ANTIMICROBIAL PEPTIDES

Abstract

The invention relates to antimicrobial peptides (AMPs). The invention also relates to uses, methods of treatment, pharmaceutical compositions and combinations with antimicrobial agents.

Claims

1. A peptide comprising amino acid sequence having at least 52% sequence identity to SEQ ID NO: 1, wherein said amino acid sequence comprises one or more of the following substitutions relative to SEQ ID NO: 1: i. K7R ii. K8R iii. V12A or V12I or V12L iv. K14R v. V16A or V16I or V16 L vi. K18R.

2. A peptide according to claim 1 comprising at least the following substitutions relative to SEQ ID NO: 1: i. K7R ii. K8R iv. K14R; and vi. K18R.

3. A peptide according to claim 1 comprising at least the following substitutions relative to SEQ ID NO: 1: iii. V12A or V12I or V12L v. V16A or V16I or V16 L.

4. A peptide according to claim 1 or claim 2 or claim 3 comprising at least the following substitutions relative to SEQ ID NO: 1: i. K7R, and ii. K8R, and iii. V12A or V12I or V12L, and iv. K14R, and v. V16A or V16I or V16 L; and vi. K18R.

5. A peptide according to any preceding claim further comprising one or more of the following substitutions relative to SEQ ID NO: 1: a) F5Y or F5W b) F6Y or F6W c) substitution of H11 for 3-methylhistidine or 1-methylhistidine or 2,3-diaminopropionic acid or (N-methyl or N,N-dimethyl derivative of 2,3-diaminopropionic acid) or E or D; d) substitution of H15 for 3-methylhistidine or 1-methylhistidine or 2,3-diaminopropionic acid or (N-methyl or N,N-dimethyl derivative of 2,3-diaminopropionic acid) or E or D e) substitution of H23 for 3-methylhistidine or 1-methylhistidine or 2,3-diaminopropionic acid or (N-methyl or N,N-dimethyl derivative of 2,3-diaminopropionic acid) or E or D.

6. A peptide according to any preceding claim wherein one or more of amino acids 9 to 21 is substituted for N-substituted glycine.

7. A peptide according to any preceding claim wherein said peptide comprises amino acid sequence having at least 76% sequence identity to SEQ ID NO: 1.

8. A peptide according to any preceding claim wherein said peptide is at least 25 amino acids in length, suitably wherein said peptide is 25 amino acids in length.

9. A peptide comprising amino acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, suitably consisting of amino acid sequence selected from SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4.

10. A peptide according to any preceding claim consisting of L-amino acids.

11. A peptide according to any preceding claim consisting of D-amino acids.

12. A peptide according to any of claims 1 to 11 for use in medicine.

13. A peptide according to any of claims 1 to 11 for use in treatment or prevention of infection, suitably bacterial infection.

14. A peptide according to any of claims 1 to 11 for use as an antimicrobial.

15. Use of a peptide according to any of claims 1 to 11 as an antimicrobial.

16. A pharmaceutical composition comprising a peptide according to any of claims 1 to 11.

17. A pharmaceutical composition according to claim 16 further comprising an antibiotic agent.

18. A method of treating or preventing infection, suitably bacterial infection, in a subject comprising administering a peptide according to any of claims 1 to 11 to said subject.

19. A method according to claim 18 further comprising administering an antibiotic agent to said subject.

20. A pharmaceutical composition according to claim 17 or a method according to claim 19 wherein said antibiotic agent comprises one or more of colistin, tobramycin and rifampin.

21. A pharmaceutical composition according to claim 17 or claim 20, or a method according to claim 19 or claim 20, wherein the bacterial infection is P.aeruginosa infection or A.baumannii infection, suitably P.aeruginosa infection.

22. A pharmaceutical composition according to claim 17 or claim 20 or claim 21, or a method according to claim 19 or claim 20 or claim 21, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2, and consists of D-amino acids.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0453] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0454] FIG. 1 shows graphs and plots

[0455] FIG. 2 shows diagrams, plots and graphs

[0456] FIG. 3 shows plots and graphs

[0457] FIG. 4 shows diagrams and plots and bar charts

[0458] FIG. 5 shows plots

[0459] Figure P1 shows graphs

[0460] Figure P2 shows plots

[0461] FIG. 6 shows graphs and a table. Parent=Pleurocidin (SEQ ID NO: 1). D-Parent=Pleurocidin (SEQ ID NO: 1)(all D-amino acids). AP1=Analogue Peptide 1—Pleurocidin-KR (PKR) (SEQ ID NO: 2). AP2=Analogue Peptide 2—Pleurocidin-VA (PVA)(SEQ ID NO: 3). D-AP1=D-Analogue Peptide 1-Pleurocidin-KR (PKR) (SEQ ID NO: 2) (all D-amino acids). D-AP2=Analogue Peptide 2—Pleurocidin-VA (PVA)(SEQ ID NO: 3) (all D-amino acids).

[0462] FIG. 7 shows a diagram, bar charts and plots. Peptides as for FIG. 6.

[0463] FIG. 8 shows plots. Peptides as for FIG. 6.

[0464] FIG. 9 shows a combined computational and experimental biophysics approach has yielded a promising AMP therapeutic. Peptides as for FIG. 6.

[0465] FIG. 10 shows plots.

[0466] FIG. 11 shows plots.

[0467] FIG. 12 shows graphs.

EXAMPLES

Example 1: Antimicrobial Peptide Analogues with Therapeutic Potential In Vitro and In Vivo

[0468] Methods: The natural AMP (termed AMP1) was modified to produce synthetic variants, AMPs 1a, 2a, 2b, 3a and 3b. Minimum inhibitory concentrations (MICs) were determined using the microbroth dilution assay with Mueller Hinton broth (MHB) or RPMI (with 5% FBS) and stability in serum was tested by conducting MICs in MHB containing 10% FBS. Synergy experiments were performed following a checkerboard protocol. Cytotoxicity was tested against HEK293 and HeLa immortalised human cell-lines using the XTT viability stain and by quantifying haemolysis of human red blood cells. Patch clamp analysis was performed using the Port-a-Patch® automated patch-clamp system on DPhPE/DPhPG (model for a Gram-negative plasma membrane) or DPhPG (Gram-positive) model membranes challenged with peptide. The murine lung infection model was performed using the agar bead model with Staphylococcus aureus strain NCTC 13616 (EMRSA-15). Mice were dosed with vancomycin or peptide in PBS 3 times over a 48-hour period; lung CFU, weight loss, immune cells in the bronchoalveolar lavage fluid and levels of the cytokines IL-6, TNFα, KC and MCP-1 were monitored.

[0469] Results: In MHB, AMP1a and AMP1b showed broad-spectrum activity against Gram-positive and Gram-negative bacteria. AMP2a and AMP2b had markedly better activity against Gram-positive and Pseudomonas species, whilst AMP3a and AMP3b maintained antibacterial activity but were much less cytotoxic against human cells. In RPMI media, the MIC results showed greater discrimination between the ‘a’ and ‘b’ enantiomers of the AMPs and the peptides were up to 16-fold more potent against S. aureus than they were in MHB. None of the AMPs showed significant loss of activity in the presence of serum or notable haemolysis at concentrations at which they were antibacterial. Patch clamp analysis revealed stark differences in channel forming ability between peptides. AMP2b was selected as the peptide with the highest therapeutic index, for testing against EMRSA-15 in a murine lung infection model. Treatment with a cumulative dose of 15 or 1.5 mg/kg of AMP2b was well tolerated and reduced lung colony forming units (CFU) by a significant level compared to vehicle, with a 1.14 log reduction for 15 mg/kg comparable to vancomycin at 600 mg/kg. No evidence of up-regulation of inflammatory markers was observed suggesting direct killing of the pathogens in the lung.

Example 2: Antimicrobial Peptide Analogues with Therapeutic Potential In Vitro and In Vivo

INTRODUCTION

[0470] Non-traditional approaches, outside the definition of a traditional antibiotic, are being investigated for their potential impact on tackling AMR. One such approach is the use of antimicrobial peptides (AMPs), although they are often dismissed as being poorly suited for systemic infections due to poor tolerability in animal models and high susceptibility to degradation.

[0471] The natural AMP, named ‘Parent’ here, has been found to have broad antimicrobial properties against Gram-positive and Gram-negative bacteria. This activity is thought to be due to its ability to damage the plasma membrane and also to disrupt cell metabolism, with several studies determining that it crosses the plasma membrane and attacks intracellular targets.

[0472] In this study we examined the altered antimicrobial activity, cytotoxicity and membrane binding properties of analogues of the parent AMP specifically designed to have increased conformational flexibility, and their all-D enantiomer counterparts. We also examined the in vivo efficacy of one analogue against a Gram-positive lung infection when delivered intravenously.

TABLE-US-00010 TABLE 1 Antimicrobial activity and cellular toxicity. MS-methicillin sensitive; EMR- epidemic methicillin resistant; VS-vancomycin sensitive; VR-vancomycin resistant. Values are given for peptides tested in Mueller-Hinton broth with values obtained in RPMI given in parentheses. The Selectivity Index (SI) is the EC.sub.50 divided by the MIC in the indicated conditions. Grey or bold values indicate respectively a significant reduction or improvement in potency in RPMI. Peptide concentration (μg/ml) Isolate Parent D-Parent AMR-1 D-AMP1 AMP-2 D-AMP-Z Gram- K. pneumoniae 4-8 (3) 4 (8-16) 2-4 (32) 2 (4) 4-8 (32) 8 (32) negative NCTC 13368 K. pneumoniae 4 (32) 4 (4-8) 2 (32) 2 (4) 4-8 (32) 4 (32) M6 A. baumannii 1-2 (4-8) 1 (2) 1-2 (8-16) 2 (2) 2 (16) 1 (8-32) AYE A. baumannii 1-2 (8) 1 (8) 1 (8) 2 (4) 1-2 (32) 1 (16) ATCC 13437 P. aeruginosa 64 (>32) 2 (16) 4 (>32) 4 (16) 16-32 (>32) 4 (32) PA01 P. aeruginosa 16-32 (>32) 8 (16-32) 8 (>32) 4 (16) 32 (>32) 32 (>32) NCTC 13437 E. coli 1-2 (16) 1 (2-4) 1 (16) 1 (2) 1 (32) 1 (8) NCTC 12923 Gram- MS S. aureus 4 (2) 2 (0.5) 2 (4) 2 (0.25-0.5) 8 (32) 4 (8) positive ATCC 9144 EMR 16 (4) 16 (1) 4 (4) 2 (0.5) 64 (32) 32 (8) S. aureas-15 EMR 16 (4) 16 (1) 4 (8) 4 (1) 64-128 (>32) 64 (32) S. aureas-16 VS E. faecalis 64 (NG) 32 (NG) 16 (NG) 8 (NG) 64-128 (NG) 64 (NG) NCTC 775 VR E. faecium 16-32 (32) 16 (4) 4 (16) 2 (2) 32-64 (32) 16 (32-64) NCTC 12204 HEK293 (87.5 ± 11.1) (61.4 ± 3.9) (40.1 ± 4.1) (36.4 ± 4.3) (~400) (217.3 ± 46.5) Toxicity HeLa (58.2 ± 3.9)  (47.1 ± 2.1) (30.7 ± 2.6) (25.3 ± 2.2) (>400) (198.2 ± 10.2) HC50 ~400 >400 >400 ~400 >>400 >>400 HEK293/ 5.5 (21.9) 3.8 (61.4) 10.0 (10.0) 18.2 (72.8) 6.25 (12.5) 6.8 (27.2) EMRSA-15

[0473] Figure P1: Activity of parent peptide and its analogues on in silico and in vitro models of bacterial plasma membranes. Patch clamp: Representative current traces illustrating membrane activity when model membranes are challenged with each peptide at the indicated concentration. The highly irregular but high amplitude conductance that diminishes over time, observed for the Parent, most notably in membranes that model the Gram-negative bacterial plasma membrane and remain intact, is consistent with an AMP crossing the bilayer without major structural disruption. Such activity is also observed in models of Gram-positive plasma membranes where, additionally, more channel like conductance is observed.

[0474] Figure P2: Systemically delivered D-AMP-1 is effective in a murine model of EMRSA-15 lung infection. C57B16J mice, challenged with 1×106 cfu/mouse EMRSA-15 in tryptic soy agar beads, were treated with vancomycin or D-AMP-1 in three intravenous doses at 4, 24 and 30 hours post infection to achieve the cumulative doses indicated. Bacterial burden in the lung (A), weight loss over the infection period (B) and BAL cells (C-E) and cytokines (F-I) reveal the effect of each intervention. Significance is indicated relative to the saline vehicle (p<0.05 *; <0.01 **; 0.001 ***; 0.0001 ****).

Conclusions

[0475] We have demonstrated that analogues of the parent AMP, in particular D-AMP-1, are potent bactericidal AMPs which can be delivered intravenously to treat bacterial lung infections without triggering the release of pro-inflammatory cytokines or stimulating recruitment of innate immune cells in the mouse model.

[0476] The biophysical analysis, taken together with previous fluorescence studies, reveal species and even strain dependent differences in the extent of membrane permeabilization caused by the peptides at their MICs. This suggests that a primary mechanism of action will be to penetrate the bacteria but at higher concentrations a secondary, membrane disruptive, effect will be observed and that optimisation of the latter function yields more robust performance in a variety of conditions.

Example 3

[0477] A patient is diagnosed with an infection caused by one of the infectious agents described herein, in one of the clinical indications identified.

[0478] A decision to treat with the compound(s) of the invention may be chosen on an empirical basis or treatment directed following initial identification/speciation of the infectious agent and/or its antibiotic resistance profile.

[0479] The treatment is selected from one of the AMP treatment options: [0480] monotherapy with an AMP as described herein, [0481] combined therapy of one or more AMP as described herein with existing antibiotics, or [0482] combination therapy with 2 or more AMPs as described herein.

[0483] The formulation and delivery method will reflect the indication, but the default will be i.v. formulation and administration.

[0484] Treatment will follow a defined schedule.

Example 4

[0485] EMRSA-15 response to challenge with the three D-pleurocidin analogues in MH varies according to metabolism, osmoregulation and cell wall composition.

[0486] FIG. 10A shows EMRSA-15 response to is grown in the presence of sub-inhibitory concentrations of peptide in MH, D-pleurocidin (MIC—16 μg/ml) triggers somewhat different responses than D-pleurocidin-KR (MIC—2 μg/ml) or D-pleurocidin-VA (MIC—32 μg/ml). Most notably, D-pleurocidin-KR alone triggers an increase in uracil and does not trigger an increase in acetate or reduction in succinate.

[0487] FIG. 10B shows EMRSA-15 response to is grown in the presence of sub-inhibitory concentrations of peptide in MH, D-pleurocidin (MIC—16 μg/ml) triggers somewhat different responses than D-pleurocidin-KR (MIC—2 μg/ml) or D-pleurocidin-VA (MIC—32 μg/ml). All three peptides induce changes in lipoteichoic acid but other cell wall components are differentially affected. D-pleurocidin-KR induces less osmotic stress as evidence by choline.

[0488] FIG. 11 shows Pleurocidin analogue is an effective therapeutic in a murine model of EMRSA-15 lung infection.

[0489] D-pleurocidin-KR delivered i.v. in three separate doses (dose shown is accumulative).

[0490] Lung CFU burden reduced by 1.1 log 10 with 15 mg/kg D-pleurocidin-KR is equivalent to that achieved with 600 mg/kg vancomycin, the current standard of care.

[0491] Weight loss and neutrophil recruitment associated with EMRSA-15 infection is mitigated in successfully treated mice.

Example 5—Summary of Potency/Toxicity Data

[0492] The bacterial strains used are as in the above examples. A table of strains is provided below to aid understanding.

[0493] The peptides in this example are as described above.

[0494] Data is provided below.

TABLE-US-00011 MIC on Gram-negative and Gram-positive initial panels Pleuro- Pleuro- Pleuro- Pleuro- D-Pleuro- D-Pleuro- D-pleuro- Pleurocidin D-Pleuro D1 D2 KR VA KR VA VAKR Gram-negatives KP13368 4-8  4 16 >128  2-4 4-8  2  8 4-8 M6  4  4 16-32 128  2 4-8  2  4  4 AYE 1-2  1  2  16 1-2  2  2  1  2 AB17978 1-2  1 2-4  16  1 1-2  2  1  2 PA01 64  2 32-64 >128   4 16-32  4  4  4 PA13437 16-32  8 32 128  8  32  4  32  32 RP73 —  8-16 — — — — 16 16-64 — EC12923 1-2  1  2  8-16  1  1  1  1  2 Gram-positives MSSA9144  4  2  8-16 16-32  2  8  2  4  8 EMRSA15 16 16  64-128 >128   4  64  2  32  32 EMRSA16 16 16 128 >128   4  64-128  4  64  64 VSE775 64 32 128 >128  16  64-128  8  64 32-64 VRE12201 NG NG >128  >128  32 >128   8 128 128 VRE12204 16-32 16 32-64  64  4 32-64  2  16 — Fungi C. albicans  8  32 Toxicity HEK293 87.5 ± 11.1 61.4 ± 3.9 >400 >400 40.1 ± 4.1 ~400 36.4 ± 4.3 217.3 ± 46.5 201.2 ± 25.1 HeLa 58.2 ± 3.9  47.1 ± 2.1 >400 >400 30.7 ± 2.6 >400 25.3 ± 2.2 198.2 ± 10.2 124.0 ± 30.4

TABLE-US-00012 Pleuro- Pleuro- Pleuro- Pleuro- D-Pleuro- D-Pleuro- D-pleuro- Pleurocidin D-Pleuro D1 D2 KR VA KR VA VAKR Gram-negatives KP13368 32  8-16  64-128 >128  32 32 4   32 M6 32 4-8  64-128 >128  32 32 4   32 AYE 4-8 2    64 128  8-16 16 2    8-32 AB17978  8 8    64 128  8 32 4   16 PA01 >32  16   >128  >128  >32  >32  16   32 PA13437 >32  16-32 >128  >128  >32  >32  16   >32  RP73 — 16-32 — — — — 33   32-64 — EC12923 16 2-4  32 128 16 32 2    8 Gram-positives MSSA9144  2 0.5 128 128  4 32 0.25-0.5   8 EMRSA15  4 1   128 128  4 32 0.5  8 EMRSA16  4 1   128 >128   8 >32  1   32 VSE775 NG NG NG NG NG NG NG NG VRE12201 NG NG NG NG NG NG NG NG VRE12204 32 4   128 128 16 32 2   32-64 D- D- Pleurocidin Pleurocidin Pleurocidin pleurocidin (MH) (RPMI) (MH) (RPMI) Klebsiella 4-8 32   4  8-16 pneumoniae KP13368 M6  4 32   4 4-8 AYE 1-2 4-8 1  2 AB17978 1-2 8   1  8 PA01 64 >32    2 16 PA13437 16-32 >32    8 16-32 EC12923 1-2 16   1 2-4

TABLE-US-00013 Stability in serum (MH) Pleurocidin D-pleurocidin D-pleurocidin-KR Pleurocidin-KR Pleurocidin-VA D-Pleurocidin-VA No No No No No No serum Serum serum Serum serum Serum serum Serum serum Serum serum Serum KP13368  4  8 4 4   2 1   2  4  8  16  8  8-32 M6  4  8 4 4   2 0.5 4  2 16  8  4  8 AYE  2  2 2 2   2 0.5 2  2  2  4  1  2 Ab17978  2  2 1 0.5 2 0.5 2  2  2  4  1  2-16 PA01 16 32 2 4   4 4   4 >128   16* >128   4 16 PA13437 16 32 8 16   4 8   4  32*  16* >128  32 32 EC12923  2  1 1 0.5 1  0.25 2  1  1  1  1  1 For the most part, the D-AMPs appear to retain activity in the presence of 10% fetal bovine serum. Activity is lost for L-AMPs for Pseudomonas but not other species.

[0495] The underlined values indicates these peptides perform at a lower level against the same strains in RPMI compared to in MHB. It does not indicate that they lack function (although in some cases activity is poor).

[0496] As noted in the description, the peptides Pleuro-D1 and Pleuro-D2 (‘mixed peptides’ containing both D- and L-amino acids) are suitably not part of the invention.

[0497] We refer to FIG. 12 which shows graphs of time kills.

TABLE-US-00014 TABLE Of Bacterial Strains Used Species Strain Antibiotic resistance profile Klebsiella NCTC 13368 AMP, PIP, GEN, TOB, pneumoniae M6 CTX, CAZ, ATM, CHL AMP, PIP, GEN, TOB Acinetobacter NCTC 17978 Sensitive baumannii AYE (ATCC AMP, GEN, CIP, TZP, BAA-1710) LVX, AMK Pseudomonas PAOI (ATCC AMP, PIP, ATM, CHL aeruginosa BAA-47) NCTC 13437 AMK, GEN, TOB, AMP, PIP, TZP, CAZ, ATM, IMP, MEM, CIP, LVX, CHL Escherichia coli NCTC 12923 AMK, GEN, TOB Enterococcus NCTC 12204 VAN, CIP faecalis NCTC 12201 VAN, CIP Enterococcus NCTC 775 Susceptible faecium Staphylococcus EMRSA-15 MET, CIP aureus (NCTC 13616) EMRSA-16 MET, CIP (NCTC 13277) ATCC 9144 Susceptible (NCTC 6571) Abbreviations as standard in the art (e.g. according to BSAC Bacteraemia Resistance Surveillance Programme'/‘BSAC Respiratory Resistance Surveillance Programme’ published by the British Society for Antimicrobial Chemotherapy, Griffin House, 53 Regent Place, Birmingham, B1 3NJ, UK)—http://www.bsacsurv.org/science/antimicrobials/

Example 6: D-PLEUROCIDIN-KR

[0498] Here we demonstrate the effectiveness of peptides according to the present invention, using D-PLEUROCIDIN-KR as an illustrative example.

[0499] We evaluate the in vitro potency of D-PLEUROCIDIN-KR. D-PLEUROCIDIN-KR is a 25 residue amphipathic peptide comprising all D-amino acids and aminated at the C-terminus. D-PLEUROCIDIN-KR has a broad spectrum activity and can act in synergy with the aminoglycoside tobramycin and with rifampin, with activity against Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli, including antibiotic resistant strains. Potency towards Pseudomonas aeruginosa is a little lower but enhanced in combination with either tobramycin or rifampin.

[0500] In this example, D-PLEUROCIDIN-KR was tested with five S. aureus strains. Three studies were conducted: (1) The MIC values were determined with a collection of five S. aureus strains using two types of medium, Mueller Hinton Broth and RPMI 1640 containing 5% fetal bovine serum. The MIC study included linezolid and vancomycin as quality control reference agents. (2) A Maximum Tolerated Dose (MTD) analysis of D-PLEUROCIDIN-KR was conducted with intravenous (IV) dose administration to determine the upper limit of dosing in immunocompetent mice.

Materials

[0501] Chemicals and Medium: Bacto agar (214040, BD, USA), Bacto agar (214040, BD, USA), Brain-heart infusion broth (BHI) (237500, BD, USA), Cyclophosphamide monohydrate (C-0768, Sigma, USA), Etomidate-®lipuro (20 mg/10 mL; B. Braun Melsungen AG, Germany), Fetal Bovine Serum (SH30084.03 HyClone, USA), Difco™ Mueller Hinton Broth II (275730, BD, USA), Linezolid (GA2609, Glentham Life Science, UK), Nutrient agar (NA) plates (CMP0101312, CMP, Taiwan), Nutrient broth (DIFCO, USA), Phosphate-buffered saline (PBS) tablet (P4417, Sigma, USA), Saline (sterilized 0.9% NaCl, 53539, SinTong, Taiwan), RPMI 1640 (SH30027.02, HyClone, USA), Vancomycin hydrochloride (V2002, Sigma, USA), and Water for injection (WFI, sterilized water, Tai-Yu, Taiwan).

[0502] Plasticware: 96-well V-bottom polypropylene plate (Nunc™ 249949, Nunc, USA), Biomek tip (717252, Beckman Coulter, USA), Counting vial (Hsin-Pei Co, Ltd., Taiwan), Disposal syringe (1 mL, Terumo, Japan), Microcentrifuge Tubes 1.5 mL Click-Cap (Treff AG, Switzerland), Pipets (Costar, USA), Sterile inoculation loop (731161, Bio-Check Laboratories, Taiwan) and Tip (Labcon, USA).

Equipment

[0503] Absorbance microplate readers (Tecan, Infinite F50, USA), Biomek 96 well liquid handling instruments (Beckman Coulter, FX and FXP, USA), Biological safety cabinet (NuAire, USA), Centrifuge (Model 5810R, Eppendorf, Germany), Centrifuge (Model 5922, Kubota, Japan), Electronic scale (Tanita, Model 1140, Japan), Incubators (Firstek, Taiwan), Individually Ventilated Cages (GM500 IVC seal safe plus cage system) (Tecniplast, Italy), Micropipettes (Gilson, France), Open-Topped cage (Allentown, USA), Orbital shaking incubator (Firstek Scientific, Taiwan), Pipetman (Rainin, USA), Pipetman (P100 Gilson, France), Polytron homogenizer (Kinematica, Switzerland), Portable weighing scale (JKH-1000, Jadever, Taiwan), Refrigerated incubator (Hotpack, USA), Shaking incubator (Firstek, Taiwan), Refrigerator-freezer (Sanyo, Japan), and Ultra-Low temperature freezer (Panasonic, Japan).

Microbial Strains

[0504] The S. aureus strains ATCC 29213, ATCC 19636, ATCC 33591 (MRSA), BAA-1556 (MRSA USA 300) were obtained from the American Type Culture Collection (ATCC). Vancomycin-resistant S. aureus VRS-2 (VanA VRSA) was obtained from the Network for Antibiotic Resistance in S. aureus (NARSA). The mouse lung infection models have been validated with these strains. The antibiotic susceptibility of these strains is summarized in the Table below and was determined following the recommend assay conditions of the Clinical Laboratory Standards Institute (CLSI) M7-A10 microdilution procedure and the CLSI M100 interpretive criteria. The species identity was confirmed with 16S rRNA analysis.

TABLE-US-00015 TABLE S. aureus strains for MIC studies, antibiotic susceptibility Source strain ID ATCC ATCC 19636 ATCC 29213 (Smith) 33591 BAA-1556* VRS-2 Resistance MRSA VanA Susceptible Susceptible MRSA USA 300 VRSA Oxacillin 0.25-0.5  S  0.25 S >64    R 64   R >64    R Cefepime 2-4 — 2   — >64    — 16-64 — >64    — Ceftriaxone 4   — 4   — >64    — ≥64    — >64    — Imipenem 0.016-0.031 —  ≤0.016 — ≥16    — 0.125-0.25  — >16    — Meropenem   0.0625 — — — — — — — — — Vancomycin 0.5-1   S 1   S 1   S 0.5-1   S >64    R Teicoplanin  0.25 S — — — — — — — — Linezolid 2-4 S 2-4 S 0.5-1   S 2   S 2   S Daptomycin 0.125-0.5  S  0.25 S 0.5 S  0.25 S  0.25 S Tigecycline 0.125-0.25  — 0.125-0.25  — 0.25-0.5  —  0.25 —  0.25 — Trimethoprim/ ≤0.5/9.5  S ≤0.5/9.5  S 0.5/9.5 S 0.25/4.75 S  >8/152 R sulfamethoxazole Clindamycin  0.125 S  0.125 S >64    R >64    R >64    R Erythromycin 0.25-1   S-I 0.25-1   S-I >64    R >64    R >64    R Gentamicin  0.25 S  0.25 S 0.25-1   S 0.25-0.5  S 32-64 R Mupirocin 0.25-2   — 1   — 1-2 — >64    — 1-2 — Levofloxacin 0.125-0.25  S 0.125 S  0.25 S 4-8 R 32   R S denotes susceptible, R denotes resistant or not susceptible, and I denotes intermediate susceptibility based on the CLSI interpretive criteria published in CLSI M100. *BAA-1556 is a mupirocin resistant strain.

[0505] D-PLEUROCIDIN-KR solutions for MIC assessment were prepared in sterilized water for injection (WFI). Solutions for MTD and efficacy assessments were prepared in normal saline (0.9% NaCl). The dose formulation for in vivo studies was freshly prepared on the day of the study, aliquoted and then stored at 4° C. for each dose administration. All solutions were prepared as described below. The dosing volume was 5 mL/kg for intravenous (IV) dosing, and 10 mL/kg for subcutaneous (SC) dosing.

[0506] For MIC assessment, the 1.6 mg/mL D-PLEUROCIDIN-KR stock solution was freshly prepared by dissolving 1 mg of D-PLEUROCIDIN-KR in 0.625 mL of WFI directly in the vial.

[0507] For MTD assessment, the 6 mg/mL D-PLEUROCIDIN-KR solution was freshly prepared by dissolving 16.2 mg of D-PLEUROCIDIN-KR in 2.7 mL of saline. A 0.9 mL of the 6 mg/mL solution was further diluted by adding 1.8 mL of saline, generating a lower 2 mg/mL dose solution. A 0.9 mL of the 2 mg/mL solution was further diluted by adding 0.9 mL of saline, generating a lower 1 mg/mL dose solution.

Experiment Design

[0508] a. Minimum Inhibitory Concentration (MIC) Assay

[0509] The MIC assay was used to assess the in vitro antimicrobial potency of D-PLEUROCIDIN-KR following the protocol M07-A10 of the Clinical and Laboratory Standards Institute (CLSI) for medium, inoculum preparation, and MIC endpoint reading. In this assay, the MIC value was the lowest concentration of the test agent that completely inhibits visible growth of the pathogen in broth culture. Two broth mediums were used, Mueller Hinton Broth and RPMI 1640+5% fetal bovine serum. Test substances were evaluated in 11-point titrations and performed in duplicate. D-PLEUROCIDIN-KR was evaluated at 32 to 0.0313 μg/mL, since D-PLEUROCIDIN-KR has a MIC of 4 μg/mL for epidemic methicillin-resistant S. aureus (EMRSA-15) in MHB (see examples above). Linezolid and vancomycin were evaluated as the historical reference compound.

[0510] b. Maximum Tolerated Dose (MTD) Assessment

[0511] Tolerability analysis was conducted to determine the upper limit of D-PLEUROCIDIN-KR dosage. Test article solutions were intravenously (IV) administered to immunocompetent ICR mice with the dose schedule and concentrations indicated in the study design in the table below.

TABLE-US-00016 TABLE Maximum tolerability assessment (MTD), Study design Dosage Test Dose Dose Conc. mg/kg/ mg/kg/ Mice Group Substance Schedule Route mg/mL mL/kg dose 24 h female 1 Vehicle TID IV NA 5 — —  4 (0.9% NaCl) 2 D- TID IV 6 5 30 90  4 PLEUROCI DIN-KR 3 D- TID IV 2 5 10 30  4 PLEUROCI DIN-KR 4 D- TID IV 1 5  5 15  4 PLEUROCI DIN-KR Immunocompetent female ICR mice: 20 The immunocompetent animals were not infected. The vehicle was 0.9% NaCl. The animals were administered test article thrice per day (TID) at 8 h intervals and clinical symptoms associated with overt toxicity were monitored 5 minutes after each dose (full observation after the 1.sup.st dose and cage-side observation after the 2.sup.nd and 3.sup.rd doses). All four groups were conducted in parallel and if overt toxicity was observed after the 1.sup.st or 2.sup.nd dose of a group, the 2.sup.nd or 3.sup.rd dose of that group was not administered.

[0512] The animals were not infected. Doses were IV administered thrice (TID) with 8 h intervals (q8h) between doses. Animals were humanely euthanized after 3 days of the last treatment or at humane endpoints.

Methods

[0513] a) Minimum Inhibitory Concentration (MIC) Study

[0514] The direct colony suspension method was used to prepare inoculated broth. Isolated colonies were taken from an 18-24 h culture plate. Optical density measurements (OD.sub.620nm) were used to estimate the bacterial density. The D-PLEUROCIDIN-KR, vancomycin and linezolid stock solutions were prepared as above. The stock solutions were diluted by 2-fold serial titrations with their respective vehicles for a total of 11 concentrations.

[0515] A 4 μL aliquot of each dilution was added to 196 μL of broth medium seeded with the organism in wells of a sterilized 96 well polypropylene plate (Nunc™ 249949) using aluminum foil as cover. The final bacterial count was 2 to 8×10.sup.5 CFU/mL. The MIC assay was conducted twice with different mediums: Mueller Hinton Broth and RPMI 1640 with 5% FBS. The final test article concentration range was 32 to 0.0313 μg/mL.

[0516] Each test substance dilution was evaluated in duplicate on one test occasion. Vehicle-control and reference control agents, linezolid and vancomycin, were used as blank and positive controls, respectively. Plates were incubated at 35-37° C. for 18 h. The test plates were visually examined and each well was visually scored for growth or complete inhibition of growth. The MIC value was recorded. The CLSI guidelines of 100% visual growth inhibition were used to call an MIC endpoint.

[0517] b) MTD Assessment of D-PLEUROCIDIN-KR

[0518] i. Animal Preparation

[0519] The MTD assessment was performed with immunocompetent female ICR mice, weighing 22 f 2 g. All animals were specific pathogen free.

[0520] ii. Treatment

[0521] Test articles were administered to animals by IV injections following the dose schedule, volumes, and concentrations indicated the Table above for MTD study. Animals were observed for 5 min after each administration to detect acute toxicity which was recorded and reported, if observed. Animals were humanely euthanized if severe acute toxicity was observed during the experimental period.

[0522] iii. Dosing and Animal Observations

[0523] Clinical examinations: animals were observed for the presence of acute toxic symptoms and autonomic effects during the first 5 minutes after the first IV administration—see table below:

TABLE-US-00017 Table: Clinical observations Decrease in Decrease in Body Weight Touch Spontaneous Low Body (B.W.) (g) Response Activity Limb Post Temperature Irritability Increase in Straub Tail Skin Color Piloerection Exploration Hyperactivity Decrease in Reactivity Respiration Increase in Exploration Palpebral Size Increase in Pinna Righting Salivation Decrease in Startle (Fluid and Palpebral Size Response Viscosity) Increase Placing Ataxia Lacrimation Death Touch Response Decrease Tremor Convulsion Diarrhea Startle (Chronic/ Response Tonic) Clinical observations were made at 5 minutes after the first dosage and recorded.

[0524] The adverse effects were monitored at 5 minutes via cage-side observation after each subsequent IV administration on Day 1. Animals were observed again for mortality at 72 h after the last treatment. Body weights were recorded before each treatment and at 72 h after the last treatment. If overt toxicity was observed after the 1.sup.st or 2.sup.nd dose of a group, the 2.sup.nd or 3.sup.rd dose of that group was not administered. Animals were humanely euthanized at earlier time points if they were found to be in distress or at a moribund state during observation.

Data and Analysis

[0525] Any adverse events observed were summarized. List of scores from observation assessments were also recorded. The maximum dose that did not result in death or a humane euthanasia endpoint was recorded and reported.

[0526] Relevant ethical approvals were sought and obtained. Animal husbandry, welfare and care was carried out according to standard methods and complied with the relevant approvals and guidelines in place.

Study Results and Discussion

[0527] a. MIC assessment with D-PLEUROCIDIN-KR

[0528] The MIC values of D-PLEUROCIDIN-KR and reference agents, linezolid and vancomycin, are summarized in Table MIC. The MIC values of D-PLEUROCIDIN-KR against the five tested S. aureus strains were all 2 μg/mL in Mueller Hinton Broth. The MIC value of D-PLEUROCIDIN-KR against the five tested S. aureus strains ranged from 0.125 to 0.5 μg/mL in RPMI 1640 with 5% fetal bovine serum. The S. aureus strains ATCC 29213 (susceptible strain) and ATCC BAA-1556 (USA300, MRSA resistant) both had a MIC value of 0.5 μg/mL, and strain ATCC 33591 (MRSA) had a MIC value of 0.25 μg/mL. The other two S. aureus strains had a MIC value of 0.125 μg/mL.

[0529] The MIC data of D-PLEUROCIDIN-KR using Mueller Hinton Broth as well as RPMI 1640 with 5% fetal bovine serum is consistent with the results in the earlier examples, with the tested S. aureus strains. The increased potency of D-PLEUROCIDIN-KR of culturing in RPMI with 5% FBS compared to Mueller Hinton Broth is also consistent with the earlier examples. The MIC of linezolid and vancomycin was determined for a quality control and the MIC value in Mueller Hinton Broth met the acceptance criteria based on PDS historical reference data.

[0530] b. MTD assessment of D-PLEUROCIDIN-KR

[0531] The MTD of D-PLEUROCIDIN-KR was assessed in immunocompetent ICR mice. The animals were not infected. D-PLEUROCIDIN-KR at 5, 10 and 30 mg/kg was IV administered thrice (TID) with 8 h intervals (q8h) for 1 day. Animals were monitored for acute symptoms (clinical observations table above) after the first dose administration. Clinical symptoms were scored after the first dose administration in Table MTD. The toxicity results of D-PLEUROCIDIN-KR in immunocompetent ICR mice are summarized in Table MTDR1. The body weight of all animal groups was recorded and summarized in Table MTDR2.

[0532] D-PLEUROCIDIN-KR was tolerated in immunocompetent ICR mice at 5 mg/kg (IP, TID, q8h), however the animals had severe cyanosis, labored respiration and edema after the first dose administration (Table MTD). We therefore infer that the 5 mg/kg dose may be close to MTD. D-PLEUROCIDIN-KR was not tolerated at 10 and 30 mg/kg dosages (Table MTDR1). All animals were found dead within 5 minutes after the .sub.1st dosing of 30 mg/kg D-PLEUROCIDIN-KR. In the 10 mg/kg D-PLEUROCIDIN-KR dosing group, one of the four animals was found dead after the 1.sup.st dosing, and the surviving three animals exhibited adverse side effects; hence the 2.sup.nd and 3.sup.rd 10 mg/kg doses were not administered.

TABLE-US-00018 TABLE MIC MIC values of D-PLEUROCIDIN-KR, linezolid and vancomycin against five S. aureus strains MIC, μg/mL PT #1235860 Reference D-PLEUROCIDIN-KR Vancomycin Linezolid RPMI + RPMI + RPMI + No. Assay # Species Strain ID Resistance MHB 5% FBS MHB 5% FBS MHB 5% FBS 1 604110 Staphylococcus aureus ATCC 29213 Susceptible 2 0.5 0.5  2 4 2 2 606000 Staphylococcus aureus ATCC 19636 (Smith) Susceptible 2  0.125 0.5  2 2 2 3 605000 Staphylococcus aureus ATCC 33591 MRSA 2  0.25 1    4 2 1 4 604055 Staphylococcus aureus ATCC BAA-1556 USA300, MRSA 2 0.5 0.5  2 2 2 5 604147 Staphylococcus aureus VRS-2 VanA, VRSA 2  0.125 4   >32  2 1 *BAA-1556 is a mupirocin resistant strain (MRSA).

TABLE-US-00019 TABLE MTD MTD results, adverse effects at 5 minutes after dose administration (full observation after the 1.sup.st dosing) Treatment PT# 1235860 Vehicle (NID-162) (0.9% NaCl) (D-PLEUROCIDIN-KR) Route IV IV Dosage 5 mL/kg, TID 5 mg/kg, TID g8h 10 mg/kg, TID q8h 30 mg/kg, TID q8h BEHAVIORAL No. 1 No. 2 No. 3 No. 4 No. 1 No. 2 No. 3 No. 4 No. 1 No. 2 No. 3 No. 4 No. 1 No. 2 No. 3 No. 4 B.W. (g) 25 24 24 24 25 25 23 25 22 23 23 22 21 22 23 24 Irritability − − − − − − − − − − − − Hyperactivity − − − − − − − − − − − − Inc. Startle − − − − − − − − − − − − Inc. Touch − − − − − − − − − − − − Dec. Startle Response − − − − − − − − ± − ± − Dec. Touch Response − − − − − − − − ± ± + ± Inc. Exploration − − − − − − − − − − − − Dec. Exploration − − − − − − − − + ± + + Pinna − − − − − − − − + ± + + Placing − − − − − − − − ± ± − − NEUROLOGIC Tremor − − − − − − − − − − − − Dec. Spont. Activity − − − − − − − − ± ± ± ± Straub Tail − − − − − − − − − − − − Reactivity − − − − − − − − − − − − Righting − − − − − − − − − − − − Ataxia − − − − − − − − ± ± ± ± Convulsion C.T.C-T − − − − − − − − − − − − Low Limb Post − − − − − − − − − ± ± ± Abdominal Tone − − − − − − ± − ± − + ± Limb Tone − − − − − − − − + + + + Grip Strength − − − − − − − − ± ± − − AUTONOMIC Skin Color − − − − C± C± C± C± C± C± C± C± Respiration − − − − D± D± D± D± D± D± D± D± Salivation F.V. − − − − − − − − − − − − Lacrimation − − − − − − − − − − − − Diarrhea − − − − − − − − − − − − Body Temperature − − − − − − − − − − − − Piloerection − − − − − − − − − − − − Inc. Palpebral Size − − − − − − − − − − − − Dec. Palpebral Size − − − − − − − − − − − − Others − − − − E E E E E E E E Death − − − − − − − − − − − − + + Notes: “−” no effects; “±”: Slight to moderate effects; Severe effects; “Inc.”: Increased; “Dec.”: Decreased; “Spont.”: Spontaneous; “C.”: Chronic; “T.”: Tonic; “C-T”: Chronic-Tonic; “F.”: Fluid; “V.:: Viscosity; “C.”: Cyanosis; “D.”: Depth; E: Edema

TABLE-US-00020 TABLE MTDR1 MTD results, mortality assessment prior and post dose administrations Mortality (total number of deaths/total number of animals) Dose 5 min of 1 h of 5 min of 1 h of 5 min of Compound Route (mg/kg) 1.sup.st dosing 1.sup.st dosing 2.sup.nd dosing 2.sup.nd dosing 3.sup.rd dosing Vehicle IV  5 mL/kg, 0/4 0/4 0/4 0/4 0/4 (0.9% NaCl) TID, q8h PT# 1235860 IV  5 mg/kg, 0/4 0/4 0/4 0/4 0/4 (NID-162) TID, q8h (D-PLEUROCIDIN-KR) 10 mg/kg, 0/4 1/4 1/4 1/4 1/4 TID, q8h 30 mg/kg, 4/4 All dead TID, q8h Mortality (total number of deaths/total number of animals) Dose 1 h of 24 h of 48 h of 72 h of Compound Route (mg/kg) 3.sup.rd dosing 3.sup.rd dosing 3.sup.rd dosing 3.sup.rd dosing Vehicle IV  5 mL/kg, 0/4 0/4 0/4 0/4 (0.9% NaCl) TID, 8qh PT# 1235860 IV  5 mg/kg, 0/4 0/4 0/4 0/4 (NID-162) TID, q8h (D-PLEUROCIDIN-KR) 10 mg/kg, 1/4 1/4 1/4 1/4 TID, q8h 30 mg/kg, All dead TID, q8h *Note: The 2.sup.nd and 3.sup.rd doses of the surviving animals in the 10 mg/kg group were not administered due to the mortality after the 1.sup.st dose.

TABLE-US-00021 TABLE MTDR2 MTD results, body weight records prior and post dose administrations Body Weight (g) Dose 24 h of 48 h of 72 h of Compound Route (mg/kg) No. 1.sup.st dosing 2.sup.nd dosing 3.sup.rd dosing 3.sup.rd dosing 3.sup.rd dosing 3.sup.rd dosing Vehicle IV  5 mL/kg, 1 25 25 25 26 26 26 (0.9% NaCl) TID q8h 2 24 24 25 25 25 26 3 24 22 23 23 24 24 4 24 24 25 25 25 26 PT #1235860 IV  5 mg/kg, 1 25 25 25 26 25 26 (NID-162) TID q8h 2 25 25 24 25 25 26 (D-PLEUROCIDIN-KR) 3 23 23 23 23 23 24 4 25 25 24 25 25 26 10 mg/kg, 1 22 21  21*  21*  21*  22* TID q8h 2 23 dead 3 23 23  23*  24*  24*  23* 4 22 21  21*  21*  21*  22* 30 mg/kg, 1 21 dead TID q8h 2 22 dead 3 23 dead 4 24 dead *Note: The 2.sup.nd and 3.sup.rd doses of the surviving animals in the 10 mg/kg group were not administered due to the mortality after the 1.sup.st dose.

TABLE-US-00022 TABLE of Sequences SEQ ID NO:1 Pleurocidin (P) SEQ ID NO:2 Pleurocidin-KR (PKR); D-Pleurocidin-KR (DPKR) when made of D amino acids SEQ ID NO:3 Pleurocidin-VA (PVA); D-Pleurocidin-VA (DPVA) when made of D amino acids SEQ ID NO:4 Pleurocidin-KRVA (PKRVA) D-Pleurocidin- KRVA (DPKRVA) when made of D amino acids SEQ ID NO: 5 See Table P To SEQ ID NO: 15 SEQ ID NO: 16 See Table V To SEQ ID NO: 23 SEQ ID NO: 24 Pleurocidin X1 to X11

[0533] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to those precise embodiments and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.