U.S. patent application number 16/470887 was filed with the patent office on 2020-01-16 for biofilm disrupting composition.
The applicant listed for this patent is The University of Sydney, Whiteley Corporation Pty Ltd. Invention is credited to Trevor Owen Glasbey, Theerthankar Das Ashish Kumar, Jim Manos, Gregory Stuart Whiteley.
Application Number | 20200016231 16/470887 |
Document ID | / |
Family ID | 62624084 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200016231 |
Kind Code |
A1 |
Kumar; Theerthankar Das Ashish ;
et al. |
January 16, 2020 |
BIOFILM DISRUPTING COMPOSITION
Abstract
A biofilm disrupting composition for use in treating
biofilm-mediated infections due to non-Pseudomonas micro-organisms
in the Cystic Fibrosis patient. One embodiment of the composition
of the invention comprises at least one biologically acceptable
thiol based antioxidant and at least one antibiotic. Another
embodiment of the composition of the invention comprises at least
one biologically acceptable thiol based antioxidant, at least one
enzyme and at least one antibiotic. The invention is also directed
to the process of preparing the composition of the invention, the
use of the composition for the manufacture of a medicament for
disrupting biofilms formed by non-Pseudomonad micro-organisms, and
a method of disrupting biofilm formed by non-Pseudomonad
micro-organisms in a patient, comprising administering to the
patient the composition of the invention.
Inventors: |
Kumar; Theerthankar Das Ashish;
(Kensington, AU) ; Manos; Jim; (Oatley, AU)
; Whiteley; Gregory Stuart; (Queenscliff, AU) ;
Glasbey; Trevor Owen; (Tanilba Bay, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Whiteley Corporation Pty Ltd
The University of Sydney |
North Sydney
The University of Sydney |
|
AU
AU |
|
|
Family ID: |
62624084 |
Appl. No.: |
16/470887 |
Filed: |
December 8, 2017 |
PCT Filed: |
December 8, 2017 |
PCT NO: |
PCT/AU2017/051349 |
371 Date: |
June 18, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/063 20130101;
A61K 9/08 20130101; A61K 38/465 20130101; A61K 9/06 20130101; A61K
45/06 20130101; A61K 31/7036 20130101; A61K 33/30 20130101; A61P
31/04 20180101; A61K 31/14 20130101; A61K 9/0014 20130101; A61K
31/496 20130101; C12Y 301/21001 20130101; A61P 27/02 20180101; A61K
33/18 20130101; A61K 47/38 20130101; A61P 31/00 20180101; A61K
38/063 20130101; A61K 2300/00 20130101; A61K 31/496 20130101; A61K
2300/00 20130101; A61K 33/30 20130101; A61K 2300/00 20130101; A61K
31/14 20130101; A61K 2300/00 20130101; A61K 33/18 20130101; A61K
2300/00 20130101; A61K 38/465 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/06 20060101
A61K038/06; A61K 31/496 20060101 A61K031/496; A61K 31/7036 20060101
A61K031/7036; A61K 38/46 20060101 A61K038/46; A61P 31/04 20060101
A61P031/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2016 |
AU |
2016905326 |
Claims
1-20. (canceled)
21. A biofilm disrupting composition when used for disrupting
biofilms formed by non-Pseudomonad micro-organisms comprising: (a)
at least one biologically acceptable thiol based antioxidant, and
(b) at least one antibiotic.
22. The composition according to claim 21 wherein the biologically
acceptable thiol based antioxidant is selected from the group
consisting of mercaptoethanol, N-acetyl cysteine, glutathione,
thiamphenicol glycinate, acetylcysteinate, sodium mercaptoethane
sulfonate, lipoic acid and erdosteine.
23. The composition according to claim 22 wherein the biologically
acceptable thiol based antioxidant is glutathione.
24. The composition according to claim 21 wherein the antibiotic is
selected from the group consisting of antibiotic classes
Penicillins, Tetracyclines, Cephalosporins, Quinolones,
Lincomycins, Macrolides, Sulfonamides, Glycopeptides,
Aminoglycosides and Carbapenems and mixtures thereof.
25. The composition according to claim 24 wherein the antibiotic is
selected from the group consisting of ciprofloxacin, dexamethasone,
amoxicillin/clavulanate, cefixime, cefaclor, clarithromycin,
levofloxacin, moxifloxacin and telithromycin.
26. A biofilm disrupting composition when used for disrupting
biofilms formed by non-Pseudomonad micro-organisms comprising: (a)
at least one biologically acceptable thiol based antioxidant, (b)
at least one enzyme and (c) at least one antibiotic.
27. The composition according to claim 26 wherein the biologically
acceptable thiol based antioxidant is selected from the group
consisting of mercaptoethanol, N-acetyl cysteine, glutathione,
thiamphenicol glycinate, acetylcysteinate, sodium mercaptoethane
sulfonate, lipoic acid and erdosteine.
28. The composition according to claim 27 wherein the biologically
acceptable thiol based antioxidant is glutathione.
29. The composition according to claim 26 wherein the antibiotic is
selected from the group consisting of antibiotic classes
Penicillins, Tetracyclines, Cephalosporins, Quinolones,
Lincomycins, Macrolides, Sulfonamides, Glycopeptides,
Aminoglycosides and Carbapenems and mixtures thereof.
30. The composition according to claim 29 wherein the antibiotic is
selected from the group consisting of ciprofloxacin, dexamethasone,
amoxicillin/clavulanate, cefixime, cefaclor, clarithromycin,
levofloxacin, moxifloxacin and telithromycin.
31. The composition according to claim 26 wherein the enzyme is
selected from the group consisting of DNase, amylase, cellulase,
and proteinase.
32. The composition according to claim 31 wherein the enzyme is
DNase.
33. A process of preparing a biofilm disrupting composition
according to claim 21, which process comprises combining at least
one biologically acceptable thiol based antioxidant and at least
one antibiotic, to form said composition.
34. A process of preparing a biofilm disrupting composition
according to claim 26, which process comprises combining at least
one biologically acceptable thiol based antioxidant, at least one
antibiotic and at least one enzyme, to form said composition.
35. The use of a composition comprising: (a) at least one
biologically acceptable thiol based antioxidant, and (b) at least
one antibiotic, for the manufacture of a medicament for disrupting
biofilms formed by non-Pseudomonad micro-organisms.
36. The use according to claim 35 wherein the biologically
acceptable thiol based antioxidant is selected from the group
consisting of mercaptoethanol, N-acetyl cysteine, glutathione,
thiamphenicol glycinate, acetylcysteinate, sodium mercaptoethane
sulfonate, lipoic acid and erdosteine.
37. The use according to claim 36 wherein the biologically
acceptable thiol based antioxidant is glutathione.
38. The use according to claim 35 wherein the antibiotic is
selected from the group consisting of antibiotic classes
Penicillins, Tetracyclines, Cephalosporins, Quinolones,
Lincomycins, Macrolides, Sulfonamides, Glycopeptides,
Aminoglycosides and Carbapenems and mixtures thereof.
39. The use according to claim 38 wherein the antibiotic is
selected from the group consisting of ciprofloxacin, dexamethasone,
amoxicillin/clavulanate, cefixime, cefaclor, clarithromycin,
levofloxacin, moxifloxacin and telithromycin.
40. The use according to claim 39 wherein the enzyme is selected
from the group consisting of DNase, amylase, cellulase, and
proteinase.
41. The use of a composition comprising: (a) at least one
biologically acceptable thiol based antioxidant, (b) at least one
enzyme and (c) at least one antibiotic, for the manufacture of a
medicament for disrupting biofilms formed by non-Pseudomonad
micro-organisms.
42. The use according to claim 41 wherein the biologically
acceptable thiol based antioxidant is selected from the group
consisting of mercaptoethanol, N-acetyl cysteine, glutathione,
thiamphenicol glycinate, acetylcysteinate, sodium mercaptoethane
sulfonate, lipoic acid and erdosteine.
43. The use according to claim 42 wherein the biologically
acceptable thiol based antioxidant is glutathione.
44. The use according to claim 41 wherein the antibiotic is
selected from the group consisting of antibiotic classes
Penicillins, Tetracyclines, Cephalosporins, Quinolones,
Lincomycins, Macrolides, Sulfonamides, Glycopeptides,
Aminoglycosides and Carbapenems and mixtures thereof.
45. The use according to claim 44 wherein the antibiotic is
selected from the group consisting of ciprofloxacin, dexamethasone,
amoxicillin/clavulanate, cefixime, cefaclor, clarithromycin,
levofloxacin, moxifloxacin and telithromycin.
46. The use according to claim 45 wherein the enzyme is selected
from the group consisting of DNase, amylase, cellulase, and
proteinase.
47. A method of disrupting biofilm formed by non-Pseudomonad
micro-organisms in a patient, which method comprises administering
to said patient a composition according to claim 21 in an amount
which effectively disrupts said biofilm.
48. A method of disrupting biofilm formed by non-Pseudomonad
micro-organisms in a patient, which method comprises administering
to said patient a composition according to claim 26 in an amount
which effectively disrupts said biofilm.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a biofilm disrupting composition
for use in treating biofilm-mediated infections due to
non-Pseudomonas micro-organisms in the Cystic Fibrosis patient.
BACKGROUND OF INVENTION
[0002] A biofilm is any group of microorganisms in which cells
stick to each other and often these cells adhere to a surface.
These adherent cells are frequently embedded within a self-produced
matrix of extracellular polymeric substance (EPS). The biofilm EPS
is typically comprised of a polymeric conglomeration generally
composed of extracellular DNA (eDNA), proteins, and
polysaccharides. Biofilms may form on living or non-living surfaces
and can be prevalent in natural, industrial and hospital settings.
The sessile microbial cells growing in a biofilm are
physiologically distinct from planktonic cells of the same
organism, which, by contrast, are single-cells that may float or
swim in a liquid medium.
[0003] Microbes form a biofilm in response to many factors, which
may include cellular recognition of specific or non-specific
attachment sites on a surface, nutritional cues, or in some cases,
by exposure of planktonic cells to sub-inhibitory concentrations of
antibiotics. When a cell switches to the biofilm mode of growth, it
undergoes a phenotypic shift in behavior in which large suites of
genes are differentially regulated.
[0004] Within a biofilm structure, microorganisms demonstrate
significantly greater resistance to both biocides and antibiotics.
This resistance feature of sessile microbes is a dual function of
the enhanced genetic expression and also shielding by the
surrounding polymeric materials of the biofilm. Traditional
antimicrobial therapies have been ineffective in the treatment of
bacterial infections when the bacteria are located within a biofilm
which is within, adherent to, or above tissue or located within a
void such as the lungs or bladder, or in nasal passages or on the
surface of a wound or burn. The additional risk of a
multi-drug-resistant-organism increases the likelihood of
significant morbidity or even death.
[0005] Inhaled glutathione (GSH) therapy has been used to reduce
oxidative stress in cystic fibrosis patients and inhibit
proliferation of Pseudomonas infections in cystic fibrosis
patients, including increasing susceptibility of the Pseudomonas to
antibiotics (Zhang Y and Duan K, "Glutathione exhibits
antibacterial activity and increases tetracycline efficacy against
Pseudomonas aeruginosa"; Sci. China Ser. C, (2009) 52:501-505.
[0006] It has also been found that GSH and DNase I can be combined
for treating chronic Pseudomonas infections in individuals with
cystic fibrosis (Klare et al., Canberra ASM meeting, July 2015).
Further, GSH and DNase I have been found to be useful in the
disruption of Pseudomonas biofilms in cystic fibrosis-like media
and increasing susceptibility of the Pseudomonas aeruginosa to
antibiotics (Klare et al., 2016 Antimicrobial Agents Chemotherapy
60 (8) 4539-4551), particularly when incorporated with an
antibiotic such as Ciprofloxacin.
[0007] The effectiveness of the combination of Glutathione and
DNase I can be ascribed to the nature of the biofilms formed by
Pseudomonas spp. The biofilm matrix consists predominantly of
polysaccharides, proteins, and nucleic acids. Despite macromolecule
heterogeneity, most research has focused on the role of bacterially
produced exopolysaccharides (EPSs) in biofilm establishment and
maturation.
[0008] The integral role of extracellular DNA in biofilm formation
was first identified in P. aeruginosa by Whitchurch et al
(Whitchurch C B, Tolker-Nielsen T, Ragas P C, Mattick J S.
"Extracellular DNA required for bacterial biofilm formation".
Science (2002) 295:1487. doi:10.1126/science.295.5559.1487), but
eDNA has since been shown to be a ubiquitous biofilm matrix polymer
across most Gram-positive and Gram-negative bacterial species. In
fact, within P. aeruginosa biofilms, eDNA is the most abundant
matrix polymer (see Matsukawa M, Greenberg E P. "Putative
exopolysaccharide synthesis genes influence Pseudomonas aeruginosa
biofilm development". J Bacteriol (2004) 186:4449-4456.
doi:10.1128/JB.186.14.4449-4456.2004. and Okshevsky M, Meyer R L.
"The role of extracellular DNA in the establishment, maintenance
and perpetuation of bacterial biofilms". Crit Rev Microbiol (2013)
41:341-352). The eDNA also serves as a structural component of the
EPS and contributes to its viscosity.
[0009] Pseudomonas aeruginosa is also known to release exoproducts
into the EPS. One of these exoproducts, the blue, redox-active
phenazine derivative called pyocyanin also contributes to the
viscosity of the EPS by intercalating directly with the EPS.
Pyocyanin has also been demonstrated to promote the release of eDNA
from P. aeruginosa (see Das T, and Manefield M, "Pyocyanin Promotes
Extracellular DNA Release in Pseudomonas aeruginosa"; Plos One
(2012), 7, e46718).
[0010] Pyocyanin also contributes to the disease processes in
Cystic Fibrosis. In vitro studies have shown that pyocyanin has
multiple deleterious effects on mammalian cells, such as inhibition
of cell respiration, ciliary function, epidermal cell growth and
prostacyclin release, disruption of calcium homeostasis, and
inactivation of catalase. Pyocyanin also induces apoptosis in
neutrophils and modulates the glutathione redox cycle in lung
epithelial and endothelial cells. It also inactivates al protease
inhibitor and contributes to the imbalance of protease-antiprotease
activity, which is readily detected in the airways of patients with
CF lung disease. More recently, it was shown that pyocyanin
inactivates the vacuolar ATPase of lung epithelial cells (See Lau G
W et al., "Pseudomonas aeruginosa Pyocyanin Is Critical for Lung
Infection in Mice". Infect. Immun. (2004) 72 4275-4278 and
references therein)
[0011] The most commonly identified organisms in respiratory
specimens taken from Cystic Fibrosis patients are various species
and forms of Pseudomonas. It can be seen that 48.5 percent of
patients tested produced positive Pseudomonas aeruginosa cultures,
with the mucoid form showing in 32.0 percent. Its prevalence is
greater in adult patients, with 60.9 percent of tested adult CF
patients producing samples indicating the mucoid form of
Pseudomonas aeruginosa, three times the corresponding proportion
for adolescents and much higher than that for children.
[0012] While prevalence of Pseudomonas organisms is lower in
children than in adults, although increasing with rising age, young
children are more likely than adult patients to produce cultures
showing presence of Staphylococcus aureus (see table 1). Half of
all child patients and adolescent patients aged 6 to 17 years had
this bacterial infection. Haemophilus influenza is also evident in
relatively high proportions of child patients, highest in children
aged from 2 to 5 years, where this organism was cultured for almost
one third of children. The youngest age groups also had the highest
proportions with positive cultures of the bacteria Escherichia
coli; 12 percent, for those in the age (source: "CYSTIC FIBROSIS IN
AUSTRALIA 2014", 17th Annual Report from the Australian Cystic
Fibrosis Data Registry, Cystic Fibrosis Australia (2016), North
Ryde, NSW, Australia).
TABLE-US-00001 TABLE 1 Percentages of other respiratory cultures by
age group (other than Pseudomonas) Age Range 0-1 2-5 6-11 12-17
18-29 30+ Total Bacteria: Staphylococcus aureus 39.0 42.6 47.6 50.3
38.6 30.7 41.8 Haemophilus influenzae 28.0 32.3 23.7 11.3 7.1 2.3
14.5 Burkholderia cepacia 0.0 0.0 1.6 2.1 4.2 2.8 2.3
Stenotrophomonas maltophilia 5.0 3.8 10.2 14.4 7.7 6.5 8.7
Escherichia coli 12.0 8.9 6.5 3.1 1.0 0.6 4.0 MRSA (Methicillin
Resistant 2.0 1.7 1.9 3.4 2.5 3.4 2.6 Staphylococcus aureus)
Alcaligenes xylosoxidans 0.0 0.4 3.0 2.6 5.0 5.6 3.5 Serratia
marcescens 2.0 1.7 0.9 1.3 0.8 0.0 0.9 Klebsiella (any species) 9.0
3.0 0.7 1.6 0.6 1.1 1.6 Non-tuberculous mycobacterium 0.0 0.0 0.0
3.1 4.6 4.2 2.5 Fungi: Candida 18.0 22.1 24.6 34.8 27.8 29.3 27.6
Aspergillus (any species) 8.0 10.2 22.0 33.5 29.2 22.3 24.0
Scediosporium (any species) 0.0 0.4 2.6 6.3 5.0 3.4 3.7 Other
organisms not listed 28.0 33.2 30.4 29.1 20.8 19.7 26.0 above
Normal flora only 63.0 79.1 82.8 77.5 38.0 28.5 59.4 No
growth/sterile culture 10.0 8.1 7.0 4.5 4.2 3.4 5.4 Patients tested
100 235 431 382 518 355 2021
[0013] It is evident therefore that whilst the bulk of respiratory
infections in Cystic Fibrosis sufferers are due to Pseudomonas, a
significant number of patients show infections due to other biofilm
generating organisms such as Staphylococcus aureus and Haemophilus
influenzae, neither of which release pyocyanin.
DESCRIPTION OF FIGURES
[0014] FIG. 1 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Pseudomonas aeruginosa both individually
and in combination (two component combination), according to
comparative Example 2.
[0015] FIG. 2 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Staphylococcus aureus (MRSA) both
individually and in combination (two component combination),
according to Example 3.
[0016] FIG. 3 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Staphylococcus aureus MSSA both
individually and in combination (two component combination),
according to Example 4.
[0017] FIG. 4 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Streptococcus agalactiae both individually
and in combination (two component combination), according to
Example 5.
[0018] FIG. 5 shows the effects of amikacin and glutathione on the
biofilm viability of Acinetobacter baumaunii multi drug resistant
(MRAB) both individually and in combination (two component
combination), according to Example 6.
[0019] FIG. 6 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Klebsiella pneumoniae both individually
and in combination (two component combination), according to
Example 7.
[0020] FIG. 7 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Enterobacter species both individually and
in combination (two component combination), according to Example
8.
[0021] FIG. 8 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Escherichia coli both individually and in
combination (two component combination), according to Example
9.
[0022] FIG. 9 shows the effects of ciprofloxacin and glutathione on
the biofilm viability of Streptococcus pyogenes both individually
and in combination (two component combination), according to
Example 10.
[0023] FIG. 10 shows the effects of ciprofloxacin, glutathione and
DNase I on the biofilm viability of Staphylococcus aureus (MRSA)
both individually, and in both dual and triple combination (three
component combination), according to Example 11.
[0024] FIG. 11 shows the effects of ciprofloxacin, glutathione and
DNase I on the biofilm viability of Staphylococcus aureus (MSSA)
both individually, and in both dual and triple combination (three
component combination), according to Example 12.
[0025] FIG. 12 shows the effects of ciprofloxacin, glutathione and
DNase I on the biofilm viability of Streptococcus agalactiae both
individually, and in both dual and triple combination (three
component combination), according to Example 13.
[0026] FIG. 13 shows the effects of amikacin, glutathione and DNase
I on the biofilm viability of Acinetobacter baumannii (MRAB) both
individually, and in both dual and triple combination (three
component combination), according to Example 14.
SUMMARY OF THE INVENTION
[0027] Whilst it may be expected that the combination of DNase I
and an antibiotic will show activity against biofilms, it has been
surprisingly found that a biologically acceptable thiol based
antioxidant can also serve to disrupt biofilms formed from
organisms other than the pyocyanin-producing Pseudomonas.
[0028] It has also been unexpectedly found that the combination of
a biologically acceptable thiol based antioxidant, an enzyme and an
antibiotic also demonstrate a synergistic effect against biofilms
formed by organisms other than Pseudomonas (ie non-Pseudomonad
organisms).
[0029] According to a first embodiment of the invention, there is
provided a biofilm disrupting composition comprising: [0030] (a) at
least one biologically acceptable thiol based antioxidant, and
[0031] (b) at least one antibiotic, [0032] wherein said composition
is capable of disrupting biofilms formed by non-Pseudomonad
micro-organisms.
[0033] According to a second embodiment of the invention, there is
provided a biofilm disrupting composition when used for disrupting
biofilms formed by non-Pseudomonad micro-organisms comprising:
[0034] (a) at least one biologically acceptable thiol based
antioxidant, and [0035] (b) at least one antibiotic.
[0036] According to a third embodiment of the invention, there is
provided a biofilm disrupting composition comprising: [0037] (a) at
least one biologically acceptable thiol based antioxidant, [0038]
(b) at least one enzyme and [0039] (c) at least one antibiotic,
wherein said composition is capable of disrupting biofilms formed
by non-Pseudomonad micro-organisms.
[0040] According to a fourth embodiment of the invention, there is
provided a biofilm disrupting composition when used for disrupting
biofilms formed by non-Pseudomonad micro-organisms comprising:
[0041] (a) at least one biologically acceptable thiol based
antioxidant, [0042] (b) at least one enzyme and [0043] (d) at least
one antibiotic.
[0044] According to a fifth embodiment of the invention, there is
provided a process of preparing a biofilm disrupting composition
according to the first and second embodiments, which process
comprises combining at least one biologically acceptable thiol
based antioxidant and at least one antibiotic, to form said
composition.
[0045] According to a sixth embodiment of the invention, there is
provided a process of preparing a biofilm disrupting composition
according to the third and fourth embodiments, which process
comprises combining at least one biologically acceptable thiol
based antioxidant, at least one antibiotic and at least one enzyme,
to form said composition.
[0046] According to a seventh embodiment of the invention, there is
provided the use of a composition comprising: [0047] (a) at least
one biologically acceptable thiol based antioxidant, and [0048] (b)
at least one antibiotic, for the manufacture of a medicament for
disrupting biofilms formed by non-Pseudomonad micro-organisms.
[0049] According to an eighth embodiment of the invention, there is
provided the use of a composition comprising: [0050] (a) at least
one biologically acceptable thiol based antioxidant, [0051] (b) at
least one enzyme and [0052] (c) at least one antibiotic, for the
manufacture of a medicament for disrupting biofilms formed by
non-Pseudomonad micro-organisms.
[0053] According to a ninth embodiment of the invention, there is
provided a method of disrupting biofilm formed by non-Pseudomonad
micro-organisms in a patient, which method comprises administering
to said patient a composition according to any one of the first,
second, third or fourth embodiments in an amount which effectively
disrupts said biofilm.
[0054] By biologically acceptable thiol based antioxidant it is
understood that this is a substance that is tolerated without ill
effect by a living body, and contains a sulfhydryl moiety, and
where said substance can inhibit the oxidation of other
molecules.
[0055] Throughout the description and claims of the specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises", is not intended to exclude other
additives, components, integers or steps.
[0056] The ingredients of the composition of the invention act
synergistically providing superior biofilm disruption.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Biologically Acceptable Thiol Based Antioxidant
[0058] The composition of the invention comprises at least one
biologically acceptable thiol based antioxidant. This is a
biologically and pharmaceutically acceptable compound capable of
reducing disulfide bonds formed within cytoplasmic proteins to
cysteines by serving as an electron donor.
[0059] Examples of suitable biologically acceptable thiol based
antioxidants include mercaptoethanol, N-acetyl cysteine (NAC),
glutathione (GSH), thiamphenicol glycinate acetylcysteinate (TGA),
sodium mercaptoethane sulfonate, dithiothreitol (DTT),
dithiobutylamine and other similar compounds. Other examples of
suitable biologically acceptable thiol based antioxidants are
compounds such as lipoic acid or erdosteine, which are capable of
generating free thiol groups in vivo following first pass
metabolism. In a preferred embodiment the biologically acceptable
thiol based antioxidant is glutathione (GSH).
[0060] Antibiotic
[0061] The composition of the invention comprises at least one
antibiotic capable of killing either or both Gram Positive or Gram
Negative organisms. The antibiotic may be selected from the
non-limiting group of antibiotic classes consisting of Penicillins,
Tetracyclines, Cephalosporins, Quinolones, Lincomycins, Macrolides,
Sulfonamides, Glycopeptides, Aminoglycosides and Carbapenems.
Specific examples of useful antibiotics include ciprofloxacin,
amoxicillin/clavulanate, cefixime, cefalosporin cefaclor,
clarithromycin, levofloxacin, moxifloxacin, gentamycin, vancomycin
and telithromycin.
[0062] Enzymes
[0063] The composition of the invention comprises one or more
enzymes capable of degrading one or more of the biopolymers that
make up the biofilm.
[0064] The enzymes may be selected from the (non-limiting) group
consisting of protease, amylase, cellulase, and DNase. In a
preferred embodiment, the composition of the invention will contain
two or more of these enzyme types. Preferably at least one of the
enzymes is DNase.
[0065] Optional Ancillary Agents
[0066] The biofilm disrupting composition may optionally contain
other ingredients such as tonicity modifiers, pH buffers,
colourants, preservatives and perfumes.
[0067] Tonicity Modifying Agent
[0068] The composition of the invention may also contain tonicity
modifying ingredients. These may comprise inorganic salts, for
example sodium bromide, potassium bromide, sodium chloride,
potassium chloride, sodium acetate, potassium acetate, sodium
citrate, potassium citrate, sodium phosphate, potassium phosphate,
or may comprise organic tonicity modifiers such as propylene
glycol, glycerol, mannitol, arabitol, glucose, fructose etc. The
composition of the invention may be isotonic (i.e. 250-350
mOsmal/Kg) or hypotonic (i.e. <250 mOsmal/Kg).
[0069] Colouring Agent
[0070] The composition of the invention may also comprise colouring
agents. The colouring agents may be added to provide a function to
the composition, such as the staining of components found within
the bacterial biofilm, or may just be added to provide an
aesthetically pleasing solution. When the colouring agent is added
to stain components of the biofilm, the resultant staining may
provide a visual cue as to the presence of the biofilm, thus also
provide a means of monitoring its removal. Suitable colouring
agents capable of staining biofilm components (for example protein,
polysaccharide or bacterial cell walls) will include Coomassie
Brilliant Blue, Crystal Violet, erythrosine and tartrazine.
[0071] Processing Aids
[0072] The biofilm disrupting composition may be in solid form, or
the composition may be a solution. In the case of a solid mixture
of ingredients, the mixture may comprise one or more processing
aids such as mannitol, starch, glucose, sucrose etc. in order to
allow the composition to be processed into micronized particles,
preferably with a mean particle size of less than 500 microns. In a
more preferred embodiment, the micronized composition will have a
mean particle size of less than 100 microns, and in a particularly
preferred embodiment, the micronized composition will have a mean
particle size of less than 40 microns. The micronized composition
of this particularly preferred embodiment is suitable for
inhalation and useful for the disruption and removal of bacterial
biofilms found in the lungs in conditions such as cystic fibrosis,
bronchitis, chronic obstructive pulmonary disease (COPD), and other
airway infections in which biofilms due to non-Pseudomonad
micro-organisms are implicated, such as recurrent rhinosinusitis or
pharyngotonsillitis.
[0073] In the case of a liquid composition, the mixture may contain
one or more processing aids such as wetting agents, defoaming
agents, antioxidants, viscosity modifiers etc.
[0074] Observed Results
[0075] Glutathione (GSH) especially at 30 milli Molar (3 times
higher than biological intracellular concentration which is 2-10
millimolar) shows significantly higher effect than antibiotics in
killing/disrupting biofilms, even against non-Pseudomonad organisms
(see FIGS. 1-13).
[0076] Combining GSH with low concentration of antibiotics (FIGS.
2-13) enhances biofilms disruptions and killing.
[0077] DNase I by itself has no effect on Staphylococcus aureus and
Streptococcus agalactiae biofilms disruption (see FIGS. 10 and 11),
but Dnase I by itself has some effect on MRAB biofilms disruption
(see FIG. 13).
[0078] Consequently 3 component combination therapy (3CT)
(GSH+DNase I+Ciprofloxacin) used for Staphylococcus aureus and
Streptococcus agalactiae has similar effect as GSH+ciprofloxacin (2
component combination therapy (2CT). 3CT use for MRAB (GSH+DNase
I+Amikacin) showed significantly better disruption/killing.
EXAMPLES
[0079] In the following examples, all clinical isolates were taken
from various hospitals in the Sydney, NSW area. Streptococcus
agalactiae from Cow mastitis, isolated on a NSW farm.
[0080] All strains sensitive to Ciprofloxacin (except for
Acinetobacter baumannii). Many strains are resistant to different
antibiotics including: penicillin, gentamicin,
amoxycillin/Clavulanate, Cefazolin, am ikacin.
Example 1: Experimental Protocol
[0081] Bacterial isolates were grown in Tryptone Soya Broth (TSB)
medium for 24 hours at 37.degree. C., in a shaking incubator set at
150 rpm. After this time the organisms were harvested by
centrifugation (5000.times.g, 5 min at 10.degree. C.). After
centrifugation, the supernatant liquid was removed and the
bacterial pellet was suspended in 1.times.Phosphate Buffered Saline
(PBS). To initiate biofilm growth, the bacterial suspension from
PBS was immediately re-suspended in TSB and 250 .mu.L of bacterial
cell suspension (OD.sub.600=0.5.+-.0.05) were added into the wells
of 96-well plates (Corning Corp. USA) and incubated at 37.degree.
C. for 48 h at 150 r.p.m.
[0082] After 48 h, biofilm were washed once with 1.times.PBS
followed by treatment (for 24 h, 37.degree. C., 150 rpm under
different conditions: either with Ciprofloxacin or Amikacin, DNase
I (40 U solution, Sigma Aldrich) or GSH (different concentration
10, 15 and 30 milliMolar solutions in PBS) individually or
combination: DNase I+ciprofloxacin or Amikacin, GSH+DNase I,
GSH+ciprofloxacin or Amikacin and GSH+DNase I+Ciprofloxacin or
Amikacin. The composition of the treatments used are given in the
subsequent examples
[0083] After 24 h, treated biofilms supernatant were replaced with
200 .mu.L of 1.times.PBS, followed by addition of 15 .mu.L of a
0.05% w/v solution of resazurin (Sigma-Aldrich), and incubated
further at 37.degree. C., 150 r.p.m.
[0084] After a further 24 h, the fluorescent intensity of the
biofilm was determined at Ex544 nm and Em590 nm (Tecan infinite
M1000 pro microplate reader). Results are plotted as percentage (%)
of bacterial vaiability calculated based on fluorescent intensity.
Control or untreated bacterial biofilm fluorescent intensity always
considered as 100% bacterial viability and % viability under rest
of treatment conditions were calculated with reference to
control.
[0085] It should be noted that the Resazurin assay works by
recording fluorescence intensity and depends upon two factors
(total number of bacteria and total number of viable bacteria in a
given biofilm sample).
Example 2: Pseudomonas aeruginosa (Clinical Isolates)
[0086] Example 2 is a comparative, prior art, example demonstrating
the use of the combination of glutathione with an antibiotic,
ciprofloxacin. As previously discussed, it is widely believed that
the role of the glutathione is to deactivate the pyocyanin released
by Pseudomonas aeruginosa. It is noted that the treatments shown in
column 1 of Table 2 were tested against three clinical isolates of
Pseudomonas aeruginosa. The treatments used are given in column 1,
and the % bacterial viability for each strain tested are given in
the subsequent columns (see also FIG. 1).
TABLE-US-00002 TABLE 2 Pseudomonas aeruginosa isolate and source
PA0053 365707 Left 364077 Scalp Treatment DFU ankle wound wound
Control 100.0 100.0 100.0 CIP 0.5 .mu.g/ml 12.2 22.0 9.7 CIP 1
.mu.g/ml 8.7 21.3 6.8 CIP2 .mu.g/ml 5.8 25.9 6.5 GSH 10 mM 89.8
103.5 95.6 GSH 15 mM 74.5 100.9 93.0 GSH 30 mM 7.3 11.2 30.3 2 part
CT 3.2 2.3 5.2 GSH 30 mM CIP 0.5 ug/ml Control = no treatment CIP =
Ciprofloxacin GHS = glutathione
[0087] The following examples illustrate the efficacy of the
combination of a biologically acceptable thiol based antioxidant,
with an antibiotic against biofilms formed by organisms other than
Pseudomonas (i.e. non-Pseudomonad organisms).
Example 3: Methicillin Resistant Staphylococcus aureus (MRSA:
Clinical Isolates)
[0088] The treatments shown in column 1 of Table 3 were tested
against three strains of methicillin-resistant Staphylococcus
aureus. The treatments used are given in column 1, and the %
bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 2).
TABLE-US-00003 TABLE 3 MRSA 27060 MRSA 30616 MRSA - 0799 Treatment
Left leg Chin vesicle DFU Control 100.00 100.00 100.00 CIP 10
.mu.g/ml 42.93 40.01 61.08 CIP 20 .mu.g/ml 25.74 31.38 54.21 CIP30
.mu.g/ml 19.53 24.56 51.60 GSH 10 mM 88.83 73.12 86.73 GSH 15 mM
65.24 58.82 75.03 GSH 30 mM 1.02 4.54 33.12 2 part CT -0.30 0.61
21.83 GSH 30 mM CIP 10 ug/ml Control = no treatment CIP =
Ciprofloxacin GHS = glutathione
Example 4: Methicillin Sensitive Staphylococcus aureus (MSSA:
Clinical Isolate)
[0089] The treatments shown in column 1 of Table 4 were tested
against three strains of methicillin-sensitive Staphylococcus
aureus. The treatments used are given in column 1, and the %
bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 3).
TABLE-US-00004 TABLE 4 MSSA - 34397 MSSA - 34654 MSSA - 0800
Treatment Left elbow Right toe DFU Control 100.00 100.00 100.00 CIP
10 .mu.g/ml 49.93 37.08 41.69 CIP 20 .mu.g/ml 36.78 32.05 32.03 CIP
30 .mu.g/ml 29.81 32.44 21.58 GSH 10 mM 88.67 86.11 82.44 GSH 15 mM
61.34 70.52 61.70 GSH 30 mM 1.06 15.33 20.09 2 part CT 0.77 5.85
9.68 GSH 30 mM CIP10 ug/ml Control = no treatment CIP =
Ciprofloxacin GHS = glutathione
Example 5: Staphylococcus agalactiae (Ex Cow Mastitis)
[0090] The treatments shown in column 1 of Table 5 were tested
against four strains of Staphylococcus agalactiae, (veterinary
strains ex Cow mastitis). The treatments used are given in column
1, and the % bacterial viability for each strain tested are given
in the subsequent columns (see also FIG. 4).
TABLE-US-00005 TABLE 5 S. S. S. S. S. agalactiae agalactiae
agalactiae agalactiae agalactiae Treatment # 30 # 44 # 70 # 88 #
106 Control 100.00 100.00 100.00 100.00 100.00 CIP 4 .mu.g/ml 74.20
63.40 74.10 71.60 72.50 CIP 8 .mu.g/ml 66.38 66.52 65.26 69.79
70.13 CIP 12 69.29 63.28 58.93 63.77 65.90 .mu.g/ml GSH 10 mM
103.78 86.99 89.17 87.95 102.63 GSH 15 mM 80.69 73.92 79.41 81.62
83.17 GSH 30 mM 35.30 29.16 38.30 25.00 29.20 2 part CT (1) 22.30
25.90 24.80 17.80 25.30 GSH 30 mM CIP 4 ug/ml Control = no
treatment CIP = Ciprofloxacin GHS = glutathione
Example 6: Multi Drug Resistant Acinetobacter baumannii
[0091] The treatments shown in column 1 of Table 6 were tested
against six clinical isolates of Multidrug resistant Acinetobacter
Baumannii, (MRAB). The treatments used are given in column 1, and
the % bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 5). As these MRAB strains were
resistant to ciprofloxacin, Amikacin was used instead.
TABLE-US-00006 TABLE 6 MRAB 1 MRAB 2 MRAB 3 MRAB 4 MRAB 5 MRAB 12
Strain ID 014754 014801 015069 015095 015103 016419 Source Urine
Catheter Skin Hospital Hospital Catheter Environ Environ Control
100.00 100.00 100.00 100.00 100.00 100.00 AMI 4 .mu.g/ml 35.91
35.99 66.60 66.36 65.23 49.66 AMI12 .mu.g/ml 24.47 14.11 48.60
42.91 26.85 22.70 AMI20 .mu.g/ml 14.67 8.36 41.70 33.92 24.60 19.80
GSH 10 mM 73.61 70.84 84.56 68.00 65.08 80.83 GSH 15 mM 55.58 41.23
63.18 57.70 51.49 49.05 GSH 30 mM 20.35 2.45 37.70 13.77 16.71
18.41 2 part CT (2) 6.90 1.53 4.69 6.85 10.96 4.30 GSH 30 mM AMI 4
.mu.g/ml Control = no treatment AMI = Amikacin GHS =
glutathione
Example 7: Klebsiella pneumoniae (Clinical Isolates)
[0092] The treatments shown in column 1 of table 7 were tested
against three clinical isolates of Klebsiella pneumoniae. The
treatments used are given in column 1, and the % bacterial
viability for each strain tested are given in the subsequent
columns (see also FIG. 6).
TABLE-US-00007 TABLE 7 Klebsiella pneumoniae isolate and source
374450 Catheter 377951 Left 385261 Neck Treatment urine hip wound
wound Control 100.00 100.00 100.00 CIP 10 .mu.g/ml 37.55 54.58
29.48 CIP 20 .mu.g/ml 22.13 40.39 21.00 CIP30 .mu.g/ml 21.29 25.39
21.97 GSH 10 mM 85.95 81.60 78.75 GSH 15 mM 72.81 70.81 62.46 GSH
30 mM 16.76 9.13 14.12 2 part CT 10.32 1.80 4.15 GSH 30 mM CIP 10
ug/ml Control = no treatment CIP = Ciprofloxacin GHS =
glutathione
Example 8 Enterobacter Species (Clinical Isolates)
[0093] The treatments shown in column 1 of Table 8 were tested
against three clinical isolates of Enterobacter species. The
treatments used are given in column 1, and the % bacterial
viability for each strain tested are given in the subsequent
columns (see also FIG. 7).
TABLE-US-00008 TABLE 8 Enterobacter species and source E. cloacae E
aerogenes E. cloacae 359315 Left 359475 Treatment 357768 Ear foot
wound Sternum Control 100.0 100.0 100.0 CIP 0.5 .mu.g/ml 39.2 64.0
14.6 CIP 1 .mu.g/ml 23.4 53.8 6.3 CIP2 .mu.g/ml 25.0 64.1 5.9 GSH
10 mM 62.8 84.7 79.0 GSH 15 mM 38.9 74.0 55.1 GSH 30 mM 0.0 19.5
14.6 2 part CT 0.0 8.5 2.3 GSH 30 mM CIP 0.5 ug/ml Control = no
treatment CIP = Ciprofloxacin GHS = glutathione
Example 9 Escherichia coli (Clinical Isolates)
[0094] The treatments shown in column 1 of Table 9 were tested
against three clinical isolates of Enterobacter species. The
treatments used are given in column 1, and the % bacterial
viability for each strain tested are given in the subsequent
columns (see also FIG. 8).
TABLE-US-00009 TABLE 9 Escherichia coli and source 362805 365714
366290 Drain Exit Wound fluid Control 100.0 100.0 100.0 CIP 0.5
.mu.g/ml 57.9 40.7 29.9 CIP 1 .mu.g/ml 48.1 36.4 29.4 CIP2 .mu.g/ml
48.7 31.3 30.8 GSH 10 mM 75.7 107.5 69.8 GSH 15 mM 71.8 93.4 68.7
GSH 30 mM 0.7 12.3 3.9 2 part CT 1.1 6.0 0.7 GSH 30 mM CIP 0.5
ug/ml Control = no treatment CIP = Ciprofloxacin GHS =
glutathione
Example 10: Streptococcus pyogenes (Clinical Isolates)
[0095] The treatments shown in column 1 of Table 10 were tested
against three clinical isolates of Enterobacter species. The
treatments used are given in column 1, and the % bacterial
viability for each strain tested are given in the subsequent
columns (see also FIG. 9).
TABLE-US-00010 TABLE 10 Streptococcus pyogenes and source 361194
Left 386596 Head 371982 Skin leg boil Wound Wound Control 100.0
100.0 100.0 CIP 4 .mu.g/ml 18.3 49.6 54.3 CIP 8 .mu.g/ml 10.1 46.8
56.5 CIP 12 .mu.g/ml 6.4 39.3 56.1 GSH 10 mM 55.6 67.7 99.8 GSH 15
mM 35.8 42.7 90.2 GSH 30 mM 4.3 22.3 15.4 2 part CT 0.1 11.8 4.8
GSH 30 mM CIP 4 ug/ml Control = no treatment CIP = Ciprofloxacin
GHS = glutathione
[0096] The following examples illustrate the efficacy of the triple
combination of a biologically acceptable thiol based antioxidant,
an antibiotic, along with an enzyme against biofilms formed by
organisms other than Pseudomonas (ie non-Pseudomonad organisms).
These represent the three part combination therapy (3 part CT).
Example 11: Methicillin Resistant Staphylococcus aureus (Clinical
Isolates)
[0097] The treatments shown in column 1 of Table 11 were tested
against three strains of methicillin-resistant Staphylococcus
aureus. The treatments used are given in column 1, and the %
bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 10).
TABLE-US-00011 TABLE 11 MRSA 27060 MRSA 30616 MRSA - 0799 Treatment
Left leg Chin vesicle DFU Control 100.00 100.00 100.00 CIP 10
.mu.g/ml 42.93 40.01 61.08 CIP 20 .mu.g/ml 25.74 31.38 54.21 CIP30
.mu.g/ml 19.53 24.56 51.60 GSH 10 mM 88.83 73.12 86.73 GSH 15 mM
65.24 58.82 75.03 GSH 30 mM 1.02 4.54 33.12 DNase I 40 U 101.74
108.30 86.56 2 part CT -0.30 0.61 21.83 GSH 30 mM CIP 10 ug/ml 3
part CT 0.00 1.70 17.11 GSH 30 mM CIP 10 ug/ml DNase I Control = no
treatment CIP = Ciprofloxacin GHS = glutathione
Example 12: Methicillin Sensitive Staphylococcus aureus (Clinical
Isolate)
[0098] The treatments shown in column 1 of Table 12 were tested
against three strains of methicillin-sensitive Staphylococcus
aureus. The treatments used are given in column 1, and the %
bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 11).
TABLE-US-00012 TABLE 12 MSSA - 34397 MSSA - 34654 MSSA - 0800
Treatment Left elbow Right toe DFU Control 100.00 100.00 100.00 CIP
10 .mu.g/ml 49.93 37.08 41.69 CIP 20 .mu.g/ml 36.78 32.05 32.03 CIP
30 .mu.g/ml 29.81 32.44 21.58 GSH 10 mM 88.67 86.11 82.44 GSH 15 mM
61.34 70.52 61.70 GSH 30 mM 1.06 15.33 20.09 DNase I 40 U 116.55
125.77 83.01 2 part CT 0.77 5.85 9.68 GSH 30 mM CIP10 ug/ml 3 part
CT 0.96 3.55 8.24 GSH 30 mM CIP 10 ug/ml DNase I Control = no
treatment CIP = Ciprofloxacin GHS = glutathione
Example 13: Staphylococcus agalactiae (Ex Cow Mastitis)
[0099] The treatments shown in column 1 of Table 13 were tested
against four strains of Staphylococcus agalactiae, (veterinary
strains ex Cow mastitis). The treatments used are given in column
1, and the % bacterial viability for each strain tested are given
in the subsequent columns (see also FIG. 12).
TABLE-US-00013 TABLE 13 S. S. S. S. S. agalactiae agalactiae
agalactiae agalactiae agalactiae Treatment # 30 # 44 # 70 # 88 #106
Control 100.00 100.00 100.00 100.00 100.00 CIP 4 .mu.g/ml 74.20
63.40 74.10 71.60 72.50 CIP 8 .mu.g/ml 66.38 66.52 65.26 69.79
70.13 CIP 12 69.29 63.28 58.93 63.77 65.90 .mu.g/ml GSH 10 mM
103.78 86.99 89.17 87.95 102.63 GSH 15 mM 80.69 73.92 79.41 81.62
83.17 GSH 30 mM 35.30 29.16 38.30 25.00 29.20 DNase I 71.50 58.50
70.60 69.60 69.30 40 U 2 part CT (1) 22.30 25.90 24.80 17.80 25.30
GSH 30 mM CIP 4 ug/ml 2 part CT (2) 36.90 28.90 34.20 21.00 28.50
GSH 30 mM DNase 1 40 U 2 part CT (3) 62.87 57.90 62.60 66.60 60.30
CIP 30 mM DNase 1 40 U 3 part CT (1) GSH 10 mM 53.10 47.90 53.10
35.00 44.80 Cip 4 ug/ml DNase 1 40 U 3 part CT (2) GSH 30 mM 20.60
17.60 27.50 16.30 24.00 CIP 10 ug/ml DNase 1 40 U Control = no
treatment CIP = Ciprofloxacin GHS = glutathione
Example 14: Multi Drug Resistant Acinetobacter baumannii
[0100] The treatments shown in column 1 of Table 14 were tested
against six clinical isolates of Multidrug resistant Acinetobacter
Baumannii, (MRAB). The treatments used are given in column 1, and
the % bacterial viability for each strain tested are given in the
subsequent columns (see also FIG. 13). As these MRAB strains were
resistant to ciprofloxacin, Amikacin was used instead.
TABLE-US-00014 TABLE 14 MRAB 1 MRAB 2 MRAB 3 MRAB 4 MRAB 5 MRAB 12
Strain ID 014754 014801 015069 015095 015103 016419 Source Urine
Catheter Skin Hospital Hospital Catheter Environ Environ Control
100.00 100.00 100.00 100.00 100.00 100.00 AMI 4 .mu.g/ml 35.91
35.99 66.60 66.36 65.23 49.66 AMI12 .mu.g/ml 24.47 14.11 48.60
42.91 26.85 22.70 AMI20 .mu.g/ml 14.67 8.36 41.70 33.92 24.60 19.80
GSH 10 mM 73.61 70.84 84.56 68.00 65.08 80.83 GSH 15 mM 55.58 41.23
63.18 57.70 51.49 49.05 GSH 30 mM 20.35 2.45 37.70 13.77 16.71
18.41 DNaseI 46.43 43.91 71.60 83.09 89.54 77.12 2 part CT (2) 6.90
1.53 4.69 6.85 10.96 4.30 GSH 30 mM AMI 4 .mu.g/ml 2 part CT (2)
18.61 27.60 29.50 62.75 46.11 39.81 DNase1 40 U AMI 4 .mu.g/ml 2
part CT (3) 9.96 2.51 36.40 5.02 9.30 15.12 GSH 30 mM DNase1 40 U 3
part CT (1) 15.67 12.06 24.20 39.98 49.98 22.24 GSH 10 mM AMI 4
ug/ml DNase1 40 U 3 part CT (2) 3.80 0.17 0.11 4.13 5.20 2.28 GSH
30 mM AMI 10 ug/ml DNase1 40 U Control = no treatment AMI =
Amikacin GHS = glutathione
* * * * *