U.S. patent application number 15/300990 was filed with the patent office on 2017-01-19 for synthetic disugar hydrocarbons as natural analogs to control microbial behaviors.
This patent application is currently assigned to Syracuse University. The applicant listed for this patent is The Research Foundation for the State Universtiy of New York, Syracuse University. Invention is credited to Yan-Yeung Luk, Guirong Wang.
Application Number | 20170014437 15/300990 |
Document ID | / |
Family ID | 54241315 |
Filed Date | 2017-01-19 |
United States Patent
Application |
20170014437 |
Kind Code |
A1 |
Luk; Yan-Yeung ; et
al. |
January 19, 2017 |
SYNTHETIC DISUGAR HYDROCARBONS AS NATURAL ANALOGS TO CONTROL
MICROBIAL BEHAVIORS
Abstract
Synthetic disaccharide hydrocarbons (DSHs) that reactive
bacterials swarming motility and inhibit bacterial adhesion and
biofilm formation. A library of DSHs were tested in several
experiment for the impact on various Pseudomonas aeruginosa
populations and compared against existing compounds to determine
efficacy and utility. Certain DSHs were also to determine the
ability to clear bacteria in a mouse pneumonia model.
Inventors: |
Luk; Yan-Yeung; (Jamesville,
NY) ; Wang; Guirong; (Syracuse, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Syracuse University
The Research Foundation for the State Universtiy of New
York |
Syracuse
Syracuse |
NY
NY |
US
US |
|
|
Assignee: |
Syracuse University
Syracuse
NY
The Research Foundation for the State University o f New
York
Syracuse
NY
|
Family ID: |
54241315 |
Appl. No.: |
15/300990 |
Filed: |
April 3, 2015 |
PCT Filed: |
April 3, 2015 |
PCT NO: |
PCT/US15/24234 |
371 Date: |
September 30, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61974812 |
Apr 3, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
Y02A 50/473 20180101; A61K 9/0078 20130101; A61P 31/04 20180101;
Y02A 50/471 20180101; A61K 31/724 20130101; A61K 31/7028 20130101;
A61K 45/06 20130101 |
International
Class: |
A61K 31/7028 20060101
A61K031/7028; A61K 45/06 20060101 A61K045/06; A61K 31/724 20060101
A61K031/724; A61K 9/00 20060101 A61K009/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0001] This invention was made with government support under Grant
No. IIP-1242505 awarded by the National Science Foundation, and
Grant No. CMMI-0845686 awarded by the National Science Foundation.
The government has certain rights in the invention.
Claims
1. A compound for treating bacteria, comprising a disugar or
disaccharide hydrocarbon selected from the group consisting of
benzyl decyl .beta.-maltoside (BDe.beta.M) and benzyl dodecyl
.beta.-maltoside (BD.beta.M), 4-tertiary butyl benzyl decyl
.beta.-maltoside (4-tBuBDe.beta.M) and 4-tertiary butyl benzyl
dodecyl .beta.-maltoside (4-tBuBD.beta.M), 3,5-dimethyl benzyl
dodecyl .beta.-maltoside (3,5-DMBD.beta.M), 4-methyl benzyl dodecyl
.beta.-maltoside (4-MBD.beta.M), benzophenonyl decyl
.beta.-maltoside (BPDe.beta.M), adamantane dodecyl .beta.-maltoside
(AD.beta.M) and 12-hydroxy decyl .beta.-maltose (12-HODe.beta.M),
dodecyl-.beta.-cellobioside (D.beta.C), dodecyl-.beta.-lactoside
(D.beta.L), dodecyl-.alpha.-rhamnoside (D.alpha.R),
.beta.-cyclodextrin (.beta.CD), dodecyl-.beta.CD-squarate
(D.beta.CDS), decyl-.beta.-cellobioside (De.beta.C),
undecyl-.beta.-cellobioside (U.beta.C),
tridecyl-.beta.-cellobioside (T.beta.C), dodecyl-.beta.-maltoside
(D.beta.M), saturated farnesyl-.beta.-maltoside (SF.beta.M),
dodecyl-.beta.-cellobioside (D.beta.C), decyl-.beta.-cellobioside
(De.beta.C), (U.beta.C), tridecyl-.beta.-cellobioside (T.beta.C),
dodecyl-.alpha.-cellobioside (D.alpha.C),
undecylenyl-.beta.-cellobioside (UD.beta.C),
farnesyl-.beta.-cellobioside (F.beta.C), saturated
farnesyl-.beta.-cellobioside (SF.beta.C), saturated
farnesyl-.alpha.-cellobioside (SF.alpha.C),
2-octyl-dodecyl-.beta.-cellobioside (2-OD.beta.C),
2-octyl-dodecyl-.alpha.-cellobioside (2-OD.alpha.C),
dodecyl-.beta.-lactoside (D.beta.L), saturated
farnesyl-.beta.-lactoside (SF.beta.L).
3. The compound of claim 1, wherein the structure of the compound
is selected from the group consisting of: ##STR00001##
##STR00002##
3. The compound of claim 1, wherein the structure of the compound
is selected from the group consisting of: ##STR00003##
##STR00004##
4. A method of treating a bacterial infection, comprising the step
of administering an effective amount of dodecyl-.beta.-maltoside
(D.beta.M).
5. The method of claim 4, wherein the bacterial infection is a
complication of cystic fibrosis.
6. The method of claim 4, wherein the step of administering an
effective amount of dodecyl-.beta.-maltoside (D.beta.M) comprises
the step of administering the effective amount of
dodecyl-.beta.-maltoside (D.beta.M) via a nebulizer.
7. The method of claim 6, further comprising the step of
administering an effective amount of an antibiotic.
8. The method of claim 7, wherein the step of administering an
effective amount of an antibiotic comprises the step of
administering the effective amount of an antibiotic via a
nebulizer.
9. The method of claim 8, wherein the steps of administering an
effective amount of dodecyl-.beta.-maltoside (D.beta.M) and
administering an effective amount of an antibiotic are performed
sequentially.
10. The method of claim 9, wherein the steps of administering an
effective amount of dodecyl-.beta.-maltoside (D.beta.M) and
administering an effective amount of an antibiotic are performed at
the same time.
11. A method of addressing a bacterial population, comprising the
step of exposing the bacterial population to an effective amount of
the compound of claim 1.
12. The method of claim 11, wherein the bacterial population has
formed a biofilm.
13. The method of claim 11, wherein the bacterial population has
not formed a biofilm.
14. The method of any one of claim 11, 12 or 13, wherein the
compound comprises dodecyl-.beta.-maltoside.
15. The method of any one of claim 11, 12 or 13, wherein the
bacterial population comprises an infection.
16. The method of claim 15, wherein the infection is associated
with cystic fibrosis.
17. The method of claim 11, further comprising the step of the step
of exposing the bacterial population to an effective amount of an
antibiotic.
18. The method of claim 17, wherein either or both of the step of
administering an effective amount of the compound of claim 1 and
the step of administering an effective amount of an antibiotic is
performed using a nebulizer.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the control of bacterial
behavior and, more particularly, to the use of synthetic
disaccharide hydrocarbons on the swarming motility of bacteria.
[0004] 2. Description of the Related Art
[0005] Microbes secrete chemicals for a wide range of functions.
These chemicals include agents that signal population density to
each other and induce a collective behavior, molecules that build
structured film in which the microbes reside, virulence factors
that are toxic to a host cell or other species, agents that enables
the swarming motility of a collection of members of the microbes.
Controlling these behaviors without killing the microbes has the
potential of developing drugs against infectious diseases without
invoking drug resistances, and enables both chemical and
bacteria-based waste cleanup for environmental bioremediation
[0006] The emerging increases in antibiotic resistant bacteria call
for fundamentally new approaches in treating wide ranges of
infectious and new diseases. The discovery of quorum sensing in
bacteria leading to biofilm formation and recognizing the
detrimental effects that biofilm formation poses has made the
exploration of non-microbicidal anti-biofilm approaches an
important area of research. To this end, important work has been
done in inhibiting the quorum sensing among the bacteria to reduce
the biofilm formation. Another approach focuses on the hypothesis
that bacterial adhesion is a major step causing various diseases,
thus inhibiting the adhesion of microbes or developing vaccines
using the microbial adhesins may be a potential therapeutics for
infectious diseases. However, this anti-adhesion strategy has not
yet reached an ultimate success of drug development, probably
because multiple adhesins are employed by the microbe for adhesion
and attachment of polymer secreted by the microbes can also
facilitate the hosting of microbes that may also lead to the
formation of biofilms.
[0007] Pseudomonas aeruginosa is an opportunistic pathogen that
causes severe infections under a wide range of immunocompromised
situations. Many bacterial activities are gene-regulated. Swarming
motility of P. aeruginosa on a soft agar gel requires the
production of a natural disaccharide derivative, rhamnolipid, that
is also gene controlled and is produced by a few other bacterial
species. Rhamnolipids control at least three different behaviors of
P. aeruginosa. First, it is necessary for making structured
biofilms with channels and pores. Second, it facilitates the
partial dispersion of mature biofilms when overproduced. Third, its
production is necessary for enabling the swarming motility of P.
aeruginosa. Deleting the gene rhlA that controls the synthesis of
rhamnolipids results in a non-swarming mutant. With all these
biological activities, the protein receptor(s), as well as its
existence, for rhamnolipid has not yet been identified.
Furthermore, the natural ligand for mediating the adhesion of P.
aeruginosa on epithelial cells of lungs in cystic fibrosis patients
is believed to be a disaccharide derivative,
GalNAc.beta.(1.fwdarw.4)Gal.beta. moiety, on the asialo-GM1
glycolipid. Synthetic molecules that tether different methylated
GalNAc.beta.(1.fwdarw.4)Gal.beta. on different aliphatic chains
have shown to be potent anti-adhesion agents for P. aeruginosa, and
that the receptor appears to be a pili protein. Structural
variation and mimics of the disaccharide glucosamine (different
stereochemistry and the presence of NAc group) has not been
extensively evaluated for inhibiting the adhesion of P.
aeruginosa.
[0008] One of the most prevalent bacterial activities for diseases
is biofilm formation, which is also gene regulated, resulting in a
dynamic surface-based multicellular organism. The film-hosting
microbes can exhibit 1000 fold higher resistance to antibiotics
than the planktonic microbes. Complete eradication of biofilms has
been a daunting challenge as these films can exhibit resistance to
many chemical agents. Interestingly, while the initial step of
biofilm formation are believed to involve microbial adhesion on
host surfaces (or on adsorbed polymer secreted by the microbes),
relatively few studies have been explored using anti-adhesion
agents to inhibit or disperse biofilm formation.
[0009] Together, the swarming activities and the ligand-mediated
adhesion suggest that disaccharide molecule are potential ligands
for one or more receptors that control the signaling processes
including swarming motility, adhesion, and biofilm formation and
dispersion.
[0010] These molecules may be useful for treating infectious
diseases, such as those associated with cystic fibrosis. Cystic
fibrosis (CF) is the most common autosomal recessive condition
present in the Caucasian population, accounting for one in 2500
births. It is a multi-system, progressive disorder characterized by
abnormal ion transport leading to viscid secretions affecting the
pancreas and the respiratory, gastrointestinal and reproductive
systems. The major cause of morbidity and mortality is the
respiratory component of the condition. Dysfunction of the cystic
fibrosis transmembrane conductance regulator in the CF airway
epithelium causes abnormal ion transport which in turn leads to
depleted airway surface liquid volume, mucus dehydration, decreased
mucus transport and mucus plugging of the airways providing an
environment for bacterial infection. This process sets up a cycle
of infection and inflammation leading to airway damage and
progressive loss of respiratory function. In 1938, 70% of babies
with CF died within the first year of life. More recent data
suggests that median survival has increased to between 37 and 41
years of age as a result of improvements in conventional therapy.
Nebulized treatment has been a key element of these therapies, but
at the cost of increased burden of care for people with CF and
their families.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention comprises a class of disugar
hydrocarbons that simulate and overpower the function of a class of
natural molecules, rhamnolipids, secreted by Pseudomonas
aeruginosa. This class of disugar hydrocarbons exhibits control
over multiple microbial behaviors. They promote the swarming
motility of Pseudomonas aeruginosa at low concentration, but
inhibit the swarming motility at high concentrations. This
capability of dual-functions dominates the effect from the
naturally existing rhamnolipids, and is vastly useful for
controlling infectious diseases. This class of molecules also
exhibits another important function of controlling the biofilm
formed by bacteria. They inhibit the formation of a biofilm by a
wide range of microbes (E. coli, Pseudomonas aeruginosa, and
Candida albicans) with a higher potency than that by rhamnolipids.
Most importantly, disugar hydrocarbons disperse already formed
biofilms whereas natural rhamnolipids extracted from bacteria do
not. Because microbial biofilms are the sources of 80% infectious
diseases, and because preformed biofilms are particularly difficult
to remove, this class of disugar hydrocarbons has applications in
for drug development and formulation.
[0012] The present invention also comprises the non-microbicidal
control of bacterial behaviors. Various synthetic disaccharide
hydrocarbons were used to explore reactivating the swarming
motility of a nonswarming mutant of Pseudomonas aeruginosa (rhlA),
and the ability of the compounds to inhibit bacterial adhesion and
biofilm formation by the wild type P. aeruginosa. Several of the
disaccharide-hydrocarbons reactivated the swarming of rhlA mutant
to its full capacity as compared to the wild type P. aeruginosa;
and the extent of reactivation was highly sensitive to the
structural details of the disaccharide-hydrocarbons. While these
disaccharide-hydrocarbons were not microbicidal at relatively high
concentrations (170 .mu.M), disaccharides with bulky hydrocarbon
groups inhibited bacterial adhesion, exhibited biofilm inhibition
and dispersion with an IC.sub.50 of 22.5 .mu.M and a DC.sub.50 of
31 .mu.M, respectively. Because the swarming motility of rhlA
mutant is abolished due to its lack of production of the natural
ligand rhamnolipid, the synthetic disaccharide-hydrocarbons may
share a common receptor as rhamnolipid. In addition, as bacterial
adhesions can also be facilitated by ligand-receptor interactions,
the results suggest a new approach of controlling bacterial
adhesion, biofilm formation and swarming motilities by a common set
of disaccharide-based molecules that targets one or more protein
receptors.
[0013] The present invention also comprises a chemical library of
disaccharide hydrocarbons (DSHs) that were made by systematically
changing the glycone as well as a glycone part of the DSH and then
investigating the effect that these structural changes have on
swarming motilities of PA and its non-swarming mutant strain. The
compounds were also tested to determine the anti-biofilm activities
of these non-microbicidal agents.
[0014] The present invention also involves the effects of Dodecyl
Maltoside (DM) on the bacterial clearance and lung inflammation in
mouse P. aeruginosa pneumonia. DM treatment significantly decreased
CFU number in the lung compared to the control and improved the
lung inflammation.
[0015] The present invention additionally includes a series of
synthetic disaccharide hydrocarbons (DSHs) that activates the
swarming motility at low concentration but inhibit swarming at high
concentration over a wide range of saccharide stereochemistries and
aliphatic structures. DSHs with a bulky aliphatic chain (3, 7,
11-trimethyl-dodecane) exhibited a dominating effect over natural
rhamnolipids extract on both activating and inhibiting the swarming
motility of Pseudomonas aeruginosa. Active DSHs inhibited biofilm
formation with a plateau of maximal inhibition as the concentration
was increased. The DSHs with a cellobiose head and a saturated
farnesol tail, saturated farnesol-.beta.-cellobioside, exhibited
higher biofilm inhibition with an IC.sub.50 of .about.9.9 .mu.M
than the maximal biofilm inhibition of rhamnolipids extract. More
importantly, active DSHs dispersed 1-day old biofilm whereas,
rhamnolipids extract did not exhibit any notable dispersion.
Together these results demonstrate that the active DSHs dominate
natural rhamnolipids at collective behaviors of swarming and
biofilm formation in Pseudomonas aeruginosa.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0016] The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
[0017] FIG. 1 is a series of chemical diagrams of maltose derived
hydrocarbons;
[0018] FIG. 2 is a series of images of nonswarming P. aeruginosa
mutant rhlA that was inoculated on M8 swarm agar (0.5% agar) plates
and homogeneously mixed with maltose derivatives to a resultant
concentration of 85 .mu.M with pictures were taken 24 h after the
inoculation of bacteria on the plates;
[0019] FIG. 3 is a graph of the inhibition of biofilm and adhesion
by selected maltose derivatives, generic surfactants (SDS,
C.sub.12EG.sub.4OH) at 110 .mu.M (anti-biofilm) and 85 .mu.M
(anti-adhesion) and known anti-biofilm agent BF8 at 110 .mu.M
(anti-biofilm) and 100 .mu.M (anti-adhesion) measured using CV dye
based and fluorescence assays respectively, where the error bar is
standard error of the mean from 6 replicates;
[0020] FIG. 4 is a graph of the dose response curve and biofilm
inhibition IC.sub.50 values for compounds 11: BPDe.beta.M and 5:
BD.beta.M using CV dye based biofilm inhibition assay, where the
error bar is standard error of the mean from 6 replicates;
[0021] FIG. 5 is a graph of the dispersion of PAO1 biofilm with
different maltose derivatives at 110 .mu.M as quantified by CV dye
based assay, where the error bar is standard error of the mean from
6 replicates;
[0022] FIG. 6 is a graph of the dose response curve and biofilm
dispersion DC.sub.50 values for compounds 11: BPDe.beta.M and 5:
BD.beta.M using crystal violet dye based biofilm inhibition assay,
where the error bar is standard error of the mean from 6
replicates;
[0023] FIG. 7 is a series of chemical diagrams of disaccharide
hydrocarbons and .beta.-cyclodextrin derivatized hydrocarbon;
[0024] FIG. 8 is a schematic showing the general synthetic scheme
for disaccharide hydrocarbons (DSHs); i) AcBr/AcOH, rt or
60.degree. C., .about.1 h; ii) ROH, FeCl.sub.3 or Hg(CN).sub.2,
MeCN, rt, .about.1 h; iii) ROH, FeCl.sub.3, MeNO.sub.2, rt,
.about.1 h; iv) MeONa/MeOH, .about.12 h, H.sup.+ amberlite resin,
Neutralize, (pH.about.6.5);
[0025] FIG. 9 is a series of images of swarm (.about.0.5% agar)
plates inoculated with PAO1 after .about.24 hours with (at 110
.mu.M) and without agents (DSHs);
[0026] FIG. 10 is a series of images of swarm (.about.0.5% agar)
plates with increasing concentration of 12: SF.beta.C. Images taken
24 h after inoculation with PAO1;
[0027] FIG. 11 is a series of images of swarm (.about.0.5% agar)
plates inoculated with non-swarming PA mutant, rhlA after .about.24
hours with (at 110 .mu.M) and without agents (DSHs);
[0028] FIG. 12 is a series of images of swarm (.about.0.5% agar)
plates containing increasing concentration of 4: D.beta.C at
different times after inoculation with non-swarming PA mutant
rhlA;
[0029] FIG. 13 is a graph of rhlA swarm area (after 24 h) on soft
agar (.about.0.5%) versus concentration of 4: D.beta.C;
[0030] FIG. 14 is a series of images of swarm (.about.0.5% agar)
plates with increasing concentration of 12: SF.beta.C with images
taken 24 h after inoculation with non-swarming PA mutant rhlA;
[0031] FIG. 15 is a graph of rhlA swarm area (after 24 h) on soft
agar (.about.0.5%) versus concentration of 12: SF.beta.C
[0032] FIG. 16 is a series of images of swarm (.about.0.5% agar)
plates containing constant concentration of 4: D.beta.C but
increasing concentration of 12: SF.beta.C after 12 h of inoculation
with non-swarming PA mutant rhlA;
[0033] FIG. 17 is a graph of the percent surface attached PAO1
biofilm remaining within the wells of microtiter plate after
biofilm was allowed to develop for 24 hours with or without various
agents. Bacterial culture media: Luria-Bertani (LB). Percent
PAO1/EGFP adhered on polystyrene surface. Resultant concentration
of each agent within wells .about.160 .mu.M;
[0034] FIG. 18 is a graph of the dose-response curve along with a
series of images of microtiter plates after CV-dye based inhibition
assay;
[0035] FIG. 19 is a graph of percent surface attached PAO1 biofilm
remaining within the wells of microtiter plate after biofilm was
allowed to develop for 24 hours without any agents and then treated
with various agents for another 24 hours. Resultant concentration
of each agent within wells .about.160 .mu.M;
[0036] FIG. 20 is a graph of a dose-response curve and images of
microtiter plates after CV-dye based dispersion assay;
[0037] FIG. 21 is a graph of surface attached PAO1 biofilm
remaining within the wells of the microtiter plate after biofilm
was allowed to develop for 24 hours with or without the agents
along with images showing the concentration of agents in the wells;
13: SF.alpha.C=85 .mu.M; PAO.sub.(ox)=85 .mu.M;
PAO.sub.(scrambled)=85 .mu.M;
[0038] FIG. 22 is a graph of the effect of Dodecyl Maltoside (DM)
on the bacterial clearance in mouse P. aeruginosa pneumonia.
[0039] FIG. 23 is a series of micrographs of the histological
analysis of representative hematoxylin and eosin-stained lung
tissue sections from P. aeruginosa pneumonia model.
[0040] FIGS. 24A and 24B are a series of schematics of structures
of synthetic saccharide-based hydrocarbons that include maltose,
cellobiose, lactose, rhamnose, and .beta.-cyclodextrin-based
stereochemistries; and hydrocarbons derived from farnesol
molecules, and "saturated" farnesols, and the structure of
di-rhamnolipid and mono-rhamnolipid is also shown;
[0041] FIG. 25 is a series of images of swarming motility of wild
type PAO1 and rhlA mutant on soft agar plates (.about.0.5% agar, M8
media) supplemented with .about.85 .mu.M DSHs. Images were taken 24
h after inoculation with bacteria. .sup.aConcentration of De.beta.C
(2) was 160 .mu.M; .sup.bRhamnolipids concentration .about.30 .mu.M
for PAO1 and .about.10 .mu.M for rhlA.
[0042] FIG. 26 is series images of swarm (.about.0.5% agar) plates
containing increasing concentrations of DSH inoculated with rhlA
mutant (left) and PAO1 (right). The concentrations in .mu.M are
indicated above the images, and the identity of the DSHs is shown
to the left. Molecules that exhibited dominance over rhamnolipids
at inhibiting or activating swarming motilities are
highlighted;
[0043] FIG. 27 is a plot of the swarm area (after 24 h) of swarming
patterns of rhlA mutant versus concentration of rhamnolipids,
D.beta.C (4), T.beta.C (5), SF.beta.M (14), D.beta.G (8) and
SF.beta.C (12) on soft agar gel (.about.0.5% agar). Insert,
expanded plot region; y-axis (0-16 cm.sup.2) and x-axis (0-25
.mu.M);
[0044] FIG. 28 is a series of images of nonswarming P. aeruginosa
mutant, rhlA after 24 h of inoculation on soft agar (.about.0.5%
agar) plates containing 40 .mu.M of D.beta.C (4), and various
concentration of SF.beta.C (12);
[0045] FIG. 29 is a graph of (A) Percent surface attached PAO1
biofilm remaining in the wells of microtiter plate after biofilm
was allowed to develop for 24 hours with or without 160 .mu.M DSHs
(filled bars). Percent PAO1/EGFP adhered on polystyrene surface
(unfilled bars). (B) Percent surface attached PAO1 biofilm
remaining within the wells of microtiter plate after 24 h old
biofilm was treated with 160 .mu.M DSHs for another 24 hours
(dispersion of 1-day old biofilm). Bacterial culture media:
Luria-Bertani (LB);
[0046] FIG. 30 is a graph of dose-response curves along with images
of microtiter plates after CV-dye based inhibition assay;
[0047] FIG. 31 is a graph of dose-response curves along with images
of microtiter plates after CV-dye based dispersion assay;
[0048] FIG. 32 is a graph of crystal violet (CV) stained PAO1
biofilm within the wells of the microtiter plate after biofilm was
allowed to develop for 24 hours in LB-media without any agents and
in LB-media that is supplemented with SF.alpha.C (13) .about.85
.mu.M; SF.alpha.C (13) .about.85 .mu.M+Pili peptide .about.85
.mu.M; SF.alpha.C (13) .about.85 .mu.M+Scrambled pili peptide
.about.85 .mu.M; Pili peptide .about.85 .mu.M; Scrambled pili
peptide .about.85 .mu.M;
[0049] FIG. 33 is a schematic of the synthesis of disugar based
polyol-derivatized hydrocarbons (PDHs); i) AcBr/AcOH, rt or
60.degree. C., .about.1 h; ii) ROH, FeCl.sub.3 or Hg(CN).sub.2,
MeCN, rt, .about.1 h; iii) ROH, FeCl.sub.3, MeNO.sub.2, rt,
.about.1 h; iv) MeONa/MeOH, .about.12 h, H.sup.+ amberlite resin,
Neutralize, (pH 6.5);
[0050] FIG. 34 is a schematic of a general structure of certain
compounds according to the present invention;
[0051] FIGS. 35A and 35B are schematics of the specific structure
of certain compounds according to the present invention;
[0052] FIG. 36 is a series of images of tests performed on certain
compounds according to the present invention;
[0053] FIG. 37 is a series of images of tests performed on certain
compounds according to the present invention;
[0054] FIG. 38 is a series of images of tests performed on certain
compounds according to the present invention;
[0055] FIG. 39 is a series of images of tests performed on certain
compounds according to the present invention;
[0056] FIG. 40 is a series of images of tests performed on certain
compounds according to the present invention;
[0057] FIG. 41 is a series of images of tests performed on certain
compounds according to the present invention;
[0058] FIG. 42 is a series of images of tests performed on certain
compounds according to the present invention;
[0059] FIG. 43 is a series of images of tests performed on certain
compounds according to the present invention;
[0060] FIG. 44 is a chart of the results of tests performed on
certain compounds according to the present invention; and
[0061] FIG. 45 is a graph of the results of tests performed on
certain compounds according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0062] Referring to the Figures, the present invention comprises a
class of disugar hydrocarbons that exhibits control over multiple
microbial behaviors. As seen in the following examples, the
compounds promote the swarming motility of Pseudomonas aeruginosa
at low concentration, but inhibit the swarming motility at high
concentrations. This capability of dual-functions dominates the
effect from the naturally existing rhamnolipids, and is vastly
useful for controlling infectious diseases. The present class of
molecules according to the present invention also exhibits control
over biofilm formation by bacteria and inhibit the formation of a
biofilm in a wide range of microbes (E. coli, Pseudomonas
aeruginosa, and Candida albicans) with a higher potency than that
of rhamnolipids.
Example 1
[0063] Swarming motility of P. aeruginosa is controlled by multiple
genes by screening studies, and, in a laboratory setting, requires
soft gels and presumably low surface tensions. For P. aeruginosa,
mutant rhlA that does not produce rhamnolipid results in
nonswarming bacteria. Externally added rhamnolipid reactivates the
swarming motility of rhlA mutant. Rhamnolipid is a biosurfactant
consisting of a disugar hydrophilic head group and two aliphatic
chains.
[0064] To evaluate the importance of the disugar head group and the
surface activities separately, the effect on different types of
generic surfactants was screened including: anionic surfactant
sodium dodecyl sulfate (SDS), cationic surfactant dodecyl trimethyl
ammonium chloride (DTAC) and nonionic surfactant tetra (ethylene
glycol) monododecyl ether (C12EG4OH), and a series of disaccharide
hydrocarbons (maltose derivatives), as seen in FIG. 1, on the
swarming motility of rhlA mutant. The disaccharide hydrocarbons
include a maltose stereochemistry (Glc.alpha.(1.fwdarw.4)Glc.beta.)
bearing different hydrocarbon tails.
[0065] To investigate the effect of aliphatic chain length, maltose
derivatives having 10 (decyl .beta.-matoside; De.beta.M) and 11
(undecyl .beta.-maltoside; U.beta.M) carbons in the aliphatic
chains were studied. To investigate the effect of terminal
hydrocarbon bulkiness, the following were synthesized: benzyl decyl
.beta.-maltoside (BDe.beta.M) and benzyl dodecyl .beta.-maltoside
(BD.beta.M), 4-tertiary butyl benzyl decyl .beta.-maltoside
(4-tBuBDe.beta.M) and 4-tertiary butyl benzyl dodecyl
.beta.-maltoside (4-tBuBD.beta.M), 3,5-dimethyl benzyl dodecyl
.beta.-maltoside (3,5-DMBD.beta.M) and 4-methyl benzyl dodecyl
.beta.-maltoside (4-MBD.beta.M), and benzophenonyl decyl
.beta.-maltoside (BPDe.beta.M). To study if nonaromatic bulky
substituents can also be effective, adamantane dodecyl
.beta.-maltoside (AD.beta.M) was synthesized. To study to what
degree a polar head group is needed or tolerated, 12-hydroxy decyl
.beta.-maltose (12-HODe.beta.M) was synthesized.
[0066] Reactivation of swarming motility of the nonswarming mutant
by specific maltose derivatives All three generic surfactants, SDS,
DTAC, C12EG4OH, did not promote or reactivate the swarming motility
of rhlA mutant (see the Supporting Information). Nine of the twelve
maltose derivatives reactivated the swarming motility of rhlA
mutant to a great extent, and with different degrees and shapes of
tendril formation, as seen in FIG. 2. The swarming motility of P.
aeruginosa is unique that it can form a pattern of tendrils that
are not seen in the swarming behavior of other bacteria. In a
laboratory setup, when the bacteria are inoculated at the center of
the soft agar plate (0.5% agar), tendrils gives a floral pattern
and occasionally further resembles a fractal pattern. For the
effect of the twelve maltose derivatives on the swarming motility
of nonswarming rhl mutants, the molecules were categorized into
three groups, swarming-activating with and without well-defined
tendril, and swarming nonactivating, as seen in FIG. 2.
[0067] On the soft agar plate (0.5% agar) with 85 .mu.M of maltose
derivatives, BPDe.beta.M, BD.beta.M, 4-tBuBDe.beta.M, U.beta.M,
BDe.beta.M caused rhlA mutant to exhibit swarming motility with
without well-defined tendrils but having small protrusions at the
periphery of the swarming circle. Among this group, all but
BDe.beta.M exhibited a swarming ring size that was comparable to
that of the swarming ring by wild-type PAO1. The maltose
derivatives D.beta.M, 4-MBD.beta.M, 3,5-DMBD.beta.M, AD.beta.M
caused long, straight and well-defined tendrils and with an overall
swarming circle that is similar in size as that by the wild-type
PAO1. Among this group of maltose derivative, AD.beta.M caused
narrow tendrils with frequent turns give a pattern similar to the
formation of fractals. The last group of maltose derivatives,
4-tBuBD.beta.M, De.beta.M, HODe.beta.M, did not exhibit any
significant activation of swarming of rhlA mutant
[0068] The mechanism of tendril formation is a challenging topic,
and has been studied by several groups. One proposal involves a
mechanism by which the dirhamnolipid is an attractant, whereas
mono-rhamnolipid and 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA)
may function as wetting agents and repellants, respectively, during
swarming of P. aeruginosa. The results and finding here are
consistent with the existence of a protein receptor for
rhamnolipid. Furthermore, as externally added rhamnolipid in the
soft gel also reactivates the swarming motilities of the rhlA
mutant, the nine maltose derivatives that also reactivated the
swarming motility of rhlA mutant may be ligands that share a common
protein receptor with rhamnolipid.
[0069] Rhamnolipid is necessary for P. aeruginosa to form channeled
biofilm at early stages, and interestingly, also for dispersion of
mature biofilm when over produced. These findings prompted an
examination of the effect of the disaccharide derivatives on
biofilm formation and dispersion, as well as an examination to see
if there is a possible correlation between activation of swarming
motility and biofilm control (dispersion and inhibition). Prior
studies showed that pilus is likely responsible for adhesion of P.
aeruginosa on biotic as well as abiotic surfaces such as
polystyrene. Using solid phase binding assays, it has been showed
that disaccharide, GalNAc.beta.(1.fwdarw.4)Gal.beta., hydrocarbons
bind to pili protein of P. aeruginosa. These results suggest that
GalNAc.beta.(1.fwdarw.4)Gal.beta. hydrocarbons are potent
inhibitors for the adhesion of P. aeruginosa. Although the maltose
derivatives have a different stereochemistry and structure than
GalNAc.beta.(1.fwdarw.4)Gal.beta., the anti-adhesion activity of
the maltose derivatives on polystyrene was examined because the
early stage of biofilm formation involves bacterial adhesion.
[0070] A crystal violet (CV) assay was used to measure the amount
of PAO1 biofilm formed after 24 h of bacterial inoculation. The
fluorescence of green fluorescent proteins (GFP) expressed by
PAO1-GFP was measured to directly quantify bacterial adhesion on
polystyrene black 96 well plates with and without maltose
derivatives after 2 h of bacterial inoculation. To evaluate the
potential of maltose derivatives as both anti-biofilm and
anti-adhesion agents, they were compared to a brominated furanone
(BF8) known to inhibit biofilm formation, and two generic
surfactants, sodium dodecyl sulfate (SDS) and tetra (ethylene
glycol) monododecyl ether (C12EG4OH). At 110 .mu.M, 5 out of 12
maltose derivatives inhibited more than 80% of PAO1 biofilm. These
maltose derivatives include BPDe.beta.M, BD.beta.M,
4-tBuBDe.beta.M, D.beta.M, and 4-MBD.beta.M. Six maltose
derivatives, AD.beta.M, 3,5-DMBD.beta.M, 4-tBuBD.beta.M,
BDe.beta.M, U.beta.M, De.beta.M, inhibited between 40 to 60% of
PAO1 biofilm, while, 12-HODe.beta.M showed insignificant amount of
inhibition, about 30% (as seen in FIG. 3). These results indicate
that the anti-biofilm activity is highly sensitive to the
structural details of the maltose derivatives, including both the
aliphatic chain length and the substituent structure. Increasing
the aliphatic chain length from 10 (De.beta.M) or 11 (U.beta.M)
units to 12 units (D.beta.M) increased the inhibition from about
50% to 70%. Incorporating a benzophenone group to a maltose
derivative with ten-carbon aliphatic chain increased the inhibition
from 50% (BDe.beta.M) to 90% (BPDe.beta.M). In general, increasing
the bulkiness of the substituents (BPDe.beta.M, BD.beta.M,
4-tBuBDe.beta.M) increased the inhibition of PAO1 biofilm. These
anti-biofilm activities of selective maltose derivatives were also
verified by fluorescence static biofilm assay. P. aeruginosa strain
PAO1-EGFP that constitutively producing green fluorescent protein
was allowed to form biofilm on sterile steel coupons. Maltose
derivatives treated biofilms showed weaker fluorescent signal than
the control of untreated steel coupons. These results indicate less
bacteria and biofilm formation (see the Supporting Information),
and is consistent with the results obtained by crystal violet
assays.
[0071] The inhibition of adhesion (anti-adhesion) of P. aeruginosa
by maltose derivatives were studied on polystyrene microtiter
plates. The anti-adhesion activities showed an overall similar
trend as the anti-biofilm activities with respect to the structures
of the maltose derivatives. However, an exact correspondence was
not obtained as seen in FIG. 3. Maltose derivatives, BPDe.beta.M,
BD.beta.M and 4-tBuBDe.beta.M exhibited high activities for both
inhibition of adhesion and biofilm formation, but BPDe.beta.M is
the most active anti-biofilm agent whereas 4-tBuBDe.beta.M is the
most active anti-adhesion agent. Comparing to the amount of
adhesion of PAO1-EGFP, 4-tBuBDe.beta.M and BD.beta.M inhibited more
than 80% of PAO1 adhesion at 85 .mu.M. Four maltose derivatives
inhibited between 30 to 70% of PAO1-EGFP adhesion, which included
BPDe.beta.M, D.beta.M, 4-tBuBD.beta.M, BDe.beta.M. Hydroxyl decyl
.beta.-maltose (12-HODe.beta.M) showed insignificant inhibition of
PAO1-EGFP adhesion, about 7% (see FIG. 3).
[0072] Examining the dose dependence of maltose derivatives on
biofilm inhibition revealed that 11: BPDe.beta.M exhibited an
IC.sub.50 of 22.5 .mu.M; and 5: BD.beta.M, 27.5 .mu.M, as seen in
FIG. 4. The anti-biofilm activities obtained from crystal-violet
(CV) dye based assays were comparable for both M9+ and LB media,
but not so for media without sodium chloride (see the Supporting
Information). It is important to note that BF8 showed no inhibition
on PAO1 adhesion, while exhibiting about .about.35% inhibition of
biofilm formation. Generic surfactants SDS and C12EG4OH did not
show any noticeable biofilm inhibition, but exhibited about 40%
inhibition of PAO1 adhesion, as seen in FIG. 3. We note that some
of the potent maltose derivatives for P. aeruginosa were further
tested for inhibition of E. coli biofilm. Mannose derivatives
BPDe.beta.M, BD.beta.M, 4-tBuBDe.beta.M and 4-tBuBD.beta.M at 85
.mu.M inhibited 70 to 75% of E. coli biofilm.
[0073] Chemical dispersion of already formed biofilm is often more
relevant to medical application, and more challenging than biofilm
inhibition assays, for which the agents are introduced at the onset
of an experiment. We screened the ability of maltose derivatives at
110 .mu.M to disperse 24 h old biofilm. A similar trend between the
anti-biofilm and dispersion activities was obtained for all twelve
maltose derivatives with BPDe.beta.M and BD.beta.M being the most
potent agents (FIG. 5). Examining the biofilm dispersion dose
dependence of BPDe.beta.M and BD.beta.M revealed the half-maximal
dispersion (DC.sub.50) values were 31 .mu.M and 32 .mu.M,
respectively (FIG. 6). The IC.sub.50 and these DC.sub.50 values of
maltose derivatives are comparable to quorum sensing-based small
molecule biofilm inhibitors and dispersers, but the mechanism of
the anti-biofilm activities of these maltose derivatives are likely
not by directly disrupting the quorum sensing of bacteria.
[0074] The maltose derivatives do not inhibit bacterial growth at
concentration (170 .mu.M) higher than those studied for biofilm
inhibition and dispersion. The active maltose derivatives are also
more potent than a brominated furanone (BF8) previously studied by
us at inhibiting biofilm formation. Furthermore, BF8 does not
inhibit the adhesion of bacteria. Together, the dual action of
anti-adhesion and anti-biofilm activities of these non-microbicidal
disaccharide hydrocarbons may offer potentials for further
therapeutic agent development.
[0075] The structural sensitivity of these maltose derivatives at
reactivating the swarming motility of rhlA mutant supported the
existence of one or more protein receptors for these disaccharide
hydrocarbons. In addition to this structural selectivity for
activating the swarming motilities, two observations also suggest
that the anti-biofilm activity is not due to a simple washing
effect because of the surface activity of the molecules. First, the
effective concentration such as IC.sub.50 and DC.sub.50 of biofilm
inhibition and dispersion is significantly lower than the critical
micelle concentration of a typical maltoside (cmc of D.beta.M is
170 .mu.M). Second, all other generic surfactants examined in this
study, including SDS, C12EG4OH and DTAC, did not show any
anti-biofilm activities (either inhibition or dispersion)
Inhibiting the bacterial adhesion alone is not likely the sole
attribute to the anti-biofilm activities because the already
adhered bacteria and formed biofilm can be effectively dispersed by
the same maltose derivatives. On the same note, the reactivation of
the swarming motility of rhlA mutant by disaccharide hydrocarbons
is also likely driven by ligand-receptor interactions rather than
merely lowering the surface tension of the soft gel. None of the
generic surfactants reactivated the swarming motility rhlA mutant
while C12EG4OH actually has a higher surface activity then known
disaccharide-derivatives.
[0076] The ability of the maltoside derivatives to antagonize the
two quorum sensing circuits las and/or rhl of P. aeruginosa was
investigated by using two reporter strains (PAO1/plasI-LVAgfp,
PAO1/prhlI-LVAgfp). These reporter strains produce natural AHL
signals, and binding of such signal molecules to the Lux-type
receptor proteins (LasI and LasR) activates the expression of green
fluorescent protein (GFP) encoded by the plasmid. Our results (see
the Supporting Information) indicated that maltose derivatives did
not compete with the natural signal to cause a decrease in
fluorescent signal. Further, the ability of the maltose derivatives
to agonize the quorum sensing circuit in P. aeruginosa in the
absence of natural AHLs was studied with a double knock-out strain
(PAO-JP2 (plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfP) that do not
produce AHLs. The maltose derivatives did not show any significant
increase in the fluorescent signals (see the Supporting
Information), indicating that they did not agonize the quorum
sensing receptors.
[0077] Many receptors exist for saccharide derivatives on bacterial
surfaces. Among these protein receptors, three systems may be
related to the anti-biofilm activities by the maltose derivatives.
First, the vast biological activities of rhamnolipids (building of
porous biofilm, dispersion of mature biofilm and enabling of
swarming motility) suggest that one (or more) receptor exists, and
the identity of which has not been discovered but maybe strongly
associated with the protein SadB (surface attachment-defective gene
product). Second, when P. aeruginosa swarm on the soft agar gel, it
appears to differentiate into at least two phenotypes, the
hyperactive swarming bacteria at the tip of the swarming tendril
and the less mobile bacteria at the center of the swarming ring and
on the stem of tendrils. Among genes screened by Deziel and
coworkers, gltK is highly up-regulated in the bacteria at the
tendril tip but not for bacteria at the swarming center.19 Gene
gltK of P. aeruginosa encodes an inner membrane component of the
ATP-binding cassette (ABC) transporter system for transporting
glucose. Interestingly, the gltK gene product in P. aeruginosa is a
member of the family of MalK proteins, which transport maltose in
E. coli. Third, 90% of the adhesion of P. aeruginosa is likely
caused by the pili proteins, and that pili protein is responsible
for the recognization of P. aeruginosa to the
GalNAc.beta.(1.fwdarw.4)Gal.beta. moieties found on human host
cells. These pili proteins are also implicated to be responsible
for adhesion on polystyrene. Together with the results that the
disaccharide hydrocarbons inhibited the adhesion of P. aeruginosa
on polystyrene, the maltose derivatives may target pili proteins
that recognize GalNAc.beta.(1.fwdarw.4)Gal.beta. on mammalian
cells. In addition, the maltose derivatives activate the swarming
motility of the nonswarming mutant--a biological function also
exhibited by rhamnolipid. Thus, these disaccharide derivatives may
also target the receptor(s) of rhamnolipids. Whether the ligands
GalNAc.beta.(1.fwdarw.4)Gal.beta. on mammalian cells and
rhamnolipids share a common receptor on bacteria is not certain,
but maltose derivatives could be promiscuous in binding multiple
receptors, or that rhamnolipid is the ligand for pili protein as
well. Both swarming reactivation and anti-biofilm activities are
highly sensitive to the structural details of the maltose
derivatives, but there is no strict correlation between the two
biofunctions. For example, for the three maltose derivatives that
do not reactivate swarming of rhlA mutant, two of them of are
sluggish biofilm inhibitors (HODe.beta.M and De.beta.M gave 32% and
41% biofilm inhibition at 110 .mu.M), but one of them is a strong
biofilm inhibitor (4-tBuBD.beta.M gave 74% biofilm inhibition at
110 .mu.M). However, the level of receptor expression is likely
different during biofilm formation and bacterial swarming, and thus
a rigorous determination of correlation is still ongoing.
[0078] Furthermore, the swarm-nonactiviting 4-tBuBD.beta.M differs
from swarming-activating 4-tBuBDe.beta.M and BD.beta.M by only two
methylene units in the aliphatic chains and a tert-butyl group,
respectively. Because of this structural sensitivity, these results
may also suggest an allosteric effect on the receptor upon binding,
which leads to different agonistic or antagonistic effect on the
further cell signaling events. The effect of disaccharide
stereochemistry and further exploration of different bulky
hydrocarbon structures on biofilm and swarming motility is an
ongoing subject of our study.
[0079] The strong biological functions (reactivation of swarming
motility, anti-adhesion and anti-biofilm activities) of this class
of maltose derivatives, and the sensitive dependence of these
bioactivities to the molecular structures suggest that one or more
protein receptors may exist, for which small molecule binding
causes a strong inhibition and dispersion effect on the biofilm and
other bacterial behavior. As biofilm formation and swarming
motilities are quite common for microbes, similar receptors for
disaccharide derivatives likely exist in different types of
bacteria. Thus, we believe that targeting these receptors form the
basis of an effective approach to control the biofilm formation and
related diseases.
Example 2
[0080] The present invention also encompasses a chemical library of
disaccharide hydrocarbons (DSHs) that were developed by
systematically changing the glycone as well as aglycone part of the
DSH (FIG. 7) and then investigated to determine the effect that
these structural changes have on swarming motilities of PA and its
non-swarming mutant strain. The library was further tested for
anti-biofilm activities. The efficacy of DSHs to inhibit biofilm
formation in PAO1 was reduced when these agents were pretreated
with a solution of synthetic pili peptide (PAO.sub.128-144)ox.
[0081] In order to understand the relationship between structural
detail and activities, a library of DSHs was synthesized as seen in
FIG. 7. The basic synthetic variations employed involved first
changing the glycone and then the aglycone part of DSHs. Glycone
variations included synthesizing 12 carbon chain DSHs with
cellobiose; Glc.beta.(1.fwdarw.4)Glc.beta.
(Dodecyl-.beta.-cellobioside; 4: D.beta.C), lactose;
Gal.beta.(1.fwdarw.4)Glc.beta. (Dodecyl-.beta.-lactoside; 7:
D.beta.L), rhamnose; rha (Dodecyl-.alpha.-rhamnoside; 9: D.alpha.R)
and .beta.-cyclodextrin; .beta.CD (Dodecyl-.beta.CD-squarate; 18:
D.beta.CDS) stereochemistries. Two commercially available DSH with
12 carbon chain bearing maltose Glc.alpha.(1.fwdarw.4)Glc.beta.
(Dodecyl-.beta.-maltoside; 1: D.beta.M) and glucose; Glc
(Dodecyl-.beta.-glucoside; 8: D.beta.G) stereochemistries were also
used for structural comparison. Next, systematic variations to
aglycone hydrocarbon chain were done. Keeping sugar stereochemistry
as cellobiose, DSHs were synthesized having 10
(Decyl-.beta.-cellobioside; 2: De.beta.C), 11
(Undecyl-.beta.-cellobioside; 3: U.beta.C), 13
(Tridecyl-.beta.-cellobioside; 5: T.beta.C) hydrocarbon chain.
Effect of terminal unsaturation was investigated by synthesizing 11
carbon cellobiose based DSH having terminal unsaturation
(Undecylene-.beta.-cellobioside; 10: UD.beta.C). Effect of
.alpha./.beta. anomers on activity was investigated by synthesizing
DSH having cellobiose stereochemistry but with 12 carbon chain at
.alpha.-position (Dodecyl-.alpha.-cellobioside; 6: D.alpha.C).
Effect of hydrocarbon bulkiness on DSHs activity was next studied
by synthesizing agents with saturated farnesyl tail (possessing
short methyl branches) with three different disaccharide
stereochemistries; cellobiose (Saturated
farnesyl-.beta.-cellobioside; 12: SF.beta.C and its .alpha.-anomer
(Saturated farnesyl-.alpha.-cellobioside; 13: SF.alpha.C), maltose
(Saturated farnesyl-.beta.-maltoside; 14: SF.beta.M and lactose
(Saturated farnesyl-.beta.-cellobioside; 15: SF.beta.L). Effect of
chain unsaturation on agent activity was investigated by
synthesizing cellobiose-derived farnesyl
(Farnesyl-.beta.-cellobioside; 11: F.beta.C). The effect of chain
bulkiness on activity was further investigated by synthesizing two
DSHs with double-branched tail (overall lipid-like structure)
(2-octyl-dodecyl-.beta.-cellobioside; 16: 2-OD.beta.C and its
.alpha.-anomer (2-octyl-dodecyl-.alpha.-cellobioside; 17:
2-OD.alpha.C).
[0082] Synthesis of mono-sugar and disugar based hydrocarbons with
systematic variation in hydrocarbon chain length and structure were
synthesized by slightly modifying a reported procedure as seen in
FIG. 8. Briefly, unprotected sugar was simultaneously acylated and
brominated (at anomeric carbon) using a binary mixture of AcBr/AcOH
to obtain aceto-bromo sugar. The aceto-bromo sugars were then
glycosidated at anomeric position using either FeCl.sub.3 or
Hg(CN).sub.2 as catalysts. For obtaining .beta.-anomer as the major
glycosidation product, MeCN was used as the solvent, while
MeNO.sub.2 primarily gave .alpha.-anomer. The resolution of
.alpha./.beta. anomers was done using column chromatography
(.beta.-anomer usually eluted first). The glycosidated products
were deprotected under basic conditions using methanolic sodium
methoxide solution followed by neutralization to a pH .about.6.5
(using H+amberlite resins) (Zemplen deacetylation).
[0083] Synthesis of di-(difluro-phenoxy) squarate (18 A) and
.beta.-cyclodextrin amine (18c: .beta.CD-NH.sub.2) were done
according to a known procedure. Dodecyl-.beta.CD-squarate (18:
D.beta.CDS) was synthesized according to FIG. 8. Briefly,
nucleophilic substitution by dodecyl amine of one phenoxy
substituent on squarate was done in THF at -78.degree. C. for 12
hours followed by substitution of second phenoxy substituent by
.beta.CD-NH.sub.2 in water/DMF/acetone ternary solvent mixture over
a period of 4 days to yield 18: D.beta.CDS.
[0084] All synthesized polyol-derivatized hydrocarbons were soluble
in water at 160 .mu.M (concentration at which these agents were
tested for anti-biofilm activity) (except 17: 2-OD.alpha.C), whose
stock solutions were prepared in a solvent mixture of water/EtOH).
In general DSH with galactose stereochemistry had poor water
solubility; (15: SF.beta.L) was not soluble in either water or in
water/EtOH mixture). All synthesized disaccharide hydrocarbons
(DSHs) were non-toxic to the growth of Pseudomonas aeruginosa at
.about.160 .mu.M.
[0085] Control of swarming motility of wild type and nonswarming
mutants of P. aeruginosa Multicellularity in bacteria manifests
through other highly organized surface associated behaviors such as
swarming motility. Swarming phenomenon has been observed in both
Gram-negative as well as Gram-positive bacteria. Under laboratory
setting, swarming motility is usually studied on a solid medium
containing .about.0.5-0.7% agar (soft agar). When a small amount of
bacterial culture is spotted on the soft agar surface, it is
essential that a certain minimum cell density is achieved in order
for swarming to happen. Bacterial quorum sensing hence is not just
critical for biofilm formation but is also closely related to
swarming process. It is argued that, that triggering a switch to
swarming mode would require a coordinated merger of many chemical
and physical cues in the bacteria. In combination, such
physio-chemical changes are capable of inducing morphological
changes that are necessary for swarming. Therefore, pathways
associated for swarming induction often share same cross-roads with
biofilm formation circuitry. Both biofilm formation and swarming
are multicellular bacterial behaviors that contribute to the
survival of the microbes in environment and in-vivo, therefore are
important life-styles necessary for maintaining virulence.
Therefore, agents that affect biofilm formation in a particular
bacterial species could potentially affect the swarming process in
that bacterium too.
[0086] The swarming bacterial cells are generally associated with
having multiple flagella on their surface. While in bacteria like
Pseudomonas aeruginosa, in addition to two-polar flagellum the
presence of Type IV pili as well as the production of a
biosurfactant, rhamnolipid is critical for promotion of swarming.
The role of rhamnolipid in Pseudomonas aeruginosa is not limited to
promotion of swarming (probably by reducing surface tension) but
they are also implicated in forming water channels with in biofilms
and maintaining mushroom shaped structures. In addition,
rhamnolipid can disperse mature biofilm by mediating detachment and
over production of rhamnolipids results in impaired biofilm
formation. Because of the important roles that rhamnolipids play in
assisting Pseudomonas aeruginosa multi-cellular behaviors, the
exploration of its putative receptor on the bacterial surface is
under constant study.
[0087] Biosynthesis of rhamnolipids involves genes that are under
the control of quorum sensing regulators RhlR and LasR. Synthesis
of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAA) precursors is
brought about by rhlA gene, following which rhlB encodes for the
enzyme (rhamnosyltransferase) that catalyzes the addition of
rhamnosyl moiety to HAA. Eventually, RhlC catalyzes the addition of
a second rhamnose moiety to generate di-rhamnolipid. Together, HAA,
mono-rhamnolipids and di-rhamnolipids are known to facilitate the
swarming of Pseudomonas aeruginosa on soft-agar to form solar-flair
like and/or dendritic/tendril swarm patterns. While the exact
mechanism of how Pseudomonas aeruginosa form tendrils is still
under investigation, some aspect of di-rhamnolipids and its
precursors have been brought to light. Namely, di-rhamnolipids are
known to attract swarm cells, whereas HAA are known to be swarm
cell repellants. Mono-rhamnolipids is known to merely serve as a
surface wetting agent.
[0088] Like rhamnolipids and its precursors, HAA and
mono-rhamnolipids (together called as rhamnolipids hence forth),
DSH are also amphiphilic molecules and we were interested to know
whether these efficient anti-biofilm agents could also undertake
functions naturally carried by rhamnolipids. Pseudomonas aeruginosa
strain PAO1 normally swarms on a soft agar surface to give a
dendritic swarm pattern. Soft-agar (0.5%) plates supplemented with
.about.7 different DSH (12: SF.beta.C; 14: SF.beta.M; 13:
SF.alpha.C; 7: D.beta.L; 2: De.beta.C; 1: D.beta.M; 4: D.beta.C) at
110 .mu.M were inoculated with PAO1 (OD600.about.0.5). Images of
the swarm agar plates 24 hours after inoculation are shown in FIG.
9. When compared to swarm agar plate with no agent, 7: D.beta.L; 2:
De.beta.C and 1: D.beta.M did not significantly affect the swarm
pattern, although 4: D.beta.C induced more tendril formation.
Importantly, DSHs that were powerful anti-biofilm and anti-adhesive
agents (12: SF.beta.C; 14: SF.beta.M and 13: SF.alpha.C) completely
inhibited the swarming of wt PAO1. These results indicate that that
12: SF.beta.C; 14: SF.beta.M and 13: SF.alpha.C could be displacing
rhamnolipids from its natural receptor.
[0089] N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone (AHL) has
been identified as a bacterial signaling molecule where naturally
isolated as well as synthetic AHLs stimulated light production in
Photobacterium fischeri and the amount of light produced increased
over the range 0.003-3 .mu.g/mL and then decreased at higher
concentrations (especially for synthetic AHL). On comparing the
effect of 12: SF.beta.C on swarming of PAO1, it is evident that
higher concentration of 12: SF.beta.C (>20 .mu.M) exhibit
inhibition of PAO1 swarming. However lower concentration of
SF.beta.C (<10 .mu.M) exhibit slight promotion of PAO1 swarming
as seen Figure [[3]]. This activity reversal as the concentration
increases is consistent with disaccharide hydrocarbons SF.beta.C
being a cell signaling molecule.
[0090] Some polyol-derivatized hydrocarbons reactivate swarming in
nonswarming PA mutant (rhlA) Transposon mutation involving gene
rhlA generates a PAO1 mutant which lacks in the biosynthesis of
di-rhamnolipids, mono-rhamnolipids and HAA hence as a consequence,
this mutant (rhlA) is non-swarming. The swarming reactivation of
non-swarming mutant rhlA by DSHs was tested (12: SF.beta.C; 14:
SF.beta.M; 13: SF.alpha.C; 7: D.beta.L; 2: De.beta.C; 1: D.beta.M;
4: D.beta.C). Agents were solubilized homogenously into the melted
agar (.about.0.5%) to a resultant concentration of 110 .mu.M and
then the agar was cooled to form a semi-solid. Non-swarming mutant
rhlA (OD600.about.0.5) was then inoculated. After 24 hours, as seen
in FIG. 11, 1: D.beta.M; and 4: D.beta.C fully restored the
swarming of rhlA with 4: D.beta.C forming fine tendrils. However,
12: SF.beta.C, 14: SF.beta.M, 13: SF.alpha.C 7: D.beta.L and 2:
De.beta.C were incapable of reactivating the swarming in
non-swarming mutant rhlA. While, the swarming results of rhlA
indicates that some DSHs like 1: D.beta.M; and 4: D.beta.C are
fully capable of mimicking the function of rhamnolipids, the theory
of swarm attractants and repellants alone cannot explain the
mechanism of tendril formation here (as there is no production of
any HAA).
[0091] As 4: D.beta.C was capable of reactivating the swarming in
non-swarming mutant rhlA, we did a concentration and time dependant
study to observe the progression of swarming. Concentration as low
as 20 .mu.M were sufficient to reactivate non-swarming mutant rhlA
to swarm (FIGS. 12 and 13). Interestingly, on increasing the
concentration from 40 .mu.M to 85 .mu.M, there was a decrease in
the swarming reactivation ability of 4: D.beta.C.
[0092] The signaling molecule like behavior of 12: SF.beta.C was
also noticed in its ability to reactivate swarming of rhlA only
between the concentration range of 5-12 .mu.M (FIGS. 14 and 15).
With rhlA mutant also, there was no reactivation at higher
concentrations of 12: SF.beta.C.
[0093] SF.beta.C is at least twice as strong a ligand than 4:
D.beta.C for the swarming receptor At 110 .mu.M 12: SFbC inhibits
the swarming of wt PAO1 (as seen in FIG. 9) whereas 4: DbC promotes
tendril formation at the same concentration. Also, at same
concentration, 12: SFbC does not reactivate the swarming of rhlA
mutant (as seen in FIG. 11) whereas 4: DbC fully restores swarming
with fine tendrils. Mechanistic understanding to consider here is
whether 12: SFbC binds swarming receptor better than other DSHs by
co-administration of two DSHs, SFbC (inhibits PAO1 swarming) and
DbC (promotes PAO1 swarming). If SFbC binds stronger than DbC to
swarming receptor, then there will be inhibition of DbC reactivated
swarming. If not, 4: DbC reactivated swarming will still be active.
Co-administration SF.beta.C (as low as 20 .mu.M) suppresses the
reactivation of rhlA swarming by D.beta.C (40 .mu.M) (FIG. 16),
suggesting that 12: SFbC is at least twice a stronger binding
ligand than 4: DbC.
[0094] The ability of DSHs was tested to inhibit Pseudomonas
aeruginosa adhesion, biofilm and to disperse pre-formed biofilm. To
quantify the amount of PAO1/EGFP attached on the surface of micro
titer plates with or without the synthesized DSHs, fluorescence
emitted by green fluorescent proteins (GFP) was quantified (see
supporting info for details). Crystal violet (CV) based static
biofilm assay (see supporting info) was used to screen the
anti-biofilm potencies of all synthesized agents. Both surface
attached (SA) (FIG. 17) and total (SA+air-liquid interface) biofilm
of P. aeruginosa was quantified. Although, only the SA biofilm is
reported here, the total biofilm quantified (not reported) also had
similar potency trend for various DSHs. CV assays were also used to
quantify the amount of P. aeruginosa biofilm remaining after
dispersion experiments (FIG. 19). Initially, all agents were screen
for anti-biofilm activities (both inhibition and dispersion) at 160
.mu.M. Agents that showed >50% inhibition or dispersion were
used further in a dose-response manner to calculate the IC.sub.50
(inhibition) or DC.sub.50 (dispersion). All reported biofilm
inhibition results are an average of 4 identical wells. Each
experiment has been repeated at least three times, each time giving
similar results (repeat results not shown).
[0095] A test of detecting the amount of bacteria adhered on
polystyrene surfaces within 2 hours of inoculation was developed.
Based on the anti-adhesive assay, DSHs having bulky aliphatic tails
were strong anti-adhesive agents that prevented Pseudomonas
aeruginosa from adhering onto poly-styrene surface as seen in FIG.
17. Agents with farnesyl or twelve carbon aliphatic chains and
bearing either cellobiose or maltose head group prevented 60-80% of
bacterial adhesion.
[0096] Stereochemistry of disaccharide is important for the
anti-biofilm activity. Monosaccharide (glucose; 8: D.beta.G and
mono-rhamnoside; 8: D.alpha.R) based hydrocarbons having twelve
carbon aliphatic chain had poor anti-biofilm activity. However
disaccharide hydrocarbons having a twelve carbon chain and a
disaccharide stereochemistry (maltose, Glc.alpha.(1a4)Glc.beta.; 1:
D.beta.M) reduced the biofilm content by .about.50% (as seen FIG.
16). Synthesized cyclic oligosaccharide (18: D.beta.CDS) was not
only incapable of exhibiting any biofilm inhibition but also
increased the biofilm content. Therefore, while DSHs with 1 sugar
head group were poor anti-biofilm agents, DSH with oligosaccharide
head group was even worse. However, DSHs with disaccharide head
group exhibited decent anti-biofilm activity.
[0097] Anti-biofilm potencies of three different DSHs each bearing
a twelve carbon aliphatic chain with either lactose
(Gal.beta.(1.fwdarw.4)Glc, 7: D.beta.L (poor solubility in water),
cellobiose (Glc.beta.(1.fwdarw.4)Glc, 4: D.beta.C) or maltose
(Glc.alpha.(1.fwdarw.4)Glc) based disaccharide head group revealed
that Glc.beta.(1.fwdarw.4)Glc stereochemistry out-performed other
stereochemistries (as seen in FIG. 16). While lactose
stereochemistry severely compromised the anti-biofilm activity,
cellobiose stereochemistry exhibited .about.60% biofilm inhibition.
In general, cellobiose hydrocarbons compared better than those of
maltose hydrocarbon.
[0098] Chain length of the hydrocarbon is important for biofilm
inhibition. Aglycone moiety of DSH was then systematically altered
keeping cellobiose based sugar head group stereochemistry constant.
Firstly, DSHs with aliphatic chain (.beta.-anomer) length having 10
(2: De.beta.C), 11 (3: U.beta.C), 12 (4: D.beta.C) and 13 (5:
T.beta.C) carbons were compared. DSH with 10 carbon chain resulted
in complete loss of anti-biofilm activity while 12 carbon chain
seemed optimum. This reliance of activity on aliphatic chain length
is reminiscent of another biochemical phenomenon exhibited by many
gram-negative as well as gram positive bacteria, quorum sensing.
Different bacterial species are able to distinguish their own
chemical signal from that of others by recognizing the difference
in the length of acyl side chain of acyl-homoserine lactone (AHL)
auto inducers (chemical signals). The AHL signals are recognized by
specific AHL-responsive receptor proteins known as "R" proteins
such as LuxR or LasR.
[0099] This recognition is primarily based on the ability of "R"
proteins to distinguish one AHL from another based on the length of
the acyl side chain. The optimization of DSH with C12 aliphatic
chain to give maximum anti-biofilm activity is therefore an
indication of a ligand-receptor phenomenon.
[0100] At 160 .mu.M, both 12: SF.beta.C and 13: SF.alpha.C were
powerful inhibitors (up to .about.80%) of P. aeruginosa biofilm
(FIG. 17). The low IC.sub.50 values of 12: SF.beta.C (.about.4.9
.mu.M) and 13: SF.alpha.C (.about.32.3 .mu.M) (see FIG. 18) are
comparable to some of the powerful synthetic agents that have been
reported to non-microbicidally inhibit P. aeruginosa biofilm. In an
attempt to further optimize anti-biofilm activity, maltose and
lactose based saturated farnesyl was synthesized (14: SF.beta.M and
15: SF.beta.L respectively). Although, only slightly less potent at
.about.160 .mu.M, 14: SF.beta.M had a much lower IC.sub.50
(.about.97.2 .mu.M) (FIG. 18). Synthetic lactose analog exhibited
poor solubility in water and hence was not tested for anti-biofilm
activity. The ability of DSHs, SF.beta.C, SF.alpha.C, D.beta.C,
De.beta.C and D.beta.L to inhibit PA biofilm formation on steel
coupons was tested. At 160 .mu.M of DSHs, CLSM micrographs reflect
the similar trend of biofilm inhibition activities obtained from
CV-dye based assay.
[0101] Polyol-derivatized hydrocarbons with the alkyl chain having
lipid-like structure are not potent anti-biofilm agents. With the
intent to further increase the bulkiness of the aliphatic chain, we
synthesized two mono-branched DSH anomers having lipid-like
structure (16: 2-OD.beta.C and 17: 2-OD.alpha.C). Unlike DSHs
having methyl branched saturated aliphatic chain (saturated
farnesyl DSHs), aliphatic chain giving a lipid-like structure were
ineffective at biofilm inhibition as seen in FIG. 17.
[0102] Unsaturated in the aliphatic chains reduced the anti-biofilm
activity of the polyol-derivatized hydrocarbons. DSH having a
farnesyl aliphatic chain (11: F.beta.C) was synthesized, only to
generate a structure with poor anti-biofilm activity as seen in
FIG. 17. Similarly, DSH having an aliphatic chain with terminal
unsaturation (10: UD.beta.C) had lesser potency than DSH with
completely saturated aliphatic chain (3: U.beta.C) as seen in FIG.
17.
[0103] DSH's possession of anti-biofilm activity was determined by
virtue of these agents interfering with bacterial cell-to-cell
communication which is under las and rhl control. One of the recent
approach for a nonmicrobicidal control of biofilm aims to develop
agents that disrupt bacterial cell-to-cell communication by acting
as mimics to the natural molecules (autoinducer) that bind lasI and
rhlI proteins, which are encoded by las and rhl gene. The binding
of these mimics with las and rhlR proteins either activates or
represses the expression of lasI or rhlI..sup.6 In gene reporter
assay, the expression of lasI and rhlI proteins is quantified by
measuring expression of green fluorescent protein (GFP) fused to
lasI and rhlI genes..sup.5 Two reporter strains of Pseudomonas
aeruginosa (PAO1/plasI_LVAgfp and PAO1/prhlI_LVAgfp) express green
fluorescence protein on activation of las or rhl quorum sensing
system. The activation or repression of las or rhl quorum sensing
system in PA was tested by various DSHs. For both las and rhl
quorum sensing systems, addition of DSHs (12: SF.beta.C and 13:
SF.alpha.C) had no significant change in the fluorescence.
[0104] Apart from preventing biofilm formation, the efficacy of an
anti-biofilm agent is further enhanced if it can also disperse
preformed biofilm. Such agents are versatile because they
effectively target different biofilm developmental stages. The
ability of DSHs to disperse pre-formed (1 day old) biofilm was
tested. The general trend of dispersion ability was consistent with
inhibition power as seen in FIG. 19. DC.sub.50 values (.about.36.4
.mu.M for 12: SF.beta.C and .about.78.4 .mu.M for 13: SF.alpha.C)
are indicative of powerful dispersion activity as seen in FIG.
20.
[0105] P. aeruginosa are reported to bind human mucosal cells via
tip of the pilus and can having binding to biotic and abiotic
surfaces that is mediated by pili. Minimum binding sequence of pili
peptide for PAO strain is the 17 AA at C-terminus PAO(128-144)ox
[Ac-A-C-K-S-T-Q-D-P-M-F-T-P-K-G-C-D-N-OH] where cysteines 129 and
142 form a disulfide bond, forming a looped peptide. These pili
specifically recognize disaccharide GalNAc-Gal. Increasing the
hydrophobicity by lengthening the alkyl chain attached at
1-position increased the binding between carbohydrate and pili
peptide. The pili peptide PAO(128-144)ox was synthesized by FMOC
synthesis. A scrambled pili peptide was also synthesized by
changing the cysteines 129 and 142 by alanines, PAO(C129A/C142)_S
[Ac-A-A-K-S-T-Q-D-P-M-F-T-P-K-G-A-D-N-OH]. When 13: SF.alpha.C was
pretreated with a solution of PAO(128-144)ox it exhibited no
inhibition of PA biofilm. However, pretreatment with
PAO(C129A/C142)_S did not compromise the efficacy of the agent as
seen in FIG. 21. DSHs are possibly preventing the initial
attachment of PA on abiotic surface, and pili-peptide may compete
with DSHs for the receptors on PA, and hence reducing the efficacy
of DSHs.
Example 3
[0106] The effects of dodecyl maltoside was tested on the bacterial
clearance in a mouse pneumonia model. SP-A and SP-D double knockout
(SP-A/D KO) mice with C57BL/6 background were used for this study.
Original SP-A/D KO mice were provided by Dr. Samuel Hawgood of The
University of California San Francisco, and these mice had been
backcrossed at least ten generations against a C57BL/6 background.
Mice used in this study were bred in the animal core facility at
SUNY Upstate Medical University under pathogen-free conditions. All
animal experiments were conducted in accordance with the
Institutional Animal Care and Use Committee guidelines of SUNY
Upstate Medical University and the National Institutes of Health
guidelines on the use of laboratory animals.
[0107] Pseudomonas aeruginosa strain PA01-BAA-47 (wild type) from
frozen stocks were streaked onto LB agar plates and incubated at
37.degree. C. for 24 hours, and a single colony was picked up and
transferred to a flask contains 20 mL of LB Broth and incubated at
37.degree. C. for 13 hours with shaking at 250 rpm. The optical
density at 600 nm (OD.sub.600) was measured and it is usually about
2.1 by this time. The bacterial cells were recovered by
centrifugation, resuspended in saline and diluted to an optical
density of 0.6 at OD.sub.600. One mL of this solution was estimated
to contain 2.times.10.sup.9 CFU. The solution was diluted 20 times
with saline for use. Based on our preliminary data, a 50 .mu.l
(containing 5.times.10.sup.6 CFU) of the diluted bacterial solution
was used to inject each mouse intratracheally.
[0108] A total of 12 male and 16 female SP-A/D KO mice (8-12 weeks
old) were used for all experiments. Three independent experiments
were performed using age and gender matched mice. The tracheal
delivery of [50 .mu.l (5.times.10.sup.6 CFU)/mouse] was
accomplished by anesthetizing mice with 30 .mu.l of the mixture of
ketamine:xylazine (100 mg/kg:10 mg/kg), making a small incision
above the trachea and directly injecting the bacterial suspension
inside the trachea. After 24 hours the incision was reopened under
anesthesia and 50 .mu.l solution containing 170 .mu.M of Dodecyl
Maltoside (DM) was injected into the trachea of the treatment group
and 50 .mu.l saline was injected into the trachea of the control
group. After 24 more hours all mice were sacrificed.
[0109] Randomly selected mice from each group were prepared for
quantitative bacteriology. The left half of the lung of each mouse
was removed aseptically and homogenized in 1 ml of sterile 0.9%
saline and 100 .mu.l of appropriately serial diluted lung
homogenates sample were plated on LB agar, incubated at 37.degree.
C. for 24 hours, and inspected for P. aeruginosa colonies.
[0110] After anesthetizing mice with ketamine:xylazine 100 mg/10
mg, a large abdominal incision was made and the intestine was
turned to the left side the inferior vena cava and Aorta were cut
using iris scissors and leave the animal was left to bleed and then
various tissues were harvested from the mice including lung, liver,
spleen, kidney and intestine. Tissues were wrapped in a labeled
aluminum foil, snap frozen in liquid nitrogen and kept in
-80.degree. C.
[0111] Randomly selected lungs were slowly inflated with 1 ml of
formalin and then completely immersed in formalin. Specimens were
embedded in paraffin and 5 .mu.m sections cut. Slides were stained
using hematoxylin and eosin for standard light microscopic
analysis.
[0112] Experimental data were analyzed by SigmaStat 3.5 software
(Systat Software, Inc., San Jose, Calif.) and presented as
means.+-.standard error. Two-group comparisons were performed using
Student's t test. A P value of <0.05 was considered to be
statistically significant. The results showed that DM treatment
significantly decreased CFU number in the lung compared to the
control, as seen in FIG. 22 and improved the lung inflammation, as
seen in FIG. 23.
Example 4
[0113] In another study, a series of disaccharide hydrocarbons
(DSHs) with different sugar stereochemistries and aliphatic chain
structures were synthesized and tested for the capability of
controlling multiple bacterial behaviors, including inhibiting and
activating the swarming motility of bacteria, inhibition of
bacterial adhesion, and inhibition and dispersion of bacterial
biofilm formation. Different structural elements and their degree
of importance have been identified for this class of molecules for
dominating natural rhamnolipids at controlling the swarming
motility of wild type Pseudomonas aeruginosa (PAO1) and a
nonswarming mutant rhlA, and inhibiting biofilm formation. In
addition, these molecules exhibit an activity reversal on
activating the swarming motility of nonswarming mutant rhlA, by
which a low concentration range of DSHs reactivates the swarming
motility, but a high concentration range inhibits the swarming
motility. The effect of causing two different phenotypes of
bacteria at the same time was also explored.
[0114] Many biological recognition events such as cell-to-cell
interactions, bacterial, fungal and viral infections of human host
cells are mediated through carbohydrate-protein interactions. While
the identity as well as the existence of the receptors for
rhamnolipids remains elusive, flagella proteins of P. aeruginosa
can bind to the sugar moieties of mucin29 and asialo-GM130, and
pilin can bind to bind D-GalNac-.beta.(1.fwdarw.4)D-Gal-.beta.
disaccharide moiety. Proteins LecA and LecB of PA, on the other
hand, are known to be more specific for galactose and fucose,
respectively.
[0115] A series of synthetic carbohydrate hydrocarbons with
different disaccharide stereochemistry, along with two
monosaccharides and one large cyclic hepta-saccharide moiety, were
screened (FIG. 24A). The disaccharide hydrocarbons include maltose
(Glc.alpha.(1.fwdarw.4)Glc.beta.)-based molecules including,
dodecyl-.beta.-maltoside (D.beta.M), 1; saturated
farnesyl-.beta.-maltoside (SF.beta.M), 14; and cellobiose
(Glc.beta.(1.fwdarw.4)Glc.beta.)-based molecules including,
dodecyl-.beta.-cellobioside (D.beta.C), 4;
decyl-.beta.-cellobioside (De.beta.C), 2;
undecyl-.beta.-cellobioside (U.beta.C), 3;
tridecyl-.beta.-cellobioside (T.beta.C), 5;
dodecyl-.alpha.-cellobioside (D.alpha.C), 6;
undecylenyl-.beta.-cellobioside (UD.beta.C), 10;
farnesyl-.beta.-cellobioside (F.beta.C), 11; saturated
farnesyl-.beta.-cellobioside (SF.beta.C), 12; saturated
farnesyl-.alpha.-cellobioside (SF.alpha.C), 13;
2-octyl-dodecyl-.beta.-cellobioside (2-OD.beta.C), 16;
2-octyl-dodecyl-.alpha.-cellobioside (2-OD.alpha.C), 17; and
lactose (Gal.beta.(1.fwdarw.4)Glc.beta.) including,
dodecyl-.beta.-lactoside (D.beta.L), 7; saturated
farnesyl-.beta.-lactoside (SF.beta.L), 15. In addition to changing
the lengths of the aliphatic chains, bulky hydrocarbons with
different degree of unsaturation (11) and methyl branches (12, 13,
14, 15) are included. These bulky hydrocarbons are derived from
farnesol and hydrogenated farnesol (3, 7, 11-trimethyl-dodecane).
The monosaccharide hydrocarbons include dodecyl-.beta.-glucoside
(D.beta.G), 8 and dodecyl-.alpha.-rhamnoside (D.alpha.R), 9. To
examine the effect of a large oligo-saccharide group,
dodecyl-.beta.-cyclodextrin (D.beta.CDS), 18, was included.
[0116] DSH are nontoxic to planktonic bacterial growth. The
toxicity of DSHs against bacterial growth in culture media was
examined. At a relatively high concentration, 160 .mu.M, none of
molecules showed inhibition to the growth of planktonic Pseudomonas
aeruginosa in LB broth. Being nonmicrobicidal, these agents have
the potential to change or control bacterial behavior without
invoking drug resistance. 35-38 We note that, however, DSH (11)
inhibited bacterial (PAO1) growth on a soft agar (.about.0.5%)
plate that was used for swarming assay (see below).
[0117] Using nonswarming mutant to screen for swarming agonists and
wild type P. aeruginosa for swarming antagonists. The transposon
mutant, rhlA (genotype, rhlA-E08::ISphoA/hah)39 of P. aeruginosa
(PAO1), that impairs the gene that is responsible for synthesizing
rhamnolipids is completely incapable of a swarming motility.40 By
the reintroducing rhamnolipids into the soft agar gel, in which
rhlA mutant was inoculated, the mutant swarms again with a radial
pattern similar to the wild type PA14. This result suggests that
the rhamnolipids is either a ligand required for activating the
swarming motility or simply a surfactant that facilitates the
swarming motility. Recent work indicate that other generic
surfactants such as sodium dodecyl sulfate (SDS), tetraethylene
glycol mono dodecyl ether (C12EG4OH) do not reactivate the swarming
motility, and small changes in disaccharide hydrocarbon structures
can lead to large differences in the degree of reactivation of the
swarming motility of rhlA. These results suggest that the
rhamnolipids are likely a class of biological ligands that bind and
trigger one or more receptors that enables swarming motility. Based
on this inference, a rhlA mutant was used in a swarming experiment
to screen the saccharide-hydrocarbons for agonists that can
reactivate the swarming motility of rhlA. Such reactivation
suggests that the active agonist may share the same active site or
a remote allosteric site of the same receptor.
[0118] In contrast, the wild type P. aeruginosa produces the
rhamnolipids and is capable of swarming on soft agar gel
(.about.0.5% agar).19 Using wild type PAO1 in a swarming experiment
facilitated an antagonist assay, by which the presence and binding
of saccharide hydrocarbons causing an antagonism of the receptor
protein will be revealed by the inhibition of the swarming
motility. We note that saccharide hydrocarbons may also promote the
swarming motility, but such promotions are less unambiguous than
the inhibition for wild type PAO1. Such promotion will be
unambiguous in the agonist assay using the nonswarming mutant
rhlA.
[0119] Disaccharide hydrocarbons and rhamnolipids exhibit dual
functions of activating and inhibiting swarming motility of
Pseudomonas aeruginosa. FIG. 25 demonstrates swarming motilities of
wild type PAO1 and rhlA mutant on a soft agar gel, in which
disaccharide hydrocarbons and rhamnolipids were homogeneously
introduced. Disaccharide hydrocarbons were introduced at 85 .mu.M,
except for D.beta.C (4), for which results from 160 .mu.M are shown
here. For rhamnolipids, a commercially available agent that was
extracted from PAO1, which consists of a 5:1 mole ratio of
dirhamnolipid and monorhamnolipid, was used. Using an average
molecular weight of 626 g/mole, we studied the swarming motility
results of PAO1 and rhlA in the presence of a range of
concentrations of (1-30 .mu.M) rhamnolipids.
[0120] Four groups of behaviors of the swarming motilities in the
presence of DSHs at the concentration of 85 .mu.M in the soft agar
plate was observed. First, two saccharide hydrocarbons, D.beta.CDS
(18) and UD.beta.C (10), showed no significant effect on the
swarming motility of both wild type PAO1 and rhlA mutants (FIG.
25A). Second, two DSHs, De.beta.C (2) and F.beta.C (11), showed
weak activation of rhlA swarming motility and weak promotion of
swarming motility of wild type PAO1 (FIG. 25B). Here, results from
160 .mu.M of De.beta.C (2) were included; at 85 .mu.M, De.beta.C
(2) did not show a noticeable effect on the swarming motility of
both strains of the bacterium. Third, five DSHs, D.beta.M (1),
U.beta.C (3), D.beta.C (4), T.beta.C (5), and D.alpha.C (6),
impacted the swarming motility of wild type PAO1 by causing
significant amount of tendril formation in the swarming pattern
(FIG. 25C). Instead of swarming outward with a circular front, the
swarming bacteria start to form protrusions radially extending from
the center of the inoculation spot. DSHs (1) and (4) caused wild
type PAO1 to swarm with tendrils containing only straight
protrusion lines of swarming bacteria, whereas DSHs (3), (5) and
(6) caused the tendril to branching from the main radial lines of
the swarming bacteria, forming a self-similar, fractal-like pattern
(FIG. 25C). Three DSHs amongst the third group (FIG. 25C) (1), (3)
and (4) showed a strong reactivation of the swarming motility in
rhlA mutant with a swarming ring of bacteria having a similar
diameter as that by the control, wild type PAO1 without agents. Two
DSHs in this group, DSHs 5 and 6 showed no apparent reactivation of
rhlA swarming. Fourth, six DSHs, D.beta.G (8), D.alpha.R (9),
SF.beta.C (12), SF.alpha.C (13), SF.beta.M (14), and 2-OD.beta.C
(16), inhibited the swarming motility of wild type PAO1 and showed
no apparent effect on rhlA (FIG. 2D). The rest of the 3 DSHs,
D.beta.L (7), SF.beta.L (15) and 2-OD.alpha.C (17), have a poor
water-solubility and were not tested for effect on swarming
motilities. We note that when testing F.beta.C (11) at higher
concentration (160 .mu.M) with both PAO1 and rhlA, there was
complete abolition of bacterial growth on the soft-agar plate. In
comparison, 160 .mu.M of F.beta.C (11) did not inhibit the
planktonic growth PAO1 in LB media.
[0121] For the rhamnolipids extract (5:1 dirhamnolipid and
monorhamnolipid), at 10 .mu.M, an activation of .about.38% of the
swarming motility was observed on rhlA; and at 30 .mu.M, an
inhibition of .about.51% of swarming motility was observed for wild
type PAO1. These values were obtained by comparing to the swarming
area to that of wild type PAO1 under the same conditions without
any added agents in the soft agar plate (FIG. 25). In comparison,
at a single concentration 85 .mu.M, DSHs exhibited different
activities: some DSHs (group 2) promoted and activated swarming of
PAO1 and of rhlA mutant, respectively; some DSHs (group 3)
inhibited swarming of PAO1 but activated rhlA mutant's swarming,
while some DSHs (group 4) inhibited swarming motility of both
strains. Because rhamnolipids and DSH De.beta.C (2) exhibited dual
functions of activating and inhibiting the swarming motilities, we
speculate that disaccharide hydrocarbons, in general, possess these
dual-functions, and thus manifest different activities at a single
concentration due to the different binding abilities.
[0122] Disaccharide hydrocarbons and rhamnolipids exhibit "activity
reversal" that transition from swam-activating to swarm-inhibiting
as concentration increases. The screening of the DSHs on their
effect on swarming of wild type PAO1 and rhlA mutant indicated a
range of two opposite bioactivities--activation and inhibition of
swarming motilities. One of the most striking observations is that
DSHs in the third group (1), (3), (4), and rhamnolipids, activated
the swarming of rhlA mutant while inducing tendril formation in the
swarming pattern of wild type PAO1. The tendril formation appears
to be caused by the development of two phenotypes, the hyperactive
swarming phenotypes at the tip of the tendril, and the less
swarming active one at the stems and in the center of the spot.
Deziel and co-workers's screening showed the over-expression of 20
genes and down regulation of 121 genes by bacteria at tip of the
tendril in comparison with bacteria in the swarm center of the
swarming pattern.43 In fact, measuring the swarming area of control
wild type PAO1 (without any DSHs) gave an area of .about.24 cm2,
and that by DSH (4) which causes tendril formation, an area of and
.about.13 cm2, a value .about.46% smaller than the control (FIG.
25C). This result indicated that while the DSHs can activate
swarming motility, they can change the phenotype of the bacteria
and cause an overall apparent inhibition. Furthermore, DSH
De.beta.C (2) showed a concentration effect by which increasing
from 85 to 160 .mu.M caused an increase of .about.38% of the
reactivated swarming ring of rhlA with respect to swarming ring of
PAO1 without agents. These observations lead us to hypothesize
that, DSHs, and also rhamnolipids, are both cell signaling
molecules, for which different concentrations of these molecules
can modulate or regulate different bacterial behavior and control
different phenotypes. To explore this hypothesis, we studied
different concentration ranges of the three groups of DSHs (not
including the first group of DSHs, which had no effect of swarming
motility).
[0123] The effect of rhamnolipids extract, which consisted of a
mixture of dirhamnolipid and monorhamnolipid in 5:1 mole ratio, was
first explored. It was found that when the concentration was
adjusted to 5 .mu.M, the rhamnolipids started to activated the
swarming motility of rhlA mutant, and increases the size of the
swarming ring of PAO1 (FIG. 26). This positive effect of swarming
activation and promotion reached a maximum at 10 .mu.M with an
swarming area of about .about.9 cm2, whereas the swarming area for
the control PAO1 is .about.24 cm2. Surprisingly, this
activation/promotion effect started to decrease at higher
concentration of rhamnolipids for both rhlA mutant and wild type
PAO1. At 20 .mu.M or higher, the swarming area of rhlA became a
constant of about 1.1 cm2. At 30 .mu.M, a clear inhibition effect
on swarming was observed for wild type PAO1, with a swarming area
of .about.11 cm2. This observation indicated an apparent activity
reversal as the concentration was increased. Such an activity
reversal was also observed in the early discovery of quorum sensing
molecules, in which acetylated homoserine lactones (AHLs)
stimulated light production in Photobacterium fischeri and the
amount of light produced increased over the range 0.003-3 .mu.g/mL
and then decreased at higher concentrations for both natural and
synthetic AHLs.
[0124] The active DSHs (Group 2, 3 and 4 of FIG. 25) all exhibited
a trend of swarming promotion at low concentration and swarming
inhibition at high concentration. FIG. 26 shows the concentration
ranges of DSHs, in which there is an impact on the swarming
motility of wild type PAO1 and rhlA mutant. For any molecules, the
transition concentrations were different for the two strains (from
activating to non-activating for rhlA mutant, and from promotion to
inhibition for PAO1). Furthermore, the transition concentrations
were also different for different DSHs, and could be roughly
categorize into two groups, early and late transition at relatively
low and high concentrations (FIG. 27 & Table 1). Whereas
rhamnolipids and DSHs (14) and (12) exhibited low transition
concentrations at 10 .mu.M, 10 .mu.M and 8 .mu.M for rhlA; 20-30
.mu.M, 7.5 .mu.M, 10-20 .mu.M for PAO1, respectively; DSHs (4), (5)
and (8) exhibited these transition at higher concentrations at
45-56 .mu.M, 40 .mu.M and 35 .mu.M for rhlA; 20-30 .mu.M, 20-30
.mu.M and 35-50 .mu.M for wild type PAO1, respectively. Table 1
shows the different transition concentrations of DSHs and
rhamnolipids for rhlA mutant and wild type PAO1. In general, the
transition concentrations for activating rhlA mutant were slightly
lower than the transition concentrations for inhibiting wild type
PAO1 swarming. The agents that exhibited low transition
concentrations (12, 14, and rhamnolipids) activated smaller rings
in the swarming pattern of the rhlA mutant than that by the agents
with high transition concentrations (FIG. 27). This result suggest
that the less potent swarming inhibitors, (4), (5) and (8), were
more capable at activating the rhlA mutant in swarming motility. At
these transition concentrations between the two opposite
bioactivities, some DSHs induced tendril formation, some did not.
For rhlA mutant, only D.beta.C (4) induced tendril formation around
.about.56 .mu.M; while for PAO1 tendrils were induced by D.beta.C
(4) at 20 .mu.M, T.beta.C (5) at .about.30 .mu.M, and SF.beta.M
(14) at .about.7.5 .mu.M.
[0125] We also note that by measuring the swarming area of
activated rhlA mutant, as the concentration increased, DSHs (4) and
(5) appeared to cause another activation of swarming at about 160
.mu.M after first inhibition at around 50 .mu.M (FIG. 27). Swarming
motilities at additional concentrations supported this observation.
Together, these results indicate that both rhamnolipids and active
DSHs have the dual function of acting as an agonist by activating
the swarming motility at low concentrations, but as an antagonist
by inhibiting the swarming motility at high concentrations.
TABLE-US-00001 TABLE 1 Concentrations of DSHs at which they exhibit
activity reversal for swarming promotion of rhlA mutant and
swarming inhibition of PAO1. Compound rhlA PAO1 Rhamnolipids ~10
.mu.M ~20-30 .mu.M SF.beta.M (14) ~10 .mu.M ~7.5 .mu.M SF.beta.C
(12) ~8 .mu.M ~10-20 .mu.M D.beta.C (4) .sup.a~45-56 .mu.M ~20-30
.mu.M T.beta.C (5) ~40 .mu.M ~20-30 .mu.M D.beta.G (8) ~35-50 .mu.M
~50-60 .mu.M
[0126] Disaccharide hydrocarbons SF.beta.C (12), SF.beta.M (14) and
D.beta.C (4) dominated rhamnolipids at inhibiting the swarming
motility of PAO1. Examining the result indicated that at 10 .mu.M,
(14) is more effective than rhamnolipids at activating rhlA
swarming, a .about.14 cm2 swarming ring was observed for (14),
whereas only a .about.9 cm2 ring was observed for
rhamnolipids--about a .about.20% reduction. Furthermore, at 20
.mu.M, (12), (14), and (4) all exhibited stronger inhibition than
rhamnolipids at inhibiting the swarming motility of wild type PAO1.
Comparing (12) and rhamnolipids at 20 .mu.M, swarming of wild type
PAO1 was completely inhibited by (12) with swarm ring of .about.1.2
cm2 (.about.5%) as compared to 24 cm2 (100%) for PAO1 without any
DSHs. At concentration lower than 20 .mu.M, all three DSHs also
exhibited stronger inhibition of swarming motility of wild type
PAO1 than rhamnolipids. Whereas (14) and (4) induced tendril
formation in wild type PAO1, rhamnolipids were not able induce such
a phenotypic bifurcation effect. These results indicated that (12),
(14) and (4) not only suppressed the effect of rhamnolipids
secreted in situ by wild type PAO1, but also had a stronger
inhibition effect when compared to the externally added
rhamnolipids in the same concentration range.
[0127] Competition assays suggests SF.beta.C (12) is a stronger
ligand than D.beta.C (4). Presumably, both of the DSHs that
activate and inhibit swarming motility of P. aeruginosa target the
same putative receptor of rhamnolipids. To further explore this
possibility, a competition assay was conducted by introducing both
SF.beta.C (12) and D.beta.C (4) at different concentrations into
the soft agar gel and observed the swarming motility of rhlA
mutant. Both SF.beta.C (12) and SF.beta.M (14) had inhibited
swarming of PAO1, which suggested that either SF.beta.C (12) and
SF.beta.M (14) block the receptor site of the in situ produced
rhamnolipids or they bound to an allosteric site of the receptor
remotely, abolishing the receptor's binding ability to
rhamnolipids. As rhlA mutant produces no rhamnolipids, this mutant
provides an ideal platform for examining the competition between
different synthetic DSHs for the putative receptor. At 40 .mu.M,
D.beta.C (4) activated the swarming motility of rhlA mutant. At 20
.mu.M, SF.beta.C (12) did not activate the swarming motility of
rhlA mutant. It is not entirely clear if SF.beta.C (12) is
inhibiting the receptor in rhlA or SF.beta.C (12) has no effect at
this concentration on rhlA mutant, although SF.beta.C (12) did
inhibit the swarming activity of wild type PAO1 at 20 .mu.M. Thus,
in this competition assay, the concentration of D.beta.C (4) was
kept at 40 .mu.M, and the concentration of SF.beta.C (12) was
increased incrementally to 0.5, 10, 20, and 40 .mu.M (FIG. 28). The
swarming motility showed that up to 10 .mu.M, SF.beta.C (12) did
not interfere with the swarming reactivation of rhlA mutant by 40
.mu.M of D.beta.C (4). But when the concentration of SF.beta.C (12)
is increased to 20 .mu.M, the swarming motility is completely
inhibited while 40 .mu.M of D.beta.C(4) was still present. This
result is consistent with the effect of SF.beta.C (12) on wild type
PAO1, for which 0.5 to 10 .mu.M promoted swarming and higher
concentrations inhibited swarming. This competition assay suggests
that SF.beta.C (12) is a stronger binding ligand (with antagonistic
activity) than D.beta.C (4), and that molecules showing early
transition (at low concentration) of swarming activating to
swarming inhibition are likely stronger binding ligands than "late"
transition molecules.
[0128] DSHs showed a similar trend of structural-activity
correlation for antiadhesion and antibiofilm (inhibition and
dispersion) activities. As rhamnolipids are known to be critically
important for building channeled biofilm by P. aeruginosa, and
under conditions that cause over production of rhamnolipids,
bacteria appears to be dispersed from the biofilm. These results
suggest that the disaccharide hydrocarbons presented in this study
may also have an impact on the biofilm formation. On the other
hand, N-acetylated disaccharides hydrocarbons have been are found
to inhibit bacteria adhesions on stainless steel and on polystyrene
surfaces. These findings suggest that disaccharide hydrocarbons may
also have an impact on the adhesion and biofilm formation by P.
aeruginosa.
[0129] The effect that DSHs have on the adhesion of PAO1-EGFP on
polystyrene surface (microtiter plate) was also examined. PAO1-EGFP
is a P. aeruginosa strain that constitutively expresses green
fluorescent protein. In the adhesion assays, PAO1-EGFP was grown in
microtiter plate for 24-hours in LB-media with (160 .mu.M) or
without DSHs. After 24-hours, the fluorescent signal obtained from
surface adhered PAO1-EGFP was measured. Percent reduction in
fluorescence was calculated by comparing fluorescent signal from
PAO1-EGFP grown in LB-media with the signal from wells that had
DSHs (FIG. 29A). To study the effect of DSHs on biofilm formation,
Pseudomonas aeruginosa (PAO1) was grown in LB-media with (160
.mu.M) or without DSHs at 37.degree. C. under static conditions
within wells of microtiter plates. After 24 hours, the surface
attached biofilms adhering to the bottom of the wells were stained
with crystal-violet dye and the biofilm was quantified by measuring
absorbance at 600 nm (OD600). The percent inhibition was calculated
by comparing biofilm content of wells with no added agents
(control) with the biofilm content from wells that had 160 .mu.M of
DSHs (FIG. 29A). Similar to inhibition, the surface attached
biofilm after dispersion with DSHs was quantified with
crystal-violet staining and absorbance measurement at 600 nm,
except that for dispersion, biofilm was first grown for 1 day in
just LB-media without any DSHs after which DSHs were added and
plates were incubated for additional 24 hours followed by
quantification (FIG. 29B).
[0130] A screening of the structures shows that, at 160 .mu.M,
SF.beta.C (12), SF.alpha.C (13), D.beta.C (4) and SF.beta.M (14)
exhibited more than 60% of inhibition of bacterial biofilm
formation on the microtiter wells; D.beta.M (1), D.beta.G (8),
T.beta.C (5), U.beta.C (3), D.alpha.R (9), 2-OD.beta.C (16) and
D.alpha.C (6) showed between 50 to 20% of biofilm inhibition, and
De.beta.C (2), D.beta.L (7), UD.beta.C (10), F.beta.C(11) and
D.beta.CDS (18) showed insignificant biofilm inhibition FIG. 29A.
Interestingly, an overall similar trend was observed for bacterial
adhesion inhibition assay at the same concentration, with the
exceptions that (9) and (18) did not show significant biofilm
inhibition, but showed 63% and 29% adhesion inhibition for P.
aeruginosa (FIG. 29A). Additionally, the biofilm formed by
PAO1-EGFP on stainless steel coupons grown in LB-media for 24-h
supplemented with 5 DSHs, SF.beta.C (12), SF.alpha.C (13), D.beta.C
(4), De.beta.C (2) and D.beta.L (7) and with no added agents were
viewed under confocal laser scanning microscope (CLSM). PAO1-EGFP
constitutively produces green fluorescent proteins in culture that
can be viewed under CLSM. CLSM micrographs show that 160 .mu.M of
SF.beta.C (12), SF.alpha.C (13) and D.beta.C (4) decreased the
fluorescence signal significantly as compared to the steel coupon
grown in just LB-media indicating that a lesser bacterial cell
density (and hence lesser biofilm) on the steel coupons. Also, the
fluorescence thickness as indicated on the Z-axis of these
micrographs indicate a much thinner bacterial coverage on steel
coupons that were placed in media containing potent DSHs. Contrary
to this, steel coupons placed in LB-media with two DSHs, De.beta.C
(2) and D.beta.L (7) had bacterial surface coverage comparable to
that of control. The findings of the CLSM-based assays were
consistent with the results of CV-dye based assay for biofilm
inhibition.
[0131] Dispersion of already formed biofilm is more challenging
than inhibiting the formation of biofilm by introducing agents at
the beginning of the assay. But dispersing preformed biofilm is
more relevant to applications for which has already formed. The
DSHs were also screened for their ability to disperse a 24 hour old
biofilm. The activities for inhibition of biofilm formation and
bacterial adhesion and for dispersion of biofilm were plotted with
the same order of agents in FIG. 29A and FIG. 29B, respectively.
With a few exceptions, an overall similar trend of
structure-activity correlation was observed between biofilm
inhibition, adhesion inhibition and biofilm dispersion. The
percentages of biofilm remaining in dispersion assays were always
higher than those in the biofilm inhibition assays. The potent
DSHs, SF.beta.C (12), SF.alpha.C (13), SF.beta.M (14) and D.beta.C
(4) that exhibited strong inhibition of biofilm formation also were
the most potent dispersing agents that dispersed more than 60% of
the 24-h old biofilm. Other DSHs, except D.beta.CDS (18), dispersed
between 20-60% of 24-h biofilm. The D.beta.CDS (18) appeared to
have promoted biofilm formation instead of causing dispersion of
24-h biofilm. The exception that broke the trend of
structure-activity correlation between inhibition and dispersion
was 2-OD.beta.C (16) with not a significant difference in the
magnitude.
[0132] Structure-activity study reveals important structure
features for strong bioactivities. Examining the structures of DSHs
and their bioactivities revealed a set of important structural
features that is common for influencing all three bacterial
behaviors swarming activation, biofilm inhibition and adhesion
inhibition. First, the size and stereochemistry is important for
activity. Glucoside, D.beta.G (8); mono-rhamnoside, D.alpha.R (9),
and .beta.-cyclodextrin, D.beta.CDS (18)-based agents, all had weak
anti-biofilm activity whereas disaccharide stereochemistry, maltose
and cellobiose showed strong activities for all three processes.
Second, for disaccharide hydrocarbons, cellobiose having a
stereochemistry of Glc.beta.(1.fwdarw.4)Glc appeared to be more
active than maltose stereochemistry-Glc.alpha.(1.fwdarw.4)Glc.
Lactose, Gal.beta.(1.fwdarw.4)Glc, based molecule, D.beta.L (7),
exhibited low activities, but had a low solubility in water. Thus,
a structural correlation conclusion cannot be drawn. Third, a chain
length of twelve carbons appeared to be optimum for showing maximum
activities for disaccharide hydrocarbons. For example at 160 .mu.M,
De.beta.C (2) and U.beta.C (3) having 10 and 11 carbons in the
aliphatic chain showed no and insignificant anti-biofilm activity,
respectively, whereas D.beta.C (4) having a 12-carbon aliphatic
chain exhibited .about.62% inhibition of biofilm. Most importantly,
bulky aliphatic chain (3, 7, 11-trimethyl-dodecane) enabled DSHs
(SF.beta.C (12) and SF.beta.M (12) to be most potent at controlling
all three bacterial activities. The structure also dominated over
natural rhamnolipids that are produced in situ by P. aeruginosa
during both inhibition and swarming activities of the bacteria. In
comparison, DSHs F.beta.C (11) and UD.beta.C (10) having
unsaturated hydrocarbons that were relatively smaller than the
saturated version showed .about.12% and .about.17% inhibition at
160 .mu.M. Interestingly, DSHs 2-OD.beta.C (16) and 2-Od.alpha.C
(17), having double aliphatic chains that were similar to
rhamnolipids were not active for biofilm inhibition or dispersion
(FIG. 29A) but 2-OD.beta.C (16) did exhibit inhibition of PAO1
swarming.
[0133] Dose-dependent study revealed "activity reversal" of
rhamnolipids on biofilm inhibition. Identifying the important
common structures for all three bioactivities, dose-dependent
studies on biofilm inhibition and biofilm dispersion with selected
DSHs that exhibited high activities, SF.beta.C (12), SF.alpha.C
(13), SF.beta.M (14) and D.beta.C (4; only inhibition) were
performed. These dose-dependence were compared to that by
rhamnolipids extract. For biofilm inhibition assays, the
rhamnolipids extract exhibited an increase in biofilm inhibition as
the concentration was increased, but surprisingly reaching a
maximum of .about.65% inhibition at 42 uM, and the inhibition %
drastically decreased as the concentration was increased further.
For instance, at 85 .mu.M, the inhibition by rhamnolipids extract
decreased to 46%. Further increase in concentration actually
promoted biofilm formation, at 160 .mu.M, 114% of PAO1 biofilm was
observed in comparison with biofilm without agents (100%). This
activity reversal of rhamnolipids on biofilm inhibition was not
observed for the other four DSHs studied (FIG. 30). All four DSHs,
SF.beta.C (12), SF.alpha.C (13), SF.beta.M (14) and D.beta.C (4)
showed an increase of biofilm inhibition, followed by a plateau.
DSH SF.beta.C (12) showed a half maximal inhibitory concentration
(IC.sub.50) .about.9.9 .mu.M; SF.alpha.C (13) .about.32 .mu.M;
D.beta.C (4) .about.72 .mu.M; and SF.beta.M .about.(14) 102 .mu.M.
Because of the activity reversal, IC.sub.50 for the rhamnolipids
was not calculated. These results also indicate that SF.beta.C (12)
was more potent than rhamnolipids at inhibiting biofilm formation,
whereas as the other three DSHs were more effective than
rhamnolipids only at higher concentrations. The lack of biofilm
inhibition activity of rhamnolipids at higher concentration also
appeared to be consistent with the results that disaccharide-based
lipid, 2-OD.beta.C (16) was not effective at inhibiting the biofilm
formation, but inhibits the swarming activities of PAO1.
[0134] Active DSHs dominates rhamnolipids in dispersing preformed
biofilm. For dispersion assays, rhamnolipids, however, were not
effective for dispersing the 24-h biofilm. About 80% of the biofilm
remained (.about.20% dispersion) after rhamnolipids were introduced
to the already formed biofilm (24-h old). As the concentration of
rhamnolipids increased, the amount of biofilm actually increased
(FIG. 31). In comparison, the three active DSHs, SF.beta.C (12),
SF.alpha.C (13) and SF.beta.M (14), showed conventional biofilm
dispersion activities, by which the percentage of dispersed biofilm
reached a plateau as the concentration increased. This
dose-dependence study showed that DSH SF.beta.C (12) exhibited a
half-maximal dispersion concentration (DC.sub.50) of .about.44
.mu.M, SF.alpha.C (13) .about.89 .mu.M and SF.beta.M (14)
.about.126 .mu.M. These DC.sub.50 were high in comparison with
other known agents that also disperse biofilm formation.
[0135] Because the structures of these class of molecules are also
surfactants, it is not clear before these results whether any
activity from these molecules is due to a mere physical effect of
the surfactant properties or a biological effect, by which a
receptor on bacterial surface is being blocked by the molecules
leading to either an antagonistic or an agonistic effect. The same
question was also posted for rhamnolipids. Several aspects of the
results in this work suggest that both rhamnolipids and DSHs likely
are ligands for certain receptors on the bacteria. First, generic
surfactants such as SDS and C12EG5OH did not incur any bioactivity
for both swarming reactivation and biofilm inhibition. Second, only
selected DSHs activated the swarming motility of the nonswarming
mutant rhlA, while all the DSHs shared surfactant properties.
Third, all the active DSHs and rhamnolipids exhibited "activity
reversal" in controlling the swarming motility of mutant rhlA, and
thus showed the dual bio-function of activating and inhibiting
swarming motility at different concentrations. This "activity
reversal" appears to be a recorded but overlooked phenomenon of the
classical bacterial signaling molecule,
N-(3-oxohexanoyl)-3-aminodihydro-2(3H)-furanone, for which the
receptor was later determined. 51-53 We note that the two important
quorum sensing systems that control P. aeruginosa are las and rhl
circuits. We conducted the antagonistic gene reporter assay to
investigate the effect of two DSHs, SF.beta.C (12) and SF.alpha.C
(13) on las and rhl quorum sensing circuits of Pseudomonas
aeruginosa, by using two reporter strains, PAO1/plasI-LVAgfp and
PAO1/prhlI-LVAgfp. The two reporter strains secrete natural
autoinducers that bind receptor proteins, lasR and rhlR activating
either lasI or rhlI genes respectively. The activation of either
lasI or rhlI genes in turn expresses green fluorescent protein that
is encoded by the plasmid.57 The results indicated that neither
SF.beta.C (12) nor SF.alpha.C (13) caused a significant change in
the fluorescence of two reporter strains, PAO1/plasI-LVAgfp and
PAO1/prhlI-LVAgfp (see Supporting Information, S6), suggesting that
DSHs do not interfere with the quorum sensing circuits of
Pseudomonas aeruginosa.
[0136] Addition of pili peptide reduces the biofilm inhibition by
DSHs. Kohler and coworkers suggested that type IV pili are
critically important for rhamnolipids-enabled swarming motility of
P. aeruginosa. On the other hand, Randall and coworkers also showed
that the adhesion of P. aeruginosa to epithelial cell surface and
polystyrene surfaces is mediated by pilus protein. While PA pili
specifically recognize D-GalNac(1.fwdarw.4) -.beta.-D-Gal
disaccharide ligand, the pili assisted epithelial cell binding
domain is located between residues 128-144 on the C-terminal region
of PilA, the pili structural protein. It has been demonstrated that
among different strains of P. aeruginosa, this 17 amino acids
region of PilA is semi-conserved and usually has a di-sulfide loop
formed by the two conserved cysteines. To explore a putative
receptor for the DSHs, the minimal 17 amino acids residues sequence
peptide that contained residues known to exist on C-terminal region
of P. aeruginosa PAO strain pilus [N'-Ac-ACKSTQDPMFTPKGCDN-OH-C']
was synthesized. This synthetic peptide is known to recognize
disaccharide GalNAc-Gal. The peptide was synthesized and the two
cysteines at positions 129 and 142 were air oxidized to form an
intra-molecular disulfide loop, resembling the natural cell binding
domain. This looped sequence was named the pili peptide, although
previously it had been described as PAO(128-144) oxidized. As a
control, a scrambled pili peptide was synthesized by changing the
cysteines at positions 129 and 142 position to alanines,
[N-Ac-AAKSTQDPMFTPKGADN-OH-C'], which has been described previously
as PAO(128-144) (C129A/C142). We found that SF.alpha.C (13) at 85
.mu.M alone inhibited about 70% of PAO1 biofilm, presence of
control scrambled pili peptide (85 .mu.M) along with SF.alpha.C
(13; 85 .mu.M) caused an inhibition of 65% of the biofilm, whereas
presence of the pili peptide (85 .mu.M) and SF.alpha.C (13; 85
.mu.M) reduced the inhibition to 30% (FIG. 32). These results
suggest that the pili peptide likely interferes with the binding
between the pili receptor and SF.alpha.C (13) whereas the control
scrambled pili peptide had little inference, and that pili protein
is a potential receptor for DSHs.
[0137] Comparing the effect of DSHs on swarming motility and
biofilm inhibition reveals that inhibiting biofilm formation had a
more stringent structure requirement than activating the swarming
motility. For example, De.beta.C (2), U.beta.C (3), D.beta.G (8)
and T.beta.C (5) all activated the swarming motility of rhlA
mutant, but did not show significant inhibition of PAO1 biofilm.
DSHs that were potent PAO1 biofilm inhibitors, SF.beta.C (12),
SF.alpha.C (13) and SF.beta.M (14) were also inhibitors for PAO1
swarming motility. But not all PAO1 swarming inhibitors, such as
2-OD.beta.C (16) and D.alpha.R (9) exhibited good anti-biofilm
activities. The DSHs that did not show anti-biofilm activities,
including De.beta.C (2), UD.beta.C (10), F.beta.C (11) and
D.beta.CDS (18) exhibited no apparent effect on PAO1 swarming. For
these agents, either there is no binding between these agents and
the putative receptor or the binding did not trigger a signaling
effect. Several DSHs, including D.beta.M (1), U.beta.C (3),
D.beta.C (4), T.beta.C (5), D.alpha.C (6) and SF.beta.M (14)
induced tendril formation in the swarming pattern of PAO1, which by
itself did not form tendril. These DSHs are likely causing a
bifurcation of PAO1 into two phenotypes at the same time; this
phenomenon is an ongoing subject of our study.
[0138] Overall, different stereochemistries of disaccharides were
effective for both biofilm and swarming inhibition, but large
oligosaccharide groups such as .beta.-cyclodextrin rendered the
molecule inactive. In contrast, the structural details of the
aliphatic chains incurred a relatively more significant impact for
high activity on controlling the swarming motility and biofilm
formation. In particular, bulky aliphatic chain involving 3, 7,
11-trimethyl-dodecane caused the highest activities among all the
DSHs for both cellobiose and maltose stereochemistry.
[0139] A class of disaccharide hydrocarbons with different
stereochemistries and aliphatic chain structures were studied for
their ability to activate and inhibit the swarming motility of P.
aeruginosa and its nonswarming mutant rhlA, and to inhibit the
bacterial adhesion and biofilm formation on polystyrene surfaces.
Among the eighteen DSHs, a common set of structures was found to be
active for controlling all these bioactivities of P. aeruginosa.
These DSHs included both cellobiose and maltose stereochemistries,
along with a bulky hydrocarbon chains comprised of 3, 7,
11-trimethyl-dodecane--a structural moiety obtained by
hydrogenation of farnesol molecules. For the influence on swarming
motility, all active DSHs exhibited dual functions that transition
from swarm-activating to swarm-inhibiting as the concentration was
increased in the soft agar plate. The DSHs with low transition
concentrations were also strong anti-adhesion and anti-biofilm
agents. This "activity reversal" in controlling swarming motility
was also observed for rhamnolipids extracts that consist of
dirhamnolipid and monorhamnolipid in 5:1 mole ratio. In contrast,
whereas active DSHs showed a plateau behavior in dose dependent
study on biofilm inhibition, rhamnolipids showed "activity
reversal", for which the biofilm inhibition activity decreased as
the concentration is increased. Three DSHs, saturated
farnesyl-.beta.-cellobioside SF.beta.C, (12) saturated
farnesyl-.alpha.-cellobioside SF.alpha.C (13) and saturated
farnesyl-.beta.-cellobioside SF.beta.M, (14), exhibited dominating
effect over rhamnolipids at inhibiting the swarming motility of
wild type P. aeruginosa. Furthermore, while active DSHs were
effective at dispersing 24-h old biofilm, rhamnolipids did not have
a significant influence on the preformed biofilm. Because this
class of DSHs do not inhibit the growth of the bacteria, their
activities at inhibiting and dispersing the biofilm formation have
the potential for further application development that does not
invoke drug resistance.
[0140] Standard solvents, and media were purchased from commercial
sources (Sigma-Aldrich, Fisher, Acros) and used as received. Column
chromatography was performed using Silicycle, Silica-P Flash Silica
Gel with 40-6 .mu.mesh size.
[0141] The synthesis of all saccharide-hydrocarbons was done
according to Scheme 1 using a literature reported synthetic route
with some modifications. Briefly, disaccharides (cellobiose,
maltose, lactose) and monosaccharide (rhamnose) were
per-O-acetylated using a binary mixture of AcBr/AcOH with
simultaneous mono-bromination at anomeric position. Glycosidation
of aceto-bromo sugars with aliphatic alcohols was done using acid
catalysts (FeCl3 or Hg(CN)2 and in either MeCN (for .beta.-anomer
as major product) or MeNO2 (for .alpha.-anomer) as solvent. The
.alpha./.beta. anomers were resolved by column chromatography and
further deacetylated by methanolic MeONa followed by neutralization
by H+amberlite resins to pH .about.6.5 (Zemplem deacetylation).
Synthesis of cyclic-hepta-saccharide hydrocarbon was done according
to Scheme 2.
[0142] R95 (5:1 di-rhamnolipids:mono-rhamnolipids, 95% pure, avg
molecular weight .about.626 g/mole) was obtained from Agae
technologies and used as purchased. Water was purified using a
Millipore Analyzer Feed System. Flat-bottomed polystyrene 96-well
microtiter plates (untreated) (Costar 3370) were used to perform
all biological assays (except assays that required fluorescence
measurements). For measuring fluorescence, flat-bottomed 96-well
microtiter plates with black walls (.mu.Clear, Greiner-One 655096)
were used. Measurement of absorbance (at 600 m, OD600) was usual
performed with 200 .mu.L of culture-media in microtiter plates on a
Biotek ELx800 .TM. absorbance microplate reader and data was
analyzed with Gen5.TM. data analysis software. Quantification of
biofilm was done using standard crystal violet (CV) dye based
static-biofilm assay. The quantified biofilm reported is the
surface attached (SA) biofilm at the well bottoms, for each assay,
total biofilm was also quantified (not reported here) and the trend
of percent biofilm inhibition for SA and total were the same. For
quantifying fluorescence, Synergy 2 multi-mode microplate reader
with Gen5 data analysis software was used to detect GFP signal at
an excitation wavelength of 500 nm and emission wavelength of 540
nm. For positive controls, wells with no-agents were used. For each
biological assay, at least 3 repeats (with similar results) were
done and data represented here for each agent is the average of
readings from 4-replicates wells from one single experiment. Graphs
were plotted using Microsoft Excel (2007) and half maximal
inhibitory concentration for both biofilm inhibition and dispersion
(IC.sub.50 and DC.sub.50) were calculated by fitting linear values
into a logarithmic equation, y=mln(x).
[0143] Pseudomonas aeruginosa strains PAO1 and PAO1-EGFP were
obtained from Dr. Guirong Wang (Upstate Medical University).
Pseudomonas aeruginosa transposon mutant strain PW6886
(rhlA-E08::ISphoA/hah) was obtained from two-allele library. PAO1
(plasI-LVAgfp), PAO1 (prhlI-LVAgfp) strains were prepared in lab
using previously reported protocol. The plasmid plasI-LVAgfp and
prhlI-LVAgfp were obtained from Dr. Hiroaki Suga (The University of
Tokyo) and were maintained by adding 300 .mu.g/mL of carbenicillin
in culture media. Freezer stocks of all strains were stored at
-80.degree. C. in LB media with .about.20% glycerol. All strains
were grown at 37.degree. C. in a rotary-shaker (at 250 rpm) in
Luria_Bertaini (LB) media (composition of LB media, 10 g/L
tryptone, 5 g/L yeast extract, and 10 g/L NaCl). All biofilm
inhibition, dispersion and adhesion assays were performed in
LB-media and plates were incubated at 37.degree. C. for 24 hours
(except for adhesion assay, 3 hours) under static conditions.
[0144] Stock solution of disaccharide hydrocarbons (DSHs). Stock
solution (.about.11.5 mM) of all synthesized and commercially
available DSHs were prepared in autoclaved water and further
sterilized by filtering through cellulose acetate syringe filter
(0.2 .mu.m pore size, GVS filter technology) into sterilized vials.
The vials containing sterilized DSHs stocks were capped and stored
at -20.degree. C. and thawed prior to each use. Appropriate volume
of sterile water was added to positive controls (no agents) in all
assays to eliminate the effect of water. Stock solution of D.beta.L
(7), SF.beta.L (15) and 2-OD.alpha.C (17) were prepared in sterile
water and 200 proof EtOH in 90:10 ratio. For positive controls
involving D.beta.L (7), SF.beta.L (15) and 2-OD.alpha.C (17)
appropriate volumes of 90:10, H2O:EtOH was added.
[0145] Swarm agar plates were based on M8 medium supplemented with
0.2% glucose, 0.5% casamino acid, 1 mM MgSO4 and 0.5% Bacto agar.17
For each set of experiment all the swarm plates were poured from
same batch of agar and allowed to dry for 1 h before inoculation of
bacteria. Bacterial culture (3 .mu.l) (wild type PAO1 or rhlA
mutant) with OD600 between .about.0.5 was inoculated on the
solidified agar plates. Swarm agar plates were incubated at
37.degree. C. for 12 h and then incubated for additional 12 h at
room temp. Chronological images of swarm plates were taken using a
camera. To measure swarming area, sizes of the petridish in all
swarming images were adjusted to a pre-assigned diameter and images
were printed on copying paper (Xerox-business 4200). Weight of a 1
cm.sup.2 copy paper square was roughly .about.8.0 mg. swarming
images on the copy paper were cut and weighed, the swarm area was
calculated by dividing the weight of image by weight of 1 cm.sup.2
square (8 mg).
[0146] Antimicrobial activities of disaccharide hydrocarbons
against planktonic growth. Overnight inoculum of PAO1 was
sub-cultured to an OD600.about.0.1 and then aliquoted (200 .mu.L)
into the wells of a micro-titer plate and DSHs (.about.160 .mu.M)
were then added to assigned wells. Wells with no-agents were
created as a positive control. The micro-titer plate was then
placed in a rotary shaker (250 rpm at 37.degree. C.) and optical
density was measured after regular intervals of time using Biotek
ELx800 .TM. absorbance microplate reader (BioTek Instruments, Inc.,
Winooski, Vt.) using Gen5.TM. data analysis software. The OD600
values were taken in sterile conditions over 24 h. The OD600 values
were plotted against time to obtain a growth curve for planktonic
growth with or without agents.
[0147] Crystal violet (CV) based-biofilm inhibition assay
Inhibitory effect of all synthesized disaccharide hydrocarbons on
biofilm formation of Pseudomonas aeruginosa (wild type PAO1) was
determined by static biofilm inhibition assay using crystal violet
staining Overnight culture of PAO1 were sub cultured in LB media to
OD600 of 0.01 and further the OD600 was allowed to reach
.about.0.1. The bacterial sub-culture (200 .mu.L) was then added
into the wells of a microtiter plate. Predetermined concentrations
of DSHs were added to respective wells containing sub culture by
pipetting out fixed volume of sterile aqueous stock solutions
(except D.beta.L (7), SF.beta.L (15) and 2-OD.alpha.C (17), which
were introduced as stocks prepared in H2O and EtOH (90:10)
solutions). The microtiter plates were then wrapped in GLAD Press
n' Seal.RTM. (saran wrap) and further incubated under stationary
conditions for 24 h at 37.degree. C. After 24 h incubation, the
culture media was discarded by pipetting carefully (without
disrupting biofilm), the plate was then washed once with sterile
water (200 .mu.L) and allowed to dry at 37.degree. C. for .about.30
mins. Microtiter plates were then stained with 0.1% aqueous
solution of crystal violet (CV) (200 .mu.L) at rt for .about.30
mins. Following this, the CV stain was removed and plate was again
washed with sterile water (200 .mu.L). Resultant CV stain in the
well was then solubilized by adding 100 .mu.L of 30% AcOH solution
(in water) and mixing gently by pipetting. The surface attached
(SA) biofilm was quantified by measuring OD600. Following this,
additional 115 .mu.L of CV solution was added to each well to
quantify total biofilm. Negative control lane, wherein no biofilm
was formed was also stained with CV and its OD600 reading was
subtracted from each well that contained culture media with DSH or
without DSH. The percent inhibition was calculated by the comparing
of the OD600 for biofilm grown in the absence of compound (No
agent) versus biofilm grown in the presence of DSH under identical
conditions.
[0148] Overnight culture of PAO1-GFP (supplemented with 300
.mu.g/mL of carbenicillin) was subcultured at 250 rpm and
37.degree. C. to an OD600 of 0.01 in LB medium. When the OD600 of
subculture reached .about.0.1, aliquot of the subculture (200
.mu.L) was transferred to the wells of black microtiter plate and
DSH (resultant conc. .about.160 .mu.M) were added to assigned
wells. Positive control were created that had no DSHs but
appropriate volume of sterile water. The black well 96-well plate
was wrapped in GLAD Press n' Seal.RTM. and then incubated at
37.degree. C. for 2 h. Following incubation for 2 h, the bacterial
culture from the wells was discarded and each well was washed once
with saline water (0.85 w/v % aqueous NaCl solution). Aliquot of
fresh LB medium (200 .mu.L) was then added into the wells and
fluorescence was measured at an excitation wavelength of 500 nm and
an emission wavelength of 540 nm by using a Synergy 2 multi-mode
microplate reader with Gen5 data analysis software. Signal from
wells containing just LB medium was subtracted from all the
wells.
[0149] Confocal laser scanning microscopy (CLSM) based biofilm
inhibition assay. Steel coupons (.about.1.times.1 cm) were washed
with EtOH, dried under a stream of nitrogen and then sterilized in
an autoclave. Overnight culture of PAO1/EGFP (OD600.about.1.0)
grown in LB media (supplemented with 300 .mu.g/mL of
carbenicillin), was sub-cultured to OD600.about.0.01 and then the
OD600 was allowed to reach .about.0.1. Aliquot (600 .mu.L) of
PAO1/EGFP subculture OD600.about.0.1 was then added into the wells
of a 24-well micro titer plate. DSHs were then introduced into the
assigned wells to reach a resultant concentration of 160 .mu.M.
Bacterial sub-culture with no agent served as positive control.
Sterile steel coupons were then placed into the wells and the
plates were wrapped with saran wrap and incubated for 24 h at
37.degree. C. under static conditions. After 24 hours, the steel
coupons were removed and washed by dipping in PBS buffer (2 washes)
and the dried by holding with a force p and gently dabbing the edge
on a bleached paper (KIM wipes). The dried steel coupons were
viewed under confocal laser scanning microscope (CLSM). Images were
taken at 4 random locations on each steel coupons. Z-stacked images
were also taken to determine the relative thickness of the
biofilm.
[0150] Crystal violet based assay for quantifying dispersion of
1-day old PAO1 biofilms were Plate for set up similar to inhibition
assay with a slight modification. Wells of the micro-titer plate
was inoculated with (200 .mu.L) bacterial subculture (PAO1
subculture OD600.about.0.1). The plates were incubated at
37.degree. C. for 24 hours to allow biofilm formation. After 24
hours, bacterial culture was gently pipetted out and appropriate
volume of DSH were added followed by addition of fresh LB medium
(200 .mu.L). The microtiter plate was incubated for an additional
24 hours at 37.degree. C., following which, similar to work up of
inhibition assay, culture was removed, wells were washed and CV-dye
was introduced, followed by washing and destaining with 30% AcOH
solution in water and quantification at 600 nm. Here again, only
the surface attached biofilm dispersed was reported by comparing
wells with DSHs with wells with no agent.
[0151] Dose dependence assays for biofilm inhibition and
dispersion. DSHs that were most potent at 160 .mu.M (exhibiting
>60% biofilm inhibition and dispersion) were selected for
determining the dose dependence. Both for biofilm inhibition and
dispersion, predetermined volumes of DSH stocks were added to the
assigned wells to span a concentration range between 0.5 .mu.M-170
.mu.M. Percent biofilm inhibition of dispersion was calculated by
comparing to wells with no agent. The percent biofilm inhibition or
dispersion was plotted versus concentration of DSHs. The linear
values were logarithmically curve fitted using equation y=mln (x).
The half maximal inhibitory concentration for both biofilm
inhibition and dispersion (IC.sub.50 and DC.sub.50) were thus
obtained.
[0152] Strains PAO1/plasI-LVAgfp and PAO1/prhlI-LVAgfp were grown
overnight in LB-media that was supplemented with 300 .mu.g/mL
carbenicillin and after 16 hours, the bacteria were sub cultured in
LB media (supplemented with 300 .mu.g/mL carbenicillin) to OD600 as
.about.0.01. The OD600 was allowed to reach .about.0.1 and then 200
.mu.L of culture was aliquoted into the wells of 96-well microtiter
plate. DSHs were then added to assigned wells at two different
concentrations (85 .mu.M and 160 .mu.M). No agent was added to the
well for positive control. The plates then incubated at 37.degree.
C. for 24 h in a rotary shaking incubator (250 rpm). The culture
from each well was then transferred to corresponding wells of a
black walled flat-bottom, 96-well plate (.mu.Clear, Greiner-One
655096). The fluorescence and OD absorbance in each well was
measured by Synergy 2 multi-mode microplate reader with a Gen5 data
analysis software. Background signals from LB medium were deducted
from all samples. Fluorescence was measured at an excitation
wavelength of 500 nm and an emission wavelength of 540 nm.
[0153] Synthesis of oxidized pili peptide PAO(128-144)ox and
scrambled pili peptide PAO(C129A/C142A)Scrambled. Synthesis of the
peptides were done according to previously reported procedure.
Fmoc-based solid phase peptide synthesis was used and the
purification was done by reversed-phase high performance liquid
chromatography (HPLC). Air oxidation was employed to oxidize the
two cysteines residues in PAO(128-144) sequence to generate a
di-sulfide linkage. For this a dilute solution (6 mg/4 mL) of
unoxidized pili peptide was made PBS buffer (pH.about.7.8)
containing 10% DMSO, and solution was stirred in air and monitored
by reversed phase HPLC and MALDI-TOF. Following the oxidation of
pili peptide, purification was done over reverse phase HPLC.
PAO(128-144)ox sequence is
N'Ac-A-C-K-S-T-Q-D-P-M-F-T-P-K-G-C-D-N-OH-C'. While for generating
scrambled pili peptide, cysteines at positions 129 and 142 were
replaced with alanines to generate the following sequence; PAO
(C129A/C142)Scrambled;
N'Ac-A-A-K-S-T-Q-D-P-M-F-T-P-K-G-A-D-N--OH-C'.
Example 5
[0154] As seen in FIGS. 34 and FIGS. 35A and 35B, several compounds
falling within a general structure were testing according to the
present invention as follows.
[0155] Plasmids plasI-LVAgfp and prhlI-LVAgfp were provided by Dr.
Hiroaki Suga (The University of Tokyo). Strains PAO-JP2
(plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfp) were obtained from Dr.
Helen Blackwell. PAO1-GFP strain was provided by Dr. Guirong Wang
(Upstate Medical University). Non-swarming mutant of P. aeruginosa,
rhlA (PW6886) was obtained from PA two-allele library. 300 .mu.g/mL
of carbenicillin was added to maintain the plasmids of strains
PAO1-GFP, PAO1 (plasI-LVAgfp), PAO1 (prhlI-LVAgfp). All the
bacterial strains were grown in Luria-Bertani (LB) medium
containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl at
37.degree. C. For biofilm inhibition and dispersion assays 95%
M9+medium with 5% of LB broth was used unless otherwise stated.
M9+medium contained 47.7 mM Na.sub.2HPO.sub.4, 21.7 mM
KH.sub.2PO.sub.4, 8.6 mM NaCl, 18.7 mM NH4Cl, 1 mM MgSO.sub.4, 0.1
mM CaCl2, 0.4% L-Arg, 0.5% CAA, 0.2% anhydrous
.alpha.-D(+)-glucose, 0.2% sodium succinate dibasic hexahydrate,
0.2% citric acid monohydrate, and 0.2% L-glutamic acid
monopotassium salt monohydrate.
[0156] Stock solution of all the agents (11.5 mM) were prepared in
autoclaved water, sterilized by filtering through 0.2 .mu.m syringe
filter, and stored at -20.degree. C. in sealed vials. Appropriate
amount of sterile water was added to controls in all assays to
eliminate solvent effect.
[0157] Swarm agar plates were made using M8 medium supplemented
with 0.2% glucose, 0.5% casamino acid, 1 mM MgSO4 and solidified
with 0.5% Bacto agar.24 Bacterial culture with OD.sub.600 between
0.4 to 0.6 was inoculated as 3 .mu.l aliquots. Swarm agar plates
were incubated at 37.degree. C. for 12 h and then incubated for
additional 12 h at rt. For each set of experiment all the swarm
plates were poured from same batch of agar and allowed to dry for 1
h before inoculation of bacteria.
[0158] Plasmids plasI-LVAgfp and prhlI-LVAgfp were transferred to
wild type P. aeruginosa (PAO1) by electroporation method. Briefly,
overnight culture of PAO1 was subcultured in 25 mL LB broth and was
grown to reach the OD600 of 0.5 to 0.8 by shaking the culture at
37.degree. C. Flask containing subculture was cooled on ice for 30
min. Cell pellet was obtained by centrifugation at 4500 rpm for 5
min. Supernatant was removed and cell pellet was resuspended in 20
mL ice-cold 300 mM sucrose solution. Cells were centrifuged at 4500
rpm for 5 min. Supernatant was discarded and cell pellet was
resuspended in 1 mL ice-cold 300 mM sucrose solution. Cell
suspension was transferred to a new 1.5 mL ice-cold microcentrifuge
tube and spun down at 13,000 rpm for 30 sec. Supernatant was
discarded and cell pellet was resuspended in 500 .mu.L ice-cold 300
mM sucrose solution. For the last time cells were spun down at
13,000 rpm for 30 sec. After removal of the supernatant, cell
pellet was resuspended in 100 .mu.L ice-cold 300 mM sucrose
solution. 50 .mu.L of the competent cell suspension along with 5
.mu.L of plasmid DNA in TE buffer was transferred to the cold 0.1
cm electroporation cuvette. Electroporator was set to Ec1 and pulse
was passed through the cells. LB medium (1 mL) was immediately
added to the electroporator cuvette and cell suspension was
transferred to a sterile microcentrifuge tube and incubated for 1 h
at 37.degree. C. with shaking at 180 rpm. Cells were then spread on
the LB agar (1.5%) plates supplemented with 300 .mu.g/mL
carbenicillin (antibiotic) and incubated over night at 37.degree.
C. to get the microcolonies of PAO1 (plasI-LVAgfp) or PAO1
(prhlI-LVAgfp).
[0159] Inhibitory effect of all the maltose hydrocarbons on P.
aeruginosa biofilm formation was determined by crystal violet dye
based biofilm inhibition assays. Overnight culture of wild type P.
aeruginosa (PAO1) was sub cultured to an OD600 of 0.01 into the
95:5 M9+:Lb medium. 200 .mu.L of the sub culture was aliquoted into
the wells of 96 well polystyrene microtiter plate when it reached
the OD.sub.600 of 0.1. Predetermined concentrations of the test
compounds were then added to the respective wells containing sub
culture. Sample plates were wrapped in GLAD Press n' Seal.RTM.
followed by incubation under stationary conditions for 24 h at
37.degree. C. After incubation the media was discarded and the
plates were washed with water and dried for 1 h at 37.degree. C.
The sample plates were stained with 200 .mu.L of 0.1% aqueous
solution of crystal violet (CV) and followed by incubation at
ambient temperature for 20 min. The CV stain was then discarded and
the plates were washed with water. The remaining biofilm adhered
stain was re-solubilized with 200 .mu.L of 30% acetic acid. After
the stain was dissolved (15 minutes), 100 .mu.L of the solubilized
CV was transferred from each well into the corresponding wells of a
new polystyrene microtiter dish. Biofilm inhibition was quantified
by measuring the OD600 of each well in which a negative control
lane wherein no biofilm was formed served as a background and was
subtracted out. The percent inhibition was calculated by the
comparison of the OD600 for biofilm grown in the absence of
compound (control) versus biofilm grown in the presence of compound
under identical conditions.
[0160] Overnight culture of PAO1-GFP was subcultured to an
OD.sub.600 of 0.01 into the 95:5 M9+:Lb medium. Subculture was
allowed to reach the OD.sub.600 of 0.1 in a rotary shaker at 250
rpm and 37.degree. C. 200 .mu.L of the subculture was then
transferred to the wells of black microtiter plate with and without
(control) maltose derivatives. This black 96 well plate was then
incubated in a shaker at 37.degree. C. for 2 h. After 2 h,
bacterial culture from the plate was discarded and each well was
washed once with saline water (0.85 w/v % aqueous NaCl solution).
Fresh 95:5 M9+:Lb medium was added to the black 96 well plate and
fluorescence of the surface adhered bacteria was measured by
Synergy 2 multi-mode microplate reader with Gen5 data analysis
software at an excitation wavelength of 500 nm and an emission
wavelength of 540 nm. Background signal from 95:5 M9+:Lb medium was
eliminated from all the samples.
[0161] Plate for biofilm dispersion assay was set up similar to
crystal-violet based-biofilm inhibition assay but without adding
any maltose derivative at the time of inoculation of bacteria in
the 96 well plate. PAO1 was allowed to grow for 24 h at 37.degree.
C. After 24 h, bacterial culture was pipetted out and replaced with
200 .mu.L of 110 .mu.M maltose hydrocarbons and sterile water
(control). After 1 h treatment with maltose hydrocarbons, biofilm
was fixed and quantified using crystal violet dye as described
above. The amount of dispersed biofilm was determined via
comparison of the amount of biofilm at 24 h in the presence of
maltose hydrocarbons versus the amount of biofilm in the "no
compound" positive control at 24 h (wells with water
treatment).
[0162] Five maltose derivatives with highest activity for biofilm
inhibition and dispersion were selected for plotting dose response
curve. Predetermined amounts maltose derivatives stock solution was
added to the 200 .mu.L bacterial culture in 96 well plate so that
the final concentration of the agent reaches the desired values of
1, 5, 10, 20, 40, 85, 113, 140 .mu.M. Biofilm inhibition and
dispersion assay was carried as described previously.
[0163] Optical density was measured using Biotek ELx800 .TM.
absorbance microplate reader (BioTek Instruments, Inc., Winooski,
Vt.) using Gen5.TM. data analysis software. The OD600 values were
taken in sterile conditions at 0, 2, 4, 6, 8, 10, 12, and 24 h
after bacterial culture inoculation in 96-well polystyrene plate
with and without agents.
[0164] For an antagonist assay, overnight culture of P. aeruginosa
PAO1/plasI-LVAgfp (las system) or PAO1/prhlI-LVAgfp (rhl system) in
LB broth (supplemented with 300 .mu.g/mL carbenicillin) was grown
from a single colony on a LB agar plate supplemented with 300
.mu.g/mL carbenicillin. The overnight culture was diluted and grown
to OD600 of 0.1 in LB broth containing 300 .mu.g/mL of
carbenicillin. Bacterial culture (200 .mu.L) was added to each well
of a polystyrene 96-well microtiter plate containing an appropriate
amount of maltose derivatives or sterile water as a control. The
plate was incubated at 37.degree. C. for 24 h in a rotary shaking
incubator (250 rpm). The culture from each well was then
transferred to a flat-bottom, 96-well plate with black wall
(.mu.Clear, Greiner-One 655096). The fluorescence and OD absorbance
in each well was measured by Synergy 2 multi-mode microplate reader
with Gen5 data analysis software. Background signals from LB broth
were eliminated from all samples. Fluorescence was measured at an
excitation wavelength of 500 nm and an emission wavelength of 540
nm. Agonist assay was also done in a similar manner except PAO-JP2
(plasI-LVAgfp) and PAO-JP2 (prhlI-LVAgfP), double knockout strains
were used instead of wt PAO1. Using the double knockout strains,
agonist activities of the maltose derivatives were obtained
(absence of natural autoinducers) and compared to the activities
exhibited by adding natural autoinducers.
[0165] There is seen in FIGS. 36 and 37, the effect of compounds
I-V on the swarming motility of rhlA mutants, where stock solution
concentration was 11.7 mM, samples were prepared with 4 dilutions
leading to 86 .mu.M, 43 .mu.M, 21.2 .mu.M, 10.3 .mu.M, and swarming
was recorded at 14 h and 38 h after inoculation.
[0166] As seen in FIGS. 38 through 41, compounds IV and V were
studied further with more concentrations. Compound IV was further
studied as seen in FIGS. 42 and 43. As seen in FIGS. 44 and 45,
certain compounds effectively inhibited and/or resulted in
anti-adhesion of the biofilm of PAO1
[0167] Compounds according to the present invention may be used to
treat infectious diseases in humans, such as bacterial infections
common in cystic fibrosis. For example, the compounds may be
administered via nebulizer. Nebulisation involves the conversion of
a drug into a very fine aerosol or `mist` so that it can be
breathed straight into the lungs. Nebulizers are commonly used for
the treatment of cystic fibrosis, asthma, COPD and other
respiratory diseases. Nebulizers are particularly useful for those
drugs which require high dose deposition to the lungs, or for
distinct patient populations, such as those with severe disease,
young children, the elderly, or mechanically-ventilated patients in
a hospital intensive care unit. When treating various respiratory
diseases, inhalation of aerosols to the lungs is the preferred
route of administration of pharmaceutical compounds. Nebulizers are
often preferred for this purpose to pressurized metered dose
inhalers (pMDIs) and multidose dry powder inhalers (DPIs), and
single-breath administration with pMDIs and DPIs. Nebulizers can be
used across a wide dose range (g-up to gram-range) without loss of
overall delivery performance, and patients can take treatment
during multiple consecutive spontaneous breathing maneuvers. Some
have higher delivery efficiency which allows for dramatically
shorter inhalation times and a substantial reduction of drug volume
and dose, as higher drug concentrations are feasible and less drug
is wasted.
[0168] Any known nebulizers and nebulizer systems, and any
nebulizer or nebulizer system developed in the future having the
appropriate performance characteristics, can be used to deliver
Dodecyl Maltoside, or the other compounds according to the present
invention, to the lungs for the treatment of cystic fibrosis or any
other disease in the lungs involving a bacterial biofilm. Nebulizer
systems are used to deliver medications to control the symptoms and
the progression of lung disease in people with cystic fibrosis.
Nebulizers change a liquid medication into a mist so it can be
breathed in. These systems decrease treatment time and deliver more
medication into the lung than other conventional nebulizers which
have slower air flows and larger medication droplets. Nebulizers
using newer technologies, e.g. adaptive aerosol delivery or
vibrating mesh technology, deliver the medication faster and may
deliver more of the medication into the lung. These systems appear
safe when used with the correct amount of medication, which may be
different to that used in a conventional nebulizer system. Some
studies suggest that people with cystic fibrosis may prefer these
newer systems and may take more of their medication when using
them.
[0169] There are several types of nebulizers currently in use for
treating cystic fibrosis, although any new nebulizer developed will
be of use in delivering the treatment of the present invention.
These types of nebulizers are briefly summarized below, and then
described individually in detail thereafter.
[0170] Conventional nebulizer systems--a machine sucks air in and
pushes it out at high speed; a tube attaches the machine to a
chamber holding the medication where the air breaks it up into a
mist. The mist of medication is delivered constantly.
[0171] Adaptive aerosol delivery nebulizer systems--use
conventional technology as described above, but also monitor
breathing and deliver the mist of medication only while the person
is breathing in.
[0172] Adaptive aerosol delivery nebulizer systems with vibrating
mesh technology--monitor breathing and deliver the mist of
medication only while the person is breathing in and use vibrating
mesh technology, as described below, to change the liquid
medication into a mist.
[0173] Vibrating mesh technology nebulizer systems--move the liquid
medication through a metal mesh to break up the liquid into a mist
where each drop is a similar size; they deliver the mist of
medication constantly.
[0174] Ultrasonic nebulizer systems--use a crystal to vibrate the
liquid medication at a high-frequency to break up the liquid
medication into a mist; they deliver the mist of medication
constantly.
[0175] Conventional nebulizer systems consist of a compressor
coupled with a nebulizer chamber. The compressor entrains roomair,
compresses it to a higher pressure and emits the air at a given
flow rate. The air enters the nebulizer chamber and passes through
a small hole, a venturi, beyond which the air expands rapidly
creating a negative pressure; this draws the medication up a
feeding tube where it is atomised into particles. The particle
sizes are variable, larger particles will impact on the baffle
within the nebulizer chamber and onto the walls of the chamber and
be returned back to the well of the chamber to be re-nebulized. The
smaller particles will be continuously released from the nebulizer
chamber during both inspiration and expiration of the person using
the nebulizer system.
[0176] There are three main types of conventional nebulizer system:
the jet nebulizer; the open-vent jet nebulizer; and the
breath-assisted open-vent jet nebulizer. The jet nebulizer works
continuously as described above. Open-vent jet nebulizers
incorporate an open vent to allow extra air to be sucked into the
chamber during inspiration. This results in greater air flow
through the chamber and so greater densities of smaller respirable
particles over a shorter period of time. Breath-assisted open-vent
jet systems use a valve system to allow air to be drawn in during
inspiration as per the open-vent design. During expiration the
valve closes and the flow of air through the chamber is decreased
to that coming from the compressor only. This decreases the amount
of particles released during expiration and therefore decreases
medication wastage. One last adaptation of compressor and nebulizer
systems is holding chambers. This is a chamber which is attached to
the nebulizer and aerosol generated continuously by the nebulizer
is held within the chamber. A negative pressure is created within
the chamber during inspiration causing a valve to open and air to
be entrained. This air picks up aerosol and delivers it to the
person breathing in. An expiratory valve diverts expired air away
from the chamber and the chamber continues to fill with aerosol.
Holding chambers are designed to reduce medication wastage. A large
number of conventional compressor and nebulizer combinations are
available and these combinations have differing characteristics in
terms of aerosol particle size, nebulization time and mass of
medication delivered. Conventional nebulization systems tend to be
cheaper than the alternatives and are less prone to reliability or
delivery problems (or both) due to poor cleaning and maintenance.
They are, however, noisy and bulky and therefore less portable;
they also produce variable particle sizes and have a larger
residual volume as compared to alternative systems, so leading to
more wastage of medication.
[0177] Commonly used combinations of compressor and nebulizer are:
a Porta-neb compressor with sidestream or ventstream nebuliser and
a PARI TurboBOY with PARI LC SPRINT nebuliser.
[0178] Two nebulizer systems, the Halolite.RTM. and Prodose.RTM.,
were the first and second generation of nebulizer systems to
utilize AAD. These systems are no longer available as they have
been superseded by an AAD nebulizer system incorporating vibrating
mesh technology (VMT); the I-Neb AAD System.RTM.. With AAD,
pressure changes relating to airflow are continuously analysed and
timed pulses of aerosol (during the first 50% to 80% of inspiration
only) are delivered based on the prior three breaths until the
preset dose; an actuation, is delivered. This eliminates wastage of
medication during exhalation which occurs with continuously
delivering nebulizers and optimizes deposition. These systems were
designed to give optimal efficiency and therefore require an
alteration in the priming dose of medication used as compared to
conventional nebulizer systems.
[0179] One nebulizer system, the I-Neb AADsystem.RTM., utilizes VMT
and AAD in combination in order to optimize deposition and
treatment times. As detailed above, AAD occurs along with the use
of VMT, as detailed below Inhalation technique is assessed; the
nebulizer system will not operate unless correctly set up and used
at the appropriate angle. The system also stores adherence and
delivery data such as treatment date, time, duration and
completeness of dose which can be downloaded by the clinician or
the person using the I-Neb using software supplied by Philips
(Insight.RTM.). These nebulizer systems were designed to give
optimal efficiency and therefore require an alteration in the
priming dose of medication used as compared to conventional
nebulizer systems. An example is the I-neb AAD System from Philips.
Adaptive aerosol delivery (AAD) devices can also adapt to the
patient's breathing pattern and stop drug delivery when a pre-set
dose has been delivered.
[0180] VMT nebulizer systems aerosolize medication utilizing a
vibrating, perforated mesh to generate particles. This is achieved
by using a piezoelectric element which either vibrates a transducer
horn or which is annular and encircles themes causing it to
vibrate. Both methods result in medication being pumped through the
perforated mesh creating homogenous particles. Some meshes are
created with an electroplating technique which forms tapered holes
and others by precision laser-drilling. Vibrating mesh systems are
silent, portable (being small and battery powered), fast and
produce more homogenously-sized particles as compared to
conventional systems. There are a number of systems available. The
Omron MicroAir.RTM., the Aerogen Aeroneb Go.RTM., and the Pari
eFlow Rapid.RTM. were designed to be similar in efficiency to
conventional breath-enhanced nebulizers by using larger particle
sizes, a system housing which causes a high residual dose within
the nebulizer system, or a medication reservoir with a larger
residual volume. Other nebulizer systems were designed to give
optimal efficiency and may therefore require an alteration in the
priming dose of medication used. The AerogenOnQ.RTM.,
Aerodose.RTM., Aeroneb Pro.RTM. and Solo.RTM., Pari eFlow.RTM. and
Philips I-Neb.RTM. aim to deliver medication more efficiently and
quicker. Some VMT systems are currently available for clinical use
while others have only been utilized in research. A number of VMT
systems use the piezoelectric crystal technology associated with
ultrasonic nebulizers (see below) to create the vibration necessary
to pump medication through a mesh. An example is a e-Flow.RTM.
rapid nebulizer system from PARI GmbH.
[0181] Ultrasonic nebulizers utilize a piezoelectric crystal which
vibrates creating standing waves within the surface of the
medication, droplets move away from the crests of these waves
becoming an aerosol. Large particles impact on a baffle to be
re-nebulized in the same way as jet nebulizers. Ultrasonic
nebulizers may be smaller and are quieter and quicker than
conventional systems. There is controversy, however, as to whether
they are suitable to nebulize certain medications.
[0182] Dodecyl Maltoside can be administered alone or in
combination with other medications such as antibiotics. It is
recommended that nebulized antibiotics be inhaled once or twice a
day as prescribed by a doctor, and that they should be inhaled
after chest physiotherapy. When nebulizing antibiotics, a filter
should be used to prevent possible environmental contamination. The
filters will have a disposable pad, which should be changed after
every treatment. Antibiotics can take longer to nebulize (15-20
mins) but the new mesh nebulizers are much quicker. In one
embodiment of the present invention, Dodecyl Maltoside is
administered using a nebulizer system first and then an antibiotic
is administered to the patient using a nebulizer system. In another
embodiment, the order of administering Dodecyl Maltoside and the
antibiotic are reversed. In one embodiment, the patient waits a
specified period of time between the administration of the Dodecyl
Maltoside, such as 1, 2, 3, 4, 5, 10, 15, 20, 30, 45, or 60
minutes, or any time period in between. In another embodiment, the
Dodecyl Maltoside and an antibiotic are mixed prior to nebulizing
and the nebulized mixture is administered to the patient using a
nebulizing system.
[0183] In one embodiment of the present invention, other
therapeutic substances are also administered to the patient, either
before or after administration of the Dodecyl Maltoside dose. These
other therapeutic substances can include hypertonic saline,
bronchodilators, corticosteroids, antibiotics, mucolytics (e.g.,
DNase [Pulmozyme]), osmotics and antifungals, including specific
substances such as tobramycin, colistin; dornase alfa, hypertonic
sodium chloride, and other aerosolised medications. Mucolytics like
Pulmozyme or hypertonic saline can make sputum thinner and
therefore easier for the patient to clear, and administering a
mucolytic before, with or after administering Dodecyl Maltoside may
improve patient outcomes. In general, substances administered using
nebulizing systems should be allowed to warm to room temperature
before inhaling.
[0184] An inhaler or puffer can also be used for delivering
medication into the body via the lungs. It is mainly used in the
treatment of asthma and Chronic Obstructive Pulmonary Disease
(COPD), although Zanamivir (Relenza), used to treat influenza, must
be administered via inhaler. To reduce deposition in the mouth and
throat, and to reduce the need for precise synchronization of the
start of inhalation with actuation of the device, MDIs are
sometimes used with a complementary spacer or holding chamber
device. The most common type of inhaler is the pressurized
metered-dose inhaler (MDI). In MDIs, medication is most commonly
stored in solution in a pressurized canister that contains a
propellant, although it may also be a suspension. The MDI canister
is attached to a plastic, hand-operated actuator. On activation,
the metered-dose inhaler releases a fixed dose of medication in
aerosol form. The correct procedure for using an MDI is to first
fully exhale, place the mouth-piece of the device into the mouth,
and having just started to inhale at a moderate rate, depress the
canister to release the medicine. The aerosolized medication is
drawn into the lungs by continuing to inhale deeply before holding
the breath for 10 seconds to allow the aerosol to settle onto the
walls of the bronchial and other airways of the lung. Dodecyl
Maltoside could be delivered, alone or with one or more of the
therapeutic compounds discussed above or other appropriate
substance, to a patient's lungs using an inhaler. A solution or
suspension containing the Dodecyl Maltoside and any substance to be
co-administered with it can be stored in a pressurized canister,
which may contain a propellant as well. As with nebulizers,
particles and droplets should be around less than 5 .mu.m in
diameter to deposit more frequently in the lower airways and
greater than 0.5 .mu.m in diameter to reduce the likelihood of
their being exhaled.
[0185] Treatment dose administered using an inhaler or nebulizing
system can be controlled using time, amount of medicine delivered
(e.g., continue inhaling until the container holding the medicine
is empty), or by the number of breaths. Depending upon the
nebulizer or inhaler used and their settings, treatment times can
vary from a minute or two up to an hour or two, with many treatment
times having a duration of about 1, 2, 3, 4, 5, 10, 15, 20, 30, 45,
60, 75, 90 and 180 minutes. The appropriate number of breaths to
complete a treatment will vary based upon the nebulizer or inhaler
used and their settings, and the patient's condition, as well as
the medication or medications being delivered. Appropriate breath
counts can be 1, 3, 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 300,
400, 500, 600 or more, and any number in between. Some devices will
precisely meter the amount of medication administered. When Dodecyl
Maltoside is delivered in combination with another therapy, the
dose of the other therapy can be a full dose or a partial dose,
ranging from 0.1, 0.25, 0.5 to 0.75 dose. Appropriate doses of
Dodecyl Maltoside for treating cystic fibrosis can be various
ranges of doses, such as mg per kg, and should be apparent to those
of skill in the art. These can be delivered as necessary once a
month, once every two weeks, once a week, twice a week, three times
a week, every other day, once a day, twice a day, three times a
day, or more or less frequently.
[0186] The compounds of the present invention may also be used to
treat any medical condition involving a bacterial pathogen that
forms a biofilm. This includes chronic wounds, C. difficile,
cholera, and Crohn's disease among others. The treatments may
consist of applying the disaccharide hydrocarbons and other
compounds of the present invention (e.g., dodecyl maltoside) either
alone, to break up the biofilm and allow the immune system of a
patient to act more effectively, or in combination with an
appropriate anti-bacterial compound/treatment, either
simultaneously administered as a mixture or in sequence (first the
disaccharide hydrocarbons, then the anti-bacterial, or vice versa).
Application could be in the form of a topical formulation (cream,
solution, spray), a support matrix (hydrogel, collagen sponge,
polymer or cotton gauze or bandage), or, for the treatment of
intestinal biofilms, as an ingestible fluid or in a capsule or pill
that releases the compounds in the gut. Alternatively, compounds of
the present invention may be sprayed onto unwanted biofilms using
an endoscope approach, such as when pathogenic biofilms in the
intestines can be identified morphologically, visually, chemically,
or fluorescently and directly targeted.
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