza dataset versus the Zaas et al., 2009, Davenport et al., 2015 an dPerth baseline datasets. Number Pathway Route of Nodes Acute myeloid FLT3-GRB2-SOS-RAS-RAF-MEK- 7 leukemia ERK1/2-cell proliferation signalling Gaq signalling GqR-Ga/b/y-PYK2-PI3K-AKT-IKK- 7 NFkB p38 MAPK TNFR/fas-TRADD/FAD-TRAF2- 7 signalling Ask1-MKK4-P38MAKa-CHOP- transcription p38 MAPK TNFR/fas-TRADD/FAD-TRAF2- 7 signalling Ask1-MKK4-P38MAKa-ELK1- transcription p38 MAPK TNFR/fas-TRADD/FAD-TRAF2- 7 signalling Ask1-MKK4-P38MAPKa-MEF2 SAPK/JNK TRADD/RIP/FADD-TRAF2-GCKs- 7 signalling MEKK1-MKK4/7-JNK-ELK-1 Sertoli cell sertoli CLDN-ZO2-factin-actinin alpha- 7 cell junction tubulin-KEAP1-Myo7a-junction signalling dynamics HIF1a signalling RTK-PI3K-AKT-HIF1a-ARNT-ET1- 6 vascular tone HIF1a signalling RTK-PI3K-AKT-HIF1a-ARNT- 6 MMPs-ECM regulation IL6 signalling TNFR-TRAF2-TAK1-MKK4/7-JNK- 6 ELK1 Protein Kinase A PKAr/PKAc-RAP1-BRAF-MEK1/2- 6 signalling ERK1/2-ELK1 SAPK/JNK TRADD/RIP/FADD-TRAF2-ASK1- 6 signalling MKK4/7-JNK-ELK1 ERK5 signalling SRC-MEKK2/3-MEK5-ERK5-SAP1 5 Glucocorticoid CYTOKINE RECEPTOR-TRAF2- 5 Receptor TAK1-MKK4/7-P38MAPK- signalling STABILIZATION OF MRNA, TRANSLATION Growth Hormone GHR-JAK2-ERK1/2-CEBPA 5 signalling Growth Hormone GHR-JAK2-ERK1/2-P90RSK- 5 signalling SRF/ELK1 HIF1a signalling RTK-PI3K-AKT-HIF1a-ARNT- 5 GLUT HIF1a signalling RTK-PI3K-AKT-HIF1a-ARNT- 5 VEGF IL-22 signalling IL22R1/2-TYK2-STAT1/3/5-SOCS3 5 IL-8 CXCR1/2-PI3K-Akt-AP1- 5 IntegrinAlphavBeta3 (Chemotaxis) IL-8 CXCR1/2-Ras-Raf-MEK1/2-ERK1/2- 5 (Neutrophil Degranulation) leptin signalling in LEPR-JAK2-STAT3-(SOCS3- 5 obesity POMC)-aMSH-anorexia Paxillin signalling Integrina/b-FAK-GRB2-SOS-Ras- 5 ERK/MAPK Role of RIG like dsRNA-RIG1-IPS1-TRAF3-TBK1- 5 receptors in IRF7-(IFNa-MDA5/LGP2/RIG1) antiviral innate immunity Role of RIG like MDA5-IPS1-TRAF3-TBK1-IRF7- 5 receptors in (IFNa-MDA5/LGP2/RIG1) antiviral innate immunity Role of RIG like TRIM25-RIG1-IPS1-TRAF3-IRF7- 5 receptors in (IFNa-MDA5/LGP2/RIG1) antiviral innate immunity CD40 signalling CD40-JAK3-STAT3-ICAM1 4 ceramide signalling EDG-SPHK-NFKB-AP1-activation of 4 inflammatory genes ceramide signalling SMPD-(ceramide)-PI3K-AKT- 4 apoptosis Eicosanoid PLA2-ALOX5-LTA4h-LTB4R- 4 signalling chemotaxis/proliferation/allergic asthma/angiogenesis/ G alpha I signalling GiCOUPLED RECEPTOR- 4 Galphai/Gbeta/Ggamma-SRC-STAT3 Germ Cell-Sertoli TGFbetaR-RAS-MEK1/2-ERK1/2- 4 Cell actin Junction signalling depolymerisation GM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 4 survival/cell proliferation GM-CSF signalling GMCSFRA-JAK2-STAT3-(BCLXL- 4 CYCLIND1) G-Protein Coupled Gicoupled receptor-GALPHAi/0- 4 Receptor SRC-STAT3 signalling IGF-1 signalling IGF1R-JAK 1/2-STAT3-SOCS3 4 IL-8 CXCR1/2-JNK-NFkB-ICAM-1 4 IL-8 CXCR1/2-PI3K-MEK1/2-ERK1/2- 4 (Neutrophil Degranulation) IL-8 CXCR1/2-Rho-NFkB-ICAM-1 4 JAK/STAT cytokine receptor-JAK-STAT-(CFOS- 4 IL6-SOCS-BCLXL) MSP-RON RON-PI3K-PKC zeta-F-ACTIN- 4 signalling phagocytic activity in macrophages pathway PI3K signalling in IL4R-IRS-P85/PI3K-P110/PI3K- 4 B Lymphocytes NFKB PPARα/RXRα ADIPOR-AMPK-P38MAPK- 4 Activation PPARalpha Production of nitric TLR2/4-PI3K-AKT-NFKB-Inos 4 oxide and ROS in macrophages Production of nitric TLR2/4-MKK4/-JNK-AP1 4 oxide and ROS in macrophages RAR activation IL-3Ra/b-JAK2-STAT5-RAR/RXR 4 Role of MAPK ASK-1-MKK4/7-JNK-CASP3- 4 signalling in the APOPTOSIS Pathogenesis of Influenza Role of RIG like dsRNA-RIG1-IPS1-TRAF3-IRF7- 4 receptors in (IFNa-MDA5/LGP2/RIG1) antiviral innate immunity Role of RIG like MDA5-IPS1-TRAF3-IRF7-(IFNa- 4 receptors in MDA5/LGP2/RIG1) antiviral innate immunity signalling by Rho Integrin-ARHGEF-RHO-FAK- 4 Family GTPases cytoskeletal reorganisation signalling by Rho Integrin-ARHGEF-RHO-PKNI-cell 4 Family GTPases trafficking Sphingosine-1- SIPR(2/3/4)-GAI-PI3K-AKT-CELL 4 phosphate SURVIVAL signalling Tec Kinase Integrin-FAK-TEC KINASE- 4 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1) Tec Kinase TCR-SRC-TEC KINASE- 4 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1) Acute myeloid FLT3-STAT3/5-PIM1-regulates 3 leukemia signalling apoptosis Antioxidant action CSF2Ralpha/beta-JAK2-STAT5-gene 3 of vitamin C expression CNTF signalling (CNTFR-LIFR-GP130)-TYK2- 3 STAT1/3-gene expression Dendritic Cell LTbetaR-IKK-RELB/NFKB-cross 3 Maturation presentation EPHRIN EPHA-JAK2-STAT3-CELL 3 RECEPTOR PROLIFERATION signalling EPHRIN EPHB-PI3KG-AKT-CELL 3 RECEPTOR MIGRATION, CELL signalling PROLIFERATION EPHRIN INTEGRIN-MEK1/2-ERK1/2-AXON 3 RECEPTOR GUIDANCE, CELL signalling PROLIFERATION FcyRIIB signalling FCyR-BTK-JNK-apoptosis 3 in B lymphocytes Glucocorticoid CYTOKINE RECEPTOR-JAK2- 3 Receptor signalling STAT1 Glucocorticoid CYTOKINE RECEPTOR-JAK3- 3 Receptor signalling STAT3/5 GNRH signalling GnRHR-Gai-NfkB 3 IL-12 signalling TLR4-p38/MAPK-IL12 3 and Production in Macrophages IL-3 signalling IL3Ralpha/beta-JAK1/2- 3 STAT1/3/5/6-gene expression IL6 signalling GP130 (IL6R)-JAK2-STAT3-gene 3 expression IL-8 CXCR1-PLD-NADPH oxidase- 3 (Superoxide production—Respiratory Burst) IL-8 CXCR1-G Protein alpha/beta/gamma- 3 PI3Ky-(Chemotaxis—Respiratory Burst) LPS stimulated TLR4-IKK-IKB-NFKB-gene 3 MAPK signalling expression mTOR signalling Nutrients-RHEB-mTORc2-AKT- 3 PI3K/AKT signalling mTOR signalling Nutrients-RHEB-mTORc2-AKT- 3 Rho/PKC)-actin organisation PDGF signalling PDGFRa/b-SPHK-CRK-mitogenesis 3 Protein Kinase A PKA-PHK-PYG-glycolysis 3 signalling Regulation of CNG-CALPAIN-RB 3 cellular mechanics by calpain protease role of JAK in IL-6 (GP130-OSMR)-JAK2-STAT1/3/5- 3 type cytokine gene expression signalling Role of JAK2 in GHR-JAK2-IRS-PI3K/AKT 3 Hormone-like SIGNALLING cytokine signalling Role of JAK2 in GHR-JAK2-STAT1/3-GENE 3 Hormone-like EXPRESSION cytokine signalling Role of JAK2 in GHR-JAK2-STAT5-GENE 3 Hormone-like EXPRESSION cytokine signalling Role of GP130-JAK2-STAT3-gene 3 Macrophages, expression Fibroblasts and Endothelial Cells in Rheumatoid Arthritis Role of Pattern NALP3-casp1-IL1b 3 Recognition Receptors in Recognition of Bacteria and Viruses Role of Pattern NOD1-Casp1-IL1b 3 Recognition Receptors in Recognition of Bacteria and Viruses Role of PI3K/AKT PI3K-AKT-IKB, NFKB 3 signalling in the Pathogenesis of Influenza Role of tissue factor PAR2-ERK1/2-HBEGF-angiogenesis 3 in cancer Role of tissue factor PAR2-ERK1/2-VEGFa-angiogenesis 3 in cancer Role of tissue factor PAR2-p38/MAPK-uPar-tumour 3 in cancer invasion Role of tissue factor PAR2-p38/MAPK-IL-1b- 3 in cancer angiogenesis Role of tissue factor PAR2-p38/MAPK-VEGFa- 3 in cancer angiogenesis STAT3 pathway cytokine receptors-TYK2/JAK2- 3 STAT3-transcription-immune response-proliferation-survival STAT3 pathway GFR-JAK2/SRC-STAT3- 3 transcription-immune response- proliferation-survival Synaptic long term AMPAR-Lyn-PKC-Phosphorylation 3 depression Tec Kinase FCeR1-TEC kinase- 3 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1) Tec Kinase TLR4-TEC kinase- 3 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT- TFII-1)

(40) TABLE-US-00004 TABLE 4 Comparison of route scores between H3N2 and H1N1. H3N2 Severe vs Severe vs Severe vs Severe vs Baseline Peak Mild Moderate Pathway Route H3N2 H3N2 H3N2 H3N2 Growth Hormone GHR-JAK2-ERK1/2-CEBPA 100.00 93.33 100.00 100 signalling PPARα/RXRα ADIPOR-AMPK-P38MAPK- 100.00 100.00 100.00 100 Activation PPARalpha-REGULATION of growth hormone genes GM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 100.00 100.00 100.00 100 survival/cell proliferation Sphingosine-1- SIPR(2/3/4)-GAI-PI3K-AKT- 100.00 100.00 100.00 100 phosphate signalling CELL SURVIVAL ceramide signalling SMPD-(ceramide)-PI3K-AKT- 100.00 100.00 100.00 100 apoptosis IL-8 CXCR1/2 - PI3K-MEK1/2- 100.00 93.33 100.00 100 ERK1/2- (Neutrophil Degranulation) Paxillin signalling Integrina/b-FAK-GRB2-SOS - 100.00 100.00 100.00 100 Ras-ERK/MAPK Tec Kinase FCeR1-TEC kinase- 100.00 100.00 100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Tec Kinase TCR-SRC-TEC KINASE- 100.00 100.00 100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Tec Kinase TLR4-TEC kinase- 100.00 100.00 100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) signalling by Rho Integrin-ARHGEF-RHO-PKNI- 100.00 100.00 100.00 100 Family GTPases cell trafficking Regulation of CNG-CALPAIN-RB 100.00 100.00 100.00 100 cellular mechanics by calpain protease Tec Kinase Integrin-FAK-TEC KINASE- 100.00 100.00 100.00 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Acute myeloid FLT3-GRB2-SOS-RAS-RAF- 100.00 100.00 100.00 85.71 leukemia signalling MEK-ERK1/2-cell proliferation signalling by Rho Integrin-ARHGEF-RHO-FAK- 100.00 95.23 100.00 100 Family GTPases cytoskeletal reorganisation IL-8 CXCR1/2 - Ras-Raf-MEK1/2- 100.00 91.67 100.00 100 ERK1/2- (Neutrophil Degranulation) JAK/STAT cytokine receptor-JAK-STAT- 100.00 91.67 100.00 100 (CFOS-IL6-SOCS-BCLXL) MSP-RON signalling RON-PI3K-PKC zeta- F-ACTIN- 100.00 91.67 100.00 100 pathway phagocytic activity in macrophages Germ Cell-Sertoli TGFbetaR-RAS-MEK1/2- 100.00 91.67 100.00 100 Cell Junction ERK1/2-actin depolymerisation signalling Role of MAPK ASK-1-MKK4/7-JNK-CASP3 - 100.00 83.33 100.00 100 signalling in the APOPTOSIS Pathogenesis of Influenza Role of PI3K/AKT PI3K-AKT-IKB, NFKB 100.00 77.78 100.00 100 signalling in the Pathogenesis of Influenza Protein Kinase A PKAr/PKAc-RAP1-BRAF- 100.00 100.00 83.33 83.33 signalling MEK1/2-ERK1/2-ELK1 IL-8 CXCR1/2 - PI3K-Akt-AP1- 100.00 80.55 80.00 100 IntegrinAlphavBeta3 (Chemotaxis) Production of nitric TLR2/4-MKK4/7-JNK-AP1 100.00 91.67 75.00 100 oxide and ROS in macrophages H1N1 Severe vs Severe vs Severe vs Severe vs Baseline Peak Mild Moderate Pathway Route H1N1 H1N1 H1N1 H1N1 Growth Hormone GHR-JAK2-ERK1/2-CEBPA 75 75 75 75 signalling PPARα/RXRα ADIPOR-AMPK-P38MAPK- 100 100 100 75 Activation PPARalpha-REGULATION of growth hormone genes GM-CSF signalling GMCSFRA-HCK-PI3K-AKT-cell 100 100 100 100 survival/cell proliferation Sphingosine-1- SIPR(2/3/4)-GAI-PI3K-AKT- 100 100 100 100 phosphate signalling CELL SURVIVAL ceramide signalling SMPD-(ceramide)-PI3K-AKT- 100 100 100 100 apoptosis IL-8 CXCR1/2 - PI3K-MEK1/2- 75 75 100 100 ERK1/2- (Neutrophil Degranulation) Paxillin signalling Integrina/b-FAK-GRB2-SOS - 100 100 100 100 Ras-ERK/MAPK Tec Kinase FCeR1-TEC kinase- 100 100 100 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Tec Kinase TCR-SRC-TEC KINASE- 100 100 100 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Tec Kinase TLR4-TEC kinase- 100 100 100 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) signalling by Rho Integrin-ARHGEF-RHO-PKNI- 75 75 75 75 Family GTPases cell trafficking Regulation of CNG-CALPAIN-RB 100 100 100 100 cellular mechanics by calpain protease Tec Kinase Integrin-FAK-TEC KINASE- 100 100 100 100 signalling (FAK, PKC, PAK, VAV, FACTIN, RHOGTPASE, NFKB, JNK, STAT-TFII-1) Acute myeloid FLT3-GRB2-SOS-RAS-RAF- 100 100 100 100 leukemia signalling MEK-ERK1/2-cell proliferation signalling by Rho Integrin-ARHGEF-RHO-FAK- 100 100 100 100 Family GTPases cytoskeletal reorganisation IL-8 CXCR1/2 - Ras-Raf-MEK1/2- 80 80 100 100 ERK1/2- (Neutrophil Degranulation) JAK/STAT cytokine receptor-JAK-STAT- 75 75 100 100 (CFOS-IL6-SOCS-BCLXL) MSP-RON signalling RON-PI3K-PKC zeta- F-ACTIN- 75 100 75 75 pathway phagocytic activity in macrophages Germ Cell-Sertoli TGFbetaR-RAS-MEK1/2- 100 100 100 100 Cell Junction ERK1/2-actin depolymerisation signalling Role of MAPK ASK-1-MKK4/7-JNK-CASP3 - 75 75 75 75 signalling in the APOPTOSIS Pathogenesis of Influenza Role of PI3K/AKT PI3K-AKT-IKB, NFKB 100 100 75 75 signalling in the Pathogenesis of Influenza Protein Kinase A PKAr/PKAc-RAP1-BRAF- 100 83.33 83.33 83.33 signalling MEK1/2-ERK1/2-ELK1 IL-8 CXCR1/2 - PI3K-Akt-AP1- 75 80 100 100 IntegrinAlphavBeta3 (Chemotaxis) Production of nitric TLR2/4-MKK4/7-JNK-AP1 100 100 100 100 oxide and ROS in macrophages

Example 2

Effects of p38MAPK Inhibition on Inflammatory Mediator Release in Key Cell Types Relevant to Severe Influenza

(41) Two p38 MAPK inhibitors were used in in vitro and ex vivo experiments: 1) PH797804, an ATP-competitive highly potent, selective and metabolically stable inhibitor of p38 (Hope HR1, et al., Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation, J. Pharmacol. Exp. Ther., 2009 (December); 331(3): 882-95); and 2) Dilmapimod (SB-681323- Betts JC1, et al., Gene expression changes caused by the p38 MAPK inhibitor Dilmapimod in COPD patients: analysis of blood and sputum samples from a randomized, placebo-controlled clinical trial, Pharmacol. Res. Perspect., 2015 (February); 3(1): e00094). The structures of these compounds are as follows:

(42) ##STR00012##

(43) Experimental testing of p38MAPK inhibition was carried out in three cell types that are key players in the pathology of severe influenza: epithelial; endothelial; and immune cells (FIG. 4). In these experiments, cells were pre-treated with simple and/or complex stimuli in order to simulate the action of inflammatory mediators that are produced from influenza-infected epithelial cells. In the case of the former, tumour necrosis factor alpha (TNFa) plus interleukin 6 (IL-6) was used to stimulate endothelial and immune cells. In the case of the latter, conditioned medium (viral “soup”) derived either from influenza virus infected A549 cells (adenocarcinoma human alveolar basal epithelial cells), or primary human bronchial epithelial cells (HBECs) was used to stimulate epithelial, endothelial and immune cells.

(44) Epithelial Cells

(45) A549 cells (adenocarcinoma human alveolar basal epithelial cells) or HBEC (Human Bronchial Epithelial Cells) were infected with A/Perth/16/2009(H3N2) virus from which viral conditioned media (or ‘viral soup’) was collected. In the experiments described here, the application of viral soup either back onto epithelial cells or onto the other cell types of interest (endothelial and immune) was performed in order to simulate the action of inflammatory mediators that are produced from Influenza infected epithelial cells.

(46) For infection, high titre stocks of Influenza (H3N2) virus were produced by infection of MDCK-RVL cells (available from ATCC as MDCK (NBL-2) (ATCC® CCL-34), derived from a kidney of an apparently normal adult female cocker spaniel, September, 1958, by S. H. Madin and N. B. Darby. The line is hyperdiploid, and there is a bi-modal chromosome number distribution. There are no consistent identifiable marker chromosomes. One normal X chromosome is present in most spreads. The cells are positive for keratin by immunoperoxidase staining). MDCK-RVL cells were plated in T175 flask and allowed to grow to 85-90% confluence. The next day, cells were washed twice with infection media. The Influenza A/Perth/16/2009 (H3N2) stock was removed from −80° C. and thawed on ice. The cells were infected with virus stock at 0.01 MOI for one hour in infection media. At the end of incubation period, unbound virus was removed from the cells. The cells were washed once with infection media and overlaid with infection media and allowed to incubate for 48 hours in 37° C. at 5% CO.sub.2/air incubator. After incubation, the flasks were frozen at −80° C. for a day. Next day, the flasks were thawed at room temperature and virus supernatant was centrifuged (2000 g, 10 min) and pooled together. The virus stock was aliquoted and stored at −80° C.

(47) Viral “soups” were prepared using the A549 cell line and primary human bronchial epithelial cells (HBECs). For the preparation of A549 soups cells were plated in a T175 flask and allowed to grow to obtain 85-90% confluence. The cells were washed twice with infection media. The Influenza A/Perth/16/2009 (H3N2)-WGC stock was removed from −80° C. and thawed on ice. The cells were infected with virus stock at 0.01 MOI for one hour in infection media. The unbound virus was removed from the cells at the end of the incubation period and cells were washed once with infection media before overlaying with infection media. The cells were incubated for 48 hours in 37° C. at 5% CO.sub.2/air incubator. After incubation, the media (‘viral soup’) was collected from all flasks, centrifuged (2000 g, 10 min) and pooled together. The viral soup was aliquoted and stored at −80° C. For the preparation of HBEC viral soup cells were cultured to passage P-3 or P-4 in Bronchial Epithelial Cell Growth Medium (BECGM; Lonza). For infection, cells were washed twice with BECGM then infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI for one hour in infection medium (BECGM containing 1.06 USP/NF units per ml TPCK trypsin). The cells were then overlaid with infection medium and incubated for 48 hours at 37° C. in 5% CO.sub.2/air. The HBEC viral soup was then processed in a similar way to A549 soup.

(48) Viral growth kinetics were optimised for generating viral soups. To determine multistep growth curves, A549 cells or HBECs were infected with virus at an MOI of 0.01 TCID.sub.50/cell at 37° C. for one hour. Following incubation, the cells were washed and overlaid with respective infection media. The samples were harvested for viral titres and measurement of cytokines at various time points for 72 hours. The viral titres were obtained by TCID.sub.50 on MDCK cells and the presence of inflammatory mediators was assessed by MSD chemiluminescence assay using methods as recommended by the vendor (https://web.archive.org/web/20160522190937/https://www.mesoscale.com/).

(49) Prior to experimental testing both A549 and HBEC soup preparations were evaluated by electrogenerated chemiluminescence for the presence of inflammatory mediators using methods as recommended by the vendor (https://web.archive.org/web/20160522190937/https://www.mesoscale.com/). Both soup preparations were found to contain elevated levels of the following cytokines (IL1-β, IL-6, IL-8, IL-10, TNFα and RANTES; FIG. 5).

(50) In vitro data were generated from A549 cells to test the effect of A549 viral soup application on p38 activation as measured by western blotting of the phosphorylation status of p38MAPK itself and downstream signalling target, HSP27. For western blotting confluent cells were washed in PBS and lysed in RIPA with protease inhibitor (Sigma-P8340), phosphatase inhibitor cocktails 2 and 3 (Sigma P5726 and P0044) and phosphatase inhibitors Na.sub.3VO.sub.4 and NaF on ice. Protein concentrations were determined using the Pierce BCA Protein Assay Kit and equal concentrations of each sample created in 4× Laemmli sample buffer (BIO-RAD 161-0747) with 2-mercaptoethanol. Samples were run on a 12% gel and then transferred on to nitrocellulose. Membranes were blocked using 5% milk powder, then hybridised with p38 MAPK rabbit antibody (Cell Signalling Technologies, cat. no. 9212S) and phosphorylated p38 MAPK rabbit antibody (Cell Signalling Technologies, cat. no. 9211S), or HSP27 antibody (Cell signalling Technologies, cat. no. 2402) and phospho-HSP27 antibody (Cell signalling Technologies, cat. no. 9709). Secondary antibodies were anti-rabbit HRP (Cell signalling Technologies catalogue no. CS7074P2) and anti-mouse HRP (Cell signalling, catalogue no. 7076S), respectively. Membranes were stripped for re-probing using Restore™ PLUS Western Blot Stripping Buffer (Life technologies #46430). The membranes were treated with Amersham ECL Prime Western Blotting Detection Reagent (GE/Amersham #RPN2232) and imaged using ChemiDoc™ Touch Imaging System (BIORAD). Analysis was performed using the Image Lab software.

(51) Induction of phosphorylation on both of these enzymes was detected indicating that p38MAPK is activated following application of A549 viral soup (FIG. 6). Furthermore, incubation with p38 MAPK inhibitors (Dilmapimod and PH 797804) was shown to dose dependently inhibit the induction of both p38MAPK and HSP27 phosphorylation by our A549 viral soup, confirming that this induction is a p38MAPK dependent process within these epithelial cells.

(52) The effect of A549 viral soup on inflammatory cytokine production in A549 cells as measured by electrogenerated chemiluminescence was also explored. As shown in FIG. 6, A549 soup was found to induce the production of key inflammatory cytokines (IL-6 and IP-10) to a greater extent compared with control uninfected A549 soup. Application of the two p38 MAPK inhibitors on A549 cells prior to A549 viral soup application significantly attenuated release of both IL-6 and IP-10 (FIG. 6 shows the data for the p38 MAPK inhibitor PH 797804). These data demonstrate that the release of inflammatory mediators in response to A549 viral soup application on A549 cells is a p38MAPK dependent process.

(53) Endothelial Cells

(54) The application of HBEC viral soup on to Human Umbilical Vein Endothelial Cells (HUVECs) was performed in order to simulate the interaction of inflammatory mediators that are produced from Influenza infected epithelial cells onto endothelial cells.

(55) In vitro data were generated from HUVEC cells to test the effect of HBEC viral soup application on p38 activation as measured by western blotting of the phosphorylation status of the p38MAPK downstream signalling target, HSP27. Induction of phosphorylation on HSP27 was detected indicating that p38MAPK is activated following application of HBEC viral soup (FIG. 7). Furthermore, pre-incubation with p38 MAPK inhibitors (Dilmapimod and PH 797804) was shown to dose dependently inhibit the induction of HSP27 phosphorylation by the HBEC viral soup, confirming that this induction is a p38MAPK dependent process within these endothelial cells.

(56) The effect of HBEC viral soup on inflammatory cytokine production in HUVEC cells as measured by electrogenerated chemiluminescence (see methods) was also explored. As shown in FIG. 7, HBEC viral soup was found to induce the production of key inflammatory cytokines (IL-6 and IP-10) to a greater extent compared with control HBEC uninfected soup. Incubation of HUVEC cells with the two p38 MAPK inhibitors prior to HBEC viral soup application was found to significantly attenuate the HBEC viral soup induced release of both IL-6 and IP-10 (FIG. 7). These data demonstrate that the release of inflammatory mediators in response to HBEC viral soup application in HUVEC endothelial cell is a p38MAPK dependent process.

(57) Immune Cells

(58) The application of A549 viral soup onto human Peripheral Blood Mononuclear Cells (PBMCs) was performed in order to simulate the interaction of inflammatory mediators that are produced from Influenza infected epithelial cells onto immune cells. PBMCs were isolated according to the manufacturers recommendations (Boyum, A., Separation of leucocytes from blood and bone marrow, Scand. J. Clin. Lab. Invest., 1968, 21, suppl. 97).

(59) Ex vivo data were generated from immune cells to test the effect of A549 viral soup application on p38 activation as measured by Western blotting (see above for method) of the phosphorylation status of the p38MAPK downstream signalling target, HSP27. Induction of phosphorylation on HSP27 was detected, indicating that p38MAPK is activated following application of A549 viral soup (FIG. 8). Furthermore, pre-incubation with p38 MAPK inhibitors (Dilmapimod and PH 797804) was shown to dose dependently inhibit the induction of HSP27 phosphorylation by the A549 viral soup, confirming that this induction is a p38MAPK dependent process within these immune cells (FIG. 8).

(60) The effect of A549 viral soup on inflammatory cytokine production in immune cells as measured by electrogenerated chemiluminescence (see above) was also explored. As shown in FIG. 8, A549 viral soup was found to induce the production of key inflammatory cytokines (TNFα, IL-1-β, IL-6 and CXCL8) to a greater extent compared with control A549 uninfected soup. Pre-incubation of p38 MAPK inhibitors (Dilmapimod and PH 797804) on immune cells prior to A549 viral soup application was found to significantly attenuate the A549 viral soup induced release of TNFα, IL-1-β, IL-6 and CXCL8 (FIG. 8). These data demonstrate that the release of inflammatory mediators in response to A549 viral soup application in immune cells is a p38MAPK driven process.

(61) Additional ex vivo data were generated from immune cells whereby the induction of inflammatory mediators in response to A549 viral soup was compared to known inflammatory stimulants (anti-CD3 and LPS). The induction of TNFα, IL-1-β, IL-6 and IL-8 by A549 viral soup was found to be greater compared with these known inflammatory stimulants (FIG. 9).

Example 3

p38 MAPK Inhibitory Activity

(62) The enzyme inhibitory activity of a compound may be determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen).

(63) Recombinant, phosphorylated p38 MAPK gamma (MAPK12:Millipore) is diluted in HEPES buffer, mixed with the candidate compound at desired final concentrations and incubated for two hours at room temperature. The FRET peptide (2 μM) and ATP (100 μM) are next added to the enzyme/compound mixture and incubated for one hour. Development reagent (protease) is added for one hour prior to detection in a fluorescence microplate reader. The site-specific protease only cleaves non-phosphorylated peptide and eliminates the FRET signal. Phosphorylation levels of each reaction are calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor) with high ratios indicating high phosphorylation and low ratios, low phosphorylation levels. The percentage inhibition of each reaction is calculated relative to non-inhibited control, and the 50% inhibitory concentration (IC.sub.50 value) then calculated from the concentration-response curve.

(64) For p38 MAPK alpha (MAPK14: Invitrogen), enzyme activity is evaluated indirectly by determining activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK a protein is mixed with its inactive target MAPKAP-K2 (Invitrogen) and the candidate compound for two hours at room temperature. The FRET peptide (2 μM), which is a phosphorylation target for MAPKAP-K2, and ATP (10 μM) are then added to the enzymes/compound mixture and incubated for one hour. Development reagent is then added and the mixture incubated for one hour before detection by fluorescence completed the assay protocol.

Example 4

P38MAPK Inhibition (p38i) Versus Inhibition of Other Potential Targets

(65) As indicated in Example 1 above, a number of targetable nodes in the 95 pathway routes highlighted by transcriptomic and bioinformatics were identified. The compound profiling experiments in Example 2 show that p38i is effective in reducing the production of inflammatory mediator release in cell types relevant to the pathology of severe influenza. This was found not to be the case for 9 other nodes that were examined: PI3K, MEK, ERK, JNK, JAK/STAT, PKC, SRC, BtK and mTor. Drug inhibition of none of these 9 nodes gave an inhibition profile as effective as p38i in epithelial, endothelial and immune cells.

(66) By way of example, data comparing p38i versus mitogen-activated protein kinase (MEK) inhibition (MEKi) by MEK inhibitors Refametinib (Iverson C et al., RDEA119/BAY 869766: a potent, selective, allosteric inhibitor of MEK1/2 for the treatment of cancer. Cancer Res., 2009; 69: 6839-6847) and Selumetinib (Huynh H et al., Targeted inhibition of the extracellular signal-regulated kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular carcinoma, Molecular Cancer Therapeutics, 2007; 6:138-146) are presented in FIG. 10 of the accompanying drawings.

(67) ##STR00013##

(68) Neither Refametinib nor Selumetinib showed dose-dependent inhibition of IP10 production in endothelial cells stimulated with HBEC viral soup and actually appeared to increase levels of IP10 at higher drug concentrations (see FIG. 10).

(69) A number of potential drug targets have been proposed for severe influenza (e.g. Liu Q et al., 2015 and Fedson DS, 2009). p38i was also compared versus drug compounds for a selection of these proposed targets.

(70) For these experiments, PH797804 was benchmarked versus corticosteroid (methyl prednisolone), macrolide (Azithromycin), PPAR agonist (Pioglitazone), PDE4 inhibitor (Roflumilast), NFκB inhibitor (EVP4593) and statin (Pravastatin) at four drug concentrations (1 nM, 10 nM, 100 nM and 1000 nM) in endothelial cells (HUVECs stimulated with HBEC viral soup), or immune cells (PBMCs plus granulocytes stimulated with A549 viral soup as described in Example 2 above).

(71) The effects of drug compound administration on IP-10, IL-8 and MCP-1 production from endothelial cells and on IL-1β, IL-6, IL-8 and TNF-α production from immune cells was assayed using electrogenerated chemiluminescence. In immune cells, corticosteroid and macrolide drug treatment showed dose-dependent inhibition of all four assayed cytokines, whereas p38i showed dose-dependent inhibition of only three of the four. The inhibitory profile of the other drugs tested was variable and did not match that of corticosteroid, macrolide, or p38i. The results are summarised in Table 5 below.

(72) TABLE-US-00005 TABLE 5 Comparison of inhibitory effects of drug compounds for literature-proposed targets for severe influenza versus p38i. PBMCs plus granulocytes were isolated as described in Example 2 and stimulated with A549 viral soup. Secreted cytokine levels were assayed by electrogenerated chemiluminescence and IC.sub.50 and iMax values were calculated from the dose responses using non-linear regression fit using a scientific 2D graphing and statistics software package (GraphPad PRISM ® version 6.07 software). Where data did not show a dose-dependent inhibition, the IC.sub.50 and iMax values were not calculated (empty boxes). Corticosteroid NFkB inhibitor p38 inhibitor Methyl Macrolide PPAR agonist PDE4 inhibitor QNZ Statin PH797804 Prednisolone Azithromycin Pioglitazone Roflumilast (EVP4593) Pravastatin IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) IL1B 9.7 79% 8.4 77% 0.5 50% IL8 2.3 87% 8.4 90% 0.8 46% 0.1 37% 0.09 31% 0.2 47% TNFa 3.9 91% 20.2 79% 86.9 63% 10.8 74% 43.6 37% IL6 36.6 57% 119 45% 0.02 41%

(73) The inhibition plots for IL1-b and TNFa for the compounds tested are shown in FIGS. 11 and 12.

(74) With endothelial cells, in contrast to immune cells, only p38 and NFκB inhibitors showed dose-dependent inhibition of the three cytokines assayed. None of the other drugs tested showed a comparable inhibitory effect. The results are summarised in Table 6 below.

(75) TABLE-US-00006 TABLE 6 Comparison of inhibitory effects of drug compounds for literature-proposed targets for severe influenza versus p38i. HUVEC cells were stimulated with HBEC viral soup. Secreted cytokine levels were assayed by electrogenerated chemiluminescence and IC.sub.50 and iMax values were calculated from the dose responses using non-linear regression fit with a scientific 2D graphing and statistics software package (GraphPad PRISM ® version 6.07 software). Where data did not show a dose-dependent inhibition, the IC.sub.50 and iMax values were not calculated. Corticosteroid NFkB inhibitor p38 inhibitor Methyl Macrolide PPAR agonist PDE4 inhibitor QNZ Statin PH797804 Prednisolone Azithromycin Pioglitazone Roflumilast (EVP4593) Pravastatin IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax IC.sub.50 iMax (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) (nM) (%) IP10 7.5 88% 3.3 72% 78 35% IL8 28.9 86% 6.5 60% MCP1 13.1 36% 3.84 44%

(76) The inhibition plots for IP10 and IL8 are shown in FIGS. 13 and 14.

(77) Based on the results obtained in the immune cell experiments, the superiority of p38i versus the other drugs was unexpected, especially methyl prednisolone, which is used routinely in clinical settings to treat a range of inflammatory diseases (e.g. asthma) and is commonly prescribed for severe influenza, although there is uncertainty over their potential benefit or harm (Rodrigo C et al., Corticosteroids as adjunctive therapy in the treatment of influenza, Cochrane Database of Systematic Reviews, 2016, Issue 3. Art. No.: CD010406. DOI: 10.1002/14651858.CD010406.pub2]. NFκB is downstream of p38, so the inhibition profile seen is not unexpected.

Example 5

Levels of a Number of Cytokines in Serum from Patients Hospitalised for Severe Influenza Were Significantly Raised Relative to Influenza-Infected Individuals Without Severe Influenza

(78) Cytokine levels in serum samples from 30 subjects hospitalised with severe influenza during the 2015 influenza season and from 28 healthy subjects infected after intranasal inoculation with influenza A/H3N2 Perth/16/2009 virus were assayed using electrogenerated chemiluminescence. In the case of the former, a serum sample prepared from a blood sample collected 24-72 hours after the subject was admitted to hospital was analysed. In the case of the latter, serum was analysed from blood samples collected at 12 pre-determined intervals (Day −1 to Day 28). Eight cytokines were observed to be significantly raised in the hospitalised versus the infected healthy subjects: IL-8, IL-7, IL-16, Eotaxin, IP10, MCP1, MCP4 and VEGF. The results for four of these are shown in FIG. 15.

(79) The results show that the hospitalised subjects are distinguishable from the healthy infected subjects in terms of their serum cytokine profiles.

Example 6

Effects of p38 MAPK Inhibition with the Compound of Formula III on Inflammatory Mediator Release in Key Cell Types Relevant to Severe or Persistent Influenza

(80) The p38 MAPK inhibitor of Formula III was used in in vitro and ex vivo experiments (Example 8 of WO 2004/076450 A1 (J. Uriach Y Compañia S. A.)).

(81) Experimental testing of p38 MAPK inhibition by the inhibitor of Formula III was carried out in three cell types that are key players in the pathology of severe and persistent influenza: epithelial; endothelial; and immune cells (FIG. 4). In these experiments, cells were pre-treated with simple and/or complex stimuli in order to simulate the action of inflammatory mediators that are produced from influenza-infected epithelial cells. In the case of the former, tumour necrosis factor alpha (TNFa) plus interleukin 6 (IL-6) was used to stimulate endothelial and immune cells. In the case of the latter, conditioned medium (viral “soup”) derived either from influenza virus infected A549 cells (adenocarcinoma human alveolar basal epithelial cells), or primary human bronchial epithelial cells (HBECs) was used to stimulate epithelial, endothelial and immune cells.

(82) Epithelial Cells

(83) For the production of conditioned medium (viral soup), A549 cells or HBECs were infected with high titre stocks of Influenza A/Perth/16/2009 (H3N2) virus. Viral stocks were produced by infection of Madin-Darby Canine Kidney [MDCK cells, available from the American Type Culture Collection as MDCK (NBL-2) (ATCC® CCL-34™)]. MDCK cells were cultured to 85-90% confluence in Minimal Essential Medium (MEM) containing 10% foetal bovine serum. Cells were washed twice with infection medium (Advanced Dulbecco's Modified Eagle Medium [DMEM] containing 1.06 USP/NF units per ml TPCK trypsin). The cells were infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI for one hour in infection medium. At the end of the incubation period, unbound virus particles were removed from the cells by washing once with infection medium. The cells were then overlaid with fresh infection medium and incubated for 48 hours at 37° C. in 5% CO.sub.2/air. After incubation, the flasks of cells were frozen at −80° C. for 24 hours, thawed at room temperature and virus supernatants were centrifuged (2000 g, 10 min) before pooling together, aliquoting and storing at −80° C.

(84) The viral stocks prepared were used for the generation of viral soups. Viral growth kinetics were optimised by determining multistep growth curves. A549 cells or HBECs were infected with virus at an MOI of 0.01 at 37° C. for one hour. Following incubation, the cells were washed and overlaid with infection medium. The samples were harvested at various time points for 72 hours for viral titre determination by TCID.sub.50 assay on MDCK cells and the presence of inflammatory mediators was assessed by MSD chemiluminescence assay using methods as recommended by the vendor (https://web.archive.org/web/20160522190937/https://www.mesoscale.com/).

(85) For the preparation of A549 viral soup, cells were cultured in MEM plus 10% foetal bovine serum to 85-90% confluence. The cells were washed twice with infection medium then infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI for one hour in infection medium. Unbound virus was removed from the cells at the end of the incubation period by washing once with infection medium before overlaying with infection medium. The cells were incubated for 48 hours at 37° C. in 5% CO.sub.2/air. After incubation, the viral soup was collected from the culture flasks, centrifuged (2000 g, 10 min) and pooled together. The viral soup was aliquoted and stored at −80° C. For the preparation of HBEC viral soup cells were cultured to passage P-3 or P-4 in Bronchial Epithelial Cell Growth Medium (BECGM; Lonza). For infection, cells were washed twice with BECGM then infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI for one hour in infection medium (BECGM containing 1.06 USP/NF units per ml TPCK trypsin). The cells were then overlaid with infection medium and incubated for 48 hours at 37° C. in 5% CO.sub.2/air. The HBEC viral soup was then processed in a similar way to A549 soup.

(86) Prior to experimental testing, levels of pro-inflammatory mediators in both A549 and HBEC soup preparations were measured by MSD chemiluminescence analysis. Both soup preparations were found to contain elevated levels of pro-inflammatory cytokines (IL1-β, IL-6, IL-8, IL-10, TNFα and RANTES; FIG. 5) and demonstrated to induce p38MAPK signalling by demonstration of phosphorylation of both p38MAPK and phosphorylation of the downstream signalling protein HSP27 by western blot analysis (FIG. 5). For western blotting, viral soup-treated cells were washed with PBS and lysed in RIPA buffer with protease inhibitor (Sigma-P8340), phosphatase inhibitor cocktails 2 and 3 (Sigma P5726 and P0044) and phosphatase inhibitors sodium orthovanadate and sodium fluoride (both, www.sigmaaldrich.com) on ice. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (www.thermofisher.com). Equivalent concentrations of each sample in 4× Laemmli sample buffer (BIO-RAD 161-0747) with 2-mercaptoethanol were analysed on a 12% polyacrylamide gel in Tris-glycine-SDS buffer and then transferred to nitrocellulose membrane. Membranes were blocked using 5% milk powder, then hybridised with p38 MAPK rabbit antibody (Cell Signalling Technologies, cat. no. 9212S) and phosphorylated p38 MAPK rabbit antibody (Cell Signalling Technologies, cat. no. 9211S), or HSP27 antibody (Cell signalling Technologies, cat. no. 2402) and phospho-HSP27 antibody (Cell signalling Technologies, cat. no. 9709). Secondary antibodies were anti-rabbit HRP (Cell signalling Technologies catalogue no. CS7074P2) or anti-mouse HRP (Cell signalling, catalogue no. 7076S), respectively. Membranes were stripped for re-probing using Restore™ PLUS Western Blot Stripping Buffer (Life technologies #46430). The membranes were treated with Amersham ECL Prime Western Blotting Detection Reagent (GE/Amersham #RPN2232) and imaged using ChemiDoc™ Touch Imaging System (BIO-RAD). Analysis was performed using Image Lab software.

(87) The effect of HBEC viral soup on inflammatory mediator production in HBEC cells was measured by MSD analysis. As shown in FIG. 16, HBEC soup induced the production of key inflammatory cytokines as exemplified by IL-6 and IP-10. Treatment of HBECs with the p38 MAPK inhibitor of Formula III prior to HBEC viral soup application attenuated release of both cytokines (FIG. 16).

(88) Endothelial Cells

(89) Both simple (TNFa plus IL-6) and complex (HBEC viral soup) stimuli were applied to Human Umbilical Vein Endothelial Cells (HUVECs) to simulate the interaction of inflammatory mediators produced by influenza-infected epithelial cells with endothelial cells.

(90) Inflammatory cytokine production by HUVEC cells treated with either TNFa plus IL-6, or HBEC viral soup as measured by MSD analysis was explored. As shown in FIG. 17, both stimuli induced the production of key inflammatory cytokines as exemplified by IL-8 and IP-10. However, treatment of HUVEC cells with p38 MAPK inhibitor of Formula III prior to stimulation was shown to attenuate this (FIG. 17).

(91) Immune Cells

(92) Both simple (TNFa plus IL-6) and complex (A549 viral soup) stimuli were applied to isolated human Peripheral Blood Mononuclear Cells (PBMCs) to simulate the interaction of inflammatory mediators that are produced from influenza-infected epithelial cells on immune cells. PBMCs were isolated from human blood according to the manufacturers recommendations (Boyum, A., Separation of leucocytes from blood and bone marrow, Scand. J. Clin. Lab. Invest., 1968, 21, suppl. 97).

(93) Inflammatory cytokine production in immune cells treated with either TNFa plus IL-6, or A549 viral soup as measured by MSD analysis was explored. As shown in FIG. 18, both stimuli induced the production of key inflammatory cytokines as exemplified by TNFα and

(94) IL-8. However, treatment of PBMCs with the p38 MAPK inhibitor of Formula III prior to stimulation was found to attenuate this (FIG. 18).

Example 7

Combination of the p38 MAPK Inhibitors with the Antiviral Drug Oseltamivir

(95) Successful therapy of severe influenza is envisaged to depend on effectively targeting both the viral infection phase (phase 1) via antiviral drugs and the later (post-infection) inflammatory phase (phase 2) with immunomodulatory drugs. As antiviral treatment is recommended as early as possible for any patient with confirmed or suspected influenza who: is hospitalised; has severe, complicated, or progressive illness; or is at higher risk for influenza complications [https://www.cdc.gov/flu/professionals/antivirals/summary-clinicians.htm], it is important that an immunomodulator does not impede the action of the antiviral drug as both will likely be given at the same time. To examine this possibility the effect of combining oseltamivir with either of two p38MAPK inhibitors (PH797804 and Dilmapimod) was investigated by examining the effect that these two drugs had on the ability of oseltamivir to suppress viral infection in HBECs.

(96) HBECs were infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI and the effect of oseltamivir in combination with either PH797804 or Dilmapimod on viral infection was examined. The oseltamivir used in this Example was in the form of oseltamivir carboxylate, which is the active metabolite of oseltamivir and pharmaceutically acceptable salts thereof (e.g. it is the active metabolite of oseltamivir phosphate). Viral titre was determined by TCID.sub.50 assay [WHO manual for the laboratory diagnosis and virological surveillance of influenza; http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf]. Whereas oseltamivir treatment reduced TCID.sub.50, neither PH797804 nor Dilmapimod treatment showed this effect. When oseltamivir was combined with either PH797804 or Dilmapimod, TCID.sub.50 was reduced to the level seen with oseltamivir on its own, indicating that p38MAPK inhibitor therapy could be used in combination with oseltamivir without impacting its antiviral activity (FIG. 19).

(97) In a clinical setting it is envisaged that reduction of viral load (phase 1) by antiviral therapy would be monitored by measuring viral RNA levels using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and effects of p38 inhibition on inflammation (phase 2) would be monitored by measuring the levels of inflammatory mediators such as those seen in patients with severe influenza (example 5) by, for example, MSD analysis of serum samples.

Example 8

Effects of p38 MAPK Inhibition with the Compound of Formula II on Inflammatory Mediator Release in Key Cell Types Relevant to Severe or Persistent Influenza

(98) The p38 MAPK inhibitor of Formula II was used in in vitro and ex vivo experiments (Example 18 of WO 2004/076450 A1 (J. Uriach Y Compañia S. A.)). As described above, the chemical structure of this compound is as follows:

(99) ##STR00014##

(100) Experimental testing of p38 MAPK inhibition by the inhibitor of Formula II was carried out in endothelial and immune cells. In these experiments, cells were pre-treated with TNFa plus IL-6, or viral soups prepared as described above in [00190] to [00193].

(101) Endothelial Cells

(102) Inflammatory cytokine production by HUVEC cells treated with either TNFa plus IL-6, or HBEC viral soup was measured by MSD analysis. As shown in FIG. 20, both stimuli induced the production of key inflammatory cytokines as exemplified by IL-8 and IP-10. However, treatment of HUVEC cells with p38 MAPK inhibitor of Formula II prior to stimulation was shown to attenuate this (FIG. 20).

(103) Immune Cells

(104) Both simple (TNFa plus IL-6) and complex (A549 virus soup) stimuli were applied to isolated human Peripheral Blood Mononuclear Cells (PBMCs) to simulate the interaction of inflammatory mediators that are produced from influenza-infected epithelial cells on immune cells. PBMCs were isolated from human blood according to the manufacturer's recommendations (Boyum, A., Separation of leucocytes from blood and bone marrow, Scand. J. Clin. Lab. Invest., 1968, 21, suppl. 97).

(105) Inflammatory cytokine production in immune cells treated with either TNFa plus IL-6, or A549 virus soup as measured by MSD analysis was explored. As shown in FIG. 21, both stimuli induced the production of key inflammatory cytokines as exemplified by MIP-1a and IL-8 (TNFa plus IL-6 as stimulus) and TNFa and IL-8 (A549 virus soup as stimulus). However, treatment of PBMCs with the p38 MAPK inhibitor of Formula II prior to stimulation was found to attenuate this (FIG. 21).

Example 9

Combination of Compound with Formula II with the Antiviral Drug Oseltamivir

(106) As it is important that an immunomodulator does not impede the action of antiviral drugs that are likely to be given at the same time, the effect of combining compound of Formula II with oseltamivir was investigated by examining the effect that this drug compound has on the ability of oseltamivir to suppress viral infection in HBECs. Conversely, the effect that this combination might have on the anti-inflammatory properties of compound with Formula II was also examined.

(107) HBECs were infected with Influenza A/Perth/16/2009 (H3N2) virus stock at 0.01 MOI and the effect of oseltamivir in combination with compound of Formula II on viral infection was examined. The oseltamivir used in this Example was in the form of oseltamivir carboxylate, which is the active metabolite of oseltamivir and pharmaceutically acceptable salts thereof (e.g. it is the active metabolite of oseltamivir phosphate).Viral titre was determined by TCID.sub.50 assay [WHO manual for the laboratory diagnosis and virological surveillance of influenza; http:/lwhqlibdoc.who.int/publications/2011/9789241548090 eng.pdf]. Whereas oseltamivir treatment alone reduced TCID.sub.50, treatment with the compound of Formula II alone did not significantly reduce the TCID.sub.50. When oseltamivir was combined with compound of Formula II, TCID.sub.50 was reduced to the level seen with oseltamivir on its own, indicating that p38MAPK inhibitor therapy could be used in combination with oseltamivir without impacting oseltamivir's antiviral activity (FIG. 22). Conversely, oseltamivir alone had no observable effect on the anti-inflammatory properties of the compound of Formula II (FIG. 23).

(108) In a clinical setting it is envisaged that reduction of viral load by antiviral therapy would be monitored by measuring viral RNA levels using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and the effects of p38 inhibition on inflammation would be monitored by measuring the levels of inflammatory mediators such as those seen in patients with severe influenza (Example 5) by, for example, MSD analysis of serum samples.

(109) Abbreviations

(110) ATP Adenosine triphosphate

(111) A549 Adenocarcinoma human alveolar basal epithelial cells

(112) BECGM Bronchial epithelial cell growth medium

(113) Btk Bruton's tyrosine kinase

(114) CXCL8 Interleukin 8

(115) CD3 Cluster of differentiation protein 3

(116) DMEM Dulbecco's modified eagle medium

(117) ERK Extracellular signal-regulated kinases)

(118) FRET Fluorescence resonance energy transfer

(119) GSK Glaxo Smith-Kline

(120) HBEC Human bronchial epithelial cells

(121) HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

(122) HSP27 Heat shock protein 27

(123) HUVEC Human vascular endothelial cells

(124) IC.sub.50 Half maximal inhibitory concentration

(125) iMax Maximal inhibition (as a %)

(126) IL1-b Interleukin 1 beta

(127) IL-6 Interleukin 6

(128) IL-8 Interleukin 8

(129) IPA Ingenuity Pathways Analysis

(130) IP-10 Interferon gamma-induced protein 10

(131) JAK/STAT Janus kinase/signal transducer and activator of transcription

(132) JNK c-Jun N-terminal kinase

(133) LPS Lipopolysaccharide

(134) MAPKAP-K2 MAP kinase-activated protein kinase 2

(135) MOI Multiplicity of infection

(136) MCP-1 Monocyte chemotactic protein-1

(137) MDCK Madin Darby canine kidney

(138) MEK Mitogen-activated protein kinase kinase

(139) MEKi MEK inhibition (by drug)

(140) MEM Minimal essential medium

(141) mTOR Mechanistic target of rapamycin

(142) MSD Mesoscale Discovery

(143) NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells

(144) P38 MAPK P38 Mitogen-activated protein kinases

(145) P38i p38 inhibition (by drug)

(146) PBMC Peripheral blood mononuclear cells

(147) PBS Phosphate buffered saline

(148) PDE4 Phosphodiesterase 4

(149) PKC Protein kinase

(150) PPAR Peroxisome proliferation-activated receptor

(151) RANTES Regulated on activation, normal T cell expressed and secreted

(152) RIPA Radio immuno precipitation assay

(153) SDS Sodium dodecyl sulphate

(154) SRC Src kinase

(155) TCID.sub.50 Tissue culture infective dose

(156) TNFa/TNFα Tumour necrosis factor alpha

(157) TPCK Tosyl phenylalanyl chloromethyl ketone

(158) USP/NF United States pharmacopeia and the national formula