U.S. patent application number 16/274896 was filed with the patent office on 2019-06-13 for inhibition of biofilm organisms.
The applicant listed for this patent is NovaBiotics Limited. Invention is credited to Cedric Charrier, Derry Mercer, Deborah O'Neil.
Application Number | 20190175528 16/274896 |
Document ID | / |
Family ID | 40671951 |
Filed Date | 2019-06-13 |
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United States Patent
Application |
20190175528 |
Kind Code |
A1 |
O'Neil; Deborah ; et
al. |
June 13, 2019 |
INHIBITION OF BIOFILM ORGANISMS
Abstract
The present invention relates to a product comprising at least
two antibiofilm agents wherein at least one of the antibiofilm
agents is an antimicrobial peptide. The second antibiofilm agent is
cysteamine. There is also provided the use of the product in the
treatment of a microbial infection.
Inventors: |
O'Neil; Deborah; (Aberdeen,
GB) ; Mercer; Derry; (Aberdeen, GB) ;
Charrier; Cedric; (Aberdeen, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NovaBiotics Limited |
Aberdeen |
|
GB |
|
|
Family ID: |
40671951 |
Appl. No.: |
16/274896 |
Filed: |
February 13, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15082976 |
Mar 28, 2016 |
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16274896 |
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13260547 |
Nov 17, 2011 |
9339525 |
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PCT/GB2010/000631 |
Mar 31, 2010 |
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15082976 |
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61165396 |
Mar 31, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61L 2300/424 20130101; A61L 2300/45 20130101; A61K 31/145
20130101; A61K 38/1729 20130101; A61K 38/08 20130101; A61P 31/00
20180101; A61L 15/46 20130101; A61L 31/16 20130101; A61L 2300/404
20130101; A61L 15/44 20130101; A61L 27/54 20130101; A61L 29/16
20130101; A61L 2300/254 20130101; A61K 31/145 20130101; A61K
2300/00 20130101; A61K 38/08 20130101; A61K 2300/00 20130101; A61K
38/1729 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/145 20060101
A61K031/145; A61L 27/54 20060101 A61L027/54; A61P 31/00 20060101
A61P031/00; A61P 31/04 20060101 A61P031/04; A61K 38/17 20060101
A61K038/17; A61L 31/16 20060101 A61L031/16; A61L 29/16 20060101
A61L029/16; A61K 38/08 20060101 A61K038/08; A61L 15/46 20060101
A61L015/46; A61L 15/44 20060101 A61L015/44 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2009 |
GB |
0905451.1 |
Claims
1. A product comprising at least two antibiofilm agents wherein at
least one of the antibiofilm agents is an antimicrobial
peptide.
2. A product as claimed in claim 1 wherein the other antibiofilm
agent is a dispersant or an anti-adhesive agent.
3. A product as claimed in claim 1 or claim 2 wherein the
antimicrobial peptide is an antibacterial peptide.
4. A product as claimed in any preceding claim wherein the
antimicrobial peptide comprises amino acids according to the
formula I: ((X).sub.l(Y).sub.m).sub.n (I) wherein l and m are
integers from 1 to 10, for example 1 to 5; n is an integer from 1
to 10; X and Y, which may be the same or different, are
independently a hydrophobic or cationic amino acid.
5. A product as claimed in claim 4 wherein the antimicrobial
peptide comprises amino acids according to the formula (I) wherein
X and Y are cationic amino acids.
6. A product as claimed in any preceding claim wherein the
antimicrobial peptide comprises between 2 and 200 amino acids.
7. A product as claimed in any preceding claim wherein X and/or Y
are cationic amino acids.
8. A product as claimed in claim 7 wherein X and/or Y are selected
from the group consisting of histidine, arginine and lysine.
9. A product as claimed in claim 8 wherein X and/or Y are selected
from arginine and lysine.
10. A product as claimed in any of claims 2 to 9 wherein the
dispersant is an agent capable of dispersing the particles of a
biofilm.
11. A product as claimed in claim 10 wherein the dispersant is a
mucolytic agent.
12. A product as claimed in any of claims 10 to 12 wherein the
dispersant is an enzyme.
13. A product as claimed in claim 12 wherein the enzyme is selected
from the group consisting of DNase, alginase, protease and
carobohydrase.
14. A product as claimed in any of claims 10 to 12 wherein the
dispersant is an amine.
15. A product as claimed in claim 14 wherein the amine is an
aminothiol.
16. A product as claimed in claim 15 wherein the amine is selected
from acetylcysteine and cysteamine.
17. A product as claimed in any of claims 10 to 12 wherein the
dispersant is an acid.
18. A product as claimed in claim 17 wherein the acid is
ethylenediaminetetraacetic acid (EDTA).
19. A product as claimed in any of claims 2 to 9 wherein the
anti-adhesive agent is an agent capable of inhibiting adhesion
between cells, proteins and organisms
20. A product as claimed in claim 19 wherein the anti-adhesive
agent is selected from the group consisting of hyaluronan, heparin
and Carbopol 934.
21. A product as claimed in any preceding claim comprising a
synergistically effective amount of (i) a first antibiofilm agent,
and (ii) a second antibiofilm agent wherein the second antibiofilm
agent is different from the first antibiofilm agent and is an
antimicrobial peptide.
22. A product as claimed in any preceding claim for use as a
disinfectant or biocide.
23. A product as claimed in any of claims 1 to 21 for use as a
medicament.
24. A substrate to which a product as claimed in any preceding
claim is applied or attached.
25. A substrate as claimed in claim 24 wherein the substrate is
selected from the group consisting of dressings, medical devices
and indwelling devices.
26. A substrate as claimed in claim 25 wherein the indwelling
device is selected from the group consisting of stents, catheters,
peritoneal dialysis tubing, draining devices, joint prostheses and
dental implants.
27. A pharmaceutical composition comprising a product as claimed in
any of claims 1 to 21 and one or more pharmaceutically acceptable
diluents, excipients and/or carriers.
28. Use of a product as claimed in any of claims 1 to 21 in the
treatment of a microbial infection or disease or condition
associated therewith.
29. Use of an antimicrobial peptide comprising amino acids
according to the formula I in the treatment of a microbial
infection or disease or condition associated therewith.
30. The use as claimed in either one of claims 28 and 29 wherein
infection, or disease or condition associated therewith, is
selected from the group consisting of skin and wound infections,
middle-ear infections, gastrointestinal tract infections,
peritoneal membrane infections, urogenital tract infections, oral
soft tissue infections, formation of dental plaque, eye infections,
endocarditis, infections in cystic fibrosis, and infections of
indwelling medical devices.
31. A method of preventing biofilm formation in an environment
comprising the step of administering to the environment a product
as claimed in any of claims 1 to 21 or a substrate as claimed in
any of claims 24 to 26.
32. A method of preventing biofilm formation in an environment
comprising the step of administering to the environment an
antimicrobial peptide comprising amino acids according to formula
I.
33. A method as claimed in either one of claims 31 and 32 wherein
the environment comprise a biofilm forming microorganism selected
from bacteria, fungi, yeast, viruses and protozoa.
34. A method as claimed in claim 33 wherein the microorganism is a
bacterium.
35. A method as claimed in claim 34 wherein the bacterium is
selected from the group consisting of may include Pseudomonas spp.,
Staphylococcus spp., Haemophilus spp., Burkholderia spp.,
Streptococcus spp., Propionibacterium spp.
36. A method as claimed in claim 35 wherein the bacterium is
selected from Pseudomonas spp., and Staphylococcus spp.
37. A method as claimed in claim 36 wherein the bacterium is
Pseudomonas aeruginosa, Staphylococcus aureus or Staphylococcus
epidermidis.
38. A method as claimed in any of claims 32 to 37 wherein the
environment is the mouth.
39. A method as claimed in claim 38 for the prevention of the
formation of plaque or caries on a human tooth or dental
implant.
40. A method of treating a microbial infection by prophylaxis or
therapy comprising the sequential or combined administration in a
therapeutically effective amount of: a first antibiofilm agent; and
a second antibiofilm agent different from the first one; wherein at
least one of the first and second antibiofilm agents is an
antimicrobial peptide.
41. A method as claimed in claim 40 wherein the first antibiofilm
agent is an antimicrobial peptide and the second antibiofilm agent
is selected from a dispersant and an anti-adhesive agent.
42. A method as claimed in claim 40 or 41 wherein the microbial
infection is a topical infection.
43. A method as claimed in claim 42 wherein the topical infection
is selected from a wound, ulcer and lesion.
44. A method as claimed in claim 40 or 41 wherein the microbial
infection is an oral infection.
45. A method as claimed in claim 44 wherein the oral infection is
selected from gingivitis, periodontitis and mucositis.
46. A method as claimed in claim 40 or 41 wherein the microbial
infection is a systemic infection.
47. A method as claimed in claim 46 wherein the systemic infection
is a mucosal infection.
48. A method as claimed in claim 47 wherein the mucosal infection
is a gastrointestinal, urogenital or respiratory infection.
49. A method as claimed in claim 47 or 48 wherein the mucosal
infection is cystic fibrosis.
50. A method of treating or preventing biofilm formation in an
environment comprising administering to said environment an
effective amount of cysteamine.
51. Use of a cysteamine, in the treatment of a microbial infection,
particularly a microbial biofilm infection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This present invention is a continuation of U.S. application
Ser. No. 15/082,976 filed Mar. 28, 2016 which is a continuation of
U.S. application Ser. No. 13/260,547 filed Nov. 17, 2011 which is a
U.S. National Phase application of PCT Patent Application No.
PCT/GB2010/000631, filed on Mar. 31, 2010 and claims priority to
United Kingdom Patent Application No. GB 0905451.1 filed on Mar.
31, 2009, which claims priority to U.S. Provisional Patent
Application No. 61/165,396, filed on Mar. 31, 2009, the disclosures
of which are incorporated by reference in their entirety for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates to products, compositions, methods and
uses which are useful in relation to the prevention and treatment
of biofilms.
BACKGROUND OF THE INVENTION
[0003] A microbial biofilm is a community of microbial cells
embedded in an extracellular matrix of polymeric substances and
adherent to a biological or a non-biotic surface. A range of
microorganisms (bacteria, fungi, and/or protozoa, with associated
bacteriophages and other viruses) can be found in these biofilms.
Biofilms are ubiquitous in nature, are commonly found in a wide
range of environments. Biofilms are being increasingly recognised
by the scientific and medical community as being implicated in many
infections, and especially their contribution to the recalcitrance
of infection treatment.
[0004] Biofilms are etiologic agents for a number of disease states
in mammals and are involved in 80% of infections in humans.
Examples include skin and wound infections, middle-ear infections,
gastrointestinal tract infections, peritoneal membrane infections,
urogenital tract infections, oral soft tissue infections, formation
of dental plaque, eye infections (including contact lens
contamination), endocarditis, infections in cystic fibrosis, and
infections of indwelling medical devices such as joint prostheses,
dental implants, catheters and cardiac implants.
[0005] Planktonic microbes (i.e., microorganisms suspended or
growing in a liquid medium) are typically used as models for
antimicrobial susceptibility research as described by the Clinical
and Laboratory Standards Institute (CLSI) and European Committee on
Antimicrobial Susceptibility Testing (EUCAST). Microbes in biofilms
are significantly more resistant to antimicrobial treatment than
their planktonic counterparts. However, there is no standardized
method for the study of antibiotic susceptibility of biofilm
microbes.
[0006] Biofilm formation is not limited solely to the ability of
microbes to attach to a surface. Microbes growing in a biofilm are
able to interact more between each other than with the actual
physical substratum on which the biofilm initially developed. For
example, this phenomenon favours conjugative gene transfer, which
occurs at a greater rate between cells in biofilms than between
planktonic cells. This represents an increased opportunity for
horizontal gene transfer between bacteria, and is important because
this can facilitate the transfer of antibiotic resistance or
virulence determinant genes from resistant to susceptible microbes.
Bacteria can communicate with one another by a system known as
quorum sensing, through which signalling molecules are released
into the environment and their concentration can be detected by the
surrounding microbes. Quorum sensing enables bacteria to
co-ordinate their behaviour, thus enhancing their ability to
survive. Responses to quorum sensing include adaptation to
availability of nutrients, defence against other microorganisms
which may compete for the same nutrients and the avoidance of toxic
compounds potentially dangerous for the bacteria. It is very
important for pathogenic bacteria during infection of a host (e.g.
humans, other animals or plants) to co-ordinate their virulence in
order to escape the immune response of the host in order to be able
to establish a successful infection.
[0007] Biofilm formation plays a key role in many infectious
diseases, such as cystic fibrosis and periodontitis, in bloodstream
and urinary tract infections and as a consequence of the presence
of indwelling medical devices. The suggested mechanisms by which
biofilm-associated microorganisms elicit diseases in their host
include the following: (i) delayed penetration of the antimicrobial
agent through the biofilm matrix, (ii) detachment of cells or cell
aggregates from indwelling medical device biofilms, (iii)
production of endotoxins, (iv) resistance to the host immune
system, (v) provision of a niche for the generation of resistant
organisms through horizontal gene transfer of antimicrobial
resistance &/or virulence determinant genes, and (vi) altered
growth rate (.i.e. metabolic dormancy) (Donlan and Costerton, Clin
Microbiol Rev 15: 167-193, 2002; Parsek and Singh, Annu Rev
Microbiol 57: 677-701, 2003; Costerton J W, Resistance of biofilms
to stress. In `The biofilm primer`. (Springer Berlin Heidelberg).
pp. 56-64.2007).
[0008] Recent experimental evidence has indicated the existence
within biofilms of a small sub-population of specialized
non-metabolising persister cells (dormant cells). It is thought
that these cells may be responsible for the high
resistance/tolerance of biofilm to antimicrobial agents.
Multi-drug-tolerant persister cells are present in both planktonic
and biofilm populations and it appears that yeasts and bacteria
have evolved analogous strategies that assign the function of
survival to this sub-population. The protection offered by the
polymeric matrix allows persister cells to evade elimination and
serve as a source for re-population. There is evidence that
persisters may be largely responsible for the multi-drug tolerance
of microbial biofilms (LaFleur et al., Antimicrob Agents Chemother.
50: 3839-46, 2006; Lewis, Nature Reviews Microbiology 5, 48-56
2007).
[0009] There remains a need for better therapies for preventing
biofilm formation and treating conditions associated with microbial
biofilms.
DESCRIPTION OF THE DRAWINGS
[0010] The invention will now be described by way of Examples only
with reference to the following Figures in which:
[0011] FIG. 1: Antibacterial activity of NP108 and NM001
(cysteamine) against P. aeruginosa ATCC BAA-47 planktonic cells
[0012] FIG. 2: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against P. aeruginosa ATCC BAA-47
planktonic cells
[0013] FIG. 3: Antibacterial activity of NP108 and NM001
(cysteamine) against S. aureus DSM 11729 planktonic cells
[0014] FIG. 4: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against S. aureus DSM 11729 planktonic
cells
[0015] FIG. 5: Activity of NP339 against biofilm cells of
Gram-positive and Gram-negative bacteria FIG. 6: Activity of NP339
against persister cells of Gram-positive and Gram-negative
bacteria
[0016] FIG. 7: Activity of NP341 against biofilm cells of
Gram-positive and Gram-negative bacteria
[0017] FIG. 8: Activity of NP341 against persister cells of
Gram-positive and Gram-negative bacteria
[0018] FIG. 9: Activity of NM001 (cysteamine) against biofilm cells
of Gram-positive and Gram-negative bacteria
[0019] FIG. 10: Activity of NM001 (cysteamine) against persister
cells of Gram-positive and Gram-negative bacteria
[0020] FIG. 11: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against P. aeruginosa ATCC BAA-47 biofilm
cells
[0021] FIG. 12: Antibacterial activity of NP108 and NM001
(cysteamine) against P. aeruginosa ATCC BAA-47 persister cells
[0022] FIG. 13: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against P. aeruginosa ATCC BAA-47
persister cells
[0023] FIG. 14A-14D: Antibacterial activity of NP339 and NM001
(cysteamine) combinations against (a) P. aeruginosa DSM 1128, (b)
P. aeruginosa ATCC BAA-47, (c) P. aeruginosa DSM 1299 and (d) P.
aeruginosa ATCC 27853 biofilm cells
[0024] FIG. 15A-15B: Antibacterial activity of NP339 and NM001
(cysteamine) combinations against (a) P. aeruginosa DSM 1128 and
(b) P. aeruginosa ATCC BAA-47 persister cells
[0025] FIG. 16: Antibacterial activity of NP108 and NM001
(cysteamine) against S. aureus DSM 11729 biofilm cells
[0026] FIG. 17: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against S. aureus DSM 11729 biofilm
cells
[0027] FIG. 18: Antibacterial activity of NP108 and NM001
(cysteamine) against S. aureus DSM 11729 persister cells
[0028] FIG. 19: Antibacterial activity of NP108 and NM001
(cysteamine) combinations against S. aureus DSM 11729 persister
cells
[0029] FIGS. 20A-20B and 21A-21B: Activity of the mucolytic agents
N-acetylcysteine
[0030] (FIGS. 20(a) and 20(b)) and NM001 (cysteamine) (FIGS. 21(a)
and 21(b)) alone and in combination with NP341 against P.
aeruginosa 27853 planktonic cells
[0031] FIG. 22A: untreated control S. aureus biofilm after 24
hours
[0032] FIG. 22B: S. aureus biofilm 24 hours following treatment
with NM001 (cysteamine) at 2 mg/ml
[0033] FIG. 22C: S. aureus biofilm 24 hours following treatment
with Colistin at 0.2 mg/ml
[0034] FIG. 22D: S. aureus biofilm 24 hours following treatment
with peptide NP108 at 2 mg/ml
[0035] FIG. 23A: untreated control S. aureus biofilm after 24
hours
[0036] FIG. 23B: S. aureus biofilm 24 hours following treatment
with NM001 (cysteamine) at 2 mg/ml
[0037] FIG. 23C: S. aureus biofilm 24 hours following treatment
with Colistin at 0.2 mg/ml
[0038] FIG. 23D: S. aureus biofilm 24 hours following treatment
with peptide NP108 at 2 mg/ml
[0039] FIG. 24A: untreated control P. aeruginosa biofilm after 24
hours
[0040] FIG. 24B: P. aeruginosa biofilm 24 hours following treatment
with NM001 (cysteamine) at 2 mg/ml
[0041] FIG. 24C: P. aeruginosa biofilm 24 hours following treatment
with Colistin at 0.2 mg/ml
[0042] FIG. 24D: P. aeruginosa biofilm 24 hours following treatment
with peptide NP108 at 2 mg/ml
[0043] FIG. 25A: untreated control P. aeruginosa biofilm after 24
hours
[0044] FIG. 25B: P. aeruginosa biofilm 24 hours following treatment
with NM001 (cysteamine) at 2 mg/ml
[0045] FIG. 25C: P. aeruginosa biofilm 24 hours following treatment
with Colistin at 0.2 mg/ml
[0046] FIG. 25D: P. aeruginosa biofilm 24 hours following treatment
with peptide NP108 at 2 mg/ml
[0047] FIG. 26: Activity of NP432 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against P.
aeruginosa PAO1 biofilm
[0048] FIG. 27: Activity of NP445 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against P.
aeruginosa PAO1 biofilm
[0049] FIG. 28: Activity of NP458 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against P.
aeruginosa PAO1 biofilm
[0050] FIG. 29: Activity of NP462 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against P.
aeruginosa PAO1 biofilm
[0051] FIG. 30: Activity of NP432 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against S. aureus
ATCC25923 biofilm
[0052] FIG. 31: Activity of NP445 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against S. aureus
ATCC25923 biofilm
[0053] FIG. 32: Activity of NP458 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against S. aureus
ATCC25923 biofilm
[0054] FIG. 33: Activity of NP462 alone and in combination with
NM001 (cysteamine) or in combination with NP108 against S. aureus
25923 biofilm
[0055] Table 1: Summary of the activity of the tested antimicrobial
agents against the Gram-negative P. aeruginosa strains and the
Gram-positive Staphylococcus spp.
[0056] Table 2: Summary of the activity of the tested antimicrobial
agents against S. epidermidis, S. aureus, and P. aeruginosa.
[0057] The present invention relates to a product comprising at
least two antibiofilm agents wherein at least one of the
antibiofilm agents is an antimicrobial peptide. The second
antibiofilm agent is generally a dispersant or an anti-adhesive
agent. There is also provided the use of the product in the
treatment of a microbial infection.
TABLE-US-00001 TABLE 1 MIC (.mu.g/ml) at pH 7 Exp#1-2 S. S. S. P.
P. P. epidermidis aureus aureus aeruginosa aeruginosa aeruginosa NP
Sequence ATCC12228 ATCC25923 DSMZ11729 DSMZ1128 D5MZ1299 ATCCBAA-47
NP432 RRRFRFFFRFRRR <7.8 31.25 62.5 62.5 15.6 15.6 NP438
HHHFRFFFRFRRR <7.8 >500 >500 >500 500 >500 NP441
HHPRRKPRRPKRHH >500 >500 >500 >500 >500 >500
NP445 KKFPWRLRLRYGRR <7.8 500 500 62.5 31.25 31.25 NP449
KKPRRKPRRPKRKK- 31.25 250 125 250 125 250 cyst NP451
HHPRRKPRRPKRHH- 125 500 500 >500 >500 >500 cyst NP457
RRRRR-cyst 31.25 125 125 >500 >500 250 NP458 RRRRRHH-cyst
31.25 250 250 250 125 62.5
TABLE-US-00002 TABLE 2 MBC (.mu.g/ml) following MIC at pH 7 Exp#3
P. S. S. S. P. P. P. aeruginosa epidermidis aureus aureus
aeruginosa aeruginosa aeruginosa NP DSMZ1299 ATCC12228 ATCC25923
DSMZ11729 DSMZ1128 DSMZ1299 ATCCBAA-47 NP432 16 250 500 250 (2) 32
(2) 250 NP438 125 >500 >500 >500 >500 >500 NP441
>500 >500 >500 >500 >500 >500 >500 NP445 62.5
>500 >500 250 125 250 NP449 125 >500 250 500 (2) 250 (2)
>500 NP451 >500 125 >500 >500 >500 >500 >500
NP457 >500 125 (2) 62.5 125 (2) >500 >500 >500 NP458
500 125 250 >500 >500 >500 Exp#3 Exp#4 MBC (.mu.g/ml)
Exp#4 MIC (.mu.g/ml) pH5.5, MBC (.mu.g/ml) pH5.5, MIC (.mu.g/ml)
pH5.5 320 mM NaCl at pH5.5 320 mM NaCl P P. P P. P. S. aeruginosa
S. aeruginosa S. aeruginosa S. aeruginosa aeruginosa aureus
ATCCBAA- aureus ATCCBAA- aureus ATCCBAA- aureus ATCCBAA- NP
DSMZ1299 11729 47 11729 47 11729 47 11729 47 NP432 >500 125
>500 125 >500 125 >500 >500 NP438 >500 125 >500
62.5 >500 >500 >500 >500 NP441 >500 >500 >500
>500 >500 >500 >500 >500 >500 NP445 >500
>500 >500 >500 >500 >500 >500 >500 NP449
>500 >500 >500 >500 >500 >500 >500 >500
NP451 >500 >500 >500 >500 >500 >500 250 >500
>500 NP457 >500 >500 >500 >500 >500 >500
>500 >500 >500 NP458 >500 >500 >500 >500
>500 >500 >500 >500
DETAILED DESCRIPTION OF THE INVENTION
[0058] According to a first aspect of the present invention there
is provided a product comprising at least two antibiofilm agents
wherein at least one of the antibiofilm agents is an antimicrobial
peptide. The other antibiofilm agent may be a dispersant or an
anti-adhesive agent.
[0059] The term "antibiofilm agent" is used herein to describe an
agent that is capable of destroying or inhibiting the growth of a
microbial biofilm. The antibiofilm agent may be capable of
disrupting the structure of the biofilm, for example the
extracellular mucous matrix, or may be capable of destroying or
inhibiting the growth of microbial cells within the biofilm.
[0060] The invention further provides a method of preventing
biofilm formation in an environment comprising the step of
administering to the environment an antimicrobial peptide.
Advantageously the method comprises the step of administering to
the environment a product according to the invention.
[0061] The invention further provides a method for treating a
microbial infection, particularly a microbial biofilm, by
prophylaxis or therapy, comprising the administration in a
therapeutically effective amount of an antimicrobial peptide, for
example a cationic peptide. Typically the method involves the
sequential or combined administration in a therapeutically
effective amount of:
[0062] a first antibiofilm agent; and
[0063] a second antibiofilm agent different from the first one;
wherein at least one of the first and second antibiofilm agents is
an antimicrobial peptide for example a cationic peptide.
[0064] The above mentioned active agents may be administered as
free or fixed combinations. Free combinations may be provided as
combination packages containing all the active agents in free
combinations. Fixed combinations are often tablets or capsules.
[0065] Included in the invention is the use in the manufacture of a
medicament for the treatment of a microbial infection, particularly
a microbial biofilm infection, by prophylaxis or therapy of the
antimicrobial peptides, or combinations of active agents outlined
above.
[0066] The products have the advantage that they demonstrate
antibacterial activity against, inter alia, the persister cells
present in the biofilms, which is an essential step towards the
eradication of biofilms.
[0067] The agents of the invention may be administered in the form
of pharmaceutically acceptable salts. The pharmaceutically
acceptable salts of the present invention can be synthesized from
the parent compound which contains a basic or acidic moiety by
conventional chemical methods. Generally, such salts can be
prepared by reacting the free acid or base forms of these compounds
with a stoichiometric amount of the appropriate base or acid in
water or in an organic solvent, or in a mixture of the two;
generally, nonaqueous media like ether, ethyl acetate, ethanol,
isopropanol, or acetonitrile are preferred. Lists of suitable salts
are found in Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton, Pa., US, 1985, p. 1418, the disclosure
of which is hereby incorporated by reference; see also Stahl et al,
Eds, "Handbook of Pharmaceutical Salts Properties Selection and
Use", Verlag Helvetica Chimica Acta and Wiley-VCH, 2002. The phrase
"pharmaceutically acceptable" is employed herein to refer to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings or, as the case may be, an
animal without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0068] The invention thus includes pharmaceutically-acceptable
salts of the disclosed compounds wherein the parent compound is
modified by making acid or base salts thereof for example the
conventional non-toxic salts or the quaternary ammonium salts which
are formed, e.g., from inorganic or organic acids or bases.
Examples of such acid addition salts include acetate, adipate,
alginate, aspartate, benzoate, benzenesulfonate, bisulfate,
butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,
pivalate, propionate, succinate, tartrate, thiocyanate, tosylate,
and undecanoate. Base salts include ammonium salts, alkali metal
salts such as sodium and potassium salts, alkaline earth metal
salts such as calcium and magnesium salts, salts with organic bases
such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts
with amino acids such as arginine, lysine, and so forth. Also, the
basic nitrogen-containing groups may be quaternized with such
agents as lower alkyl halides, such as methyl, ethyl, propyl, and
butyl chloride, bromides and iodides; dialkyl sulfates like
dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides
such as decyl, lauryl, myristyl and stearyl chlorides, bromides and
iodides, aralkyl halides like benzyl and phenethyl bromides and
others.
[0069] The invention therefore includes pharmaceutical products
generally comprising at least:
[0070] a first antibiofilm agent; and
[0071] a second antibiofilm agent different from the first one
wherein at least one of the first and second antibiofilm agents is
an antimicrobial peptide for example a cationic peptide.
The First Antibiofilm Agent
[0072] The first antibiofilm agent may be an antimicrobial peptide
for example an antibacterial peptide. Preferably the first
antibiofilm agent is an antimicrobial peptide, hereinafter referred
to as "the first antimicrobial agent". The first antimicrobial
agent may comprise amino acids according to the formula I:
((X).sub.l(Y).sub.m).sub.n (I)
wherein l and m are integers from 1 to 10, for example 1 to 5; n is
an integer from 1 to 10; X and Y, which may be the same or
different, are independently a hydrophobic or cationic amino
acid.
[0073] Preferably the first antimicrobial agent comprises amino
acids according to the formula (I) wherein X and Y are cationic
amino acids.
[0074] The antimicrobial peptide may comprise from 2 to 200 amino
acids, for example 3, 4, 5, 6, or 7 up to 100 amino acids,
including 3, 4, 5, 6, or 7 up to 10, 15, 20, 25, 30, 35, 40, 45 or
50 amino acids. According to one embodiment, the antimicrobial
peptide comprises 3 or 4 to 50 amino acids. Alternatively the
peptide may comprise more than 27 amino acids, typically 27 to 300
amino acids, suitably 27 to 200 amino acids.
[0075] The peptide may comprise 100 to 200 amino acids, 20 to 100,
20 and 45 amino acids such as 20, 25, 30, 35, 40, 42 or 45 amino
acids. The peptide may comprise between 3 and 15 amino acids, for
example 5 to 15 amino acids.
[0076] As used herein, the term "hydrophobic" refers to an amino
acid having a side chain that is uncharged at physiological pH,
that is not polar and that is generally repelled by aqueous
solution.
[0077] As used herein, the term "cationic" refers to amino acids
having a net charge that is greater than or equal to 0. Generally
the term "cationic" refers to amino acids having a net charge that
is greater than zero.
[0078] Generally a hydrophobic amino residue has a hydrophobicity
greater than or equal to -1.10 and a charge greater than or equal
to 0.
[0079] Hydrophobic amino acids may include, leucine phenylalanine,
proline, alanine, tryptophan, valine, isoleucine and
methionine.
[0080] Preferably X and/or Y are cationic amino acids for example
selected from the group consisting of histidine, arginine and
lysine. Preferably still X and/or Y are arginine or lysine. X
and/or Y may be selected from non-naturally occurring amino acids
for example the cationic amino acid ornithine.
[0081] X and/or Y may be optical isomers of a cationic amino acid
as defined herein for example D or L-amino acids. Moreover, X
and/or Y may be alternating amino acids.
[0082] The amino acids may be naturally occurring or synthetic. The
invention also includes known isomers (structural, stereo-,
conformational & configurational) and structural analogues of
the above amino acids, and those modified either naturally (e.g.
post-translational modification) or chemically, including, but not
exclusively, phosphorylation, glycosylation, sulfonylation and/or
hydroxylation.
[0083] According to one embodiment the peptide may include one or
more substitution of the cationic or hydrophobic amino acids X and
Y. However, the peptide would predominantly comprise the cationic
or hydrophobic amino acids X and Y. Typically the peptide may
comprise 1 to 5 substitutions, suitably 1 to 3 substitutions,
generally one substitution. The substitutions may be terminal or
non-terminal.
[0084] The substitutions may consist of amino acids, or non-amino
acids. The substitutions may be charged or uncharged. Typically one
or more of the substitutions are uncharged amino acids.
Alternatively or additionally one or more of the substitutions may
be non-amino acids such as cysteamine.
[0085] Preferably X and Y are the same and are lysine or
arginine.
[0086] According to one embodiment, the peptide comprises
predominantly arginine amino acids which may be substituted with
one or more amino acids which are not arginine.
[0087] Generally the peptide comprises 7 to 20 arginine amino
acids, optionally substituted with 1 to 5 non-arginine amino acids,
typically 3 to 5 non-arginine substitutions.
[0088] Alternatively the peptide may comprise 7 to 20 lysine amino
acids, optionally substituted with 1 to 5 non-lysine amino acids,
typically 3 to 5 non-lysine substitutions.
[0089] According to a further embodiment, the peptide may comprise
27 to 300 lysine amino acids, generally 27 to 200 lysine amino
acids. Typically the peptide comprises no non-terminal
substitutions with non-lysine amino acids.
[0090] In the peptide of formula (I) l and m may be 1, 2, 3, 4, 5,
6, 7, 8, 9 or 10 and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0091] In the peptide of formula (I) l may be 1, n may be 1 and m
may be between 4 and 9, for example, m may be 3, 4, 5, 6, 7, 8 or
9.
[0092] In the peptide of formula (I) 1, n and/or m may be between 1
and 5, for example, 1, 2, 3, 4 or 5.
[0093] In the peptide of formula (I) l and m may be an integer
between 0 and 7 and n may be an integer between 1 and 10.
[0094] In the peptide of formula (I) l and m may be 0, 1 or 2 and n
may be an integer between 1 and 10.
[0095] In the peptide of formula (I) X and Y may be the same, l may
be 0, m may be 1 and n may be 3, 4, 5, 6, 7, 8, 9 or 10.
[0096] In the peptide of formula (I) X and Y may be the same, l and
m may be 1 and n may be 2, 3, 4 or 5.
[0097] In the peptide of formula (I) X and Y may be the same, l may
be 1, m may be 2 and n may be 1, 2, 3 or 4.
[0098] In the peptide of formula (I) X and Y may be the same, l and
m may be 2 and n may be 1, 2, 3 or 4.
[0099] Preferably the first antimicrobial agent comprises a peptide
sequence selected from the group consisting of polylysine and
polyarginine.
[0100] In one embodiment, the first antimicrobial agent comprises a
polylysine.
[0101] In an alternative embodiment, the first antimicrobial agent
comprises polyarginine.
[0102] According to a further aspect of the present invention there
is provided the use of the first antimicrobial agent in the
treatment of prevention of a biofilm.
[0103] Typically the first antimicrobial agent is in the form of
the product of the invention as described below.
The Second Antibiofilm Agent
[0104] The second antibiofilm agent may be any agent which inhibits
biofilm formation. By way of example, the second antibiofilm agent
may inhibit bacterial adhesion, hydrophobicity or slime production.
The second antibiofilm agent may be selected from a dispersant and
an anti-adhesive agent.
[0105] According to one embodiment of the present invention the
second antibiofilm agent is not a peptide.
[0106] The term "dispersant" is intended to include any agent
capable of dispersing the particles of a biofilm. In particular,
the dispersant may promote the dispersion of slime produced by
microbes such as bacteria, mucous which forms part of the biofilm
for example mucous produced by the cells to which the biofilm
microbes adheres, and biofilm microbes such as bacteria.
[0107] The dispersant may be a mucolytic agent. The mucolytic agent
may be an enzyme for example a DNase, alginase, protease or
carobohydrase. Alternatively the mucolytic agent may be a small
molecule for example an amine such as an aminothiol or an acid such
as ethylenediaminetetraacetic acid (EDTA). The amine may be
selected from acetylcysteine and cysteamine.
[0108] The term "anti-adhesive agent" is intended to include any
agent capable of inhibiting adhesion between cells, proteins and
organisms e.g. microbes thereby preventing biofilm formation or
promoting biofilm self-destruction. In particular, the
anti-adhesive agent may prevent the adhesion to a surface or
substrate of all cell types encountered in microbial biofilms in
particular free living microbes i.e. planktonic cells.
Anti-adhesive agents may include, but are not limited to,
hyaluronan, heparin or Carbopol 934.
[0109] The second antibiofilm agent may be an antibacterial agent.
The antibacterial agent may be a mucolytic agent for example a
mucolytic agent having both mucolytic and antibacterial activity.
Preferably the antibacterial agent is cysteamine.
The Products of the Invention
[0110] The product of the present invention may comprise an
antimicrobial peptide.
[0111] A preferred product comprises an antimicrobial peptide and a
mucolytic agent.
[0112] The ratio of the first antibiofilm agent to the second
antibiofilm agent in the products of the invention may be from 1:10
to 10:1; generally at least 2:1 for example at least 3:1 or 4:1.
According to one embodiment, the ratio of first antibiofilm agent
to the second antibiofilm agent is approximately 1:1. Preferably
the first antibiofilm agent is a cationic peptide and the second
antibiofilm agent is a mucolytic agent and the ratio of cationic
peptide:mucolytic agent ranges from 2:1 up to 4:1. According to a
further embodiment the ratio may be approximately 1:1.
[0113] The active agents may be administered simultaneously,
sequentially or separately. The active agents may be provided as a
combination package. The combination package may contain the
product of the invention together with instructions for
simultaneous, separate or sequential administration of each of the
active agents. For sequential administration, the active agents can
be administered in any order.
[0114] The active agents of the product of the invention may be
provided as pharmaceutical compositions additionally containing one
or more pharmaceutically acceptable diluents, excipients and/or
carriers. This applies to both fixed and free combinations.
[0115] The active agents of the present invention may be
administered by any suitable route known to those skilled in the
art, preferably in the form of a pharmaceutical composition adapted
to such a route, and in a dose effective for the treatment
intended. The active compounds and composition may, for example, be
administered parenterally, orally, intranasal, intrabronchial,
enterally, transdermally, sublingually, rectally, vaginally,
ocularly, or topically. Both local and systemic administration is
contemplated.
[0116] For the purposes of parenteral administration ("parenteral"
as used herein, refers to modes of administration which include
intravenous, intramuscular, enteral, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion of which
intravenous (including continuous intravenous administration) is
most preferred) solutions in aqueous propylene glycol can be
employed, as well as sterile aqueous solutions of the corresponding
water-soluble salts. Such aqueous solutions may be suitably
buffered, if necessary, and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These aqueous solutions
are especially suitable for intravenous, intramuscular,
subcutaneous and intraperitoneal injection purposes. In this
connection, the sterile aqueous media employed are all readily
obtainable by standard techniques well-known to those skilled in
the art.
[0117] The products of the invention can also be administered
intranasally or by inhalation and are conveniently delivered in the
form of a dry powder inhaler or an aerosol spray presentation from
a pressurised container, pump, spray, atomiser, nebuliser, with or
without the use of a suitable propellant.
[0118] Alternatively the products of the invention can be
administered in the form of a suppository or pessary, or they may
be applied topically in the form of a gel, hydrogel, lotion,
solution, cream, ointment or powder. The products of the invention
may be dermally or transdermally administered, for example, by use
of a skin patch, depot or subcutaneous injection. They may also be
administered by pulmonary or rectal routes.
[0119] For oral administration, the pharmaceutical composition may
be in the form of; for example, a tablet, capsule, suspension or
liquid. The pharmaceutical composition is preferably made in the
form of a dosage unit containing a particular amount of the active
ingredient. Examples of such dosage units are capsules, tablets,
powders, granules or a suspension, with conventional additives such
as lactose; mannitol, corn starch or potato starch; with binders
such as crystalline cellulose, cellulose derivatives, acacia, corn
starch or gelatins; with disintegrators such as corn starch, potato
starch or sodium carboxymethylcellulose; and with lubricants such
as talc or magnesium stearate. The active ingredient may also be
administered by injection as a composition wherein, for example,
saline, dextrose or water may be used as a suitable carrier.
[0120] The products of the invention may also find application
as/in an oral formulation wherein the product is formulated in a
carrier, for example selected from films, tapes, gels,
microspheres, lozenges, chewing gum, dentrifices and mouthwash.
[0121] The amount of therapeutically active compound that is
administered and the dosage regimen for treating a disease
condition with the compounds and/or compositions of this invention
depends on a variety of factors, including the age, weight, sex and
medical condition of the subject, the severity of the disease, the
route and frequency of administration, and the particular compound
employed, as well as the pharmacokinetic properties of the
individual treated, and thus may vary widely. The dosage will
generally be lower if the compounds are administered locally rather
than systemically, and for prevention rather than for treatment.
Such treatments may be administered as often as necessary and for
the period of time judged necessary by the treating physician. One
of skill in the art will appreciate that the dosage regime or
therapeutically effective amount of the inhibitor to be
administrated may need to be optimized for each individual. The
pharmaceutical compositions may contain active ingredient in the
range of about 0.1 to 2000 mg, preferably in the range of about 0.5
to 500 mg and most preferably between about 1 and 200 mg. A daily
dose of about 0.01 to 100 mg/kg body weight, preferably between
about 0.1 and about 50 mg/kg body weight and most preferably from
about 1 to 20 mg/kg body weight, may be appropriate. The daily dose
can be administered in one to four doses per day.
[0122] The products of the invention are preferably administered to
the respiratory tract. Thus, the present invention also provides
aerosol pharmaceutical formulations comprising a product of the
invention. Also provided is a nebuliser or inhaler containing a
product of the invention.
[0123] Additionally, the products of the invention may be suited to
formulation as sustained release dosage forms and the like. The
formulations can be so constituted that they release the active
agents, for example, in a particular part of the intestinal or
respiratory tract, possibly over a period of time. Coatings,
envelopes, and protective matrices may be made, for example, from
polymeric substances, such as polylactide-glycolates, liposomes,
microemulsions, microparticles, nanoparticles, or waxes. These
coatings, envelopes, and protective matrices are useful to coat
indwelling devices, e.g. stents, catheters, peritoneal dialysis
tubing, draining devices and the like.
[0124] The products of the invention may include synergistically
effective amounts of each active agent defined herein. The
invention therefore includes products comprising a synergistically
effective amount of (i) a first antibiofilm agent, (ii) a second
antibiofilm agent which is different from the first antibiofilm
agent and is typically an antimicrobial peptide. The product may be
for use in the manufacture of a medicament, for simultaneous,
separate or sequential administration said agents in the treatment
of a microbial infection for example a biofilm infection.
"Synergistically", as used herein, may describe the action of the
two or more agents of the product of the invention working together
to produce an effect greater than the expected combined effect of
the agents used separately.
[0125] In a further aspect of the invention there is provided a
substrate to which a product of the invention is applied or
attached. Preferably, the substrate is suitable for application to
wounds or delivery to wound sites. Preferably, the substrate allows
for the transfer of the active agents of the product of the
invention from the substrate to a wound bed to achieve their
antibiofilm effect. The substrate may be a dressing, for example,
wound dressing. The dressing may comprise a fabric material or it
may be a collagen-like material. The substrate may be in any
suitable form for application to a wound, typically the substrate
may be in the form of a hydrogel, colloid, ointment, cream, gel,
foam or spray.
[0126] The products of the invention may also find application
as/in a disinfectant or biocide. In this context, the peptide or
pharmaceutical compositions of the invention may be applied, either
alone or in combination with other disinfecting agents, to a
surface to be treated. As used herein a "surface to be treated" may
be a substrate as defined herein and may include medical devices
and indwelling devices, e.g. stents, catheters, peritoneal dialysis
tubing, draining devices, joint prostheses, dental implants and the
like.
Methods and Use
[0127] The invention provides a method of preventing biofilm
formation in an environment comprising the step of administering to
the environment a product according to the invention. The method
may be in vivo or ex vivo.
[0128] According to one embodiment, the method comprises the step
of administering an antimicrobial peptide.
[0129] Advantageously the method comprises the step of
administering
a first antibiofilm agent; and a second antibiofilm agent different
from the first one wherein at least one of the first and second
antibiofilm agents is an antimicrobial peptide for example a
cationic peptide.
[0130] The environment may comprise any biofilm forming
microorganism selected from bacteria, fungi, yeast, viruses and
protozoa.
[0131] Typically the microorganism is a bacterium for example a
Gram-positive or Gram-negative bacterium. A bacterial pathogen may
be derived from a bacterial species selected from the group
consisting of: Staphylococcus spp., e.g. Staphylococcus aureus,
Staphylococcus epidermidis; Enterococcus spp., e.g. Enterococcus
faecalis; Streptococcus pyogenes; Listeria spp.; Pseudomonas spp.;
Mycobacterium spp., e.g. Mycobacterium tuberculosis; Enterobacter
spp.; Campylobacter spp.; Salmonella spp.; Streptococcus spp., e.g.
Streptococcus Group A or B, Streptoccocus pneumoniae; Helicobacter
spp., e.g. Helicobacter pylori; Neisseria spp., e.g. Neisseria
gonorrhea, Neisseria meningitidis; Borrelia burgdorferi; Shigella
spp., e.g. Shigella flexneri; Escherichia coli; Haemophilus spp.,
e.g. Haemophilus influenzae; Chlamydia spp., e.g. Chlamydia
trachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Francisella
fularensis; Bacillus spp., e.g. Bacillus anthraces; Clostridia
spp., e.g. Clostridium botulinum; Yersinia spp., e.g. Yersinia
pestis; Treponema spp.; Burkholderia spp.; e.g. Burkholderia mallei
and B pseudomallei.
[0132] In particular the bacterium may include Pseudomonas spp.,
for example Pseudomonas aeruginosa; Staphylococcus spp., for
example Staphylococcus aureus and Staphylococcus epidermidis;
Haemophilus spp., for example Haemophilus influenza; Burkholderia
spp., for example Burkholderia cepacia; Streptococcus spp.,
Propionibacterium spp., for example Propionibacterium acnes.
Preferably the bacterium is selected from Pseudomonas spp., for
example Pseudomonas aeruginosa and Staphylococcus spp., for example
Staphylococcus aureus and Staphylococcus epidermidis.
[0133] A viral pathogen may be derived from a virus selected from
the group consisting of: Human Immunodeficiency Virus (HTV1 &
2); Human T Cell Leukaemia Virus (HTLV 1 & 2); Ebola virus;
human papilloma virus (e.g. HPV-2, HPV-5, HPV-8 HPV-16, HPV-18,
HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56); papovavirus;
rhinovirus; poliovirus; herpesvirus; adenovirus; Epstein Barr
virus; influenza virus, hepatitis B and C viruses, Variola virus,
rotavirus or SARS coronavirus.
[0134] A parasitic pathogen may be derived from a parasitic
pathogen selected from the group consisting of Trypanosoma spp.
(Trypanosoma cruzi, Trypansosoma brucei), Leishmania spp., Giardia
spp., Trichomonas spp., Entamoeba spp., Naegleria spp.,
Acanthamoeba spp., Schistosoma spp., Plasmodium spp.,
Crytosporidiwn spp., Isospora spp., Balantidium spp., Loa Loa,
Ascaris lumbricoides, Dirofilaria immitis, Toxoplasma ssp., e.g
Toxoplasma gondii. A fungal pathogen may be derived from a fungal
pathogen which is of the genus Candida spp., (e.g. C. albicans),
Epidermophyton spp., Exophiala spp., Microsporiim spp.,
Trichophyton spp., (e.g T. rubrum and T. interdigitale), Tinea
spp., Aspergillus spp., Blastomyces spp., Blastoschizomyces spp.,
Coccidioides spp., Cryptococcus spp., Histoplasma spp.,
Paracoccidiomyces spp., Sporotrix spp., Absidia spp.,
Cladophialophora spp., Fonsecaea spp., Phialophora spp., Lacazia
spp., Arthrographis spp., Acremonium spp., Actinomadura spp.,
Apophysomyces spp., Emmonsia spp., Basidiobolus spp., Beauveria
spp., Chrysosporium spp., Conidiobolus spp., Cunninghamella spp.,
Fusarium spp., Geotrichum spp., Graphium spp., Leptosphaeria spp.,
Malassezia spp., Mucor spp., Neotestudina spp., Nocardia spp.,
Nocardiopsis spp., Paecilomyces spp., Phoma spp., Piedraia spp.,
Pneumocystis spp., Pseudallescheria spp., Pyrenochaeta spp.,
Rhizomucor spp., Rhizopus spp., Rhodotorula spp., Saccharomyces
spp., Scedosporium spp., Scopulariopsis spp., Sporobolomyces spp.,
Syncephalastrum spp., Trichoderma spp., Trichosporon spp.,
Ulocladium spp., Ustilago spp., Verticillium spp., Wangiella
spp.
[0135] According to a further embodiment the microorganism may be a
fungi, in particular Candida.
[0136] The method of the invention may be used to minimise and,
preferably, prevent the formation of biofilms in a variety of
environments including, but not limited to, household, workplace,
laboratory, industrial environment, aquatic environment (e.g.
pipeline systems), medical devices including indwelling devices
such as defined herein, dental devices or dental implants, animal
body for example human body.
[0137] The method of the invention may thus be used in the mouth to
prevent the formation of plaque or caries on a human tooth or
dental implant for example a denture.
[0138] The method of the invention may be used to prevent or
restrict the formation of a biofilm in the human body especially in
the treatment of microbial infections. Conditions associated with
biofilm infections may include topical infections, oral infections
and systemic infections. Topical infections may include wounds,
ulcers and lesions for example, cutaneous wounds such cuts or
burns, and conditions associated therewith.
[0139] Oral infections may include gingivitis, periodontitis and
mucositis.
[0140] Systemic infections may include cystic fibrosis and other
conditions associated with mucosal infections, for example,
gastrointestinal, urogenital or respiratory infections.
[0141] Another aspect of the invention resides in methods of
treating, preventing or delaying the progression of a disease or
condition associated with the presence of a microbial biofilm
infection in a mammal, especially a human, by administering a
therapeutically effective amount of a product of the invention to
the mammal.
[0142] By an "effective" amount or "therapeutically effective
amount" is meant an amount of one or more active substances which,
within the scope of sound medical judgment, is sufficient to
provide a desired effect without excessive toxicity, irritation,
allergic response, or other problem or complication, commensurate
with a reasonable benefit/risk ratio.
[0143] According to one aspect of the present invention the method
comprises the step of administering an antimicrobial peptide.
[0144] Advantageously the method comprises the step of
administering
[0145] a first antibiofilm agent; and
[0146] a second antibiofilm agent different from the first one
wherein at least one of the first and second antibiofilm agents is
an antimicrobial peptide for example a cationic peptide.
[0147] The invention further provides the use of a product of the
invention in the manufacture of a medicament for the treatment of a
microbial infection, particularly a microbial biofilm infection, by
prophylaxis or therapy of the combinations of active agents
outlined above.
[0148] Additionally the present invention provides the use of the
antimicrobial peptide described above in the manufacture of a
medicament for the treatment of a microbial infection, particularly
a microbial biofilm infection, by prophylaxis or therapy.
[0149] Thus the product of the invention may be useful in the
prevention of, delay of progression of, or treatment of a disease
or condition selected from the group consisting of skin and wound
infections, middle-ear infections, gastrointestinal tract
infections, peritoneal membrane infections, urogenital tract
infections, oral soft tissue infections, formation of dental
plaque, eye infections (including contact lense contamination),
endocarditis, infections in cystic fibrosis, and infections of
indwelling medical devices such as described herein.
[0150] The invention also includes methods of treatment in which a
product of the invention is administered to a mammal together with
one or more other antibacterial agents for example an
antibiotic.
[0151] The inventors have surprisingly found that certain
dispersants, in particular mucolytic agents, inhibit the growth of
biofilm persister cells. Thus the invention also includes a method
of treating/preventing biofilm formation in an environment
comprising administering to said environment a mucolytic agent, for
example cysteamine. The mucolytic agent may be administered alone
or in combination with another antimicrobial agent for example an
antimicrobial peptide.
[0152] The invention also provides a method for treating a
microbial infection, particularly a microbial biofilm, by
prophylaxis or therapy, comprising the administration in a
therapeutically effective amount of a dispersant, in particular a
mucolytic agent, for example cysteamine.
[0153] The invention further provides the use of a dispersant, in
particular a mucolytic agent, for example cysteamine, in the
manufacture of a medicament for the treatment of a microbial
infection, particularly a microbial biofilm infection.
[0154] The active agents mentioned in this specification can exist
in different forms, such as free acids, free bases, esters and
other prodrugs, salts and tautomers, for example, and the invention
includes all variant forms of the agents.
[0155] Throughout the description and claims of this specification,
the singular encompasses the plural unless the context otherwise
requires. In particular, where the indefinite article is used, the
specification is to be understood as contemplating plurality as
well as singularity, unless the context requires otherwise.
[0156] Features, integers, characteristics, compounds, chemical
moieties or groups described in conjunction with a particular
aspect, embodiment or example of the invention are to be understood
to be applicable to any other aspect, embodiment or example
described herein unless incompatible therewith.
[0157] Throughout the description and claims of this specification,
the words "comprise" and "contain" and variations of the words, for
example "comprising" and "comprises", mean "including but not
limited to", and are not intended to (and do not) exclude other
moieties, additives, components, integers or steps.
[0158] Generally the term "approximately" is intended to encompass
a range of 10% or less of any numerical value to which it is
applied.
[0159] Further aspects and embodiments of the invention are set
forth in the following description and claims.
Examples
Activity of Antimicrobial Agents Against Bacterial Biofilms
1. Materials and Methods
1.1 Bacterial Strains
[0160] Pseudomonas aeruginosa ATCC27853, P. aeruginosa BAA-47
(PAO1), P. aeruginosa DSM1128, P. aeruginosa DSM1299 and S.
epidermidis ATCC35984, S. epidermidis ATCC12228 Staphylococcus
aureus 25923 and methicilin-resistant Staphylococcus aureus DSM
11729 (MRSA) (DSMZ, Braunschweig, Germany) were used in this study.
Four P. aeruginosa clinical isolates (NH57388A-D, Hoffmann et al.,
2005, 2007) were obtained and used for antimicrobial susceptibility
testing.
1.2 Preparation of Antimicrobial Compounds
[0161] The antimicrobial agents tested in this study were the
cationic peptide NP108, which corresponds to a 10-20 kDa
poly-L-lysine, hydrobromide and cysteamine (NM001). Both agents
were obtained from Sigma-Aldrich (Gillingham, UK) and stock
solutions were prepared at 20 mg/ml in 14-18 M.OMEGA.cm pure water
(Purite HP40 water purification system, Oxon, UK). Once dissolved,
the preparations were filter-sterilized using 0.22 .mu.m filters
(Millipore, Watford, England) and stored at -20.degree. C.
The following NovaBiotics antimicrobial peptides were also
investigated
TABLE-US-00003 NP339 dRdRdRdRdRdRdRdRdRdRdRdRdR NP340
Ac-dRdRdRdRdRdRdRdRdRdRdRdRdR-CONH NP341
dRdRdRdRdRdRdRdRdRdRdRdRdR-CONH NP352 RRRRRRRRRRRRRRR NP432
RRRFRFFFRFRRR NP438 HHHFRFFFRFRRR NP441 HHPRRKPRRPKRRHH NP445
KKFPWRLRLRYGRR NP449 KKPRRKPRRPKRKK-cysteamine NP451
HHPRRKPRRPKRHH-cysteamine NP457 RRRRR-cysteamine NP458
RRRRRHH-cysteamine
[0162] NovaBiotics antimicrobial peptides were synthesized by
NeoMPS (Strasbourg, France) using Fmoc synthesis and were at least
95% pure.
1.3 Preparation of the Bacterial Inoculum
[0163] The bacterial inoculum was established by the dilution
method from actively-growing cultures in Mueller-Hinton broth,
standardized with 0.5 McFarland turbidity standard as described in
the CLSI method M26-A.
1.4 Determination of the Minimum Inhibitory Concentration (MIC)
[0164] To determine the prevention of biofilm formation, both the
bacterial inoculum and the antimicrobial agents were added
simultaneously to the plates. The plates were incubated at
37.degree. C. for 24 h and the optical density was read at 625 nm
on a microtitre plate reader (BioTek Powerwave XS, Winooski, USA).
The MIC was obtained as the lowest concentration of antimicrobial
showing total inhibition of bacterial growth.
1.5 Determination of the Fractional Inhibitory Concentration
(FIC)
[0165] The FIC corresponds to an interaction coefficient indicating
whether the combination of antimicrobial agents is synergistic,
additive, antagonist or neutral. The FIC is determined by comparing
the activity of an agent in combination (MIC of agent A+agent B)
with the activity of the agent alone (MIC of agent A or agent B) as
follow (Singh et al., 2000):
FIC=MIC.sub.A[combination]/MIC.sub.A[alone]+MIC.sub.B[combination]/MIC.s-
ub.B[alone]
[0166] Additive combinations of two antimicrobial agents are
indicated by a FIC index of 1, whereas a FIC index <1 indicates
synergistic combinations. Neutral combinations would give a FIC
between 1 and 4, a FIC index higher than 4 indicates antagonist
effects between the two antimicrobial agents.
[0167] The FIC was also calculated to assess the interaction of two
antimicrobial agents in combination against bacterial biofilms. The
same formulae applied, using MBEC instead of MIC.
1.6 Determination of the Minimum-Biofilm Eradication Concentration
(MBEC)
[0168] A total volume of 100 .mu.l bacterial inoculum in
Mueller-Hinton was added to each well of 96-well plates (challenge
plates) and the plates were incubated at 37.degree. C. for 24 h on
a gyrorotary shaking platform (Grant-bio PS-3D, Shepreth, England)
at 24 rpm to allow for biofilm formation.
[0169] The challenge plates were then rinsed once with sterile PBS
(lx) and two-fold serial dilutions of antimicrobial agents in
Mueller-Hinton were added to the challenge plates. The challenge
plates were incubated at 37.degree. C. for 24 h on a gyrorotary
shaking platform (Grant-bio PS-3D, Shepreth, England) at 24
rpm.
[0170] The supernatants from each of the challenge plates were
transferred into fresh plates and the optical density was measured
at 625 nm on a microtitre plate reader (BioTek Powerwave XS,
Winooski, USA). The MBEC was obtained by the lowest concentration
of antimicrobial showing no bacterial growth.
1.7 Estimation of the Persister Cells in the Biofilms
[0171] Following transfer of the supernatant from the challenge
plates, the biofilms were rinsed once with sterile PBS (lx) and 100
.mu.l of BacLight live/dead fluorescent staining solution
(Invitrogen, Paisley, UK) containing 4 .mu.M SYTO9 and 20 .mu.M
propidium iodide (PI) in sterile PBS (lx) were added to the wells
of the challenge plates. The plates were incubated at room
temperature in the dark for 15 min and the fluorescence was read at
485(ex)/528(em) and 485(ex)/645(em) for SYTO9 and PI fluorescence,
respectively on a fluorescence microtitre plate reader (BioTek
Synergy HT, Winooski, USA) with the sensitivity set at 50 and
bottom optics position was selected. Direct observation of the
biofilms with an Axiovert 40 fluorescence microscope (Zeiss,
Gottingen, Germany) allowed identifying the presence of live and
dead bacteria and pictures of the biofilms were taken at 100 to
400-fold magnification.
[0172] The relative viability of persister cells was determined by
the live/dead fluorescence measurements ratio and microscopic
observations were used to confirm the presence or absence of live
cells.
2. Results
2.1 Prevention of Biofilm Formation
[0173] In order to assess for the prevention of biofilm formation
by both Gram-positive and Gram-negative bacteria, the bacterial
inoculum and antimicrobial agents were added simultaneously in the
plates. The range of concentrations of antimicrobial agents was
0-500 .mu.g/ml NP108 and 0-320 .mu.g/ml cysteamine against the
Gram-negative bacteria P. aeruginosa ATCC BAA-47 and 0-1000
.mu.g/ml NP108 and 0-320 .mu.g/ml cysteamine against the
Gram-positive MRSA.
2.1.1 Activity Against P. aeruginosa ATCC BAA-47
[0174] The MIC of NP108 was 62.5 .mu.g/ml and 320 .mu.g/ml for
cysteamine. NP108 was bactericidal at 250 .mu.g/ml whereas
cysteamine was not bactericidal at up to 320 .mu.g/ml (data not
shown).
[0175] In the presence of 160 .mu.g/ml cysteamine the MIC of NP108
was reduced to 31.25 .mu.g/ml. When the concentration of cysteamine
was doubled (ie. 320 .mu.g/ml) no growth was observed regardless of
the concentration of NP108.
[0176] Determination of the FIC for this combination indicates that
the antimicrobial agents have additive effects (FIC=1). Moreover,
bactericidal activity was obtained in the presence of 125 .mu.g/ml
NP108 and 320 .mu.g/ml cysteamine (data not shown), which confirms
the additive effect of these agents.
2.1.2 Activity Against S. aureus DSM 11729
[0177] The MIC of NP108 was 125 .mu.g/ml and more than 320 .mu.g/ml
for cysteamine. NP108 was bactericidal at 125 .mu.g/ml whereas
cysteamine was not bactericidal at up to 320 .mu.g/ml (data not
shown).
[0178] Increasing concentrations of cysteamine showed a higher
inhibition of growth for any given concentration of NP108. In the
presence of 40 .mu.g/ml cysteamine the MIC of NP108 was reduced to
31.25 .mu.g/ml and down to 15.625 .mu.g/ml when 320 .mu.g/ml
cysteamine were added.
[0179] Determination of the FIC for this combination indicates that
the antimicrobial agents have at least additive effects (FIC<1).
Moreover, bactericidal activity was obtained in the presence of
31.25 .mu.g/ml NP108 and .gtoreq.160 .mu.g/ml cysteamine as well as
62.5 .mu.g/ml NP108 and .gtoreq.80 .mu.g/ml cysteamine (data not
shown), which confirms the additive effect of these agents.
[0180] Appendix 1 shows the time course activity of the short
linear arginine peptides (NP339, NP340, NP341 and NP352) against S.
aureus DSM 11729 planktonic cells.
[0181] Appendix 2 provides a summary of the activity of NP108,
cysteamine, both compounds in combination as well as the activity
of NP339 and NP341 against S. aureus DSM 11729 and P. aeruginosa
BAA-47 planktonic cells.
2.2 Destruction of Formed Biofilms
[0182] The assessment of the activity of NP108 and cysteamine
against bacterial biofilms was carried out with 24 h-old biofilms
and the activity of both compounds in combination was also
determined. The activity of the antimicrobial agents against
bacterial biofilms was determined by their activity against the
biofilm cells and against the persister cells.
2.2.1 Activity of NP339 Against Bacterial Biofilms
[0183] FIG. 5 shows the high activity of NP339 against biofilms of
3 Staphylococcus species, resulting in MBEC of 156 to 625 .mu.g/ml.
The increase in optical density at the highest dose of NP339
against S. aureus 25923 is likely to be an artefact due to the
complex and heterogeneous nature of microbial biofilms. In contrast
NP339 reduced the growth P. aeruginosa BAA-47 (PAO1), but even the
highest dose tested (i.e. 5 mg/ml) was not sufficient to inhibit
100% of the biofilm cells.
[0184] FIG. 6 provides evidence that NP339 is active against
persister cells. In contrast to the activity of NP339 against the
biofilm cells of the 4 strains tested, it was less active against
the persister cells of Staphylococcus species than those of P.
aeruginosa BAA-47 (PAO1). NP339 was able to inhibit the viability
of P. aeruginosa BAA-47 (PAO1) persister cells at 625 .mu.g/ml.
2.2.2 Activity of NP341 Against Bacterial Biofilms
[0185] Similarly to NP339 (FIG. 5), NP341 showed significant
reduction in biofilm cells viability. The MBEC for MRSA 11729 and
S. epidermidis 12228 was 625 .mu.g/ml. NP341 reduced the viability
of biofilm cells of MRSA 11729 and P. aeruginosa BAA-47 (PAO1) by a
2 to 3-fold factor.
[0186] As seen with NP339, the viability of P. aeruginosa persister
cells was totally inhibited at 625 .mu.g/ml NP341. The viability of
the persister cells of the 3 Staphylococcus species was decreased
by 25 to 50%.
2.2.3 Activity of Cysteamine Against Bacterial Biofilms
[0187] FIG. 9 provides evidence that cysteamine has antimicrobial
activity against biofilm cells of the Gram-positive and
Gram-negative bacteria tested.
[0188] FIG. 10 shows the activity of cysteamine against persister
cells of the Gram-negative and Gram-positive bacteria tested.
[0189] The results presented here show the antimicrobial activity
of the linear short cationic peptides NP339 and NP341 against
biofilms of Gram-positive and Gram-negative bacteria. These
compounds appear more effective against the biofilm cells of
Gram-positive bacteria than Gram-negative bacteria, whereas it is
the opposite against persister cells. Cysteamine showed activity
against biofilm cells at high concentrations, however, it
suppressed the viability of both Gram-positive and Gram-negative
persister cells at the lowest concentration tested (i.e. 6.25
mg/ml).
2.2.4 Activity of NP108 and Cysteamine in Combination Against P.
aeruginosa ATCC BAA-47
[0190] The combination of these two antimicrobial agents showed
complete inhibition of bacterial growth in the presence of 250
.mu.g/ml NP108 and 62.5 to 500 .mu.g/ml cysteamine. The addition of
31.25 .mu.g/ml cysteamine to 500 .mu.g/ml NP108 had a similar
effect, whereas 31.25 .mu.g/ml cysteamine plus 250 .mu.g/ml NP108
showed only partial inhibition of bacterial growth.
[0191] The FIC obtained with those MBEC values
(MBEC.sub.NP108[alone]>500 .mu.g/ml,
MBEC.sub.NP108[combination]=250 .mu.g/ml,
MBEC.sub.cysteamine[combination]=62.5 .mu.g/ml,
MBEC.sub.cysteamine[alone]=>100.00 .mu.g/ml,) was .about.0.5,
which indicates a synergistic effect between these two
antimicrobial agents. This is consistent with the observations made
from the activity of NP108/cysteamine combination against the
planktonic cells (FIG. 2).
[0192] The activity of NP108 and cysteamine against the persister
cells was assessed using a fluorescence staining method to
determine the relative viability of the cells. The nucleic
acid-binding fluorescent molecules used were SYTO9 and PI, which
penetrate all bacterial cells (green fluorescence) and
membrane-disrupted cells (red fluorescence), respectively.
Therefore the ratio green (live)/red (dead) fluorescence emitted
gives an indication of the relative viability of the bacterial
population and is used to estimate the presence of residual live
cells corresponding to persister cells within the biofilm.
[0193] FIG. 12 shows that the relative viability of the biofilms
treated with either NP108 or cysteamine remained significant,
indicating the lack of activity of these compounds against the
persister cells of P. aeruginosa ATCC BAA-47.
[0194] FIG. 13 provides evidence that the combination of NP108 and
cysteamine showed higher activity against the persister cells of P.
aeruginosa ATCC BAA-47 than either compound alone (FIG. 12). The
most efficient combinations against those cells were 250-500
.mu.g/ml NP108 and 62.5-500 .mu.g/ml cysteamine. These combinations
showed the lowest relative viability within the biofilms. Similar
results were obtained with 31.25 .mu.g/ml NP108 and 500 .mu.g/ml
cysteamine with only partial inhibition observed with 250 .mu.g/ml
cysteamine.
[0195] The activity of these compounds against persister cells
shows similarities to the profile of optimum combinations obtained
against the biofilm cells (FIG. 11). Moreover, direct microscopic
observations of the fluorescently-stained biofilms confirmed the
activity of these combinations against the persister cells as no
live cells could be observed in the presence of 250-500 .mu.g/ml
NP108 and 62.5-500 .mu.g/ml cysteamine (data not shown).
2.2.5 Activity of NP339 and Cysteamine in Combination Against P.
aeruginosa
[0196] FIG. 14(a)-(d) show the activity of 3 concentrations of
NP339: 1 .mu.g/ml, 10 .mu.g/ml and 100 .mu.g/ml in combination with
increasing concentrations of cysteamine up to 10 mg/ml against 4
strains of Pseudomonas aeruginosa.
[0197] These data clearly demonstrate the increased antimicrobial
activity against P. aeruginosa biofilm cells of NP339 in
combination with cysteamine. The following figures show examples of
the activity of these combinations against persister cells of 2 of
these strains.
[0198] FIG. 16 shows the activity of NP108 and cysteamine against
S. aureus DSM 11729 biofilm cells. The MBEC for cysteamine was 250
.mu.g/ml, whereas NP108 inhibited the growth of those cells at 125
.mu.g/ml.
[0199] The combination of NP108 and cysteamine showed complete
inhibition of bacterial growth in the presence of 31.25 .mu.g/ml
NP108 and 62.5 .mu.g/ml cysteamine and partial inhibition with
lower concentrations of either compound (FIG. 17). Hence, the FIC
obtained with those MBEC (MBEC.sub.NP108[alone] 125 .mu.g/ml,
MBEC.sub.NP108[combination]=31.25 .mu.g/ml,
MBEC.sub.cysteamine[alone]=250 .mu.g/ml,
MBEC.sub.cysteamine[combination]=62.5 .mu.g/ml) was 0.5 thereby
indicating a synergistic effect between these two antimicrobial
agents against biofilm of these Gram-positive bacteria. Similar
results were observed for Gram-negative bacterial biofilm (FIG.
11). This is also consistent with the observations made from the
activity of NP108/cysteamine combination against the planktonic
cells of S. aureus DSM 11729 (FIG. 4).
[0200] Similarly to the lack of activity observed against P.
aeruginosa ATCC BAA-47 persister cells (FIG. 12), the relative
viability of the S. aureus DSM 11729 biofilms treated with either
NP108 or cysteamine remained significant, indicating the lack of
activity of these compounds at low concentrations against the
persister cells of these Gram-positive bacteria (FIG. 18).
[0201] The combination of NP108 and cysteamine showed higher
activity against the persister cells of S. aureus DSM 11729 than
either compound alone (FIG. 19). The most efficient combinations
against those cells were 250-500 .mu.g/ml NP108 and 125-250
.mu.g/ml cysteamine. These combinations showed the lowest relative
viability within the biofilms. Similar results were obtained with
62.5 .mu.g/ml NP108 and 500 .mu.g/ml cysteamine. Combinations with
lower concentrations of either compound showed high relative
viability within the biofilms.
[0202] Unlike the Gram-negative persister cells, direct microscopic
observations of the fluorescently-stained S. aureus DSM 11729
biofilms indicated the presence of residual live cells at the
highest combined concentrations of NP108 and cysteamine (data not
shown).
[0203] Table 1 provides a summary of the activity of the short
arginine peptides NP339, NP341, the poly-L-lysine NP108, cysteamine
and the combination of NP108 with cysteamine against a
Gram-positive and Gram-negative bacteria.
[0204] Table 1:
[0205] Summary of the activity of the tested antimicrobial agents
against the Gram-negative P. aeruginosa strains and the
Gram-positive Staphylococcus spp. The number into brackets
indicates the maximum number of strains tested. MIC: minimum
inhibition concentration; MBEC: minimum biofilm eradication
concentration; FIC: fractional inhibitory concentration.
TABLE-US-00004 TABLE 1 P. aeruginosa strains Staphylococcus spp (7)
(4) MIC (.mu.g/ml) NP108 31.25-500 16-125 NP339 62.5 4-128 NP341
31.25 250 Cysteamine 300-2,500 300-625 NP108/ Cysteamine 31.25/160
31.25/40 FIC: NP108/ Cysteamine 1 0.6 MBEC (.mu.g/ml) NP108
250->500 125-250 NP339 >5,000 156-625 NP341 >5,000
625->5,000 Cysteamine >5,000 >25,000 NP108/ Cysteamine
125/125-250/62.5 31.25/62.5-125/125 FIC: NP108/ Cysteamine
.ltoreq.0.75 0.5-1 Persisters (.mu.g/ml) NP108 250->500 >500
NP339 625 625->5,000 NP341 625 625->5,000 Cysteamine
500-6,250 6,250-12,500 NP108/ Cysteamine 62.5/250-250/62.5
>250/>250 FIC: NP108/ Cysteamine 0.75-1 .ltoreq.0.5
[0206] Notes:
[0207] appendix 1 shows the MIC of the tested short arginine
antimicrobials against Staphylococcus aureus DSM 11729.
[0208] appendix 2 shows the activity of the mucolytic agents
cysteamine and N-acetylcysteine in combination with NP341 against
P. aeruginosa ATCC27853.
APPENDIX 1
[0209] The data (not shown) demonstrates the activity of short
linear arginine peptides over a 48-h period against planktonic
cells of methicillin-resistant S. aureus (MRSA) DSM 11729. The
range of concentrations tested as shown in the legends is in mg/ml.
The data (not shown) demonstrates the activity of short linear
arginine peptides over a 48-h period against planktonic cells of
methicillin-resistant S. aureus (MRSA) DSM 11729. The time course
activity demonstrates that bacterial growth inhibition is
associated with the dose of antimicrobial and to the time of
exposure to the cells. Complete bactericidal activity was observed
for NP339, NP 340 and NP352 at concentrations above 0.5 mg/ml for
the 48-h period; 0.125 and 0.25 mg/ml showed complete inhibition
for at least 24 h, and lower concentrations such as 0.06 and 0.03
mg/ml showed complete inhibition for at least 20 h and 15 h,
respectively. Similar results were obtained with NP341, except that
0.25 mg/ml showed complete inhibition for the 48-h period.
APPENDIX 2
[0210] In combination with 3-6 mg/ml of N-acetylcysteine, however,
only 205 .mu.g/ml of NP341 are needed to reach the MBEC (FIG. 20a).
Similar increased activity for the combination of these 2 compounds
was observed against persister cells: 1024 .mu.g/ml NP341+3128
.mu.g/ml N-acetylcysteine inhibited approximately 75% of the
persister cells, which is a much higher inhibition than that
obtained with either of the two compounds alone (FIG. 20b).
[0211] The combination of cysteamine or N-acetylcysteine with NP341
shows increased antibacterial activity compared to the activity of
either compound alone. The MBEC of NP341 alone against P.
aeruginosa ATCC27853 was more than 2 mg/ml and more than 100 mg/ml
for cysteamine (FIG. 21a). This indicates that there is no
cooperative effect between the two compounds against the biofilm
cells of P. aeruginosa ATCC27853. However, such cooperation was
observed against the persister cells: 205 .mu.g/ml NP341+3 mg/ml
cysteamine inhibit approximately 75% of the persister cells, which
is much higher than any of the two compounds alone (FIG. 21b).
[0212] When used in combination with NP339, we observed that the
addition of cysteamine even in small amounts helps reducing the
MBEC values of NP339 (FIG. 14a-d). More interestingly, the
combination of NP339 and cysteamine also showed increased activity
against persister cells of P. aeruginosa DSM1128 and P. aeruginosa
BAA-47 (FIG. 15a-b).
* * * * *