Use of triazolo(4,5-d)pyrimidine derivatives for prevention and treatment of bacterial infection

Abstract

A method for treatment of a bacterial infection in a host mammal in need of such treatment or a method of administering to the host mammal an effective amount of a Triazolo(4,5-d)pyrimidine of formula(I): ##STR00001##
wherein R.sup.1 is C.sub.3-5 alkyl optionally substituted by one or more halogen atoms; R.sup.2 is a phenyl group, optionally substituted by one or more halogen atoms; R.sup.3 and R.sup.4 are both hydroxyl; R is XOH, wherein X is CH.sub.2, OCH.sub.2CH.sub.2, or a bond, and wherein when X is a bond, R is OH; or a pharmaceutical acceptable salt, provided that when X is CH.sub.2 or a bond, R.sup.1 is not propyl; when X is CH.sub.2 and R.sup.1 is CH.sub.2CH.sub.2CF.sub.3, butyl or pentyl, the phenyl group at R.sup.2 must be substituted by fluorine; when X is OCH.sub.2CH.sub.2 and R.sup.1 is propyl, the phenyl group at R.sup.2 must be substituted by fluorine.

Claims

1. A method for treatment of a bacterial infection in a host mammal in need of such treatment comprising administering to the host mammal an effective amount of a Triazolo(4,5-d)pyrimidine of formula (1): ##STR00014## wherein R.sup.1 is C.sub.3-5 alkyl optionally substituted by one or more halogen atoms; R.sup.2 is a phenyl group, optionally substituted by one or more halogen atoms; R.sup.3 and R.sup.4 are both hydroxyl; R is XOH, wherein X is CH.sub.2, or a bond, and wherein when X is a bond, R is OH; or a pharmaceutical acceptable salt, provided that when X is CH.sub.2 or a bond, R.sup.1 is not propyl; when X is CH.sub.2 and R.sup.1 is CH.sub.2CH.sub.2CF.sub.3, butyl or pentyl, the phenyl group at R.sup.2 must be substituted by fluorine.

2. The method according to claim 1 wherein R.sup.2 is phenyl substituted by fluorine atoms.

3. The method according to claim 1, wherein R is OH.

4. The method according to claim 1, wherein the Triazolo(4,5-d)pyrimidine of formula (1) is selected from the group consisting of: (1R-(1, 2, 3(1R*, 2*),5))-3-(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)-5-((3,3,3-trifluoropropyl)thio)3H-1,2,3-triazolo(4,5d)pyrimidin-3-yl)5(hydroxy)cyclopentane-1,2-diol; (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-y]-1,2-3cyclopentanetriol; and a pharmaceutical acceptable salt.

5. The method according to claim 1, wherein the Triazolo(4,5-d)pyrimidine of formula (1) is (1S,2R,3 S,4R)-4-[7-[[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also called Fluometacyl.

6. The method according to claim 1, wherein the effective amount to be administered to the host mammal is less than 1.8 g per day.

7. A method of killing bacteria or reducing bacterial growth in a biofilm formation comprising applying on a surface an effective amount of a Triazolo(4,5-d)pyrimidine of formula (1): ##STR00015## wherein R.sup.1 is C.sub.3-5 alkyl optionally substituted by one or more halogen atoms; R.sup.2 is a phenyl group, optionally substituted by one or more halogen atoms; R.sup.3 and R.sup.4 are both hydroxyl; R is XOH, wherein X is CH.sub.2, OCH.sub.2CH.sub.2, or a bond, and wherein when X is a bond, R is OH; or a pharmaceutical acceptable salt, provided that when X is CH.sub.2 or a bond, R.sup.1 is not propyl; when X is CH.sub.2 and R.sup.1 is CH.sub.2CH.sub.2CF.sub.3, butyl or pentyl, the phenyl group at R.sup.2 must be substituted by fluorine; when X is OCH.sub.2CH.sub.2 and R.sup.1 is propyl, the phenyl group at R.sup.2 must be substituted by fluorine.

8. The method according to claim 7 wherein R.sup.2 is phenyl substituted by fluorine atoms.

9. The method according to claim 7, wherein R is OH or OCH.sub.2CH.sub.2OH.

10. The method according to claim 7, wherein R is OH.

11. The method according to claim 7, wherein the Triazolo(4,5-d)pyrimidine derivative of formula (1) is selected from the group consisting of: (1R-(1, 2, 3(1R*, 2*),5))-3-(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)-5-((3,3,3-trifluoropropyl)thio)3H-1,2,3-triazolo(4,5d)pyrimidin-3-yl)5(hydroxy)cyclopentane-1,2-diol; (1S-(1, 2, 3(1R*, 2*),5))-3-(7-((2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-1,2,3-triazolo(4,5d)pyrimidin-3-yl)5(2-hydroxyethoxy)cyclopentane-1,2-diol; (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol; (1S,2S,3R,5S)-3-[7-[1R,2S)-2-(4-fluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol; (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-y]-1,2-3 cyclopentanetriol; and a pharmaceutical acceptable salt.

12. The method according to claim 7, wherein the Triazolo(4,5-d)pyrimidine derivative of formula (1) is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, also called Triafluocyl.

13. The method according to claim 7, wherein the Triazolo(4,5-d)pyrimidine of formula (1) is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also called Fluometacyl.

14. The method according to claim 7, wherein the effective amount is between 0.1 and 1000 g/ml.

15. The method according to claim 7, wherein the surface is located on a medical device.

16. The method according to claim 7 wherein the surface is located on a biomaterial.

17. The method according to claim 15, wherein the medical device is a heart valve.

18. The method according to claim 15, wherein the medical device is a catheter.

19. A method of reducing risk of acquiring a bacterial infection in a host mammal comprising administering to the host mammal an effective amount of a Triazolo(4,5-d)pyrimidine of formula (1): ##STR00016## wherein R.sup.1 is C.sub.3-5 alkyl optionally substituted by one or more halogen atoms; R.sup.2 is a phenyl group, optionally substituted by one or more halogen atoms; R.sup.3 and R.sup.4 are both hydroxyl; R is XOH, wherein X is CH.sub.2 or a bond, and wherein when X is a bond, R is OH; or a pharmaceutical acceptable salt, provided that when X is CH.sub.2 or a bond, R.sup.1 is not propyl; when X is CH.sub.2 and R.sup.1 is CH.sub.2CH.sub.2CF.sub.3, butyl or pentyl, the phenyl group at R.sup.2 must be substituted by fluorine.

20. The method according to claim 19 wherein the Triazolo(4,5-d)pyrimidine of formula (1) is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also called Fluometacyl.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Staphylococcus aureus. Growth curves (A) and viable counts (B) in the presence of different concentrations of Triafluocyl or DMSO as vehicle are shown.

(2) FIG. 2 illustrates an inhibition of Staphylococcus aureus biofilm formation by Triafluocyl at stage 2.

(3) FIG. 3 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Enterococcus faecalis. Growth curves (A) and viable count (B) in the presence of different concentrations of Triafluocyl or DMSO as vehicle are shown.

(4) FIG. 4 illustrates an inhibition of Enterococcus faecalis biofilm formation by Triafluocyl at stage 2.

(5) FIG. 5 illustrates a bacteriostatic and bactericidal effect of Triafluocyl on Staphylococcus epidermidis. Growth curve (upper panel) and viable count (lower panel) in the presence of different concentrations of Triafluocyl or DMSO as vehicle.

(6) FIG. 6 illustrates an inhibition of Staphylococcus epidermidis biofilm formation at stage 2 by Triafluocyl.

(7) FIG. 7 illustrates a destruction of mature biofilm (stage 3: 24-hour biofilm) by Triafluocyl. Viable count of S. epidermidis biofilm after a 24h treatment with Triafluocyl (upper panel). Percentage of live cells in the biofilm (lower panel).

(8) FIG. 8 illustrates bactericidal activity against MRSA, GISA and VRE strains of Triafluocyl as compared to Vancomycin and Mynocycline:

(9) (A) illustrates a killing curve for methilcillin-resistant S. aureus (MRSA).

(10) (B) illustrates a killing curve for Glycopeptide intermediate-resistant S. aureus (GISA).

(11) (C) illustrates a killing curve for vancomycin resistant E. faecalis (VRE).

(12) FIG. 9 illustrates bactericidal activity of Fluometacyl against S. aureus MRSA.

(13) FIGS. 10 (A) and (B) illustrate the antibacterial effect of different concentrations of Fluometacyl on S. aureus and S. epidermidis biofilm formation respectively.

(14) FIG. 11 illustrates an Antibacterial effect of Triafluocyl together with Polymyxin B nonapeptide on Escherichia coli (ATCC 8739). Effect of Triafluocyl and polymixin B nonapeptide on the growth of E. coli (ATCC 8739) determined by measuring the optical density (A) for MIC determination or the CFU (B) for minimal bactericidal concentration (MBC) after 24 hr incubation at 37 C.

(15) FIG. 12 illustrates an Antibacterial effect of Fluometacyl together with Polymyxin B nonapeptide on Escherichia coli (ATCC 8739).

(16) Effect of Fluometacyl and polymixin B nonapeptide on the growth of E. coli (ATCC 8739) determined by measuring the optical density (A) for MIC determination or the CFU (B) for minimal bactericidal concentration (MBC) after 24 hr incubation at 37 C.

(17) FIG. 13 illustrates an Antibacterial effect of Triafluocyl together with C12(7)K12 on Escherichia coli (ATCC 8739). Effect of Triafluocyl and C12(7)K12 on the growth of E. coli (ATCC 8739) is determined by measuring the optical density (A) for MIC determination or the CFU (B) for minimal bactericidal concentration (MBC) after 24 hr incubation at 37 C.

(18) FIG. 14 illustrates an Antibacterial effect of Fluometacyl together with C.sub.12(7)K.sub.12 on Escherichia coli (ATCC 8739). Effect of Fluometacyl and C.sub.12(7)K.sub.12 on the growth of E. coli (ATCC 8739) is determined by measuring the optical density (A) for MIC determination or the CFU (B) for minimal bactericidal concentration (MBC) after 24 hr incubation at 37 C.

(19) FIG. 15 illustrates an Antibacterial effect of Triafluocyl together with C.sub.12(7)K.sub.12 on Pseudomonas aeruginosa (ATCC 27853). Effect of Triafluocyl and C.sub.12(7)K.sub.12 on the growth of Pseudomonas aeruginosa is determined by measuring the optical density for MIC determination.

(20) FIG. 16 illustrates an Antibacterial effect of Fluometacyl together with C.sub.12(7)K.sub.12 on Pseudomonas aeruginosa (ATCC 27853).

(21) Effect of Fluometacyl and C.sub.12(7)K.sub.12 on the growth of Pseudomonas aeruginosa (ATCC 27853) is determined by measuring the optical density for MIC determination.

DESCRIPTION

Examples

(22) The invention is illustrated hereafter by the following non limiting examples.

(23) We have conducted in vitro experiments, using S. aureus, S. epidermidis, and E. faecalis as clinically relevant Gram-positive bacterial strains.

(24) We have also conducted in vitro experiments, using Escherichia coli and Pseudomonas aeruginosa as clinically relevant Gram-negative bacterial strains.

(25) The tests were performed in accordance with the recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).

(26) Fluometacyl (1S,2R,3S,4R)-4-(7-(((1R,2S)-2-(3,4-Difluorophenyl)cyclopropyl)amino)-5-((propyl)thio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2,3-triol may be synthesized according to the process described in WO 99/05143.

Example 1

Use of (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol) or Triafluocyl (Cayman, item No 15425)

(27) S. aureus (American Type Culture Collection, ATCC 25904) was grown overnight in Tryptic Soy Broth (TSB) medium, diluted 1:100 in fresh TSB, and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.25-0.3).

(28) Increasing concentrations of Triafluocyl (Cayman Chemical, Item No. 15425) or vehicle (DMSO) was then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals (20-100 min) by spectrophotometry (OD.sub.600) and by counting the colony-forming units after plating appropriate culture dilutions on TS agar plates.

(29) Bacteriostatic and bactericidal effects were measured with Triafluocyl. In FIG. 1 kinetics of S. aureus growth in the presence of an increasing concentrations (1 g/ml to 20 g/ml) of Triafluocyl were measured by turbidity measurement (upper graph), and viable count (lower graph). Data represent mediansrange (n=3). *p<0.05; **p<0.01, ***p<0.001, Triafluocyl vs vehicle.

(30) As shown in FIG. 1, while a concentration of 10 g/ml Triafluocyl was able to inhibit bacterial growth, 20 g/ml Triafluocyl displayed potent bactericidal effect.

Example 2

Use of (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl) cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol) or Triafluocyl as Inhibitor of Biofilm Formation

(31) S. aureus (ATCC 25904) was grown overnight in TSB medium, before being diluted 100 fold in fresh TSB, and incubated aerobically at 37 C. until bacteria culture reached an OD.sub.600 of 0.6 (corresponding to approximately 1-310.sup.8 CFU/ml). Bacteria cultures were then diluted to 110.sup.4 CFU/ml in fresh TSB. 800 l aliquots of diluted bacteria suspensions were distributed in each well of a 24-well plate. Bacteria were allowed to adhere for 4 hours under static conditions at 37 C. After removing media, wells were rinsed 2 times with PBS to eliminate planktonic bacteria and re-filled with TSB supplemented with 0.5% glucose

(32) Triafluocyl or DMSO as vehicle was then added at desired concentration and plates were incubated at 37 C. for 20 hours. After incubation, wells were washed and stained with 0.5% (w/v) crystal violet for 30 minutes, washed again and the dye was solubilized by adding 20% acetic acid (v/v in water) before reading absorbance at 595 nm.

(33) S. aureus biofilms were formed on polystyrene surface in the presence of increasing concentrations of Triafluocyl or DMSO as vehicle. In FIG. 2, Biofilm mass is presented as percentage of values obtained in the presence of DMSO (*P<0.05; **P<0.01; ***P<0.001, Triafluocyl versus DMSO, n=4).

(34) Triafluocyl significantly reduces S. aureus biofilm formation at all concentrations tested. In the presence of 10 g/ml Triafluocyl, no biofilm could form on polystyrene surface.

Example 3

Use of (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol) or Triafluocyl (Cayman Chemical, Item No. 15425)

(35) E. faecalis (ATCC 29212) was grown overnight in Brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.25-0.3).

(36) Increasing concentrations of Triafluocyl (Cayman Chemical, Item No. 15425) or DMSO as vehicle was then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals (30-120 min) by spectrophotometry (OD.sub.600) and by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates.

(37) Bacteriostatic and bactericidal effects were measured with Triafluocyl. In FIG. 3 kinetics of E. faecalis growth in the presence of an increasing concentrations (5 g/ml to 40 g/ml) of Triafluocyl were measured by turbidity measurement (upper graph), and viable count (lower graph). Data represent mediansrange (n=3).

(38) As shown in FIG. 3, while a concentration of 10 g/ml Triafluocyl was able to inhibit bacterial growth, 20 g/ml Triafluocyl and more importantly 40 g/ml displayed potent bactericidal effects.

Example 4

Use of Triafluocyl as Inhibitor of Biofilm Formation

(39) E. faecalis (ATCC 29212) was grown overnight in BHI medium, before being diluted 100 fold in fresh TSB, and incubated aerobically at 37 C. until bacteria culture reached an OD.sub.600 of 0.6 (corresponding to approximately 2-510.sup.8 CFU/ml). Bacteria cultures were then diluted to 110.sup.4 CFU/ml in fresh TSB. 800 l aliquots of diluted bacteria suspensions were distributed in each well of a 24-well plate. Bacteria were allowed to adhere for 4 hours under static conditions at 37 C. After removing media, wells were rinsed 2 times with PBS to eliminate planktonic bacteria and re-filled with TSB supplemented with 0.5% glucose

(40) Triafluocyl or DMSO as vehicle was then added at desired concentration and plates were incubated at 37 C. for 20 hours. After incubation, wells were washed and stained with 0.5% (w/v) crystal violet for 30 minutes, washed again and the dye was solubilized by adding 20% acetic acid (v/v in water) before reading absorbance at 595 nm.

(41) E. faecalis biofilms were formed on polystyrene surface in the presence of increasing concentrations of Triafluocyl or DMSO as vehicle. In FIG. 2, Biofilm mass is presented as percentage of values obtained in the presence of DMSO (*P<0.05; **P<0.01; ***P<0.001, Triafluocyl versus DMSO, n=4).

(42) Triafluocyl significantly reduces E. faecalis biofilm formation at a starting concentration of 10 g/ml. In the presence of 40 g/ml Triafluocyl, no biofilm could form on polystyrene surface.

Example 5

Time-Kill Study of Triafluocyl Against S. epidermidis

(43) To evaluate Triafluocyl antibacterial effect we have tested S. epidermidis liquid growth in the presence of different Triafluocyl concentrations in logarithmic phase. In this phase usually bacteria are highly susceptible to agents with bactericidal activity because they are rapidly dividing.

(44) A 1:100 inoculum in 50 ml TSB of an O/N culture of S. epidermidis was cultured for 3 hr up to its logarithmic phase (OD.sub.600=0.26 and 310.sup.8 CFU/ml).

(45) Bacteria were split in several tubes containing different concentrations of DMSO as vehicle alone or in combination with Triafluocyl in TSB and grown for 100 min at 37 C. with 220 rpm shaking, the OD.sub.600 was measured every 20 min.

(46) Compared to the growth with DMSO (0.25%) we observed a dose-dependent inhibition of S. epidermidis growth between 10 g/ml and 20 g/ml Triafluocyl (FIG. 5). At 50 g/ml we observed a slight bacteriostatic activity, confirmed by the number of viable cells at 80 min, 310.sup.8 CFU/ml, equal to the number of bacteria in the untreated control at the beginning of the assay (FIG. 5).

(47) Moreover, we have tested the effect of Triafluocyl on a low-density inoculum, 0.0810.sup.6 CFU/ml, from a culture of S. epidermidis in logarithmic phase. We have followed the growth for 4 hr with or without Triafluocyl and measured the OD.sub.600: already 5 g/ml of Triafluocyl decreased the OD by 50% compared to the growth in absence of Triafluocyl at the same time point; 10 g/ml and 20 g/ml inhibited growth (OD value equal to OD at the beginning of the growth) (data not shown).

(48) This means that the lower the inoculum density the lower the concentration of Triafluocyl to slow down growth or kill bacteria.

Example 6

Triafluocyl Prevents S. epidermidis Biofilm Formation

(49) To study the effect of Triafluocyl on biofilm formation, S. epidermidis in early logarithmic phase (510.sup.8 CFU/ml) was plated in a 24-well plate and let to adhere at the bottom of the well for 4 hr at 37 C. in static conditions. After 4 hr incubation, planktonic bacteria were removed and adherent bacteria were washed twice in TSB. Fresh TSB medium supplemented or not with 0.25% glucose was added to the well with 5 different concentrations of Triafluocyl and incubated for 24 hours. Wells were washed 3 times with NaCl 0.9% and incubated for 1 hr at RT with Crystal Violet 1% solution in dH.sub.2O to stain the biofilm.

(50) Wells were washed 3 times with dH.sub.2O to eliminate unbound crystal violet, then 400 l Acetic Acid 10% was added and incubated at RT for 10 min. Absorbance was measured in triplicate at 570 nm, reflecting total biomass of the biofilm (live and dead bacteria).

(51) Triafluocyl affected biofilm formation (FIG. 6): already at 5 g/ml, in the absence of glucose, it inhibited biofilm formation by 50%, while in presence of glucose we reach 50% biofilm reduction only at 20 g/ml Triafluocyl.

(52) The concentration of Triafluocyl that inhibits at least 90% biofilm formation is called minimum biofilm inhibitory concentration (MBIC). Triafluocyl MBIC for S. epidermidis is 50 g/ml both in the presence and in the absence of glucose.

Example 7

Triafluocyl Destroys S. epidermidis Mature Biofilm

(53) In another experiment we let adhere 0.510.sup.8 CFU/ml S. epidermidis cells for 4 hr and let the biofilm form for additional 24 hr in presence of 0.25% glucose, at this point we treated the biofilm with several concentrations of Triafluocyl for 24 hr in TSB with 0.25% glucose and determined the viable count (FIG. 7) as well as the percentage of live cells using the BacLight bacterial viability kit (Molecular Probes).

(54) For biofilm analysis, we first washed the biofilm to eliminate all planktonic bacteria and then the biofilm was detached mechanically using a scraper. To assure that the aggregates from the biofilm were completely dissociated, the suspension of cells was passed through a needle (0.516 mm) and a dilution was plated on TSA plates.

(55) Only the highest concentration of Triafluocyl, 50 g/ml, was effective in reducing the number of viable cells in the biofilm with a reduction of almost 3 log (from 1.110.sup.8 CFU/ml in the control to 1.510.sup.5 CFU/ml).

(56) In the same experiment we also determined the percentage of live and dead bacteria. To do so we followed the procedure of the kit LIVE/DEAD from Molecular Probes. Briefly, the biofilm was resuspended in a solution of 0.9% NaCl and cells were stained with a mixture of SYTO9 (green fluorescence) and propidium iodide (PI) (red fluorescence) for 15 min in the dark. Stained cells were transferred in a 96-well plate and fluorescence was measured using the Enspire Spectrophotometer with excitation wavelength of 470 nm and emission spectra in the range of 490-700 nm. SYTO9 dye (green fluorescence 500-520 nm) penetrates all the cells (dead and live) and binds to DNA, while PI (red fluorescence in the range 610-630 nm) enters only in dead cells with a damaged cell membrane. When PI and SYTO9 are in the same cell the green fluorescence intensity decreases. Therefore, in a population of cells with high percentage of dead cells there is a reduction in the emission spectra of the green fluorescence, because there is more PI staining. The ratio of fluorescence intensity (green/live) is plotted against a known percentage of live cells to obtain a standard curve and the percentage of live cells in our samples is obtained by extrapolation (FIG. 7). Triafluocyl, at concentrations of 20 g/ml and 50 g/ml reduced the percentage of live bacteria to 80% and 30%, respectively.

Example 8

Triafluocyl Antibacterial Effects on S. epidermidis: Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

(57) The Minimal Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) of Triafluocyl were determined in Staphylococcus epidermidis (ATCC 35984, also known as RP62A) according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) recommendations.

(58) Briefly, a single colony grown on a Tryptic Soy Agar (TSA) plate was resuspended and cultured in Tryptic Soy Broth (TSB) overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:50 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3 hr and an inoculum of 1:100 dilution, corresponding to 310.sup.5 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO (vehicle). After O/N growth the OD of each culture was measured at 600 nm in a spectrophotometer (OD.sub.600). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. OD at 600 nm equal to zero (blank is the medium alone).

(59) We have also determined the MBC, i.e. the concentration at which the liquid culture, when spread on TSA plates, will not produce any colony.

(60) The MIC for Triafluocyl against S. epidermidis is equal to 123 g/ml and the MBC is 173 g/ml (two biological replicates, detection limit 10.sup.3).

(61) The Minimum Duration for killing 99.9% S. epidermidis (MDK99.9) by Triafluocyl, a tolerance metric according to the EUCAST, was 2 hours.

Example 9

Triafluocyl Antibacterial Effects on S. aureus: Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

(62) Further experiments were conducted using different strains of S. aureus, as clinically relevant Gram-positive bacterial strains: ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide intermediate-resistant (GISA) S. aureus Mu-50 (ATCC 700695) in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal concentration required to prevent bacterial growth; the Minimal Bactericidal Concentration (MBC) which determines the lowest concentration at which an antimicrobial agent kill a particular microorganism and a Minimum Duration for killing 99.9% bacteria (MDK99.9) which is a tolerance metric according to the EUCAST.

(63) MIC determination: A single colony selected from the different strains of S. aureus is resuspended and cultured in the appropriate medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3 hr (OD=0.08-0.1) and an inoculum of 1:300 dilution, corresponding to 310.sup.5 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600 nm in a spectrophotometer (OD.sub.600). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. OD at 600 nm equal to zero (blank is the medium alone). MIC for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 20, 20, 15, and 20 g/ml, respectively.

(64) MBC and MDK99.9 determination: A single colony selected from the different strains of S. aureus is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic conditions for 2 h. The culture is then challenged with triafluocyl at MIC concentration or higher concentrations. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. The concentration that kill at least 99.9% of the started inoculum in 24 h is defined as the MBC. And the real time needed is defined as the MDK.sub.99.9. MBC for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 20 g/ml for each of them. MDK.sub.99.9 for Triafluocyl against S. aureus ATCC 25904, ATCC 6538, methilcillin-resistant S. aureus (MRSA) ATCC BAA-1556, Glycopeptide intermediate-resistant (GISA), and S. aureus Mu-50 (ATCC 700695) were 10, 6, 2, and 14 hours, respectively.

Example 10

Triafluocyl Antibacterial Effects on E. faecalis: Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

(65) Further experiments were conducted using different strains of E. faecalis, as clinically relevant Gram-positive bacterial strains: E. faecalis vancomycin-resistant (VRE) ATCC BAA-2365, and E. faecalis ATCC 29212 in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal concentration required to prevent bacterial growth; the Minimal Bactericidal Concentration (MBC) which determines the lowest concentration at which an antimicrobial agent kill a particular microorganism and a Minimum Duration for killing 99.9% bacteria (MDK99.9) which is a tolerance metric according to the EUCAST.

(66) MIC determination: A single colony selected from the different strains of E. faecalis is resuspended and cultured in the appropriate medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3 hr (OD=0.08-0.1) and an inoculum of 1:300 dilution, corresponding to 310.sup.5 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600 nm in a spectrophotometer (OD.sub.600). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. OD at 600 nm equal to zero (blank is the medium alone). MIC for Triafluocyl against E. faecalis vancomycin-resistant (VRE) ATCC BAA-2365, and E. faecalis ATCC 29212 were 20 and 40 g/ml, respectively.

(67) MBC and MDK99.9 determination: A single colony selected from the different strains of E. faecalis is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic conditions for 2 h. The culture is then challenged with triafluocyl at MIC concentration or higher concentrations. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. The concentration that kill at least 99.9% of the started inoculum in 24h is defined as the MBC. And the real time needed is defined as the MDK.sub.99.9. MBC for Triafluocyl against E. faecalis vancomycin-resistant (VRE) ATCC BAA-2365 was 20 g/ml. MDK.sub.99.9 for Triafluocyl against E. faecalis vancomycin-resistant (VRE) ATCC BAA-2365 was 24 hours.

Example 11

Triafluocyl Antibacterial Effects on Streptococcus agalactiae: Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC)

(68) Further experiments were conducted using S. agalactiae (ATCC 12386), as clinically relevant Gram-positive bacterial strains in order to determine the Minimal Inhibitory Concentration (MIC) which is the minimal concentration required to prevent bacterial growth; the Minimal Bactericidal Concentration (MBC) which determines the lowest concentration at which an antimicrobial agent kill a particular microorganism and a Minimum Duration for killing 99.9% bacteria (MDK99.9) which is a tolerance metric according to the EUCAST.

(69) MIC determination: A single colony selected from the different strains of S. agalactiae (ATCC 12386) is resuspended and cultured in the appropriate medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in Mueller-Hinton broth (MHB) was incubated in aerobic conditions for 3 hr (OD=0.08-0.1) and an inoculum of 1:300 dilution, corresponding to 310.sup.5 CFU/ml, was incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO. After O/N growth the OD of each culture was measured at 600 nm in a spectrophotometer (OD.sub.600). The MIC represents the concentration at which there is no visible growth of bacteria, i.e. OD at 600 nm equal to zero (blank is the medium alone). MIC for Triafluocyl against S. agalactiae (ATCC 12386) was 40 g/ml.

(70) MBC and MDK99.9 determination: A single colony selected from S. agalactiae (ATCC 12386) is resuspended and cultured in the appropriate medium (TSB, or BHI) overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum in the appropriate medium was incubated in aerobic conditions for 2 h. The culture is then challenged with triafluocyl at MIC concentration or higher concentrations. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. The concentration that kill at least 99.9% of the started inoculum in 24 h is defined as the MBC. And the real time needed is defined as the MDK.sub.99.9. MBC for Triafluocyl against S. agalactiae (ATCC 12386) was 40 g/ml. MDK.sub.99.9 for Triafluocyl against S. agalactiae (ATCC 12386) was 1 hour.

(71) The results of all experiments are illustrated in Table 1 and in FIGS. 8 A,B,C, wherein the effect of Triafluocyl on resistant strains such as MRSA: methilcillin-resistant S. aureus; GISA: Glycopeptide intermediate-resistant S. aureus; VRE: vancomycin-resistant E. faecalis is shown.

(72) TABLE-US-00001 TABLE 1 MIC: minimal inhibitory concentration; MBC: minimal bactericidal concentration (cut-off = 99.9% reduction in CFU); MDK99.9: time(h) needed to kill 99.9% of the started inoculum; nd: not determined. MIC MBC MDK 99.9 Strains Resistance g/ml g/ml Time (h) S. aureus 20 20 10 (ATCC25904) S. aureus 20 20 6 (ATCC6538) S. aureus MRSA 15 20 2 S. aureus-Mu50 GISA 20 20 14 S. epidermidis 15 20 2 E. faecalis 40 nd nd E. faecalis VRE 20 20 24 S. agalactiae 40 40 1

(73) FIG. 8A also illustrates a comparison between the antibacterial effects of Triafluocyl, Vancomycin and Minocycline on MRSA.

(74) S. aureus MRSA (ATCC BAA-1556) was grown overnight in brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.25-0.3).

(75) Triafluocyl (Cayman Chemical, Item No. 15425) (20 g/ml), Vancomycin (Sigma, 4 g/ml or 10 g/ml), Minocycline (Sigma, 8 g/ml) or a solvent (DMSO) were added to 5 ml of S. aureus MRSA suspensions.

(76) Bacterial growth for S. aureus MRSA was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates.

(77) One clearly observes that Triafluocyl causes a decrease of S. aureus MRSA viable count as early as after the first two hours, at which time doses of Vancomycin and Minocycline equal to 10- and 8-fold MIC, respectively, were ineffective. Over the 24 h-experiment, the bactericidal effect of Vancomycin and Minocyclin remained less efficient than the one of Triafluocyl.

(78) FIG. 8B) illustrates a comparison between the antibacterial effect of Triafluocyl and Minocycline on S. aureus GISA.

(79) S. aureus Mu50 GISA was grown overnight in brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.25-0.3).

(80) Triafluocyl (Cayman Chemical, Item No. 15425) (20 g/ml), Minocycline (Sigma, 8 g/ml) or vehicle (DMSO) were then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates.

(81) Here again Triafluocyl (20 g/ml) had a quicker and more efficient antibacterial effect than a high dose of Minocycline (10 g/ml).

(82) FIG. 8C illustrates a comparison between Triafluocyl and Minocycline on E. faecalis VRE.

(83) E. faecalis VRE (ATCC BAA-2365) was grown overnight in brain heart infusion (BHI) medium, diluted 1:100 in fresh BHI, and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.2-0.25).

(84) Triafluocyl (Cayman Chemical, Item No. 15425) (20 g/ml), Minocycline (Sigma, 10 g/ml) or vehicle (DMSO) was then added in 5 ml of bacteria suspensions. Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates.

(85) Here Triafluocyl (20 g/ml) showed bactericidal effect while a high dose of Minocycline (10 g/ml) was only bacteriostatic.

Example 12

Fluometacyl Antibacterial Effects on Gram-Positive Bacteria Strains: S. aureus, S. epidermidis, E. faecalis

(86) Susceptibility Testing: MIC and MBC Determination:

(87) The Minimal Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) of Fluometacyl were determined on several gram-positive strains (Table 2) following EUCAST (European Committee on Antimicrobial susceptibility Testing) recommendations.

(88) For MIC determination a single colony was resuspended and cultured in the appropriate bacteria-specific medium (TSB: Tryptic Soy Broth for S. aureus atcc 25904 and S. epidermidis and BHI: brain-heart infusion medium for all the other strains) overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:100 inoculum was incubated in Mueller-Hinton broth (MHB) in aerobic conditions for 3 hr (OD.sub.600=0.08-0.1). A further inoculum, 1:300 dilution of the MHB culture, corresponding to 310.sup.5 CFU/ml, was grown in presence or absence of different concentrations of Fluometacyl, 1% DMSO in MHB for 20 hr.

(89) For MBC determination a 1:100 inoculum of an O/N culture (prepared like before) was incubated in aerobic conditions for 2 h in bacteria-specific medium. The culture was then challenged with Fluometacyl at the MIC concentration or higher. Bacterial growth was measured after different time intervals by counting the colony-forming units (CFU) after plating appropriate culture dilutions on bacteria-specific medium agar plates. The concentration that kills at least 99.9% of the started inoculum in 24 h is defined as the MBC.

(90) TABLE-US-00002 TABLE 2 MIC and MBC determination for different strains. MRSA: methilcillin-resistant S. aureus; GISA: Glycopeptide intermediate- resistant S. aureus; VRE: vancomycin-resistant Enteroccocus. MIC: minimal inhibitory concentration; MBC: minimal bactericidal concentration (cut-off = 99.9% reduction in CFU). MIC MBC Strains Resistance M M S. aureus 30-38 38 (ATCC25904) S. aureus MRSA 20-30 38 S. aureus-MU50 GISA 30-38 38 S. epidermidis 30 38 E. faecalis VRE 38 38

(91) Time-Kill Study of Fluometacyl against Methilcillin-Resistant S. aureus

(92) S. aureus MRSA (ATCC BAA-1556) was grown overnight in BHI medium, then a 1:100 inoculum was diluted in fresh BHI and incubated aerobically at 37 C. until bacteria growth reached a logarithmic phase (OD.sub.600=0.25-0.3). The culture was split into two and challenged with 38 M Fluometacyl (=18.2 g/ml) or DMSO (Ctrl). Bacterial growth was measured after different time intervals by counting the colony-forming units after plating appropriate culture dilutions on BHI agar plates. (N=2)

Example 13

Fluometacyl Antibacterial Effects on Biofilm Formation

(93) Staphyloccocus aureus (ATCC 25904) or Staphyloccocus epidermidis (ATCC 35984) were grown overnight in TSB medium, before being diluted 100 fold in fresh TSB, and incubated aerobically at 37 C. until bacteria culture reached an OD.sub.600 of 0.6 (corresponding to approximately 1-310.sup.8 CFU/ml). Bacteria cultures were then diluted to 110.sup.4 CFU/ml in fresh TSB. Aliquots of 800 l diluted bacteria suspensions were distributed in each well of a 24-well plate. Bacteria were allowed to adhere for 4 hours under static conditions at 37 C. After removing the media, wells were rinsed 2 times with PBS to eliminate planktonic bacteria and re-filled with TSB supplemented with 0.5% glucose containing Fluometacyl at desired concentration or DMSO alone (Ctrl). The 24-well plates were incubated at 37 C. for 20 hours. Wells were then washed and stained with 0.5% (w/v) crystal violet for 30 minutes and rinsed with PBS 4 times. The dye was solubilized by adding 20% acetic acid (v/v in water) before reading absorbance at 595 nm. FIG. 10A and FIG. 10B show Fluometacyl effect on S. aureus and S. epidermidis biofilm formation respectively at all concentrations tested. In presence of 38 M Fluometacyl, both S. aureus and S. epidermidis could not form any biofilm.

Example 14

Triafluocyl Antibacterial Effects on Escherichia coli (ATCC 8739) Together with Polymyxin B Nonapeptide (PMBN) as Membrane Penetrating Agent

(94) Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of Triafluocyl (Cayman Chemical, Item No. 15425) in presence of polymyxin B nonapeptide (PMBN) (Sigma-Aldrich Merk, Item No. P2076) against Escherichia coli (ATCC 8739).

(95) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria after 24 h incubation (no pellet or cloudiness), i.e. OD at 600 nm equal to zero wherein OD is the difference between the resulting optical density (OD) with the molecule together with PMBN, and the optical density (OD) of the blank (blank is the medium alone).

(96) The MBC represents the lowest concentration of drug required to kill the bacteria in a liquid culture.

(97) Triafluocyl stock solution of 100 mg/ml is prepared by adding to 50 mg Triafluocyl vial, provided by Cayman Chemical, 500 l of DMSO (Sigma-Aldrich Merk, Item No. D2650). Following dissolution in DMSO Triafluocyl is further diluted in DMSO to 20 mg/ml working solution. Both the stock and working solution are stored at 20 C.

(98) PMBN stock solution is prepared in water at 10 mg/ml and stored at 20 C.

(99) A single colony grown on a Luria-Bertani Agar (LB) plate was resuspended and cultured in LB medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:50 inoculum in Mueller-Hinton broth (MHB) is incubated in aerobic conditions for 1 h30 (OD=0.08-0.1) and an inoculum corresponding to 310.sup.6 colony forming unit (CFU)/ml, is incubated in presence or absence of different concentrations of Triafluocyl in 1% DMSO with or without 40 g/ml Polymyxin B nonapeptide (PMBN) for 18 hr in aerobic conditions (37 C. with 220 rpm shaking). Growth of Escherichia coli in 7 conditions depicted in FIG. 11 is evaluated by reading the OD of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Victor 3-Perkin Elmer).

(100) The MIC for Triafluocyl in presence of 40 g/ml PMBN against Escherichia coli (ATCC 8739) is equal to 5 g/ml. Triafluocyl taken alone in concentrations up to 15 g/ml or PMBN alone up to 40 g/ml are unable to inhibit E. coli growth (FIG. 11A).

(101) The Minimal Bactericidal Concentration (MBC) of Triafluocyl in presence of 40 g/ml PMBN against Escherichia coli (ATCC 8739) is equal to 5 g/ml (FIG. 11B). To determine the MBC, several dilutions of each culture are spread on LB agar plates to evaluate the number of colony forming unit (CFU) after 24 hr incubation at 37 C.

Example 15

Fluometacyl Antibacterial Effects on Escherichia coli (ATCC 8739) Together with Polymyxin B Nonapeptide (PMBN) as Membrane Penetrating Agent

(102) Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of Fluometacyl in presence of polymyxin B nonapeptide (PMBN) (Sigma-Aldrich Merk, Item No. P2076) against Escherichia coli (ATCC 8739).

(103) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria (no pellet or cloudiness), i.e. OD at 600 nm equal to zero wherein OD is the difference between the resulting optical density (OD) with the molecule together with PMBN, and the optical density (OD) of the blank (blank is the medium alone).

(104) The MBC represents the lowest concentration of drug required to kill the bacteria in a liquid culture.

(105) Fluometacyl stock solution of 20 mg/ml is prepared by adding 250 l DMSO (Sigma-Aldrich Merk, Item No. D2650) to 5 mg Fluometacyl powder. The stock is stored at 20 C.

(106) PMBN stock solution is prepared in water at 10 mg/ml and stored at 20 C.

(107) A single colony grown on a Luria-Bertani Agar (LB) plate is resuspended and cultured in LB medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking), next day a 1:50 inoculum in Mueller-Hinton broth (MHB) is incubated in aerobic conditions for 1 h30 (OD=0.08-0.1) and an inoculum corresponding to 310.sup.6 colony forming unit (CFU)/ml is incubated in presence or absence of different concentrations of Fluometacyl in 1% DMSO (vehicle) with or without 40 g/ml Polymyxin B nonapeptide (PMBN) for 18 hr in aerobic conditions (37 C. with 220 rpm shaking). Growth of bacteria in the 7 individual cultures (FIG. 12A) is evaluated by reading the OD of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Victor 3-Perkin Elmer).

(108) The MIC for Fluometacyl in presence of 40 g/ml PMBN against Escherichia coli (ATCC 8739) is equal to 5 g/ml. Fluometacyl taken alone in concentrations up to 15 g/ml or PMBN alone up to 40 g/ml is unable to inhibit E. coli growth (FIG. 12A).

(109) The Minimal Bactericidal Concentration (MBC) of Fluometacyl in presence of 40 g/ml PMBN against Escherichia coli (ATCC 8739) is equal to 5 g/ml (FIG. 12B). To determine the MBC, several dilutions of each culture are spread on LB agar plates to evaluate the number of colony forming unit (CFU) after 24 hr incubation at 37 C.

Example 16

Triafluocyl Antibacterial Effects on Escherichia coli (ATCC 8739)

(110) (together with a Oligo-Acyl-Lysine (OAK) as membrane penetrating agent: the C.sub.12(7)K.sub.12, also called cis-7-dodecenoyl-lysyl-lysyl-aminododecanoyl-lysyl-amide).

(111) Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of Triafluocyl (purchased from Cayman Chemical, Item No. 15425) in presence of C.sub.12(7)K.sub.12 (purchased from biomers.net, Germany) against Escherichia coli (ATCC 8739). The C.sub.12(7)K.sub.12 was synthesized by solid-phase method as described by I. Radzishevsky in Antimicrobial Agents and Chemotherapy, May 2007 (1753-1759) (https://aac.asm.org/content/51/5/1753).

(112) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria after 24 hours incubation (no pellet or cloudiness), i.e. OD at 600 nm equal to zero, wherein OD is the difference between the resulting optical density (OD) with Triafluocyl together with C.sub.12(7)K.sub.12, and the optical density (OD) of the blank (blank being the medium alone).

(113) The Minimal Bactericidal Concentration (MBC) represents the lowest concentration of Triafluocyl together with C.sub.12(7)K.sub.12 required to kill over a period of 24 hours at least 99.9% of bacteria present at time zero in a liquid culture. Viable count in the liquid culture is estimated by counting the colony forming units (c.f.u.) on a Luria-Bertani (LB) agar plateafter 24 h incubation at 37 C.and calculating the c.f.u. per milliliter of the liquid culture (c.f.u./ml).

(114) Triafluocyl stock solution of 100 mg/ml is prepared by adding to 50 mg Triaflyocyl vial, provided by Cayman Chemical, 500 l of DMSO (Sigma-Aldrich Merk, Item No. D2650). Following dissolution in DMSO Triafluocyl is further diluted in DMSO to 20 mg/ml working solution. Both the stock and working solution are stored at 20 C. C.sub.12(7)K.sub.12 stock solution of 10 mg/ml is prepared by adding to 14.3 mg C12.sub.(7)K.sub.12 dried powder, provided by biomers.net, 1.43 ml of water (Thermo Fisher Item No. AM9930) and stored at 20 C. In both graphs of FIG. 13 C.sub.12(7)K.sub.12 is referred as OAK.

(115) A single colony of Escherichia coli grown on a Luria-Bertani (LB) agar plate is resuspended and cultured in LB medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking in the New Brunswick Innova 4330 Shaker incubator). An aliquot of 100 l such overnight Escherichia coli culture is diluted 1:50 in 5 ml Mueller-Hintom broth (MHB) and is incubated in aerobic conditions during 1 hour 30 minutes (OD=0.08-0.1) to reach the exponential phase of their growth curve. An inoculum of the exponentially growing Escherichia coli, corresponding to around 110.sup.6 c.f.u./ml is then incubated in presence or absence of different concentrations of Triafluocyl in 2% DMSO with or without OAK for 24 hr in aerobic conditions (37 C. with 220 rpm shaking in the New Brunswick Innova 4330 Shaker incubator). Growth of Escherichia coli in each condition depicted in FIG. 13A is evaluated by reading the Optical Density of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Victor-3, Perkin Elmer).

(116) The MIC for Triafluocyl in presence of 5 g/ml OAK against Escherichia coli (ATCC 8739) is equal to 5 g/ml. Triafluocyl taken alone at a concentration of 10 g/ml or OAK alone at a concentration up to 10 g/ml are unable to inhibit Escherichia coli growth (FIG. 13A).

(117) The Minimal Bactericidal Concentration (MBC) of Triafluocyl in presence of 5 g/ml OAK against Escherichia coli (ATCC 8739) is equal to 10 g/ml (FIG. 13B). To determine the MBC, 20 l of a dilution 1:100,000 in 0.9% NaCl of cultures with OD=0.2 or 20 l of a dilution 1:10 in 0.9% NaCl of cultures with OD=0 are spread on LB agar plates to evaluate the number of colony forming unit (c.f.u.) after 24 hr incubation at 37 C.

Example 17

Fluometacyl Antibacterial Effects on Escherichia coli (ATCC 8739)

(118) (together with a Oligo-Acyl-Lysine (OAK) as membrane penetrating agent: the C.sub.12(7)K.sub.12, also called cis-7-dodecenoyl-lysyl-lysyl-aminododecanoyl-lysyl-amide)

(119) Determination of Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of Fluometacyl (prepared according to WO 99/05143) in presence of C.sub.12(7)K.sub.12 (purchased from biomers.net, Germany) against Escherichia coli (ATCC 8739). The C.sub.12(7)K.sub.12 was synthesized by solid-phase method as described by I. Radzishevsky in Antimicrobial Agents and Chemotherapy, May 2007 (1753-1759) (https://aac.asm.org/content/51/5/1753).

(120) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria after 24 hours incubation (no pellet or cloudiness), i.e. OD at 600 nm equal to zero, wherein OD is the difference between the resulting optical density (OD) with Fluometacyl together with C.sub.12(7)K.sub.12, and the optical density (OD) of the blank (blank being the medium alone).

(121) The Minimal Bactericidal Concentration (MBC) represents the lowest concentration of Fluometacyl together with C.sub.12(7)K.sub.12 required to kill over a period of 24 hours, at least 99.9% of bacteria present at time zero in a liquid culture. Viable count in the liquid culture is estimated by counting the colony forming units (c.f.u.) on a Luria-Bertani (LB) agar plateafter 24 h incubation at 37 C.and calculating the c.f.u. per milliliter of the liquid culture (c.f.u./ml).

(122) Fluometacyl stock solution of 20 mg/ml is prepared by adding 250 l DMSO (Sigma-Aldrich Merk, Item No. D2650) to 5 mg Fluometacyl powder. The stock solution is stored at 20 C.

(123) C.sub.12(7)K.sub.12 stock solution of 10 mg/ml is prepared by adding to 14.3 mg C.sub.12(7)K.sub.12 dried powder, provided by biomers.net, 1.43 ml of water (Thermo Fisher Item No. AM9930) and stored at 20 C. In both graphs of FIG. 14 C.sub.12(7)K.sub.12 is referred as OAK.

(124) A single colony of Escherichia coli grown on a Luria-Bertani (LB) agar plate is resuspended and cultured in LB medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking in the New Brunswick Innova 4330 Shaker incubator). An aliquot of 100 l such overnight Escherichia coli culture is diluted 1:50 in 5 ml Mueller-Hintom broth (MHB) and is incubated in aerobic conditions during 1 hour 30 minutes (OD=0.08-0.1) to reach the exponential phase of their growth curve. An inoculum of the exponentially growing Escherichia coli, corresponding to around 110.sup.6 c.f.u./ml is then incubated in presence or absence of different concentrations of Fluometacyl in 2% DMSO with or without OAK for 24 hr in aerobic conditions (37 C. with 220 rpm shaking in the New Brunswick Innova 4330 Shaker incubator). Growth of Escherichia coli in each condition depicted in FIG. 14A is evaluated by reading the Optical Density of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Victor-3, Perkin Elmer).

(125) The MIC for Fluometacyl in presence of 5 g/ml OAK against Escherichia coli (ATCC 8739) is equal to 5 g/ml. Fluometacyl taken alone at a concentration of 10 g/ml or OAK alone at a concentration up to 10 g/ml is unable to inhibit E. coli growth (FIG. 14A).

(126) The Minimal Bactericidal Concentration (MBC) of Fluometacyl in presence of 5 g/ml OAK against Escherichia coli (ATCC 8739) is equal to 5 g/ml (FIG. 14B). To determine the MBC, 20 l of a dilution 1:100,000 in 0.9% NaCl of cultures with OD=0.2 or 20 l of a dilution 1:10 in 0.9% NaCl of cultures with OD=0 are spread on LB agar plates to evaluate the number of colony forming unit (c.f.u.) after 24 hr incubation at 37 C.

Example 18

Triafluocyl Antibacterial Effects on Pseudomonas aeruginosa (ATCC 27853)

(127) (together with a Oligo-Acyl-Lysine (OAK) as membrane penetrating agent: the C.sub.12(7)K.sub.12, also called cis-7-dodecenoyl-lysyl-lysyl-aminododecanoyl-lysyl-amide). The C.sub.12(7)K.sub.12 was synthesized by solid-phase method as described by I. Radzishevsky in Antimicrobial Agents and Chemotherapy, May 2007 (1753-1759) (https://aac.asm.org/content/51/5/1753).

(128) Determination of Minimal Inhibitory Concentration (MIC) of Triafluocyl (Cayman Chemical, Item No. 15425) in presence of C.sub.12(7)K.sub.12 (provided by Custom synthesis Biomers Germany) against Pseudomonas aeruginosa (ATCC 27853).

(129) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria after 24 hours incubation (no pellet or cloudiness), i.e. OD at 600 nm equal to zero wherein OD is the difference between the resulting optical density (OD) with the molecule together with C.sub.12(7)K.sub.12, and the optical density (OD) of the blank (blank is the medium alone).

(130) Triafluocyl stock solution of 100 mg/ml is prepared by adding to 50 mg Triaflyocyl vial, provided by Cayman Chemical, 500 l of DMSO (Sigma-Aldrich Merk, Item No. D2650). Following dissolution in DMSO, Triafluocyl is further diluted in DMSO to 20 mg/ml working solution. Both the stock and working solution are stored at 20 C.

(131) C.sub.12(7)K.sub.12 stock solution is prepared in water at 10 mg/ml and stored at 20 C. In FIG. 15A, C.sub.12(7)K.sub.12 is referred as OAK.

(132) A single colony of Pseudomonas aeruginosa grown on a Tryptic Soy agar (TSA) plate is resuspended and cultured in Tryptic Soy Broth (TSB) medium overnight (O/N) in aerobic conditions (37 C. with 190 rpm shaking in New Brunswick Innova 4200 Incubator Shaker). Next day the resulting inoculum is diluted again at 1:100 in Mueller-Hinton broth (MHB) and then incubated in aerobic conditions for 1 h30 (OD=0.08-0.1) to reach an exponential phase in the grown curve. A resulting inoculum corresponding to around 510.sup.5 colony forming unit (CFU)/ml, is further incubated in presence or absence of different concentrations (10 and 20 g/ml) of Triafluocyl in 2% DMSO with or without C.sub.12(7)K.sub.12 for 18 hr in aerobic conditions (37 C. with 190 rpm shaking in New Brunswick Innova 4200 Incubator Shaker). Growth of Pseudomonas aeruginosa in each condition depicted in FIG. 15A is evaluated by reading the OD of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Fisher Scientific, cell density meter model 40).

(133) The MIC for Triafluocyl in presence of 10 g/ml OAK against Pseudomonas aeruginosa (ATCC 27853) is equal to 10 g/ml. Triafluocyl taken alone at a concentration of 10 g/ml or OAK alone at a concentration up to 10 g/ml are unable to inhibit P. aeruginosa growth (FIG. 15A).

Example 19

Fluometacyl Antibacterial Effects on Pseudomonas aeruginosa (ATCC 27853)

(134) (together with a Oligo-Acyl-Lysine (OAK) as membrane penetrating agent: the C.sub.12(7)K.sub.12, also called cis-7-dodecenoyl-lysyl-lysyl-aminododecanoyl-lysyl-amide)

(135) Determination of Minimal Inhibitory Concentration (MIC) of Fluometacyl (prepared according to WO99/0514) in presence of C.sub.12(7)K.sub.12 (provided by Biomers.net Germany) against Pseudomonas aeruginosa (ATCC 27853). The C.sub.12(7)K.sub.12 was synthesized by solid-phase method as described by I. Radzishevsky in Antimicrobial Agents and Chemotherapy, May 2007 (1753-1759) (https://aac.asm.org/content/51/5/1753)

(136) The Minimal Inhibitory Concentration (MIC) represents the concentration, in a liquid culture, at which there is no visible growth of bacteria after 24 hours incubation (no pellet or cloudiness), i.e. OD at 600 nm equal to zero wherein OD is the difference between the resulting optical density (OD) with the molecule together with C.sub.12(7)K.sub.12, and the optical density (OD) of the blank (blank is the medium alone).

(137) Fluometacyl stock solution of 20 mg/ml is prepared by adding 250 l DMSO (Sigma-Aldrich Merk, Item No. D2650) to 5 mg Fluometacyl powder. The stock solution is stored at 20 C.

(138) C.sub.12(7)K.sub.12 stock solution is prepared in water at 10 mg/ml and stored at 20 C. In FIG. 16A, C.sub.12(7)K.sub.12 is referred as OAK.

(139) A single colony of Pseudomonas aeruginosa grown on a Tryptic Soy agar (TSA) plate is resuspended and cultured in Tryptic Soy Broth (TSB) medium overnight (O/N) in aerobic conditions (37 C. with 220 rpm shaking in New Brunswick Innova 4200 Incubator Shaker). Next day the resulting inoculum is diluted again at 1:100 in Mueller-Hinton broth (MHB) and then incubated in aerobic conditions for 1 h30 (OD=0.08-0.1) to reach an exponential phase in the grown curve. A resulting inoculum corresponding to around 510.sup.5 colony forming unit (CFU)/ml, is further incubated in presence or absence of different concentrations (10 and 20 g/ml) of Fluometacyl in 2% DMSO with or without C.sub.12(7)K.sub.12 for 18 hr in aerobic conditions (37 C. with 190 rpm shaking in New Brunswick Innova 4200 Incubator Shaker). Growth of Pseudomonas aeruginosa in each condition depicted in FIG. 16A is evaluated by reading the Optical Density of each culture at 600 nm (OD.sub.600) in a spectrophotometer (Fisher Scientific, cell density meter model 40).

(140) The MIC for Fluometacyl in presence of 10 g/ml C.sub.12(7)K.sub.12 against Pseudomonas aeruginosa (ATCC 27853) is equal to 20 g/ml. Fluometacyl taken alone at a concentration of 20 g/ml or C12(7)K12 alone at a concentration up to 10 g/ml are unable to inhibit P. aeruginosa growth (FIG. 16A).