U.S. patent application number 15/966088 was filed with the patent office on 2018-11-01 for biofilm penetrating compositions and methods.
This patent application is currently assigned to Nevada Naturals Inc.. The applicant listed for this patent is Nevada Naturals Inc.. Invention is credited to Anthony J. Sawyer, Richard F. Stockel.
Application Number | 20180310566 15/966088 |
Document ID | / |
Family ID | 63915433 |
Filed Date | 2018-11-01 |
United States Patent
Application |
20180310566 |
Kind Code |
A1 |
Sawyer; Anthony J. ; et
al. |
November 1, 2018 |
Biofilm Penetrating Compositions and Methods
Abstract
Compositions are provided that have at least two of three active
ingredients. The active ingredients maybe a salt having a cation
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester, a glycerol monoester of a fatty acid and a sugar ester of a
fatty acid. The compositions are useful in methods of killing or
inhibiting planktonic bacteria or fungi and bacteria or fungi
embedded in a biofilm and prevention of biofilm formation on
surfaces. The composition may further comprise a hydrogel and a
benefit agent such as an antibiotic that can be solubilized by the
hydrogel and supplied to the biofilm matrix by the active
ingredients of the composition. Devices such as chronic wound
coverings coated with the composition are also provided. Methods of
preserving products with the composition are also provided.
Inventors: |
Sawyer; Anthony J.;
(Albuquerque, NM) ; Stockel; Richard F.;
(Bridgewater, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nevada Naturals Inc. |
Albuquerque |
NM |
US |
|
|
Assignee: |
Nevada Naturals Inc.
Albuquerque
NM
|
Family ID: |
63915433 |
Appl. No.: |
15/966088 |
Filed: |
April 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62492131 |
Apr 29, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 29/145 20130101;
A01N 2300/00 20130101; A61L 2300/406 20130101; A61L 31/145
20130101; A01N 43/16 20130101; A61L 26/0066 20130101; A01N 47/44
20130101; A61L 29/16 20130101; A61L 27/52 20130101; A61L 27/54
20130101; A61L 2400/04 20130101; A61L 17/005 20130101; A61L 15/20
20130101; A61L 31/10 20130101; A01N 37/36 20130101; A61L 27/34
20130101; A61L 15/60 20130101; A61L 31/16 20130101; A61L 29/085
20130101; A61L 15/46 20130101; A01N 47/44 20130101; A01N 25/30
20130101; A01N 47/44 20130101; A01N 25/30 20130101; A01N 37/12
20130101; A01N 37/36 20130101; A01N 43/16 20130101 |
International
Class: |
A01N 47/44 20060101
A01N047/44; A01N 43/16 20060101 A01N043/16; A01N 37/36 20060101
A01N037/36 |
Claims
1. A method of killing or inhibiting planktonic bacteria or fungi
and bacteria or fungi embedded in a biofilm comprised of at least a
matrix and bacteria or fungi, the method comprising: applying to a
surface of the biofilm a composition having an active ingredient
comprising at least two or more of: a) a salt having a cation
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester and an anion selected from the group consisting: of halide,
nitrite, nitrate, linolenate, laurate, oleoate, phenolate,
polyphenolate, carboxylate, hydroxycarboxylate, hyaluronate,
antibiotic anion, resveratrol, and an amino acid, the salt being
present in an amount from about 0.025 wt % to about 10 wt %; b) a
glycerol monoester of a fatty acid being present in an amount from
about 0.05 wt % to about 20 wt %; and c) a sugar ester of a fatty
acid being present in an amount from about 0.075 wt % to about 30
wt %; and optionally comprising one or more of: d) a solvent being
present in an amount from about 20 wt % to about 99.9 wt %; or e) a
thickener or carrier or gelling agent being present in an amount
from about 20 wt % to about 75 wt %; or f) a sacrificial agent
being present in an amount from about 0.05 wt % to about 5 wt %; or
g) a hydrogel having a three-dimensional hydrophilic polymer
network, the active ingredient of the composition killing or
inhibiting planktonic bacteria or fungi and penetrating the biofilm
matrix and killing or inhibiting biofilm bacteria or fungi.
2. The method of claim 1, further characterized by: the a)
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or the b)
glycerol monoester a fatty acid being monolaurin; or the c) sugar
ester of a fatty acid being sucrose laurate; or the d) solvent
being at least one of: water, 1,2-propylene glycol or 1,3-propylene
glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol, polyethylene
glycol, polypropylene glycol, butylene glycol, pentylene glycol,
hexylene glycol; or the e) thickener or carrier or gelling agent
being at least one of: a polymer, a hydrocolloid, an acrylate, an
acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
trimethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
3. The method of claim 1, the biofilm further characterized as
covering a wound, or in medical tubing, or on medical instruments,
or in devices, or in wound drainage tubes, or in human or animal
food processing or packaging equipment, or on food conveyor belts,
or on pet chew toys, or in animal water bowls, or on floating toys,
or in piping or in or on contact lens.
4. The method of claim 1 further comprising delivering an
antibiotic, an antimicrobial, or a benefit agent to planktonic
bacteria or fungi or biofilm bacteria or fungi, the method
comprising: adding to the composition comprising at least two of
a), b) or c) and optionally d)-g): h) a benefit agent comprising an
antibiotic, an antimicrobial, or a drug; and applying the
composition comprising a)-h) to a biofilm, the composition of a)
through g) acting as a delivery means for the benefit agent of h)
to both planktonic bacteria or fungi and to biofilm bacteria or
fungi by penetrating the biofilm matrix to deliver the benefit
agent.
5. The method of claim 4, the benefit agent being water soluble or
water insoluble and the benefit agent being solubilized in the g)
hydrogel and added in combination with the composition comprising
at least two of a), b) or c) and optionally d)-f).
6. A method of preserving a surface or product by preventing or
inhibiting biofilm formation by bacteria or fungi, the method
comprising: applying to a surface or adding to a product a
composition having an active ingredient comprising at least two or
more of: a) a salt having a cation N.sup..alpha.C8-C16 alkanoyl-L
di-basic amino acid --C1-C4 alkyl ester and an anion selected from
the group consisting of: halide, nitrite, nitrate, linolenate,
laurate, oleoate, phenolate, polyphenolate, carboxylate,
hydroxycarboxylate, hyaluronate, antibiotic anion, resveratrol, and
an amino acid, the salt being present in an amount from about 0.025
wt % to about 10 wt %; b) a glycerol monoester of a fatty acid
being present in an amount from about 0.05 wt % to about 10 wt %;
and c) a sugar ester of a fatty acid being present in an amount
from about 0.075 wt % to about 20 wt %; and optionally comprising
one or more of: d) a solvent being present in an amount from about
20 wt % to about 99.9 wt %; or e) a thickener or carrier or gelling
agent being present in an amount from about 20 wt % to about 75 wt
%; or f) a sacrificial agent being present in an amount from about
0.05 wt % to about 5 wt %; or g) a hydrogel having a
three-dimensional hydrophilic polymer network; the active
ingredient of the composition preventing or inhibiting bacteria or
fungi from forming a biofilm on a surface or in a product.
7. The method of claim 6, further characterized by the a)
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or the b)
glycerol monoester a fatty acid being monolaurin; or the c) sugar
ester of a fatty acid being sucrose laurate; or the d) solvent
being at least one of: water, ethanol, 1,2-propylene glycol or
1,3-propylene glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol,
polyethylene glycol, polypropylene glycol, butylene glycol,
pentylene glycol, hexylene glycol; or the e) thickener or carrier
or gelling agent being at least one of: a polymer, a hydrocolloid,
an acrylate, an acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
triethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
8. The method of claim 6, the surface being selected from the group
consisting of: microcapsules, wound dressings, implants, wound
closures, staples, meshes, controlled drug delivery systems, wound
coverings, fillers, sutures, tissue adhesives, tissue sealants,
absorbable and non-absorbable hemostats, catheters, wound drainage
tubes, arterial grafts, soft tissue patches, gloves, shunts,
stents, guide wires and prosthetic devices, contact lens, medical
devices, food processing equipment, food conveyor belts, food
packaging equipment, pet or animal food, pet chew toys, pet or
animal water bowls, and floating toys.
9. The method of claim 6, the product being selected from the group
consisting of: cosmetics and personal care items.
10. A composition for penetrating a biofilm matrix and killing both
planktonic and biofilm bacteria or fungi, the composition having an
active ingredient comprising at least two or more of: a) a salt
having a cation N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid
--C1-C4 alkyl ester and an anion selected from the group consisting
of: halide, nitrite, nitrate, linolenate, laurate, oleoate,
phenolate, polyphenolate, carboxylate, hydroxycarboxylate,
hyaluronate, antibiotic anion, resveratrol, and an amino acid, the
salt being present in an amount from about 0.025 wt % to about 10
wt %; b) a glycerol monoester of a fatty acid being present in an
amount from about 0.05 wt % to about 10 wt %; and c) a sugar ester
of a fatty acid being present in an amount from about 0.075 wt % to
about 20 wt %; and optionally comprising one or more of: d) a
solvent being present in an amount from about 20 wt % to about 99.9
wt %; or e) a thickener or carrier or gelling agent being present
in an amount from about 20 wt % to about 75 wt %; or f) a
sacrificial agent being present in an amount from about 0.05 wt %
to about 5 wt %; or g) a hydrogel having a three-dimensional
hydrophilic polymer network.
11. The composition of claim 9, further characterized by: the a)
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or the b)
glycerol monoester a fatty acid being monolaurin; or the c) sugar
ester of a fatty acid being sucrose laurate; or the d) solvent
being at least one of: water, 1,2-propylene glycol or 1,3-propylene
glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol, polyethylene
glycol, polypropylene glycol, butylene glycol, pentylene glycol,
hexylene glycol; or the e) thickener or carrier or gelling agent
being at least one of: a polymer, a hydrocolloid, an acrylate, an
acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
triethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
12. The composition of claim 10 further comprising h) at least one
bioactive agent.
13. A treated device or treated product comprising a device or
product and the composition of claim 10, the treated device made by
a process comprising impregnating, dipping, coating, brushing or
soaking the device with the composition or the treated product made
by a process of mixing the composition with the product.
14. The device of claim 10, the device being selected from the
group consisting of: microcapsules, wound dressings, surgical
implants, wound closures, staples, meshes, controlled drug delivery
systems, wound coverings, medical fillers, sutures, tissue
adhesives, tissue sealants, absorbable and non-absorbable
hemostats, catheters, wound drainage tubes, arterial grafts, soft
tissue patches, gloves, shunts, stents, surgical guide wires,
prosthetic devices, contact lens, endoscopes, dentures, medical
devices, food processing equipment, food conveyor belts, food
packaging equipment, pet or animal food, pet chew toys, pet or
animal water bowls, and floating toys.
15. The device of claim 14 being a wound covering for chronic
wounds that does not adhere to the wound surface, is held stable at
the wound site, and has water absorbing properties, the wound
covering further comprising: an outer flexible, water-resistant
protective covering that does not contact the surface of the
chronic wound; means for securing dressing to a wound site; and a
surface that is in contact with the chronic wound comprising
synthetic polymers, natural polymers or a combination thereof that
absorb water and release the composition to the surface of the
chronic wound.
Description
PRIORITY
[0001] This application claims priority to U.S. Provisional Pat.
App. No. 62/492,131, filed on Apr. 29, 2017, which is incorporated
by reference herein in its entirety.
BACKGROUND
[0002] Previous patents WO 2013/169231 A1, U.S. Pat. No. 9,023,891,
U.S. Pat. No. 9,271,495, U.S. Pat. No. 8,834,857, U.S. Pat. No.
8,926,997, U.S. Pat. No. 8,795,638, U.S. Pat. No. 8,734,879 and
U.S. Pat. No. 8,193,244 have disclosed salts having a cation
N.sup..alpha. C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester and various anions selected from the group consisting of
halide, nitrite, nitrate, phenolate, polyphenolate, carboxylate,
hydroxycarboxylate, hyaluronate, antibiotic anion and an amino
acid.
[0003] U.S. Pat. No. 8,604,073 disclose medical devices
incorporated with a biofilm inhibiting composition that comprises
lauric arginate (LAE) and an antibiotic. U.S. Pat. No. 8,604,073
discloses an antimicrobial composition comprising lauric arginate
(LAE) and one or more antibiotic.
[0004] Gil et al. (Antimicrobial Agents and Chemotherapy, July 2017
Vol. 61 Is.7) report the use of monolaurin stainless steel K-wires
were coated with monolaurin solubilized in ethanol using a simple
but effective dip-coating method.
[0005] LAE has been disclosed as inhibiting biofilm formation on
surgical implants and catheters (WO2012013577). U.S. Pub. Appl. No.
2015/0010715 discloses antimicrobial coatings are composed of a
hydrogel and a bioactive agent including a substantially
water-insoluble antimicrobial metallic material (silver
sulfadiazine) that is solubilized within the coating. U.S. Pat. No.
6,638,978 lists a preservative formulation for food and cosmetics
consisting of glyceryl mono-laurate (monolaurin, or "ML"), a
mixture of caprylic and capric acid and propylene glycol in an
aqueous base.
[0006] U.S. Pat. No. 4,002,775 discloses the discovery that highly
effective and yet food-grade microbicides are provided by
mono-esters of a polyol and a twelve-carbon atom aliphatic
carboxylic fatty acid.
[0007] Biofilm
[0008] It is accepted that biofilms are a ubiquitous problem in
industry, dentistry and medicine (Rhoads et al., J. of Wound Care,
Vol. 17, No 11, November 2008). Phillips et al. (Wounds
International, Vol 1, Issue 3 May 2010) described biofilms as
complex microbial communities containing bacteria and fungi (yeast
and molds). The microorganisms synthesize and secrete a protective
matrix that attaches the biofilm firmly to a living or non-living
surface. Biofilms are dynamic heterogeneous communities that are
continuously changing. At the most basic level a biofilm can be
described as bacteria or fungi embedded in a thick, barrier of
sugars and proteins. The biofilm barrier protects the
microorganisms from external threats. Biofilms have long been known
to form on surfaces of medical devices, such as urinary catheters,
endotracheal and tympanostomy tubes, orthopedic and breast
implants, contact lenses, intrauterine devices (IUDs) and sutures.
They are a major contributor to diseases that are characterized by
an underlying bacterial infection and chronic inflammation, e.g.
periodontal disease, cystic fibrosis, chronic acne and
osteomyelitis. Kaplan, et al. (J. of Bact. December 2004, p.
8213-8220) write that the extracellular polymeric substances (EPS)
matrix may also contribute to the increased resistance to
antibiotics and host defenses exhibited by biofilm cells.
Polysaccharide is a major component of the EPS matrix in most
bacterial biofilms.
[0009] A serious potential problem not addressed by many wound
healing techniques is the presence of biofilms, particularly
biofilms containing Pseudomonas aeruginosa (P. aeruginosa or
Pseudomonas a. or Pseudomans a.). The microbial cells growing in a
biofilm are physiologically distinct from planktonic cells of the
same organism, which by contrast are single-cells that may float or
move in a liquid medium. When a cell switches to the biofilm mode
of growth, it undergoes a phenotypic shift in behavior in which
large suites of genes are differentially regulated. A critical
factor in the development of biofilm is that a specific type of
signal molecule by microorganisms is important for the switching on
and off of various properties such as virulence factor and biofilm
production. This type of property is called quorum sensing.
Wounds, Burns, and Biofilm
[0010] Biofilm is a very serious problem and is responsible for
persistent infections when treating burn wounds or wounds in
general (Costertan et al., Science 284: p 1318-1322 (1999)). It is
suggested that biofilms contain anoxic regions where the metabolic
activity and also the susceptibility to antimicrobials of aerobes
such as P. aeruginosa is reduced (Walters et al, Antimicrob. Agents
Chemother, 47: p 317-323 (2003).
Livestock and Companion Animals
[0011] It has been reported that biofilm formation by bacterial
pathogens of veterinary or zoonotic importance has surprisingly
received relatively little attention. For example, animals have
problems with plaque biofilm formation on their teeth. Publications
have reported that chew toys as well as water bowls are a source of
biofilm that results from the saliva enzymes. Biofilm bacteria can
also cause systemic inflammation, cardiovascular diseases, urinary
tract infections and chronic kidney disease in pets, especially
cats. Zambori et al. (Scientific Papers: Animal Science and
Biotechnologies, 2012, 45(2)) report that the importance of biofilm
in disease processes in humans and animals is now widely
recognized.
SUMMARY
[0012] This invention discloses a composition and method of use
comprising the combination of several green and naturally derived
ingredients and suitable carriers in a form suitable for use as
biofilm penetrating and inhibiting composition and a wound healing
composition for treating wounds containing biofilm and the methods
of use thereof.
[0013] Based on the experimental data in the instant invention,
combinations of LAE (N.sup..alpha.C8-C16 alkanoyl-L di-basic amino
acid --C1-C4 alkyl ester being
N.sup..alpha.-lauroyl-L-arginine-ethyl ester)-HCl, SL (sucrose
laurate), and ML (glycerol monolaurate) penetrate established P.
aeruginosa biofilm and kill sessile (anchored) and planktonic (free
floating) bacteria.
[0014] The instant invention discloses the use of a composition to
inhibit biofilm formation and kill planktonic bacteria or fungi as
well as biofilm bacteria or fungi on medical devices as well as
contact lens, food preparation surfaces and the like.
[0015] It is the objective of this invention to present a novel and
unanticipated approach using green and naturally derived food
ingredients that can effectively penetrate and reach bacteria
imbedded in biofilm that may be found in a wound, in surgical
devices, in body cavities such as nasal passages, vaginal areas, or
on animal and human food processing equipment.
[0016] Another objective of this invention is to present a system
to penetrate and reach the bacteria in mature biofilm cells and
kill it, while also killing the planktonic cells that form the
biofilm.
[0017] A third object of this invention is to provide means treat
wounds with a system that does not require daily changes. A fourth
object of this invention is to provide a safe and non-cytotoxic
system for biofilm penetration and inhibition. A fifth object of
this invention is to reduce biofilm and inhibit the growth or
reconstitution of additional biofilm.
[0018] Biofilm containing bacteria can occur in or on the body,
e.g. in wounds, burns, in oral care whereby plaque is considered
biofilm, in the nasal cavity, in skin acne, in the ear, in contact
lens, etc. The compositions can be used as storage solutions or a
disinfecting system for contact lenses or to inhibit or kill
planktonic and sessile cells because contact lenses frequently have
biofilm. Biofilms can be found in or on medical or dental devices
or equipment, or surgical instruments used for procedures where it
is difficult to reach/penetrate biofilm, e.g. in surgical
instruments such as endoscopes, etc. These compositions are used as
a coating on existing medical devices or dental devices, e.g.
surgical implants and similar devices, medical/dental/surgical
equipment, etc. to inhibit the formation of biofilms.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention may be obtained by
reference to the following detailed description that sets forth
illustrative embodiments in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0020] FIG. 1A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 1.
[0021] FIG. 2A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 2.
[0022] FIG. 3A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 3.
[0023] FIG. 4A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 4.
[0024] FIG. 5A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 5.
[0025] FIG. 6A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 6.
[0026] FIG. 7 depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 7.
[0027] FIG. 8 A-B depicts the formulas tested and results after
treatment with the compositions of the present invention according
to Example 8.
DETAILED DESCRIPTION
[0028] One embodiment of the invention is a method of killing or
inhibiting planktonic bacteria or fungi and bacteria or fungi
embedded in a biofilm comprised of at least a matrix and bacteria,
the method comprising: applying to a surface of the biofilm a
composition having an active ingredient comprising at least two or
more of: a) a salt having a cation N.sup..alpha.C8-C16 alkanoyl-L
di-basic amino acid --C1-C4 alkyl ester and an anion selected from
the group consisting: of halide, nitrite, nitrate, linolenate,
laurate, oleoate, phenolate, polyphenolate, carboxylate,
hydroxycarboxylate, hyaluronate, antibiotic anion, resveratrol, and
an amino acid, the salt being present in an amount from about 0.025
wt % to about 10 wt %; b) a glycerol monoester of a fatty acid
being present in an amount from about 0.05 wt % to about 20 wt %;
and c) a sugar ester of a fatty acid being present in an amount
from about 0.075 wt % to about 30 wt %. To this active ingredient
composition can optionally be added one or more of: d) a solvent
being present in an amount from about 20 wt % to about 99.9 wt %;
or e) a thickener or carrier or gelling agent being present in an
amount from about 20 wt % to about 75 wt %; or f) a sacrificial
agent being present in an amount from about 0.05 wt % to about 5 wt
%; or g) a hydrogel having a three-dimensional hydrophilic polymer
network. In this method, the active ingredient of the composition
killing or inhibiting planktonic bacteria or fungi and penetrating
the biofilm matrix and killing or inhibiting biofilm bacteria or
fungi.
[0029] Even more specifically, the method may be characterized by:
the a) N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4
alkyl ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or
the b) glycerol monoester a fatty acid being monolaurin; or the c)
sugar ester of a fatty acid being sucrose laurate; or the d)
solvent being at least one of: water, 1,2-propylene glycol or
1,3-propylene glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol,
polyethylene glycol, polypropylene glycol, butylene glycol,
pentylene glycol, hexylene glycol; or the e) thickener or carrier
or gelling agent being at least one of: a polymer, a hydrocolloid,
an acrylate, an acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
triethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
[0030] The compositions and methods in at least one embodiment of
the invention treat biofilm covering a wound, or in medical tubing,
or on medical instruments, or in devices, or in wound drainage
tubes, or in human or on animal food processing or packaging
equipment, or on food conveyor belts, or on pet chew toys, or in
animal water bowls, or on floating toys, or in piping or in or on
contact lens.
[0031] In yet another embodiment, methods of delivering an
antibiotic, an antimicrobial, or a benefit agent to planktonic
bacteria or fungi or biofilm bacteria or fungi are provided.
Specifically, a composition is formed by adding to the composition
comprising at least two of a), b) or c) and optionally d)-g) the
ingredient h) a benefit agent comprising an antibiotic, an
antimicrobial, or a drug. The benefit agent may be solubilized in a
hydrogel and then added to the remaining ingredients of the
composition. When this novel composition is applied to a biofilm,
the composition of a) through f) acts as a delivery means for the
benefit agent of h) to both planktonic bacteria or fungi and to
biofilm bacteria or fungi by penetrating the biofilm matrix to
deliver the benefit agent.
[0032] In yet another embodiment, a method of preserving a surface
or product by preventing or inhibiting biofilm formation by
bacteria or fungi is provided that comprises applying to a surface
or adding to a product a composition having an active ingredient
comprising at least two or more of: a) a salt having a cation
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester and an anion selected from the group consisting of: halide,
nitrite, nitrate, linolenate, laurate, oleoate, phenolate,
polyphenolate, carboxylate, hydroxycarboxylate, hyaluronate,
antibiotic anion, resveratrol, and an amino acid, the salt being
present in an amount from about 0.025 wt % to about 10 wt %; b) a
glycerol monoester of a fatty acid being present in an amount from
about 0.05 wt % to about 10 wt %; and c) a sugar ester of a fatty
acid being present in an amount from about 0.075 wt % to about 20
wt %. To the active ingredients of the composition a d) a solvent
being present in an amount from about 20 wt % to about 99.9 wt %;
or e) a thickener or carrier or gelling agent being present in an
amount from about 20 wt % to about 75 wt %; or f) a sacrificial
agent being present in an amount from about 0.05 wt % to about 5 wt
%; or g) a hydrogel having a three-dimensional hydrophilic polymer
network may be added. In this method, the active ingredient of the
composition acting as a preservative by preventing or inhibiting
bacteria or fungi from forming a biofilm on a surface or in a
product.
[0033] The methods may also be characterized by the a)
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or the b)
glycerol monoester a fatty acid being monolaurin; or the c) sugar
ester of a fatty acid being sucrose laurate; or the d) solvent
being at least one of: water, ethanol, 1,2-propylene glycol or
1,3-propylene glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol,
polyethylene glycol, polypropylene glycol, butylene glycol,
pentylene glycol, hexylene glycol; or the e) thickener or carrier
or gelling agent being at least one of: a polymer, a hydrocolloid,
an acrylate, an acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
triethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
[0034] The methods may be applied to a surface being selected from
the group consisting of: microcapsules, wound dressings, implants,
wound closures, staples, meshes, controlled drug delivery systems,
wound coverings, fillers, sutures, tissue adhesives, tissue
sealants, absorbable and non-absorbable hemostats, catheters, wound
drainage tubes, arterial grafts, soft tissue patches, gloves,
shunts, stents, guide wires and prosthetic devices, contact lens,
medical devices, food processing equipment, food conveyor belts,
food packaging equipment, pet or animal food, pet chew toys, pet or
animal water bowls, cosmetics, and floating toys.
[0035] The methods may be used for preserving the products selected
from the group consisting of: cosmetics and personal care
items.
[0036] Compositions for penetrating a biofilm matrix and killing
both planktonic and biofilm bacteria or fungi that have an active
ingredient comprising at least two or more of: a) a salt having a
cation N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4
alkyl ester and an anion selected from the group consisting of:
halide, nitrite, nitrate, linolenate, laurate, oleoate, phenolate,
polyphenolate, carboxylate, hydroxycarboxylate, hyaluronate,
antibiotic anion, resveratrol, and an amino acid, the salt being
present in an amount from about 0.025 wt % to about 10 wt %; b) a
glycerol monoester of a fatty acid being present in an amount from
about 0.05 wt % to about 10 wt %; and c) a sugar ester of a fatty
acid being present in an amount from about 0.075 wt % to about 20
wt %; and optionally comprising one or more of: d) a solvent being
present in an amount from about 20 wt % to about 99.9 wt %; or e) a
thickener or carrier or gelling agent being present in an amount
from about 20 wt % to about 75 wt %; or f) a sacrificial agent
being present in an amount from about 0.05 wt % to about 5 wt % are
provided; or g) a hydrogel having a three-dimensional hydrophilic
polymer network.
[0037] The composition may be further characterized by: the a)
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester being N.sup..alpha.-lauroyl-L-arginine-ethyl ester; or the b)
glycerol monoester a fatty acid being monolaurin; or the c) sugar
ester of a fatty acid being sucrose laurate; or the d) solvent
being at least one of: water, 1,2-propylene glycol or 1,3-propylene
glycol, 1,2-pentanediol, sorbitol, glycerol, xylitol, polyethylene
glycol, polypropylene glycol, butylene glycol, pentylene glycol,
hexylene glycol; or the e) thickener or carrier or gelling agent
being at least one of: a polymer, a hydrocolloid, an acrylate, an
acrylamide, a carboxylated cellulose, lecithin,
poly(lactic-co-glycolic acid) (PLGA), polymeric ethers, polymeric
aliphatic alcohols, polyalkoxylated alcohols, naturally occurring
high molecular weight substances such as sodium alginate, gums,
xanthan gum, gum tragacanth, starch, collagen aluminum silicate,
quince seed extract, semi-synthetic high molecular substances such
as methyl cellulose, carboxymethyl cellulose,
hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethyl
cellulose (HPMC), soluble starch and cationized cellulose,
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol, arabic gum, carbomer, polyethylene oxide,
poloxamer; or the f) sacrificial agent being at least one of:
triethyl citrate, trimethyl citrate, or zinc glycinate; or the g)
hydrogel being at least one of: polyvinyl alcohol,
polyvinylpyrrolidone, polyethyleneimine, polyacrylic acid,
polyhydroxyethyl-methacrylate, polyvinyl alcohol-glycine
co-polymer, or polyvinyl alcohol-lysine co-polymer.
[0038] In yet another embodiment a device or product treated with
the composition is provided. The device may be made by a process
comprising impregnating, dipping, coating or soaking the device
with the composition. The device being selected from the group
consisting of: microcapsules, wound dressings, surgical implants,
wound closures, staples, meshes, controlled drug delivery systems,
wound coverings, medical fillers, sutures, tissue adhesives, tissue
sealants, absorbable and non-absorbable hemostats, catheters, wound
drainage tubes, arterial grafts, soft tissue patches, gloves,
shunts, stents, surgical guide wires, prosthetic devices, contact
lens, endoscopes, dentures, medical devices, food processing
equipment, food conveyor belts, food packaging equipment, pet or
animal food, pet chew toys, pet or animal water bowls, and floating
toys. The product made by a process of mixing the composition with
the product.
[0039] In yet another embodiment, a wound covering for chronic
wounds that does not adhere to the wound surface, is held stable at
the wound site, and has water absorbing properties, the wound
covering further comprising: an outer protective covering that does
not contact the surface of the chronic wound; means for securing
dressing to a wound site; and a surface that is in contact with the
chronic wound comprising synthetic polymers, natural polymers or a
combination thereof that absorb water and release the composition
to the surface of the chronic wound.
[0040] In yet another embodiment the composition may further
comprise h) at least one bioactive agent. The bioactive agent may
be substantially water-insoluble antimicrobial or drug. The
bioactive material may be solubilized by the g) hydrogel.
[0041] Some embodiments refer to planktonic and biofilm bacteria or
fungi. Planktonic bacteria or fungi are understood to be bacteria
or fungi that are free floating or are otherwise unattached to a
matrix. These bacteria may be located on the surface of a biofilm
matrix or between a biofilm matrix and a surface, such as a wound
bed or a device. These planktonic bacteria or fungi are
phenotypically distinguishable from bacterial that are located in
the biofilm matrix. The term biofilm bacteria or fungi, or sessile
bacteria or fungi or embedded bacteria or fungi refer generally to
bacteria or fungi that are either physically attached to a surface
or a biofilm matrix or trapped therein.
[0042] The compositions and methods described herein are useful for
killing or inhibiting both planktonic and biofilm bacteria or
fungi. It is not necessary to kill bacteria or fungi for the
compositions to be effective, merely that the bacteria or fungi are
inhibited from form a biofilm or forming an attachment to a surface
or in a product so as to enable biofilm formation to begin.
[0043] The compositions described must have at least two active
ingredients. However, all three active ingredients may also be used
in combination and at any concentration.
[0044] The compositions described are useful for prevention and
treatment of biofilm on an unlimited number of surfaces.
Practically, any surface upon which a biofilm may form is
encompassed by the present invention. This is particularly the case
because of the beneficial features of the compositions, namely
being nontoxic, generally recognized as safe, and consumable, they
may be used on any surface.
[0045] The compositions described are also useful as preservatives
when mixed with personal care items or cosmetics as preventing
biofilm formation.
[0046] With respect to hydrogels, a hydrogel is a network of
polymer chains that are hydrophilic, absorbent, flexible and are
made of natural or synthetic polymeric networks. The hydrogel is
not limited to a specific shape or form. A hydrocolloid is a
substance that forms a gel in the presence of water.
[0047] While various embodiments described herein refer to killing
or inhibiting bacteria or preventing bacteria from forming a
biofilm, it is fully appreciated that the inventive methods and
compositions also encompass killing or inhibiting yeasts, fungi,
molds, and any type of bacteria or other microorganism that can
adhered or otherwise become attached to a surface and form a
microorganism/matrix complex likened to biofilm. A biofilm may
contain a mixture of different types of microorganisms, such as
yeast and mold and bacteria.
[0048] Various embodiments herein may recite the term "including"
or in the claims the term "comprising", and their grammatical
variants. For each such embodiments, corresponding additional
embodiments are explicitly contemplated where the term "comprising"
is replaced with "consisting essentially of" and "consisting of".
For example, a composition comprising a) a salt having a cation
N.sup..alpha.C8-C16 alkanoyl-L di-basic amino acid --C1-C4 alkyl
ester, b) a glycerol monoester of a fatty acid, c) a sugar ester of
a fatty acid, d) a solvent, e) a thickener, a sacrificial agent.
May consist essentially of the listed ingredients a)-f) or may
consist of only the listed ingredients a)-f). Similar terminology
would also apply to compositions further comprising g) a hydrogel
and h) a benefit agent.
[0049] The term "wt %" is equivalent to wt %, or wt. %, or wt. %,
or % of the final formulation. The term wt % represents the amount
of an ingredient in comparison with the weight of the total
formulation. As the chemical structure of some compounds is known,
and therefore the molecular weight of specific compounds is known,
the mol % may also be calculated if desired.
[0050] It must be noted that to kill bacteria in biofilm, the
biofilm must be penetrated. In order to kill bacteria in biofilm,
the biofilm exopolysaccharide matrix needs to be penetrated in
order to reach the bacteria. In the Examples of the instant
invention the complete kill of the planktonic and biofilm bacteria
(also referred to as "bioburden") is shown by compositions of the
instant invention.
[0051] It is well known that Pseudomonas aeruginosa can produce
biofilm on wounds, which is difficult to treat effectively. The ex
vivo model used for data generation in the instant invention was
reported by Phillips et al. (International Wound Journal, ISSN
1742-4801, J. Wiley and Sons, 2013).
[0052] Two GRAS approved food additives were experimentally found
to be unexpectedly active in the presence of LAE-HCl as penetrating
biofilm whereby it has been shown to reduce Pseudomonas aeruginosa
in both planktonic and sessile cells by up to a ten log reduction
as reported in the examples. These two GRAS approved food additive
are monolaurin (also referred in this disclosure as "glycerol
monolaurate" or "GML" or "ML") and sucrose mono-fatty esters
(C8-C18), e.g. sucrose laurate (referred to in this disclosure as
"SL"), sucrose myristate, sucrose palmitate, or sucrose stearate.
Also unexpectedly the data in the examples show that a combination
of at least two of the three ingredients in the instant invention,
i.e. LAE, ML, and SL, will produce clinically significant biofilm
penetration and kill in the Phillips et al. ex vivo model. The
compositions described herein are capable of diminishing or
eliminating biofilm formation by complete kill with no regrowth of
the microorganism.
[0053] The amount of N.sup..alpha.C8-C16 alkanoyl-L di-basic amino
acid --C1-C4 alkyl ester salts, can range from about 0.025 wt % to
about 10.0 wt. % based on the total weight of the final
formulation. The preferred amount of N.sup..alpha.C8-C16 alkanoyl-L
di-basic amino acid --C1-C4 alkyl ester salts may also range from
about 0.05 wt % to about 10.0 wt % or between about 0.1 wt % to
about 10.0 wt %; or between about 0.2 wt % to about 10.0 wt %; or
between about 5 wt % to about 10.0 wt %; or between about 0.05 wt %
to about 5 wt %; or between about 0.05 wt % to about 1 wt %. The
preferred amount of N.sup..alpha.C8-C16 alkanoyl-L di-basic amino
acid --C1-C4 alkyl ester salts may also include any single wt %
encompassed by the range of between about 0.05 wt % to about 10.0
wt %, including for example, 0.05 wt %, 0.1 wt %, 1 wt. % or the
like. The preferred amount of LAE salts can range from about 0.05
wt % to about 5.0 wt % based on the total weight of the final
formulation. The invention encompasses any individual amount
encompassed by this range, including but not limited to for example
about 5.0 wt %, about 1.0 wt % etc., the weight percent being based
on the total weight of the final formulation.
[0054] The amount of glycerol monoester of a C8-C14 fatty acid,
such as for example monolaurin (ML) can range from about 0.05 wt %
up to about 20.0 wt. % based on the total weight of the final
formulation. The preferred amount of a glycerol monoester of a
C8-C14 fatty acid may also range from about 0.1 wt % to about 20.0
wt % or between about 1 wt % to about 20.0 wt %; or between about
0.05 wt % to about 18.0 wt %; or between about 0.05 wt % to about
10.0 wt %; or between about 0.05 wt % to about 5 wt %. The
preferred amount of glycerol monoester of a C8-C14 fatty acid may
also include any single wt % encompassed by the range of between
about 0.05 wt % to about 20.0 wt %, including for example, 0.05 wt
%, 1 wt. %, 10 wt %, or the like. The invention encompasses any
individual amount encompassed by this range, including but not
limited to for example about 2.0 wt %, about 1.0 wt % etc., the
weight percent being based on the total weight of the final
formation.
[0055] The range of the sucrose C8-C18 fatty acid monoesters can
range from about 0.075 wt % to about 30.0 wt % based on the total
formulation. The preferred amount may also of the sucrose C8-C18
fatty acid monoesters may also range from about 0.075 wt % to about
10.0 wt % based on the total formulation, or between about 0.075 wt
% to about 10.0 wt %; or between about 0.075 wt % to about 5.0 wt
%, or between about 3 wt % to about 30.0 wt %, or about 10 wt % to
about 30.0 wt % The invention encompasses any individual amount
encompassed by this range, including but not limited to for example
about 10.0 wt % or about 1.0 wt % etc., the weight percent being
based on the total weight of the final formation.
Major Ingredients
[0056] A combination of two GRAS approved food additives were
experimentally found to be active as penetrating biofilm whereby it
has been shown to reduce Pseudomonas aeruginosa in both planktonic
and sessile cells.
[0057] LAE-HCl
[0058] N.sup..alpha.-long chain alkyl di-basic amino acid alkyl
ester acid salts have been known since the 1960'. One of the first
patents to recommend these amino acids, specifically for food
applications was U.S. Pat. No. 3,825,560. A number of derivatives
are disclosed include N.sup..alpha.-cocoyl-L-arginine ethyl ester
pyrolidone carboxylate and N.sup..alpha.-lauroyl-L-arginine methyl
ester hydrochloride.
[0059] Extensive toxicological and metabolic experiments are
reported for N.sup..alpha.-lauroyl L-arginine ethyl ester
monohydrochloride (LAE-HCl)(Food and Chemical Toxicology, 42
(2004), p 242-259).
[0060] US Pub. Appln. No. 2011/10230558 discloses LAE compounds are
known to destroy endotoxins produced by some bacteria. Another
advantage of the instant invention is that L-arginine derivatives
of LAE has a positive charge and will react with anionic
hydrocolloids that are used for wound healing dressings.
Biofilm Inhibition by LAE
[0061] Musk et al. (Chemistry & Biology, Vol. 12, 789-796,
July, 2005) write that bacterial biofilms are thought to aid in the
survivability of a variety of intractable infections in humans.
Ferric ammonium citrate inhibited biofilm formation in a
dose-dependent manner. P. aeruginosa strains taken from the sputum
of 20 CF patients showed a similar response to elevated iron
levels. Cai et al (Brazilian Journal of Microbiology, ISSN
1517-8382), indicate that Pseudomonas aeruginosa is one of the
major causes of nosocomial infections. In addition, P. aeruginosa
is a leading pathogen among patients with cystic fibrosis, diffuse
panbronchiolitis, and chronic obstructive pulmonary disease. In
patients with these underlying diseases, it can cause chronic
infections characterized by the formation of biofilms. Therefore,
infections with biofilm-forming bacteria are persistent and
difficult to treat with antibiotics. Iron is essential for most
pathogens because iron is an indispensable component of many
proteins, especially some enzymes in bacteria. Therefore, iron
acquisition from environment is important for the growth and
metabolism of P. aeruginosa. Recently, many studies revealed that
iron also play an important role in biofilm formation. In vitro
experiments showed both iron-depletion (<1 .mu.M) and
iron-repletion (>100 .mu.M) retarded biofilm formation.
Furthermore, some reports showed that the level of free iron is
increased in airway secretions of cystic fibrosis patients, and
this might be one of the possible reasons for the frequent
identification of biofilms in the lungs of these patients.
According to Braun et al. (Springer Briefs in Biometals, DOI:
10.1007/978-94-007-6088-2_2), iron is an essential element for many
key redox systems. It is difficult to acquire for cells under oxic
conditions, since Fe3+ forms insoluble hydroxides.
[0062] Kim et al. (Frontiers in Microbiology, Vol. 8, May 2017)
report that Pseudomonas aeruginosa is a ubiquitous gram-negative
bacterium capable of forming a biofilm on living and non-living
surfaces, which frequently leads to undesirable consequences. They
found that lauroyl arginate ethyl (LAE), a synthetic non-oxidizing
biocide, inhibited biofilm formation by P. aeruginosa at a
sub-growth inhibitory concentration under both static and flow
conditions. Thus LAE generated iron-limiting conditions, and in
turn, blocked iron signals necessary for Pseudomonas aeruginosa
biofilm development. As destroying or blocking signals leading to
biofilm development would be an efficient way to mitigate
problematic biofilms, these findings suggest that LAE can aid in
reducing Pseudomonas aeruginosa biofilms for therapeutic and
industrial purposes. LAE activated the genes involved in iron
acquisition (e.g., the pyoverdine and pyochelin related genes) and
increased twitching motility, due to the low availability of iron
to P. aeruginosa because LAE chelated the iron.
[0063] It was found in the experimental results that an effective
amount of antimicrobial agent like LAE-HCl was between about 0.05
to about 5.0 wt % based on the total amount of the formula. If the
LAE salt has an anion other than a halide, e.g. C8-C23 carboxylate
or polyphenolate anion, then the amount is proportional to the
molecular weight of the anion.
[0064] While this instant invention discloses the antimicrobial LAE
salts, both in water-soluble, and lesser water-soluble form having
a controlled release property as disclosed by allowed patents as
listed previously, other antimicrobials can be used such as
chlorhexidine salts, cetylpyridinium halide, monomeric or polymeric
quats, PHMB salts, diallyl dimethyl ammonium halide (Merquat.TM.),
defensins, cationic antibiotics, monovalent silver, or combinations
thereof may be used in the disclosed methods of biofilm penetrating
to deliver other antimicrobials, antibiotics, and silver and
nano-silver.
[0065] The preferred di-basic amino acid derivative in this
invention is N.sup..alpha. lauroyl-L-arginine ethyl ester. Some
preferred salts of LAE are the HCl, linolenate, laurate, oleoate,
nitrate, nitrite salts and various salts containing antioxidants
having a phenolate or polyphenolate anion and/or carboxylate
functionalities. Another lesser preferred compound is the L-lysine
corresponding compound. LAE has excellent antimicrobial activity of
a broad nature including gram positive, gram negative, molds, yeast
and other type microorganisms, and it is also effective against
endotoxins. Other desirable properties of LAE are non-toxic,
biodegradable and its metabolic breakdown to form arginine, lauric
acid and ethanol, which all of these compounds are found in the
human body as natural materials and present no toxicological
problems.
[0066] LAE metabolizes to arginine which is a semi essential amino
acid. Arginine is non-essential because the body can produce it,
however, under period of growth, illness and metabolic stress not
enough arginine is produced by the body. Arginine regulates many
metabolic and physiologic body functions and has several attributes
that support wound repair like the following: has 32% nitrogen; is
a precursor to proline, which is converted to hydroxyproline, then
to collagen; has a positive influence on the body's levels of
insulin like growth factor (IGF-I), a hormone that promotes wound
healing; is the only amino acid substrate for nitric oxide
synthesis (Nitric oxide has a beneficial effect on circulatory
status and increases blood supply to the wound); contains immune
enhancing properties that reduce the risk of wound complications;
will break down to form NO, a desirable compound which stimulates
the healing process.
[0067] Solubilizing and Surfactants
[0068] Solubilizing the biofilm matrix is very important because it
is been proven that dead cells are inflammatory (U.S. Pub. App. No
2010/0183519). The use of a poloxamer, e.g. poloxamer-188 or
poloxamer-407, as thickening agents or carriers or gelling agents
for the instant invention can be used. Any suitable and/or
acceptable gelling or thickening or carrier agents can be used in
the instant invention including polymers like hydrocolloids,
acrylates, acrylamides, carboxylated celluloses.
[0069] U.S. Pat. No. 9,283,2782 describe a method for treating a
microbial biofilm on a patient including the steps of contacting
the microbial biofilm with a composition comprising a surface
active agent and a sub-lethal amount of an antimicrobial agent. The
surface active agent of embodiments may be a poloxamer, meroxapol,
poloxamine or combinations thereof.
[0070] Surfactants disrupt biofilm structural integrity by causing
structural disturbance of proteinaceous matrix components.
Surfactants might also disrupt the cell membrane and thereby weaken
or release extracellular polymeric substance (EPS) molecules that
are putatively anchored to the cell via a membrane interaction
(U.S. Pat. No. 9,283,278 and Montana State University thesis by
Xiao Chen "Chemically Induced Biofilm Detachment" (1998)).
Lecithin
[0071] It has been found that lecithin can aid in penetrating
biofilms. Lecithins are mixtures of glycerophospholipids including
phosphatidylcholine, phosphatidylethanolamine,
phosphatidylinositol, and phosphatidic acid. Lecithin has
emulsification and lubricant properties, and is a surfactant. It
can be totally metabolized by humans, so is well tolerated by
humans and nontoxic when ingested; some other emulsifiers can only
be excreted via the kidneys. The major components of commercial
soybean-derived lecithin are: 33-35% soybean oil, 20-21% inositol
phosphatides, 19-21% phosphatidylcholine, 8-20%
phosphatidylethanolamine, 5-11% other phosphatides, 5% free
carbohydrates, 2-5% sterols, and 1% moisture. Lecithin is used for
applications in human food, animal feed, pharmaceuticals, paints,
and other industrial applications.
[0072] Hydrophilic-Lipophilic Balance or "HLB" is an index of the
predicted preference of an emulsifier for oil (O) or water (W)--the
higher the HLB, the more hydrophilic the molecule; the lower the
HLB, the more hydrophobic the molecule. Typical usage levels of
lecithin in an emulsion system are: 1-5% of the fat for W/O; 5-10%
of the fat for O/W. The amount of lecithin used is dependent upon
factors such as the pH, the inclusion of proteins and, and the salt
concentration.
Coating Implant with LAE-Hyaluronic Salt
[0073] In the instant invention an implant can be coated with
LAE-hyaluronic acid salt as the LAE component of the instant
invention. Also, the implant can be a metal, a metal alloy, a
ceramic, or a combination thereof. Also a multi-coated implant
comprising: (a) a first layer residing on the surface of the
implant; and (b) a second layer comprising LAE-hyaluronic acid
residing on the first layer.
[0074] The coated implant resists microbial growth. Examples of
microbial growth that can be resisted include, but are not limited
to Staphylococcus aureus and Staphylococcus epidermidis.
[0075] The coated implants of the invention can be bioabsorbable,
resorbable, or permanent. The implants of the invention can be used
in osteointegrative, osteosynthetic, orthopedic, and dental
applications. Representative implants include, but are not limited
to, void fillers (e.g., bone void fillers), adjuncts to bone
fracture stabilization, intramedullary fixation devices, joint
augmentation/replacement devices, bone fixation plates (e.g.,
craniofacial, maxillofacial, orthopedic, skeletal, and the like),
screws, tacks, clips, staples, nails, pins, rods, anchors (e.g.,
for suture, bone, or the like), scaffolds, stents, meshes (e.g.,
rigid, expandable, woven, knitted, weaved, etc.), sponges, implants
for cell encapsulation or tissue engineering, drug delivery devices
(e.g., antivirals; antibiotics; carriers; bone ingrowth induction
catalysts such as bone morphogenetic proteins, growth factors,
peptides, and the like), monofilament or multifilament structures,
sheets, coatings, membranes (e.g., porous, microporous, and
resorbable membranes), foams (e.g., open cell and closed cell
foams), screw augmentation devices, cranial reconstruction devices,
a heart valve, and pacer lead.
[0076] The term "hyaluronic acid," as used herein includes a
(co)polymer of acetylglucosamine (C.sub.8H.sub.15NO.sub.6) and
glucuronic acid (C.sub.6H.sub.10O.sub.7) occurring as alternating
units.
[0077] Representative materials for the implant include, but are
not limited to, metals and metal alloys (e.g., titanium, titanium
alloy, nickel-titanium alloy, tantalum, platinum-iridium alloy,
gold, magnesium, stainless steel, chromo-cobalt alloy); ceramics;
and biocompatible plastics or polymers (e.g., polyurethanes and/or
poly(.alpha.-hydroxy ester)s such as polylactides, polyglycolides,
polycaprolactones, and the like, and combinations and/or copolymers
thereof). Other non-limiting examples of implants include those
made from materials disclosed in any of the following U.S. Pat.
Nos. 4,503,157; 4,880,610; 5,047,031; 5,053,212; 5,129,905;
5,164,187; 5,178,845; 5,279,831; 5,336,264; 5,496,399; 5,569,442;
5,571,493; 5,580,623; 5,683,496; 5,683,667; 5,697,981; 5,709,742;
5,782,971; 5,820,632; 5,846,312; 5,885,540; 5,900,254; 5,952,010;
5,962,028; 5,964,932; 5,968,253; 6,002,065; 6,005,162; 6,053,970;
6,334,891. The implant can be substantially free of a polymeric
component (i.e., a plastic or polymer).
[0078] Non-limiting examples useful implants substantially free of
plastic or polymer include a bone void filler, an adjunct to bone
fracture stabilization, an intramedullary fixation device, a joint
augmentation/replacement device, a bone fixation plate, a screw, a
tack, a clip, a staple, a nail, a pin, a rod, an anchor, a
scaffold, a stent, a mesh, a sponge, an implant for cell
encapsulation, an implant for tissue engineering, a drug delivery
device, a bone ingrowth induction catalyst, a monofilament, a
multifilament structure, a sheet, a coating, a membrane, a foam, a
screw augmentation device, a cranial reconstruction device, a heart
valve, or a pacer lead.
[0079] The LAE-hyaluronic acid salt provides an in vivo resistance
to absorption, adhesion, and/or proliferation of a bacteria, such
as Staphylococcus aureus or Staphylococcus epidermidis. Any method
capable of forming a coating of the LAE-hyaluronic acid salt can be
utilized to make the coated implants of the instant invention
including, but not limited to dip-coating, application by a brush,
spray coating, and any combination thereof. Examples of coating
methods can be found in, e.g., U.S. Pat. Nos. 4,500,676, 6,187,369
and 6,106,889 and U.S. Pub. App. Nos. 2002/0068093 and
2003/0096131. Typically, a composition comprising the
LAE-hyaluronic acid salt and an organic solvent is applied to the
implant, and the resultant coated implant is allowed to dry or
cure.
[0080] In the instant invention a multi-coated implant comprising:
(a) a first coat residing on the surface of the implant; and (b) a
second coat comprising the LAE-hyaluronic acid salt residing on the
first coat is disclosed. Non-limiting examples useful first coats
include metals (e.g., titanium, gold, or platinum), ceramic
materials (e.g., hydroxyapatite or tricalcium phosphate, or
polymers (e.g., an acrylic polymer base coat), or any combination
thereof.
[0081] The first coat can be the same as, or different from, the
implant material. Non-limiting examples of useful implant materials
include metals, metal alloys, or ceramics as described above;
and/or plastics or polymers, e.g., polyurethanes and/or
poly(.alpha.-hydroxy ester) such as polylactides, polyglycolides,
polycaprolactones, and the like; or any combination thereof.
[0082] Methods for coating the implant with a ceramic or polymer
include those describe above for coating the implant with the
LAE-hyaluronic acid salt. In certain embodiments, the
LAE-hyaluronic acid salt coating can comprise one or more polymer
additives. Without being limited by theory, the addition of a
polymer, e.g., an elastic film forming polymer, can improve the
structural characteristics of the LAE-hyaluronic acid salt coating
such as can impart improved flexibility, adhesion and/or as
resistance to cracking. Any polymer can be used provided the
polymer is biocompatible and does not significantly interfere with
the desired characteristics of the hyaluronic acid component.
Typically, the polymer, when used, is bioadsorbable or erodible.
More preferably, the polymer, when used, is bioadsorbable. A
non-limiting examples of a useful polymers include polyurethane
(see U.S. Pat. No. 4,500,676, the entire disclosure of which is
incorporated herein as reference); polylactides; polyglycolides;
homopolymers or copolymers of monomers selected from the group
consisting of L-lactide; L-lactic acid; D-lactide; D-lactic acid;
D,L-lactide; glycolide; .alpha.-hydroxybutyric acid;
.alpha.-hydroxyvaleric acid; .alpha.-hydroxyacetic acid;
.alpha.-hydroxycaproic acid; .alpha.-hydroxyheptanoic acid;
.alpha.-hydroxydecanoic acid; .alpha.-hydroxymyristic acid;
.alpha.-hydroxyoctanoic acid; .alpha.-hydroxystearic acid;
hydroxybutyrate; hydroxyvalerate; .beta.-propiolactide;
.beta.-propiolactic acid; .gamma.-caprolactone;
.beta.-caprolactone; .gamma.-butyrolactone; pivalolactone;
tetramethylglycolide; tetramethylglycolic acid; dimethylglycolic
acid; trimethylene carbonate; dioxanone; those monomers that form
liquid crystal (co)polymers; those monomers that form cellulose;
those monomers that form cellulose acetate; those monomers that
form carboxymethylcellulose; those monomers that form
hydroxypropylmethyl-cellulose (HPMC); polyurethane precursors
comprising macrodiols selected from the group consisting of
polycaprolactone, poly(ethylene oxide), poly(ethylene glycol),
poly(ethylene adipate), poly(butylene oxide), and a mixture
thereof, isocyanate-functional compounds selected from the group
consisting of hexamethylene diisocyanate, isophorone diisocyanate,
cyclohexane diisocyanate, hydrogenated methylene diphenylene
diisocyanate, and a mixture thereof, and chain extenders selected
from the group consisting of ethylenediamine, 1,4-butanediol,
1,2-butanediol, 2-amino-1-butanol, thiodiethylene diol,
2-mercaptoethyl ether, 3-hexyne-2,5-diol, citric acid, and a
mixture thereof; collagen, alginates (e.g., sodium or calcium
alginate), polysaccharides such as chitin and chitosan,
poly(propylene fumarate); and any mixture thereof. Similarly, the
instant invention compositions that include N.sup..alpha.C8-C16
alkanoyl-L di-basic amino acid --C1-C4 alkyl ester salts, glycerol
monoester of a C8-C14 fatty acid, and sucrose C8-C18 fatty acid
monoesters can be incorporated into the biocompatible bioactive
biomaterial for biofilm inhibition and penetration and bacteria
kill. The instant invention discloses a combination of at least two
out of the three ingredients, i.e. LAE/ML, LAE/SL, or ML/SL, as a
coating onto surfaces, e.g., surgical implants, wires, catheters,
etc., that can be solubilized in a non-aqueous solvent, e.g.
ethanol, and then coated onto the surface to be inhibited. There
will be hydrogen bonding between each ingredient and the surface to
be coated as well as between each ingredients, thus developing
bonds to improve adhesion to the surface and to each other.
Monolaurin and Sugar Esters of Fatty Acids
[0083] It has been shown in the examples that the combination of
LAE salts with both sucrose monolaurate and monolaurin can kill
both Pseudomonas aeruginosa planktonic cells as well as Pseudomonas
aeruginosa biofilm bacteria cells. However it was unanticipated
that, as shown in the experimental section of the disclosure, 1)
sucrose monolaurate in combination with glycerol monolaurate w/o
LAE salts can penetrate and result in clinically significant
Pseudomonas aeruginosa planktonic and biofilm bacteria kill and
also 2) sucrose monolaurate in combination with LAE salts w/o
glycerol monolaurate can also penetrate and result in clinically
significant Pseudomonas aeruginosa planktonic and biofilm bacteria
kill.
[0084] Both glycerol monofatty esters and sugar monofatty esters
have been reported as having preservative characteristics,
primarily in foods but also in cosmetics ("Handbook of
Preservatives")
[0085] Both monolaurin ("ML") and sucrose fatty esters have been
reported to have some degree of biofilm inhibition and penetration
properties (U.S. Pat. No. 5,284,833).
[0086] There are two factors to consider when choosing the glycerol
monoester of a fatty acid. The ester part could be from C8-C14
saturated hydrocarbon, however the C12 has been consistently shown
to be the optimal choice, since when esterifying glycerin it is
possible to obtain di- and tri-esters as well as the monoester.
Therefore in order to achieve the best antibacterial and biofilm
dispersion, the monoester of monolaurin should be greater or equal
to 70 wt % of the total ester content, the higher being the better.
Preferred monoester level is 90 wt %.
Sugar Esters
[0087] In the United States, interest in the synthesis of sugar
esters of fatty acids began in 1952, when the Sugar Research
Foundation saw their surfactant potential. They are used as
non-ionic surfactants, bleaching boosters and food additives.
[0088] Sucrose mono fatty esters according to the previous
experiments are not active at 500 ppm or lower. Monolaurin is
synergistic with LAE salts as while SL appears to enhance the
antibacterial performance of LAE and of ML. For monolaurin this
synergy is described in U.S. Pat. Nos. 8,193,244 and 9,023,891 and
WO 2013/169231. For sucrose mono fatty esters the results are in
the Examples.
Additional Ingredients
[0089] The instant invention discloses the use of certain chemicals
to penetrate/disperse existing biofilm and/or prevent and/or
inhibit biofilm formation and also that have antimicrobial
activity. Additionally certain antimicrobials and antibiotics can
be added to the wound healing and biofilm penetrating compositions
of the instant invention. For Na.sub.2EDTA, where a salt between
the LAE based arginine derivative and the anions of ferulic acid,
gallic acid, Na.sub.2EDTA can be formed in a 1:1 or 2:1 molar
ratio, where the LAE based arginine derivative is either 1 or 2
molar equivalents to the Na.sub.2EDTA. The Na.sub.2EDTA can also be
used as a chelating agent and also as an additive with the arginine
derivative.
[0090] Solvents
[0091] In some cases it may be necessary to include up to a maximum
of about 40 wt % of a safe, green, and non-toxic solvent to the
aqueous gel formulation to act as a solubilizer to dissolve all of
the ingredients in the biofilm penetrating/wound healing
composition. A partial list might include 1,2-propylene glycol or
1,3-propylene glycol, glycerol, polyethylene glycols, polypropylene
glycols, butylene glycol, pentylene glycol, hexylene glycol or
combinations thereof. Even though sorbitol or xylitol are solids,
they form very concentrated aqueous solutions that can be used in
this invention. In fact xylitol is a known anti-adhesive for
bacteria binding to a variety of surfaces. A solubilizer such as
propylene glycol or similar is necessary for monolaurin and LAE
salts under certain conditions. However the level of the
solubilizer is important as cytotoxicity is a concern with higher
levels of solubilizer. For example, propylene glycol can be used to
solubilize the LAE salts and monolaurin, and then this phase can be
added to the water phase containing the sucrose laurate. Sucrose
fatty esters can also used in the instant invention as solubilizers
for monolaurin. Ethylene glycol is a known toxin, but propylene
glycol has an acute oral toxicity of 20.0 g/kg (LD.sub.50) as
reported by R. J. Louis Sr ("Dangerous Properties of Industrial
Materials", eighth addition, Van Nostrand, Reinhold, N.Y.
1992).
[0092] Solvents can also reinforce the antimicrobial agents and
help penetrate the active ingredients into the skin. So the
selection of the proper solvent system can play an important role.
For example in Acta. Derma. Venereal., 1991, 71 (2), pp 148-150, it
was reported that 10 wt % of hexylene glycol was equivalent to 30
wt % of 1,3 butylene glycol or propylene glycol in vitro against
Streptococcus pyogenes, Streptococcus mitis, Staphylococcus
epidermidis, and E. coli in terms of killing power. 1,2-pentanediol
is another solvent which has desirable properties such as excellent
moisturizing, broad-spectrum antimicrobial activity, excellent as a
solubilizer, as well as a dissolution ability.
Sacrificial Enzyme Inhibitors
[0093] In some wounds it may be necessary to include a sacrificial
enzyme inhibitor to maintain or enhance the efficacy of the
antimicrobials and biofilm disruptors.
[0094] Ohkawa (J. of Biochem., 1979 v. 86, C31, pages 643-656)
found that all 11 strains of Pseudomonas aeruginosa had esterases
on the cell envelope. The enzymes to have specificity for long
chain esters with hydrophilic groups. Some antimicrobials like LAE
salts, the glycerol monoesters of C8-C18 fatty acids, and the
sucrose mono fatty esters C8-C16 can be hydrolyzed by esterases
present on bacterial cells. In order to inhibit enzymatic
hydrolysis and maintain efficacy, a natural or synthetic enzyme
inhibitor can be added to the formulation. In addition to the other
ingredients of the instant invention, it has been found
experimentally that the addition of triethyl citrate ("TEC") can
act as an inhibitor of esterases and can act to enhance and prolong
the antibacterial activity of LAE-HCl, monolaurin and SL. Triethyl
citrate ("TEC") will also enhance and prolong the antibacterial
activity of other LAE salts.
Esterase inhibitors, e.g. triethyl citrate, trimethyl citrate, and
zinc glycinate will prolong activity of LAE salts, sucrose fatty
esters, and monolaurin. This invention prefers the use of a
sacrificial enzyme inhibitor such as a triester (C.sub.1-C4)
citrate like triethyl citrate. The oral LD.sub.50 in rats is 7.0
cc/kg, a low toxicity molecule. In general the usage range of
triethyl citrate is from about 0.05 to about 5.0 wt % based on the
total weight of the formulation. The water solubility of triethyl
citrate at 25.degree. C. is 6.5 g/100 g of solution.
Wound Dressings or Covering
[0095] An effective wound covering must have 1) a positive effect
for promoting wound healing, 2) exhibit a sufficient water
absorbing property, thus can absorb a wound exudates, 3) does not
adhere to the wound surface and 4) can be held stable at the
affected part. Wound dressings can be comprised of either synthetic
or natural polymers, or combinations of the two. For serious wounds
the medical profession usually use hydrophilic or cross-linked
hydrogels having good oxygen permeable. Many different polymers can
be used for example polyacrylate and salts thereof,
polyvinylpyrrolidone (PVP) and copolymers, polyalkylenes,
polymethyl vinyl ether-maleic anhydride or dicarboxylate and
copolymers, polyacrylamide and copolymers, alginate, gum Arabic,
tragacanth gum, carrageenans, xanthan gum or other natural
gums.
[0096] It is understood that many other synthetic or natural
polymers with these desirable properties can be substituted by one
skilled in the art. The hydrogel layer in U.S. Pat. No. 8,604,073
comprises a three-dimensional network formed by a hydrophilic
polymer by ionic or chemical cross-linking, cryogel formation, or
by an interpenetrating polymeric network using polyfunctional water
soluble polymers, such as polyvinyl alcohol, polyvinylpyrrolidone,
polyethyleneimine, polyacrylic acid, polyhydroxyethylmethacrylate,
polylactic acid, polylactide, polyglycolide, poly
epsilon-caprolactone, copolymers and mixtures thereof, poly vinyl
alcohol-glycine co-polymer, and polyvinyl alcohol-lysine
co-polymer. Ionic or chemical crosslinking of the hydrophilic
polymers can be accomplished in the polyfunctional polymers
included in the antimicrobial coatings of the invention.
[0097] For example, a hydrogel layer coating a substrate material
with the antimicrobial coating is applied, dried to a
pre-determined extent, and reacted with a suitable ionic or
chemical crosslinking agent or agents known in the art.
[0098] For example U.S. Pat. No. 6,399,092 discloses an anhydrous,
hydrophilic wound dressing containing a superabsorbent polymer and
an antimicrobial agent. It's anhydrous nature allows it, when
applied to a wound site, to absorb wound fluid and slowly release
its water-soluble active microbial agent into the wound. The
combination is an anhydrous, hydrophilic gel base carrier which may
be a poloxamer, e.g. block copolymers of ethylene oxide and
propylene oxide, etc. or polyethylene glycol with a superabsorbent
polymer, which may be a starch polymer, a graft copolymer of starch
polyacrylonitrile and non-starch homopolymers of polyacrylonitrile
or a poly(2-propenamide-co-2-propenoic acid sodium salt), a
homopolymer, or a cellulose base superabsorbent polymer. The
importance of the initial composition being anhydrous is that such
is essential and critical to the consistent release of the
effective concentration of the soluble active of the formulation as
it interfaces with an open wound. Such is less likely to occur if
the formulation initially contains water.
[0099] Another aspect of the instant invention involves the salt
formation by reacting the N.sup..alpha.-alkanoyl-L basic amino acid
ethyl ester water soluble salts of this invention with a variety of
ingredients commonly found in wound dressings having
functionalities like carboxylic groups. Examples of suitable
anionic polymers are: Alginates, oxidized celluloses, chitosan
water soluble salt derivatives, poly acrylic acid or polyacrylate
acid copolymers which incorporate an acid comonomer like itaconic
acid, carboxyethylcellulose, hyaluronic acid, or combinations
thereof. It is understood that many other synthetic or natural
polymers with these desirable properties can be substituted by one
skilled in this art.
[0100] For wound dressings the amino acid derivatives of this
invention can react with the carboxylate groups of the dressing
under basic conditions (NaOH solution) to yield a salt, which will
slowly release by contact with the extuate of the wound. Both bound
and unbound biocidals will be beneficial to healing. For burn
wounds a combination of Ag.sup.+1 and the compositions of the
instant invention can be employed. Other salt anions of the di
basic amino acid derivatives disclosed can have anions which can
also have wound healing properties.
[0101] The usefulness of lucuma nut oil material (LNO) (J. of
Cosmetic Dermatology, Vol. 9 Is. 3 p. 185-195 September 2010) has
been described. One or more of these fatty acids can be utilized as
the anionic portion of the long chain alkyl di-basic amino acid
alkyl ester acid salts disclosed in the instant invention are
preferred if controlled release of the cation is desired as well as
the benefit of the anionic component.
[0102] Some of the fatty carboxylates of the invention, for example
linolenic acid, are very expensive. These fatty acids exist in
nature and after refining can be utilized. For example soy bean
oil, corn oil, canola oil, safflower oil, sunflower oil and others
have multiple fatty acid mixtures. The importance of lauric,
myristic, palmitic, stearic, oleic, linoleic and linolenic as
counter ions to make a low water soluble salts to the long chain
alkyl di-basic amino acid alkyl ester acid cations are preferred if
controlled release of the cation is desired. The above mentioned
fatty acids have these requirements suitable for the purposes of
the anionic portion of the LAE salts disclosed in the instant
invention. Other long chain carboxylates include omega 3, 6 and 9
acids. EPA (eicosapentaenoic acid) and DHA (docosapentaenoic acid),
both omega-3 acids found in fish oil, are also preferred.
[0103] A particular useful group of anions for the cationic dibasic
amino acid derivatives of the instant invention are both natural
and synthetic dietary flavonoids and phenolic and polyphenolic
compounds such as: flavonals, flaovones, flavanones, resveratrol,
chalcones, anthocyanidins, anthocyanins, isoflavones, phenolic
acids, hydroxycinammates, stilbenes, and rutin. As the counter ion
of the antibacterial cations of this invention, the flavonoids as
listed above will have excellent antioxidant properties, which are
useful for wound healing. Once a wound (burn) occurs,
concentrations of reactive oxygen species such as hydroxyl singlet
oxygen, hydroperoxyl, superoxide anions radicals increase in
damaged tissue producing a condition known as oxidative stress.
Hydrogen peroxide behaves similarly. Thus the healing of chronic
wounds can be assisted by the use of antioxidants which is a part
of the N.sup..alpha.-alkanoyl-L basic amino acid ethyl ester
antimicrobial agents of this invention, as an anion of the salt.
This invention also teaches that mixtures of the preferred di-basic
amino acid derivatives and the conjugate acid form of the
antioxidant can be effective. Previous literature examples of using
antioxidant as wound healing compositions include U.S. Pat. No.
5,667,501, U.S. Pat. No. 5,612,321, and U. S. Pub. Appl. No.
2006/0159732.
[0104] Some illustrative examples of flavonoids but not an
exclusive list are the following: kaernpferal, quercetin,
epicotechin, hesperatin, cyanidin, genistein, gallic acid, ferulic
acid, salicylic acid, trans or cis resveratrol, catechin, syringic
acid, toxifolin, epigallocatechin, curcumin and myricetin. Many
more flavonoids exist which can be utilized to form biocidal
flavonoids salts taught in this invention can be found in a text
book entitled, "Plant phenolics and Human Health, Biochemistry,
Nutrition and Pharmacology".
[0105] Alpha keto propionic acid is another compound useful in
wound healing. It is commonly known as pyruvic acid. As with other
enhancers of this invention it can be employed as: 1) an anion of
the di-basic amino acid derivatives of this invention, or 2) as an
admixture.
[0106] Pyruvic acid supplies energy to living cells through the
citric acid cycle (Krebs cycle) when oxygen is present (aerobic
respiration) and alternatively ferments to produce lactic acid when
oxygen is lacking.
[0107] The salts of this invention can be easily prepared by a
simple metathesis reaction, e.g., a water soluble cationic biocide
like N.sup..alpha.-alkanoyl-L-di basic amino acid ethyl ester and a
water soluble anion. These metathesis reactions can be performed in
water or alcohol, however, absolute alcohol is required for the
resulting NaCl to precipitate.
Coatings
[0108] The compositions of the instant invention can be adhered to
a substrate, e.g. a surgical implant, endoscope, medical device,
catheter, suture, food processing conveyor belt, a food carrying
conduit, etc.
Thickeners, Gelling Agents, Carrier Agents
[0109] Any medical or food grade suitable and/or acceptable gelling
or thickening or carrier agents can be used in the instant
invention. Examples of thickening agents include smectite gelling
agent is a synthetic magnesiosilicate that is free of any heavy
metal contaminants; naturally occurring high molecular substances
such as sodium alginate, various gums, xanthan gum, gum tragacanth,
starch, collagen aluminum silicate, quince seed extract;
semi-synthetic high molecular substances such as methyl cellulose,
carboxymethyl cellulose, soluble starch and cationized cellulose;
synthetic high molecular substances such as carboxyvinyl polymer
and polyvinyl alcohol; polymers like hydrocolloids, acrylates,
acrylamides, carboxylated celluloses, arabic gum, carbomer,
polyethylene oxide, poloxamer and mixtures thereof; hydrogels;
hydrophilic synthetic polymers, sugars, glycerol, propylene glycol
(PG), derivatives thereof, and combinations.
[0110] The use of a poloxamer, e.g. poloxamer-188 or poloxamer-407,
as thickening agents or carriers or gelling agents for the instant
invention can be used. In an embodiment, the said biofilm
penetrating compositions and wound healing compositions comprises a
gelling or thickening agent in the range of 0.2 to 75.0 wt %,
together with one or more pharmaceutically acceptable
carriers/excipients. The thickener may preferably be contained in
an amount of 0.5 to 50 wt % with respect to the total weight of the
composition. The thickener may more preferably be contained in an
amount of 1.0 to 10 wt % with respect to the total weight of the
composition
[0111] Examples of carrier liquids include Poly(lactic-co-glycolic
acid) (PLGA), polymeric ethers, polymeric aliphatic alcohols,
either together or alone, polyalkoxylated alcohols, dextrin or
carboxymethyl dextrin cross linked to epichlorohydrin, propylene
glycol, hexylene glycol, dipropylene glycol, tripropylene glycol,
glycerin, ethanol, propylene glycol methyl ether, dipropylene
glycol methyl ether, dipropylene glycol, tripropylene glycol,
ethanol, n-propanol, n-butanol, t-butanol, 2-methoxyethanol,
2-ethoxyethanol, ethylene glycol, isopropanol, isbutanol,
1,4-butylene glycol, 2,3 butylene glycol,
2,4-dihydroxy-2-methylpentane, trimethylene glycol, 1,3-butanediol,
1,4,-butanediol, and combinations thereof. Specific examples of
pharmaceutically acceptable carriers that may be used are described
in the Handbook of Pharmaceutical Excipients.
[0112] Any suitable gelling agent can be used to prepare the gels
of the invention. As used herein, the term "gelling agent" includes
any natural or synthetic material that will provide the yield point
and viscosity defined herein. Examples of gelling agents found in
nature are polysaccharides and carrageenans, alginates and agars,
guar gum, gelatin, and locust bean (carob) gum. Also synthetic
organics such as polyethylene glycols, particularly the ultra-high
molecular weight polyethylene glycols, polyvinyl alcohol-boric acid
gels, polyacrylamides, crosslinked polyvinylpyrrolidones, and
polyacrylic acids can be used.
[0113] Some preferred gelling agents include hydroxyethylcellulose
hydroxypropylcellulose cross-linked acrylic acid polymers MVE/MA
decadiene crosspolymer, PVM/MA copolymer, ammonium
acrylates/acrylonitrogens, carboxymethylcellulose and
polyvinylpyrrolidone. It is preferred that the gelling agent
comprise between about 0.5% to about 10% by weight with respect to
the total weight of the composition.
[0114] Film forming polymers are selected from hydroxyethyl
cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose
(HPMC), hydroxyethyl methyl cellulose, polyvinyl alcohol,
polyethylene glycol, polyethylene oxide, ethylene oxide-propylene
oxide co-polymers, collagen and derivatives, gelatin, albumin,
polyaminoacids and derivatives, polyphosphazenes, polysaccharides
and derivatives, or chitin and chitosan, alone or in combination,
and a bioadhesive polymer selected from polyacrylic acid, polyvinyl
pyrrolidone, or sodium carboxymethyl cellulose, alone or in
combination.
[0115] If anionic hydrophilic polymers are utilized for enhancing
viscosity, the overall polymer negative charge may
electrostatically attract and accumulate the cationic LAE biocide
and a greater concentration of LAE will then be needed to provide
biocidal efficacy comparable to the utilization of a neutral or
cationic water-soluble polymer. Thus, preferred water soluble
polymers are neutral in charge, such as hydroxyethylcellulose,
hydroxypropylcellulose, hydroxypropylmethylcellulose, guar,
hydroxypropylguar, hydroxypropylmethylguar, poly(ethylene oxide),
and poly(N-vinylpyrrolidone), or cationic in charge, such as
cationic chitosans, cationic cellulosics, and cationic guar.
Chitosan polymers may also enhance the antimicrobial behavior of
the antimicrobial composition. More preferred hydrophilic polymers
comprise hydroxypropylmethylcellulose, hydroxypropylcellulose,
hydroxypropylguar, hydroxymethylchitosan, poly(ethylene oxide),
N-[(2-hydroxy-3-trimethylammonium)-propyl]chitosan chloride, with
hydroxymethylpropylcellulose being most preferred.
[0116] Chelating agents enhance the susceptibility of bacteria and
other organisms to the biocidal effects of the antimicrobial agent,
thus rendering a wound care solution or device containing a
chelating agent more effective in combating infection.
Additionally, chelating agents deactivate matrix metalloproteases
(MMPs), enzymes that can impede tissue formation and healing by
breaking down collagen. MMPs are often found at elevated levels in
chronic wounds. Chelating agents bind to zinc ions, which are
necessary for MMP activity, disrupting the MMP, causing
deactivation, and thus facilitating healing.
[0117] The chelating agent is selected from any compound that is
able to sequester monovalent or polyvalent metal ions. The cations
of the chelating agent are more preferably disodium, trisodium or
tetrasodium salts of EDTA, and most preferably disodium EDTA and
trisodium EDTA.
[0118] The concentration of chelating agent can range from 0.0025
to 1.0 wt %, or from 0.005 to 0.5 weight %, or from 0.0075 to 0.15
weight % and can also be any specific wt % found within this
range.
Applications
[0119] Potential applications for the compositions of the instant
invention include the following: water treatment, potable water,
waste water, the inside of pipes carrying either potable or
non-potable water or other liquids, flushes for pipes carrying
either potable or non-potable water or other liquids, food
processing equipment and surfaces, drains, drilling equipment and
drilling processes.
[0120] Compositions of this invention can be used for many
applications, e.g. to penetrate biofilm and/or to kill pathogenic
and other microbials in conduits, tubes, etc. used in the dental
office, hospital, medical facilities, household, or industry.
Non-limiting examples of applications for this invention include
antimicrobial products, household products and cleaners, fabric
detergents, dish detergents, cleansers, soaps, bubble baths,
disinfectants, deodorizers, human and animal foods, food products,
beverages, preservative compositions, antimicrobial packaging,
pharmaceutical products, medical devices, e.g. catheters, wound
dressings, ophthalmic uses, contact lenses and storage containers,
cosmetics, feminine hygiene compositions, vaginal douches, infant
care products, antimicrobial soaps, hand sanitizers, deodorants,
antiperspirants, anti-microbial coatings, dental compositions,
toothpastes, mouth rinses and washes, oral swabs and sponges,
lipsticks, dental appliances and devices, skin swabs, medications,
athlete's foot treatments, cold sore treatments, herpes virus
treatments, medicated chewing gums, wound care compositions,
dermatological compositions, acne treatments, skin conditioners,
skin moisturizers, anti-wrinkle formulations, skin whiteners
sunscreens, tanning lotions, hair products, shampoos, shower gels,
bubble baths, conditioners, shaving creams, spermicides. Also
included are microbial-resistant fabrics and apparel,
anti-microbial condoms, surgical gowns, microbial-resistant
hospital equipment, anti-microbial paper products, animal care
products, antimicrobial plastics, antimicrobial plastic devices,
rubbers and other fabrication materials, appliances with
antimicrobial constituents or coatings, etc. Activity against gram
negative organisms is increased if the pH is about 5.0 or lower.
Other incipients to enhance antimicrobial activity against gram
negative organisms would be the addition of organic acids, e.g.
lactic, citric, etc. and small amounts of EDTA, e.g. about 25-50
ppm.
[0121] Additionally, compositions of the invention can also be
added to articles from where it can release the compositions of
this invention. Generally where added to food packaging, the
amounts of composition needed to effect food preservation would be
higher than the amount needed when incorporated directly into food.
Typically, from about 100 ppm to about 5% by weight of the food
packaging food products would be used. Also the compositions of the
instant invention can be used to coat and/or be added to human or
animal food, e.g. kibble.
[0122] Additionally, plastics and miscellaneous products can be
coated and/or impregnated with or used to deliver the compositions
of the invention, including: medical items, thermometers,
catheters, surgical sutures, blood lines, implants, bandages,
surgical dressings, surgical apparel, respirators, fluid-dispensing
tubing; drug and cosmetic packaging, eating utensils shower
curtains; bath mats; sponges; mops; toilet seats, rubber gloves;
contact lenses; hearing aids; shelving paper; carpet pads; pool
covers; animal bedding and cat litter; computer covers and computer
keys; doorknobs; tampons and sanitary napkins; adult novelties;
sexual aids; sex toys; pregnancy barriers; dental chairs; dryer
sheets; dishcloths; paints and coatings; deodorizing liquids,
solids, sprays, gels and powders; filters; foams; hair brushes;
combs; diaper rash preventer; plasma bag treatment; disposable
glove treatment; additive to pasteurized cow milk; additive to
blood sample tubes to inactivate HIV, HCMV, and other viruses
(safety measure for lab technicians and healthcare providers);
additives for condoms, band-aids, or bandages; additive for paint;
or animal or plant treatment for microbial infections; animal chew
toys, children chew toys, children floating toys, e.g. "rubber
ducky", animal and pet food coatings and ingredients, and the
like.
[0123] Additionally, fibers and fabrics can be coated and/or
impregnated with the compositions of the invention, including
natural and synthetic fibers and fabrics manufactured from such
fibers; wipes, cloths; surgical gauze; crib covers; bassinet
covers; bed linens; towels and wash cloths; tents; draw sheets;
cubicle curtains; shower curtains; wall coverings; wood and wood
products; hospital clothing such as examination robes, physicians'
coats, nurses uniforms, etc.; apparel; paper, non-woven fabric,
knitted fabric, woven fabric, brick, stone, plastic, polymer,
latex, metal, tile, walls, floors, gurneys, tables, or trays; shoes
and the like. Regarding the use of ML types and SL types into food
packaging or other plastic or polymer films, ML and SL types have
outstanding thermal melt stability during melt processing, e.g. in
extrusion, injection molding, blow molding, or the like.
[0124] Cleaning products can usefully incorporate the compositions
of the invention for the purposes of sanitizing or deodorizing
surfaces. Typically, the compositions would be added to aqueous
cleaning formulations in concentrations between about 100 to about
2000 ppm. Other cleaning agents can be added at the concentrations
needed to make the products effective which will depend on usage
concentration. Most cleaning formulations contain surfactants. As
mentioned previously, virtually all nonionic, amphoteric and
cationic surfactants are generally compatible with the enhanced
combinations of the invention. Most anionic surfactants will cause
the N.sup..alpha.-long chain alkanoyl dibasic amino acid alkyl
ester salts to precipitate from solution. One advantage of using
SL/ML types in combination is that there is no interaction with
cationic or anionic species, so the possibility of more stable
systems can be realized.
[0125] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention encompassed by the appended
claims.
Data Generation and Methodology
[0126] Ingredients were chosen for highest purity. Sucrose
monolaurate was obtained from Mitsubishi Kagaku, RYOTO Sugar Ester
(Food grade) L-1995 (90% monoester). LAE-HCl solid (>89% purity)
was obtained from A&B Ingredients Inc., Fairfield, N.J.
Glycerol monolaurate was obtained from Colonial Chemical (90%
monoester, 1 position).
Biofilm Porcine Explant Model
[0127] The porcine explant model used in the examples of the
specification is fully described by Phillips et al. (Wounds
International, Vol 1, Is. 3 May 2010). Briefly, the ex vivo model
of biofilm on porcine skin explants consisted of 12-mm biopsied
explants (3-4 mm thick) prepared from freshly harvested, shaved and
cleaned porcine skin. The mechanically created `wound bed` was 3 mm
in diameter and approximately 1.5 mm in depth. The `wound bed` of
the explants was inoculated with early-logarithmic (log)-phase
Pseudomonas aeruginosa biofilm ("PA01") suspension culture
(10.sup.6 CFU) and cultured at 37.degree. C. with 5% CO.sub.2 and
saturated humidity until biofilm maturity was achieved. Typically
day 3 for the Examples. An explant thus treated serves as the
"total" bacterial count. Some explants were submerged in TSB media
containing 200 .mu.g/ml gentamicin for 24 hours to kill planktonic
PAO1 and to generate the "biofilm" bacterial count. In yet another
set of explants, the explants were treated with compositions of the
disclosed invention for 24 hours. The bacterial load of the
explants was determined in each of the assays of this study as
follows: each explant was aseptically placed into a 15-ml sterile
tube (on ice) containing cold 7-ml sterile phosphate-buffered
saline (PBS) with 5 .mu.l/l Tween-80. The explants in the tubes
were sonicated. Serial dilutions of the bacterial suspension were
plated in triplicate on TSA plates and incubated overnight at 37.0
with 5% CO.sub.2 and saturated humidity. Colonies were counted from
the plates to determine the CFU/ml of the sonicated explant
bacterial suspension. The following examples were tested according
to the Phillips et al. ex vivo model protocol. The accuracy of the
test is .+-.1 log reduction.
[0128] Without being bound by theory it is suggested that the test
involves killing of planktonic bacteria that recolonize a debrided
wound bed as well as expansion of any biofilm bacteria that were
not killed or removed by previous treatment or debridement. In
wound healing, many times the wound is subjected to debridement
which is a mechanical scraping of the wound surface to open up the
biofilm surface. This is painful for many patients and also can
remove healthy cells. Furthermore, the ability of a formulation to
kill mature biofilm without killing all the wound cells is the most
valuable clinical property of a wound treatment. The instant
invention formulation also kills planktonic bacteria so it would be
predicted to prevent reconstitution of a biofilm after initial
debridement and treatment.
EXAMPLES
[0129] The following examples are set forth to assist in
understanding the invention and should not, of course, be construed
as specifically limiting the invention described and claimed
herein. Such variations of the invention, including the
substitution of all equivalents now known or later developed, which
would be within the purview of those skilled in the art, and
changes in formulation or minor changes in experimental design, are
to be considered to fall within the scope of the invention
incorporated herein.
[0130] The following abbreviations may be found throughout the
Examples and Figures: "LAE HCL" is N.sup..alpha.C8-C16 alkanoyl-L
di-basic amino acid --C1-C4 alkyl ester being
N.sup..alpha.-lauroyl-L-arginine-ethyl ester HCL salt; "ML" is
monolaurin; "SL" is sucrose laurate; "HPC" is Dow Methocell.TM. K4M
hydroxypropyl cellulose; "TEC" is Triethyl citrate; "CDM" is Croda
Arlasilk.TM. CDM Sodium Coco PG-dimonium Chloride Phosphate
phospholipid; "PG" is propylene glycol; "DW" is deionized water;
"MIC" is Minimum Inhibitory Concentration; CFUs is colony forming
units; Avg is average; ppm is parts per million; Std Dev is
standard deviation.
Example 1
[0131] FIG. 1A provides the formulation of 4 different compositions
that were tested in the ex vivo porcine skin explant model as
described above. The results were measured as colony forming units
(CFUs) as follows: Total, an average 1.20.times.10.sup.8 (Std Dev
7.85.times.10.sup.7); Biofilm, an average 1.12.times.10.sup.7 (Std
Dev 5.45.times.10.sup.6); Treatment #1, an average
7.28.times.10.sup.1 (Std Dev 6.35.times.10.sup.1); Treatment #2, an
average 0.00.times.10.sup.0 (Std Dev 0.00.times.10.sup.0);
Treatment #3, an average 1.74.times.10.sup.4 (Std Dev
2.00.times.10.sup.4); Treatment #4, an average 3.63.times.10.sup.2
(Std Dev 132.7222). These results are also shown in a bar graph in
FIG. 1B. Three samples give >4 log reduction which is clinically
significant. Sample #2 gives complete kill (7 log reduction).
Example 2
[0132] FIG. 2A lists the formulations tested, R-170131-1 through
R-170131-6. The results were measured as colony forming units
(CFUs) as follows: Total, an average 4.71.times.10.sup.8 (Std Dev
2.94.times.10.sup.8); Biofilm, an average 4.55.times.10.sup.7 (Std
Dev 2.32.times.10.sup.7); Treatment # R-170131-1, an average
7.47.times.10.sup.6 (Std Dev 3.68.times.10.sup.6); Treatment #
R-170131-2, an average 1.28.times.10.sup.6 (Std Dev
1.13.times.10.sup.6); Treatment # R-170131-3, an average
1.80.times.10.sup.4 (Std Dev 1.21.times.10.sup.4); Treatment #
R-170131-4, an average 1.98.times.10.sup.3 (Std Dev
2.08.times.10.sup.3); Treatment # R-170131-5, an average
2.00.times.10.sup.5 (Std Dev 1.73.times.10.sup.5); Treatment #
R-170131-6, an average 3.09.times.10.sup.3 (Std Dev
5.53.times.10.sup.3). FIG. 2B represents this data as a bar chart
of the log CFUs remaining after the treatment. Several samples give
>2 log reduction which is clinically significant. Samples
R-170203-4 and R-170205-6 give >4 log reduction.
Example 3
[0133] FIG. 3A lists the formulations tested. The results were
measured as colony forming units (CFUs) as follows: Total, an
average 4.53.times.10.sup.9 (Std Dev 3.42.times.10.sup.8); Biofilm,
an average 2.80.times.10.sup.8 (Std Dev 1.73.times.10.sup.8);
Treatment # R-170310-1, an average 9.40.times.10.sup.5 (Std Dev
7.58.times.10.sup.5); Treatment # R-170310-2, an average
4.89.times.10.sup.4 (Std Dev 7.95.times.10.sup.4); Treatment #
R-170310-3, an average 1.25.times.10.sup.1 (Std Dev
2.50.times.10.sup.1); Treatment # R-170310-4, an average
0.00.times.10.sup.0 (Std Dev 0.00.times.10.sup.0).
[0134] FIG. 3B is a bar chart of this data and demonstrates that
all four solutions reduced biofilms at clinically significant
levels, but # R-170310-3 produced >7-log reduction and #
R-170310-4 totally eliminated the biofilm and planktonic
bacteria.
Example 4
[0135] FIG. 4A lists the formulations tested. The results were
measured as colony forming units (CFUs) as follows: Total, an
average 9.62.times.10.sup.10 (Std Dev 6.08.times.10.sup.10);
Biofilm, an average 5.69.times.10.sup.9 (Std Dev
2.99.times.10.sup.9); Treatment A, an average 1.67.times.10.sup.0
(Std Dev 3.33.times.10.sup.0); Treatment B, an average
0.00.times.10.sup.0 (Std Dev 0.00.times.10.sup.0); Treatment C, an
average 5.00.times.10.sup.0 (Std Dev 6.38.times.10.sup.0);
Treatment D, an average 0.00.times.10.sup.0 (Std Dev
0.00.times.10.sup.0); Treatment E, an average 5.38.times.10.sup.6
(Std Dev 1.05.times.10.sup.7); Treatment F, an average
1.51.times.10.sup.6 (Std Dev 3.01.times.10.sup.6); Treatment G, an
average 2.49.times.10.sup.4 (Std Dev 1.07.times.10.sup.4). FIG. 4B
is a bar chart of the log CFUs remaining after the treatment.
[0136] FIG. 4B show that all seven formulations reduced biofilms,
and all seven gave clinically significant results. Also samples A
and C produced 9-log reduction and samples B and D totally
eliminated the biofilm. The data of Example 4 demonstrate that a
combination of two of the three active ingredients (LAE, ML, or SL)
will penetrate and kill biofilm bacteria in a clinically
significant level. The data demonstrate that a combination of at
least two of the three ingredients in the instant invention, i.e.
LAE, ML, and SL, will produce clinically significant biofilm
penetration and bacteria kill when tested in the Phillips ex vivo
test.
Example 5
[0137] FIG. 5A lists the content of four formulations. Each
formulation was treated for 24 hr, then analyzed for samples
1,2,3,4 A. For samples 1,2,3,4 B, explants are treated as in A and
then flipped over and immersed again; recovery was in 24 hours, no
debridement.
[0138] The results were measured as colony forming units (CFUs) as
follows: Total, an average 4.37.times.10.sup.8 (Std Dev
5.49.times.10.sup.8); Biofilm, an average 1.93.times.10.sup.6 (Std
Dev 2.83.times.10.sup.6); Treatment #1A, an average
7.00.times.10.sup.1 (Std Dev 3.00.times.10.sup.1); Treatment #1B,
an average 1.67.times.10.sup.1 (Std Dev 2.08.times.10.sup.1);
Treatment #2A, an average 0.00.times.10.sup.0 (Std Dev
0.00.times.10.sup.0); Treatment #2B, an average 0.00.times.10.sup.0
(Std Dev 0.00.times.10.sup.0); Treatment #3A, an average
2.67.times.10.sup.1 (Std Dev 2.52.times.10.sup.1); Treatment #3B,
an average 0.00.times.10.sup.0 (Std Dev 0.00.times.10.sup.0).
Treatment #4A, an average 2.27.times.10.sup.2 (Std Dev
1.91.times.10.sup.2); Treatment #4B, an average 8.33.times.10.sup.1
(Std Dev 1.12.times.10.sup.2). FIG. 5B is a bar chart of the log
CFUs remaining after the treatment. The formulations of Example 5
produce hydrogel like consistencies.
[0139] In Examples 1-5, the data show that varying amounts of LAE,
ML, and SL produce clinically significant results, i.e. >2 log
reduction. Propylene glycol may aid in solubilization of the ML,
and help to penetrate the biofilm.
[0140] In Example 5, Sample 2 has the best penetration and kill
resulting in >6 log reduction. The result is greater than
99.9999% kill. Sample 3 gave the second best results. This resulted
in >4 log drop or 99.99% kill.
[0141] The absence of any LAE-HCl in sample #2 clearly shows that
the combination of ML and SL are responsible for the penetration of
the biofilm and reduction in CFUs. This was also demonstrated in
Examples 4 and 5. The levels of the three main ingredients are also
much lower in Example 5 when compared to Examples 1 and 4.
[0142] Inorganic nitrates can be added as a salt to increase the
amount of NO if a reducing sugar is present like sucrose, glucose
or sucrose laurate (SL). Triethyl citrate ("TEC") can act as an
inhibitor of esterases and can act as a synergist to enhance and
prolong the antibacterial activity of both LAE-HCl, ML, and SL.
Example 6
[0143] In the previous biofilm testing in Examples 2 and 4, it was
unanticipated that a combination of LAE and SL w/o ML reduced or
killed both planktonic and biofilm bacteria in the Phillips et al.
ex vivo test. Also in Example 4 it was unanticipated that a
combination of SL and ML w/o LAE reduced or killed both planktonic
and biofilm bacteria. Further testing was performed to show the
relationship between LAE and SL. Because the combinations of LAE/SL
and SL/ML were also active on planktonic bacteria alone, this would
support potential uses for these two combination of LAE/SL or SL/ML
as preservatives, e.g. in food, personal care, or cosmetic
applications. Example 6 demonstrates combinations of SL/ML show
that these two combinations both have activity using time
kill/recovery as well as Minimum Inhibitory Concentration (MIC)
testing.
[0144] Time-Kill Kinetics Test is a method of testing Antimicrobial
Efficacy also known as the "suspension tests or suspension time
kill analysis", determines the time required by the antimicrobial
agent to kill the challenge test microorganism. This test is
utilized in microbiological studies to assess a test article's in
vitro antimicrobial activity in relation to time. The test
essentially perform the following steps: the undiluted and/or
diluted test compound is introduced to a particular test bacteria
at time zero. This mixture is grown at a set temperature and at
specified time intervals, samples are taken out of the inoculum,
put into a neutralization buffer, and then the microbe population
is enumerated. The resulting data for the Time-Kill test is
typically presented graphically, where the colony counts for each
antimicrobial agent is plotted against the concentration tested at
each time point when the subcultures were performed (usually at 0,
4, 8, 12, and 24 hours). Generally, in a Time-Kill test, a
3-log.sup.10 reduction is considered the minimum level that would
indicate a product has significant killing activity against a
particular test microorganism. In contrast, in the minimal
bactericidal concentration (MBC) test, bactericidal activity is
defined as a 99.9% or greater killing efficacy at a specified
time.
[0145] Time kill and MIC values of various mixtures of sucrose
laurate and glyceryl monolaurate (monolaurin) on Staphylococcus
aureus and Candida alibans MIC values were determined and are shown
in FIGS. 6A and B. ML may be solubilized in propylene glycol (PG),
or DMSO. The use of DMSO is well documented in the literature as a
solubilizing agent. FIGS. 6A and 6B provide the wt % of the active
ingredient of each formulation. Samples in FIG. 6A were tested at
100 ppm each; recovery was at 24 hours. In FIG. 6A samples were
supplied at 1 wt % (active ingredient) and solubilized in distilled
water with 5% propylene glycol ("PG"). In FIG. 6B samples were
supplied at 1 wt % and solubilized in water and PG. All of the
above solutions were conditioned at about 40-45.degree. C. prior to
testing and then incubated at 35.degree. C.
[0146] In FIG. 6A, the time kill data demonstrates that the
combination of 50 wt % SL and 50 wt % ML gives higher log reduction
than that of 100 wt % ML. At the data point of 25 wt % SL/75 wt %
ML, the log reduction indicates a trend for enhanced biocidal
activity. Because the ML was solubilized in FIG. 6A using 95 wt %
DW and 5 wt % PG, the slight difference between the average log
reduction of 50/50 wt % SL/ML being 2.6 and the average log
reduction of 25/75 wt % SL/ML being 1.8 can be explained by the
lower solubilizing effect of the SL. Specifically, with an increase
of ML from 50 to 75 wt %, we would expect that the log reduction
would be similar. This slightly lower average log reduction shows a
trend in the enhancement of ML alone. Using only 5% PG to
solubilize the 100% ML sample, the PG has not completely
solubilized the ML. However in the 50/50 and 25/75 wt % SL/ML
samples, the SL contributes to the solubilization of the ML.
[0147] In FIG. 6B, lwt % ML and lwt % SL were supplied as
solubilized in PG. In FIG. 6B, the MIC data of combinations of ML
and SL show a similar trend as the results in FIG. 6A in the ratios
of from 75 wt % ML:25 wt % SL to 25 wt % ML:75 wt % SL all show a
degree of enhancement. SL has been reported in the literature to
have very high MIC values when tested alone. This data shows that
SL enhances the antimicrobial activity of ML possibly through
improved solubilization with or without PG. This effect is also
shown in the results in Experiments 4 and 5.
Example 7
[0148] Example 7 demonstrates the effect of combining sugar esters
of fatty acids with N.sup..alpha.-long chain alkanoyl dibasic amino
acid alkyl ester salts. MIC values of various mixtures of
N.sup..alpha.-lauroyl arginine ethyl ester HCl salt and sucrose
monolaurate in preventing the growth of Candida alibicans were
determined. In FIG. 7, the MIC data of combinations of SL and LAE
are identical to that of 100 wt % LAE. In comparing ratios of wt %
of SL to wt % of LAE, ratios from 2:1 to 1:2 show enhancement
compared to 100 wt % LAE.
Example 8
[0149] In Example 8, different pathogens were tested for MIC
(Minimum Inhibitory Concentration) with formulations that comprise
combinations of LAE and SL. In the previous biofilm testing in
Examples 2 and 4, the combination of LAE and SL without ML reduced
or killed both planktonic and biofilm bacteria. FIG. 8A-B shows the
results of further testing with LAE and SL. In FIGS. 8A and 8B the
active ingredients LAE and SL were solubilized in DW as indicated
and were tested. FIG. 8A reports MIC combination of LAE and SL
tested on Candida albicans fungi in duplicate and for gram positive
S. aureus. FIG. 8B reports MIC on combinations of LAE/SL exposed to
S. epidermidis.
[0150] In FIG. 8A, ratios of LAE/SL from 25 wt % LAE/75 wt % SL to
75 wt % LAE/25 wt % SL have similar MIC (minimum inhibitory
concentration) values when tests as 1 wt % (active ingredient) in
distilled water. This indicates an enhanced relationship regarding
antimicrobial performance for various levels of LAE. Noted are that
the levels of 100 wt % SL alone have much higher MIC values than
LAE or combinations of LAE/SL.
[0151] In FIG. 8B, ratios of LAE/SL solubilized in DW from 40 wt %
LAE/60 wt % SL to 60 wt % LAE/40 wt % SL have similar MIC values
tested against S. epidermidis. This indicates an enhanced
relationship regarding antimicrobial performance for various levels
of LAE and confirms the data in FIG. 8A. Noted are that the levels
of 100 wt % SL alone have much higher MIC values than LAE or
combinations of LAE/SL. This high MIC of SL is in agreement with
several publications.
[0152] It is well known that LAE and ML both have activity against
pathogens separately. However ML does not have similar broad
activity against all pathogens that LAE does. Using combinations of
LAE/SL and of SL/ML show improved activity using lower level of ML
with SL, while SL alone has very low activity. Similarly
performance of lower levels of LAE alone can be improved with SL.
This provides an advantage in cost performance basis as LAE is many
times more expensive than cosmetic versions of SL or other sucrose
fatty acid monoesters.
[0153] As stated above, while the present application has been
illustrated by the description of embodiments thereof and while the
embodiments have been described in considerable detail, it is not
the intention of the applicants to restrict or in any way limit the
scope of the appended claims to such detail. Additional advantages
and modifications will readily appear to those skilled in the art,
having the benefit of the present application. Therefore, the
application, in its broader aspects, is not limited to the specific
details of the illustrative examples shown. Departures may be made
from such details and examples without departing from the spirit or
scope of the general inventive concept.
* * * * *