1. health and fitness
2. disease

US 200602283 84A1
(19) United States
(12) Patent Application Publication (10) Pub. No.: US 2006/0228384 A1
Eldridge
(43) Pub. Date:
Oct. 12, 2006
(54)
CONTROL OF BIOFILM WITH A BIOFILM
INHIBITOR
Publication Classi?cation
(51)
(75) Inventor: Gary R. Eldridge, Encinitas; CA (US)
Correspondence Address:
THOMPSON COBURN, LLP
ONE US BANK PLAZA
SUITE 3500
(52)
Int. Cl.
A611; 31/7034
A611; 31/202
A611; 31/496
A611; 31/65
A611; 31/43
A611; 31/545
A611; 31/4745
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
(2006.01)
US. Cl. .......................... ..424/400;514/35;514/154;
514/200; 514/192; 514/291;
514/253.08; 514/312; 514/560;
ST LOUIS, MO 63101 (US)
514/210.09
(73) Assignee: Sequoia Sciences, Inc.
(21) Appl.No.:
11/099,800
(57)
ABSTRACT
The present invention provides methods for preventing;
reducing; inhibiting or removing bacterial bio?lm. The
present invention also provides methods for controlling acne
and chronic bacterial infections. The present invention fur
ther provides a method for identifying agents that prevent;
(22) Filed:
Apr. 6, 2005
reduce; inhibit or remove bacterial bio?lm.
Patent Application Publication Oct. 12, 2006 Sheet 1 0f 5
US 2006/0228384 A1
FIGURE 1
Beta-Galactoglycerolipids from Sambucus mexicana and Penslemon centranthifolius
CR1
0 R5
R4
OH
R30
0
0 R2
R1
R2
R3
R4
R5
SP3 82
hexadecadieno yl
Ac
Ac
H
Bz
SP3 83
linolenoyl
Ac
H
H
Bz
SF386
linolenoyl
H
H
Ac
Bz
SF 3 9O
linolenoyl
H
H
H
Bz
SF391
linolenoyl
H
H
H
H
SP3 97
linolenoyl
H
H
Ac
Ac
Patent Application Publication Oct. 12, 2006 Sheet 2 of 5
US 2006/0228384 A1
FIGURE 2
HPLC/UV Puri?cation Chromatograms for Hit 39 from Penstemon centranthz?ilius
0 mn _
nan:
0 mm _
0 man
0.0M;
0907:!
0.0050 -
09050
0025
Patent Application Publication Oct. 12, 2006 Sheet 3 of 5
US 2006/0228384 A1
FIGURE 3
1D Proton NMR Spectra for Hit 39 from Penstemon centranthifolz'us
SF382
I
I
l'
as
so
1.5
l
I
v
I
I
L0
7.5
7.0
6.5
‘.0
70
6.5
l
:0
SF383
|
8.5
H n
55
so
is
.o
w
Patent Application Publication Oct. 12, 2006 Sheet 4 of 5
US 2006/0228384 A1
FIGURE 4
HPLC/UV Puri?cation Chromatograms for Hit 37 from Sambucus mexicana
mi
SF391
up»
one
Q
'
a
a
’
m
n
m
Iliul-x
m
n
m
z:
*
Patent Application Publication Oct. 12, 2006 Sheet 5 0f 5
US 2006/0228384 A1
FIGURE 5
1D Proton NMR Spectra for Hit 37 from Sambucus mexicana
I
I
I
I
I
I
I
|
I
I
|
i
as
so
75
10
as
5.0
55
so
45
4o
:5
so
8.0
7.5
7.0
6.5
5.0
5.5
5.0
4.5
4.0
3.5
3.0
$F390
8.5
2.5
.D
1.5
.0
In
SF391
I
I
I
l
I
I
I
I
I
I
I
l
I
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
I
.0
I
1.5
l
.0
p:
Oct. 12, 2006
US 2006/0228384 A1
CONTROL OF BIOFILM WITH A BIOFILM
INHIBITOR
GOVERNMENTAL SUPPORT
[0001]
Part of this Work Was supported by the US.
a bio?lm. Presently, current antibiotics, speci?cally antibac
terial compounds, cannot provide e?‘ective treatment for
bio?lms of chronic infections, because antibiotic therapy
fails to eradicate the bio?lm. The Cystic Fibrosis Foundation
estimated that approximately 30,000 patients have cystic
?brosis in the United States. These patients desperately need
National Institutes of Health under the grant no. R42
RR0l6363-02. The United States Government has certain
neW medications that prevent the formation of bio?lms in
rights in this invention.
tics that interfere With the formation of bio?lms might assist
in the treatment of these patients.
their lungs. Research supports the conclusion that therapeu
FIELD OF THE INVENTION
[0007] As treatments for patients With gastritis, bio?lm
[0002] The present invention generally relates to methods
and compounds useful for inhibiting, reducing, preventing,
or removing bio?lms produced by microorganisms. In par
ticular, the invention provides a method for the control of
bio?lms using palmitoleic acid or its derivative as a bio?lm
inhibiting agent.
inhibitors can be used to prevent the attachment of Helico
baclor pylori to gastric epithelial cells preventing their
eventual invasion into these cells or inhibiting or reducing
subsequent virulence factors, Which can result in in?amma
tion. Bio?lm inhibitors can also be used to prevent or reduce
the risks associated With the virulence factors of H. pylori,
for example, by reducing arterial damage, Which can lead to
BACKGROUND
an increased risk of stroke. As treatments for urinary tract
[0003] Bacterial bio?lms exist in natural, medical, and
infections, bio?lm inhibitors can be used to prevent, control,
reduce, or eradicate the population of E. Coli that reside
engineering environments. Bio?lms o?fer a selective advan
tage to a microorganism to ensure its survival, or alloW it a
certain amount of time to exist in a dormant state until
suitable groWth conditions arise. Unfortunately, this selec
tive advantage poses serious threats to animal health, espe
intracellularly in bladder cells, Which resist conventional
antibiotics and evade a host’s immune systems.
[0008]
Each year in the United States, over 7 million
patients receive medical device implants, Which include
cially human health. For example, bio?lms are involved in
central venous catheters (CVC), endotracheal tubes,
65% of human bacterial infections. Bio?lms are involved in
mechanical heart valves, pacemakers, and prosthetic joints.
Approximately one-half of these patients develop nosoco
prostatitis, biliary tract infections, and urinary tract infec
tions, cystitis, lung infections, sinus infections, ear infec
mial infections, and approximately 80,000 deaths per year
tions, acne, rosacea, open Wounds, and chronic Wounds.
are attributed to nosocomial infections.
[0004] Chronic bacterial infections are serious medical
problems in the United States. In chronic bacterial infec
tions, bio?lms protect bacteria from antibiotics and the
[0009] According to the guidelines of the Center for
host’s immune response mechanisms, thus increasing the
rates of recurring symptoms and resistance to antibiotics.
Examples of antibiotics are Macrobid for the treatment of
acute UTI (cystitis) and Tobramycin for the treatment of
lung infections of cystic ?brosis patients. In addition to
cystitis and cystic ?brosis, chronic bacterial infections
include, but are not limited to dental caries, periodontitis,
otitis media, and nosocomial infections due to contact lens
Disease Control (CDC), the level of CVC bacterial coloni
Zation should be determined by the semi-quantitative or
quantitative method (Centers for Disease Control and Pre
vention, “Guidelines for the Prevention of Intravascular
Catheter-Related Infections”, MMWR. 5lzRR-l0, 2002). In
these guidelines, a positive blood culture and more than 103
colony-forming units (CFU) per catheter segment, as mea
sured by the quantitative method, suggest that the CVC is
the cause of the bloodstream infection (BSI). These guide
lines have been used in peer-revieWed studies that examine
and central venous catheters.
nosocomial BSI. The results of these studies suggest a direct
[0005]
relationship betWeen the probability of obtaining a BSI and
the CPU per catheter segment. Therefore, reducing the
Bio?lm inhibitors Will have a substantial medical
impact by treating many chronic infections, reducing cath
eter- and medical device-related infections, and treating lung
and ear infections. The potential market for potent bio?lm
inhibitors is exempli?ed by the sheer number of cases in
Which bio?lms cause medical problems. The bio?lm inhibi
tors may be used to control microorganisms existing in
extracellularly or intracellularly of living tissues. They may
be used to cure, treat, or prevent a variety of conditions, such
as, but are not limited to, arterial damage, gastritis, urinary
tract infections, otitis media, leprosy, tuberculosis, benign
prostatic hyperplasia, chronic prostatitis, chronic lung infec
tions of humans With cystic ?brosis, osteomyelitis, blood
stream infections, skin infections, open or chronic Wound
infections, and any other acute or chronic infection that
involves or possesses a bio?lm.
[0006] Cystic ?brosis patients have chronic lung infec
bacterial coloniZation of a catheter should loWer the inci
dence of nosocomial BSI.
[0010]
Clinical studies shoW that the use of antiseptic
coated or antibiotic-impregnated polyurethane catheters sig
ni?cantly reduces bacterial coloniZation of CVC and noso
comial BSI. A study in 1997 shoWed that in comparison to
untreated catheters, the use of polyurethane catheters coated
With chlorhexidine and silver sulfadiaZine resulted in statis
tically signi?cant reductions in the level of catheter coloni
Zation and the incidence of BSI (Maki, D. G. et. al.,
“Prevention of Central Venous Catheter-Related Blood
stream Infection by Use of an Antiseptic-Impregnated Cath
eter”, Ann. Int. Med. l27(4):257-266, 1997). In a peer
revieWed clinical study, the superiority of the antibiotic
impregnated catheters versus the anti-septic impregnated
tions that are currently treated With the antibiotic tobramy
catheters Was demonstrated (Darouiche, R. et. al., “A Com
cin. The principal organism found in the lungs of cystic
?brosis patients is Pseudomonas aeroginosa, existing Within
parison of TWo Antimicrobial-Impregnated Central Venous
Catheters”, NeW Engl. Jour. Med. 340(1): 1-8, 1999). In
Oct. 12, 2006
US 2006/0228384 A1
2003, a study in Which polyurethane catheters impregnated
infections often requires an administration of a combination
With minocycline and rifampin Were used in both medical
and surgical intensive care units demonstrated BSI reduc
tions of 8.3 to 3.5 and 4.8 to 1.3 per 1,000 days ofinpatient
tin/dalfopristin, or administration of tWo antibiotics simul
hospitalization, respectively (Hanna, H. A., et. al., “Antibi
otic-Impregnated Catheters Associated With Signi?cant
of products, such as amoxicillin/clavulanate and quinupris
taneously. The present invention provides an alternative
approach to treating chronic infection; a subject is admin
istered a combination of a bio?lm inhibitor and antibiotic.
Decrease in Nosocomial and Multidrug-Resistant Bacter
The tWo compounds provide different mechanisms of action.
emias in Critically Ill Patients”, Chest. 124(3):1030-1038,
2003). This study also shoWed a statistically signi?cant
decrease in bacteremia caused by vancomycin-resistant
[0016]
Enterococci.
[0011]
The results of these and other studies have led the
CDC to strongly recommend the use of antimicrobial- or
antiseptic-impregnated CVC in adult patients Whose cath
eters are anticipated to remain in place for more than 5 days
(Centers for Disease Control and Prevention, “Guidelines
for the Prevention of Intravascular Catheter-Related Infec
tions”, MMWR. 51:RR-10, 2002). Antimicrobial catheters
have also been recommended for use in patients Who have
a high risk of developing nosocomial BSI, such as patients
With burns or resistance to bacterial infections, patients
undergoing cancer treatments, hemodialysis, or transplanta
tion, and patients receiving emergency care. With a large
number of patients falling into these high-risk categories,
reducing nosocomial BSI Would offer a signi?cant societal
bene?t. Another approach for reducing BSI is to directly
inhibit the bacterial mechanisms that form bio?lms.
Bacteria have no knoWn resistance to bio?lm
inhibitors. Minocycline and rifampin have been used Widely
to treat infections, and microbial resistance to these antibi
otics is emerging. A study of an intensive care unit revealed
that six out of seven vancomycin-resistant enterococci Were
resistant to rifampin (Hanna, H. A., et. al, “Antibiotic
Impregnated Catheters Associated With Signi?cant Decrease
in Nosocomial and Multidrug-Resistant Bacteremias in
Critically Ill Patients”, Chest. 143(3):1030-1038). Bio?lm
inhibitors could extend the product life of current antibiot
ics. To date, doxycycline, an analog of minocycline, is a
?rst-line therapy for inhalation and cutaneous anthrax. With
the dramatically increased threat of bioterrorism, prevention
of the emergence of resistance to this class of antibiotic is
crucial. Recently, it has been reported that Bacillus anthracis
are resistant to cipro?oxacin (Price, L. B., et. al., “In vitro
selection and characteriZation of Bacillus anthracis mutants
With high-level resistance to cipro?oxacin”, Antimicrob.
Agents Chemother. 57:2362-2365, 2003). Clearly, microbes
acquire antibiotic resistance quickly, challenging research
In the United States, the market for antibiotics is
ers to develop neW antibiotics. Bio?lm inhibitors provide an
greater than $8.5 billion. After cardiovascular therapeutics,
alternate strategy to control dangerous, and possibly lethal,
antibiotics are the second largest drug market in the United
States. The siZe and groWth of this market are continually
fueled by an increased resistance to conventional antibac
terial therapies, With approximately 70% of bacteria found
in hospitals resisting at least one of the most commonly
prescribed antibacterials. Bio?lms are involved in 65% of
human bacterial infections. Accordingly, bio?lm inhibitors
could signi?cantly affect the antibiotic market.
bacterial infection. Bio?lm inhibitors are not likely to trigger
groWth-resistance mechanisms or affect the groWth of the
normal human ?ora.
[0012]
[0013] Antibiotics traditionally have been discovered
using the susceptibility test methods of the National Com
mittee for Clinical Laboratory Standards (NCCLS), Which
identify compounds that speci?cally effect groWth or the
killing of bacteria. These methods involve inoculation of
bacterial species in a suitable groWth medium, folloWed by
the addition of a test compound and plot of the bacterial
groWth over a time period post-incubation. These antibiotics
[0017] Bio?lm inhibitors act speci?cally on the biological
mechanisms that provide bacteria protection from antibiot
ics and a host’s immune system. In one study of urinary
catheters, rifampin Was unable to eradicate methicillin
resistant Staphylococcus aureus in a bio?lm but Was effec
tive against planktonic, or suspended cells (Jones, S. M., et.
al., “Effect of vancomycin and rifampicin on methicillin
resistant Staphylococcus aureus bio?lms”, Lancet. 357:40
41, 2001). A bio?lm inhibitor, co-administered With an
antibiotic that alone is unable to clear a bio?lm, for example,
can disrupt the bio?lm protection sufficiently to alloW the
antibiotic to reach the bacteria, eliminate them and prevent
a recurring infection.
are not effective therapeutics for chronic infections involv
ing bio?lms because these NCCLS methods do not test
[0018]
compounds in a preformed bio?lm. Accordingly, the meth
treatment of acne. Acne vulgaris is the most common
Bio?lm inhibitors can also be employed for the
ods of the NCCLS used to identify current antibiotics do not
cutaneous disorder. Propionibaclerium acnes, Which is the
apply to chronic infections involving bio?lms.
[0014] When compounds are employed as bio?lm inhibi
tors, they inhibit, reduce, prevent, or remove bio?lms pro
duced by microorganisms. The bio?lm may offer a selective
predominant microorganism occurring in acne, reside in
advantage to the microorganism to ensure its survival or
alloW it an undetermined amount of time to exist dormant
until suitable groWth conditions arise. The bio?lm may
directly or indirectly cause a disease or unfavorable health
condition in a host. Bio?lm inhibitors used to treat or
prevent chronic infections from microorganisms or bio?lm
inhibitor-impregnated or coated medical devices offer
numerous advantages.
[0015] Bio?lm inhibitors have an alternative mechanism
of action. Currently, successful treatment of nosocomial
bio?lms. Its existence in a bio?lm matrix provides a pro
tective, physical barrier that limits the effectiveness of
antimicrobial concentrations (Burkhart, C. N. et. al., “Micro
biology’s principle of bio?lms as a major factor in the
pathogenesis of acne vulgaris”, International J. of Derma
tology. 42:925-927, 2003). Bio?lm inhibitors may be used to
prevent, control, reduce, or eradicate R acnes bio?lms.
[0019] Furthermore, bio?lm inhibitors can be employed to
improve contact lens hygiene. Investigations on the occur
rence of bacterial bio?lms on contact lens have shoWn that
up to 80 percent of contact lens Wearers experienced con
tamination of lens cases. Bacteria protected by the bio?lm
can be resistant to current lens disinfectant systems
Oct. 12, 2006
US 2006/0228384 A1
(McLaughlin, B. L. et. al., “Bacterial Bio?lm on contact
lenses and in lens storage cases in Wearers With microbial
keratitis”, J. ofApplied Microbiology 84(5):827-838, 1998).
The use of bio?lm inhibitors may prevent, control, reduce,
or eradicate bacterial bio?lms on contact lens.
[0020]
Accordingly, for the reasons discussed above and
others, there continues to be a need for a means to control
bio?lm and its formation.
SUMMARY
[0021] Accordingly, the present invention provides a
method for preventing, reducing, inhibiting or removing a
bio?lm comprising contacting the bio?lm or contacting a
cell capable of forming a bio?lm With an effective amount
of palmitoleic acid, Wherein the composition prevents,
[0030] FIG. 2 shoWs the HPLC/UV puri?cation chro
matograms for Hit 39 from Penstemon centranthifolius.
[0031] FIG. 3 shoWs the proton NMR spectra for Hit 39
from Penstemon centranthifolius.
[0032] FIG. 4 shoWs the HPLC/UV puri?cation chro
matograms for Hit 37 from Sambucus mexicana.
[0033]
FIG. 5 shoWs the proton NMR spectra for Hit 37
from Sambucus mexicana.
DETAILED DESCRIPTION
[0034] Accordingly, the present invention provides a
method for preventing, reducing, inhibiting or removing a
bio?lm comprising contacting the bio?lm or contacting a
reduces, inhibits or removes the bio?lm.
cell capable of forming a bio?lm With an effective amount
[0022] Still, the present invention provides a method for
controlling acne, comprising administering to a subject
of palmitoleic acid, Wherein the composition prevents,
a?licted With acne an effective amount of an anti-acne
composition comprising palmitoleic acid, Wherein the
amount of the palmitoleic acid in the anti-acne composition
is suf?cient to prevent, reduce, inhibit or remove a bio?lm.
[0023] The present invention also provides a method for
controlling a chronic bacterial infection, comprising admin
reduces, inhibits or removes the bio?lm.
[0035]
In an embodiment, the cell is selected from the
group consisting of Streptococcus spp. including, but not
limited to, Streptococcus mutans, Streptococcus sobrinus,
Streptococcus pyogenes, and Streptococcus pneumoniae,
and Escherichia coli, Pseudomonas aeruginosa, Staphylo
istering to a subject in need thereof an effective amount of
coccus aureus, Staphylococcus epidermidis, Propionibacte
rium acnes, Enterococcus faecium, Enterococcus faecalis,
a composition comprising palmitoleic acid, Wherein the
and Haemophilus in?uenzae.
amount is effective to prevent, inhibit, reduce, or remove a
[0036] Palmitoleic acid does not appear to be toxic as it
can be readily found in foods. Palmitoleic acid can be found
bio?lm.
[0024] In addition, the present invention provides a
method for identifying an agent capable of preventing,
reducing, inhibiting or removing a bio?lm, comprising a)
in, for example, animal fat products, vegetable oil, olive oil,
macadamia nut oil, sea buckthom oil and peanut butter
products.
contacting a bio?lm With the agent, Wherein the agent is a
derivative of a palmitoleic acid; b) quantifying the bio?lm
before and after contacting the bio?lm With the agent; and c)
selecting the agent that prevents, reduces, inhibits or
removes at least about 50% of the bio?lm, Wherein the agent
does not inhibit bacterial groWth When the concentration
amount of the agent is at least tWice the amount needed to
prevent, reduce, inhibit, or remove at least 50% of the
bio?lm.
[0025] Furthermore, the present invention provides a phar
maceutical composition for reducing, preventing, inhibiting
or removing bio?lm formation comprising a pharmaceuti
cally acceptable carrier or diluent and a therapeutically
effective amount of 0.01 to 50% of palmitoleic acid.
[0026]
The present invention also provides an animal food
composition comprising palmitoleic acid, Wherein the ani
mal food composition promotes animal dental hygiene by
preventing, reducing, inhibiting or removing a bio?lm.
[0027]
In addition, the present invention provides a den
tifrice composition comprising palmitoleic acid, Wherein the
dentifrice composition promotes dental hygiene by prevent
ing, reducing, inhibiting or removing a bio?lm.
[0028] The present invention further provides a nebuliZer
for administration of a composition to a subject, Wherein the
composition comprises palmitoleic acid and Wherein the
TABLE 2
Examples of palmitoleic acid in plant oils and food.
% Palmitoleic Acid
Animal fat, bacon grease
Olive oil
Vegetable oil, soybean lecithin
Peanut Butter
Sea buckthom seed oil
Macadamia nut oil
2.7
0.3*3.5
0.4
0*8
9.0
12*22
[0037] Palmitoleic acid can also be found in macadamia
nuts. Macadamia nuts consist of approximately 75% fat, of
Which palmitoleic acid is approximately 12% to 22%. A
reasonable serving of macadamia nuts Would be approxi
mately 50 grams. Therefore, at a minimum, an individual
Would consume approximately 4.5 grams of palmitoleic acid
While eating a reasonable portion of macadamia nuts. An
enriched diet containing macadamia nuts signi?cantly
decreases the plasma total cholesterol, loW density lipopro
tein cholesterol, and triacylglycerol concentrations in
plasma (Yang, Baoru et. al., “Effects of dietary supplemen
tation With sea buckthom seed and pulp oils on atopic
dermatitis”, J. Nutr. Biochem. 10:622-630, 1999).
composition prevents, reduces, inhibits or removes a bio
?lm.
[0038] Palmitoleic acid can also be found in sea buckthom
oil. A topical application of sea buckthom oil on burned,
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 shoWs the chemical structure of the com
humans and experimental animals have shoWn healing and
anti-in?ammatory effects (Yang, Baoru et al., “Effects of
dietary supplementation With sea buckthom seed and pulp
pound of the present invention and its analogs or derivatives.
scaled, Wounded, and radioactively damaged skins of both
Oct. 12, 2006
US 2006/0228384 A1
oils on atopic dermatitis”, J. Nutr. Biochem. 10:622-630,
1999). Further, treatment With sea buckthom reduces the
serum levels of laminin, hyaluronic acid, total bile acid,
collagen types III and IV in patients With liver cirrhosis
(Gao, Ze-Li et al., “Effect of Sea buckthorn on liver ?brosis:
A clinical study”, World J Gastroenterol. 9(7):l615-1617,
2003).
[0039] As can be appreciated, palmitoleic acid has many
bene?cial medicinal values. Its prevalence in food suggests
that it is safe and non-toxic to animals at high doses. The
disclosure herein describes a novel use for palmitoleic acid
in the control of bio?lm and/or its formation. Palmitoleic
acid may potentially provide a safe means to control, remedy
or treat a variety of conditions involving bio?lms.
tobramycin, clindamycin, cipro?oxacin, tetracyclines,
rifampin, triclosan, ox?oxacin, macrolides, penicillins,
cephalosporins, amoXicillin/clavulante, quinupristin/dalfo
pristin, amoXicillin/sulbactum, metronidaZole, ?uoroquino
lones, quinolones, ketolides, or aminoglycosides.
[0044] In still another embodiment, the dentifrice compo
sition is selected from the group consisting of toothpastes,
toothpoWders, liquid dentifrices, mouth detergents, mouth
Washes, troches, cheWing gums, dental or gingival massage
creams, dental strips, dental gels, and gargle tablets. In many
instances, an antimicrobial or the agent controlling the
bio?lm alone does not resolve the medical condition or
disease. The disclosure herein describes the discovery that,
When used in combination With at least one antimicrobial
agent, palmitoleic acid provides a signi?cant synergistic
[0040] Plaque bio?lms contribute to cavities and and
periodontitis. Plaque bio?lm accumulates because of bacte
e?fect.
rial coloniZation by Slreplococci spp. such as S. mulans, S.
[0045]
sobrinas, S. gordonii, and Porphyromonas gingivalis, and
minant Directs the Species-Species Adherence of Porphy
order. Propionibaclerium acnes, Which is the predominant
microorganism present in acne, reside in bio?lms. The
bio?lm matrix provides a protective, physical barrier that
romonas gingivalis to Oral Streptococci, Infection and
limits the e?fectiveness of antimicrobial against R acnes.
Aclinomyces spp (Demuth, D. et al. Discrete Protein Deter
Immunity, 2001, 69(9) p5736-5741; Xie, H., et al. Interge
neric Communication in Dental Plaque Bio?lms. J. Bacte
Acne vulgaris is the most common cutaneous dis
[0046] The present invention, therefore, provides a
riol. 2000, 182(24), p 7067-7069). The primary coloniZing
method for controlling acne, comprising administering to a
bacteria of plaque accumulation are Slreplococci spp. and R
gingivalis is a leading cause of periodontitis (Demuth, D. et
al. Discrete Protein Determinant Directs the Species-Species
acne composition comprising palmitoleic acid, Wherein the
Adherence of Porphyromonas gingivalis to Oral Strepto
is su?icient to prevent, reduce, inhibit or remove a bio?lm.
cocci, Infection and Immunity, 2001, 69(9) p5736-5741).
[0047] As used in herein, “control” or “controlling” refers
to the treatment and prevention of acne. Preferably, palmi
Bio?lm inhibitors can be employed to prevent microorgan
isms from adhering to surfaces like teeth that may be porous,
subject al?icted With acne an e?fective amount of an anti
amount of the palmitoleic acid in the anti-acne composition
toleic acid prevents, reduces, inhibits or removes a bio?lm
soft, hard, semi-soft, semi-hard, regenerating, or non-regen
of Propionibaclerium acnes. Preferably, the antimicrobial
erating. These surfaces can be the polyurethane material of
agent is an antibiotic. The antibiotic may be selected from
central venous catheters, or metal, alloy, or polymeric sur
faces of medical devices, or regenerating proteins of cellular
the group consisting of tobramycin, clindamycin, ciprof
loxacin, tetracyclines, rifampin, triclosan, ox?oxacin, mac
membranes of mammals, or the enamel of teeth. These
rolides, penicillins, cephalosporins, amoXicillin/clavulante,
inhibitors can be coated on or impregnated into these
surfaces prior to use, or administered at a concentration
quinupristin/dalfopristin, amoXicillin/sulbactum, metronida
Zole, ?uoroquinolones, quinolones, ketolides, or aminogly
surrounding these surfaces to control, reduce, or eradicate
the microorganisms adhering to these surfaces.
cosides.
[0048] Preferably, the anti-acne composition comprises
[0041] Therefore, another embodiment of the present
betWeen about 0.01% to about 50% of palmitoleic acid.
invention is a method for preventing, reducing, inhibiting or
removing a bio?lm comprising contacting the bio?lm or
contacting a cell capable of forming a bio?lm With an
e?fective amount of a composition comprising palmitoleic
acid, Wherein the composition further comprises a dentifrice.
[0049] The anti-acne composition may be topically
administered. Preferably, the anti-acne composition is
applied to the skin of the subject. The anti-acne composition
[0042]
The present invention also provides a dentifrice
composition comprising palmitoleic acid, Wherein the den
tifrice composition promotes dental hygiene by preventing,
reducing, inhibiting or removing a bio?lm. In one embodi
may be a cream, gel, emollient or soap. Preferably, the
subject is a mammal. More preferably, the mammal is a
mouse or rat. Even more preferably, the mammal is a human.
[0050] In another embodiment, the concentration of
palmitoleic acid in the anti-acne composition is about
consisting of toothpaste, toothpoWder, liquid dentifrice,
0.01%, more preferably about 0.1% to about 5.0%. Because
palmitoleic acid can be readily found in food and poses
mouth detergent, mouthWash, troches, cheWing gum, dental
limited toxicity, the present invention contemplates that
or gingival massage cream, dental strip, dental gel, and
gargle tablet. Preferably, the dentifrice composition com
prises about 0.01% to about 20% of palmitoleic acid. More
about 1.0% to 50% of palmitoleic acid in the anti-acne
composition may be administered to control acne.
ment, the dentifrice composition is selected from the group
preferably, the dentifrice composition comprises about 2%
of palmitoleic acid.
[0051] In yet another embodiment, the anti-acne compo
[0043] In another embodiment, the dentifrice composition
sition further comprises at least one antimicrobial agent.
Preferably, the antimicrobial agent is an antibiotic. The
antibiotic may be selected from the group consisting of
further comprises at least one antimicrobial agent. Prefer
ably, the antimicrobial agent is an antibiotic. The antibiotic
may be selected from the folloWing group consisting of
tobramycin, clindamycin, cipro?oxacin, tetracyclines,
rifampin, triclosan, ox?oxacin, macrolides, penicillins,
cephalosporins, amoXicillin/clavulante, quinupristin/dalfo
Oct. 12, 2006
US 2006/0228384 A1
pristin, amoxicillin/sulbactum, metronidaZole, ?uoroquino
lones, quinolones, ketolides, or aminoglycosides
[0052] Akamatsu et al., Arch Dermatol Res (1990)
282:449-454 described that acne could be treated with
anti-in?ammatory compounds, such as free fatty acids and
metronidaZole. The article taught that while a combination
of palmitoleic acid and metranidaZole reduces in?ammatory
mediators of neutrophils, neither compound alone could
effectively inhibit the growth of Propionibacterium aches.
The authors used approximately 2 ug/ml of palmitoleic acid
with 100 ug/ml of metranidaZole to inhibit the in vitro
growth of Propionibacterium acnes. The authors applied
methods similar to that set forth by the National Committee
for Clinical Laboratory Standards (NCCLS). Only a very
small amount of palmitoleic acid was used because the
authors believed that while metranidaZole affected the grow
of R aches, palmitoleic acid merely reduced the in?amma
tion associated with acne. The present inventors determined
that 2 ug/ml of palmitoleic acid would be ineffective in
controlling bio?lm, especially for in vivo use. This may
explain why the application of an antibiotic alone to the skin
of a subject with acne (even assuming that the skin naturally
has palmitoleic acid), does not resolve the acne.
[0053] Willie et al., Skin Pharmacol. Appl. Skin Physiol.
(2003) 16:176-187 offer another perspective on the role of
palmitoleic acid. Willie et al. taught that palmitoleic acid
could inhibit the growth of Gram-positive bacteria (typical
minimal inhibitory concentration (MIC) value of 10-20
ug/ml), but could not inhibit the growth of Gram-negative
bacteria such as Pseudomonas aeruginosa, and Escherichia
coli, or Propionibacterium acnes and several methicillin
resistant strains of Staphylococcus aureus. For example,
palmitoleic acid failed to inhibit the growth of R aches even
at a high concentration, such as 125 ug/ml, using methods
similar to that prescribed by the NCCLS. Willie et al.
described that palmitoleic acid could inhibit the growth of
gram-positive bacteria, such as strains of Staphylococcus
aureus and Streptococcus salivarius, but concluded that it
had no inhibitory effect on clinical strains of Staphylococcus
aureus at 100 ug/ml, suggesting that palmitoleic acid might
crobial, which usually indicates the presence of bio?lms, it
has been only recently that bio?lms have been implicated as
the reason these infections are very difficult to treat and
eradicate (Harrison-Balestra, C. et al. A Wound-isolated
Pseudomonas aeruginosa Grow a Bio?lm In Vitro Within
10 Hours and Is VisualiZed by Light Microscopy. Dermatol
Surg 2003, 29, p. 631-635; Edwards, R., et al. Bacteria and
wound healing. Curr Opin Infect Dis, 2004, 17, p. 91-96). In
fact, it is estimated that approximately 140,000 amputations
occur each year in the United States due to chronic wound
infections that cannot be successfully treated with conven
tional antibiotics. This is another clear unmet medical con
dition that conventional antibiotics have been unable to
resolve. It has also been suggested that treating these infec
tions with high doses of antibiotics over long time periods
has been a contributor to the development of antibiotic
resistance (Howell-Jones, R. S., et al. A review of the
microbiology, antibiotic usage and resistance in chronic skin
wounds. J. Antimicrob. Ther. January 2005). Bio?lm inhibi
tors in combination therapy with antibiotics have great
potential to make an impact at treating chronic wounds.
[0056] With bio?lms, microbes can resist antibiotics at
concentrations ranging from 1 to 1.5 thousand times higher
than conventional antibiotic therapy. Similarly, during an
infection, bacteria surrounded by bio?lms are rarely
resolved by the immune defense mechanisms. Costerton,
Stewart, and Greenberg, leaders in the ?eld of bio?lms, have
proposed that in a chronic infection, a bio?lm gives bacteria
a selective advantage by reducing the penetration of an
antibiotic into the depths needed to completely eradicate the
bacteria’s existence. Clearly, bio?lms play a signi?cant role
in chronic infections, the treatment of which represents a
large market whose needs have not been met.
[0057] A typical chronic lung infection is in the lungs of
cystic ?brosis patients. The principal organism found in the
lungs of cystic ?brosis patients is Pseudomonas aeroginosa.
The bacteria exist within a bio?lm. Presently, current anti
biotics, speci?cally antibacterial compounds, cannot provide
effective treatment of chronic infections, because antibiotic
therapy fails to eradicate the bio?lm. The Cystic Fibrosis
not be useful in a medical treatment. Interestingly, no
Foundation estimated that approximately 30,000 patients
synergistic effect was observed when palmitoleic acid and
an antimicrobial agent, mupirocin, was used against Gram
have cystic ?brosis in the United States. Research supports
the conclusion that therapeutics that interfere with the for
mation of bio?lms might assist in the treatment of these
positive bacteria.
[0054] The present invention also provides a method for
controlling a chronic bacterial infection, comprising admin
patients.
[0058]
Bio?lm inhibitors can be used to prevent the
istering to a subject in need thereof an effective amount of
attachment of Helicobactor pylori to gastric epithelial cells
a composition comprising palmitoleic acid, wherein the
preventing their eventual invasion into these cells or inhib
amount is effective to prevent, inhibit, reduce, or remove a
iting or reducing subsequent virulence factors which result
bio?lm.
in in?ammation. Bio?lm inhibitors can also be used to
prevent or reduce the risks associated with the virulence
[0055]
Chronic wound infections are problematic because
they are difficult to eradicate or routinely recur. Diabetic foot
ulcers, venous leg ulcers, arterial leg ulcers, and pressure
ulcers are the most common types of chronic wounds. Of
these four types of chronic wounds, diabetic foot ulcers
appear to be the most prevalent. These wounds are typically
factors of H. pylori, for example, by reducing arterial
damage which can lead to an increased risk of stroke. As
treatments for urinary tract infections, bio?lm inhibitors can
be used to prevent, control, reduce, or eradicate the popu
lation of E. coli that reside intracellularly in bladder cells.
coloniZed by multiple species of bacteria including Staphy
[0059] Examples 1, 2, 4 and 8 herein demonstrates that
lococcus spp., Streptococcus spp., Pseudomonas spp. and
palmitoleic acid acts as a bio?lm inhibitor by reducing the
Gram-negative bacilli (Lipsky, B. Medical Treatment of
Diabetic Foot Infections. Clin. Infect. Dis. 2004, 39, p.S
104-14). Even though clinical evidence has shown for quite
attachment of Pseudomonas aeruginosa and Staphylococcos
epidermidis, Escherichia coli, Streptococcus mutans, and
some time that these chronic wound infections are polymi
Streptococcus sobrinas to surfaces. The invention demon
strates that palmitoleic acid exhibits bio?lm inhibition and
Oct. 12, 2006
US 2006/0228384 A1
reduction against bacteria that are genetically diverse from
infection may result from an infection of bacteria selected
each other as listed in the examples. These bacteria are
from the group consisting of Streptococcus spp. including
Gram-positive and Gram-negative and from clinical and
laboratory strains. Due to the diversity of the bacteria shoWn
in the examples of the speci?cation, palmitoleic acid exhib
its broad spectrum bio?lm inhibition. Therefore, the
examples are not meant to limit the scope of the invention
against only the bacteria examined, but only to provide
su?icient evidence that palmitoleic acid exhibits broad spec
trum bio?lm inhibition and reduction. The examples also
speci?cally demonstrate that palmitoleic acid can reduce a
mature bio?lm With and Without antibiotic. Examples 5 and
6 shoWs that palmitoleic acid can be applied to reduce the
sustainability of pre-formed bio?lms With an antibiotic.
Because bio?lm contributes to many chronic bacterial infec
tions, these examples strongly support the use of palmitoleic
acid and possibly its analogs or derivatives described herein
in the treatment of chronic bacterial infections, such as lung
and ear infections. The results of the examples and descrip
tions in the speci?cation also teach the distinct difference
betWeen the methods used to discover bio?lm inhibitors like
palmitoleic acid and the NCCLS methods used to discover
conventional antibiotics. Furthermore, it becomes clear to
those skilled in the art Why chronic infections are not
resolved by conventional antibiotics: conventional antibiot
ics Were identi?ed according to the NCCLS methods Which
do not represent the bio?lms found in chronic infections.
[0060] As demonstrated in Example 5, palmitoleic acid
may be administered alone to a subject for a treatment
period. After this treatment period, an antibiotic With the
compound could then be administered to the subject for a
but not limited to Streptococcus pyogenes, Streptococcus
pneumoniae, Escherichia coli, Pseudomonas aeruginosa,
Staphylococcus aureus, Staphylococcus epidermidis, Propi
onibacterium aches, and Haemophilus in?uenzae.
[0064]
The present invention further provides a nebuliZer
for administration of a composition to a subject, Wherein the
composition comprises palmitoleic acid and Wherein the
composition prevents, reduces, inhibits or removes a bio
?lm. Preferably, the composition prevents, reduces, inhibits
or removes a bio?lm in the lungs of the subject. Still
preferably, the composition comprises about 0.1% to about
2.0% palmitoleic acid.
[0065] In addition, the present invention provides a
method for identifying an agent capable of preventing,
reducing, inhibiting or removing a bio?lm, comprising a)
contacting a bio?lm With the agent, Wherein the agent is a
derivative of a palmitoleic acid; b) quantifying the bio?lm
before and after contacting the bio?lm With the agent; and c)
selecting the agent that prevents, reduces, inhibits or
removes at least about 50% of the bio?lm, Wherein the agent
does not inhibit bacterial groWth When the concentration
amount of the agent is at least tWice the amount needed to
prevent, reduce, inhibit, or remove at least 50% of the
bio?lm. Preferably, the bio?lm is quanti?ed by staining the
bio?lm With a dye and measuring the stained bio?lm using
a spectrophotometer to obtain the quantitative value for the
mass of each bio?lm. The bio?lm is preferably a mature
combined treatment period. Alternatively, the compound
bio?lm. That is, “mature” refers to a bio?lm that is fully
developed and shoWs resistance to knoWn antibiotics.
could be repeatedly administered alone to the subject to
continually reduce the formation of neW bio?lms.
[0066] The present application also describes novel com
pounds that alfect bio?lm formation. (See FIG. 1). The
[0061]
compounds can prevent or reduce the formation of bio?lms
or reduce the sustainability of a bio?lm. The methods
In an embodiment, the composition to control the
chronic bacterial infection further comprises at least one
antimicrobial agent. Preferably, the antimicrobial agent is an
antibiotic. The antibiotic may be selected from the group
consisting of tobramycin, clindamycin, cipro?oxacin, tetra
cyclines, rifampin, triclosan, ox?oxacin, macrolides, peni
cillins, cephalosporins, amoxicillin/clavulante, quinupristin/
dalfopristin,
amoxicillin/sulbactum,
metronidaZole,
?uoroquinolones, quinolones, ketolides, or aminoglyco
sides. Preferably, the subject is a mammal. More preferably,
the mammal is a mouse or rat. Even more preferably, the
mammal is a human.
[0062]
Bio?lms provide a structural matrix that facilitates
bacterial adhesion to the inert surfaces of medical device
implants and venous catheters. The bacteria also attach to
mammalian cells. For the 5 million central venous catheters
that are implanted each year, microscopic studies con?rm
that all of these catheters are coated by bacteria embedded
in bio?lms. More than 1 million patients develop urinary
tract infections from catheters.
[0063] Accordingly, in an embodiment of the present
invention, the chronic bacterial infection is selected from the
group consisting of urinary tract infection, gastritis, lung
infection, cystitis, pyelonephritis, arterial damage, otitis
media, leprosy, tuberculosis, benign prostatic hyperplasia,
chronic prostatitis, osteomyelitis, bloodstream infection,
skin infection, rosacea, open Wound infection, chronic
Wound infection, and sinus infection. The chronic bacterial
described in the speci?cation and the examples involve ?rst,
identifying compounds that inhibit the attachment of bacte
ria to surfaces (plastic or epithelial cells), Which involve
bio?lms, and second, testing the compounds against a pre
formed bio?lm that is resistant to an antibiotic. This proce
dure simulates chronic mammalian infections, by exposing
the bacteria to the bio?lm inhibitor and determining if the
inhibitor can reduce or modify the bio?lm With or Without
the presence of an antibiotic.
[0067] Other analogs and derivatives of palmitoleic could
potentially be used to control bio?lm. Accordingly, the
present inventor has produced a compound library using the
method previously described (Eldridge, G. R. et. al., 2002;
Cremen, Peadar A. et. al., 2002). The compound samples
have corresponding preparative HPLC elution conditions
that are tracked With a bar code system, so that the exact
gradient elution conditions of any sample in the library
could be recalled. The ability to rapidly isolate the com
pounds in the active samples is achieved by utiliZing starting
conditions from the preparative HPLC separation and apply
ing these to the semi-preparative HPLC system. With this
information, precise starting HPLC conditions are used to
loWer the gradient elution conditions to rapidly develop a
method to isolate the compounds in the active samples. The
individual compounds are then submitted for re-screening in
the bio?lm inhibition assays to identify the active com
pound(s). By “bio?lm inhibition”, it is meant that the
Oct. 12, 2006
US 2006/0228384 A1
compound prevents, reduces, inhibits or removes a bio?lm
formation or a bio?lm that is fully developed.
[0068] After screening 12,000 samples produced from a
compound library of plants according to similar methods as
described in Example 1, samples that exhibited bio?lm
[0072] FIGS. 2 and 4 illustrate the HPLC collection
chromatograms of the compounds isolated from tWo
samples, Hits 39 and 37. The active compounds from Hit 39
Were isolated from Penstemon cenlranlhifolius (Scorphu
lariales) collected in North America. The active compounds
Inhibiton Assay results presented in Table 1, one compound
from Hit 39, SF382 con?rmed bio?lm inhibition after puri
from Hit 37 Were isolated from Sambucus mexicana (Capri
foliaceae) collected in North America. The chemical struc
tures of the bio?lm inhibitors are determined using a 600
?cation. As can be seen from the adjacent column (bacterial
MHZ NMR spectrometer equipped With a CapNMR probe.
inhibition Were identi?ed. As is evident from the Bio?lm
growth prior to rinsing the assay Wells), this inhibition is not
due to the inhibition of bacterial groWth. Thus, the percent
age of bacteria attached (Within the bio?lm) versus free
?oating compared to untreated (negative) control has shifted
toWard free-?oating upon addition of the treatment. Com
[0073] Hit 37 and 39 Were generated from Sambucus
mexicana Presl. (leaf and ?oWer extract) and from Penste
mon cenlranlhifolius Benth. (Whole plant extract), respec
tively. The common names for the source plants are Blue
pound SF382 Was particularly active, reducing the formation
of bio?lm by 50% compared to negative control. Compound
SF382 is represented by structure (I).
Elderberry and Scarlet bugler (or Beardtongue), respec
tively. Sambucus is in the Family Caprifoliaceae, Order
Dipsacales; Penstemon is from the family Scrophulariaceae,
[0069]
As can be seen in the second part of Table 1, the
Asteridae. The Asteridae also contains Asters, Dogbanes,
Detachment Assay results, the selected compounds detached
signi?cant portions of a preformed bio?lm When compared
Mints and other taxa knoWn to produce a Wide range of
to the untreated (negative) control. Compound SF397 Was
particularly active With a detachment of 27% after one hour
incubation time.
[0070] FIGS. 3 and 5 shoW the analysis of an NMR data;
the data reveal that the active compounds isolated from Hits
37 and 39 are structurally similar. They all consist of a
beta-galactoglyceryl moiety esteri?ed via the terminal glyc
erol hydroxy functionality to a fatty acid. Aromatic func
tionality has not been previously reported. An extensive
literature search has revealed that the benZoyl containing
compounds SF382, SF383, SF386, SF390, and SF397 are
Order Scrophulariales. Both orders are in the Class
chemical repellants. Because Classes and Orders are fairly
large taxonomic groupings it is safe to suggest that While the
tWo plants are clearly related, the relationship is someWhat
distant.
[0074]
In addition to being from the same Class, the tWo
plants share similar biogeography. Both are Woodland plants
from California and both tend to groW in open spaces beloW
3000 meters. The Elderberry tends toWards moister climates
than the Beardtongue, but neither is found in a truly arid
environment. It Would not be surprising to ?nd that tWo
plants in the same taxonomic Class and sharing the same
environment Would evolve similar compounds.
TABLE 1
Bio?lm Inhibition and Detachment of the Isolated
Compounds from the Active Samples
Bio?lm Inhibition
Assay
% Growth
Isolated
Sample
Compound
Hit 39
SF382
SF383
SP3 84
SF390
7
—4
2
—4
50
4
19
21
4
1
5
0
SF391
—7
—1
—4
3
11
SF397
0
5
3
9
27
100
0
100
100
0
Negative
Control
novel structures. While compound SF391 is knoWn in the
art, the present inventors have shoWn herein that this com
pound can prevent, reduce, inhibit or remove a bio?lm.
Similarly, none of structures described herein have previ
ously been reported as bio?lm inhibitors.
[0071] Compound SF382 exhibited potent bio?lm inhibi
% Bio?lm
% GroWth
Active
Hit 37
%
Detachment Assay
GroWth Inhibition
Before
After
%
Compound
Compound
Detachment
14
1
11
4
15
15
19
15
[0075] Analogs and moieties of palmitoleic acid Were
tested as mentioned in Example 1. The analogs also include
oleic acid, ricinoleic acid, 1-O-hexyadecyl-2,3-dipalmitoyl
rac-glycerol, and 1-palmitoyl-2-oleyl-3-linoleoyl-rac-glyc
erol, Which each exhibited bio?lm inhibition from approxi
mately 20% to 40%. Preferably, the analog has a 16-carbon
conjugated chain. Unlike palmitoleic acid and compound
SF382, palmitic acid lacks the double bond conjugation and
tion. SF382 contains a hexadecadienoyl fatty acid side
chain. The other compounds carry a linolenoyl fatty acid
side chain. The hexadecadienoyl appears to be a signi?cant
contributor to bio?lm inhibition. This hypothesis lead to the
[0076] FIG. 1 lists the compounds prepared and tested for
discovery of palmitoleic acid.
bio?lm inhibition as described in Example 1. The com
does not inhibit the formation of bio?lms.
Oct. 12, 2006
US 2006/0228384 A1
pounds Were prepared based on the discovery of the novel
function of palmitoleic acid in controlling bio?lm as
described herein. The compounds are represented by the
composition may further comprise at least one antimicrobial
agent. Preferably, the antimicrobial agent is an antibiotic.
The antibiotic may be selected from the group consisting of
following structures:
tobramycin, clindamycin, cipro?oxacin, tetracyclines,
rifampin, triclosan, ox?oxacin, macrolides, penicillins,
cephalosporins, amoxicillin/clavulante, quinupristin/dalfo
pristin, amoxicillin/sulbactum, metronidaZole, ?uoroquino
SF 382
lones, quinolones, ketolides, or aminoglycosides.
[0078] In another embodiment, the composition further
KEY.
comprises a pharmaceutically acceptable carrier and diluent.
This pharmaceutical composition may further comprise at
least one antimicrobial agent. Preferably, the antimicrobial
agent is an antibiotic. The antibiotic may be selected from
the group consisting of tobramycin, clindamycin, ciprof
loxacin, tetracyclines, rifampin, triclosan, ox?oxacin, mac
rolides, penicillins, cephalosporins, amoxicillin/clavulante,
quinupristin/dalfopristin, amoxicillin/sulbactum, metronida
Zole, ?uoroquinolones, quinolones, ketolides, or aminogly
cosides.
[0079] Furthermore, the present invention provides an
animal food composition comprising palmitoleic acid,
Wherein the animal food composition promotes animal den
tal hygiene by preventing, reducing, inhibiting or removing
a bio?lm. The bio?lm may be a dental plaque. In an
embodiment, the animal food composition is a dry or
SF 386
@YO
A00
EXAMPLES
[0080] The folloWing examples illustrate the testing of
palmitoleic acid and related compounds and the preparation
of formulations comprising palmitoleic acid. The examples
0
OH
O
HO
semi-moist pet food composition. For example, the pet food
composition is a pet biscuit. Alternatively, the pet food
composition is applied to the surface of a cheWable pet toy.
Preferably, the pet food composition is a dog or cat food.
O
demonstrate the many uses of palmitoleic acid and are not
O
intended to limit the scope of the present invention.
OH
,
Example 1
O
SF 390
\JYO
HO
Effect of Palmitoleic Acid on Bio?lm Inhibition
[0082] In vitro bio?lm inhibition Was evaluated using a
high-throughput method. This method uses standard pre
steriliZed 96-Well polystyrene microtiter plates. Each Well
O
OH
Was ?lled to a ?nal volume of 200 pl. Initially, a concen
O
HO
[0081]
With Pseudomonas aeruginosa PAOl and Staphylococcus
epidermidis ATCC 29641.
O
trated compound solution is transferred into each Well,
O
except those Which are used as controls, to achieve an
OH
,
O
appropriate testing concentration, typically 10 ug/ml. 150 pl
of sterile media Was then added, folloWed by 50 ul of
or
SF 397
A00
[0083] After the allotted groWth time, the samples Were
OAc
removed from the shaker, rinsed and stained. The rinse
OH
HO
O
bacterial inoculum. The plates Were then placed on a shaker
for 24 hours.
O
involves ?rst draining the Wells, ?lling each Well With PBS,
and draining each Well again. The rinse removes suspended
O
OH
,
O
[0077] A composition comprising one or more of these
compounds may be used to affect bio?lm formation. The
cells from the system. The bio?lm Was then stained With
crystal violet for 20 minutes. The Wells Were each rinsed 4
times to remove any excess stain from the system and then
eluted With 250 pl of ethanol. The elution step improves the
detection of the stain during the analyses. The plate Was then
immediately analyZed With a microtiter plate reader. The
Oct. 12, 2006
US 2006/0228384 A1
samples and controls Were analyzed in triplicate. Each plate
contains negative and positive controls. The test samples
Were compared to the positive and negative controls to
detect any reduction in the total amount of bio?lm.
Example 4
[0090]
[0091]
[0084] Palmitoleic acid at 10 ug/mL resulted in 55%
bio?lm inhibition for R aeruginosa PA01 and 80% bio?lm
inhibition for S. epidermidis ATCC 29641.
Effect of Palmitoleic Acid on Bio?lm Inhibition of
a clinical strain of Escherichia coli UT189.
Dose response experiments Were conducted on
palmitoleic acid as described in Example 2 at 4, 8, 16, and
32 ug/mL With E. coli UT189 in LB media.
[0092]
Palmitoleic acid inhibited bio?lm of E. coli UT189
by approximately 10% (4 ug/mL), 55% (8 ug/mL), 70% (16
Example 2
[0085] Palmitoleic acid and Bio?lm Formation With Wild
Type Escherichia coli JM109.
[0086] Bio?lm inhibition experiments Were conducted
using an assay adapted from the reported protocol described
in Pratt and Kolter, 1998, Molecular Microbiology, 30:
285-293; Li et al., 2001, J. Bacteriol., 183: 897-908. E. coli
J M109 Was groWn in LB With or Without 0.2% glucose in 96
Well plates at 370 C. for tWo days Without shaking. To
quantify the bio?lm mass, the suspension culture Was
poured out and the bio?lm Was Washed three times With
Water. The bio?lm Was stained With 0.1% crystal violet for
20 minutes. The plates Were then Washed three times With
Water. OD reading at 540 nm Was measured to quantify the
bio?lm mass at the bottom of the Wells. Then 95% ethanol
Was added to dissolve the dye at the bottom and on the Wall
and the OD reading at 540 nm Was measured to quantify the
total bio?lm mass. To study the overall effect of palmitoleic
acid (3.6 mg/mL in 100% ethanol as stock solution), it Was
added With the inoculation and a time course of bio?lm mass
Was measured. To study if the compound can cause bio?lm
removal, it Was added one day after inoculation. Appropriate
amounts of 100% ethanol Were added to each sample to
eliminate the effect of solvent. Four plates Were prepared for
each study to give a time course. Each condition had 3-4
replicates on each plate.
ug/mL), and 80% (32 ug/mL) compared to controls.
Example 5
[0093] Effect of palmitoleic acid and Tobramycin on Bio
?lm formation With Pseudomonas aeruginosa.
[0094] Bio?lm formation of R aeruginosa Was evaluated
using a standardiZed bio?lm method With a rotating disk
reactor (RDR). This method provides a model resembling
the formation of bio?lms in cystic ?brosis patients. The
rotating disk reactor consists of a one-liter glass beaker ?tted
With a drain spout. The bottom of the vessel contains a
magnetically driven rotor With six 1.27 cm diameter cou
pons constructed from polystyrene. The rotor consists of a
star-head magnetic stir bar upon Which a disk Was af?xed to
hold the coupons. The vessel With the stir bar Was placed on
a stir plate and rotated to provide ?uid shear. A nutrient
solution (AB Trace Medium With 0.3 mM glucose, see Table
3 beloW for composition) Was added through a stopper in the
top of the reactor at a How rate of 5 ml/min. The reactor
volume Was approximately 180 ml and varied slightly
betWeen reactors depending on the placement of the drain
spout and the rotational speed of the rotor. At a volume of
180 ml, the residence time of the reactors Was 36 minutes.
The reactors Were operated at room temperature (ca. 260
C.).
TABLE 3
Composition of the AB Trace Medium used for the RDR test.
Component
Formula
groWth of E. coli JM109 When compared to controls, dem
Disodium phosphate
Na2HPO4
6.0
onstrating that palmitoleic acid is not an antibacterial com
Monopotassium phosphate
KH2PO4
3.0
3.0
[0087]
Palmitoleic acid had no inhibitory effect on the
pound. HoWever, When palmitoleic acid Was added With
inoculation of E. coli, the compound, at approximately 20
uM, inhibited the total bio?lm formation by about 60%
reduction as compared to the controls.
Concentration (g/l)
Sodium Chloride
NaCl
Ammonium sulfate
(NH4)2SO4
2.0
Magnesium chloride
MgCl2
0.2
Glucose
Calcium chloride
Sodium sulfate
Ferric chloride
C6O12H6
CaCl2
NaZSO4
FeCl3
0.054
0.010
0.011
0.00050
Example 3
[0088] Antibacterial effect of palmitoleic acid on methi
cillin-resistant Staphylococcus aureus (ATCC 33591 and
78-13607A).
[0089] Using the appropriate NCCLS procedures, the anti
bacterial elfect of palmitoleic acid on tWo strains of methi
cillin-resistant Staphylococcus aureus (ATCC 33591 and
[0095] For each test, tWo RDRs Were operated in parallel
With one receiving palmitoleic acid and the other serving as
an untreated control. The RDRs Were steriliZed by auto
clave, then ?lled With sterile medium and inoculated With
Pseudomonas aeruginosa strain PAOl. The reactors Were
then incubated at room temperature in batch mode (no
medium ?oW) for a period of 24 hours, after Which the How
78-13607A) Was studied at 32 ug/mL. For both S. aureus
ATCC 33591 and S. aureus ATCC 78-13607A, palmitoleic
Was initiated for a further 24 hour incubation. Palmitoleic
acid Was dissolved in 10 ml ethanol to achieve a concen
acid had no inhibitory effect represented by an MIC (mini
tration of 1.8 mg/ml. After the 48 hours of bio?lm devel
mal inhibitory concentration) of greater than 32 ug/mL.
These results along With the results described in Example 2,
palmitoleic acid Was added to the reactor to achieve a ?nal
opment described above, the 10 ml of ethanol containing
further supports that palmitoleic acid is not an antibacterial
concentration of approximately 100 ug/ml. Control reactors
compound.
received 10 ml of ethanol. The reactors Were then incubated
Oct. 12, 2006
US 2006/0228384 A1
for an additional 24 hours in batch (no How) mode. After this
incubation period, the six coupons Were removed from each
reactor and placed in 12-Well polystyrene tissue culture
plates With Wells containing either 2 ml of a 100 ug/ml
tobramycin solution or 2 ml of phosphate-buffered saline
(PBS). These plates Were incubated at room temperature for
tWo hours. The coupons Were then rinsed by three transfers
to plates containing 2 ml of fresh PBS. For each tWo RDR
reactors run in parallel, four sets of three coupons Were
obtained: one set With no test compound treatment and no
tobramycin treatment, one set With no test compound treat
ment and tobramycin treatment, one set treated With a test
compound treatment and no tobramycin treatment, and one
set treated With a test compound treatment and tobramycin.
After rinsing, one coupon of each set of three Was stained
With a LIVE/DEAD® BacLightTM Bacterial Viability Kit
[0098]
Peg lids Were rinsed three times in sterile Water,
placed onto ?at-bottom microtiter plates containing bio?lm
inhibitors at 10, 20, and 30 ug/ml in 100 pl of CAMHB per
Well and incubated for 40 to 48 h at 37° C.
[0099] Pegs Were rinsed, placed in a 0.1% (Wt/vol) crystal
violet solution for 15 min, rinsed again, and dried for several
hours. To solubiliZe adsorbed crystal violet, pegs Were
incubated in 95% ethanol (150 pl per Well of a ?at-bottom
microtiter plate) for 15 min. The absorbance Was read at 590
nm on a plate reader. The Wells containing palmitoleic acid
Were compared to negative controls. Negative controls Were
prepared as stated above but Without palmitoleic acid.
[0100]
Palmitoleic acid caused approximately 50% to
70% detachment of the mature bio?lms at 10, 20, and 30
ug/ml against three clinical strains of R aeruginosa.
(Molecular Probes, Eugene Oreg.) and imaged using epif
luorescent microscopy. The remaining tWo coupons Were
placed in 10 ml of PBS and sonicated for ?ve minutes to
remove and disperse bio?lm cells. The resulting bacterial
suspensions Were then serially diluted in PBS and plated on
Example 7
[0101] In Vitro Cytoxicity Assessment of palmitoleic acid
in Human Hepatocytes.
tryptic soy agar plates for enumeration of culturable bacte
[0102]
ria. The plates Were incubated for 24 hours at 37° C. before
ity in human hepatocellular carcinoma cells (HepG2).
colony forming units (CFU) Were determined.
Palmitoleic acid Was tested at tWo concentrations of 30 and
[0096]
The combined treatment of palmitoleic acid and
tobramycin resulted in a 60% reduction of CFUs for R
aeruginosa compared to treatment With tobramycin alone.
This clearly demonstrates that palmitoleic acid increased
bio?lm susceptibility to tobramycin by modifying the bio
?lm. As a comparison to multiple published clinical studies,
these results With palmitoleic acid and tobramycin demon
strate that improved lung function (FEV or forced expiratory
volume) and decreased average CFU (density) in sputum
from patients With cystic ?brosis Would be observed in a
combination therapy involving these compounds (Ramsey,
Bonnie W. et. al., “Intermittent administration of inhaled
Palmitoleic acid Was tested to assess its cytotoxic
60 uM in HepG2 cells in triplicate. Gross cytotoxicity Was
assayed by ?uorometric detection in response to mitochon
drial activity according to Nociari M M, et al. (1998) J.
Immunol. Met. 213:157. ChlorpromaZine Was used as a
positive control.
[0103] Cytotoxicity of HepG2 cells Was not observed at
palmitoleic acid concentrations of 30 or 60 uM.
Example 8
[0104] Effect of Palmitoleic Acid on Bio?lm GroWth and
Inhibition With Streptococcus mutans 25175 and Strepto
tobramycin in patients With cystic ?brosis”, NeW England I.
coccus sobrinus 6715.
Medicine 340(1):23-30, 1999; Saiman, L. “The use of
[0105] Palmitoleic acid Was tested against S. mutans
25175 and S. sobrinus 6715 at a concentration of 80 ug/ml
using the method described in Example 2. The use of 1 mL
polycarbonate tubes Were used in place of 96 Well polys
terene microtiter plates.
macrolide antibiotics in patients With cystic ?brosis”, Curr
Opin Pulm Med, 2004, 10:515:523; PirZada, O. et al.
“Improved lung function and body mass index associated
With long-term use of Macrolide antibiotics.”, J. Cystic
Fibrosis, 2003, 2, p. 69-71). Using the endpoints listed in
these publications and used in Cystic Fibrosis clinical trials,
this example demonstrates that a combined treatment of
tobramycin and palmitoleic acid Would provide bene?t to
Cystic Fibrosis patients or other people suffering from
chronic lung infections. The research results of this example
also demonstrate that palmitoleic acid in combination With
an antibiotic Would remove bio?lms from teeth, skin, and
other surfaces.
Example 6
[0097] Effect of Palmitoleic Acid on Mature Bio?lms of
clinical strains of R aeruginosa clinical isolates isolated
from cystic ?brosis patients Were passed tWice on tryptic soy
agar With 5% sheep blood after retrieval from —80° C. and
then groWn overnight in CAMHB. After dilution of a culture
to 0.5 McFarland in broth medium, 100 pl Was transferred in
triplicate to Wells of a ?at-bottom 96-Well microtiter plate.
Bacterial bio?lms Were formed by immersing the pegs of a
[0106] Testing palmitoleic acid at 80 ug/mL against S.
mutans 25175 and S. sobrinus 6715 shoWed greater than
75% bio?lm groWth inhibition.
Example 9
[0107]
In Vivo PK Study of Palmitoleic Acid in Rats.
[0108] TWo groups of animals Were prepared to evaluate
palmitoleic acid. Palmitoleic acid Was evaluated for tWo
dose routes consisting of 50 mg/kg (IV) and 100 mg/kg
(Oral). Three animals Were assigned to the IV route group
and tWo animals Were assigned to the oral route group. Prior
to dosing, a baseline blood sample Was taken from each
animal. At time Zero, a single bolus dose of palmitoleic acid
in 10% Cremophor (BASF) Was given to each animal. Blood
samples Were taken from each animal at 1, 4, 12 and 24
hours post dosing. All blood samples Were processed to
plasma.
modi?ed polystyrene microtiter lid into this bio?lm groWth
[0109] The plasma samples Were extracted by adding
plate, folloWed by incubation at 37° C. for 20 hours With no
internal standard at a concentration of 10 ug/mL to achieve
a ?nal concentration of 2 ug/mL When reconstituted. A
movement.
Oct. 12, 2006
US 2006/0228384 A1
protein precipitation. The samples Were then vortexed and
[0117] Formulations A and B Were stored for thirty days at
2° C. to 8° C., room temperature (approximately 22° C.), and
centrifuged. The supernatant Was decanted and the solvent
Was evaporated olf. Samples Were then analyZed and quan
remained homogenous for thirty days.
solution of 80:20 methanolzchloroform Was used to cause
at 30° C. Both formulations at each storage condition
ti?ed by LC/MS.
[0110] The reported AUC for IV dosed rats Was 123
ug/ml/hr and the AUC for the orally dosed rats Was 219.71
ug/ml/hr. These data demonstrate that palmitoleic acid is in
Example 12
[0118] Palmitoleic Acid, 2% Toothpaste Formulation
sufficient quantities in serum of rats to demonstrate bio?lm
[0119] Toothpaste preparations Were prepared containing
inhibition or reduction.
2% palmitoleic acid With and Without antibiotic and With
and Without polymer. Polymer, GantreZ S-97, Was added to
improve retention of palmitoleic acid and antibiotic on teeth.
Example 10
[0111] Palmitoleic Acid, 2% Topical Bio?lm Inhibitor
Formulation.
[0112] This invention provides topical bio?lm inhibitor
preparations containing 2% by Weight palmitoleic acid. A
similar formulation could be prepared as listed in this
example With an antibiotic.
[0120] All of the dry ingredients Were mixed together.
Glycerin Was sloWly added While mixing. An aliquot of
Water Was added sloWly and thoroughly mixed. Peppermint
extract Was added and then the rest of the Water Was added
While mixing. Palmitoleic acid and antibiotic Were then
added until homogenous.
[0113] Components of the oil phase are heated to 65° C.,
With constant stirring, until molten. Palmitoleic acid is added
to the oil phase and stirred for 5 minutes. The aqueous phase
is heated to 65° C. and stirred until complete solution is
achieved. The aqueous phase is mixed With oil phase at the
Formulation A
Ingredients
same temperature for 10 minutes. The cream is stirred and
cooled to 40-45° C., before a vacuum of 1 bar is pulled.
Stirring is continued until the cream reaches 250 C.
Parts By Weight
Sorbitol
20.0
Glycerin
22.0
Silica
20
Sodium lauryl sulfate
2.0
Xanthum gum
1
2.0
1.0
0.3
Palmitoleic Acid
Peppermint extract
Sodium ?uoride
Water
Formulation A
Ingredients
31.7
Parts By Weight
[0121]
Oil Phase
Mineral Oil
50.9
Stearyl Alcohol
Cetyl Alcohol
Phenoxy Ethanol
BenZyl Alcohol
3.5
3.5
0.5
1.0
Palmitoleic Acid
2.0
Formulation B
Ingredients
Aqueous Phase
Polyethylene Glycol
6.0
Xanthum Gum
Water
0.2
32.4
Parts By Weight
Sorbitol
20.0
Glycerin
22.0
Silica
20
Sodium lauryl sulfate
2.0
Xanthum gum
Palmitoleic Acid
1
2.0
Peppermint extract
Sodium ?uoride
1.0
0.3
2.5
29.2
GantreZ S-97
Water
Example 11
[0114] Topical antibacterial gels Were prepared containing
5% of palmitoleic acid by Weight With and Without antibi
otic.
[0122]
[0115] Formulation A. 0.5 gram of palmitoleic acid Was
dissolved in 5.5 grams of ethanol. 0.2 grams of hydroxypro
pyl methylcellulose Was added With gentle stirring until a
homogenous solution Was obtained. 3.74 grams of Water Was
then added With gentle shaking.
[0116] Formulation B. 0.5 gram of palmitoleic acid Was
dissolved in 5.5 grams of ethanol. 0.2 grams of aZithromycin
Was dissolved in this solution. 0.2 grams of hydroxypropyl
methylcellulose Was added With gentle stirring until a
homogenous solution Was obtained. 3.74 grams of Water Was
then added With gentle shaking.
M
Ingredients
Parts By Weight
Sorbitol
20.0
Glycerin
22.0
Silica
20
Sodium lauryl sulfate
2.0
Xanthum gum
Palmitoleic Acid
Triclosan
1
2.0
0.3
Oct. 12, 2006
US 2006/0228384 A1
strating that nebuliZation can be used to deliver this com
pound to patients With lung infections.
-continued
Formulation C
Ingredients
Example 16
Parts By Weight
Peppennint extract
Sodium fluoride
1.0
0.3
Gantrez S-97
Water
2.5
28.9
[0131] Palmitoleic Acid, 2% Liquid Soap Formulation
[0132] This invention provides a liquid soap preparation
containing 2% by Weight palmitoleic acid With the following
formulation. This liquid soap could be used daily on the face
prior to going to sleep to prevent and reduce facial acne.
[0123] Formulations A, B, and C Were prepared and stored
for thirty days at 2° C. to 8° C., room temperature (approxi
mately 22° C.), and at 30° C.
Ingredients
Example 13
[0124]
[0125]
Parts By Weight
Palmitoleic Acid
Ethanol
2.0
1.5
Propylene glycol
3.0
Palmitoleic Acid, 2% Mouth Rinse Formulation.
Cellulose
1.5
Glycerol
16.0
This invention provides an oral care mouth rinse
Tetrasodium EDTA
preparation containing 2% by Weight palmitoleic acid With
the following formulation.
0.30
Triethylalnine
10.0
Water
65.7
Example 17
Ingredients
Parts By Weight
Palmitoleic Acid
Ethanol
2.00
10.00
[0133] Palmitoleic Acid, 2% Topical Burn and Wound
Healing Formulation
7.00
[0134] This invention provides a topical preparation for
Sorbitol (70%)
Glycerol
SodiuIn lauryl sulfate
10.00
9.00
0.30
treating burns and healing Wounds containing 2% by Weight
Tauranol
Flavor
Water
0.20
0.10
61.4
Propylene glycol
palmitoleic acid With the folloWing formulation.
Ingredients
Palmitoleic Acid
Vitalnin E
Example 14
2.0
0.3
Polyethylene glycol 400
Polyethylene glycol 4000
[0126] Palmitoleic Acid, Pharmaceutical Suspension For
Water
mulation for Aerosol Administration
[0127]
Parts By Weight
49.8
44.9
3.0
This invention provides a suspension aerosol for
mulation comprising a therapeutically effective amount of
about 0.1 to 0.5% by Weight of palmitoleic acid, about 5 to
12% by Weight of ethanol, about a 0.1 to 0.5% by Weight of
oleic acid, and a propellant comprising HFC 227.
[0135]
[0128] The palmitoleic acid suspension aerosol formula
containing palmitoleic acid.
tion of this invention can be prepared by ?rst placing the
palmitoleic acid in an aerosol vial. A mixture of the propel
lant, oleic acid and ethanol can then be added, and the
palmitoleic acid dispersed in the mixture. This formulation
could be used to assist in the reduction and prevention of
bacterial bio?lms in the lungs.
[0137] It is preferred that the pet food of the present
Example 15
Example 18
Dental Care Pet Food With Palmitoleic Acid
[0136] This invention provides a pet food composition
invention be a dry or semi-moist pet food, preferably a
biscuit, but the description applies generally to dry pet foods
such as pelletiZed dog or cat foods.
[0138] The manufacture of such pet foods, particularly pet
biscuits, generally involves the step of shaping and baking
a dough containing the desired ingredients. The dental care
biscuits of the present invention can be made from any
[0129] Palmitoleic Acid, Pharmaceutical Formulation for
suitable dough according to conventional techniques knoWn
NebuliZation
in the art.
[0130] Solutions Were prepared comprising 2 mg/ml and
10 mg/ml of palmitoleic acid in ethanol/propylene glycol/
to palmitoleic acid, at least one ?our, meal, fat and Water. A
[0139] The dough compositions may comprise, in addition
conventional dough for dog biscuits may also comprise
Water (85:10:5). These solutions Were nebuliZed separately
by a ProNeb Ultra nebuliZer manufactured by PARl. The
nebuliZed solutions Were collected in a cold trap, processed
appropriately, and detected by mass spectrometry. Palmi
particles of meat and/or meat byproducts and fat solids.
Alternatively, the palmitoleic acid can be applied to the
toleic acid Was recovered from both formulations demon
Water.
surface of the biscuit after baking, as a 1-50% solution in
Oct. 12, 2006
US 2006/0228384 A1
[0140] The dough ingredients are admixed at a tempera
ture of about 15° C. to about 300 C. The dough pieces can
be baked using any suitable or conventional equipment and
conditions in the range of about 100° C. to about 3000 C.
With subsequent drying to produce the desired moisture
content in the ?nal product.
REFERENCES
[0141] Costerton, J. W. et. al. 1999. Bacterial Bio?lms: A
Common Cause of Persistent Infections. Science.
284:1318-1322.
[0142] Centers for Disease Control and Prevention 2002.
Guidelines for the Prevention of Intravascular Catheter
Related Infections. MMWR. 51:RR-10.
[0143] Maki, D. G. et. al. 1997. Prevention of Central
Venous Catheter-Related Bloodstream Infection by Use of
an Antiseptic-Impregnated Catheter. Ann. Int. Med.
127(4):257-266.
[0144] Darouiche, R. et. al. 1999. A Comparison of TWo
Antimicrobial-Impregnated Central Venous Catheters.
NeW Engl. Jour. Med. 340(1):1-8.
[0145] Raad, I, et. al. 1997. Central Venous Catheters
Coated With Minocycline and Rifampin for the Prevention
of Catheter-Related ColoniZation and Bloodstream Infec
tions. Ann. Int. Med. 127:267-274.
[0146] Hanna, H. A., et. al. 2003. Antibiotic-Impregnated
Catheters Associated With Signi?cant Decrease in Noso
comial and Multidrug-Resistant Bacteremias in Critically
Ill Patients. Chest. 124(3):1030-1038.
[0147]
Pendland, S. L. et. al. 2002. In vitro synergy testing
of levo?oxacin, o?oxacin, and cipro?oxacin in combina
tion With aZtreonam, ceftaZidime, or piperacillin against
Pseudomonas aeruginosa. Diag. Micro. InfDis. 42:75
McLaughlin, Borlace L. et. al. 1998. Bacterial
Bio?lm on contact lenses and in lens storage cases in
Wearers With microbial keratitis. J. of Applied Microbi
ology 84(5):827-838.
[0155] Landa, H. C. et. al. 1998. Ef?cacy of ophthalmic
solutions to detach adhering Pseudomonas aeruginosa
from contact lenses. 17(3):293-300.
[0156]
Akamatsu, H. et. al. 1990. The inhibition of free
radical generation by human neutrophils through the
synergistic effects of metronidaZole With palmitoleic acid:
a possible mechanism of action of metronidaZole in
rosacea and acne. Archives of Dermatological Research
282:449-454.
[0157] Wille, J. J. et. al. 2003. Palmitoleic Acid Isomer
(C16:1A6) in Human Skin Sebum Is Effective against
Gram-Positive Bacteria. Skin Pharmacol Appl Skin
Physiol. 16:176-187.
[0158] Yang, Baoru et. al. 1999. Effects of dietary supple
mentation With sea buckthom (Hippophae rhamnoides)
see and pulp oils on atopic dermatitis. J. Nutr. Bichem.
10:622-630.
[0159] Gao, Ze-Li et. al. 2003. Effect of Sea buckthorn on
liver ?brosis: A clinical study. World J. Gastroenterol.
9(7):1615-1617
[0160] Eldridge, G. R. et. al. 2002. High-throughput
method for the production and analysis of large natural
product libraries for drug discovery. Analytical Chemis
try. 74(16):3963-3971.
[0161]
Cremen, Peadar A. et. al. 2002. High-Throughput
Analysis of Natural Product Compound Libraries by
Parallel LC-MS Evaporative Light Scattering Detection.
Analytical Chemistry. 74(21):5492-5500.
[0162]
Slack, J. M. 2000. Stem Cells in Epithelial Tissues.
Science. 287:1431-1433.
78.
[0148] Jones, S. M., et. al. 2001. Effect ofvancomycin and
rifampicin on methicillin-resistant Staphylococcus aureus
bio?lms. Lancet. 357:40-41.
[0149] CDC. Update: Investigation of Bioterrorism-Re
lated Anthrax and Interim Guidelines for Exposure Man
agement and Antimicrobial Therapy. October 2001.
MMWR 2001. 50:909-919.
[0150] Price, L. B., et. al. 2003 In vitro selection and
characteriZation of Bacillus anthracis mutants With high
level resistance to cipro?oxacin. Antimicrob. Agents
Chemother. 57:2362-2365.
[0151] Ramsey, Bonnie W. et. al. 1999. Intermittent
Administration of Inhaled Tobramycin in Patients With
Cystic Fibrosis. NeW England J. Medicine. 340(1):23-30.
[0152]
[0154]
Ramage, Gordon et. al. 2003. Formation of Propi
onibacterium aches bio?lms on orthopaedic biomaterials
and their susceptibility to antimicrobials. Biomaterials.
24:3221-3227.
What is claimed:
1. A method for preventing, reducing, inhibiting or
removing a bio?lm comprising contacting the bio?lm or
contacting a cell capable of forming a bio?lm With an
effective amount of a composition comprising palmitoleic
acid, Wherein the composition prevents, reduces, inhibits or
removes the bio?lm.
2. The method of claim 1, Wherein the cell is selected from
the group consisting of Streptococcus mutans, Streptococcus
sobrinus, Streptococcus pyogenes, Streptococcus pneumo
niae, Escherichia coli, Pseudomonas aeruginosa, Staphylo
coccus aureus, Staphylococcus epidermidis, Propionibacte
rium aches, Enterococcus faecium, Enterococcus faecalis,
and Haemophilus in?uenzae.
3. The method of claim 1, Wherein the composition
comprises about 0.1% to about 20% of palmitoleic acid.
4. A method for controlling acne, comprising administer
ing to a subject a?licted With acne an effective amount of an
anti-acne composition comprising palmitoleic acid, Wherein
the amount of the palmitoleic acid in the anti-acne compo
sition is suf?cient to prevent, reduce, inhibit or remove a
[0153] Burkhart, Craig N. et. al. 2003. Microbiology’s
bio?lm.
5. The method of claim 4, Wherein the composition
principle of bio?lms as a major factor in the pathogenesis
of acne vulgaris. International J. of Dermatology. 42:925
prevents, reduces, inhibits or removes a bio?lm of Propi
927.
onibacterium aches.
Oct. 12, 2006
US 2006/0228384 A1
6. The method of claim 4, wherein the antimicrobial agent
and palmitoleic acid or its derivative provide a synergistic
effect.
7. The method of claim 4, Wherein the antimicrobial agent
is an antibiotic.
8. The method of claim 7, Wherein the antibiotic may is
selected from the group consisting of tobramycin, clinda
30. The method of claim 29, Wherein the antimicrobial
agent is an antibiotic.
31. The method of claim 30, Wherein the antibiotic may
is selected from the group consisting of tobramycin, clin
damycin, cipro?oxacin, tetracyclines, rifampin, triclosan,
ox?oxacin, macrolides, penicillins, cephalosporins, amox
icillin/clavulante, quinupristin/dalfopristin, amoXicillin/sul
mycin, cipro?oxacin, tetracyclines, rifampin, triclosan,
bactum, metronidaZole, ?uoroquinolones, quinolones,
ox?oxacin, macrolides, penicillins, cephalosporins, amox
icillin/clavulante, quinupristin/dalfopristin, amoXicillin/sul
ketolides, or aminoglycosides.
bactum, metronidaZole, ?uoroquinolones, quinolones,
ketolides, or aminoglycosides.
9. The method of claim 4, Wherein the amount of palmi
toleic acid is betWeen about 0.5% to about 50%.
10. The method of claim 4, Wherein the anti-acne com
position is topically administered.
11. The method of claim 10, Wherein the anti-acne com
position is applied to the skin of the subject.
12. The method of claim 4, Wherein the anti-acne com
position is a cream, gel, emollient, or soap.
13. The method of claim 4, Wherein the subject is a
mammal.
14. The method of claim 13, Wherein the mammal is a
mouse or rat.
15. The method of claim 13, Wherein the mammal is a
human.
16. The method of claim 4, Wherein the anti-acne com
32. The method of claim 28, Wherein the subject is a
mammal.
33. The method of claim 32, Wherein the mammal is a
mouse or rat.
34. The method of claim 32, Wherein the mammal is a
human.
35. The method of claim 28, Wherein the chronic bacterial
infection is selected from the group consisting of urinary
tract infection, gastritis, lung infection, ear infection, cysti
tis, pyelonephritis, arterial damage, leprosy, tuberculosis,
benign prostatic hyperplasia, chronic prostatitis, osteomy
elitis, bloodstream infection, skin infection, rosacea, open
Wound infection, chronic Wound infection, and sinus infec
tion.
36. The method of claim 28, Wherein the chronic bacterial
infection results from an infection of bacteria selected from
the group consisting of Slreplococcuspyogenes, Streptococ
position further comprises at least one antimicrobial agent.
cus pneumoniae, Escherichia coli, Pseudomonas aerugi
17. The method of claim 16, Wherein the anti-acne com
position prevents, reduces, inhibits or removes a bio?lm of
Propionibaclerium aches.
18. The method of claim 16, Wherein the antimicrobial
agent and palmitoleic acid or its derivative provide a syn
nosa, Staphylococcus aureus, Staphylococcus epidermidis,
ergistic effect.
19. The method of claim 16, Wherein the antimicrobial
agent is an antibiotic.
20. The method of claim 19, Wherein the antibiotic may
Propionibaclerium aches, and Haemophilus in?uenzae.
37. A method for identifying an agent capable of prevent
ing, reducing, inhibiting or removing a bio?lm, comprising
a. contacting a bio?lm With the agent, Wherein the agent
is a derivative of a palmitoleic acid;
b. quantifying the bio?lm before and after contacting the
bio?lm With the agent;
is selected from the group consisting of tobramycin, clin
damycin, cipro?oxacin, tetracyclines, rifampin, triclosan,
ox?oxacin, macrolides, penicillins, cephalosporins, amox
icillin/clavulante, quinupristin/dalfopristin, amoXicillin/sul
bactum, metronidaZole, ?uoroquinolones, quinolones,
ketolides, or aminoglycosides.
c. selecting the agent that prevents, reduces, inhibits or
removes at least about 50% of the bio?lm, Wherein the
agent does not inhibit bacterial groWth When the con
centration amount of the agent is at least tWice the
amount needed to prevent, reduce, inhibit, or remove at
least 50% of the bio?lm.
21. The method of claim 16, Wherein the amount of
palmitoleic acid is betWeen about 0.5% to about 50%.
22. The method of claim 16, Wherein the anti-acne com
38. The method of claim 37, Wherein in step b) the bio?lm
is stained With a dye and measured using a spectrophotom
position is topically administered.
eter to obtain the quantitative value for the mass of each
bio?lm.
23. The method of claim 22, Wherein the anti-acne com
position is applied to the skin of the subject.
39. A pharmaceutical composition for reducing, prevent
ing, inhibiting or removing bio?lm formation comprising a
24. The method of claim 16, Wherein the anti-acne com
position is a cream, gel, emollient, or soap.
25. The method of claim 16, Wherein the subject is a
mammal.
26. The method of claim 25, Wherein the mammal is a
pharmaceutically acceptable carrier or diluent and a thera
peutically effective amount of 0.01 to 50% of palmitoleic
acid.
mouse or rat.
comprising at least one antimicrobial agent.
27. The method of claim 25, Wherein the mammal is a
human.
28. A method for controlling a chronic bacterial infection,
comprising administering to a subject in need thereof an
effective amount of a composition comprising palmitoleic
acid, Wherein the amount is effective to prevent, inhibit,
reduce, or remove a bio?lm.
29. The method of claim 28, Wherein the composition
further comprises at least one antimicrobial agent.
40. The pharmaceutical composition of claim 39, further
41. The pharmaceutical composition of claim 40, Wherein
the antimicrobial agent is an antibiotic.
42. The pharmaceutical composition of claim 41, Wherein
the antibiotic may is selected from the group consisting of
tobramycin, clindamycin, cipro?oxacin, tetracyclines,
rifampin, triclosan, ox?oxacin, macrolides, penicillins,
cephalosporins, amoXicillin/clavulante, quinupristin/dalfo
pristin, amoXicillin/sulbactum, metronidaZole, ?uoroquino
lones, quinolones, ketolides, or aminoglycosides.