BIOFILM DISRUPTION

Abstract

The present invention relates to methods of dispersing biofilms comprising Gram-negative bacteria, the methods comprising exposing the biofilm to an epoxytiglienone compound or a salt thereof. Methods of treating infections comprising the localised administration, for example, topically or by injection, of an epoxytiglienone compound into or onto an established biofilm comprising Gram-negative bacteria to disrupt the structure of that biofilm and methods of preventing biofilms comprising Gram-negative bacteria forming or dispersing biofilms comprising Gram-negative biofilms that have formed on medical devices are also described.

Claims

1. A method of dispersing a biofilm comprising Gram-negative bacteria comprising exposing the biofilm to an epoxytiglienone compound of formula (I): ##STR00012## wherein R.sub.1 is selected from hydrogen and C.sub.1-6alkyl; R.sub.2 is selected from —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.3 is selected from —OH, —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is selected from hydrogen, —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —OC(O)C.sub.2-6alkynylaryl; R.sub.7 is selected from hydroxy, —OC.sub.1-6alkyl, —OC.sub.2-6alkenyl, —OC.sub.2-6alkynyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl, —OC(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —OC(O)aryl, —OC(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —C(O)C.sub.2-6alkynylaryl; R.sub.8 is selected from hydrogen or C.sub.1-6alkyl; or a salt thereof.

2. The method according to claim 1 wherein one or more of the following applies: i) R.sub.1 is C.sub.1-3 alkyl; ii) R.sub.2 is selected from —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; iii) R.sub.3 is selected from —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; iv) R.sub.4 and R.sub.5 are each methyl; v) R.sub.6 is selected from hydrogen, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl and —C(O)aryl; vi) R.sub.7 is hydroxyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl or —OC(O)C.sub.2-6alkynyl; and vii) R.sub.8 is C.sub.1-3alkyl.

3. (canceled)

4. The method according to claim 2 wherein R.sub.2 is selected from —OC(O)C.sub.3-6alkyl and —OC(O)C.sub.3-6alkenyl.

5. (canceled)

6. The method according to claim 2 wherein R.sub.3 is selected from —OC(O)C.sub.3-6alkyl, —OC(O)C.sub.3-6alkenyl and —OC(O)C.sub.3-6alkynyl.

7-8. (canceled)

9. The method according to claim 2 wherein R.sub.6 is selected from hydrogen, —C(O)CH.sub.3, —C(O)CH.sub.2CH.sub.3, —C(O)CH(CH.sub.3).sub.2 or —C(O)CH.sub.2CH.sub.2CH.sub.3.

10-11. (canceled)

12. The method according claim 1 wherein the alkyl or alkenyl group of R.sub.2 and/or R.sub.3 are branched alkyl or alkenyl groups.

13. The method according to claim 1 wherein the alkyl or alkenyl group of R.sub.2 and/or R.sub.3 are linear alkyl or alkenyl groups.

14. The method according to claim 1 wherein the compound of formula (I) is selected from: 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 1); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 2); 12-hexanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 3); 12,13-dihexanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 4); 12-tigloyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13-pentahydroxy-20-acetyloxy-1-tiglien-3-one (Compound 5); 12-propanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 6); 12,13-ditigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 7); 12-(2-methylbutanoyl)-13-tigloyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 8); 12-butanoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 9); 12-(3,3-dimethylbut-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 10); 12-hex-2,4-dienoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 11); 12-tigloyl-13-(2-methylpropanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 12); 12-but-2-enoyl-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 13); 12-tigloyl-13-butanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 14); 12,13-dibutanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 15); 12,13-dipentanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 16); 12,13-di-(2E,4E)-hexa-2,4-dienoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 17); 12,13-di-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one (Compound 18); 12-(2-methylprop-2-enoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (Compound 19); 12,13-di-heptanoyl-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (Compound 20); and 12,13-di-(3-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tigliaen-3-one (Compound 21); or a salt thereof.

15. The method according to claim 1 wherein the biofilm comprising Gram-negative bacteria comprises at least one Gram-negative bacteria selected from Pseudomonas species, Acinetobacter species, Aeromonas species, Bacteroides species, Bordetella species, Borrelia species, Burkholderia species, Citrobacter species, Campylobacter species, Escherichia species, Enterobacter species, Flavobacterium species, Fusobacterium species, Klebsiella species, Leptospira species, Neisseria species, Helicobacter species, Hemophilus species, Legionella species, Moraxella species, Yersinia species, Oligella species, Pantoea Species, Porphyromonas species, Prevotella species, Proteus species, Raoutella species, Salmonella species, Serratia species, Shigella species, Sphingomonas species, Stenotophomonas species, Treponema species, Veillonella species and Vibrio species.

16. The method according to claim 15 wherein the Gram-negative bacteria is selected from Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Hemophilus influenzae, Legionella pneumophila, Yersinia pestis, Yersinia enterocolitica, Salmonella enterica, Salmonella bongori, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Bacteroides fragilis, Fusobacterium necrophorum, Burkholderia cepacian and Prevotella intermedia.

17. A method of treating a bacterial infection comprising a biofilm comprising Gram-negative bacteria, said method comprising locally administering to the bacterial infection an epoxytiglienone compound of formula (I): ##STR00013## wherein R.sub.1 is selected from hydrogen and C.sub.1-6alkyl; R.sub.2 is selected from —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.3 is selected from —OH, —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is selected from hydrogen, —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —OC(O)C.sub.2-6alkynylaryl; R.sub.7 is selected from hydroxy, —OC.sub.1-6alkyl, —OC.sub.2-6alkenyl, —OC.sub.2-6alkynyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl, —OC(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —OC(O)aryl, —OC(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —C(O)C.sub.2-6alkynylaryl; R.sub.8 is selected from hydrogen and C.sub.1-6alkyl; or a pharmaceutically acceptable salt thereof.

18. The method according to claim 17 wherein the local administration is topical administration.

19. The method according to claim 17 wherein the infection is a post-surgical infection or an infection at the site of insertion of a medical device or implantation of an implant.

20. The method according to claim 17 wherein the bacterial infection is a chronic infection.

21. The method according to claim 17 wherein the administration is in combination with an antibiotic to which the Gram-negative bacteria is susceptible when in planktonic state.

22. A method of preventing a biofilm comprising Gram-negative bacteria forming on a medical device or dispersing a biofilm comprising Gram-negative bacteria on a medical device, said method comprising applying an epoxytiglienone compound of formula (I) to the medical device: the compound of formula (I) comprising ##STR00014## wherein R.sub.1 is selected from hydrogen and C.sub.1-6alkyl; R.sub.2 is selected from —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.3 is selected from —OH, —OC.sub.1-8alkyl, —OC.sub.2-8alkenyl, —OC.sub.2-8alkynyl, —OC(O)C.sub.1-7alkyl, —OC(O)C.sub.2-7alkenyl and —OC(O)C.sub.2-7alkynyl; R.sub.4 and R.sub.5 are independently selected from hydrogen and C.sub.1-6alkyl; R.sub.6 is selected from hydrogen, —C.sub.1-6alkyl, —C.sub.2-6alkenyl, —C.sub.2-6alkynyl, —C(O)C.sub.1-6alkyl, —C(O)C.sub.2-6alkenyl, —C(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —C(O)aryl, —C(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —OC(O)C.sub.2-6alkynylaryl; R.sub.7 is selected from hydroxy, —OC.sub.1-6alkyl, —OC.sub.2-6alkenyl, —OC.sub.2-6alkynyl, —OC(O)C.sub.1-6alkyl, —OC(O)C.sub.2-6alkenyl, —OC(O)C.sub.2-6alkynyl, —C(O)C.sub.3-8cycloalkyl, —C(O)C.sub.1-6alkylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkenylC.sub.3-8cycloalkyl, —C(O)C.sub.2-6alkynylC.sub.3-8cycloalkyl, —OC(O)aryl, —OC(O)C.sub.1-6alkylaryl, —C(O)C.sub.2-6alkenylaryl and —C(O)C.sub.2-6alkynylaryl; R.sub.8 is selected from hydrogen and C.sub.1-6alkyl; or a pharmaceutically acceptable salt thereof.

23. The method according to claim 22 wherein the medical device is a surgical instrument, catheter or medical implant.

24. A method according to claim 23 wherein the medical device is a catheter.

25. The method according to claim 23 wherein the medical device is a dental implant.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0096] FIG. 1 provides confocal laser scanning microscopy (CLSM) images showing biofilm disruption of E. coli IR57 following treatment with Compounds 1, 4 and 6 compared to the comparator compound CC-1, untreated and ethanol equivalent (blank) controls. Upper panel in each image group is overhead view, small lower panel is cross-sectional view.

[0097] FIG. 2 provides A: biofilm biomass volume or bio-volume (μm.sup.3/μm.sup.2) that was quantified and confirmed with COMSTAT image analysis after treatment of the E. coli IR57 biofilm with Compounds 1, 4 and 6, compared to the comparator compound CC-1, untreated and ethanol equivalent (blank) controls. Decreases in bio-volume were evident in biofilms treated with these compounds, which were significant with Compound 1 treatment (p<0.05). B: there was no significant differences between any of the compounds and control treatments in the DEAD/LIVE bacterial ratio, demonstrating disruption to the E. coli IR57 biofilm density and bio-volume in these experiments was not related to a direct antibiotic activity.

[0098] FIG. 3 provides a graph of the mean squared displacement (MSD) of 200 nm FluoSpheres® over time (in seconds) within E. coli IR57 biofilm structures. The disruption and alteration of the E. coli IR57 biofilm structure is illustrated by substantially higher mean squared displacement of the FluoSphere® particles following treatment with Compounds 1, 4 and 6, compared with an untreated and ethanol equivalent (blank) controls.

[0099] FIG. 4 provides a graph of increased biofilm creep compliance (demonstrating) decreased resistance to mechanical deformation of the biofilm medium derived from the MSD vs lag time as seen in FIG. 3) following treatment E. coli IR57 biofilm with Compounds 1, 4 and 6, compared to the untreated and ethanol equivalent (blank) controls.

[0100] FIG. 5 provides A: the CLSM images when Compounds 1, 4, 6 and the comparator compound CC-1 are applied to biofilms of A. baumannii 7789 compared to an untreated and ethanol equivalent (blank) control. Upper panel in each image is overhead view, small lower panel is cross-sectional view. B: the CLSM images when compounds 1, 4, 6 and the comparator compound CC-1 are applied to biofilms of P. aeruginosa PAO1 compared to an untreated and ethanol equivalent (blank) control. Upper panel in each image is overhead view, small lower panel is cross-sectional view.

[0101] FIG. 6 provides A: biofilm biomass volume or bio-volume (μm.sup.3/μm.sup.2) that was quantified and confirmed with COMSTAT image analysis after treatment of the A. baumannii 7789 biofilms with Compounds 1, 4, 6 and the comparator compound CC-1 compared to the untreated and ethanol equivalent (blank) controls. Significant decreases in bio-volume were evident in biofilms treated with compounds 1, 4 and comparator compound CC-1 (p<0.05). B: biofilm biomass volume or bio-volume (μm.sup.3/μm.sup.2) that was quantified and confirmed with COMSTAT image analysis after treatment of the P. aeruginosa PAO1 biofilms with Compounds 1, 4, 6 and the comparator compound CC-1 compared to the untreated and ethanol equivalent (blank) controls. Only Compound 4 had a significant effect on biofilm bio-volume compared to other treatments (P<0.05). C & D: there were no significant differences in the DEAD/LIVE bacterial ratio, that was quantified and confirmed with COMSTAT image analysis, after treatment of the A. baumannii 7789 and P. aeruginosa PAO1 biofilms with Compounds 1, 4, 6 and the comparator compound CC-1 compared to the untreated and ethanol equivalent (blank) controls.

[0102] FIG. 7 provides cell membrane permeabilization data when Compounds 1, 4 and 6 were applied to planktonic cells of E. coli IR57, P. aeruginosa PAO1 and S. aureus (1004A; MRSA) compared to the untreated control and positive control of 70% Isopropanol. The Gram-negative strains (E. coli IR57 and P. aeruginosa PAO1) only showed significant increases in cell permeabilization when treated with compounds 1 and 4 at concentration ≥512 μg/mL unlike the Gram-positive strain (MRSA 1004A) which demonstrated significant cell permeabilization at concentrations as low as 32 μg/mL.

[0103] FIG. 8 provides representative images showing the induction of NETosis and necrosis of neutrophils treated with six concentrations of Compound 4 at two times after treatment, 3 and 6 hours. NETosis/necrosis was first observed at 3 hours after treatment in the two highest concentrations (50 and 500 μM) and at 6 hours in the four lower concentrations. At 500 μM the chromatin in the cells is condensed and indicates necrosis has occurred. At 50 μM the chromatin is more diffuse indicating that NETosis has occurred.

[0104] FIG. 9 provides a graph with mean values and standard deviations for in vitro release of human defence peptide LL-37 from neutrophils at 3 hours after application of four therapeutically relevant concentrations of Compound 4. Release of LL-37 from neutrophils increased in a concentration dependent manner.

EXAMPLES

[0105] The compounds of the present invention may be obtained by isolation from a plant or plant part, or by derivatisation of the isolated compound, or by derivatisation of a related compound. Isolation procedures and derivatisation procedures may be found in WO 2007/070985 and WO2014/169356.

Example 1: Epoxy-Tigilanes have No Direct Antibiotic Activity Against Gram-Negative Bacteria in Planktonic Culture Systems

[0106] The effects of five epoxytiglienones (Compounds 1, 2, 3, 4 and 6) and two comparator comparator compounds (epoxytiglienones having longer carbon chains at the C.sub.12 position) on six human pathogenic bacteria (two Gram-positive and four Gram-negative species) were measured in conventional planktonic culture systems. A minimum inhibitory concentrations (MIC) assay (the standard assay employed to define and quantify antibiotic activity) was performed to determine antibacterial activity for each epoxytiglienone against each bacterial strain.

[0107] The Gram-positive bacteria used in this study were methicillin resistant Staphylococcus aureus (MRSA) 1004A and Streptococcus pyogenes. The Gram-negative bacteria used in this study were Pseudomonas aeruginosa PAO1, Escherichia coli IR57 (V7), Klebsiella pneumoniae, and Acinetobacter baumannii 7789 (V19). MIC assays were performed on planktonic cultures of each bacteria grown in Mueller-Hinton broth using the standard broth dilution method described by Jorgensen et al. (1999) using epoxytiglienones dissolved in ethanol. The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Minimum inhibitory concentrations (μg/mL) determined for Compounds 1, 2, 3, 4 and 6 and comparator compounds CC-1 and CC-2 against two Gram-positive bacteria and four Gram-negative bacteria. C = confluent growth, i.e. no MIC could be determined at any concentration tested. Data presented represent the mean of three replicate tests. Compound number Comparator* Bacteria 1 2 3 4 6 CC-1 CC-2 Gram-positive species S. aureus 256 256 256 512 C 8 16 S. pyogenes 128 128 128 256 C 8 8 Gram-negative species E. coli C C C C C C C P. aeruginosa C C C C C C C K. pneumoniae C C C C C C C A. baumannii C C C C C C C *Comparator compounds are epoxytiglienones having longer carbon chains at the C12 position. CC-1 is 12-(2,4-decadienoyl)-13-(2-methylbutanoyl)-6,7-epoxy-4,5,9,12,13,20-hexahydroxy-1-tiglien-3-one and CC-2 12R-(2,4-decadienoyl)-13R-(2-methylbutanoyl)-6,7-epoxy-4R,5R,9S,12R,13R,20-hexahydroxy-1-tiglien-3-one.

[0108] The results demonstrated that the direct antimicrobial properties of the epoxytiglienones in planktonic culture were restricted to the Gram-positive bacteria. No MIC value could be determined for any of the compounds with any of the Gram-negative bacteria tested, all of which were unaffected (with confluent growth of bacteria at concentrations of the test compounds exceeding 512 μg/mL).

Example 2: Epoxytiglienones Disrupt Established Biofilm of Gram-Negative E. coli

[0109] The effects of administration of Compounds 1, 4 and 6 and comparator compound CC-1 on disruption of established in vitro biofilms of Escherichia coli (E. coli) was investigated using methods described in Powell et al. (2018).

[0110] Biofilms of E. coli IR57 were grown on 96-well glass-bottomed plates in Mueller-Hinton (MH) broth for 24 h, before 50% of the supernatant was replaced with fresh MH broth with or without the epoxytiglienone compound added to a final concentration of 256 μg/mL (vehicle is ethanol). An ethanol equivalent blank was used as a further control treatment. The plates were then incubated for a further 24 h at 37° C.

[0111] Confocal laser scanning microscopy (CLSM) was then used to image the biofilms, following staining with Live/Dead® Baclight stain with phosphate buffered saline (PBS) added to each well before imaging with z-stack CLSM. The resultant images were analysed by COMSTAT software to produce measurements of (i) biofilm biomass volume or bio-volume (μm.sup.3/μm.sup.2) and (ii) DEAD/LIVE bacterial ratio.

[0112] CLSM demonstrated marked differences between treatments in the distribution of the bacteria in the biofilms. Compounds 1, 4 and 6 induced significant changes the distribution and density of bacteria in the biofilm compared to the untreated and ethanol equivalent (blank) controls (FIG. 1); this was quantified and confirmed with COMSTAT image analysis. Significant decreases in bio-volume were evident in biofilms treated with Compound 1 (p<0.05; FIG. 2A). The comparator compound CC-1 had no apparent effect on biofilm distribution and density or bio-volume. Interestingly, there were no significant differences between any of the epoxytiglienones and the two control treatments in the DEAD/LIVE bacterial ratios (FIG. 2B), demonstrating the effect of the epoxytiglienones on biofilm density and bio-volume in these experiments was unrelated to direct antibiotic activity.

Example 3: Epoxytiglienones Disrupt the Extracellular Matrix and Increase Particle Diffusion Through an Established Biofilm of the Gram-Negative E. coli Bacteria

[0113] The effect of three epoxytiglienones (Compounds 1, 4 and 6) on the assembly and permeability of established biofilms of E. coli was assessed using multiple particle tracking (MPT). MPT is a recently described technique, allowing simultaneous tracking of micron-size particles through biofilms using microscopy, from which the diffusion-based parameters of embedded particles within the extracellular polymeric matrix (EPS) of the biofilm can be determined (Cao et al. 2016). MPT measurements also allows for calculation of micro-rheological properties of the biofilm structure following treatment with test compounds.

[0114] E. coli biofilms were established and epoxytiglienone and control treatments applied as described in Example 2 above. Twenty-four hours after application of the treatments, the biofilms were stained with SYTO9® and 0.0025% of negatively-charged, carboxylate-modified FluoSpheres® (200 nm) added onto the biofilms and incubated for a further 2 hours. FluoSphere® particle movements within the biofilms were then captured on video using epifluorescence microscopy with a high frame-rate camera (33 ms). Particle trajectories were tracked using ImageJ softwaren (Mosaic) before calculation of three parameters of the 200 nm FluoSphere® particles: (i) ensemble diffusion coefficient (Deff), (ii) ensemble mean squared displacement (MSD) and (iii) creep compliance (a measure of resistance to mechanical deformation derived from the MSD vs lag time). Each treatment was replicated three times.

[0115] All three epoxytiglienone compounds that were tested increased particle diffusion through the established E. coli biofilms by between 80 and 420 times compared to the control treatments (Table 2).

TABLE-US-00002 TABLE 2 Diffusion coefficients for 200 nm negatively-charged, carboxylate-modified FluoSpheres ® particles in E. coli IR57 biofilms treated with epoxytiglienones. Diffusion coefficient (Deff) Compounds (cm.sup.2 .Math. s.sup.−1 × 10.sup.−9) ± standard deviation Compound 4 0.8449 ± 0.1250 Compound 1 0.6018 ± 0.0901 Compound 6 0.1607 ± 0.0104 No treatment controls  0.002 ± 0.0014 Ethanol only control 0.0155 ± 0.0052

[0116] Significant disruption and alteration to the structure of the E. coli biofilm matrix by the epoxytiglienones were illustrated by substantially higher mean squared displacement values of the FluoSphere® particles following treatment (FIG. 3) and decreased resistance to mechanical deformation in the treated biofilms was evident in the increase of the creep compliance of the epoxytiglienone treated biofilms versus controls (FIG. 4).

Example 4: Epoxytiglienones Significantly Decrease Biomass in Established Biofilms of Two Other Gram-Negative Pathogens P. aeruginosa and A. baumannii

[0117] The effects of the epoxytiglienones (Compounds 1, 4, 6 and the comparator compound CC-1) on disruption of established in vitro biofilms were further investigated with two other species of Gram-negative bacteria (Pseudomonas aeruginosa PA01 and Acinetobacter baumannii 7789) using methods described in Example 2 above. Cell permeabilisation following Compound 1, 4 and 6 treatment was also determined using SYTOX™ Green Nucleic Acid Stain with untreated control and positive control of 70% isopropanol.

[0118] Consistent with the results of the E. coli study, the epoxytiglienone compounds significantly changed the distribution and density of bacteria in the A. baumannii biofilms compared to the untreated and ethanol equivalent (blank) controls (FIG. 5A). This was also reflected in their effects as measured by bio-volume for A. baumannii biofilms (FIG. 6A), where significant reductions were seen for Compounds 1, 4 and CC-1. For P. aeruginosa only Compound 4 had a significant effect on biofilm distribution (FIG. 5B) and on bio-volume compared to other treatments (FIG. 6B). DEAD/LIVE bacterial ratios were also assessed and, as in Example 2 with E. coli, there were no differences between treatments in either A. baumannii or P. aeruginosa (FIGS. 6C & D). Cell permeabilization studies on E. coli and P. aeruginosa bacteria (FIG. 7) only showed significant increases in permeabilization when treated with Compounds 1 and 4 at concentrations ≥512 μg/mL, which is greater than the compound concentration used in the biofilm disruption assays (256 μg/mL). This further confirms that the effects of the epoxytiglienones on biofilm density and bio-volume in these experiments was unrelated to any direct, antibiotic activity in killing bacteria within the biofilms.

Example 5: Epoxytiglienones Resolve Established Biofilm In Vivo in a Mouse Model of Chronic Biofilm Infection

[0119] The effects of administration of Compound 4 on biofilm infection in vivo was studied in a diabetic murine model of chronic biofilm infection (Zhao et al. 2010). In this study we used methods described by Dhall et al. (2014) in which bacterial biofilm infections develop spontaneously following wound creation.

[0120] Briefly, db/db diabetic mice (>6 months old) were housed for 4-5 weeks in non-sterile conditions prior to the creation of wound on the back of each mouse with a 6 mm diameter excisional punch biopsy. Mice were then administered a catalase inhibitor intra-peritoneally (1 g/kg aminotriazole) and a glutathione peroxidase (GPx) inhibitor topically around the edge of the wound site (1 g/kg mercaptosuccinic acid) prior to dressing with Tegaderm. After 24 h, biofilms were evident at the wound site on all mice. The mice where divided into two groups (7 mice per group) and treated with either Compound 4 (0.3 mg/mL in a hydrogel vehicle) or a Control treatment (hydrogel vehicle only). The Tegaderm dressing was the then replaced. Two further administrations of Compound 4 or the Control (vehicle only) were performed at 8 and 15 days. No antibiotics or other antibacterial treatments were applied during the study.

[0121] The presence of biofilm and the surface area of the wound were assessed over the course of the study. Biopsies were taken from Compound 4-treated and vehicle-only (control) treated mice between day 21 and 28 post wounding, for histological and histochemical analysis of the wound site. Only 1 of the 7 wounds in the Control group healed within this period, with the presence of a fibrinous slough and bacterial infiltration of the wound site clearly evident. In epoxytiglienone-treated infected wounds, complete wound healing was evident (with complete re-epithelialisation, resolution of inflammation and an absence of bacteria within the dermis) in 6 of the 7 mice treated with Compound 4.

Example 6: Epoxytiglienone Treatment of Human Adult Keratinocytes and Fibroblasts In Vitro Induces Upregulation of Chemokines/Cytokines Involved in Neutrophil Recruitment

[0122] In addition to disrupting the structure of biofilms comprising Gram-negative bacteria, effects of epoxytiglienones on regulation of genes involved in host response to bacterial infection were investigated in microarray studies with human adult epidermal keratinocytes (HEKa) and human adult dermal fibroblasts (HDF) in vitro.

[0123] For these studies, HEKa were cultured in EpiLife® Medium supplemented with Supplement S7 (both from Life Technologies, Carlsbad, Calif., USA), while HDF were cultured in Medium 106 supplemented with LSGS (Low Serum Growth Supplement) (both from Life Technologies). Cells where then treated with vehicle or 170 nM Compound 4 for 0, 0.5, 1, 2, 4, 8, 24, 48 and 72 hours. RNA was extracted using a Qiagen RNeasy mini kit and biotinylated using an Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, Tex., USA). Labelled RNA was hybridised to HumanHT-12 v4 BeadChip Arrays (Illumina Inc, San Diego, Calif., USA) and scanned according to standard Illumina protocols. Data were extracted in GenomeStudio (Illumina) using default analysis settings and no normalisation method. Resulting data were imported into GeneSpring GX (Agilent, Santa Clara, Calif., USA. Expression values were normalised using quantile normalisation with default settings.

[0124] In both HDF and HEKa in vitro, treatment with Compound 4 (170 nM) induced significant upregulation of two critical chemokines/cytokines involved in PMNL recruitment (IL8, CXCL1) within 2 to 4 hours. In HEKa, Compound 4 significantly upregulated production of host defence peptides (DEFB2, DEFB3, DEFB4, RNASE7) from 8 to 72 hours post treatment.

Example 7: Epoxytiglienone Treatment Induces NETosis/Necrosis and Release of the Antimicrobial Peptide Cathelicidin LL-37 In Vitro from Isolated Human Neutrophils

[0125] Neutrophils are the most abundant leukocyte in the blood and constitute the first line of host defense against infectious pathogens. Central to their function is their ability to be recruited to sites of infection, to recognize microbes, and then be activated to kill pathogens through a combination of phagocytotic and cytotoxic mechanisms. Amongst these mechanisms used by neutrophils to kill pathogens are: (i) the production of reactive oxygen species, (ii) the expulsion of their nuclear chromatin contents (coated with histones, proteases and granular and cytosolic proteins) to immobilise and catch pathogens (the process of NETosis), and (iii) the release of antimicrobial peptides.

[0126] Subsequent to identifying the effect of epoxytiglienone compounds in upregulating production in HDF and HEKa of chemokines and cytokines involved in neutrophil recruitment (see Example 6 above), the effect of Compound 4 on two aspects of neutrophil function, induction of NETosis/necrosis and antimicrobial peptide release, were examined.

[0127] For these assays, neutrophils were isolated from fresh blood of a healthy human donor by lysis of a red blood cell pellet that had been obtained by Ficoll-Paque sedimentation. The neutrophils (˜4×10.sup.6 cells/mL) were incubated with 10 μg/mL dihydroethidium (DHE) (Sigma-Aldrich) in complete culture medium at 37° C. for 15 min alongside an aliquot of unstained cells to be tested as unstained control.

[0128] NETosis/necrosis assays followed methods fully described by Brinkmann et al. 2010. Isolated neutrophils were plated into 96-well plates (RPMI1640, 10% FCS) and incubated with a 1:50,000 dilution of Hoescht (10 mg/mL) and Sytox® Green (5 mM). Compound 4 was added at six concentrations (0.005, 0.05, 0.5. 5, 50, 500 μM) and the cells incubated at 37° C., 5% CO.sub.2. Vehicle treated controls were included in all assays and there were three replicates of each assay. Hoescht/Sytox® Green fluorescence images were recorded for each well at 3 and 6 hours.

[0129] NETosis and necrosis of neutrophils was initially observed at 3 hours after treatment in the two highest concentrations (50 μM and 500 μM) of Compound 4 and was evident at 6 hours in the four lower concentrations (0.005 μM-5 μM; FIG. 8).

[0130] To examine antimicrobial peptide release by neutrophils in response to treatment with Compound 4, isolated neutrophils were incubated with either vehicle or Compound 4 at the four concentrations (62.5, 125, 250 and 500 μM). At 3 h, cell culture supernatants were removed and tested for LL-37 content using a LL-37-directed ELISA kit (Hycult Biotech). ELISA readings were normalised to vehicle only controls to determine fold increases in LL-37 release.

[0131] Release of LL-37 from neutrophils at 3 hours after treatment with Compound 4 increased in a concentration dependent manner (FIG. 9). At concentrations of Compound 4 of between 125 and 500 μM, LL-37 release was 3- to 5-fold higher than the control treatment (FIG. 9).

[0132] The data from this Example show that at therapeutically relevant concentrations in vitro, Compound 4 induces suicidal neutrophil NETosis transitions to necrosis, leading to the release of the potent antimicrobial defence peptide LL-37.

[0133] The data from Examples 6 and 7 above also demonstrate that in addition to their direct effects in disrupting the structure of Gram-negative biofilms, epoxytiglienones can also induce local innate immune responses in both migratory/resident myeloid cells (e.g. neutrophils) and in dermal and stromal cell types. Such responses suggest that the effect of epoxytiglienones alone may be adequate to resolve many biofilm infections without the need for conventional antibiotics.

REFERENCES

[0134] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. [0135] Brinkmann V et al. 2010. Neutrophil extracellular traps: How to generate and visualize. Them. J. Vis. Exp. 36: e 1724. [0136] Cao, H. et al. (2016) Revealing region-specific biofilm viscoelastic properties by means of a micro-rheological approach. npj Biofilms and Microbiomes, 2(1), pp. 1-7. [0137] Cepas V et al. 2019. Relationship between biofilm formation and antimicrobial resistance in Gram-negative bacteria. Microb. Drug Resist. 25: 72-79. [0138] Dhall S et al. 2014. “Generating and Reversing Chronic Wounds in Diabetic Mice by Manipulating Wound Redox Parameters.” J. Diabetes Res. 562625. [0139] Doi et al. 2017. Gram-negative bacterial infections: Research priorities, accomplishments and future directions. Clin Infect Dis. 64 (S1): S30-S35 Fleming D & Rumbaugh K P 2017. Approaches to dispersing medical biofilms. Microorganisms 2017 5, 15. [0140] Gunn J S et al. 2016. What's on the outside matters: the role of extracellular polymeric substance of Gram-negative biofilms in evading host immunity and as a target for therapeutic intervention. J. Biol. Chem. 291: 12538-12546. [0141] Hoiby et al. 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 35: 322-332. [0142] Hoiby N et al. 2015. ESCMID guideline for the diagnosis and treatment of biofilm infections. Clin. Microbiol. Infect. 21 (Suppl. 1): S1-S25. [0143] Ho J et al. 2010. Multiresistant Gram-negative infections: a global perspective. Curr. Opin. Infect. Dis. 23: 546-53. [0144] Jorgensen J J H et al. 1999. Antibacterial susceptibility tests: dilution and disk diffusion methods. In Murray P R et al. (Eds.), Manual of Clinical Microbiology (pp 1526-1543). Washington, D C: ASM Press. [0145] Koo H et al. 2017. Targeting microbial biofilms: current and prospective therapeutic strategies. Nat. Rev. Microbiol. 15: 740-755. [0146] Powell et al. 2018 Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides. NPJ Biofilms Microbiomes. 4: 13.

[0147] Zhao et al. 2010. Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds. Wound Repair Regen. 18: 467-477.