Combinations with a backbone-cyclized peptide

Abstract

A novel combination comprising a β-hairpin peptidomimetic of the formula cyclo(-Thr-Trp-Ile-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Dab-Ala-Ser-.sup.DPro-Pro) (I), and a further compound with antibiotic activity, that enable therapeutic control of specific bacterial infections in human or animals at doses of the individual compounds lower than either of the compounds administered alone. The combination can be used as a medicament to treat e.g. skin or soft tissue infections; eye, ear, blood stream, or intra-abdominal infections; infections related to respiratory diseases, to bone diseases, to cardiovascular diseases, to genitourinal diseases, or to gastrointestinal diseases.

Claims

1. A combination comprising a β-hairpin peptidomimetic of the formula cyclo(-Thr-Trp-Ile-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Dab-Ala-Ser-.sup.DPro-Pro) (I), wherein Dab is (S)-2,4-diaminobutanoic acid; .sup.DDab is (R)-2,4-diaminobutanoic acid; Orn is (S)-2,5-diaminopentanoic acid; and a further antibiotic compound selected from ciprofloxacin, levofloxacin, moxifloxacin, gemifloxacin, ceftaroline, ceftobiprole, ceftazidime, ceftriaxone, cefepime, daptomycin, ramoplanin, vancomycin, colistin, polymyxin B, ertapenem, meropenem, doripenem, imipenem, aztreonam, piperacillin, amikacin, rifampicin, neomycin, gentamicin, tobramycin, fosfomycin, azithromycin, minocycline, doxycycline, or tetracycline, or a pharmaceutically acceptable salt thereof.

2. The combination according to claim 1 wherein the further antibiotic compound is selected from ertapenem, azithromycin, ciprofloxacin, or amikacin, or a pharma-ceutically acceptable salt thereof.

3. The combination according to claim 1 wherein the further antibiotic compound is selected from the class of macrolides, or pharmaceutically acceptable salts thereof.

4. The combination according to claim 1 wherein the further antibiotic compound is selected from the class of quinolones, or pharmaceutically acceptable salts thereof.

5. The combination according to claim 1 wherein the further antibiotic compound is selected from the class of aminoglycosides, or pharmaceutically acceptable salts thereof.

6. The combination according to claim 1 wherein the further antibiotic compound is ertapenem, or a pharmaceutically acceptable salt thereof.

7. The combination according to claim 1 wherein the further antibiotic compound is azithromycin, or a pharmaceutically acceptable salt thereof.

8. The combination according to claim 1 wherein the further antibiotic compound is ciprofloxacin, or a pharmaceutically acceptable salt thereof.

9. The combination according to claim 1 wherein the further antibiotic compound is amikacin, or a pharmaceutically acceptable salt thereof.

10. The combination according to claim 1 for use in medicine.

11. The combination according to claim 1 for the treatment of bacterial infections or diseases related to such infections in human or animals.

12. A method for manufacturing a pharmaceutical compositing comprising the combination according to claim 1, comprising: formulating the pharmaceutical composition for the treatment of bacterial infections or diseases related to such infections in human or animals.

13. A pharmaceutical composition containing a combination according to claim 1 and at least one pharmaceutically inert carrier.

14. The pharmaceutical composition according to claim 13 in a form suitable for oral, topical, transdermal, injection, infusion, buccal, transmucosal, rectal, vaginal, pulmonary or inhalation administration, especially in the form of tablets, dragees, capsules, solutions, liquids, gels, plaster, creams, ointments, syrup, slurries, powders, suspensions, spray, nebulizer or suppositories.

15. A method for the treatment of bacterial infections or diseases related to such infections in a human or an animal, comprising: administering a therapeutically effective amount of the pharmaceutical composition according to claim 13 to the human or the animal.

16. A kit comprising a part containing a β-hairpin peptidomimetic of the formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof.

17. A kit comprising a part containing a β-hairpin peptidomimetic of the formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof and a part containing a compound with antibiotic activity selected from the classes of β-lactams, carbapenems, macrolides, quinolones, or aminoglycosides, or pharmaceutically acceptable salts thereof.

18. A kit according to claim 16 wherein the compound with antibiotic activity is selected from ertapenem, meropenem, azithromycin, ciprofloxacin, amikacin, neomycin, tobramycin, colistin, polymyxin B, minocycline, or tetracycline, or a pharmaceutically acceptable salt thereof.

19. A kit according to claim 16 wherein the compound with antibiotic activity is selected from ertapenem, azithromycin, ciprofloxacin, or amikacin, or a pharmaceutically acceptable salt thereof.

20. A kit according to claim 16 wherein the compound with antibiotic activity is ertapenem, or a pharmaceutically acceptable salt thereof.

21. A kit according to claim 16 wherein the compound with antibiotic activity is azithromycin, or a pharmaceutically acceptable salt thereof.

22. A kit according to claim 16 wherein the compound with antibiotic activity is ciprofloxacin, or a pharmaceutically acceptable salt thereof.

23. A kit according to claim 16 wherein the compound with antibiotic activity is amikacin.sup.AG, or a pharmaceutically acceptable salt thereof.

24. A method of treating an bacterial infection or disease related to such infection in human or animals comprising administering to a subject in need thereof an adequate amount of a combination according to claim 1.

Description

EXAMPLES

(1) In Vivo Efficacy Test:

(2) Efficacy in Murine Pneumonia Model Against Pseudomonas aeruginosa PAX11045 and Estimation of ED.sub.50

(3) Reference 1:

(4) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) was determined against Pseudomonas aeruginosa clinical isolate PAX11045 in a pneumonia model in mice. Colony counts in the lungs and spleen were determined at 20 hours post treatment.

(5) Infection of Mice

(6) Fresh overnight colonies of PAX11045 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 5×10.sup.7 CFU/ml. Female mice (DBA/2, outbred, 18-22 g, Charles River) were anesthetized with 0.08 ml of Zoletil (tiletamine+zolazepam) and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 10.sup.6 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(7) Treatment of Mice with Compound 1

(8) Two vials containing 10 mg of active compound 1 were dissolved in 2.25 ml 0.9% sterile saline each to a concentration of 4.5 mg/ml. One vial was further 2-fold diluted with saline to 2.25, 1.125, 0.56 and 0.28 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As positive control ciprofloxacin was used in the same manner with a fixed dose of 19 mg/kg.

(9) Sampling

(10) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs and spleens were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10-fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-48 hours at 35° C. in ambient air.

(11) CFU Counts

(12) The CFU/ml in the inoculum was determined to 7.92 log.sub.10 CFU/ml corresponding to 6.62 log.sub.10 CFU/mouse.

(13) At 4 hours after infection the mean log.sub.10 CFU/lung was 5.28 and the CFU level remained at a similar level after 24 hours in the vehicle-only group. Analog baseline data were collected for the spleen with a mean log.sub.10 CFU/spleen of 1.96 at 4 hours, which increased to 2.60 after 24 hours in the vehicle-only group.

(14) Treatment with compound 1 resulted in both organs in a concentration dependent significant reduction of the CFU levels compared to vehicle treatment (p<0.001 for the higher concentrations). Also ciprofloxacin (19 mg/kg) had a significant effect on reducing the bacterial loads (p<0.001).

(15) Evaluation of the dose-response curve for ED.sub.50 of compound 1 against PAX11045 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 4.33 mg/kg. Table 1 below summarizes the relevant efficacy values.

Example 1

(16) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) in combination with ertapenem was determined against Pseudomonas aeruginosa clinical isolate PAX11045 in a pneumonia model in mice. Colony counts in lung were determined at 20 hours post treatment.

(17) Infection of Mice

(18) Fresh overnight colonies of PAX11045 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 5×10.sup.7 CFU/ml. Female mice (DBA/2, outbred, 18-22 g, Charles River) were anesthetized with 0.1 ml of Zoletil and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 10.sup.6 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(19) Treatment of Mice with Ertapenem

(20) 1 g of ertapenem (Invanz™, MSD Denmark Aps) was dissolved in 10 ml 0.9% sterile saline to a concentration of 100 mg/ml and further diluted with saline to 5 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 3 hours post infection corresponding to 50 mg/kg on the basis of a mean animal weight of 20 g.

(21) Treatment of Mice with Compound 1

(22) One vial containing 10 mg of active compound 1 was dissolved in 2 ml 0.9% sterile saline to a concentration of 5 mg/ml and further diluted with saline to 2, 1, 0.55, 0.275 and 0.137 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As positive control ciprofloxacin was used in the same manner with a fixed dose of 20 mg/kg.

(23) Sampling

(24) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10 fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-24 hours at 35° C. in ambient air.

(25) CFU Counts

(26) The CFU/ml in the inoculum was determined to 7.65 log.sub.10 CFU/ml corresponding to 6.35 log.sub.10 CFU/mouse.

(27) At 4 hours after infection the mean log.sub.10 CFU/lung was 5.14 and the CFU level remained at a similar level after 24 hours in the vehicle-only group.

(28) Treatment with a combination of compound 1 and ertapenem resulted in a concentration dependent significant reduction of the CFU levels compared to vehicle treatment (p<0.01-p<0.001). Also ciprofloxacin treatment (20 mg/kg), compound 1 (2.75 mg/kg) alone and ertapenem (50 mg/kg) alone had a significant effect on reducing the bacterial loads (p<0.001).

(29) Evaluation of the dose-response curve for ED.sub.50 of compound 1 in presence of a fixed dose of ertapenem (50 mg/kg) against PAX11045 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 1.24 mg/kg. Table 1 below summarizes the relevant efficacy values.

(30) TABLE-US-00002 TABLE 1 Efficacy values of compound 1 compound 1 in presence compound 1 of 50 mg/kg ertapenem Top level 1.3 log.sub.10 CFU/ml −0.34 log.sub.10 CFU/ml Bottom level −2.2 log.sub.10 CFU/ml  −2.32 log.sub.10 CFU/ml E.sub.max 3.5 log.sub.10 CFU/ml  1.98 log.sub.10 CFU/ml ED.sub.50 4.33 mg/kg 1.24 mg/kg Static dose 1.55 mg/kg 0.63 mg/kg 1 log killing dose 8.1 mg/kg 1.14 mg/kg 2 log killing dose 20 mg/kg 1.48 mg/kg R.sup.2 0.55-0.75 0.54

Example 2

(31) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) in combination with azithromycin was determined against Pseudomonas aeruginosa clinical isolate PAX11045 in a pneumonia model in mice. Colony counts in lung were determined at 20 hours post treatment.

(32) Infection of Mice

(33) Fresh overnight colonies of PAX11045 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 5×10.sup.7 CFU/ml. Female mice (DBA/2, outbred, 17-23 g, Charles River) were anesthetized with 0.1 ml of Zoletil and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 10.sup.6 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(34) Treatment of Mice with Azithromycin

(35) 480 mg of azithromycin (Zitromax™, Pfizer) were dissolved in 4.8 ml 0.9% sterile saline to a concentration of 100 mg/ml and further diluted with saline to 5 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 3 hours post infection corresponding to 50 mg/kg on the basis of a mean animal weight of 20 g.

(36) Treatment of Mice with Compound 1

(37) One vial containing 10 mg of active compound 1 was dissolved in 2 ml 0.9% sterile saline to a concentration of 5 mg/ml and further diluted with saline to 2, 1, 0.55, 0.275 and 0.137 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As positive control ciprofloxacin was used in the same manner with a fixed dose of 20 mg/kg.

(38) Sampling

(39) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10 fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-24 hours at 35° C. in ambient air.

(40) CFU Counts

(41) The CFU/ml in the inoculum was determined to 7.3 log.sub.10 CFU/ml corresponding to 6.0 log.sub.10 CFU/mouse.

(42) At 4 hours after infection the mean log.sub.10 CFU/lung was 5.84 and the CFU level remained at a similar level after 24 hours in the vehicle-only group.

(43) Treatment with a combination of compound 1 and azithromycin resulted in a concentration dependent significant reduction of the CFU levels compared to vehicle treatment (p<0.01-p<0.001). Also ciprofloxacin treatment (20 mg/kg) and treatment with compound 1 (5.5 mg/kg) alone had a significant effect on reducing the bacterial loads (p<0.001). Treatment with azithromycin (50 mg/kg) alone had no effect on the bacterial loads.

(44) Evaluation of the dose-response curve for ED.sub.50 of compound 1 in presence of a fixed dose of azithromycin (50 mg/kg) against PAX11045 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 1.74 mg/kg. Table 2 below summarizes the relevant efficacy values.

(45) TABLE-US-00003 TABLE 2 Efficacy values of compound 1 compound 1 in presence compound 1 of 50 mg/kg azithromycin Top level 1.3 log.sub.10 CFU/ml −0.10 log.sub.10 CFU/ml Bottom level −2.2 log.sub.10 CFU/ml  −2.59 log.sub.10 CFU/ml E.sub.max 3.5 log.sub.10 CFU/ml  2.49 log.sub.10 CFU/ml ED.sub.50 4.33 mg/kg 1.74 mg/kg Static dose 1.55 mg/kg 0.63 mg/kg 1 log killing dose 8.1 mg/kg 1.2 mg/kg 2 log killing dose 20 mg/kg 2.4 mg/kg R.sup.2 0.55-0.75 0.52

(46) Efficacy in Murine Pneumonia Model Against Pseudomonas aeruginosa PA9349 and Estimation of ED.sub.50

(47) Reference 2:

(48) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) was determined against Pseudomonas aeruginosa clinical isolate PA9349 in a pneumonia model in mice. Colony counts in the lungs were determined at 18-20 hours post treatment.

(49) Infection of Mice

(50) Fresh overnight colonies of PA9349 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 5×10.sup.6 CFU/ml. Female mice (DBA/2, outbred, 18-22 g, Charles River) were anesthetized with 0.1 ml of Zoletil (tiletamine+zolazepam) and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 5×10.sup.5 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(51) Treatment of Mice with Compound 1

(52) One vial containing 10 mg of active compound 1 was dissolved in 5 ml 0.9% sterile saline to a concentration of 2 mg/ml and was further 2-fold diluted with saline to 1, 0.5, 0.25, 0.125 and 0.06 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As positive control colistin was used in the same manner with a fixed dose of 40 mg/kg.

(53) Sampling

(54) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10-fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-48 hours at 35° C. in ambient air.

(55) CFU Counts

(56) The CFU/ml in the inoculum was determined to 7.29 log.sub.10 CFU/ml corresponding to 5.98 log.sub.10 CFU/mouse.

(57) At 4 hours after infection the mean log.sub.10 CFU/lung was 3.47 and the CFU level increased to 4.92 at 20 hours post inoculation in the vehicle-only group.

(58) A reduction of the mean CFU level compared to the vehicle group was observed in the 10 mg/kg compound 1 treatment group whereupon a significant reduction was observed in the 20 mg/kg compound 1 treatment group.

(59) Evaluation of the dose-response curve for ED.sub.50 of compound 1 against PA9349 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 7.35 mg/kg. Table 3 below summarizes the relevant efficacy values.

Example 3

(60) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) in combination with ciprofloxacin was determined against Pseudomonas aeruginosa clinical isolate PA9349 in a pneumonia model in mice. Colony counts in lung were determined at 20 hours post treatment.

(61) Infection of Mice

(62) Fresh overnight colonies of PA9349 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 10.sup.7 CFU/ml. Female mice (C57BL/6 male, outbred, 20-25 g, Hellenic Pasteur Institute) were anesthetized with ether and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 10.sup.6 CFU. After inoculation, mice were treated with paracetamol suppositories as a pain relief.

(63) Treatment of Mice with Ciprofloxacin

(64) 400 mg of ciprofloxacin (Sigma) was dissolved in 0.9% sterile saline to a concentration of 10 mg/ml and further diluted with saline to 2 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 3 hours post infection corresponding to 20 mg/kg on the basis of a mean animal weight of 20 g.

(65) Treatment of Mice with Compound 1

(66) One vial containing 5 mg of active compound 1 was dissolved in 2.5 ml 0.9% sterile saline to a concentration of 2 mg/ml. 1 vial was further diluted with saline to 1, 0.8 and 0.4 mg/ml. The mice were treated subcutaneously with 0.25 ml (25 mg/kg dose) or 0.2 ml (for all other doses) in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As controls colistin and ciprofloxacin were used in the same manner with a fixed dose of 20 mg/kg.

(67) Sampling

(68) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10 fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-24 hours at 35° C. in ambient air.

(69) CFU Counts

(70) The CFU/ml in the inoculum was determined to 7.0 log.sub.10 CFU/ml corresponding to 5.8 log.sub.10 CFU/mouse.

(71) At 4 hours after infection the mean log.sub.10 CFU/lung was 5.63 and the CFU level remained at a similar level after 24 hours in the vehicle-only group.

(72) Treatment with a combination of compound 1 at 1.88-25 mg/kg and ciprofloxacin resulted in a significant reduction of the CFU levels compared to vehicle treatment (p<0.001). Treatment with compound 1 (5.5 mg/kg) alone had a significant effect on reducing the bacterial loads (p<0.001) whereupon colistin treatment (20 mg/kg) alone and ciprofloxacin treatment (20 mg/kg) alone had no or only slight effects on the bacterial loads.

(73) Evaluation of the dose-response curve for ED.sub.50 of compound 1 in presence of a fixed dose of ciprofloxacin (20 mg/kg) against PA9349 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 1.55 mg/kg. Table 3 below summarizes the relevant efficacy values.

(74) TABLE-US-00004 TABLE 3 Efficacy values of compound 1 compound 1 in presence compound 1 of 20 mg/kg ciprofloxacin Top level 1.59 log.sub.10 CFU/ml −0.21 log.sub.10 CFU/ml Bottom level −0.80 log.sub.10 CFU/ml  −4.17 log.sub.10 CFU/ml E.sub.max −2.4 log.sub.10 CFU/ml  3.96 log.sub.10 CFU/ml ED.sub.50 7.35 mg/kg 1.55 mg/kg Static dose 9.15 mg/kg nd 1 log killing dose nd 0.45 mg/kg 2 log killing dose nd 1.00 mg/kg 3log killing dose nd 1.82 mg/kg R.sup.2 0.67 0.54 nd: not determined

(75) Efficacy in Murine Pneumonia Model Against Pseudomonas aeruginosa PA18298 and Estimation of ED.sub.50

(76) Reference 3:

(77) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) was determined against Pseudomonas aeruginosa clinical isolate PA18298 in a pneumonia model in mice. Colony counts in the lungs were determined at 18-20 hours post treatment.

(78) Infection of Mice

(79) Fresh overnight colonies of PA18298 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 4×10.sup.7 CFU/ml. Female mice (DBA/2, outbred, 18-22 g, Charles River) were anesthetized with 0.1 ml of Zoletil (tiletamine+zolazepam) and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 1×10.sup.6 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(80) Treatment of Mice with Compound 1

(81) One vial containing 10 mg of active compound 1 was dissolved in 2 ml 0.9% sterile saline to a concentration of 5 mg/ml and was further diluted with saline to 2, 1, 0.75, 0.55, 0.275 and 0.137 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. As positive control colistin was used in the same manner with a fixed dose of 20 mg/kg.

(82) Sampling

(83) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10-fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-48 hours at 35° C. in ambient air.

(84) CFU Counts

(85) The CFU/ml in the inoculum was determined to 7.49 log.sub.10 CFU/ml corresponding to 6.20 log.sub.10 CFU/mouse.

(86) At 4 hours after infection the mean log.sub.10 CFU/lung was 5.05 and the CFU level declined to 2.62 at 24 hours post inoculation in the vehicle-only group.

(87) Treatment with POL7080 at 11-20 mg/kg resulted in significant reduction of the CFU levels compared to vehicle treatment (p<0.01-p<0.001). Also colistin treatment (20 mg/kg) had some effect on reducing the bacterial loads (p<0.001).

(88) Evaluation of the dose-response curve for ED.sub.50 of compound 1 against PA18298 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 26.6 mg/kg. Table 4 below summarizes the relevant efficacy values.

Example 4

(89) The efficacy and ED.sub.50 of the compound of formula (I) (“compound 1”) in combination with amikacin was determined against Pseudomonas aeruginosa clinical isolate PA18298 in a pneumonia model in mice. Colony counts in lung were determined at 20 hours post treatment.

(90) Infection of Mice

(91) Fresh overnight colonies of PA18298 from a 5% Horse Blood Agar plate were suspended in 0.9% sterile saline to approximately 10.sup.8 CFU/ml and further diluted to approximately 5×10.sup.7 CFU/ml. Female mice (DBA/2, outbred, 18-22 g, Charles River) were anesthetized with 0.1 ml of Zoletil and inoculated via the nose with a pipette with 0.05 ml of the bacteria suspension containing approximately 10.sup.6 CFU. 4 hours after inoculation, the mice were treated orally with 45 μl neurophen (20 mg ibuprofen/ml corresponding to approximately 30 mg/kg) as pain relief.

(92) Treatment of Mice with Amikacin

(93) 175 mg of amikacin (Sigma) were dissolved in 5 ml 0.9% sterile saline to a concentration of 35 mg/ml and further diluted with saline to 3 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 3 hours post infection corresponding to 30 mg/kg on the basis of a mean animal weight of 20 g.

(94) Treatment of Mice with Compound 1

(95) One vial containing 10 mg of active compound 1 was dissolved in 2 ml 0.9% sterile saline to a concentration of 5 mg/ml and further diluted with saline tot, 1, 0.55, 0.275 and 0.137 mg/ml. The mice were treated subcutaneously with 0.2 ml in the neck region with a single dose at 4 hours post infection with a dose calculation based on a mean animal weight of 20 g. Colistin was used as a control in the same manner with a fixed dose of 20 mg/kg.

(96) Sampling

(97) Colony counts were determined post inoculation at 4 hours (untreated mice) and 24 hours (treated and vehicle-only treated mice). Immediately after the mice were sacrificed, the lungs were collected and frozen at −20° C. After thawing, the organs were homogenized in 1 ml 0.9% saline. Each sample was then 10 fold diluted in saline and 20 μl spots were applied on blood agar plates. All agar plates were incubated 18-24 hours at 35° C. in ambient air.

(98) CFU Counts

(99) The CFU/ml in the inoculum was determined to 7.4 log.sub.10 CFU/ml corresponding to 6.17 log.sub.10 CFU/mouse.

(100) At 4 hours after inoculation the mean log 10 CFU/lung was 5.06 and the CFU level declined to 1.55 mean log 10 CFU/lung after 24 hours in the vehicle group. Colistin treatment (20 mg/kg) as well as amikacin treatment (30 mg/kg) alone had some effects on reducing the bacterial loads.

(101) Evaluation of the dose-response curve for ED.sub.50 of compound 1 in presence of a fixed dose of amikacin (30 mg/kg) against PA18298 in murine lungs using a sigmoidal dose-response model (variable slope) revealed an estimation of 9.1 mg/kg. Table 4 below summarizes the relevant efficacy values.

(102) TABLE-US-00005 TABLE 4 Efficacy values of compound 1 compound 1 in presence compound 1 of 30 mg/kg amikacin Top level −3.60 log.sub.10 CFU/ml −3.03 log.sub.10 CFU/ml Bottom level −2.48 log.sub.10 CFU/ml −3.82 log.sub.10 CFU/ml E.sub.max  1.12 log.sub.10 CFU/ml  0.79 log.sub.10 CFU/ml ED.sub.50 26.6 mg/kg 9.1 mg/kg Static dose 9.15 mg/kg nd 1 log killing dose nd nd 2 log killing dose nd nd R.sup.2 0.26 0.05 nd: not determined