Anti-Microbial Compositions

Abstract

The present invention relates to compounds, compositions, methods of forming/preparing such compounds and compositions, and uses for sanitising and/or substantially removing biofilms and microorganisms living within or around biofilms. The present invention, in particular, relates to compounds of formula Mn(P) as described herein which can be used in wound treatments, wound dressings, medical devices, water treatments, food processing and dental care biofilms.

Claims

1. A compound of formula M.sub.n(P), wherein n is an integer from 2 to 6; each M is independently a metal ion; Mn comprises at least two different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions; and P is an aminopolycarboxyl component comprising an optionally substituted alkylene amino backbone containing from 3 to 5 nitrogen atoms in the backbone and 5 or 6 carboxyl groups appended to the backbone, wherein the aminopolycarboxyl component contains from 10 to 20 atoms in the longest linear chain.

2. The compound according to claim 1, wherein P is a compound according to formula (I): ##STR00003## wherein: X is 1 or 2 each Y group is independently H or a negative charge wherein at least two Y groups are negative charges; and R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are each independently optionally substituted C.sub.1-3alkylene.

3. The compound according to claim 2, wherein R.sub.3 and R.sub.4 are each optionally substituted ethylene and/or wherein R.sub.1, R.sub.2, R.sub.5, R.sub.6 and R.sub.7 are each optionally substituted methylene or ethylene.

4. The compound according to claim 3, wherein R.sub.3 and R.sub.4 are each ethylene and R.sub.1, R.sub.2, R.sub.5, R.sub.6 and R.sub.7 are each methylene.

5. The compound according to claim 1, wherein M.sub.n comprises at least one Ag ion and at least one Cu ion or wherein Mn comprises at least one Ag ion and at least one Zn ion.

6. The compound according to claim 1, wherein the compound is selected from AgCu(DPTA), AgZn(DPTA), AgAl(DPTA), AgBi(DPTA), AgMo(DPTA), AgSr(DPTA), Ag.sub.2Cu(DPTA), Ag.sub.2Zn(DPTA), Ag.sub.2Al(DPTA), Ag.sub.2Bi(DPTA), Ag.sub.3Cu(DPTA), Ag.sub.3Zn(DPTA), AgCu(TTHA), AgZn(TTHA), AgAl(TTHA), AgBi(TTHA), AgMo(TTHA), AgSr(TTHA) Ag.sub.2Cu(TTHA), Ag2Zn(TTHA), Ag.sub.2Al(TTHA), Ag.sub.2Bi(TTHA), Ag.sub.3Cu(TTHA), Ag.sub.3Zn(TTHA), Ag.sub.3Al(TTHA) Ag.sub.3Bi(TTHA), Ag.sub.4Cu(TTHA) and Ag.sub.4Zn(TTHA).

7. A composition comprising the compound of claim 1 and one or more pharmaceutically acceptable excipients.

8. The composition according to claim 7, wherein the composition is a hydrogel or an aqueous solution, a lotion, an ointment, a cream, a balm, a gel, a paste or a solid.

9. The composition according to claim 7, wherein the composition comprises fibres which are in contact with the one or more compounds, wherein the one or more compounds are provided on the surface of the fibres, and-/-or wherein the one or more compounds are incorporated within the fibres.

10. The composition according to claim 7, wherein the composition further comprises a non-metal ion anti-microbial agent, surfactant, and/or further metal ion chelator.

11. A method of forming a compound of formula M.sub.n(P), wherein n is an integer from 2 to 6; each M is independently a metal ion; Mn comprises at least two different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions; and P is an aminopolycarboxyl component comprising an optionally substituted alkylene amino backbone containing from 3 to 5 nitrogen atoms in the backbone and 5 or 6 carboxyl groups appended to the backbone, wherein the aminopolycarboxyl component contains from 10 to 20 atoms in the longest linear chain, the method comprising: providing a solution comprising P; contacting the solution comprising P with a first metal ion source comprising a first metal ion M and a second metal ion source comprising a second metal ion M to form the compound of formula M.sub.n(P); wherein the first metal ion M and second metal ion M are independently selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions, and the first metal ion M is different to the second metal ion M.

12. The method according to claim 11, wherein: the first metal ion source is an Ag metal ion source and the second metal ion source is a Cu metal ion source; or the first metal ion source is a Cu metal ion source and the second metal ion source is a Ag metal ion source; or the first metal ion source is an Ag metal ion source and the second metal ion source is a Zn metal ion source; or wherein the first metal ion source is a Zn metal ion source and the second metal ion source is a Ag metal ion source.

13. A method of preparing an anti-microbial composition comprising at least two different metal ions selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions and a component P, wherein P is an aminopolycarboxyl component comprising an optionally substituted alkylene amino backbone containing from 3 to 5 nitrogen atoms in the backbone and 5 or 6 carboxyl groups appended to the backbone, wherein the aminopolycarboxyl component contains from 10 to 20 atoms in the longest linear chain, the method comprising: providing a solution comprising component P; contacting the solution comprising component P with a first metal ion source comprising a first metal ion M selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions; and a second metal ion source comprising a second metal ion M that is different to the first metal ion M, the second metal ion M selected from Ag, Al, Au, Ba, Bi, Tl, Ce, Co, Cu, Fe, Ga, Ge, Ir, Mo, Rh, Ru, Sb, Se, Sn, Sr, Ti and Zn ions, to form the anti-microbial composition; wherein the molar ratio of the component P, first metal ion source and second metal ion source (P : first metal ion : second metal ion) is from about 1 : 2 : 2 to about 1 : 10 : 10 or from about 1 : 0.1 : 0.1 to about 1 : 10 : 10.

14. An anti-microbial composition obtained by the method according to claim 13.

15. A wound dressing or medical device comprising the compound according to claim 1.

16. A method for sanitising or substantially removing a biofilm from a substrate, the method comprising contacting the substrate with the compound according to claim 1, wherein the method excludes using the compound or composition in a method for treatment of the human or animal body by surgery or therapy.

17. (canceled)

18. The method of claim 16, wherein the substrate is a wound on a human or animal body.

19. A method for treating infections of cuts, bruises, surgical sites, lacerations, abrasions, punctures, incisions, gunshots, bums, pyoderma, atopic dermatitis, eczema, pressure ulcers, venous and artery leg ulcers, diabetic foot ulcers, cystic fibrosis (CF)-associated infections, mastitis, otitis, community or hospital acquired infections, or food-borne diseases in a subject in need thereof the method comprising treating the subject with a pharmaceutically effective amount of the compound according to claim 1.

Description

DESCRIPTIONS OF THE FIGURES

[0192] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying Figures in which:

[0193] FIG. 1 is a powder X-Ray diffractogram of Ag.sub.3CuDTPA.

[0194] FIG. 2 is a power X-Ray diffractogram of copper sulphate.

[0195] FIG. 3 is a powder X-Ray diffractogram of silver nitrate.

[0196] FIG. 4 is a powder X-Ray diffractogram of DPTA.

EXAMPLES

[0197] The invention, and the surprising benefits afforded over the prior art, will be illustrated with reference to the following non-limiting examples.

[0198] As mentioned above, it has been unexpectedly observed that the efficacy of the metal aminopolycarboxyl compounds of the present invention are significantly greater than the EDTA based compounds described in WO2017/191453. This is evidenced by the minimum inhibitory concentration (MIC) data provided below.

Preparation of Compounds of the Present Invention

[0199] Compounds of the present invention may be suitably synthesised according to the following exemplary synthesis methods.

Method 1 - Preparation of Ag.SUB.3.Cu(DTPA) (Analogous to the Methods Disclosed in WO2017/191453A1)

[0200] A stock solution of DTPA sodium salt was made by dissolving 1.68 g DTPA in 18.12 g demineralised water plus 1.2 g of 40% aqueous NaOH solution. 3.6 g of the DTPA solution was added to a solution of 0.6 g silver nitrate (AgNO.sub.3) made up to 3.5 g with demineralised water and the mixture shaken for 30 seconds. The resulting milky white liquid was then centrifuged at 2500 rpm for 3 minutes. The supernatant liquid containing sodium nitrate by-product was then removed using a pipette leaving a solid containing the silver/DPTA complex. The silver/DPTA solid was then dissolved in a solution of 0.47 g copper sulphate pentahydrate (CuSO.sub.4.Math.5H.sub.2O) made up to 9.34 g with demineralised water to form the final compound Ag.sub.3Cu(DPTA).

Method 2 - Preparation of Ag.SUB.3.Cu(DTPA) Without the Step of Centrifugation Separation

[0201] A stock solution of DTPA sodium salt was made by dissolving 1.68 g DTPA in 18.12 g demineralised water plus 1.2 g of 40% aqueous NaOH solution. 3.6 g of the DTPA solution was added to a solution of 0.6 g silver nitrate (AgNO.sub.3) made up to 3.5 g with demineralised water and the mixture shaken for 30 seconds. The resulting solution was then directly combined with a solution of 0.47 g copper sulphate pentahydrate (CuSO.sub.4.Math.5H.sub.2O) made up to 9.34 g with demineralised water to form the final compound Ag.sub.3Cu(DPTA).

Method 3 - Preparation of Ag.SUB.3.Cu(DTPA) Where Copper Sulphate (CuSO.SUB.4..Math.5H.SUB.2.O) is Introduced Before Silver Nitrate (AgNO.SUB.3.)

[0202] 0.84 g DTPA was dissolved in 2.66 g NaOH solution containing demineralised water and 0.58 g 40% of NaOH to produce a DTPA sodium salt solution. 1.8 g silver nitrate (AgNO.sub.3) was dissolved in 1.7 g demineralised water to produce a silver nitrate solution. 1.41 g copper sulphate pentahydrate (CuSO.sub.4.Math.5H.sub.2O) was dissolved in 7.93 g demineralised water to produce a copper sulphate solution. Copper sulphate solution was added to the DTPA solution with vigorous stirring. A dark blue clear solution was formed. Silver nitrate solution was added to the clear blue solution to form the final compound Ag.sub.3Cu(DTPA). After stirring the mixture remained as a dark blue clear solution.

[0203] It was found that when performing a method using identical starting materials and reagents to that of method 3 above, but instead introducing the silver nitrate solution to the DTPA sodium salt solution before then adding the copper sulphate solution, a milky white dispersion was formed containing a solid DPTA/silver intermediate species. Method 3 involves the addition of the copper sulphate solution to the DTPA sodium salt solution first forms a dark blue clear solution containing a DPTA/copper intermediate species. This sequence of method steps is advantageous since all materials remain in solution during the entire method, which as a result, improves reaction times and yields. In addition, this is advantageous when working on larger scales since there is no need for an intermediate dissolution step to aid the solubility of the DPTA silver intermediate species. Higher concentrations of final compound solutions are achievable when using method 3 described above.

[0204] A further variant of the preparation process may also involve pre-mixing the two or more metal salt solutions (e.g. silver nitrate solution and copper nitrate solution) be adding the mixed metal salt solution to the DTPA sodium salt solution. The skilled person understand that this is only desirable where the two or more metal salts solutions are miscible. One particular example of this has been observed using a combined mixture of copper nitrate solution and silver nitrate solution which forms a miscible mixture of silver and copper. This can then be used with a DTPA sodium salt solution to successfully form a silver/copper DTPA compound according to the present invention.

Concentration Studies

Ag.SUB.3.CuDPTA

[0205] 1.53 g DTPA was dissolved in 4.37 g demineralised water plus 2.10 g 40% NaOH to produce a DTPA sodium salt solution. 3.29 g silver nitrate was dissolved in 4.71 g demineralised water to produce a silver nitrate solution. 2.58 g copper sulphate pentahydrate was dissolved in 16.09 g demineralised water to produce a copper sulphate solution. Silver nitrate solution was added to the DPTA solution with vigorous stirring. A milky white dispersion was formed. Copper sulphate solution was added to the white dispersion. With vigorous stirring the white precipitate dissolved to give a clear, blue liquid, signifying the formation of the silver/copper DTPA mixed metal complex (MMC). The total soluble solids content of the solution was measured and found to be 22.5% w/w. The theoretical concentration of silver/copper DTPA MMC is 9.2% (0.15 mol/Kg). Comparative concentration studies performed on Ag/Cu EDTA MMC examples provided theoretical concentration values of 0.07-0.09 mol/Kg.

Ag.SUB.3.ZnDPTA

[0206] 1.11 g DTPA was dissolved in 4.59 g demineralised water plus 1.3 g 40% NaOH to produce a DTPA sodium salt solution. 2.39 g silver nitrate was dissolved in 4.61 g demineralised water to produce a silver nitrate solution. 2.08 g zinc sulphate monohydrate was dissolved in 15.92 g demineralised water to produce a zinc sulphate solution. Silver nitrate solution was added to the DTPA solution with vigorous stirring. A milky white dispersion was formed. Zinc sulphate solution was added to the white dispersion. With vigorous stirring the white precipitate dissolved to give a clear, light brown liquid, signifying the formation of silver/zinc-DTPA mixed metal complex. The total soluble solids content of the solution was measured and found to be 24.7% w/w. The theoretical concentration of silver/copper DTPA MMC is 6.8% (0.11 mol/Kg). Comparative concentration studies performed on Ag/Zn EDTA MMC examples provided theoretical concentration values of 0.04-0.06 mol/Kg

[0207] These concentration studies illustrate that the DPTA complexes of the present invention is capable of being loaded into an aqueous solution at a high concentration as compared to their EDTA equivalents.

Preparation of Ag.SUB.2.Al(DTPA)

[0208] 0.29 g DTPA was dissolved in 2.93 g demineralised water plus 0.28 g of 40% NaOH to produce a DTPA sodium salt solution. 0.24 g silver nitrate (AgNO.sub.3) was dissolved in 3.26 g demineralised water to produce a silver nitrate solution. 0.66 g aluminium sulphate hexadecahydrate (Al.sub.2H.sub.32O.sub.28S.sub.3) was dissolved in 8.34 g demineralised water to produce an aluminium sulphate solution. Silver nitrate solution was added to the DTPA solution with vigorous stirring. A milky white dispersion was formed. Aluminium sulphate solution was added to the white dispersion. With vigorous stirring the white precipitate dissolved to give a clear, colourless liquid, signifying the formation of Ag.sub.2Al(DTPA) mixed metal complex.

[0209] The total soluble solids content of the solution was measured and found to be 7.2% w/w. The theoretical concentration of Ag.sub.2AlDTPA is 2.9%.

Preparation of Ag.SUB.2.Bi(DPTA)

[0210] A DTPA sodium salt solution was formed by dissolving 1 g DTPA in 10.4 g demineralised water plus 0.6 g of 40% NaOH solution. 0.15 g bismuth nitrate (Bi(NO.sub.3).sub.3)0.5H.sub.2O was added to the DTPA solution and stirred for 1 hour until the bismuth nitrate dissolved to form a bismuth/OPTA solution. Silver nitrate solution was formed by dissolving 0.24 g silver nitrate (AgNO.sub.3) in 3.26 g demineralised water. 0.62 g of the silver nitrate solution was added dropwise to 3 g of the bismuth/DTPA solution whilst stirring. At this point a clear and colourless solution was formed. A theoretical total solids concentration is 9.7%.The theoretical concentration of Ag.sub.2BiDTPA is 0.02 mol/Kg.

General Outline of MIC Measurement Method

[0211] MIC measurements were performed using an adapted method described in the Clinical and Laboratory Standards Institute (CLSI) guidelines M07-A11. Compounds were serial diluted two-fold in Mueller Hinton Broth (MHB) in 96 well plates. A positive growth control and a negative growth control were also included in the well plates.

[0212] A colony suspension of S. aureus (ATCC 29213), P. aeruginosa (ATCC 15442) and C. albican was prepared by taking several colonies from a fresh agar plate and suspending them in MHB. The suspension was adjusted to 0.5 McFarland (~1 × 10.sup.8 CFU/mL) and diluted 1:100. The inoculum was then added to wells at a final concentration of ~5 × 10.sup.6 CFU/mL. Plates were incubated overnight at 37° C. The following day, MICs were determined as the lowest concentration that no growth could be visually observed.

Anti-Biofilm Ability - MIC Measurements

MIC Measurements for Uncomplexed DPTA and EDTA Compounds

[0213] An 80 mg/ml solution of T-EDTA was prepared by dissolving T-EDTA (99% pure, Acros Organics) in sterile distilled water. The pH of this solution was measured at 10.5, using a pH electrode. A 16 mg/ml solution of DTPA was prepared by dissolving DTPA (98%+ Acros Organics) in distilled water, and adjusting pH to 10.5 using the addition of sodium hydroxide.

[0214] Table 1 shows the MIC results measured for both T-EDTA and DTPA compounds (which are not complexed to metal ions) as well as the metal aminopolycarboxyl compounds Ag.sub.2Cu(EDTA), Ag.sub.3Cu(DPTA), Ag.sub.2Al(DTPA) and Ag.sub.2Bi(DTPA). It can be seen that only modest increases in potency against P. aeruginosa and S. aureus are observed (2.5 and 1.25 fold respectively) for the uncomplexed DTPA as compared to the uncomplexed T-EDTA. In contrast, the results show that when comparing MIC potency measurements for Ag.sub.2Cu(EDTA) and Ag.sub.3Cu(DPTA) there is a significant increase in potency for the DPTA compound. In particular, there is an approximately 16 fold increase against S. aureus and an approximately 4 fold increase against P. aeruginosa when comparing the MIC values for Ag.sub.3Cu(DPTA) versus the prior art compound Ag.sub.2Cu(EDTA). This would not have been expected based on the MIC results for both T-EDTA and DTPA compounds.

TABLE-US-00001 P. aeruginosa (ATCC 15442) S. aureus (ATCC 29213) C. albican (ATCC 10231) MIC (.Math.g/ml) MIC (.Math.g/ml) MIC (.Math.g/ml) T-EDTA 5000 1250 - DTPA 2000 1000 - Ag.sub.2Cu(EDTA) 6 98 - Ag.sub.3Cu(DPTA) 1.6 6 - Ag.sub.2Al(DTPA) 450 370 530 Ag.sub.2Bi(DTPA) 94 190 11

Minimum Inhibitory Concentration (MIC) Assay for AgZnDTPA and AgCuDTPA Complexes With Non-Metal Additives

[0215] The MIC values for AgZnDTPA and AgCuDTPA (with and without additives) were determined against Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 15442. The DTPA complexes compositions with and without additives were serial diluted two fold in Mueller Hinton Broth (MHB) in 96 well plates as mentioned above. The plates were then inoculated with the bacterial strains as described above and the following day, MICs were determined as the lowest concentration that no growth could be visually observed.

Results

[0216] AgZnDTPA and AgCuDTPA showed potent antimicrobial activity against both bacterial strains with MICs 0.016 against S. aureus and 0.0009 against P. aeruginosa. Following combination of additives such as Tris and Urea an 80 fold and 9 fold increase in MIC of both DTPA complexes were found against S. aureus and P. aeruginosa, respectively. Additionally, combination of the DTPA complexes with antimicrobials at subtherapeutic concentrations (0.25x MIC) was found to increase the potency of the complexes, with iodine increasing the MIC by ≥160 fold against both bacterial strains and hypochlorous acid (Suprox) increasing the potency 9 and 16 fold against P. aeruginosa and S. aureus, respectively.

TABLE-US-00002 Bacterial Strain Complex Additive MIC Fold Change S. aureus ATCC 29213 AgZnDTPA No additive 0.016 - 1.5% Tris 0.0002 80 25% Suprox 0.001 16 0.04% Iodine ≤0.0001 ≥160 AgCuDTPA No additive 0.016 - 1.5% Tris 0.0002 80 25% Suprox 0.001 16 0.04% Iodine ≤0.0001 ≥160 P. aeruginosa ATCC 15442 AgZnDTPA - 0.0009 - 1.5% Urea 0.0001 (9 fold) 9 50% Suprox 0.0001 (9 fold) 9 0.04% Iodine ≤0.00002 ≥45 fold AgCuDTPA - 0.0009 - 1.5% Urea 0.0001 (9 fold) 9 50% Suprox 0.0001 (9 fold) 9 0.04% Iodine ≤0.00002 ≥45 fold

Log Reduction Assay for AgZnDTPA and AgCuDTPA Complexes With Non-Metal Additives

[0217] The speed of kill of two DTPA metal complexes, AgZnDTPA and AgCuDTPA was evaluated against S. aureus ATCC 29213 and P. aeruginosa ATCC 15442. The DTPA complexes were diluted to 8x and 16x MIC in MHB and inoculated with the strains at a final bacterial cell density of 1 × 106 CFU/mL. Tubes were incubated at 37° C. and 125 rpm for 24 hours and samples were collected at 3 hours and 24 hours. Collected samples were added to Dey-Engley neutralising broth at a 1:10 ratio, vortexed and serial diluted 1:10 in PBS before spot plating the dilutions onto trypticase soy agar (TSA). Plates were incubated overnight at 37° C. and the following day bacterial colonies were enumerated. The log reduction was calculated as the difference in bacterial cell density in treated samples compared to untreated growth controls.

Results

[0218] At 16x MIC (0.256%) both DTPA complexes showed complete eradication of S. aureus by 24 hours, with a 9.1 log reduction in bacterial cell density. At 16x MIC (0.0072%) both DTPA complexes showed complete eradication of P. aeruginosa by 3 hours, with a 7 log reduction in bacterial cell density. Following addition of 1.5% Tris an enhanced speed of kill was found with AgZnDTPA at 8x MIC, with complete eradication of S. aureus by 3 hours (7.1 log reduction).

Conclusion

[0219] The mixed metal complexes AgZnDTPA and AgCuDTPA both showed potent antimicrobial activity against representative Gram-positive and Gram-negative strains S. aureus and P. aeruginosa. Enhanced antimicrobial activity was found when additives or antimicrobials at subtherapeutic concentrations such as Tris, Urea, Hypochlorous Acid and Iodine were included.

[0220] Additionally, the DTPA complexes demonstrated rapid speed of kill, with eradication of P. aeruginosa by 3 hours and S. aureus by 24 hours. The speed of kill of S. aureus was increased following inclusion of an additive 1.5% Tris, with eradication found by 3 hours.

Structural Characterisation of the Compounds of the Present Invention

[0221] Compounds of the present invention have been characterised using X-Ray Powder Diffraction (XRD) techniques known to the person skilled in the art.

[0222] FIG. 1 illustrates an X-Ray powder diffractogram of Ag.sub.3CuDTPA prepared according to the preparation method 3 described above with the additional steps of drying the product in a recirculating oven at 60° C. to constant weight, then turning the raw solid into a powder for diffraction analysis. FIGS. 2 to 4 show X-Ray diffractograms measured for the starting materials copper sulphate, silver nitrate and DPTA. When comparing the diffractogram of Ag.sub.3CuDTPA with the raw materials DTPA, copper sulphate, silver nitrate it is evident that the diffractogram for the Ag.sub.3CuDTPA complex contains peaks at different positions to the raw materials and that the peaks present in the starting material diffractograms are generally absent. For example, the major reflections of DTPA at 20.5, 24.9 and 32.5 2-theta degrees are substantially absent from the Ag.sub.3CuDTPA diffractogram and the major reflections of silver nitrate at 29.8 two-theta degrees and copper sulphate at 20.2 two-theta degrees are completely absent. The X-Ray powder diffractogram data confirms the formation of the Ag.sub.3CuDTPA complex according to the present invention.