Template-fixed peptidomimetics with antimicrobial activity

Abstract

Template-fixed -hairpin peptidomimetics of the general formula ##STR00001##
wherein Z is a template-fixed chain of 12 -amino acid residues which, depending on their positions in the chain (counted starting from the N-terminal amino acid) are Gly, or Pro, or of certain types which, as the remaining symbols in the above formula, are defined in the description and the claims, and salts thereof, have the property to selectively inhibit the growth of or to kill microorganisms such as Pseudomonas aeruginosa. They can be used as disinfectants for foodstuffs, cosmetics, medicaments or other nutrient-containing materials, or as medicaments to treat or prevent infections. These -hairpin peptidomimetics can be manufactured by processes which are based on a mixed solid- and solution phase synthetic strategy.

Claims

1. A method of treating a disease or infection caused by a Pseudomonas aeruginosa comprising administering to a subject in need thereof an effective amount of a compound represented by formula (I): ##STR00037## wherein ##STR00038## is .sup.DPro-.sup.LPro and Z is a chain of 12 -amino acid residues, wherein the positions of the amino acid residues in the chain are counted starting from the N-terminal amino acid, wherein the amino acid residues are, in positions P1 to P12 of the chain: P1: Thr; P2: Trp; P3: Ile; P4: Dab; P5: Orn; P6: .sup.DDab; P7: Dab; P8: Trp; P9 Dab; P10: Dab; P11: Ala; and P12: Ser, or a pharmaceutically acceptable salt or enantiomer thereof.

2. The method of claim 1, wherein the subject is suffering from at least one disease selected from the group consisting of respiratory diseases, skin or soil tissue diseases, gastrointestinal diseases, eye diseases, ear diseases, CNS diseases, bone diseases, cardiovascular diseases, gastrourinal diseases, cancer and HIV.

3. The method of claim 1, wherein the subject is suffering from at least one disease selected from the group consisting of cystic fibrosis, emphysema, asthma, surgical wounds, traumatic wounds, burn wounds, epidemic diarrhea, necrotizing enterocolitis, typhlitis, keratitis, endophthalmitis, otitis, brain abscess, meningitis, osteochondritis, osteomyelitis, endocartitis, pericarditis, epididymitis, prostatitis, urethritis, cancer and HIV.

4. The method of claim 1, wherein the Pseudomonas aeruginosa is multi-drug resistant.

5. The method of claim 1, wherein the compound represented by formula (I) is administered in the form of a pharmaceutically acceptable composition, wherein the pharmaceutically acceptable composition comprises one or more physiologically acceptable carriers, diluents, excipients or auxiliaries.

6. The method of claim 1, wherein at least one additional pharmaceutical agent is administered to the subject.

7. The method of claim 1, wherein the additional pharmaceutical agent is selected from the group consisting of antimicrobial agents, antibiotic agents, anti cancer agents and antiviral agents.

8. The method of claim 1, wherein the compound of formula (I) is administered topically.

9. The method of claim 1, wherein the compound of formula (I) is administered by injection.

10. The method of claim 1, wherein the compound of formula (I) is administered transdermally, transmucosally, orally or by pulmonary administration.

Description

EXAMPLES

(1) 1. Peptide Synthesis

(2) Coupling of the First Protected Amino Acid Residue to the Resin

(3) 0.5 g of 2-chlorotritylchloride resin (Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) (0.83 mMol/g, 0.415 mmol) was filled into a dried flask. The resin was suspended in CH.sub.2Cl.sub.2 (2.5 ml) and allowed to swell at room temperature under constant stirring for 30 min. The resin was treated with 0.415 mMol (1 eq) of the first suitably protected amino acid residue (see below) and 284 l (4 eq) of diisopropylethylamine (DIEA) in CH.sub.2Cl.sub.2 (2.5 ml), the mixture was shaken at 25 C. for 4 hours. The resin was shaken (CH.sub.2Cl.sub.2/MeOH/DIEA: 17/2/1), 30 ml for 30 min; then washed in the following order with CH.sub.2Cl.sub.2 (1), DMF (1), CH.sub.2Cl.sub.2 (1), MeOH (1), CH.sub.2Cl.sub.2(1), MeOH (1), CH.sub.2Cl.sub.2 (2), Et.sub.2O (2) and dried under vacuum for 6 hours.

(4) Loading was typically 0.6-0.7 mMol/g.

(5) The following preloaded resin was prepared: Fmoc-Pro-2-chlorotritylresin.

(6) Synthesis of the Fully Protected Peptide Fragment

(7) The synthesis was carried out using a Syro-peptide synthesizer (Multisyntech) using 24 to 96 reaction vessels. In each vessel were placed 60 mg (weight of the resin before loading) of the above resin. The following reaction cycles were programmed and carried out:

(8) TABLE-US-00013 Step Reagent Time 1 CH.sub.2Cl.sub.2, wash and swell (manual) 3 1 min. 2 DMF, wash and swell 1 5 min 3 40% piperidine/DMF 2 5 min. 4 DMF, wash 5 2 min. 5 5 equiv. Fmoc amino acid/DMF + 2 60 min. 5 eq. HCTU + 5 eq. DIEA 6 DMF, wash 4 2 min. 7 CH.sub.2Cl.sub.2, wash (at the end of the 3 2 min. synthesis)

(9) Steps 3 to 6 are repeated to add each amino-acid.

(10) After the synthesis of the fully protected peptide fragment had been terminated, then subsequently the cleavage, cyclization and work up procedure as described hereinbelow, was used for the preparation of the peptides.

(11) Analytical Methods:

(12) Method 1: Analytical HPLC retention times (RT, in minutes) were determined using an Jupiter Proteo column (90A, 1502.0 mm, cod. 00F4396-B0Phenomenex) with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.1% TFA) and the gradient: 0 min: 95% A, 5% B; 20 min: 40% A 60% B; 21-23 min: 0% A, 100% B; 23.1-30 min: 95% A, 5% B.

(13) Method 2: Analytical HPLC retention times (RT, in minutes) were determined using an Aquity UPLC BEH C18 column (1.7 m, 1002.1 mm, cod. 186002352Waters) with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.085% TFA) and the gradient: 0 min: 95% A, 5% B; 0.2 min: 95% A 5% B; 4 min: 35% A, 65% B; 4.2 min: 5% A, 95% B; 4.25 min: 95% A, 5% B; 4.9 min: 95% A, 5% B.

(14) Procedure: Cleavage, Cyclization and Work Up of Backbone Cyclized Peptides

(15) Cleavage, Backbone Cyclization and Purification of the Peptide

(16) After assembly of linear peptides, the resin was suspended in 1 ml (0.39 mMol) of 1% TFA in CH.sub.2Cl.sub.2 (v/v) for 3 minutes and filtered, and the filtrate was neutralized with 1 ml (1.17 mMol, 3 eq.) of 20% DIEA in CH.sub.2Cl.sub.2 (v/v). This procedure was repeated twice to ensure completion of the cleavage. The resin was washed with 2 ml of CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layer was evaporated to dryness.

(17) The fully protected linear peptide was solubilised in 8 ml of dry DMF. Then 2 eq. of HATU in dry DMF (1 ml) and 4 eq. of DIPEA in dry DMF (1 ml) were added to the peptide, followed by stirring for 16 h. The volatiles were evaporated to dryness. The crude cyclic peptide was dissolved in 7 ml of CH.sub.2Cl.sub.2 and extracted with 10% acetonitrile in water (4.5 ml) three times. The CH.sub.2Cl.sub.2 layer was evaporated to dryness. To fully deprotect the peptide, 3 ml of cleavage cocktail TFA:TIS:H.sub.2O (95:2.5:2.5) were added, and the mixture was stirred for 2.5 h. The volatile was evaporated to dryness and the crude peptide was dissolved in 20% AcOH in water (7 ml) and extracted with diisopropyl ether (4 ml) for three times. The aqueous layer was collected and evaporated to dryness, and the residue was purified by preparative reverse phase LC-MS.

(18) After lyophilisation the products were obtained as white powders and analysed by HPLC-ESI-MS analytical methods as described above. The analytical data comprising purity after preparative HPLC and ESI-MS are shown in Table 1.

(19) Examples 1-50, are shown in Table 1. The peptides were synthesized starting with the amino acid L-Pro which was grafted to the resin. Starting resin was Fmoc-Pro-2-chlorotrityl resin, which was prepared as described above. The linear peptides were synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro-.sup.DPro-P12-P11-P10-P9-P8-P7-P6-P5-P4-P3-P2-P1. Ex. 1-50, were cleaved from the resin, cyclized, deprotected and purified as indicated by preparative reverse phase LC-MS.

(20) After lyophilisation the products were obtained as white powders and analysed by HPLC-ESI-MS methods as described above.

(21) HPLC-retention times (minutes) were determined using the analytical methods as described above. Examples 1 to 39 were analysed with method 1, for Examples 40-50 method 2 was used: Ex. 1 (8.87), Ex. 2 (9.26), Ex. 3 (9.34), Ex. 4 (9.45), Ex. 5 (9.48), Ex. 6 (9.44), Ex. 7 (10.11), Ex. 8 (9.99), Ex. 9 (10.22), Ex. 10 (9.76), Ex. 11 (10.56), Ex. 12 (11.37), Ex. 13 (9.13), Ex. 14 (9.34), Ex. 15 (8.80), Ex. 16 (9.23); Ex. 17 (9.65), Ex. 18 (9.18), Ex. 19 (8.37), Ex. 20 (8.86), Ex. 21 (8.78), Ex. 22 (9.32), Ex. 23 (9.58), Ex. 24 (9.27), Ex. 25 (9.31), Ex. 26 (9.24), Ex. 27 (9.23), Ex. 28 (9.34), Ex. 29 (9.66), Ex. 30 (9.88), Ex. 31 (9.62), Ex. 32(8.86), Ex. 33 (9.73), Ex. 34 (10.46), Ex. 35 (9.21), Ex. 36 (9.80), Ex. 37 (9.73), Ex. 38 (9.20), Ex. 39 (9.53), Ex. 40 (2.07), Ex. 41 (1.77), Ex. 42 (1.66), Ex. 43 (1.67), Ex. 44 (1.81), Ex. 45 (1.87), Ex. 46 (1.81), Ex. 47 (1.83), Ex. 48 (1.79), Ex. 49 (1.88), Ex. 50 (2.17).

(22) TABLE-US-00014 TABLE 1 Examples Purity [M + Example Sequ. ID P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 Template %.sup.a) 2H]/2 1 SEQ ID NO: 1 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 790.9 2 SEQ ID NO: 2 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Gly Dab .sup.DPro.sup.LPro 95 783.9 3 SEQ ID NO 3 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab .sup.DAla Dab .sup.DPro.sup.LPro 95 791.1 4 SEQ ID NO 4 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab .sup.DVal Dab .sup.DPro.sup.LPro 95 805.2 5 SEQ ID NO: 5 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Aib Dab .sup.DPro.sup.LPro 86 798.0 6 SEQ ID NO: 6 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Abu Dab .sup.DPro.sup.LPro 86 797.9 7 SEQ ID NO: 7 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Leu Dab .sup.DPro.sup.LPro 95 812.5 8 SEQ ID NO: 8 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Ile Dab .sup.DPro.sup.LPro 89 812.6 9 SEQ ID NO: 9 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Nle Dab .sup.DPro.sup.LPro 39 812.3 10 SEQ ID NO: 10 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Nva Dab .sup.DPro.sup.LPro 87 805.1 11 SEQ ID NO: 11 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Chg Dab .sup.DPro.sup.LPro 82 825.7 12 SEQ ID NO: 12 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Cha Dab .sup.DPro.sup.LPro 95 831.9 13 SEQ ID NO: 13 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Gln Dab .sup.DPro.sup.LPro 87 819.6 14 SEQ ID NO: 14 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Asp Dab .sup.DPro.sup.LPro 80 813.5 15 SEQ ID NO: 15 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Glu Dab .sup.DPro.sup.LPro 83 820.1 16 SEQ ID NO: 16 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Thr Dab .sup.DPro.sup.LPro 95 806.1 17 SEQ ID NO: 17 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Pro Dab .sup.DPro.sup.LPro 85 804.0 18 SEQ ID NO: 18 Cit Trp Ile Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 819.6 19 SEQ ID NO: 19 Thr Tyr Ile Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 779.9 20 SEQ ID NO: 20 Thr Trp Ile Dab Lys Dab Dab Tyr Dab Dab Ala Dab .sup.DPro.sup.LPro 95 779.5 21 SEQ ID NO: 21 Thr Trp Ile Dab Lys Dab His Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 810.0 22 SEQ ID NO: 22 Ala Trp Ile Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 776.2 23 SEQ ID NO: 23 Ala Trp Ile Dab Dab Dab Dab Trp Dab Dab Val Dab .sup.DPro.sup.LPro 95 776.4 24 SEQ ID NO: 24 Thr Trp Ile Lys Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 87 805.1 25 SEQ ID NO: 25 Thr Trp Ile Dab Lys Lys Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 805.0 26 SEQ ID NO: 26 Thr Trp Ile Dab Lys Dab Lys Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 87 805.2 27 SEQ ID NO: 27 Thr Trp Ile Dab Lys Dab Dab Trp Lys Dab Ala Dab .sup.DPro.sup.LPro 95 805.1 28 SEQ ID NO: 28 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Ala Lys .sup.DPro.sup.LPro 95 805.0 29 SEQ ID NO: 29 Thr Trp Ile Gln Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 86 805.2 30 SEQ ID NO: 30 Thr Trp Ile Gln Lys Dab Dab Trp Dab Dab Val Dab .sup.DPro.sup.LPro 88 819.1 31 SEQ ID NO: 31 Thr Trp Nle Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 791.1 32 SEQ ID NO: 32 Thr Trp Val Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 784.0 33 SEQ ID NO: 33 Thr Trp Chg Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 804.4 34 SEQ ID NO: 34 Thr Trp Cha Dab Lys Dab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 811.5 35 SEQ ID NO: 35 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Hse Dab .sup.DPro.sup.LPro 95 806.4 36 SEQ ID NO: 36 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab t-BuG Dab .sup.DPro.sup.LPro 95 812.5 37 SEQ ID NO: 37 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Cpa Dab .sup.DPro.sup.LPro 89 811.3 38 SEQ ID NO: 38 Thr Trp Ile Dab Lys Dab Dab Trp Dab Dab Asn Dab .sup.DPro.sup.LPro 95 813.1 39 SEQ ID NO: 39 Thr Trp Ile Dab Lys Dab Gln Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 95 805.2 40 SEQ ID NO: 40 Thr Trp Ile Gln Lys Dab Dab Trp Dab Dab Ala Gln .sup.DPro.sup.LPro 95 819.6 41 SEQ ID NO: 41 Thr Trp Ile Dab Lys .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 91 791.2 42 SEQ ID NO: 42 Asp Tyr Ile Dab Lys .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 85 786.6 43 SEQ ID NO: 43 Asp Tyr Ile Dab Orn .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 85 779.6 44 SEQ ID NO: 44 Glu Trp Ile Dab Lys .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 78 805.1 45 SEQ ID NO: 45 Glu Trp Ile Dab Lys .sup.DDab Dab Trp Dab Dab Ala Gln .sup.DPro.sup.LPro 82 819.2 46 SEQ ID NO: 46 Glu Trp Ile Dab Lys .sup.DDab Dab Trp Dab Dab Ala Dap .sup.DPro.sup.LPro 83 798.0 47 SEQ ID NO: 47 Glu Trp Ile Dab Orn .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 85 798.3 48 SEQ ID NO: 48 Thr Trp Ile Dab Orn .sup.DDab Dab Trp Dab Dab Ala Dab .sup.DPro.sup.LPro 86 784.1 49 SEQ ID NO: 49 Thr Trp Ile Dab Orn .sup.DDab Dab Trp Dab Dab Ala Ser .sup.DPro.sup.LPro 91 777.7 50 SEQ ID NO: 50 Glu Trp Ile Gln Lys Dab Dab Ser Dab Dab Ala Ser .sup.DPro.sup.LPro 95 805.8 .sup.a)%-puritity of compounds after prep. HPLC.
2. Biological Methods
2.1. Preparation of the Peptide Samples.

(23) Lyophilized peptides were weighed on a Microbalance (Mettler MT5) and dissolved in sterile water to a final concentration of 1 mg/ml unless stated otherwise. Stock solutions were kept at +4 C., light protected.

(24) 2.2. Antimicrobial Activity of the Peptides.

(25) The selective antimicrobial activities of the peptides were determined in 96-well plates (Nunclon polystyrene) by the standard NCCLS broth microdilution method (see ref 1, below) with slight modifications. Innocula of the microorganisms were diluted into Mueller-Hinton (MH) broth+0.02% BSA and compared with a 0.5 Mcfarland standard to give appr. 10.sup.6 colony forming units (CFU)/ml. Aliquots (50 l) of inoculate were added to 50 l of MH broth+0.02% BSA containing the peptide in serial two-fold dilutions. The following microorganisms were used to determine antibiotic selectivity of the peptides: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa ATCC 27853, P8191900, P1021903, P1021913, IMP 1 Livermore 3140). Antimicrobial activities of the peptides were expressed as the minimal inhibitory concentration (MIC) in g/ml at which no visible growth was observed after 18-20 hours of incubation at 37 C.

(26) 2.3. Cytotoxicity Assay

(27) The cytotoxicity of the peptides to HELA cells (Acc57) and COS-7 cells (CRL-1651) was determined using the MTT reduction assay [see ref 2 and 3, below]. Briefly the method was as follows: HELA cells and COS-7 cells were seeded at 7.010.sup.3 and, respectively, 4.510.sup.3 cells per well and grown in 96-well microtiter plates for 24 hours at 37 C. at 5% CO.sub.2. At this point, time zero (Tz) was determined by MTT reduction (see below). The supernatant of the remaining wells was discarded, and fresh medium and the peptides in serial dilutions of 12.5, 25 and 50 M were dispensed into the wells. Each peptide concentration was assayed in triplicate. Incubation of the cells was continued for 48 hours at 37 C. at 5% CO.sub.2. Wells were then washed once with phosphate buffered saline (PBS) and subsequently 100 l MTT reagent (0.5 mg/ml in medium RPMI1640 and, respectively, DMEM) were added to the wells. This was incubated at 37 C. for 2 hours and subsequently the medium was aspirated and 100 l isopropanol were added to each well. The absorbance at 595 nm of the solubilized product was measured (OD.sub.595 peptide). For each concentration averages were calculated from triplicates. The percentage of growth was calculated as follows: (OD.sub.595peptide-OD.sub.595Tz-OD.sub.595Empty well)/(OD.sub.595Tz-OD.sub.595Empty well)100% and was plotted for each peptide concentration.

(28) The LC 50 values (Lethal Concentration, defined as the concentration that kills 50% of the cells) were determined for each peptide by using the trend line function of EXCEL (Microsoft Office 2000) for the concentrations (50, 25, 12.5 and 0 M), the corresponding growth percentages and the value 50, (=TREND(C50:C0,%50:%0,50)). The GI 50 (Growth Inhibition) concentrations were calculated for each peptide by using a trend line function for the concentrations (50, 25, 12.5 and 0 g/ml), the corresponding percentages and the value 50, (=TREND (C.sub.50:C.sub.0,%.sub.50:%.sub.0,50).

(29) 2.4. Hemolysis

(30) The peptides were tested for their hemolytic activity against human red blood cells (hRBC). Fresh hRBC were washed three times with phosphate buffered saline (PBS) by centrifugation for 10 min at 2000g. Peptides at a concentration of 100 M were incubated with 20% v/v hRBC for 1 hour at 37 C. The final erythrocyte concentration was approximately 0.910.sup.9 cells per ml. A value of 0% and, respectively, 100% cell lysis was determined by incubation of the hRBC in the presence of PBS alone and, respectively, 0.1% Triton X-100 in H.sub.2O. The samples were centrifuged, the supernatant was 20-fold diluted in PBS buffer and the optical density (OD) of the sample at 540 nM was measured. The 100% lysis value (OD.sub.540H.sub.2O) gave an OD.sub.540 of approximately 1.3-1.8. Percent hemolysis was calculated as follows: (OD.sub.540peptide/OD.sub.540H.sub.2O)100%.

(31) 2.5. Plasma Stability

(32) 405 l of plasma/albumin solution were placed in a polypropylene (PP) tube and spiked with 45 l of compound from a 100 mM solution B, derived from 135 l of PBS and 15 l of 1 mM peptide in PBS, pH 7.4. 150 l aliquots were transferred into individual wells of the 10 kDa filter plate (Millipore MAPPB 1010 Biomax membrane). For 0 minutes controls: 270 l of PBS were placed in a PP tube and 30 l of stock solution B was added and mixed. 150 l of control solution were placed into one well of the filter plate and served as filtered control.

(33) Further 150 l of control solution were placed directly into a receiver well (reserved for filtrate) and served as not-filtered control. The entire plate including evaporation lid was incubated for 60 min at 37 C. Plasma samples (rat plasma: Harlan Sera lab UK, human plasma: Blutspendezentrum Zrich) were centrifuged at least for 2 h at 4300 rpm (3500 g) and 15 C. in order to yield 100 l filtrate. For serum albumin-samples (freshly prepared human albumin: Sigma A-4327, rat albumin: Sigma A-6272, all at 40 mg/ml concentration in PBS) approximately 1 hour of centrifugation was sufficient. The filtrates in the receiver PP plate were analysed by LC/MS as follows: Column: Jupiter C18 (Phenomenex), mobile phases: (A) 0.1% formic acid in water and (B) acetonitrile, gradient: 5%-100% (B) in 2 minutes, electrospray ionization, MRM detection (triple quadrupole). The peak areas were determined and triplicate values were averaged. The binding was expressed in percent of the (filtered and not-filtered time point 0 min) control 1 and 2 by: 100(100T.sub.60/T.sub.0). The average from these values was then calculated.

(34) 2.6. Pharmacokinetic Study (PK)

(35) Pharmacokinetic Study after Single Intravenous, Subcutaneous and Intraperitoneal Administration in Mice

(36) Pharmacokinetic study after single intravenous (i.v.) and subcutaneous (s.c.) administration was performed for the compound of Example 1 (Ex. 1). CD-1 mice (20-25 g) were used in the study. Physiological saline was used a vehicle. The volume was 2 ml/kg i.v., and 5 ml/kg s.c. and the peptide Ex. 1 was injected to give a final intravenous dose of 1 mg/kg, and a subcutaneous dose of 5 mg/kg. Approximately 200-250 l of blood was removed under light isoflurane anesthesia by cardiac puncture at predetermined time intervals (0, 5, 15, 30 min and 1, 2, 3, 4 and 5 hours for the i.v. study and 0, 15, 30 min and 1, 2, 4, 6, 8 and 10 hours for the s.c. study) and added to heparinized tubes. Plasma was removed from pelleted cells upon centrifugation and frozen at 80 C. prior to HPLC-MS analysis.

(37) Preparation of the Plasma Calibration Samples

(38) Blank mouse plasma from untreated animals was used. Aliquots of plasma of 0.2 ml each were spiked with 50 ng of propranolol (Internal Standard, IS), (sample preparation by solid phase extraction on OASIS HLB cartridges (Waters)) and with known amounts of Ex. 1 in order to obtain 9 plasma calibration samples in the range 10-5000 nM. The OASIS HLB cartridges were conditioned with 1 ml of methanol and then with 1 ml of 1% NH.sub.3 in water. Samples were then diluted with 700 l of 1% NH.sub.3 in water and loaded. The plate was washed with 1 ml of methanol/1% NH.sub.3 in water 5/95. Elution was performed using 1 ml of 0.1% TFA in methanol.

(39) The plate containing eluates was introduced into the concentrator system and taken to dryness. The residues were dissolved in 100 L of formic acid 0.1%/acetonitrile, 95/5 (v/v) and analysed in the HPLC/MS on a reverse phase analytical column (Jupiter C 18, 502.0 mm, 5 m, Phenomenex), using gradient elution (mobile phases A: 0.1% formic acid in water, B: Acetonitrile; from 5% B to 100% B in 2 min.).

(40) Preparation of Plasma Samples

(41) Samples coming from animal treatments were pooled in order to obtain an appropriate volume for the extraction. If the total volume obtained was less than 0.2 ml the appropriate amount of blank mouse plasma was added in order to keep the matrix identical to the calibration curve. Samples were than spiked with IS and processed as described for the calibration curve.

(42) Pharmacokinetic Evaluation

(43) PK analysis was performed on pooled data (generally n=2 or 3) using the software Win Nonlin (Pharsight). The area under the curve AUC was calculated by the linear trapezoidal rule. Elimination half-life was calculated by the linear regression on at least three data points during the elimination phase. The time intervals selected for the half-life determinations were evaluated by the correlation coefficient (r.sup.2), which should be at least above 0.85 and most optimally above 0.96. In case of i.v. administration the initial concentration at t.sub.zero was determined by extrapolation of the curve through the first two time points. Finally bioavailability after i.p. administration was calculated from the normalised AUCinf_D_obs ration after s.c. versus i.v. administration.

(44) 2.7. In vivo Septicemia Assay

(45) Groups of 6 CD-1 (Crl.) derived male mice weighing 242 g were used. The mice were each inoculated intravenously (IV) with an LD90-100 of Pseudomonas aeruginosa (ATCC 27853)(9106 CFU/0.5 ml/mouse) in brain heart infusion broth without 5% mucin. Compound at doses of 5, 2.5, 1, 0.5, 0.25 and 0.1 mg/kg, vehicle (0.9% NaCl, 10 ml/kg) was administered subcutaneously (SC) to test animals at 1 hour after bacterial inoculation. Also, an additional group was treated twice with compound at a dose of 5 mg/kg at 1 and 6 hours after bacterial inoculation. Mortality was recorded once daily for 7 days following the bacterial inoculation and an increase of survival of the animals by 50 percent or more (.sup.350%) after the bacterial inoculation, relative to vehicle control, indicates significant antimicrobial effect. The MED (ED50) was determined by nonlinear regression using Graph-Pad Prism (Graph Pad Software, USA).

(46) 2.8. Results

(47) The results of the experiments described in 2.2, 2.3 and 2.4, above, are indicated in Table 2, herein below

(48) TABLE-US-00015 TABLE 2 Minimal inhibitory concentrations (MIC in g/ml) in Mueller-Hinton broth, cytotoxicity and percentage hemolyses at a concentration of 100 g/ml of peptide P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa P. aeruginosa Average Cytotoxicity Hemolysis Ex. ATCC 27853 P8191900 P1021903 P1021913 IMP1 Livermore 3140 MIC GI.sub.50 Hela Cells at 100 g/ml 1 0.03 0.07 0.07 0.13 0.18 0.10 30 0.5 2 0.04 0.10 0.16 0.18 0.24 0.15 50 0.3 3 0.09 0.30 0.30 0.31 2.0 0.6 34 0.6 4 0.75 1.50 1.50 2.50 >2.0 >1 40 0.6 5 0.08 0.30 0.21 0.21 0.75 0.31 50 0.6 6 0.05 0.10 0.09 0.16 0.24 0.13 50 0.2 7 0.13 0.50 1.50 0.65 >2 >1 13 0.8 8 0.05 0.18 0.18 0.31 0.43 0.23 50 0 9 0.21 0.75 1.50 0.75 >2 >1 50 0.7 10 0.05 0.18 0.13 0.37 0.43 0.23 50 0 11 0.30 1.00 2.00 0.75 >2 >1 50 0.5 12 0.40 1.00 2.00 0.75 >2 >1 50 0.7 13 0.21 0.30 0.40 0.31 0.75 0.40 50 0.4 14 0.21 0.40 0.31 0.65 >2 >1 12 0.3 15 0.50 2.50 1.25 2.25 >2 >1 50 0.3 16 0.05 0.09 0.05 0.10 0.24 0.11 50 0.1 17 0.75 1.50 2.00 1.25 >2 >1 29 0.5 18 0.06 0.10 0.10 0.81 0.30 0.28 50 0.4 19 0.03 0.05 0.03 0.10 0.10 0.06 50 0.2 20 0.21 0.40 0.21 0.31 1.25 0.48 50 0.2 21 0.09 0.40 0.21 0.31 1.00 0.40 50 0.4 22 0.02 0.09 0.09 0.16 0.24 0.12 50 0.2 23 1.00 1.00 1.50 1.25 >2 >1 13 0.5 24 0.05 0.16 0.08 0.16 0.24 0.14 50 0.1 25 0.05 0.10 0.05 0.13 0.24 0.11 50 0.1 26 0.08 0.30 0.13 0.18 0.40 0.22 50 0.8 27 0.09 0.30 0.30 0.21 0.50 0.28 50 0.6 28 0.04 0.10 0.07 0.16 0.24 0.12 50 0.1 29 0.09 0.13 0.13 0.21 0.40 0.19 50 0.5 30 0.09 0.13 0.30 0.50 2.00 0.60 46 0.5 31 0.03 0.16 0.40 0.16 0.24 0.20 50 0.5 32 0.02 0.05 0.05 0.10 0.22 0.09 50 0 33 0.05 0.18 0.29 0.31 0.43 0.25 50 0.1 34 0.05 0.24 0.29 0.48 0.60 0.33 50 0 35 0.05 0.22 0.10 0.24 0.31 0.18 48 0.2 36 0.06 0.18 0.18 0.24 0.43 0.22 50 0.1 37 0.06 0.18 0.31 0.37 1.17 0.42 50 0.5 38 0.09 0.17 0.17 0.27 1.06 0.35 50 0.5 39 0.13 0.21 0.31 0.31 0.75 0.34 50 0.4 40 0.25 0.50 0.50 0.50 1.00 0.55 50 0.7 41 0.02 0.05 0.03 0.25 0.25 0.12 50 0.2 42 0.25 0.06 0.125 0.50 0.50 0.29 50 0.2 43 0.13 0.50 0.125 2.00 2.00 0.95 50 0.5 45 0.25 0.50 0.50 1.00 1.00 0.65 50 0.2 46 0.06 0.25 0.25 0.50 0.50 0.31 50 0.3 47 0.09 0.13 0.13 0.50 0.75 0.32 50 0.2 48 0.02 0.03 0.02 0.13 0.19 0.08 50 0.2 49 0.03 0.06 0.03 0.25 0.50 0.17 50 0.1 50 0.09 0.13 0.06 0.50 2.00 0.56 50 n.d. n.d. = not determined

(49) The results of the experiment described in 2.5, above, are indicated in Table 3 herein below.

(50) TABLE-US-00016 TABLE 3 Stability human Stability rat Ex. Plasma t.sub.1/2 (min) Plasma t.sub.1/2 (min) 1 300 300 2 300 300 6 300 300 16 300 300 22 300 300 24 300 300 25 300 300 28 300 300 29 300 300 32 300 300 35 300 300

(51) The results of the experiment described in 2.6 (PK), above, are indicated in Table 4 herein below.

(52) TABLE-US-00017 TABLE 4 Administration route Parameters Dimensions I.V. S.C. HL_Lambda_z hr 0.53 0.95 Tmax hr 0.08 0.58 Cmax ng/mL 1268.0 2333.3 Cmax_D kg*ng/mL/mg 1268.0 466.7 C0 ng/mL 2174.0 AUCINF_obs hr*ng/mL 679.5 4016.5 AUCINF_D_obs hr*kg*ng/mL/mg 679.5 803.3 Vz_obs mL/kg 1136.1 1705.6 Cl_obs mL/hr/kg 1539.1 1249.8 Bioavailability % 100 118.2

(53) After intravenous administration of Ex. 1 at a dose level of 1 mg/kg body weight, Ex. 1 followed intravenous kinetic characteristics. After PK analysis, Ex. 1 showed an extrapolated C.sub.initial of 2174 ng/ml and a C.sub.max observed of 1268 ng/ml at 5 min. Plasma levels rapidly decreased to 575 and 177 ng/ml at 15 min and 1 hour respectively. From 0.5 to 2 h plasma levels decreased with an elimination half-life of 0.53 h to 10.6 ng/ml at 3 h. The AUCINF_obs amounted to 679.5 ng.Math.h/ml.

(54) After subcutaneous administration of Ex. 1 at a dose level of 5 mg/kg body weight, plasma levels of Ex. 1 increased the first 0.5-1 h and showed a C.sub.max of 2333 ng/ml. From 0.5 to 8 h plasma levels decreased with an elimination half-life of 0.95 h to 7.3 ng/ml at 8 h. The AUCINF_obs amounted to 4016.5 ng.Math.h/ml.

(55) As compared to the normalized AUC value after i.v. administration (100% absorbed, 679 ng.Math.h/ml) of Ex. 1 absorbed after s.c. administration amounted to 118% (803 ng.Math.h/ml). The value above 100% may partially reflect an impaired reliability caused by the limited number of points or is caused by a non-linearity in dose.

(56) The results of the experiment described in 2.7 (Septicaemia Assay), above, are indicated in Table 5-7 herein below.

(57) Septicaemia experiment in mice: LD90-100 of Pseudomonas aeruginosa (ATCC 27853) (9106 CFU/0.5 ml/mouse IV and after 1 h Ex. 1 s.c.

(58) TABLE-US-00018 TABLE 5 Compound/dose Dead Survivors Negative control 5 1 Gentamycin, 1 mg/kg 0 6 Compound of Example 1 in following doses (mg/kg) 10 (2 5) 0 6 5 0 6 2.5 0 6 1 1 5 0.5 2 4 0.25 5 1 0.1 5 1

(59) Septicaemia experiment in mice: LD90-100 of Pseudomonas aeruginosa (ATCC 27853) (9106 CFU/0.5 ml/mouse N), and after 1 and 5 h Ex. 40 s.c.

(60) TABLE-US-00019 TABLE 6 Compound/dose Dead Survivors Negative control 6 0 Gentamycin, 2 1 mg/kg 1 5 Compound of Example 40 in following doses (mg/kg) 2 10 0 6 2 3 0 6 2 1 2 4 2 0.3 6 0 2 0.1 6 0

(61) Septicaemia experiment in mice: LD90-100 of Pseudomonas aeruginosa (ATCC 27853) (9106 CFU/0.5 ml/mouse IV), and after 1 and 5 h Ex. 50 s.c.

(62) TABLE-US-00020 TABLE 7 Compound/dose Dead Survivors Negative control 5 1 Gentamycin, 2 1 mg/kg 1 5 Compound of Example 50 in following doses (mg/kg) 2 10 0 6 2 3 3 3 2 1 5 1 2 0.3 6 0 2 0.1 6 0

REFERENCES

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