U.S. patent application number 13/510361 was filed with the patent office on 2012-11-15 for antimicrobial compositions containing free fatty acids.
Invention is credited to Michael Anthony Folan.
Application Number | 20120289591 13/510361 |
Document ID | / |
Family ID | 43412541 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120289591 |
Kind Code |
A1 |
Folan; Michael Anthony |
November 15, 2012 |
Antimicrobial Compositions Containing Free Fatty Acids
Abstract
The invention concerns antimicrobial compositions comprising
free fatty acids emulsified with membrane lipids or hydrolysed
derivatives thereof, and pharmaceutical formulations comprising
same. The compositions can be used in the treatment or prophylaxis
of microbial infections. They can also regulate the rate of blood
clotting rendering them suitable for incorporation in catheter
locking solutions and for use in wound care.
Inventors: |
Folan; Michael Anthony;
(Donegal Town, IE) |
Family ID: |
43412541 |
Appl. No.: |
13/510361 |
Filed: |
November 17, 2010 |
PCT Filed: |
November 17, 2010 |
PCT NO: |
PCT/EP2010/067710 |
371 Date: |
July 31, 2012 |
Current U.S.
Class: |
514/474 ;
514/557; 514/558; 514/560; 514/574 |
Current CPC
Class: |
A61K 2800/74 20130101;
A61Q 11/00 20130101; A61P 31/22 20180101; A61K 47/12 20130101; A61P
31/10 20180101; A61K 8/06 20130101; A61K 8/361 20130101; A61K 47/24
20130101; A61K 9/0014 20130101; A61K 8/553 20130101; A61K 31/20
20130101; A61K 31/685 20130101; A61P 31/04 20180101; A61K 31/201
20130101; A61K 9/107 20130101; A61K 31/20 20130101; A61K 2300/00
20130101; A61K 31/685 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/474 ;
514/557; 514/558; 514/560; 514/574 |
International
Class: |
A61K 31/19 20060101
A61K031/19; A61K 31/201 20060101 A61K031/201; A61K 31/202 20060101
A61K031/202; A61K 31/194 20060101 A61K031/194; A01P 1/00 20060101
A01P001/00; A61K 31/375 20060101 A61K031/375; A01N 37/02 20060101
A01N037/02; A01N 37/06 20060101 A01N037/06; A61P 31/04 20060101
A61P031/04; A61K 31/20 20060101 A61K031/20; A61K 31/191 20060101
A61K031/191 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2009 |
IE |
200900872 |
Claims
1.-31. (canceled)
32. An antimicrobial and blood-clotting regulatory composition for
use in blood contact applications selected from the group
consisting of surgical irrigation, wound care, catheter locking
solutions, and the coating of catheters and other medical devices
for insertion through the skin or into a bodily orifice or cavity,
the composition comprising: (a) one or more saturated or
unsaturated free fatty acids having from 4 to 22 carbon atoms or a
pharmaceutically acceptable salt or ester thereof; and (b) one or
more membrane lipids or a hydrolysed derivative thereof, as
emulsifying agent for the free fatty acid(s) or the salt or ester
thereof.
33. The composition of claim 32, wherein the free fatty acid is
selected from valeric, caproic, caprylic, pelargonic, capric,
undecanoic, undecylenic, lauric, myristic, palmitic, stearic,
oleic, linoleic and linolenic acids and mixtures thereof, and
pharmaceutically acceptable salts and esters thereof.
34. The composition of claim 32, wherein the free fatty acid is
selected from one or more of caproic, caprylic, pelargonic, capric,
undecylenic and lauric acids, especially caprylic acid.
35. The composition of claim 32, wherein the membrane lipid is
selected from one or more of phospholipids, lecithin,
glycerophospholipids, sphingolipids, glycosphingolipids,
glycoglycerolipids and cholesterols, and hydrolysed derivatives
thereof.
36. The composition of claim 32, wherein the membrane lipid is
selected from one or more of phosphatidic acid,
phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,
lecithin, ceramide, sphingomyelin, glycolipids, glycosphingolipids,
cerebrosides, gangliosides, glycoglycerolipids, mono-galactosyl
diglyceride, lanosterol and cholesterol.
37. The composition of claim 32, wherein the membrane lipid is a
phosopholipid selected from one or more of phosphatidic acid,
phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and
lecithin, especially lecithin.
38. The composition of claim 32, wherein the membrane lipid or
hydrolysed derivative thereof is delipidised.
39. The composition of claim 32, comprising a free fatty acid
selected from one or more of caproic, caprylic, pelargonic, capric,
undecylenic and lauric acids, in combination with a membrane lipid
selected from one or more phosopholipids, especially caprylic acid
in combination with delipidised lecithin.
40. The composition of claim 32, wherein the ratio of component (a)
to component (b) is from about 0.25:1 to about 10:1; or from about
0.5:1 to about 10:1, or from about 0.5:1 to about 5.0:1, or from
about 1.0:1 to about 2.5:1, or from about 1.25:1 to about 2.5:1, on
a weight for weight basis.
41. The composition of claim 32, further comprising one or more
pharmaceutically acceptable organic acids or a pharmaceutically
acceptable salt or ester thereof and/or one or more
pharmaceutically acceptable inorganic acid salts.
42. The composition of claim 41, wherein the organic acid is
selected from acetic, pyruvic, propionic, glycolic, oxalic, lactic,
glyceric, tartronic, malic, maleic, ascorbic, fumaric, tartaric,
malonic, glutaric, propenoic, cis or trans butenoic and citric
acids and mixtures thereof, and pharmaceutically acceptable salts
and esters thereof.
43. The composition of claim 41, wherein the organic acid is citric
or lactic acid or the sodium or potassium salt thereof or wherein
the organic acid salt is sodium citrate.
44. The composition of claim 32 for use in a catheter locking
solution.
45. The composition of claim 32 for use as an antimicrobial and
blood-clotting regulatory agent in a formulation for coating the
surface of a catheter or other medical device for insertion through
the skin or into a bodily orifice or cavity.
46. The composition of claim 32 for use in a surgical irrigation
fluid.
47. An antimicrobial and blood-clotting regulatory catheter locking
solution comprising: (a) one or more saturated or unsaturated free
fatty acids having from 4 to 22 carbon atoms or a pharmaceutically
acceptable salt or ester thereof; and (b) one or more membrane
lipids or a hydrolysed derivative thereof, as emulsifying agent for
the free fatty acid(s) or the salt or ester thereof.
48. The catheter locking solution of claim 47, wherein the membrane
lipid or hydrolysed derivative thereof is delipidised.
49. The catheter locking solution of claim 47, wherein the free
fatty acid is selected from one or more of caproic, caprylic,
pelargonic, capric, undecylenic and lauric acids, and the membrane
lipid is selected from one or more phosopholipids; or wherein the
free fatty acid is caprylic acid and the membrane lipid is
delipidised lecithin.
50. The catheter locking solution of claim 47, further comprising a
pharmaceutically acceptable viscosity-enhancing agent wherein,
preferably, the viscosity-enhancing agent is dextran.
51. The catheter locking solution of claim 47, further comprising
one or more pharmaceutically acceptable organic acids or a
pharmaceutically acceptable salt or ester thereof, wherein the
organic acid is citric acid or lactic acid or the sodium or
potassium salt thereof; or wherein the organic acid salt is sodium
citrate.
Description
[0001] This invention relates to antimicrobial compositions
containing free fatty, acids and to pharmaceutical formulations
containing same.
[0002] Free fatty acids are essentially insoluble in water. Their
insolubility and the fact that they are incompatible with many
conventional excipients have seriously restricted their medicinal
use to, date. While salts of free fatty acids are soluble in water,
they are known to have greatly reduced antimicrobial effect.
Several methods have been used to `solubilise` free fatty acids,
including the use of alcohols and surfactants and derivitisation by
re-esterification to form mono-glycerides and/or ethoxylation and
propoxylation procedures.
[0003] The antimicrobial properties of free fatty acids have been
known for many years (Kabara J. et al. Antimicrobial Agents and
Chemotherapy, July 1972; 2(1): pp 23-28).
[0004] Bergson et al. (Antimicrobial Agents and Chemotherapy,
November 2001, pp 3209-3212), reported that both capric and lauric
acid were effective in killing the yeast Candida albicans.
[0005] Sun et al. (Chemico-Biological Interactions 140 (2002), pp
185-198), identified the superior microbicidal properties of
caprylic, capric and lauric acid, concluding that lauric was most
potent against gram positive bacteria while caprylic was optimal
against gram negative organisms.
[0006] The anti-viral properties of free fatty acids were reported
by Halldor et al. (Antimicrobial Agents and Chemotherapy; January
1987, pp 27-31).
[0007] WO 03/018049 discloses the antimicrobial activity of milk
serum apo-proteins in combination with free fatty acids from milk
fat. It is illustrated that the adhesion inhibitory properties of
milk extracts are exclusively attributable to the water soluble
protein fraction and that the lipid component makes no contribution
to this effect.
[0008] WO 2009/072097 discloses properties of compositions of free
fatty acids such as melting point depression and sequestration
which affect antimicrobial potency. Also disclosed are
emulsification methods used to incorporate blends of free fatty
acids in a milk whey protein isolate.
[0009] Sprong et al. (Antimicrobial Agents and Chemotherapy, Vol
45, No 4, 2001, pp 1298-1301), report microbicidal effects for
sphingosine and sphingomyelin and some slight effect from
lyso-phosphatidyl ethanolamine and lyso-phosphatidyl choline, but
no effect was observed from any of the unmodified phopshollpids. It
is notable that these results are reported for exposure times in
excess of 2 hours at 37.degree. C., in contrast to the present
invention where microbicidal effects are shown for combinations of
membrane lipids and free fatty acids, for exposure times of less
than 5 minutes.
[0010] Jones et al: Journal of Pharmacy and Pharmacology, 2003, Vol
55, No 1. pp 43-52 discloses the use of lecithin in combination
with cholesterol as a surface coating to inhibit bio-film formation
on medical devices.
[0011] It is an object of the invention to provide improved
antimicrobial compositions containing free fatty acids.
[0012] The invention provides an antimicrobial composition
comprising: [0013] (a) one or more saturated or unsaturated free
fatty acids having from 4 to 22, preferably 6 to 18 or 6 to 12,
carbon atoms or a pharmaceutically acceptable salt or ester
thereof; and [0014] (b) one or more membrane lipids or a hydrolysed
derivative thereof, as emulsifying agent for the free fatty acid(s)
or the salt or ester thereof.
[0015] The free fatty acid, may be selected from: butyric (C4:0),
valeric (C5:0), caproic (C6:0), heptanoic (C7:0), caprylic (C8:0),
pelargonic (C9:0), capric (C10:0), undecanoic (C11:0), undecylenic
(C11:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0),
palmitoleic (C16:1), margaric (C17:0), stearic (C18:0), oleic
(C18:1), linoleic (C18:2), linolenic (C18:3), arachidonic (C20:4)
and euricic (C22:1).
[0016] The free fatty acid is preferably selected from: valeric,
caproic, caprylic, pelargonic, capric, undecanoic, undecylenic,
lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic
acids and mixtures thereof, and pharmaceutically acceptable salts
and esters thereof. Particularly preferred are caproic; caprylic,
pelargonic, capric, undecylenic and lauric acids, especially
caprylic acid.
[0017] Where combinations of fatty acids are, used including higher
melting point entities such as lauric acid, lower melting point
entities such as caprylic or oleic are preferably included to
depress the melting point of the combination to less than normal
physiological temperatures.
[0018] Membrane lipids are ubiquitous components of all cell
membranes in the plant and animal kingdoms. They are
characteristically made up of one or in most cases two long chain
hydrocarbon molecules attached to a highly polar head group, which
is a derivative of either, glycerol-3-phosphate, a long chain amino
alcohol (sphingosine), a sugar, or a derivative of a steroid
(cholesterol). The properties of each membrane lipid are mainly
dictated by the variation in the polar head group. Nearly all are
amphoteric in so far as they behave as both acid and base and more
importantly all are amphipathic, having a water soluble,
hydrophilic end at the polar head group and a fat soluble
lipophylic end at the hydrocarbon tail.
[0019] The physico-chemical properties of membrane lipids are well
known and a suitable review of their occurrence and biological
properties may be found in Biochemistry, 3.sup.rd edition: Mathews,
Van Holde & Ahern: ISBN 0-8053-3066-6, from which Table 1 has
been collated.
TABLE-US-00001 TABLE 1 Major classes of membrane lipids. Polar Head
Class of Membrane Lipids group Membrane Lipids
Lecithin/Phospholipids/ Glycerol Phosphatidic acid
Glycerophospholipids Phosphatidylcholine Phosphatidylethanolamine
Phosphatidylglycerol phosphatidylinositol Phosphatidylserine
Sphingolipids & Sphingosine Ceramide glycosphingolipids
Sphingomyelin Glycoglycerolipids Saccharide Glycolipids
Glycosphingolipids Cerebrosides Gangliosides Glycoglycerolipids
Mono-galactosyl diglyceride Cholesterol Steroids Lanosterol
[0020] Of the four major classes of membrane lipids those
containing phosphate in the polar head group are the most common.
Described as glycerophospholipids, phosphoglycerides or more
frequently as phospholipids this group makes up the major portion
of all membrane lipids in the bacteria, plant and animal kingdoms.
All phospholipids are based on a glycerol backbone with two
hydrophobic acyl side chains on carbons at sn1 and sn2 and a
phosphate moiety at sn3. Variations in the phosphate head
differentiate six separate types of commonly occurring
phospholipids, the simplest being phosphatidic acid and progressing
with increasing complexity through phosphatidylcholine,
phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine,
and phosphatidyl inositol, phosphatidylcholine being the most
prevalent in animals and bacteria.
[0021] All six of the above mentioned phospholipids are found in
lecithin, a membrane lipid of commerce which is extracted on
industrial scale from a variety of sources including but not
limited to soya bean, sunflower, canola, palm oil, egg yolk and
butterfat. The term `lecithin` is frequently used synonymously with
the major phospholipid component phosphatidylcholine, which may be
purified from crude lecithin, as is usual when rigorous control of
quality is required in pharmaceutical applications. It is also
possible to enzymatically modify lecithin or any of its component
phospholipids by, for example, removing one of the hydrocarbon side
chains to form `lysophospholipids` all of which are equally
suitable for use in this invention.
[0022] Hydrolysed derivatives of membrane lipids include
lyso-phospholipids and lyso-sphingolipids which may be produced
enzymatically using pancreatic phospholipases available from
Novozyme, Denmark. The process involves preparation of an aqueous
dispersion of phospholipids or sphingolipid, addition of
phopsholipase enzyme at 2% W/V and incubation at an elevated
temperature of from about 50.degree. C. to about 60.degree. C. for
about 2 hours. Yield of hydrolysed membrane lipid may be up to 70%,
and the product may be separated from the mixture by water
partition based on increased hydrophilicity of the lyso
derivative.
[0023] The membrane lipids used herein may be extracted from plant
or animal sources including oil bearing seeds, animal fat, wool,
milk and eggs.
[0024] The membrane lipids and derivatives thereof used in the
present invention are preferably delipidised. As used herein, the
term "delipidised" is intended to mean that substantially all of
the conjugated extraneous lipid material, such as oil, fat or
triglyceride material, with which membrane lipids are normally
associated, in nature, is removed. "Substantially all" in this
context is intended to mean that the delipidised membrane lipid
contains less than 10%, preferably less than 5%, more preferably
less than 3%, of conjugated extraneous lipid material.
[0025] The membrane lipid is preferably selected from one or more
of phospholipids, lecithin, glycerophospholipids, sphingolipids,
glycosphingolipids, glycoglycerolipids and cholesterols. Suitable
membrane lipids, include lecithin, phosphatidic acid,
phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol, phosphatidylserine,
ceramide, sphingomyelin, glycolipids, glycosphingolipids,
cerebrosides, gangliosides, glycoglycerolipids, mono-galactosyl
diglyceride, lanosterol or cholesterol or any combination thereof.
Preferred are lecithin and other phospholipids selected from
phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine,
phosphatidylglycerol, phosphatidylinositol and phosphatidylserine
and mixtures thereof. Lecithin is particularly preferred.
[0026] In the antimicrobial composition, of the invention, a
combination of one or more of caproic, caprylic, pelargonic,
capric, undecylenic and lauric acids with one or more phospholipids
is particularly preferred, especially caprylic acid with lecithin
that is preferably delipidised.
[0027] The ratio of free fatty acid or pharmaceutically acceptable
salt or ester thereof to membrane lipid may be from about 0.25:1 to
about 10:1; or from about 0.5:1 to about 10:1, or from about 0.5:1
to about 5.0:1, or from about 1.0:1 to about 2.5:1, or from about
1.25:1 to about 2.5:1, on a weight for Weight basis.
[0028] Free fatty acids are negatively affected when in contact
with bodily fluids, such as blood, serum or mucus. However, it was
unexpectedly found, that this effect could be ameliorated in the
presence of an organic acid or salt or ester thereof and/or an
inorganic acid salt. Thus, the antimicrobial composition of the
invention may also comprise one or more pharmaceutically acceptable
organic acids or a pharmaceutically acceptable salt or ester
thereof; and/or one or more pharmaceutically acceptable inorganic
acid salts.
[0029] The organic acid is preferably selected from acetic,
pyruvic, propionic, glycolic, oxalic, lactic, glyceric, tartronic,
malic, maleic, ascorbic, fumaric, tartaric, malonic, glutaric,
propenoic, cis or trans butenoic and citric acids and mixtures
thereof, and pharmaceutically acceptable salts and esters thereof.
The inorganic acid salt is preferably selected from chloride,
sulphate and nitrate.
[0030] Particularly preferred organic acids are citric and lactic
acids and the sodium and potassium salts thereof, especially sodium
citrate.
[0031] The molar concentration of the organic acid is preferably
from about 25 mM to about 500 mM, more preferably from about 50 mM
to 250 mM, and more preferably from about 50 mM to 150 mM. The pH
of the organic acid salt is preferably between 4.0 and 6.5,
preferably between 4.0 and 5.5 or between 4.0 and 5.0.
[0032] In the antimicrobial composition of the invention, the
membrane lipid emulsifies the fatty acid, rendering it
water-dispersible. Thus, the membrane lipid-fatty acid combination
is an emulsion, preferably an oil-in-water emulsion, although a
water-in-oil emulsion is also possible.
[0033] In a particularly preferred embodiment. the composition of
the invention comprises an emulsion of 0.5% caprylic acid in 0.4%
de-lipidised lecithin, which is then diluted to 50% of its
concentration using 200 mM sodium citrate buffer at pH 4.5, the
final concentration being 0.25% caprylic acid emulsified in 0.2%
de-lipidised lecithin and dispersed in 100 mM sodium citrate at pH
4.5. This composition is referred to hereinafter as the "standard
formulation" and may conveniently be used to demonstrate the
antimicrobial effects of the inventive compositions.
[0034] The antimicrobial compositions of the invention exhibit a
dual antimicrobial effect, in that they exhibit inhibition of
microbial adhesion to host cell surfaces and achieve a microbicidal
effect through dissolution of microbial cell membranes.
[0035] It has unexpectedly been found that when membrane lipids are
separated from conjugated lipid moieties, different membrane lipids
exhibit strikingly different adhesion inhibitory properties on
microbial cells in planktonic culture. While not wishing to be
bound by theory, it is suggested that these differences arise from
variable hydrophobic/hydrophilic characteristics. Furthermore it
has been discovered that the same amphipathic variability affects
the tenacity of emulsions formed with individual membrane lipids
and that these differences can be exploited to manipulate the rate
of release of emulsified microbicidal free fatty acids, thereby
greatly facilitating the design of fast or slow acting microbicidal
formulations with superior utility in medical applications.
[0036] As described herein, superior and adjustable adhesion
inhibitory properties may be obtained using membrane lipids,
particularly phospholipids, collectively described as lecithins,
after they have been extracted and freed of conjugated lipid with
which they are normally associated in their natural environment.
Inhibition of microbial adhesion requires that the lipophylic end
of the membrane lipid is free to orientate with and conjugate to
lipid moieties on the microbial cell surface. This process is
impeded if the lipophylic sites are occupied by non-membrane lipids
(triglycerides for example).
[0037] A particular utility afforded by the inventive compositions
is the discovery that the different classes and different members
of each class of membrane lipids have different release
characteristics. The same dose of the same fatty acid in the same
amount of different membrane lipid will deliver the same
microbicidal effect over a faster or slower period depending on the
individual membrane lipid. This peculiar characteristic facilitates
the use of combinations of different membrane lipid emulsions of
the same fatty acid to achieve a sustained release effect over
relatively useful periods.
[0038] A feature of free fatty acids and particularly the short to
medium chain saturated fatty acid, such as caprylic acid, is that
they are rapidly absorbed through skin and mucosal membranes. Rapid
absorption depletes the dose at the epithelial surface and
consequently impairs the microbicidal effect at that surface. For
this reason, membrane lipids delivering the most immediate
microbicidal effect are not necessarily the most effective in
therapeutic applications. As demonstrated hereinafter in Example 9,
certain individual membrane lipids are more tenacious than others,
which restricts the availability of their emulsified free fatty
acid, and consequently restricts the rate of its microbicidal
effect, which equally restricts its absorption at the epithelial
surface. It should be noted, however, that the overall microbicidal
effect (log numbers of microorganisms killed) is not affected.
[0039] It has also unexpectedly been found that the antimicrobial
compositions of the invention can be used to regulate the rate of
blood clotting. By varying the ratio of free fatty acid to membrane
lipid and/or by incorporation of pharmaceutically acceptable salts
of organic or inorganic acids, or oligosaccharides or other
polymers, the formulations will either catalyse the rate of blood
clotting or suppress it altogether. This property gives great
advantage in use as a blood contact antimicrobial agent.
[0040] As disclosed herein, an aqueous dispersion Of de-lipidised
membrane lipid added to fresh sheep blood at a volume of 20% will
accelerate the normal rate of blood clotting, reducing the time to
clot from 6 minutes to less than one minute. Addition of a fatty
acid, such as caprylic acid, by emulsification up to a ratio of
about 1.0-1.3 times the weight of de-lipidised membrane lipid, such
as lecithin, will further reduce the time to clot formation, but
thereafter as the ratio of fatty acid increases, the time to clot
formation is increased and an anti-coagulation effect is observed
when the weight of emulsified fatty acid exceeds about 1.0-1.3
times the weight of de-lipidised membrane lipid. A skilled person
will appreciate that the blood regulatory effect will depend not
only on the ratio of free fatty acid to de-lipidised membrane
lipid, but also on the nature of the membrane lipid and free fatty
acid used.
[0041] The presence of an organic salt in the inventive
composition, as might be required for amplification of microbicidal
effect, will also destroy the pro-coagulation effect, and as
illustrated herein the use of a viscosity-enhancing agent, such as
dextran, will extend the anti-coagulation effects to the point
where they are comparable with a solution of heparin.
[0042] In a further embodiment, an emulsion of free fatty acid not
exceeding about 1.0-1.3 times the weight, of de-lipidised membrane
lipid may be used to enhance the rate of blood clotting and exert
an antimicrobial effect at the site of bleeding, while an emulsion
of free fatty acid exceeding about 1.0-1.3 times the weight of
de-lipidised membrane lipid with or without added salts of organic
acids may be used to inhibit blood clot formation and exert a
microbicidal effect at the site of bleeding.
[0043] The compositions of the invention containing membrane lipid
emulsified free fatty acids exert vastly superior dual
antimicrobial effects: both inhibition of adhesion and microbicidal
effect. One of the significant advantages provided by dual
antimicrobial effect is that the adhesion inhibitory properties
prevail long after the microbicidal pay-load has been exhausted.
Most microbicides are chemically reactive with the target organism
and most microbicidal reactions are irreversible under
physiological conditions; a fixed dose of a microbicide therefore
has a limited reactive capability. In combination with an adhesion
inhibitory substance however, the adhesion inhibitory properties
persist after the microbicide has been depleted and additional
protection is afforded against any residual viable pathogens.
[0044] In addition to superior and adjustable inhibition of
adhesion combined with superior microbicidal effect and intrinsic
blood-clotting regulatory effects, the compositions of the
invention are compatible with systemic administration (blood
contact), in contrast to milk protein compositions disclosed in WO
03/018049, and WO 2009/072097. Membrane lipids and free, fatty
acids are natural metabolites in the human and animal body, their
antimicrobial effects are concentration dependent and when they
enter the systemic circulation they are rapidly diluted,
metabolized and excreted as natural metabolites.
[0045] The use of membrane lipids in combination with an
antimicrobial agent is counter-intuitive as most conventional
antimicrobial agents are inactivated by lecithin and related
membrane lipids; lecithin is listed as an approved antiseptic
neutralizing agent for use in European Standard EN 1499 and 1500
testing for evaluating the microbicidal effect of hand soaps and
gels.
[0046] The compositions of the invention may be used in the
treatment or prophylaxis of microbial infections in humans or
animals. Due to the regulatory effect on blood-clotting, the
compositions may be used in, for example, catheter locking
solutions and in wound care.
[0047] The invention also provides a pharmaceutical formulation
comprising a composition according to the invention and a
pharmaceutically acceptable carrier, diluent or excipient therefor.
The composition may be present in the pharmaceutical formulation in
an amount of from about 0.1% to about 25% (w/v); or from about 0.1%
to about 10% (w/v); or from about 0.1% to about 1.0% (w/v).
[0048] The pharmaceutical formulation may be in any form suitable
for administration to a human or animal, including for example,
forms suitable for oral, topical, enteral, parenteral or mucosal
administration.
[0049] The formulation of the invention can be used as a topical
antimicrobial agent for prevention and treatment of infections of
the skin, hair, nails and external membranes of the body
orifices.
[0050] The inventive composition or formulation may be used in the
construction of a liquid soap or hand gel for elimination of
asymptomatic carriage of potentially pathogenic bacteria such as
methicilin resistant Staphylococcus aureus and other species
commonly associated with nosocomial or hospital acquired
infections. It may be used as a gel for treatment of acne. It may
be used in the form of an ointment for treatment of dermatophytic
fungal infections of the hair follicles including that caused by
Trychophyton species commonly known as ringworm. It may be
constructed in the form of a spray for delivery to the skin as a
treatment for infections caused by the enveloped viruses including
Herpes varieties causing cold sores and shingles. It may be
prepared in the form of liquid drops for treatment of infections of
the eye and ear. It may be prepared in a variety of
pharmaceutically acceptable delivery systems for treatment of
infections of the mucosa including the mucosal surfaces of the
nose, mouth, throat, bronchiole and lungs and the gastro-intestinal
tract and genitalia. It may for example be prepared in the form of
a toothpaste and mouthwash to facilitate improved dental hygiene
and eliminate the burden of organisms causing dental caries, gum
disease, aphthous ulcer and halitosis. It may be prepared in a form
suitable as a saliva supplement for alleviation of the symptoms of
xerostomia. It may be prepared as a spray for de-colonisation of
the mouth, throat and nasal membranes, particularly for eradication
of asymptomatic carriage of antibiotic resistant species. It may be
prepared, in the form of a gel for the prevention and treatment of
microbial infections of the genitalia caused by a variety of
bacteria, yeast and viruses including those known to be the
causative agents of Candidiasis, non-specific bacterial vaginosis,
the herpes virus and HIV. It may be prepared in the form of an
enema for prevention and treatment of infections of the bowel and
lower intestinal tract including those caused by anaerobic
Clostridium species and Desulfovibrio medically known as
pseudomembraneous colitis and pouchitis.
[0051] Membrane lipid emulsified free fatty acids may be used to
prepare foods that exert an antimicrobial effect in the
gastro-intestinal tract, serving to protect against enteric
pathogens such as Helicobacter, E. coli, Salmonella, Campylobacter,
Clostridium species, Protozoa and enveloped viruses.
[0052] A suitable food carrier for membrane lipid emulsified free
fatty acids is dairy milk, preferably fat free dairy milk and more
preferably skim milk powder such as Marvel, available commercially
from Premier International Foods (UK) Ltd, Spalding, Lincolnshire,
England.
[0053] Separate emulsions of, for example, caprylic, capric and
lauric acids may be prepared as 5.0% W/V emulsions in 4.0% W/V
de-lipidised lecithin as described in the methods. The individual
emulsions may be combined in a ratio of 1:1:1 or any other suitable
ratio such as 1:2:3. Membrane lipid emulsions of other fatty acids
such as butyric and or emulsions of essential oils such as
peppermint or vanilla may be added to impart flavour and improve
taste.
[0054] Marvel skim milk powder may be re-constituted by adding the
specified amount to potable water (4 heaped teaspoons to one pint).
To this an amount from about 1% W/V to 15% W/V of a selected ratio
of membrane lipid emulsified free fatty acids may be added and
mixed by stirring. Preferably the amount of emulsified fatty acid
will be from about 1% W/V to 10% W/V and more preferably about 5%
W/V. It will be clear to a skilled person that skim milk powder may
be re-constituted in less than the optimal volume of water for use
as milk, in which case a concentrated dairy cream is formed to
which amounts of emulsified fatty acids may be added.
[0055] Alternatively, a dairy whey protein isolate may be used as a
suitable food carrier: Provon 190 from Glanbia PLC is a suitable
whey protein isolate. The whey protein isolate may be re-hydrated
in potable water in an amount of from about 10% W/V to 20% W/V,
preferably about 15% W/V. Once fully hydrated, an amount of
membrane lipid emulsified free fatty acid or blend thereof may be
added and mixed by stirring. When whey protein isolate is used as a
carrier, the amount of added membrane lipid emulsified free fatty
acid may be from about 1% W/V to 20% W/V, preferably from about 5%
W/V to 15% W/V and more preferably about 10% W/V.
[0056] The products of the invention have utility as blood contact
antimicrobial agents where their combined blood clotting or
anti-clotting capability is combined with their antimicrobial
effects. Such uses include surgical irrigation, wound care,
catheter locking solutions, the coating of catheters and other
tubular surgical devices for insertion into a bodily orifice or
cavity, and in food safety.
[0057] A significant advantage of the products of the invention is
that very short exposure times are required to achieve an
antimicrobial effect, generally less than 1 hour or less than 30
minutes or less than 10 minutes.
[0058] As used herein and in conventional use, the term
`antimicrobial` refers to any substance, component or composition
of components which exhibit an antagonistic effect towards
protozoa, gram positive and negative bacteria (both, aerobes and
anaerobes), yeast, fungi, mycoplasma and/or viruses and in
particular those microbial species that are capable of causing
disease in humans and animals.
[0059] Infectious disease arises either from ingress of pathogenic
microbial species (microorganisms) from the external environment or
as a result of aberrant growth of microorganisms that are normally
present in the natural micro-flora of the skin, hair, and mucosal
membranes of the eye, nose, mouth, gastro-intestinal tract and the
genitalia.
[0060] Whether exogenous or endogenous it is widely understood
among healthcare professionals that the first stage of microbial
pathogenesis involves adhesion of the microorganism to the surface
of human or animal tissue. Once adhered, colonization takes place
by way of proliferation and further adherence after which toxin
production inflammation and destruction of host tissue give rise to
the classical symptoms of infectious disease. It is also widely
accepted that if adhesion can be prevented, initiation of the
pathogenic process can be inhibited and many infectious diseases
could be prevented or at least limited in the scope of their
proliferation.
[0061] The novel properties of membrane lipid emulsified free fatty
acids present wide ranging utility in human and animal healthcare.
In blood contact applications these include surgical irrigation
fluids, haemostatic antimicrobials in wound care, catheter coatings
and catheter locking solutions for prevention of catheter related
bacteraemia; in mucosal healthcare they can be used to replicate
the natural antimicrobial mechanism of healthy mucus; in topical
skin care they can be used to amplify the natural antimicrobial
defenses of the skin; in food safety because they are natural
metabolites they can be applied directly to a food surface to
eradicate food borne pathogens; and as medical foods they can be
used to supplement a normal diet to prevent or treat infections of
the gastro-intestinal tract.
Surgical Irrigation and Wound Care
[0062] During surgical intervention it is common practice to employ
a variety of techniques to minimize the ingress of potentially
pathogenic bacteria, yeasts, moulds and viruses. A common and
growing problem is the potential infection of an open wound by
antibiotic resistant bacteria such as Methicillin Resistant
Staphylococcus Aureus (MRSA), and many others including but not
limited to Enterococcus species, Pseudomonas, and the yeast Candia
albicans. In many cases open wounds are irrigated to clear them of
loose tissue, blood and other body fluids and in many cases a
solution of sterile saline is currently in use, although this
offers no antimicrobial effect. The use of surgical irrigating
fluids containing antibiotics is not recommended simply because
these have the potential to generate further antibiotic resistant
species. Equally, many conventional antiseptic agents are
unsuitable and potentially toxic in direct systemic contact with an
open wound. There is therefore a great need for a safe and
effective antimicrobial irrigating fluid, preferably one which has
the additional optional properties of inhibiting blood clotting
during microsurgery and/or a companion product which can promote
blood clotting and healing after surgery. Compositions based on
optionally de-lipidised membrane lipids combined with free fatty
acids and/or derivatives thereof as described herein provide such
utility. Also in trauma care after accidental injury or during
military operations, wounds are frequently jagged, dirty and
possibly hemorrhaging profusely. There is a great need for
emergency intervention with antimicrobial products that promote
blood clotting and which can be applied safely to an open
wound.
Prevention of Catheter Related Infection
[0063] In simplistic terms a catheter is a tube inserted through
the skin, into an artery or vein or through a natural orifice for
the purpose of draining body fluids, or for administration of drugs
or for the purpose of monitoring disease or manipulation of
surgical instruments. Some catheters remain in place for relatively
long periods of time and in many cases these are susceptible to
microbial contamination causing bio-film on the inside and outside
surfaces and potentially leading to disseminated infection of the
patient.
[0064] The most common catheter is a urinary tract tube with a
collecting vessel to drain urine from the bladder; these are
recognized as being significant sources of hospital acquired
infection. A more elaborate and complicated system is a central
venous catheter (CVC) inserted either through the jugular vein in
the neck, and threaded through to the superior vena cava at the
heart or inserted through a peripheral vein and threaded through to
the same vein draining into the heart. CVCs are designed to be left
in place for extended periods of up to 90 days or more and are
intended for long term repetitive infusion of drugs and/or
nutritional formulations; in such applications they are routinely
opened for administration and closed again for intermittent periods
during which fluid in the lumen of the catheter is static and
susceptible to microbial growth if contaminated. Accidental
contamination of CVCs is relatively common and frequently gives
rise to catheter related blood stream infections which are
potentially fatal.
[0065] Blood enters the lumen of a CVC during routine medical
procedures, where it clings to the inside and may clot blocking the
lumen. A blocked CVC lumen necessitates catheter replacement under
surgery which adds greatly to time, cost and patient mortality.
[0066] A catheter locking solution (CLS) is a volume of fluid
sufficient to fill the lumen of the catheter between medical
procedures. An optimal CLS will exert an antimicrobial and
anti-coagulation effect. Currently there are a number of
proprietary CLS formulations on the market that are designed to
achieve both prevention of blood clotting and an antimicrobicidal
effect. The selection of active ingredients to construct these CLS
formulations is constrained by the risk of accidental intra-venous
infusion. The void volume of a CVC may be as much as 3.0 ml or
more. In the event that a medical administrator forgot that a
locking solution had been inserted and accidentally added a second
dose, the first void volume would be infused directly into the
blood stream where it could, have serious toxicological
implications for the patient.
[0067] The formulations herein may be in the form of catheter
locking solutions, which can achieve optimal microbicidal effect in
less than 1 hour and have anti-clotting properties similar to
heparin without the use of that material in the formulation. The
catheter locking solutions preferably have a viscosity
approximating to that of whole blood, to minimize the potential for
dilution and mixing at the catheter tip. The desired viscosity can
be achieved using viscosity-enhancing agents.
[0068] Viscosity-enhancing agents routinely used in pharmaceutical
preparations include a wide range of hydrogels of natural or
synthetic origin. Included among these are derivatives of cellulose
such as carboxymethyl cellulose and hydroxyethyl cellulose. Other
natural polymers of plant origin include dextran, alginate, pectin,
guar gum and acacia gum; synthetic polymers include a range of
carbomers (acrylates/C10-30 alkyl acrylate cross polymers) and
Poloxamers (triblock copolymers of polyoxyethylene and
polyoxypropylene). For reasons of residual toxicity and metabolic
clearance, few of these are approved for routine systemic use in
human medicine. The Poloxamer, Pluronic F68 has been used in
parenteral nutrition formulations, but the naturally occurring
dextran has achieved more extensive use in surgery.
[0069] In the formulations of the invention, dextran is the
preferred viscosity-enhancing agent. Dextran is a naturally
occurring polysaccharide. A complex branched glucan of glucose
monomers, it is produced by many bacteria including Leuconstoc
species, and it has been used as an irrigant and anti-clotting
agent in micro-surgery. Dextran is available commercially in a
range of molecular weights ranging from 10 Kilo daltons (Kd) to 150
Kd. In the invention, 20 Kd to 60 Kd dextran is preferred. More
preferably, 40 Kd dextran is used to adjust the viscosity of a
membrane lipid/free fatty acid-based catheter locking, solution to
approximate to that of whole blood: 3.6-6.5 cP.
[0070] When 40 Kd dextran is used as viscosity-enhancing agent, its
concentration in formulations should preferably be from about 5% to
50% on a weight by volume basis, more preferably from about 10% to
40%, and more preferably still from about 15% to 30% weight by
volume based on the entire formulation.
[0071] Table 2 below provides a summary of the more common
proprietary CLS formulations currently in use:
TABLE-US-00002 TABLE 2 Proprietary CLS Formulations currently in
use or under development Known Active Proprietary Brand Ingredients
Reported Efficacy Duralock 47.6% Tri-sodium Anti-coagulant,
MedComp, Philadelphia citrate microbistatic not USA microbicidal
Zuragen 7% sodium citrate Anti-coagulant Ash Access 0.05% Methylene
Blue Microbicidal over Technologies 0.15% Methyl Parabens 12 hours
Lafayette, Indiana, 0.015% Propyl Parabens USA Taurolock 1.35%
Taurolidine Anti-coagulant Tauropharm AG, 4% sodium citrate
Microbicidal over Waldebuttelbrunn 12 hours Germany Canusal 0.9%
saline Anti-coagulant Wockhardt UK LTD 200 I.U. heparin/ml Not
microbicidal Wrexham Wales
[0072] Most of the lock solutions in use currently have relatively
low microbicidal effect: reduction in microbial viability of 3-4
logs in more than one hour exposure. As disclosed herein, a CLS
based on membrane lipid emulsified free fatty acids exhibits
superior antimicrobial effect eradicating greater than 6 logs in
less than 6 minutes. Optimal anti-coagulation effects are achieved
by modifying the ratio of membrane lipid and free fatty acid and
the optional use of a viscosity-enhancing agent eliminates
migration of blood into the catheter tip. Greater safety in the
event of accidental double locking is assured by the fact that the
components used in this invention are natural metabolites.
Antimicrobial Surface Coating
[0073] The compositions of this invention can be used to create
dual action anti-adhesion and antimicrobial surface coatings by
trapping a film of free fatty acid, on any animate or inanimate
surface, including but not limited to skin, plastic, rubber, metal
or glass, wherein they exert a microbicidal effect at the surface
in addition to repelling adhesion of microbial species.
[0074] Active antimicrobial surface coatings have particular
application in healthcare, for prevention of bio-film formation on
medical instruments and on catheter surfaces, and also as a surface
coating for work-stations, procedural trays and all patient contact
surfaces, including the hands of healthcare workers. Similarly,
active antimicrobial surface coatings have extensive application in
the food industry where they can be applied to food preparation and
packaging surfaces to minimize carriage of food borne pathogens
such as Salmonella, Campylobacter, Listeria and E. coli and as
demonstrated herein, they can also be applied to the surface of
food products, particularly post-slaughter meal, to eradicate these
pathogens at source.
[0075] Conventional surface antisepsis is achieved using an
antiseptic wipe which may contain triclosan, chlorhexidine,
quaternary ammonium compounds or a concentrated solution of
alcohol. However, the residual toxicity of many conventional
antiseptics limits their use on food and animate surfaces.
[0076] When used in surface coating applications, the composition
of the invention may be applied in a suitable pharmaceutically
acceptable delivery system, with or without other excipients.
Pharmaceutically acceptable delivery systems include, but are not
limited to, organic solvents designed to evaporate on application
leaving a dry residue, liquids, creams, gels, pastes, ointments,
powders or sprays and include combinations of these with insoluble
materials such as fibrous wipes.
Mucosal Fortification:
[0077] The mucus membranes of humans and animals are
characteristically moist surfaces at the interface of natural body
orifices, and the lining of the gastro-intestinal tract and the
genitalia, they include the eye, nose, inner ear, mouth,
naso-pharyngeal surfaces, trachea, bronchioli, esophagus, stomach,
large and small intestines, rectum, vagina and external labia, the
glans and the lining of the urinary tract. In addition to these
anatomically related surfaces that are common to humans and
animals, there are mucosal structures that are unique to particular
species such as the guttural pouch in equids.
[0078] Mucus membranes are covered by a layer of mucus, a viscous
hydrocolloid comprised mainly of mucins, a group of heavily
glycosylated high molecular weight proteins which act as a matrix
within which many other biologically active materials are dispersed
including secretory antibodies and components of the innate immune
system such as histatins and statherins in saliva. In addition to
hydration and lubrication, mucus is essential for prevention of
ingress of potential microbial pathogens and for prevention of
adhesion and colonization of the mucus membranes themselves.
[0079] Impairment of mucosal secretions and debilitation of the
integrity of the mucus itself may arise as a consequence of
disease, or as a side effect of medical and/or pharmaceutical
intervention or as a consequence of life-style. Where this happens,
those afflicted suffer from recurring mucosal infection including
but not limited to dental caries, gum-disease, oral thrush, yeast
vaginitis, bacterial vaginosis, enteritis and infectious colitis.
The complexity of mucus has defied all attempts to construct exact
replicas which may be used in replacement therapy. There are a
number of commercial substitutes, which are designed to relieve the
pre-dominant physical symptoms of oral dryness. Most of these are
based on hydro-gels such as carboxymethyl cellulose and a
composition of salts to affect buffering and re-mineralisation. One
is based on pig-gut mucin (Saliva Orthana, AS Pharma, Eastbourne,
UK), but none approximate to the natural adhesion inhibitory
aspects of saliva and mucus in general. The combined adhesion
inhibitory and microbicidal properties of the inventive
formulations, particularly tailored release formulations as
disclosed herein, offer superior mimicry of the antimicrobial
properties of natural MUCUS.
Topical Disinfection and Skin Care
[0080] The skin, hair and nails of mammals, being the external body
surfaces are subject to constant environmental contamination.
Healthy mammalian skin has intrinsic antimicobial properties based
on naturally occurring free fatty acids in the skin, and these may
be advantageously fortified by topical application of delipidised
membrane lipid-emulsified free fatty acids as disclosed herein.
[0081] Bacteria causing skin infection include, but are not limited
to, Staphylococcus aureus, Streptococus pyogenes and Pseudomonas
areruginosa, causing Impetigo, Folliculitis, Erysipilis, Cellulitis
and Necrotising Fasciitis. Propionibacteriium acne is the causative
agent of juvenile acne. Fungal skin infections are caused mainly by
Trichophyton, Microsporum and Epidermophyton species and the
diseases are known collectively as Tinnea (pedis, cruris, capitis,
corporis and unguinium) which include ringworm and nail infections.
Yeast including Candida albicans cause Intertrigo and Paronychia;
Malassezia furfur causes Tinnea versicolor (seborrheic dermatitis
and infectious dandruff) and it is also a common ailment in dogs'
ears. Enveloped viral infections include Herpes Simplex Type 1
affecting orofacial regions and type 2 affecting the genital
regions. Herpes zoster causes shingles and the poxvirus causes
Molluscum contagiosum. Superficial asymptomatic carriage of HIV,
SARS, Hepatitis, Swine flu, bird flu and many other zoonotic viral
infections may also be eliminated using the compositions
herein.
[0082] There is significant public concern about the increasing
incidence of hospital acquired infection (HAI). HAI include
infections caused by antibiotic resistant bacteria such as
Methicilin Resistant Staphylococcus aureus (MRSA), and Vancomycin
Resistant Enterococci (VRE) and other multi-drug resistant species
such as Clostridium difficile and other less well known
opportunistic pathogens including Pseudomonas and Candida.
[0083] It is well known that hand antisepsis is critical in
preventing cross-contamination between patients and patient care
staff. Most patient care establishments routinely use an alcohol
gel for hand antisepsis. Alcohol is an immediate and potent
microbicidal agent. It evaporates within seconds however, leaving
no persistent effect to protect against accidental contamination
that might happen immediately after evaporation. Membrane lipid
emulsions of free fatty acids may be tailored in a manner that
provides a sustained release of microbicidal effect and when
formulated with alcohol, the effect is both immediate and
persistent.
Food Safety
[0084] It is well known that many food animals harbour food borne
pathogens, primarily in their gut, but also on their skin from
contamination with faeces. Conventional antiseptics and antibiotics
are not suitable for liberal topical or systemic application to
animals before slaughter, or to their meat after slaughter. The
individual components that make up the products of this invention,
however, have the advantage of being used routinely in parenteral
nutrition; in separate constructs therefore they have been deemed
to be non-toxic, and their suitability for use topically on fresh
meat is disclosed herein.
[0085] Food borne pathogens include Salmonella, Escherichia,
Campylobacter and Clostridium species. The efficacy of the products
of this invention in vitro is demonstrated in the Examples. Methods
of eradication of food borne pathogens at primary processing of
meat include the proposed use of carcass washing with a suitable
antimicrobial agent. Other methods of application that are relevant
to food safety include the use of membrane lipid-emulsified free
fatty acids as surface coatings for food packaging using procedures
similar to those described for application of antimicrobial surface
coatings to medical devices.
Methods and Materials:
Emulsification Procedures
[0086] Free fatty acids greater than C6 are insoluble in aqueous
media. Membrane lipids are equally insoluble in water but may be
dispersed therein, and under appropriate conditions combinations of
free fatty acids and membrane lipid may be induced to form
emulsions in water. These are either oil-in-water or water-in-oil
emulsions, the latter having the higher relative concentration of
fatty acid by weight.
[0087] A water-in-oil (free fatty acid) emulsion may be prepared by
dissolving e.g. about 1 gram of membrane lipid, such as optionally
delipidised lecithin, in about 2 grams of free fatty acid. Stir for
about 10 minutes and then add about 2 grams of sterile distilled
water, agitate vigorously by shaking for 30 seconds and allow to
hydrate for 30 minutes. When fully hydrated the emulsion has a
thick creamy consistency which may be further diluted with up to 20
ml of water, added in 5 ml aliquots with intermediate agitation by
shaking. An excess of water added to this emulsion will cause it to
break and invert in part or in whole and become unsuitable for
further processing.
[0088] Oil-in-water emulsions are better suited to high water
content applications such as wound dressings, catheter locking
solutions and surface disinfection, as described hereinafter.
Methods of preparing oil-in-water emulsions are well known to those
skilled in the art and are disclosed in for example,
WO2009/072097.
[0089] A suitable example of an oil-in-water emulsion of free fatty
acid in membrane, lipid may be prepared by suspending a suitable
quantity of membrane lipid, such as optionally delipidised
lecithin, in sterile distilled water, and stirring this at room
temperature for about 30 minutes with a magnetic stirrer to ensure
even dispersion. Using a suitable dispersing device, the desire
amount of free fatty acid, such as caproic, or caprylic, or
pelargonic acid, is slowly added while vigorously agitating the
volume of dispersed membrane lipid. The amount of membrane lipid
(e.g. lecithin) is conveniently 0.4 g in 100 ml water, to which 0.5
g of free fatty acid can be added using e.g. a 5-ml syringe or
pipette. A laboratory homogenizer such as an Ultra-Turax Model T18
(IKA Works, Wilmington, N.C. 28405, USA) fitted with an S18N-19G
dispersing tool, operating at 6,000 to 8,000 RPM, is a preferred
method of agitation. The resulting emulsion is a thin white liquid
suspension of free fatty acid, which is stable and can be diluted
many-fold without de-stabilising the emulsion.
[0090] A similar procedure may be used to prepare an oil-in-water
emulsion of, for example, capric acid or undecylenic acid, provided
the constituents are first equilibrated at 37.degree. C., or above
their melting point. Once emulsified, the emulsion remains stable
on cooling below the melting point of the incorporated free fatty
acid and may be diluted further for use in formulation.
[0091] A membrane lipid emulsion of lauric acid may be prepared. in
a similar manner provided all constituents are first equilibrated
at 50.degree. C. or above the melting point of lauric acid, and
again this emulsion will remain stable on cooling and in further
dilution for use in formulation.
[0092] Free fatty acids have limited microbicidal effect at
temperatures below their individual melting point. This fact has
little significance for those acids with melting points below
normal physiological temperatures (37.degree. C.), except in
situations where such emulsions may be required to exhibit an
effect in hypothermic conditions.
[0093] Blends of high and low melting point free fatty acids have
depressed melting points due to the solvent effect of the lower
melting point constituent. A combination of lauric acid with a
melting point of 44.degree. C. and caproic acid with a melting
point of -3.4.degree. C. in a 50:50 ratio, will exhibit a combined
melting point of 26.degree. C., and the blend may be emulsified as
described above at temperatures above 26.degree. C.
[0094] Higher melting point free fatty acids may also be dissolved
in low melting point blends of essential oils or other neutral oils
to achieve a depression of melting point suitable for antimicrobial
use under physiological conditions. Essential oils are hydrophobic
constituents extracted from many different plants by distillation
or solvent extraction. They are widely used commercially as
perfumes and in fringe medical practice such as aromatherapy. Most
notable examples include oil of clove, orange, lemon, lavender,
juniper, and rose. Many of these are known to exhibit microbicidal
effect in their own right; oil of lemon balm for example is
recognized as being an effective viricidal agent and when used as a
solvent for higher melting point fatty acids such as lauric acid,
the combined microbicidal and viricidal properties provide expanded
utility in medical applications.
[0095] Oil of lemon balm will dissolve up to 40% by weight of
lauric acid at 20.degree. C. and the blend may be emulsified in
membrane lipid such as lecithin, as described above.
[0096] Oil-in-water emulsions of free fatty acids or blends thereof
may be prepared in concentrations of optionally de-lipidised
membrane lipid ranging from about 0.1% to 10% and emulsified free
fatty acid loadings of from 01 to 10%.: The loading may
conveniently be 4% membrane lipid such as lecithin, and 5% free
fatty acid or blend thereof. More preferably still a loading of
0.4% optionally de-lipidised lecithin and 0.5% free fatty acid or
blend thereof is used, which may be diluted further for use in
formulation.
[0097] The microbicidal efficacy of any membrane lipid emulsion of
free fatty acid according to the invention is amplified by
increasing surface area of the emulsified droplet relative surface
area is increased by decreasing the droplet size. Droplet size is a
function of the amount of energy imparted by the dispersing tool
during the emulsification procedure. Generally, finer droplets may
be produced using higher homogenization speeds and the use of an
ultrasonic probe adjacent to the emulsification head will also
facilitate smaller droplets and increased surface area.
De-Lipidisation of Membrane Lipids
[0098] De-lipidisation can be achieved by suspending the membrane
lipid or hydrolysed derivative thereof in a polar solvent, such as
an alcohol or ketone, at a concentration of about 10% w/v and
stirring for about 30 minutes during which time extraneous lipid is
dissolved. Membrane lipids are insoluble in polar solvents and will
readily settle out after stirring allowing the polar solvent with
its dissolved lipid to be decanted. Residual solvent is allowed to
evaporate in e.g. a fume hood. Suitable solvents include acetone
and ethanol.
Adhesion Assay:
[0099] A suitable model of microbial adhesion and inhibition of
adhesion is provided by the interaction between the yeast Candida
albicans and human Buccal Epithelial Cells freshly harvested from
the inside the cheek.
[0100] In the assay procedure, a standardized population of fresh
late log-phase Candida are exposed to a variable concentration of
test substance for 10 minutes, then combined with a fixed ratio of
buccal epithelial cells (BECs) for 60 minutes, during which time
yeast will adhere to the BECs to a greater or lesser degree
depending on the potency of the inhibitory substance used in
pre-treatment: After the adhesion period, the combined yeast and
BECs are diluted by a factor of 2.times. in sterile buffer and
agitated briefly (5 seconds) on a laboratory vortex. Wet mount
samples of the mix were examined under a microscope using
400.times. magnification. Yeast cells adhering to BECs are clearly
visible and enumeration of these is facilitated using dark field
conditions; the use of a Neubauer hemocytometer slide facilitates
counting. In total 100 BECs are evaluated and the total number of
yeast adhering to these is used as the sample count. The control is
equivalent to 100% adhesion and reduction in this number in
response to test item or protein blank is reported as percent
inhibition of adhesion.
[0101] Buccal epithelial cells are harvested from the inner cheek
mucosa of volunteers by rubbing a sterile tongue depressor in a
circular fashion and rinsing the collected cells into 5 ml of
sterile phosphate buffered saline(PBS). BECs should preferably be
collected in the morning before eating or brushing teeth. Donations
from 4-5 volunteers are needed to achieve five assay points.
[0102] BECs are pooled and counted using direct microscopic count
with a hemocytometer slide; a count of 500/ml (for example) may be
achieved. The pooled sample is centrifuged at 1,500 RPM for 3
minutes, and re-suspended in a volume of sterile PBS, calculated to
achieve a concentration of 500 BECs per ml. This concentrate was
divided into 2.5ml aliquots in 10 ml centrifuge tubes and
centrifuged again at 1,500 RPM for 3 minutes. The supernatant is
discarded and the cell pellets held on ice pending their use in the
rest of the assay.
[0103] The yeast used in these assays is a fresh clinical isolate
of Candida albicans; ATCC 10231 may be used as an alternative, but
all culture collection `Type` strains have lost some virulence and
do not adhere as well as fresh isolates.
[0104] Yeast cells are maintained on yeast extract peptone dextrose
(YEPD) agar, and a loop-full of a pure culture is used to inoculate
a 250 ml Erlenmeyer flask containing 100 ml of sterile YEPD broth.
The inoculated flasks are incubated overnight (10-14 hours) at
37.degree. C. in an orbital incubator at 100 RPM. YEPD is: 2% W/V
glucose, 1% W/V Yeast Extract, 1% W/V bacteriological peptone, Agar
where required at 1-2% W/V, all from Oxoid, UK.
[0105] The late log-phase cells are counted with a hemocytometer
slide and the concentration adjusted to 100.times. the test
concentration of BECs (40,000/ml in these examples), using 50 mM
sodium lactate buffer pH 4.5. 2.5 ml aliquots of the standardized
suspension of yeast are washed once by centrifugation with sodium
lactate buffer at 4,000 RPM and held as a pellet pending the assay
procedure as described below. A solution of test substance and
protein blank at the required concentration is prepared in 50/100
mM sodium lactate buffer pH 4.5 or sodium citrate buffer pH 4.5;
unless otherwise stated, citrate or lactate buffers on their own
were used as controls to determine full adhesion. Bovine serum
albumin (BSA) is used as a protein `blank`, Sigma-Aldrich A-7030;
St Louis, Mo., USA. The material is crude, "initial fractionation
by heat-shock" as non-heat shocked fractions may contain active
serum immunoglobulin which may contribute to the inhibition of
adhesion.
[0106] Washed yeast pellets from above are re-suspended in 2.5 ml
volumes of test, blank or control solutions and held at 37.degree.
C. for 10 minutes pre-treatment. Each 2.5 ml volume of test, blank
or control (with yeast in suspension) is then used to re-suspend a
pellet of washed BECs from above. The combined suspensions are then
incubated with gentle agitation at 37.degree. C. for 60 minutes.
The use of an orbital incubator at 50 RPM provides a suitable
environment.
[0107] After incubation, 2.5 ml of 50 mM sodium lactate buffer at
pH 4.5 is added to each of the test solutions mixed and subjected
to a 5 second pulse on a laboratory vortex. The purpose of the
final dilution and vortexing is to separate loosely adhering yeast
and yeast cells that are lying adjacent to, but not attached to,
BECs. Further dilution may be necessary to facilitate counting of
adherent yeast.
[0108] The assay may be performed as a pre-treatment of BECs by
reversing the order of re-suspension described above. BECs are
first re-suspended in 2.5 ml volumes of control buffer, test or
protein blank, held at 37.degree. C. for 10 minutes and then used
to re-suspend a pellet of washed yeast and the procedure completed
as described above.
Assay of Microbicidal/Microbistatic Effect:
[0109] The assay is a standard microbiological suspension test
wherein known concentrations of late log phase bacteria, yeast or
fungi are inoculated into a fixed, volume or weight of a test
substance, blank or control. After a set period of time a
neutralizing solution is added to stop the antimicrobial effect and
the residual population of viable microorganisms is enumerated by
serial dilution and plate counting. The counting procedure is a
standard and basic microbiological procedure for enumerating viable
microorganisms and will be well known to those skilled in the
art.
[0110] In its generic form the method requires inoculation of 1
gram or 1 ml of test sample with 0.1 ml of 18 hour (late log phase)
followed by vigorous agitation to mix. After the pre-determined
exposure time has elapsed, 9.0 ml of neutralizing buffer is added
and mixed. This has the effect of stopping the microbicidal effect
which allows reliable estimates to be made of the percentage kill
achieved by a particular test sample in the period between
inoculation and neutralization. Typically exposure periods will
range from 30 seconds up to 30 minutes and may progress to several
hours where that time period is required to measure the effect. In
order to enumerate residual viable cells and from that to compute
percentage kill, serial dilutions and plate counts are carried out
on the 10 ml sample plus neutralizing buffer.
[0111] In the assays herein, stocks of bacteria, yeast and fungi
are routinely stored on beads in 50% glycerol at -80.degree. C.
When required for viability/microbicidal assay, small aliquots from
these stocks are spread on an appropriate nutrient agar, grown and
sub-cultured to ensure purity. Where broth cultures are required,
250 ml Erlenmeyer flasks containing 100 ml of broth are inoculated
with a transfer loop from pure agar cultures and incubated under
constant agitation in a rotary incubator at 37.degree. C.
[0112] Bacteria are routinely cultured using brain heart infusion
(BHI) broth and agar or tryptone soya agar or broth (TSB), both of
which may be acquired commercially from Oxoid, UK. TSB has the
following constituents: 1.5% W/W tryptone (pancreatic digest of
casein), 0.5% W/W peptone (papaic digest of soybean meal), 0.5% W/W
sodium chloride, and agar at 1.5% W/W when required as a solid
medium. Yeast are grown on YEPD agar or broth (see adhesion assay
method hereinabove).
[0113] The diluting and neutralizing buffers used in the present
method are phosphate buffered saline (PBS), containing 137 mM
sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate, to
which is added 3% polysorbate Tween 80 (anionic surfactant), 0.3%
lecithin, and 0.5% histidine as neutralising agents. These
`neutralising` agents are those prescribed under EU Guidelines for
ISO certification of microbicidal efficacy and were validated as
suitable for neutralizing free fatty acids and chorhexidine at the
concentrations used herein.
[0114] Some microbial species are extremely difficult to culture,
requiring specialized media and/or anaerobic conditions which are
not easily achieved in liquid culture. Clostridium difficile for
example is an anaerobe and also aero-intolerant dying off quickly
in the presence of oxygen. Fungal pathogens such as the
Trychophyton species grow in hyphal mode and cannot be enumerated
using the standard serial dilution procedure. In order to evaluate
the microbicidal effect of the inventive product against these
species, agar dilution techniques were used and plate culture
evaluated to find the minimum inhibitory concentration, i.e. that
concentration of product above which no growth was observed.
[0115] The technique requires preparation of 10.times.
concentrations of the test formulation for dilution in 9 volumes of
agar. A 4 ml aliquot of this 10.times. concentrate was added to 16
ml of cooled sterile agar and dispensed to a Petri dish--this
represented the full strength (100%) formulation in the agar
medium, which may then be expressed as the percentage concentration
of microbicidal fatty acid in agar. Further dilutions of the
10.times. concentrate with sterile distilled water and the use of 4
ml aliquots of these dilutions in 16 ml of cooled agar allowed the
preparation of a series of decreasing concentrations of the product
in agar. The test organisms were inoculated onto these agars with a
sterile loop and the minimum inhibitory concentration was
determined as the lowest dilution of test item at which no growth
was detectable.
Inhibition of Biofilm Formation:
[0116] Bio-film is an attachment and accretion of planktonic
bacteria, yeast and/or fungi to solid surfaces. The attachment is
usually facilitated through exo-polysaccharides secreted by the
microorganisms and studies suggest that it happens more easily on
hydrophobic surfaces, such as plastic, and less easily on
hydrophilic surfaces such as steel. The formation of bio-film is
highly significant in medical science, particularly its formation
on the surface of indwelling catheters, where it can be the source
of catheter-related blood stream infections. Many different
microbial species are capable of forming bio-film, in medicine.
However, those of greatest significance are Staphylococcus aureus,
Staphylococcus epidermidis, Enterococcus faecalis, Escherichia
coli, Klebsiella pneumoniae, Pseudominas aeruginosa and the yeast,
Candida albicans.
[0117] The model of biofilm formation used herein is based on the
method of Christensen et al. J. Clin. Microbiol. 22: 996-1006,
where growth of a selected organism and its biofilm formation in
microtitre plate wells is measured by staining with crystal violet
and measuring the intensity (Optical Density) of the stained
biofilm in a microtitre plate reader at 570 nm wavelength. The
intensity of the stain is a measure of the extent of biofilm
formation and its reduction in the presence of inhibitory
substances is evaluated on the basis of reduction of the stain
intensity.
[0118] The organism used in this assay is Staphylococcus aureus
RN4220, a restriction deficient variant derived originally from
NCTC 8325 known for its avidity in biofilm formation, particularly
in the presence of excess levels of sodium chloride which is added
to the culture media to promote that feature, culture media is 3.7%
BHI from Oxoid UK to which 4% W/V NaCl is added.
[0119] Nunc microtitre plate wells (Nunc International, Rosskilde,
Denmark) are coated with the test sample and 1:100 dilutions of
late log phase bacteria in fresh saline supplemented BHI are
dispensed to each test well and incubated for 24 hours to
facilitate biofilm formation.
[0120] On completion of the incubation period, the weirs are rinsed
three times with sterile distilled water, and dried for one hour at
60.degree. C. to fix the adhering biofilm. Staining is achieved
using a 0.4% solution) of crystal violet which is added to each
well and agitated thereby rocking the plate for 4 minutes. Excess
dye is removed and the plates are rinsed three times with sterile
distilled water and dried. When dry, the intensity of the stain in
each well is measured using a ASYS Hitech UVM-340 plate reader at
570 nm (ASYS, Eugendorf, Germany).
Manipulation of Hemostasis: Clotting/Anti-Coagulation of Blood
[0121] Hemostasis is the inherent ability of the blood to react to
traumatic damage by forming a clot to plug the wound and so prevent
excessive loss of blood and facilitate tissue repair and healing of
the wound. Hemostasis also refers to the inherent ability of the
blood to remain liquid in undamaged blood vessels and so fulfill
its primary physiological transport functions. Both of these
hemostatic aspects can become defective in disease and both can be
overwhelmed by traumatic damage, either accidentally or by default
during surgical intervention.
[0122] The ability to manipulate the hemostatic mechanism by either
amplifying the rate of clotting to prevent blood loss after
accidental trauma or to prevent clotting during surgical repair
and/or to prevent clot formation on, or in, surgically inserted
medical instruments including catheters, is of great importance in
medicine. To measure the rate of blood clotting herein, use is made
of a whole blood clotting meter, the Hemochron Signature 11,
manufactured by ITC, International Technidyne Corporation, Edison,
N.J., USA. The meter uses customized cuvettes into which one drop
of fresh blood may be added and the time taken for clot formation
is measured optically and reported electronically by the
device.
[0123] In the assay herein, fresh sheep blood was used, which was
drawn from a jugular vein using an 18 gauge-needle and suitable
volume syringe. Fresh blood samples are immediately mixed with a
volume of test material at varying concentrations and one drop
inserted in the cuvette. Sterile phosphate buffered saline is used
as a blank to obviate the effects of dilution and heparin is used
as a control anti-coagulant. Measurement of the anti-clotting
effects of the products of the invention are evident in seconds
compared to untreated whole blood, and compared to the
anti-clotting effect of heparin and other proprietary anti-clotting
(anti-coagulation) products. In optimal configuration the products
of the invention achieve an anti-coagulation effect lasting in
excess of 1,000 seconds, which is comparable to 5,000 I.U of
heparin under similar test conditions.
[0124] Measurement of accelerated clotting time (amplified
clotting) is not possible using the Hemochron device, as in most
cases the products of the invention achieve clotting in under 60
seconds, which is `Off scale` for the meter.
[0125] Comparative measurement of accelerated clotting times is
achieved using appropriate volumes of fresh blood mixed with a
volume of test material, where the combined volumes totalled 5 ml
in each case in a 15 ml graduated Greiner centrifuge tube (Greiner
Bio-one, North Carolina, USA). Immediately after addition of fresh
blood, the tubes are inverted twice to mix and allowed to stand
without further agitation. Clot formation is visibly apparent when
the tubes are tilted slightly, and once a clot formed its integrity
was such that it would remain suspended when the tubes were
inverted. In each test a 5 ml sample of fresh whole blood
(undiluted) is used as a control.
Analysis of Membrane Lipid and Free Fatty Acid
[0126] Routine analytical High Performance Liquid Chromatography is
a suitable method of analyzing the individual membrane lipid
components in a composition such as lecithin. The procedure is well
known to those skilled in the art of analytical chromatography. A
Waters 2420 ELSD HPLC system from Waters Corporation, Milford,
Mass., USA is used herein. A Symmetry C8 column (3.0.times.150 mm,
5 micron column is suitable with a non-linear gradient of 82%
methanol in water containing 0.1% tri-fluoroacetic acid and gives
adequate separation of membrane lipid components.
Mammalian Cell Viability.
[0127] The reduction in viability of mammalian cells in the
presence of membrane lipid emulsified free fatty acids was
evaluated using Raji B lymphocytes grown in RPMI 1640 media
containing 10% fetal calf serum and Gibco Penstrep 15140 antibiotic
supplement. Mature cells are harvested by centrifugation at 1,000
RPM for 3 minutes and re-suspended in RPMI 1640 without supplements
for test purposes. Toxicity is assessed by evaluating uptake of
trypan blue dye by dead cells using an Invitrogen Countess
Automated Cell Counter (Invitrogen Inc, Carlsbad, Calif., USA). The
procedure involves exposing a population of Raji B cells to the
test solution by mixing 100 .mu.l of test and cell suspension in a
microtitre plate well. After a pre-determined period of exposure,
10 .mu.l of cell and test mix are combined with 10 .mu.l of 0.4%
trypan blue and 10 .mu.l of this added to the chamber of a cell
cytometer cuvette and evaluated in the cell counter described
above. Results are provided as a total cell count and numbers of
these that are alive or dead are based on uptake or exclusion of
the dye; a percentage viability is computed automatically.
BRIEF DESCRIPTION OF THE DRAWINGS
[0128] FIG. 1 illustrates the antimicrobial inhibitory effect of
blood serum and the counteraction of this effect in combination
with salts of organic acids, as described in Example 3.
[0129] FIG. 2 illustrates blood clotting and anti-dotting
properties of varying components of a product according to the
invention, as described in Example 5.
[0130] FIG. 3 illustrates anti-clotting properties of a catheter
locking solution according to the invention, as described in
Example 7.
[0131] FIG. 4 illustrates comparative microbicidal potency of a
catheter locking solution prepared according to the invention and
compared with alternative commercial products, as described in
Example 7.
[0132] FIG. 5 illustrates the biofilm inhibitory properties of a
product according to the invention, as described in Example 8.
[0133] FIG. 6 illustrates the variable release of microbicidal
effect achieved using different membrane lipids and caprylic acid
products according to the invention, as described in Example 9.
[0134] FIG. 7 illustrates reduction in viability of Candida
albicans in response to tailored release characteristics of
products according to the invention, as described in Example
10.
[0135] FIG. 8 illustrates inhibition of adhesion of Candida
albicans by tailored release products according to the invention,
as described in Example 10.
[0136] FIG. 9 illustrates the eradication of Staphylococcus aureus
and Streptococcus pyogenes from an ex-vivo wound model, as
described in Example 12.
[0137] FIG. 10 is a comparison of the ex-vivo efficacy of a product
of the invention in sodium citrate with conventional wound care
antimicrobials, chlorhexidine and silver sulfadiazine, as described
in Example 12.
[0138] FIG. 11 illustrates comparative eradication of established
biofilm and the superior efficacy of a product of the invention
over existing commercially available catheter locking solutions, as
described in Example 13.
[0139] The practical applications and benefits of this invention
are illustrated further in the following Examples. Unless otherwise
stated, the membrane lipids used in the Examples are delipidised
and contain less than 3% conjugated extraneous lipid material as
shown by HPLC analysis as described in the Methods.
EXAMPLE 1
Preparation of Adhesion Inhibitory Compositions of Membrane
Lipids
[0140] The following membrane lipids were purchased from Sigma
Aldrich, Poole, Dorset, UK.
TABLE-US-00003 TABLE 3 Membrane lipids Original Membrane lipid
Compound/Component source Code Phospholipid Crude Lecithin Soya
Bean LC Phospholipid Phosphatidylcholine Soya Bean PTC Phospholipid
Phosphatidylethanolamine Soya Bean PTE Phospholipid
Phosphatidylglycerol Soya Bean PTG Phospholipid
phosphatidylinositol Soya Bean PTI Phospholipid Phosphatidylserine
Soya Bean PTS Sphingolipid Ceramide Bovine Brain C Sphingolipid
Sphingomyelin Bovine Brain SGM Glycoglycerolipid Mono-galactosyl
Wheat flour MGDG diglyceride Cholesterol Lanosterol Sheep wool
L
[0141] Crude lecithin was purchased from GR Lane Ltd, Gloucester,
UK.
[0142] Aqueous dispersions of each of the above membrane lipids
were prepared by suspending 1.0 grams in 100 ml of sterile
distilled water (10 mg/ml) and agitating therein for 60 minutes
with the aid of a magnetic stirrer. Dispersion of ceramide,
sphingomyelin and lanosterol was achieved with the aid of
sonication and stirring, while dispersion of all other membrane
lipids was relatively easily achieved with stirring alone.
Phospholipid and glycoglycerolipid dispersions were relatively
stable, but others had a tendency to separate on standing and so
all, dispersions were subjected to 30 seconds homogenization using
a laboratory homogenizer immediately prior to, use in the adhesion
inhibitory assay. A suitable homogeniser is an Ultra-Turax Model
T18 (IKA Works, Wilmington, N.C. 28405, USA) fitted with an
S18N-19G dispersing tool, operating at 6,000 to 8,000 RPM.
[0143] The adhesion inhibitory properties of each phospholipid were
tested as described in the methods at concentrations of 10, 5, and
2.5 mg/ml; the lower dilutions were achieved by diluting the
original dispersion with sterile distilled water. Due to logistical
limitations of collecting sufficient buccal epithelial cells at any
one time, the assays were conducted in blocks of five individual
membrane lipids on separate days, and the results shown in Table 4
are a composite of these; the Control is zero concentration of test
item and Bovine Serum Albumin (BSA) is used as a protein blank.
TABLE-US-00004 TABLE 4 Candida albicans per 100 Buccal Epithelial
Cell Item % inhibition Code 0 mg/ml 2.5 mg/ml 5.0. mg/ml 10 mg/ml
at 2.5 mg/ml LC 540 387 291 92 28 PTC 540 127 59 0 76 PTE 496 167
74 0 66 PTG 496 114 62 0 77 PTI 519 201 87 0 61 PTS 519 233 102 39
55 C 484 363 282 109 25 SGM 484 379 277 126 21 MGDG 540 261 128 66
51 L 519 306 236 133 41 BSA 496 400 379 303 19
[0144] In preliminary evaluation of crude lecithin from various
sources (egg yolk, soya and sunflower), it was noted that the
adhesion inhibitory properties varied considerably, despite the
reported purity being approximately the same. It was reasoned that
the purchased material might contain extraneous fat or
tri-glyceride and to remove this, the original material was
suspended in acetone at 10% W/V and stirred for 30 minutes after
which the insoluble membrane lipid was allowed to settle and the
acetone decanted. The `de-lipidised` lecithin (DLL) was dried in a
fume hood, and its adhesion inhibitory properties evaluated as
previously described. The remaining membrane lipids listed in Table
4 were delipidised in the same manner. The delipidised membrane
lipids were analysed by HPLC as described in the Methods, and all
were shown to contain less than 3% conjugated extraneous lipid
material. The results are shown in Table 5 below, including the
incremental increase in adhesion inhibitory effect achieved by
de-lipidisation of individual membrane lipids.
TABLE-US-00005 TABLE 5 Candida albicans per 100 Buccal Epithelial
Cell Item % inhibition Code 0 mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml at
2.5 mg/ml LC 540 387 291 92 28 DLL 489 138 48 0 71 (+43) DL PTC 508
132 0 0 84 (+8) DL PTE 515 139 0 0 73 (+7) DL PTG 474 57 0 0 88
(+11) DL PTI 519 68 0 0 77 (+16) DL PTS 519 145 62 0 72 (17) DL C
493 305 118 41 38 (+13) DL SGM 511 301 133 44 41 (+20) DL MGDG 516
206 116 0 60 (+9) DL L 536 247 91 49 54 (+13) DL BSA 513 431 393
329 16 (-3)
[0145] Solvent de-lipidisation increases the adhesion inhibitory
properties of membrane lipids, especially lecithin by a factor of
2.5, bringing it in line with that of its individual components:
PTC, PTE, PTG, PTI and PTS. Further de-lipidisation of the
individual components of lecithin achieves further improvement in
the adhesion inhibitory properties of these, although less dramatic
compared to the effect achieved in de-lipidising crude lecithin. It
is assumed here that the commercial process of isolation used to
extract individual components of lecithin results in almost
complete de-lipidisation, hence the optimal inhibitor effect of
these relative to the `crude` lecithin and the smaller incremental
effect achieved by additional de-lipidisation.
[0146] In many of the applications for the product of this
invention the adhesion inhibitory substance will be in with blood
serum, mucus and other body fluids. It has been discovered that
de-lipidised lecithin and most of its constituents lose their
adhesion inhibitory properties in the presence of Bovine Serum
Albumin (BSA). It has also been discovered however, that adding an
organic acid salt counteracts this negative effect and restores
most of the adhesion inhibitory properties to the combined membrane
lipid and BSA.
[0147] In this example, 100 mM solutions of sodium lactate and
sodium citrate were used. The salts were prepared first as 200 mM
solutions and the pH adjusted to 4.5, and aliquots of both were
used to prepare 1% solutions of bovine serum albumin (i.e. 1% BSA
in 200 mM sodium salt of citrate or lactate at pH 4.5). The salt
solutions were used to dilute a 1% suspension of de-lipidised
lecithin (DLL) and a 1% suspension of phosphatidyl choline (PTC),
to achieve preparations of 0.5% DLL and 0.5% PTC in 100 mM sodium
lactate/sodium citrate at pH 4.5 with 5% BSA in each. A further
dilution of the membrane lipid suspension to 0.5% with water and
the use of this in halving dilutions of salt solution containing
0.5% BSA gave a composition of 0.25% membrane lipid in 100 mM salt
with 0.25% BSA. Similar procedures were used to prepare 0.25% and
0.5% solutions of BSA in water and in 100 mM organic salt solution:
the, results are presented in Table 6.
TABLE-US-00006 TABLE 6 Candida albicans per 100 Buccal Epithelial
Cell Item % inhibition Code 0 mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml at
5 mg/ml LC: water 540 387 291 92 46 DLL: water 489 138 48 0 90 DLL
+ BSA 396 320 280 ND 30 water DLL + BSA 396 290 144 ND 63 Sod'
Citrate DLL + BSA 396 340 164 ND 58 Sod' Lactate PTC: water 540 127
59 0 90 PTC + BSA 474 392 344 ND 28 water PTC + BSA 474 273 162 ND
66 Sod' citrate PTC + BSA 474 291 138 ND 70 Sod' Lactate BSA 474
393 369 ND 22 Sod' citrate BSA 474 408 372 ND 21 Sod' Lactate BSA:
water 496 438 400 362 12
[0148] In this Example, with the exception of crude lecithin, all
of the membrane phospholipids tested were shown to have superior
inhibitory properties preventing Candida albicans adhesion to
Buccal Epithelial Cells. When de-lipidised with acetone, crude
lecithin is as inhibitory as its main membrane phospholipid
Components. In combination with bovine serum albumin, the adhesion
inhibitory property is eradicated, but it can be restored in the
presence of 100 mM solutions of organic acid salts at acid pH.
EXAMPLE 2
Preparation and Testing of Microbicidal Combinations of Membrane
Lipid and Free Fatty Acid
[0149] The microbicidal effect of 0.5% caprylic acid in 0.4%
de-lipidised lecithin, prepared as described in the Methods,
against a range of microbial species may be demonstrated using the
microbicidal suspension test described in the Methods. The potency
of this material is such that complete eradication of an inoculum
in excess of 6 logs may be anticipated in less than 6 minutes. Gram
negative species are slightly more resistant than gram positives,
particularly those known to be slime producers, such as Escherichia
coli and Pseudomonas fluorescens, where it is though that the slime
layer protects from contact with the fatty acid for some additional
minutes.
[0150] Table 7 below illustrates the reduction in viability of a
range of bacteria and yeast. Because the inoculums vary, the latest
time to achieve 50% in viability of the inoculum is provided in the
last column (L. T. 50%), as a comparative measure of potency
against that particular species.
TABLE-US-00007 TABLE 7 Microbicidal Effect: 0.5% Caprylic in 0.4%
DLL Exposure Time L.T. Organism Gram 0 60 120 180 240 300 360 50%
Staph aureus RN4220 +ve 8.53 5.22 3.71 1.61 0 0 0 <120 Staph
epidermidis +ve 8.27 4.96 2.67 0 0 0 0 <120 NCTC 11047 Strep
pyogenes +ve 7.91 5.32 3.33 1.49 0 0 0 <120 NCTC 8198 Strep
faecalis +ve 8.11 4.98 2.54 1.66 0 0 0 <120 NCTC 12697 E. coli
-ve 8.43 7.17 6.2 5.28 3.64 1.79 0 <240 ATCC 11698 Salmonella
typhimurium -ve 7.76 5.94 3.52 2.27 0 0 0 <120 NCTC 74
Klebsiella aerogenes -ve 6.93 5.15 3.37 1.99 0 0 0 <120 NCTC
9528 Proteus mirabilis C.I. -ve 7.41 6.61 5.89 3.75 1.59 0 0
<240 Enterobacter Cloacae C.I. -ve 8.62 6.54 4.94 2.32 0 0 0
<180 Pseudomanas aeruginosa -ve 8.33 7.13 5.48 4.78 2.92 1.23 0
<240 ATCC 27853 Pseudomanas fluorescens -ve 7.29 6.42 5.13 3.63
1.29 0 0 <240 NCTC 10038 Candida albicans C.I. Yeast 6.86 4.39
2.96 0 0 0 0 <120 Candida glabrata Yeast 6.74 4.19 2.23 0 0 0 0
<120 NCPF 8750 Crypto' neoformans C.I. Yeast 5.97 4.11 2.96 0 0
0 <120 Note C.I = Clinical Isolate Note: L.T 50% is Latest Time
to achieve 50% reduction in viability in this test
[0151] The numerical data presented are log numbers where the
integer is the log and the decimal the colony count at that log:
6.3.times.10.sup.5 for example is presented as 5.63 and this
convention will be used throughout this document.
[0152] For reasons of difficulty in culturing or because of special
growth requirements the potency of the formulation against
anaerobes is determined by the Minimum Inhibitory Concentration
procedure as described in the Methods, and the results are
presented in Table 8.
TABLE-US-00008 TABLE 8 Minimum Inhibitory Concentration of 0.5%
Caprylic in 0.4% DLL against anaerobes and fungal pathogens using
agar dilution technique: MIC value is the % of Caprylic acid in the
test plate Organism Notes MIC Clostridium perfringens G -ve
anaerobe >0.1 ATCC 43150 Clostridium difficile G -ve anaerobe
>0.1 ATCC 43598 (aero intolerant) Bacterioides fragilis G -ve
obligate anaerobe >0.2 ATCC 43859 Fusobacterium nucleatum G -ve
anaerobe: >0.3 NCTC 10652 3 weeks Columbia blood agar
Desulfovibrio desulfuricans G -ve anaerobe >0.1 Porphyromonas
gingivalis G -ve obligate anaerobe >0.1 Campylobacter jejunii G
-ve microaerophilic >0.075 Actinobacillus G -ve microaerophilic
>0.1 actinomycetemcomitans Corynebacterium diphtheriae G + ve
facultative anaerobe >0.1 Treponema denticola Obligate anaerobe
>0.2 Mycobacterium tuberculosis Aerobic acid fast bacillus
>0.2 6 weeks on Lowenstein-jensen medium
EXAMPLE 3
Membrane Lipid Antimicrobials in Blood Contact Applications
[0153] In common with the negative effect on adhesion inhibitory
properties reported in Example 1, the presence of bovine serum
albumin also impacts on the microbicidal effect. However as in
Example 1, this can be overcome by incorporation of an organic acid
salt, at acid pH.
[0154] An oil-in-water emulsion of 0.5% caprylic acid in 0.4%
de-lipidised lecithin is used in this Example. 200 mM solutions of
glycolic, acetic, lactic, and citric acids were adjusted to pH 4.5
using 200 mM sodium hydroxide. 10% W/V solutions of Bovine Serum
Albumin were prepared in each of the organic acid salts and in
water as a control. 5 ml aliquots of the 0.5% caprylic emulsion
were added to 5 ml aliquots of each salt solution with and without
BSA, and in water with and without BSA, resulting in dispersions of
0.25% caprylic emulsion in 0.2% de-lipidised lecithin dispersed in
100 mM salt solution at pH 4.5 in each test sample.
[0155] E. coli was selected for this Example as it has been
demonstrated to be one of the most resistant species. The bacterium
was cultured in Brain Heart Infusion broth for 18 hours at
37.degree. C. 10 ml fractions of each test sample were inoculated
at time zero with 1.1 ml of the overnight culture, and 1.0 ml
samples removed from this at 3 minute intervals for residual
viability determination using serial dilution and plate counting
procedures.
TABLE-US-00009 TABLE 9 Antimicrobial effect is impaired by blood
components and re-instated by combination with salts of organic
acids 0.25% Caprylic in 0.2% De-lipidised Lecithin +/- 5% Bovine
Seum Albumin kill 0 3 6 9 12 15 18 21 24 27 30 time Water 8.37 4.97
2.23 0 0 0 0 0 0 0 0 9 Water + BSA 8.37 7.51 6.42 5.98 5.17 4.93
3.72 3.33 2.21 1.49 0 30 Glycolate 8.37 5.25 2.68 0 0 0 0 0 0 0 0 9
Glycolate + BSA 8.37 6.62 5.35 3.79 2.41 1.56 0 0 0 0 0 18 Acetate
8.37 5.94 4.21 1.28 0 0 0 0 0 0 0 12 Acetate + BSA 8.37 7.41 6.93
5.24 4.53 3.47 2.18 1.34 0 0 0 24 Citrate 8.37 4.9 2.7 0 0 0 0 0 0
0 0 12 Citrate + BSA 8.37 6.2 4.5 1.93 0 0 0 0 0 0 0 12 Lactate
8.37 5.29 3.19 0 0 0 0 0 0 0 0 9 Lactate + BSA 8.37 7.1 4.9 1.8 0 0
0 0 0 0 0 12
[0156] In a water diluent the effect of 5% W/V BSA is to reduce
potency of 0.25% caprylic acid by a factor of 3. Kill Time is
extended from 9 minutes to 27. In combination with 100 mM sodium
salts of citrate and lactate acids at pH 4.5, the kill time is 12
minutes, glycolate is 18 minutes and acetate is 24. The results of
combination with citrate and lactate acid salts with and without
BSA are presented in Table 9 above and illustrated in FIG. 1.
EXAMPLE 4
Use in Food Safety
[0157] In this Example 1 cm.sup.3 sections of fresh beef were
deliberately contaminated with late log phase cultures of
Salmonella enterica and Escherichia coli O157:H7 at room
temperature and allowed to adhere there for 60 minutes.
Confirmation of contamination was obtained by mechanically
macerating the meat sections in sterile phosphate buffered saline
(PBS) and enumeration by serial dilution and plate counting.
[0158] A suitable but not optimal carcass wash was prepared using
0.5% caprylic acid in 0.4% de-lipidised lecithin and diluting that
.times.2 with 200 mM sodium citrate at pH 4.5 as described in
Example 3. The wash was sprayed onto contaminated sections of fresh
beef, and the antimicrobial effect evaluated by mechanical
maceration, serial dilution and plate counting. The results are
presented in Table 10 below:
TABLE-US-00010 TABLE 10 Reduction in Viability of Pathogens on
Fresh Beef Time Escherichia coli Salmonella enterica minutes
Untreated Treated Untreated Treated 0 7.14 6.96 6.91 6.61 30 6.98
5.61 6.37 4.59 60 6.56 3.89 6.71 7.73 90 6.82 2.19 6.52 1.29 120
6.69 ND 6.28 ND Note: ND = Not detected
[0159] It should be appreciated that the rate of kill on a porous
surface such as meat is extended due to the nature of the surface
and the time required to permeate it.
EXAMPLE 5
Use of Membrane Lipids to Manipulate Blood Clotting Time
[0160] As described in this Example the membrane lipid products
used may be tailored to affect the rate of blood clotting in
addition to contributing a significant antimicrobial effect.
[0161] Rate of blood clotting was determined using freshly
aspirated sheep blood and apparatus and procedures described in the
Methods section. An activated blood clotting meter was used to
measure anti-clotting effects and a visual tube comparison to
evaluate reduced clotting times.
[0162] It was unexpectedly discovered that an aqueous dispersion of
de-lipidised lecithin (DLL) will amplify 2-5 the rate of blood
clotting in a concentration dependent manner. Varying
concentrations of dispersions of DLL were prepared by suspending
the required weight in a volume of water, stirring for 30 minutes
to hydrate and homogenizing the suspension with a laboratory
homogenizer.
[0163] Blood clotting and/or anti-clotting effects were assessed
using a 20% ratio of test item to fresh blood: in practice 4.0 ml
of fresh sheep blood was added to tubes containing 1.0 ml of test
item, inverted twice to mix and then either left standing for
visual assessment of reduced clotting time or a single drop was
applied to the cuvette of an activated blood clotting apparatus for
assessment of anti-clotting effect (extended time to clot
formation).
[0164] As the concentration of DLL in the test item increases, the
observed rate of blood clotting increases, i.e. time to clot
formation decreases from 360 seconds for whole blood to
approximately 60 seconds or less at DLL concentrations in excess of
1%.
[0165] The addition of caprylic acid by emulsification in DLL will
also amplify the dotting effect up to a point Where the
concentration by weight equals or slightly exceeds the
concentration of DLL by weight. At 0.5% DLL on its own, the
clotting time is approximately 125 seconds. Addition of emulsified
caprylic acid up to 0.5% does not significantly affect the dotting
time. Between 0.5% and 0.75% caprylic acid there is a further
depression of clotting time from 125 seconds to approximately 40
seconds (70% less). Thereafter, however, the anti-clotting effect
is reversed and clotting time increases with increasing
concentration of caprylic acid.
[0166] When the concentration of DLL is reduced to 0.25% with
increasing concentration of caprylic acid, there is no significant
reduction of clotting over and above that attributable to DLL on
its own. In fact, as the concentration of caprylic increases to
between 0.75% and 1.0%, the clotting time is restored to normal
(360 seconds). Further increasing the relative ratio of caprylic
acid in 0.25% DLL, has an anti-clotting effect.
[0167] Using an emulsion of 0.25% caprylic acid in 0.25% DLL as a
standard (STD), it can also be observed that addition of increasing
concentrations of sodium citrate salts at pH 4.5 suppresses the
anti-clotting effect of DLL in combination with caprylic acid. At
0.5% concentration, the anti-dotting effect of 0.25% caprylic acid
in 0.25% DLL has been suppressed completely and clotting time has
been restored to above normal (400 seconds). A further increase of
sodium citrate in this composition has a progressively increasing
anti-clotting effect--up to 600 seconds at 2% sodium citrate in
0.25% caprylic emulsified in 0.25% DLL. The results are illustrated
in FIG. 2.
EXAMPLE 6
Use of Antimicrobial Membrane Lipids in Amplified Clotting for
Wound Care
[0168] The combined antimicrobial and clot forming capability of
the products of the invention are demonstrated in this Example. A
suitable, example of an antimicrobial membrane lipid preparation
with enhanced clot forming properties may be selected from the
combinations prepared in Example 3. A 0.5% dispersion of DLL in
sterile distilled water with 1.0% caprylic acid was used here. It
will be noted that no organic acid salt is included in this example
and further noted that the absence of such reduces the
antimicrobial potency but does not limit the clotting effect. The
use of a relatively high caprylic acid load compensates for the
interference of blood components with the antimicrobial effect.
[0169] A bacterial inoculum of E. coli was grown in Brain Heart
Infusion broth for 18 hours and a 10ml volume of this was
sedimented by centrifugation at 4,000RPM for 10 minutes, the pellet
was re-suspended in 1.0 ml of the supernatant (concentration
.times.10).
[0170] Two 4.0 ml samples of fresh blood were dispensed to two 15
ml Greiner centrifuge tubes and both were inoculated with 0.1 ml of
concentrated E. coli suspension: a blank comprising 5.0 ml of 5%
Bovine Serum Albumin in sterile distilled water was inoculated at
the same time.
[0171] A 1.0 ml of volume of 4001. U. heparin and 500 I.U.
streptokinase in water was added to the first tube, and 1.0 ml of
test preparation to the second. Both tubes were mixed by inversion
and incubated at 37.degree. C. for 45 minutes. The tube containing
the test preparation clotted in approximately 60 seconds; no clot
was detectable in the heparin/streptokinase tube after 45
minutes.
[0172] At the end of the 45 minute incubation a 1.0 ml of volume of
400 I. U. heparin and 500 I. U. streptokinase in water was added to
the clotted tube with test preparation and 1.0 ml of sterile
distilled water added to the second. Both samples were homogenized
at 1,000 RPM for 2 minutes using an Ultra Turax homogenizer. The
clot disruption procedure took approximately 15 minutes (60 minutes
exposure in total), after which serial dilution and plate counting
procedures were used to enumerate residual bacterial viability in
all three tubes.
[0173] The BSA blank contained 6.4.times.10.sup.7 viable E. coli
cells per ml, the control blood (heparin/streptokinase treated)
sample. contained 8.3.times.10.sup.5, and no viable bacteria were
recovered from the test sample.
[0174] It will be clear to those skilled in the art that
preparations such as described in this Example may be applied to
wounds in the form of a liquid, spray, gel, powder, or wet-bandage.
It will also be clear to those skilled in the art that the
preparations described may be added to other pro-coagulants such as
chitin, kaolin or alginate to enhance their pro-coagulation effect
and add a microbicidal effect.
EXAMPLE 7
Use of Antimicrobial-Membrane Lipids with Anti-Clotting Effect in
Surgical Procedures
[0175] Ingress of potentially infectious agents during surgical
procedures is a major cause for concern among healthcare
professionals. The use of irrigating fluids with antimicrobial
effects facilitates prevention of this. There are also many
surgical procedures where the ability to prevent blood clotting is
advantageous, micro-surgery procedures for example, where
pre-emptive clotting may be exacerbated by the implements used and
where the use of an irrigating solution to wash out blood and body
fluids to prevent occlusion of the site is desirable. A specialized
application is the use of anti-clotting liquids to fill the void
volume of indwelling catheters during periods when the catheter is
not in use.
[0176] In this Example, a dispersion of 0.4% de-lipidised lecithin
(DLL) with 0.5% caprylic acid emulsified therein is prepared as
described in the Methods. The emulsion is diluted to 50% of its
initial concentration with 200 mM sodium citrate at pH 4.5, the
result being 0.25% caprylic acid, 0.2% DLL in 100 mM sodium
citrate, or approximately 2.5% W/V sodium salt of citric acid at pH
4.5. Sodium citrate is prepared by adjusting the pH of 200 mM
citric acid with 200 mM sodium hydroxide to 4.5; this is not the
same as `tri-sodium citrate` which is commonly used as an
anti-clotting agent, because not all of the carboxylic acid
residues have been salted out.
[0177] As described hereinabove, the use of a viscosity-enhancing
agent to adjust the viscosity of the formulation to approximate to
that of whole blood provides a distinct advantage in preventing
dilution of a catheter locking solution at the catheter tip due to
blood, flow turbulence. Dextran 40 is used here as a
viscosity-enhancing agent where it has been found that 20% W/V
inclusion provides a viscosity of approximately 4 cP, the viscosity
of human blood being between 3.6 and 6 cP.
[0178] The emulsified free fatty acid/membrane lipid catheter
locking solution used here (ML CLS) has the following constituents:
0.2% W/V de-lipidised lecithin; 0.25% caprylic acid; 20% dextran
40, in 100 mM (2.5% W/V) sodium citrate, pH 4.5. Aliquots of this
formulation were dispensed to 15 ml Greiner centrifuge tubes in the
following amounts: 0, 0.25, 0.5, 0.75 and 1.0 ml amounts. To each
of these 5.0, 4.75, 4.5 and 4.25 ml aliquots of fresh sheep blood
were added, respectively. Each tube was evaluated consecutively
with fresh blood added immediately after it was aspirated. The
respective volume dilutions represent 0, 5%, 10%, 15% and 20% by
volume of blood. Immediately after addition of blood, the tubes
were inverted twice to mix and a single drop added to the test well
of a Hemochron Signature Activated Blood Clotting Meter.
[0179] A similar procedure was adopted using a solution of 25,000
I.U. of Heparin, which at 5%, 10, 15% and 20% gave sample
concentrations of 1,250, 2,500, 3,750 and 5,000 units per ml of
test volume.
[0180] Also tested were a commercially available Catheter Locking
solution, Duralock from MedComp, which contains 47% sodium citrate
only, and a composition of 0.05% Methylene Blue, 0.15% Methyl
Parabens, and 0.015% Propyl Parabens in 7% (0.24M) sodium citrate,
being a replica of the reported formulation for Zuragen; and
Taurolock from Tauropharm AG comprising 1.35% Taurolidine in 4%
sodium citrate: (see Table 2). Phosphate Buffered Saline (PBS) is
used as a dilution control.
[0181] It should be noted here that the Hemochron blood clotting
system relies on a clot activation process which accelerates time
to clot formation; whole blood without additives clots in less than
200 seconds in this apparatus. The timelines in this experiment are
not directly comparable therefore with clotting times reported in
Example 5 where the baseline for normal clot time is shown as 360
seconds being the observed time for normal (non-activated)
clotting. It should also be noted here that the Hemochron meter
goes `out of range` at 1,000 seconds of Activated Clotting Time and
more extended time recordings are not available.
[0182] The results are reported in table 11 below and illustrated
in FIG. 3.
TABLE-US-00011 TABLE 11 Activated Blood Clotting Times for Various
Catheter Locking Solutions % Incorporation in whole blood 0% 5% 10%
15% 20% ML CLS 159 347 537 769 1018 Heparin .sup.1 159 284 423 601
1023 Duralock 159 300 364 484 722 Zuragen 159 219 265 394 614
Taurolock 159 230 310 380 510 PBS control 159 163 165 233 296 Note
.sup.1: Heparin concentrations range from 1,250 I.U. at 5% to 5.000
I.U. per ml at 20%
[0183] In terms of metered anti-clotting effect, the inventive
product of this Example is better than 25,000 I.U. of Heparin and
considerably better than Duralock, Zuragen or Taurolock. It should
be noted again however, that these are `Activated` clotting times.
In practice, none of the treated samples--apart from control
PBS--showed any visual sign of clotting even after several
hours.
[0184] The antimicrobial effect of the formulation in this Example
was tested using procedures similar to those used in Example 6,
with the exception that the clot disrupting agents, heparin and
streptokinase, were used to break the clots in the control
untreated bloods.
[0185] 18 hour Brain Heart Infusion Broth cultures of
Staphylococcus aureus, Streptococcus epidermidis, Escherichia coli
and an 18 hour culture of Candida albicans grown in Yeast Extract
Peptone Dextrose broth were concentrated .times.10 by
centrifugation and re-suspension in one tenth volume of
supernatant.
[0186] 2.5 ml aliquots of the bacterial concentrates were used to
inoculate 22.5 ml volumes of freshly aspirated sheep blood, mixed
and the blood dispensed as 5.0 ml, 4.75 ml, 4.5 ml, 4.25 ml and 4.0
ml volumes to Greiner centrifuge tubes containing 0, 0.25 ml, 0.5
ml, 0.75 ml and 1.0 ml volumes of the test formulation of this
Example.
[0187] The inoculated tubes were incubated for 45 minutes at
37.degree. C. following which time, a 1.0 ml of volume of 400 I.U.
heparin and 500 I. U. Streptokinase in water was added to all tubes
and each was subjected to slow speed homogenization at 1,000 RPM
for 2 minutes: the only visible clotting was in the control tubes.
Immediately after 60 minutes had elapsed, serial dilution and plate
count methods were used to assess residual viability in all
samples. For comparative purposes, the same procedure was repeated
with Taurolock, Duralock, Zuragen and Heparin at the maximum dose
loading of 1.0 ml in 4.0 ml of blood only. The results are
presented in Table 12 and illustrated in FIG. 4.
TABLE-US-00012 TABLE 12 Comparative Microbicidal Effect of Catheter
Locking Solutions in whole blood Escherichia Staphylococcus
Streptococcus Candida coli aureus epidermidis albicans Time zero
7.69 8.71 8.12 6.67 Blank Time 8.19 8.9 8.51 6.33 60 min ML CLS 5%
4.83 3.62 4.17 3.9 ML CLS 2.58 1.44 1.97 0 10% ML CLS 1.62 0 0 0
15% ML CLS 0 0 0 0 20% Taurolock 6.53 4.15 4.72 5.72 20% Duralock
6.97 6.49 7.78 6.54 20% Zuragen 5.56 4.91 5.73 4.28 20% Heparin
7.92 8.39 8.22 6.94 25,000 I.U. 20%
[0188] In this test the 20% by volume ML CLS according to the
invention achieved, complete eradication of E. coli (8 logs), Staph
aureus (8 logs), Strep epidermidis (8 logs) and Candida albicans (6
logs) in one hour in whole blood: a 5% volume achieved
approximately 4 log reduction of the test inoculums in the same
time. Of the four comparative preparations (Duralock, Taurolock,
Zuragen or Heparin), only Zuragen and Taurolock had an appreciable
microbicidal effect achieving a reduction of between 3 and 4 logs
in viability of the test inoculum in one hour; Duralock appears to
have a microbistatic effect while no microbial inhibition could be
attributed to Heparin.
[0189] Thus, the above catheter locking solution according to the
invention exhibits significantly better anti-clotting effects and
much greater microbicidal effect than existing conventional
products.
EXAMPLE 8
Use of Membrane Lipids in antimicrobial Surface Coating
[0190] A suitable method of applying a persistent coating of a
product according to the invention involves emulsification of 1%
caprylic acid in 0.8% de-lipidised lecithin prepared as described
in the Methods. An equivalent volume of 100 mM sodium citrate
buffer pH 4.5 is added to the emulsion to obtain a final
concentration of 0.5% caprylic acid, 0.4% de-lipidised lecithin in
50 mM sodium citrate. Eight Volumes of absolute ethanol are then
added slowly to two volumes of the emulsion with constant vigorous
stirring to make the final coating material in 80% ethanol.
[0191] In order to coat a plastic surface it is preferable to use
some form of surface conditioning which may include a process known
as corona discharge wherein an electrical field is generated across
the Surface imparting a residual charge which facilitates adhesion
of the applied coat. Following corona treatment, the ethanol
solution described above is sprayed on the surface and dried under
forced air conditions at 60.degree. C. Several coats may be applied
to construct a layer of antimicrobial coating.
[0192] A base layer of membrane lipid may first be applied to an
inert surface, and once dried and annealed it is used to `anchor` a
second layer of de-lipidised membrane lipid emulsified free fatty
acid according to the invention.
[0193] In this Example an organic solvent solution of a membrane
lipid is applied to the surface of a microtitre plate well, dried
and fixed by heating at 60.degree. C. followed by a further
application of an aqueous suspension of caprylic acid emulsified in
de-lipidised lecithin prepared as described in the Methods. The
de-lipidised lecithin emulsion was dried and annealed to the first
lecithin coating by heating at 60.degree. C. for 3 hours. Plates
treated with de-lipidised lecithin, without caprylic acid were used
as a control and untreated plates were used to determine optimum
biofilm formation.
[0194] A base layer of de-lipidised, lecithin (DLL) was prepared by
suspending 5% by weight DLL in 80% ethanol: water, and dispensing
100 .mu.l aliquots of this to the test wells. Wells for
determination of optimum biofilm were left untreated. The ethanol
fractions were dried in a fume hood and annealed at 60.degree. C.
for one hour in an oven. 1% W/V dispersion of de-lipidised lecithin
was prepared in sterile distilled water as_described in Example 1,
and a volume of this used to emulsify 0.25% caprylic acid. 100
.mu.l aliquots of DLL or DLL+0.25% caprylic were transferred to
DLL, pre-coated wells and dried in an oven at 60.degree. C. for 3
hours.
[0195] A 10 hour culture of Staphylococcus aureus RN 4220 grown in
Brain Heart Infusion broth (BHI), supplemented with 4% sodium
chloride was used as inoculum. The mid log phase culture was
diluted to 1% with fresh sodium chloride supplemented BHI and 200
.mu.l of this added to the microtitre plate wells, covered and
incubated.
[0196] At each sample point, 100 .mu.l from each well was
transferred to a fresh plate for assessment of growth by Optical
Density at 570 nM and the plate was then decanted and washed with
copious volumes of sterile distilled water, dried and annealed in
an oven at 60.degree. C.
[0197] When dry, 100 .mu.l of 0.4% crystal violet was added to each
well--including untreated controls. After 4 minutes, excess crystal
was drained off, and the plates were again washed with copious
volumes of water to remove excess dye and again dried with the aid
of an oven at 60.degree. C. The results are presented in table 13
and illustrated in FIG. 5.
TABLE-US-00013 TABLE 13 Inhibition of Biofilm Formation: 24 hour
culture Optical Density 570 nM Control growth 2.8 Coated planktonic
growth 2.4 Uncoated biofilm 0.9 DLL coated biofilm 0.7 DLL + Cap
Inhibited biofilm 0.1
[0198] Uncoated planktonic growth is largely unaffected by the
coating of inhibitory DLL+caprylic add. Wells coated with DLL alone
(DLL coated biofilm) are slightly reduced, but in comparison, wells
coated with the product of this invention (DLL+Cap Inhibited
Biofilm) are essentially free of any biofim: the coating itself
takes up some of the crystal violet dye which accounts for a small
increase in Optical Density in the DLL+Cap wells.
EXAMPLE 9
The Use of Membrane Lipids to achieve Sustained Release of
Microbicidal Free Fatty Acids
[0199] In therapeutic applications considerable advantage may be
gained from using combinations of membrane lipid emulsions with
`tailored` release characteristics, which facilitates sustained
microbicidal effect at the epithelial surface.
[0200] In this Example individual membrane lipids were selected
from each of the four classes presented in Table 1 and were the
same as those used in adhesion inhibitory studies in Example 1.
0.4% Aqueous dispersions of each were prepared and 0:2% caprylic
emulsified in each using procedures described in the Methods for
de-lipidised lecithin (DLL). A 0.4% sample of DLL with 0.2%
caprylic was also prepared. Each of the membrane lipid preparations
was diluted to 50% of its volume with 200 mM sodium citrate at pH
4.5, and therefore each preparation then consisted of 0.1% caprylic
acid and 0.2% membrane lipid in 100 mM sodium citrate at pH 4.5. It
should be noted here that this is less than half of the
microbicidal caprylic-acid content of the test item used in Example
3, Table 9.
[0201] The yeast Candida albicans was used in this Example, and the
inoculum prepared as an 18 hour YEPD broth culture as described in
the Methods. The late log phase culture was centrifuged at 4,000
RPM for 5 minutes and re-suspended in one tenth volume of
supernatant to concentrate .times.10.
[0202] 12.0 gram samples of each test preparation were dispensed to
Sterilin tubes and each of these was inoculated with 1.2 ml
aliquots of concentrated yeast culture. Samples of 1.0 ml volume
were withdrawn form these inoculated tubes at timed intervals over
the course of one hour and added to 9.0 ml of diluting buffer
containing 3% Tween 80 to neutralize. Serial dilutions and plate
counting was undertaken to enumerate residual viability as
described in the Methods. The results are presented in Table 14
below and illustrated in FIG. 6.
TABLE-US-00014 TABLE 14 Variable Release Characteristics of
Membrane Lipids: Kill time for > 6 logs; Candida albicans
Minutes 0 5 10 15 20 25 30 35 40 45 50 55 60 Blank 7.13 6.92 6.86
6.69 7.19 6.67 6.93 7.21 6.73 7.17 6.89 6.78 6.84 DLL 6.39 5.64
4.53 3.21 1.96 0 PTC 6.5 4.84 2.57 1.22 0 0 PTE 7.32 5.41 3.67 1.88
0 PTG 6.76 5.93 5.17 4.53 3.68 2.82 1.95 1.14 0 PTI 7.41 6.11 4.83
3.79 2.32 1.16 0 PTS 6.8 6.36 5.85 5.23 4.53 3.79 3.13 2.61 1.88
1.1 0 C 7.24 7.28 6.89 6.54 6.14 5.96 5.32 4.97 4.65 4.29 3.87 3.74
3.32 SGM 6.84 6.55 6.12 5.85 5.42 4.87 4.22 3.76 3.13 2.79 2.22
1.84 1.33 MGDG 7.3 6.66 6.1 5.16 4.34 3.32 1.84 0 L 7.39 6.45 5.86
5.34 4.78 4.21 3.67 2.98 2.33 1.74 1.17 0
[0203] It is evident from the results that the slowest acting
emulsions are Ceramide and Sphingomyelin followed by Lanosterol,
Phosphatidylserine and Phosphatidylglycerol; the fastest acting
emulsions are Phosphatidylcholine, Phosphatidylethanolamine and the
combination of phospholipids in de-lipidised lecithin (DLL).
EXAMPLE 10
The use of Combinations of Antimicrobial Membrane Lipid Emulsions
to Fortify Mucus and achieve a `Tailored` Microbicidal Effect at
the Mucosal Surface
[0204] Mucosal fortification involves complementary hydration,
lubrication and enhanced antimicrobial effect of mucosal secretions
of the eye, nose, mouth, naso-pharyngeal surfaces, the
gastro-intestinal tract and the genitalia. This Example describes a
preparation suitable for fortification of the mucosal secretions of
the mouth and vagina and most particularly suitable for use by
individuals susceptible to recurring oral and/or vaginal thrush and
other common bacterial and viral infections responsive to the
products of this invention.
Examples of Mucosal Fortificants:
[0205] Part A: A fast acting membrane lipid emulsion of caprylic
acid (Cap) is based on 0.2% W/V Phosphatidylcholine (OTO) dispersed
in sterile distilled water, hydrated and homogenized as described
in the Methods and used to emulsify 0.25% W/V caprylic acid by the
procedure described. [0206] Part B: A slow acting membrane lipid
emulsion of caprylic acid (Cap) is based on 0.2% Sphingomyelin
(SGM), dispersed, hydrated and homogenized as described and then
used to emulsify 0.25% W/V caprylic acid as described in the
methods. [0207] Part C: A 200 mM solution of citric acid is
adjusted to pH 4.5 with 200 mM sodium hydroxide. A cellulose based
viscosity-enhancing agent (hydroxypropylmethylcellulose, Methocel
E4M from Dow Gmbh, Germany) is added at 1% W/V: the polymer is
sifted in while vigorously stirring the sodium citrate solution and
allowed to hydrate for 30 minutes.
[0208] A test preparation of PTC+Caprylic acid (PTC+Cap) was
prepared by mixing equal amounts of Part A and Part C, yielding an
emulsion of 0.125% caprylic acid with 0.1% PTC in 100 mM sodium
citrate containing 0.5% W/V Methocel.
[0209] A test preparation of SGM+Caprylic acid (SGM+Cap) was
prepared by mixing equal amounts of part B and Part C, yielding an
emulsion of 0.125% caprylic acid with 0.1% SGM in 100 mM sodium
citrate containing 0.5% W/V Methocel.
[0210] A test combination preparation comprising fast and slow
acting emulsions was prepared by mixing 30% PTC+Cap with 70%
SGM+Cap and combining this with an equal volume of part C (30:70
blend). The 30:70 blend is an emulsion of 0:03% caprylic acid in
0.075% PTC combined with 0:07% caprylic acid in 0.0875% SGM in 100
mM sodium citrate containing 0.5% Methocel.
[0211] The microbicidal and adhesion inhibitory properties of all
three test preparations were assessed using the standard viability
and adhesion inhibition assay described in the Methods. The
inoculum for both assays was an 18 hour broth culture of Candida
albicans, grown in YEPD medium and concentrated .times.10 by
centrifugation and re-suspension in one tenth volume of
supernatant.
[0212] 12.0 gram samples of each test Preparation were dispensed to
Sterilin tubes and each of these was inoculated with 1.2 ml
aliquots of concentrated yeast culture. Samples of 1.0 ml volume
were withdrawn form these inoculated tubes at timed intervals over
the course of one hour and added to 9.0 ml diluting buffer
containing between 80 to neutralize. Serial dilutions and plate
counting was undertaken to enumerate residual viability as
described in the Methods. The results are presented in Table 15 and
illustrated in FIG. 7.
TABLE-US-00015 TABLE 15 Mucosal Fortification: Viability of Candida
albicans in Tailored Release Preparations Minutes 0 5 10 15 20 25
30 35 40 45 50 55 Blank 6.81 6.92 6.79 6.85 6.97 6.76 6.84 6.99
6.69 6.95 6.78 6.9 P T C + Cap 6.5 4.84 3.13 1.72 0 SGM + Cap 6.84
6.55 6.12 5.85 5.42 4.87 4.22 3.76 3.13 2.79 2.22 1.84 30:70 blend
6.73 5.21 4.14 3.56 3.12 2.8 2.42 2.16 1.89 1.55 1.33 0.89
[0213] The fast acting PTC+Cap behaved as expected reducing
viability to zero detectable cells in 20 minutes. The slow acting
SGM+Cap was also as expected with viability being reduced by 5 logs
in 55 minutes. The combination 30:70 blend gives a good example of
a `tailored release` profile, viability ,was reduced by more than 2
logs in 10 minutes, a rate which paralleled the fast acting
PTC+Cap, thereafter the microbicidal rate slowed considerably, and
approximated to that of the slow acting SGM+Cap. The blend however
had achieved 1 log greater reduction in viability at 55 minutes
compared to SGM+Cap alone.
[0214] Assessment of adhesion inhibitory properties of the three
test preparations was undertaken using the Buccal Epithelial Cell
model described in the Methods and used in Example 1. For
comparison, preparations of the two membrane lipids, PTC and SGM,
at 0.1% in 100 mM sodium citrate buffer pH 4.5 with 0.5% Methocel
were also prepared and included in the test procedure.
[0215] Washed yeast cell pellets were re-suspended in 2.5 ml
volumes of the test preparations (PTC, PTC+Cap, SGM, SGM+Cap, 30:70
blend described above and BSA blank). 100 mM sodium citrate at pH
4.5 was used for determination of control adhesion. After 10
minutes pre-exposure, the yeast suspensions were used to re-suspend
washed Buccal Epithelial Cell pellets which had been harvested and
prepared as described in the Methods. The combined yeast and Buccal
Epithelial cell in test, blank or control were incubated with
gentle agitation for 60 minutes at 37.degree. C., after which
direct microscopic counts using a Hemocytometer slide were used to
enumerate the numbers of yeast adhering to 100 Buccal Epithelial
cells: The results are presented in Table 16 and illustrated in
FIG. 8.
TABLE-US-00016 TABLE 16 Mucosal fortification: Inhibition of
Adhesion in Tailored release Preparations: Candida albicans to
Buccal Epithelial Cell % % Count/ Adhe- Inhibi- 100 BEC sion tion
Blank 509 100 0 PTC 239 47 53 SGM 374 73 27 PTC + Cap 20 4 96 SGM +
Cap 266 52 48 30:70 Blend 88 17 83 0.5% BSA 425 83 17
[0216] The adhesion inhibitory properties of 0.1% PTC on its own
and with 0.125% caprylic acid are considerably better than
equivalent preparations of SGM: as might be expected the 30:70
blend lies midway between the two.
[0217] From the data in Table 16 it is also evident that the
emulsified fatty acid has a synergistic effect on the adhesion
inhibitory properties of the membrane lipid. The percentage
reduction in adhesion achieved with PTC alone (53%), is reduced by
a further 43% in combination with caprylic acid.
[0218] It should be noted that according to the viability data in
Table 16 and as illustrated in FIG. 7, none of the yeast cells in
PTC +Cap is viable after 20 minutes exposure, and approximately 50%
to 60% of those in the other two samples are dead. Under the
microscope however, yeast cells appear to be intact and while
greatly reduced there are still a few apparent that are adhering to
Buccal Epithelial cells, suggesting that dead cells are capable of
adhesion and biofilm formation.
EXAMPLE 11
The Use of Membrane Lipids in Skin Antisepsis and Prevention of
Cross Contamination in Hospitals and Patient Care
Establishments
[0219] Methods of evaluating skin antiseptic agents in wash and gel
formulations are well established and are fully described in the
official procedures of the EU for ISO Certification under EN 1500
(hand gel) and EN 1499 (liquid soap).
[0220] In this Example, the relatively non-pathogenic Escherichia
coli K12 NCTC 10538 was used (The National Collections of
Industrial and Marine Bacteria Ltd, UK: Catalogue of Type Strains
ISBN No 0 9510269 3 3). The bacterium is routinely cultured and
maintained on tryptone Soya agar or broth (TSB), which may be
acquired commercially from Oxoid, UK and has the following
constituents: 1.5% W/W tryptone (pancreatic digest of casein), 0.5%
W/VV peptone (papaic digest of soybean meal), 0:6% WM/ sodium
chloride; and agar at 1.5% VV/W when required as a solid
medium.
[0221] Prior to the test volunteers wash their hands with a mild
non-antiseptic soap; a suitable product is E45 Emollient Wash Cream
from Boots Healthcare, UK. After washing and drying, the hands are
dipped into a 2 litre beaker containing 1 litre of 24 hour culture
of E. coli grown in TSB and containing not less than
2.times.10.sup.6 viable cells per ml as confirmed by serial
dilution and plate counting. Both hands are immersed in the
contaminating suspension up to the mid-metacarpals and held there
for 5 seconds, and then removed. Excess contaminating fluid is
allowed to run-off and then the hands are air dried in the
horizontal position for 3 minutes.
[0222] To ensure adequate contamination and to establish a
pre-value for enumerating reduction, the hands are sampled by
dipping the tips of the fingers and thumb of each hand into 10 ml
of sterile PBS in a Petri-dish, and rubbed against the base of the
plate for 1 minute. After sampling, the hands are treated with
either the product of this invention or Spirigel; 4 ml of either
preparation is applied to the hands and manipulated over the
surface area of both hands. The hands are then rinsed under clean
(potable) running water, which is lukewarm at approximately
37.degree. C. for a timed period of 20 seconds. After rinsing, the
hands are held in an upright position while an assistant dries the
palms and wrists with a paper towel. The finger tips and thumb are
then sampled by immersion in 10 ml of PBS as described above.
[0223] Immediately after sampling, prior to or after washing, 1 ml
of the sampling fluid was aseptically transferred to and spread on
the surface of a TSB agar plate and another 1 ml is transferred
aseptically to 9 ml of sterile PBS and mixed and the process of
serial dilution and plate counting proceeds as described
previously.
[0224] A test preparation of the product of this invention was
prepared, being a combination of caprylic acid in phosphatidyl
choline at 30% and caprylic acid in sphingomyelin at 70% prepared
as described in the Methods. In this Example the membrane lipids
were prepared as 1% concentrates with 1% caprylic acid and blended
at 30:70 ratio.
[0225] A viscosity-enhancing agent is employed to improve the
rheology of the test preparation. In this case a Carbopol
co-polymer, Pemulen TR-1 from Noveon Inc, Cleveland, Ohio was used
at 0.45%. The polymer was added to a volume of absolute ethanol
equivalent to 70% of final preparation volume and allowed to
disperse therein for 30 minutes. A 30% volume of 30:70 blend of the
product of this invention as described above was then added to the
ethanol and polymer with constant stirring to facilitate rapid
dispersion.
[0226] The Final Test Product Contains:
[0227] 0.045% Caprylic acid in 0.045% Phosphatidylcholine: 0.105%
Caprylic acid in 0.105% Sphingomyelin: 0.45% Carbopol polymer;
29.25% water: 70% ethanol.
[0228] Spirigel is reported to contain 70% ethanol, 30% water and
an unknown amount Of an unknown Viscosity-enhancing agent.
[0229] In this Example both test product and Spirigel were used
immediately after experimental contamination of volunteers' hands
to evaluate de-contamination, and both were used on experimentally
re-contaminated hands 10 minutes after application of Spirigel and
test preparation according to the invention; the results are
presented in Table 17.
TABLE-US-00017 TABLE 17 Persistent Effect of tailored release
membrane lipids in skin antisepsis Spirigel Test item Pre-treatment
contamination 6.42 6.69 Post treatment contamination, 3.23 (-3.19)
3.42 (-3.27) i.e. residual viability Pre-application 10 min Not
Applicable Not Applicable pre-contamination (treatment 10 min
pre-contamination) Contamination: 10 min post 6.42 6.59 treatment
Contamination: 10 min Post 5.97 2.67 (-3.3) application, i.e.
viable cells recovered for contamination 10 mins post
application
[0230] As might be anticipated from the alcohol content, when used
immediately after contamination both products achieved greater than
3 log reductions of the applied contamination. When used on hands
10 minutes prior to contamination however the alcohol content of
both products had evaporated by the time the contamination was
applied, and the results show that Spirigel had no significant
residual effect (0.45 log reduction). The persistent nature of the
membrane lipid emulsion of fatty acid was still present on the
hands in the test item, and achieved greater than 3 log reduction.
Spirigel has an immediate but no persistent antimicrobial effect,
while the test item according to this invention is both immediate
and persistent microbicidal effects.
EXAMPLE 12
Use of Membrane Lipid Emulsions in Wound Care
[0231] To illustrate the potential utility of the product of this
invention in wound care an ex-vivo model employing freshly
slaughtered sections of beef brisket is used. Brisket has an
optimal distribution of lean muscle, fat and collagen, and is
consequently considered to be representative of all potentially
infected wound surfaces. Brisket sections are excised immediately
post slaughter, without chilling and with the external fascia
membrane intact, these are divided under aseptic conditions into
cubes of approximately 1 cm square. Prepared cubes are `infected`
by submerging them in late log phase cultures of bacteria for one
minute, dried and suspended in a 37.degree. C. environment for one
hour to facilitate bacterial adherence and colonization of the meat
surfaces.
[0232] A suitable example of a wound care formulation is the
"standard formulation" of this invention (containing 0:5% caprylic
acid in 0.4% DLL) diluted 1:1 in 200 mM sodium citrate buffer at pH
4.5, Which then consists of 025% caprylic acid in 0.2% DLL in 100
mM sodium citrate buffer at pH4.5
[0233] Test sections of infected meat are treated by spraying the
wound care formulation directly onto the infected surface and
evaluating the test samples for residual bio-burden at
predetermined exposure times infection and its eradication are
confirmed by mechanical maceration of treated and untreated
sections in sterile phosphate buffered saline (PBS) and enumeration
of the bio-burden by standard microbiological serial dilution and
plate counting techniques.
[0234] Typical results are presented in FIG. 9, where greater than
7 logs of the common wound infecting bacteria, Staphylococcus
aureus and Streptococcus pyogenes are shown to be eradicated in
less than 120 minutes. It should be appreciated that the rate of
kill on a fissured surface such as a wound is extended due to the
nature of the surface and the time required for the formulation to
permeate to the seat of the infection.
[0235] A comparison of the relative potency of the standard
formulation in citrate buffer as above with the alternative and
commonly used antimicrobials, chlorhexidine gluconate and silver
sulphadiazine is presented in FIG. 10. At 100 minutes exposure the
product of this invention has virtually eradicated 7 logs of
Staphylococcus aureus: 1 log remains under chlorhexidine treatment
and 3.5 logs with silver sulphadiazine.
EXAMPLE 13
The Surgical Use of Membrane Lipids in Prevention and Disruption of
Biofilm
[0236] Staphylococcus aureus RN4220 is noted for its ability to
form tenacious biofilm under laboratory conditions when stress
cultured in the presence of sodium chloride. The organism was
cultured to mid log phase (10 hours) in Brain Heart Infusion broth
and 20 micro-liter volumes used to inoculate wells of a 96 well
microtitre plate containing 180 .mu.l of BHI supplemented with 4%
sodium chloride. When incubated at 37.degree. C. under these
conditions for 6 hours an appreciable biofilm is formed at the base
of each well. The biofilm is quantified by decanting the culture
and washing the wells with sterile distilled water, after which the
plates are dried and stained with 0.4% crystal violet, re-washed
and dried; the Optical Density of the stained biofilm is measured
at 570 nm.
[0237] From previous Examples it will be clear that incorporation
of the membrane lipid emulsified product of this invention will
have a microbicidal effect, preventing growth and biofilm
formation. As illustrated here where biofilm already exists,
however, contact with an emulsion of membrane lipid and free fatty
acid will effectively kill all viable bacteria in the biofilm and
disrupt the film itself.
[0238] Assessment of reduction of viability of an established
biofilm prepared as described above was achieved by incorporating
Alamar Blue at 10% by volume in fresh BHI broth and re-charging the
wells of a 96 well Microtitre plate with pre-formed biofilm. Alamar
Blue is a redox indicator from Invitrogen Ltd, Paisley, UK. It
imparts a deep blue color to the media, and when reduced by
microbial metabolic activity the Color changes from non-fluorescent
blue to a highly fluorescent red; absorbance and emerging
fluorescence may be measured at 570 nm and 600 nm.
[0239] Microtitre plate wells containing pre-formed biofilm were
treated with the membrane lipid CLS (ML CLS) used in Example 7 and
similar wells were also treated with Zuragen, Duralock and
Taurosept for time periods ranging from zero to 60 minutes. At the
end of each exposure period, the plates were decanted and washed
once with phosphate buffered saline containing 3% Tween 80 and
twice with sterile distilled water. 200 .mu.l of BHI containing 10%
Alamar Blue was added to the wells and the plates incubated for 60
minutes. There was no detectable color change in any of the wells
treated with the membrane lipid CLS of this invention indicating
complete eradication of viability within the biofilm in less than
one hour. All of the wells treated with Zuragen, Duralock or
Taurosept had changed from blue to red demonstrating little or no
reduction in viability in the established biofilm.
[0240] The efficacy of the membrane lipid CLS of this invention,
and the comparable ineffectiveness of the other three formulations
in reducing the actual amount of pre-formed biofilm may be
demonstrated using similar procedures.
[0241] Microtitre plate went containing pre-formed biofilm were
treated with the four formulations for time periods from zero to 60
minutes, decanted and washed as described previously. The treated
plates were dried and stained with 0.4% crystal violet, dried and
the intensity of stain being a measure of residual biofilm was
recorded at 570 nm. The results are illustrated in FIG. 11 wherein
it is evident that although some reduction in biofilm was achieved
with Zuragen, Duralock and Taurosept, it is inconsequential in
comparison to the near total eradication of biofilm achieved with
the membrane lipid CLS (ML CLS) of this invention.
EXAMPLE 14
Comparative Adhesion Inhibitory Properties
[0242] De-lipidised lecithin is a more potent adhesion inhibitory
substance compared to milk serum apo-proteins in WO 03/018049,
wherein the apo-proteins are lipase hydrolysis of whey proteins.
For adhesion inhibitory comparison, a whey protein hydrolysate was
prepared As described in WO 03/018049 using Carbelac 80 whey
protein concentrate. A whey protein isolate (Provon 190 from
Glanbia PLC) was also tested contemporaneously. For full comparison
purposes a formulation of 0.5% W/V caprylic acid in 0.4% W/V
de-lipidised lecithin was prepared in 100 mM sodium citrate pH 4.5
as described in the Methods and included in the test sequence. All
test items were dispersed in 100 mM sodium lactate buffer at pH
4.5. The results are presented in Table 18 below.
TABLE-US-00018 TABLE 18 Candida albicans per 100 Buccal Epithelial
Cells 0 2.5 5.0 10 15 % inhibition mg/ml mg/ml mg/ml mg/ml mg/ml at
5 mg/ml Carbelac 80 483 395 320 260 189 33 Provon 190 514 313 277
113 53 46 Lipase digest of Carbelac 80 491 330 154 48 0 68
De-lipidised lecithin (DLL) 504 130 33 0 0 93 0.5% Caprylic in 0.4%
DLL 517 46 0 0 0 100
[0243] While de-lipidised lecithin on its own is significantly
better than the three diary based preparation, the
emulsified-combination of de-lipidised lecithin and caprylic acid
is the most potent.
EXAMPLE 15
Comparative Microbicidal Effect
[0244] The MIC by agar dilution technique is also used here to
demonstrate the superior potency of the "standard formulation"
described hereinabove over the product disclosed in WO2009/072097
comprising a blend of free fatty acids emulsified in the whey
protein isolate, Provan 190 from Glanbia PLC. The product of
WO2009/072097 contains 28% by weight of free fatty acid blend,
while the standard formulation of this inventions contains just
0.5% by weight. In order to make a suitable comparison between the
two products the product of WO2009/072097 was diluted by 5/28 in
sterile distilled water to obtain a dispersion comprising 5.0% free
fatty acid which was then diluted further and used to prepare agar
plates ranging from 1.0% free fatty acid to 0.5%, 0.4%, 0.3%, 0.2%,
0.1%, 0.075%, 0.05% and 0.025% (of fatty acid content).
[0245] For further comparison an emulsion was constructed using
0.5% caprylic acid in 0.4% Provon 190, using the emulsification
agent from WO2009/072097 instead of de-lipidised lecithin. The
results are shown in Table 19 below.
TABLE-US-00019 TABLE 19 Comparative MIC values Formulation of
invention 0.5% Caprylic containing Product of in 0.5% caprylic
WO/2009/ 0.4% Provon acid Test organism 072097 190 in 0.4% DLL
Staph aureus RN4220 >0.4% >0.7% >0.1% Staph epidermidis
>0.4% >0.7% >0.1% NCTC 11047 Strep pyogenes >0.4%
>0.7% >0.075% NCTC 8198 Strep faecalis >0.4% >0.7%
>0.075% NCTC 12697 E. coli >1.0% >1.0% >0.4% ATCC 11698
Salmonella typhimurium >0.8% >0.8% >0.3% NCTC 74
Pseudomonas aeruginosa >1.0% >1.0% >0.5% ATCC 27853
Pseudomonas fluorescens >1.0% >1.0% >0.5% NCTC 10038
Candida albicans C.I. >0.4% >0.7% >0.075% Candida glabrata
>0.4% >0.7% >0.1% NCPF 8750
[0246] For the two Staphylococci, the two Streptococci and the
Candida sp, the measured MIC for the standard formulation is one
quarter or less than that of the product of WO2009/072097,
indicating a four times greater potency. In the case of the two
Pseudomonas sp. E. coli and Salmonella, the measured MIC is one
half to one third less than the product of WO2009/072097 again
clearly demonstrating significantly greater potency. The measured
MIC's for the emulsion of caprylic acid in Provon 190 are
significantly greater (significantly less potent) than the standard
formulation of this invention.
EXAMPLE 16
Comparative Microbicidal and Adhesion Inhibitory Effect of
Different Free Fatty Acids in both Emulsified and Free Form
[0247] Fatty acids in free form (not emulsified) have relatively
little microbicidal effect primarily because of their insolubility
in aqueous medium. Emulsification in membrane lipids as described
herein expands the relative surface area of the fatty acid and
facilitates its dispersal in aqueous medium. The membrane lipid
emulsification agent also facilitates contact and transfer of the
fatty acid to a microbial cell surface. As illustrated in previous
Examples, variable lipophylicity between different membrane lipids
affects the rate of microbicidal effect. In general, membrane
lipids are superior emulsification agents, exhibiting superior
microbicidal effect as demonstrated in Example 15. As demonstrated
here the superior potency of membrane lipid emulsified free fatty
acid extends across a range of microbicidal fatty acids.
[0248] Seven separate emulsions of seven different free fatty acids
at 0.5% W/V were prepared in 0.4% W/V de-lipidised lecithin as
described, in the methods. Emulsions were prepared at temperatures
above the melting points of the individual free fatty acids:
caproic, caprylic and pelargonic at room temperature (20.degree.
C.); capric and undecylenic acid were prepared at 37.degree. C.,
lauric acid was emulsified at 50.degree. C. and myristic at
60.degree. C. A blend of 50% caproic and 50% lauric acid has a
melting point of less than 28.degree. C. as does a blend of 49%
leuric acid in oil of lemon balm, and these were emulsified at
37.degree. C.
[0249] The comparative microbicidal effect of each individual fatty
acid and blend thereof in free non-emulsified form and emulsified
in de-lipidised lecithin was evaluated using the microbicidal
suspension test described in the methods. Evaluation of the
microbicidal effect of free non-emulsified fatty adds is frustrated
by their insolubility. Inclusion of the non-emulsified form was
considered necessary to illustrate the exponential increase in
potency in the emulsified form. Each free fatty acid was prepared
at 0.5% W/V in water and agitated vigorously to disperse followed
by immediate pipetting of 1.0 ml aliquots to test containers, prior
to inoculation with the test organism. The test organism was
Staphylococcus aureus RN 4220 grown to late log phase on Brain
Heart Infusion Broth. The results are presented in Table 20
below.
TABLE-US-00020 TABLE 20 Comparative Microbicidal Effect Free fatty
acids or blends thereof at 0.5% W/V non-emulsified in the test and
at 0.5% W/V emulsified in 0.4% W/V de-lipidised lecithin in the
test. Exposure Time in Seconds at 37.degree. C. Fatty acid or blend
0 60 120 180 240 300 360 Free caproic acid 7.87 7.17 6.82 7.57 6.93
6.47 6.73 Emulsified caproic acid 7.87 6.21 4.89 2.53 0 0 0 Free
caprylic acid 6.94 6.79 6.83 6.21 6.33 5.89 6.17 Emulsified
caprylic acid 6.94 5.32 3.71 0 0 0 0 Free pelargonic acid 6.94 6.55
6.31 5.96 5.67 5.83 5.27 Emulsified pelargonic acid 6.94 4.63 2.99
1.81 0. 0 0 Free capric acid 6.94 6.32 6.48 6.73 6.36 5.97 6.11
Emulsified capric acid 6.94 4.79 3.44 2.19 0 Free undecylenic acid
7.87 7.19 6.99 6.57 6.83 6.67 6.31 Emulsified undecylenic 7.87 4.97
2.69 0 0 0 0 Free lauric acid 7.87 7.35 7.77 7.41 7.98 7.23 7.65
Emulsified lauric acid 7.87 5.79 4.63 3.86 2.55 0 0 Free myristic
acid 7.38 7.27 7.84 6.93 7.11 6.26 6.84 Emulsified myristic 7.38
6.46 5.81 5.21 4.98 3.69 2.99 Free lauric caproic 50:50 7.38 6.99
7.18 6.74 6.37 6.95 6.81 Emulsified Lauric: caproic 7.38 6.76 5.32
3.17 2.98 0 0 Free 40% lauric in oil of lemon balm 7.38 7.21 7.76
7.39 6.95 6.87 6.49 Emulsified 40% Lauric in Oil of Lemon Balm 7.38
5.92 3.13 2.77 0 0 0 De-lipidised Lecithin 0.4% W/V 7.33 7.92 7.41
6.83 7.14 7.66 6.95 Blank determinations were conducted in all
assays by suspending the inoculum in phosphate buffered saline at
37.degree. C. and no reduction in inoculum viability was detected
over the six minute exposure period in any test sequence.
[0250] Free fatty acids in non-emulsified form achieve at best 1
log reduction in viability over the test period of 6 minutes. The
membrane lipid emulsification agent (de-lipidised lecithin) equally
exerts little or no microbicidal effect In comparison, with the
exception of emulsified myristic acid, equivalent weight emulsions
of all other fatty acids and blends thereof in de-lipidised
lecithin reduce residual viability in a test inoculum of 6.94 to
7.87 logs to zero in less than 4 minutes.
[0251] Neither free fatty acids on their own nor the de-lipidised
membrane lipid on its own have any detectable microbicidal effect
within the time-frame of this test. An emulsified combination of
the two enables the microbicidal effect of the fatty acid
synergistically.
[0252] The same test items used in microbicidal evaluation above
were also tested for their adhesion inhibitory properties using the
Buccal Epithelial Cell assay described in the Methods.
TABLE-US-00021 TABLE 21 Comparative Adhesion Inhibitory Effect
Blank Test % % Count/ Count/ Adhe- Inhibi- Fatty acid or blend 100
BEC 100 BEC sion tion Free caproic acid 429 380 89 11 Emulsified
caproic acid 503 0 0 100 Free caprylic acid 429 400 93 7 Emulsified
caprylic acid 503 0 0 100 Free pelargonic acid 429 360 84 16
Emulsified pelargonic acid 503 2 0.3 99.7 Free capric acid 429 386
90 10 Emulsified capric acid 503 0 0 100 Free undecylenic acid 521
448 86 14 Emulsified undecylenic 508 0 0 100 Free lauric acid 474
450 95 5 Emulsified lauric acid 508 56 11 89 Free myristic acid 474
436 92 8 Emulsified myristic 508 68 13 87 Free lauric caproic 50:50
474 450 95 5 Emulsified Lauric:caproic 508 0 0 100 Free 40% lauric
in oil of 526 460 87 13 lemon balm Emulsified 40% Lauric in 526 0 0
100 Oil of Lemon Balm De-lipidised Lecithin 497 96 19 81 0.4% W/V
Free fatty acids or blends thereof at 0.5% W/V non-emulsified in
the test and at 0.5% W/V emulsified in 0.4% W/V de-lipidised
lecithin in the test.
[0253] De-lipidised lecithin on its own in the test achieves 81%
inhibition of adhesion. Emulsions of caproic, caprylic, pelargonic,
capric and undecylenic achieve greater than 99% inhibition, and
emulsions of lauric and myristic achieve greater than 86%
inhibition. All non-emulsified free fatty acids achieve less than
17% inhibition. The adhesion inhibitory effect of caprylic acid for
example is amplified by a factor of 14 in emulsified form.
[0254] At the concentrations of test item used in Table 21, the
adhesion inhibitory effect is essentially swamped by the intrinsic
microbicidal effect. It has been demonstrated that the majority of
microbial cells will be dead as a result of exposure to the
microbicidal effect of the emulsified fatty acid, and it must be
assumed that this will influence adhesion.
[0255] A more measure of the amplified adhesion inhibitory effect
attributable to emulsions vs free acid or non-emulsified membrane
lipids may be obtained using a concentration below the Minimum
inhibitory Concentration (MIC) of the emulsified free fatty acid.
The MIC of caprylic acid in the formulation of this invention is
greater than 0.1%.
[0256] A formulation of 0.5% caprylic in 0.4% de-lipidised lecithin
(as used above) is diluted by a factor of 5 in sterile distilled
water to achieve a concentration of 0.1% caprylic in 0.08%
de-lipidised lecithin and further diluted by half to achieve 0.05%
caprylic in 0.04% DLL. These dilutions together with the same
concentrations of DLL and non-emulsified free caprylic acid were
tested in the Buccal Epithelial Cell assay as above. The results
are presented in Table 22.
TABLE-US-00022 TABLE 22 Low Dose Adhesion Inhibitory Effect Blank
Test % % Count/ Count/ Adhe- inhibi- 100 BEC 100 BEC sion tion Free
caprylic acid 0.1% 489 466 95 5 Free caprylic acid 0.05% 487 459 94
6 DLL 0.08% 533 421 85 15 DLL 0.04% 509 453 87 13 0.1% caprylic in
0.08% DLL 499 255 51 49 0.05% caprylic in 0.04% DLL 513 303 59 41
Caprylic acid at 0.1% W/V and 0.05% W/V non-emulsified in the test
and at 0.1% W/V and 0.05% W/V emulsified in 0.08% W/V and 0.04%%
W/V DLL respectively in the test, together with 0.08% and 0.04%
non-emulsified DLL.
[0257] As illustrated in Table 22, the addition of caprylic acid at
concentrations below its MIC (0.1% and 0:05%) amplifies the
adhesion inhibitory effect of 0.08% DLL and 0.04% DLL by more than
a factor of three in both cases.
EXAMPLE 17
Reduction of Antagonistic Effect on Mammalian Cells
[0258] Mammalian cell membranes are susceptible to disruption by
free fatty acids in a manner not dissimilar to their effect on
microbial cell membranes. The effect is not classical cell toxicity
as it relates to superficial cell surface damage and not
interference with metabolic process or nucleic acid replication. It
is ameliorated significantly by body fluids in vivo. Protection
against mammalian cell membrane damage can be enhanced by adding
additional amounts of free membrane lipid to a membrane lipid
emulsion of a free fatty acid. The protective effect is not
confined to the use of membrane lipids. As illustrated here, milk
serum whey protein isolate also serves as a suitable, although not
optimal barrier against mammalian cell damage.
[0259] The test item is an emulsion of 0.5% caprylic acid in 0.4%
de-lipidised lecithin prepared as described in the Methods, and
combined with an equal volume of 200 mM sodium citrate at pH 4.5
also prepared as described in the Methods. Dispersions of 0.4% and
0.8% de-lipidised lecithin were prepared in 200 mM sodium citrate
at pH 4.5, and combined in equal volumes with aliquots of the
emulsion of caprylic acid in de-lipidised lecithin to achieve
emulsions of 0.25% caprylic acid in 0.2% de-lipidised lecithin
suspended in an aqueous solution of 100 mM sodium citrate at pH 4.5
in which further-amounts of either 0.2% or 0.4% de-lipidised
lecithin were dispersed, these are described as Test+0.2% DLL or
Test+0.4% DLL
[0260] Similar suspensions of the same emulsion were prepared in
sodium citrate dispersions of a Whey Protein Isolate (WPI) (Provon
190 from Glanbia PLC) and with Bovine Serum Albumin for comparison
purposes. These are described as Test+0.2% or Test+0.4% WPI or
BSA.
[0261] Raji B lymphocytes were grown as described in the Methods
and viability after a 60 minute exposure to the various test
solutions was assessed using an Invitrogen Countess Cell Viability
meter as described in the Methods. The results are presented in
Table 23 below.
TABLE-US-00023 TABLE 23 Reduction in cell viability at 60 minute
exposure to test items Raji B Lymphocytes Buffer Test + Test + Test
+ Test + Test + Test + control blank Test 0.2% DLL 0.4% DLL 0.2%
WPI 0.4% WPI 0.2% BSA 0.4% BSA % Viable T zero 78 72 75 77 73 76 79
75 71 % Viable T 60 min 72 69 12 59 68 48 53 19 21 % cell survival
92 96 16 77 93 63 67 25 30
[0262] The % cell survival in the test item is just 16% after 60'
minutes. By comparison the control consisting of Raji B cells in
cell culture media lost just 8% viability and the buffer blank
being 100 mM sodium citrate at pH 4.5 was even less at 4%
reduction. With the addition of 0.2% and 0.4% free membrane lipid
(de-lipidised lecithin) % cell survival in the test was increased
from 16% to 77% and 93%, and although not quite as effective, the
addition of free WPI increased cell survival from 16% to 63% and
67%. Bovine serum albumin was considerably less effective in
protecting against cell damage.
[0263] The inclusion of free membrane lipid in an aqueous
dispersion of membrane lipid emulsion has no significant effect on
the microbicidal properties of the membrane lipid emulsion. A late
log phase culture of the yeast Candida albicans grown as described
in the Methods contained 1.33.times.10.sup.7 viable cells per ml.
This was used to inoculate aliquots of each test item from above at
a dilution of 1:10 such that each ml of test item contained in
excess of 6 logs of yeast cells. Samples were withdrawn over time
periods of up to 10 minutes and assessed for residual viability
using the procedures described in the Methods. As illustrated in
Table 24 below, detectable viability was eradicated in less than 5
minutes by all test items.
TABLE-US-00024 TABLE 24 Microbicidal Effect of Free Membrane Lipid
in Membrane Lipid Emulsions Time to kill greater than 6 logs
Candida albicans. Buffer Test + Test + Test + Test + Test + Test +
control blank Test 0.2% DLL 0.4% DLL 0.2% WPI 0.4% WPI 0.2% BSA
0.4% BSA Time Mins NA NA <5 <5 <5 <5 <5 >5
>5
EXAMPLE 18
Use in a Medical Food
[0264] Separate emulsions of caprylic, capric and lauric acid were
prepared as 5.0% W/V fatty acid emulsified in 4.0% de-lipidised
lecithin as described in the Methods. The individual emulsions were
mixed in a ratio of 1:1:1.
[0265] Marvel skim milk powder from Premier International Foods
(UK) Ltd, Spalding, Lincolnshire, England was re-constituted using
90% of the water volume according to the manufacturer's
instructions. Once fully hydrated 10% by volume of the combined mix
of separately emulsified free fatty acids was added and mixed by
stirring, bringing the total volume to 100%.
[0266] Helicobacter pylori was grown on Columbia Blood Agar
supplemented with 5% de-fibrinated sheep blood in an anaerobic jar
using Anaerogen low oxygen gas packs from Oxoid UK. Salmonella
typhimurium and E. coli K12 were grown on Brain Heart Infusion agar
as described in the Methods.
[0267] The microbicidal efficacy of the re-constituted milk
supplemented with the three individually emulsified free fatty
acids was determined using the Minimum Inhibitory Concentration
(MIC) method of agar dilution described in the methods. Dilutions
of the re-constituted skim milk were prepared in sterile distilled
water such that further dilutions of aliquots of these in cooled
agar provided an agar with combined free fatty acid concentrations
ranging from 1% to 0.1% in 0.1% increments and from 0.1% to 0.01%
in increments of 0.01%. Cultures of the three test organisms were
inoculated onto these plates and incubated according to culture
requirements: Helicobacter under low oxygen tension, Salmonella and
E. coli under aerobic conditions all at 37.degree. C.
[0268] The minimum Inhibitory Concentration, being the lowest
dilution where no growth was observed was greater than 0.5% for
Helicobacter and greater than 0.1% for both Salmonella and E.
coli.
* * * * *