U.S. patent application number 17/402535 was filed with the patent office on 2021-12-02 for methods for using antimicrobial compositions.
The applicant listed for this patent is Next Science IP Holdings Pty Ltd. Invention is credited to Matthew F. Myntti.
Application Number | 20210368788 17/402535 |
Document ID | / |
Family ID | 1000005779676 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210368788 |
Kind Code |
A1 |
Myntti; Matthew F. |
December 2, 2021 |
METHODS FOR USING ANTIMICROBIAL COMPOSITIONS
Abstract
A composition that can be used to treat a wide spectrum of gram
positive and gram negative bacteria, including but not limited to
those in biofilm form, and other microbes can be used in
cilia-containing areas such as the sinus cavities and middle/inner
ear, while resulting in no, or very minimal amounts of,
deciliation. Where such a targeted treatment area includes a
biofilm, the composition often can detach and assist in removing
the biofilm from affected tissue. Many embodiments of the
composition are biocompatible.
Inventors: |
Myntti; Matthew F.; (Ponte
Vedra Beach, FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Next Science IP Holdings Pty Ltd |
Chatswood |
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AU |
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|
Family ID: |
1000005779676 |
Appl. No.: |
17/402535 |
Filed: |
August 14, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16314168 |
Dec 28, 2018 |
11090369 |
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PCT/US17/39836 |
Jun 28, 2017 |
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17402535 |
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62357147 |
Jun 30, 2016 |
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62524522 |
Jun 24, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 63/50 20200101;
A61K 47/10 20130101; A61K 47/12 20130101; A61K 38/47 20130101; A61P
31/04 20180101; A01N 63/00 20130101; A61K 9/0043 20130101; C12Y
302/01017 20130101; A01N 37/02 20130101; A61K 9/0046 20130101; A61K
9/08 20130101; A61K 47/20 20130101 |
International
Class: |
A01N 37/02 20060101
A01N037/02; A61P 31/04 20060101 A61P031/04; A61K 9/00 20060101
A61K009/00; A61K 9/08 20060101 A61K009/08; A61K 38/47 20060101
A61K038/47; A61K 47/10 20060101 A61K047/10; A61K 47/12 20060101
A61K047/12; A61K 47/20 20060101 A61K047/20; A01N 63/50 20060101
A01N063/50; A01N 63/00 20060101 A01N063/00 |
Claims
1. A method for treating a cilia-containing area of an animal body,
said method comprising: a) introducing to said area a composition
having a pH of from 6 to 8 and an effective solute concentration of
no more than 400 mOsm/L, wherein said composition consists of (1) a
solvent component that comprises water and at least 2% (w/v) of one
or more organic liquids, and (2) a solute component that comprises
organic acid, no more than 1% (w/v) anionic surfactant, and at
least 0.005% (w/v) of one or more enzymes which are active at
6.ltoreq.pH.ltoreq.8; and b) permitting said composition to lyse
bacteria contained in said area.
2. The method of claim 1 wherein said composition has an effective
solute concentration of no more than 250 mOsm/L.
3. The method of claim 1 wherein said composition has an effective
solute concentration of no more than 200 mOsm/L.
4. The method of claim 1 wherein said organic acid is a
polyacid.
5. The method of claim 4 wherein said polyacid is citric acid.
6. The method of claim 1 wherein said solute component further
comprises a buffer precursor.
7. The method of claim 6 wherein said buffer precursor comprises a
salt of said organic acid.
8. The method of claim 7 wherein said salt of an acid is a sodium
salt of citric acid.
9. The method of claim 1 wherein said solute component comprises no
more than 0.8% (w/v) anionic surfactant.
10. The method of claim 9 wherein said solute component comprises
no more than 0.7% (w/v) anionic surfactant.
11. The method of claim 10 wherein said solute component comprises
no more than 0.5% (w/v) anionic surfactant.
12. The method of claim 1 wherein said cilia-containing area is a
sinus cavity.
13. The method of claim 1 wherein said solute component is free of
anionic surfactant.
14. The method of claim 13 wherein said cilia-containing area is
the middle or inner ear.
15. The method of claim 1 wherein said one or more enzymes is
lysozyme.
16. The method of claim 1 wherein said one or more organic liquids
comprises DMSO.
17. The method of claim 1 wherein said one or more organic liquids
comprises ethanol.
18. A process for treating a sinus cavity of an animal, said method
comprising: a) introducing to said sinus cavity a composition
having a pH of from 6 to 8 and an effective solute concentration of
from 100 to 275 mOsm/L, wherein said composition consists of (1) a
solvent component that comprises water and from 2 to 20% (w/v)
DMSO, and (2) a solute component that comprises 0.1 to 50 g/L
organic acid, from 10 to 65 g/L anionic surfactant, from 60 to 400
ppm of one or more enzymes which are active at
6.ltoreq.pH.ltoreq.8, and a buffer precursor; and b) permitting
said composition to lyse bacteria contained in said sinus
cavity.
19. A method for treating the middle or inner ear of an animal,
said method comprising: a) introducing to the ear cavity of said
animal a composition having a pH of from 6 to 8 and an effective
solute concentration of from 100 to 275 mOsm/L, wherein said
composition consists of (1) a solvent component that comprises
water and from 2 to 9% (w/v) ethanol, and (2) a solute component
that comprises 0.1 to 50 g/L organic acid, less than 0.5 g/L
anionic surfactant, from 60 to 400 ppm of one or more enzymes which
are active at 6.ltoreq.pH.ltoreq.8, and a buffer precursor; and b)
permitting said composition to lyse bacteria contained in said ear
cavity.
20. The method of claim 19 wherein said one or more enzymes is
lysozyme.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
16/314,168, having a 35 U.S.C. .sctn. 371(c) date of 28 Dec. 2018
and issued as U.S. Pat. No. 11,090,369 on 17 Aug. 2021, which was a
national stage entry of international application
PCT/US2017/039836, filed 28 Jun. 2017, which claims priority to
U.S. provisional patent application nos. 62/357,147, filed 30 Jun.
2016, and 62/524,522, filed 24 Jun. 2017, which are incorporated
herein by reference.
BACKGROUND INFORMATION
[0002] Microbes are found virtually everywhere, often in high
concentrations, and are responsible for a significant amount of
disease and infection. Eliminating these microorganisms from
targeted tissues is often desirable and sometimes critically
important.
[0003] Bacteria present special challenges because they can exist
in a number of forms, including planktonic, spore and biofilm, and
their various self-preservation mechanisms make treating and/or
eradicating them extremely difficult. For example, the bacteria in
biofilms or spores are down-regulated (sessile), making them
resistant to attack by a large group of antibiotics and
antimicrobials that are effective only during the active parts of a
bacterium's lifecycle, e.g., cell division.
[0004] In a biofilm, microbes such as bacteria or fungi interact
with and adhere to surfaces, forming colonies which facilitate
continued growth. The microbes produce exopoly-saccharide (EPS)
and/or extracellularpolysaccharide (ECPS) macromolecules that keep
them attached to a surface and form a protective barrier effective
against many forms of attack. The small diameter of flow channels
in the EPS/ECPS macromolecular matrix, which restricts the size of
molecules that can reach the underlying microbes, and consumption
of biocides through interactions with portions of the EPS/ECPS
macromolecular matrix and microbe secretions and waste products
contained therein probably play roles in the protective barrier
function.
[0005] Due to the protection afforded by the macromolecular matrix
and their down-regulated state, microbes in a biofilm state are
very difficult to treat. The types of biocides and antimicrobials
effective in treating microbes in this form often are strongly
acidic or caustic, and often oxidizing due to the presence of
halogen atoms, oxygen atoms, or both. Large dosages of such
chemicals must be allowed to contact the biofilm for extended
amounts of time to be effective, which makes them impractical for
many applications.
[0006] Compositions intended for use in connection with compromised
animal/human tissue which solvate a biofilm matrix so that it can
be rinsed or otherwise removed from infected tissue have been
described in, e.g., U.S. Pat. Nos. 7,976,873, 7,976,875, 7,993,675,
and 7,959,943. Compositions based on similar components but
intended for other uses have been described in U.S. Pat. Nos.
8,940,792 and 9,314,017 and U.S. Pat. Publ. Nos. 2010/0086576,
2014/0242188, and 2016/0073628.
[0007] However, animal testing has shown that compositions such as
those described in the preceding paragraph, when applied to
ciliated tissue such as exist in the sinus cavities and inner ear,
tend to result in deciliation, i.e., the loss in functionality
and/or removal of cilia, which are the relatively thick protruding
organelles found in and projecting from the bod of eukaryotic
cells. Sinus cavity cilia facilitate clearance of the sinuses,
while those in the ear act as sound receptors.
[0008] Testing suggests that contributing factors in deciliation
likely include the presence of ionic surfactants, high effective
solute concentrations, and pH. Unfortunately, compositions
described in the documents listed in the preceding paragraph all
call for at least 0.2% (w/w) surfactant, and many require or prefer
low pH and very high effective solute concentrations, i.e.,
osmolarities.
[0009] Because biofilms deciliate affected tissue anyway and
because cilia in the sinus cavities can regrow within days, limited
deciliation which does not impede clearing of the sinuses might be
an acceptable side effect in a product intended for use in those
sinus cavities not connected to the inner ear via a Eustachian
tube. However, compositions intended for use in treating portions
of the ear inward of the tympanic membrane (or treating a sinus
cavity that is connected to the inner ear) cannot be permitted to
result in deciliation because inner ear cilia do not regrow,
resulting in irreparable hearing loss.
[0010] That which is desirable is a composition that can
effectively treat microbes present in cilia-containing areas such
as the sinus cavities and middle/inner ear, particularly microbes
in a biofilm form, without resulting in unacceptable levels of
deciliation. A composition that can accomplish the foregoing while
also causing or facilitating detachment of biofilms from affected
tissue is particularly desirable.
[0011] Such an antimicrobial composition that can be provided in a
variety of viscosities and introduced to the targeted area via more
than one delivery route is particularly desirable.
[0012] Further desirable is a composition that is sufficiently
biocompatible so as to not require removal via irrigation using,
for example, a saline rinse.
SUMMARY
[0013] The present invention is directed to compositions that can
be used to treat microbes including but not limited to bacteria,
including those in biofilm form. The term "treat" includes killing,
inactivating and/or removal.
[0014] A composition according to the present invention is
effective against a wide spectrum of gram positive and gram
negative bacteria and exhibits lethality toward other microbes such
as viruses, fungi, molds, and yeasts.
[0015] Advantageously, this composition can effectively kill
microbes present in cilia-containing areas such as the sinus
cavities and middle/inner ear, even microbes in a biofilm form,
while resulting in no, or very minimal amounts of, deciliation.
Where such a targeted treatment area includes a biofilm, the
composition often can detach and assist in removing the biofilm
from affected tissue.
[0016] The composition typically is provided as a liquid having a
viscosity similar to that of water, but can be thickened to provide
a variety of forms with a range of viscosities. It also can be
delivered via techniques employing a variety of presently available
equipment.
[0017] All embodiments of the composition are biocompatible, while
many embodiments are ciliacompatible.
[0018] The composition has a near-neutral pH, typically from 6 to
8, and includes a moderate amount of osmotically active solutes,
often having an effective solute concentration of no more than 400
mOsm/L, commonly no more than 250 mOsm/L and more commonly no more
than 200 mOsm/L. Embodiments intended for use in the sinus cavities
can include no more than 1.0, 0.8, 0.7 or 0.5% (w/v) of one or more
anionic surfactants, while other embodiments intended for use in
the middle or inner ear can include no added surfactant(s). The
composition includes at least 2% (w/v) of one or more non-aqueous
liquids, with the upper limit determined in large part by the
intended end use of the composition. The composition also includes
at least 0.005% (w/v) of one or more enzymes which are active at
6.ltoreq.pH.ltoreq.8 so as to facilitate microbial cell wall
rupture by catalyzing and/or easing the breaking of chemical bonds
present in or between molecules in those cell walls. (Because
enzymes vary so widely in terms of chemical structure and targeted
utility, the upper limit often is based on the amount of a
particular enzyme, if any, that results in a level of ciliotoxicity
deemed to be unacceptable for a particular end use.)
[0019] The composition is effective at interrupting or breaking
ionic crosslinks in the macromolecular matrix of a biofilm, which
facilitates passage of the solutes, surfactant (if present) and
enzyme through the matrix to the microbes (e.g., bacteria)
entrained therein and/or protected thereby. The composition thus
bypasses and/or disables the biofilm defenses, allowing previously
protected microbes to be accessed and killed, typically by
processes that include inducing membrane leakage in bacteria,
leading to cell lysis.
[0020] Also provided are methods for treating affected areas
including application of non-solid compositions can be applied to
an affected area. The composition can be non-flowing if intended to
be left in place or can be a liquid if intended to irrigate or
otherwise flow over or around a treatment area.
[0021] Embodiments of the composition can be used to treat chronic
otitis media, cholesteatoma and other bacterial ear conditions, as
well as chronic rhinosinusitis and other bacterial sinus
conditions.
[0022] Often, a flowable form of the composition is introduced
peri- or post-surgery performed on an affected area. For example, a
composition can be introduced into the middle ear via a
tympanostomy tube immediately or soon after its insertion or, if
deemed necessary desirable, during a post-surgical follow-up
evaluation.
[0023] To assist in understanding the following description of
various embodiments, certain definitions are provided immediately
below. These are intended to apply throughout unless the
surrounding text explicitly indicates a contrary intention: [0024]
"comprising" means including but not limited to those ingredients
which follow the term; [0025] "consisting of" means including only
those ingredients which follow the term as well as minor amounts of
inactive additives or adjuvants; [0026] "consisting essentially of"
means including only the listed ingredients, minor amounts (less
than 2%, 1%, 0.5%, 0.25%, or 0.1%, all w/v) of other ingredients
that supplement the antimicrobial activity and/or provide a
secondary effect (e.g., antifogging, soil removal, wound cleaning,
etc.) that is desirable in view of the intended end use, and/or
inactive additives or adjuvants; [0027] "microbe" means any type of
microorganism including, but not limited to, bacteria, viruses,
fungi, viroids, prions, and the like; [0028] "antimicrobial agent"
means a substance having the ability to cause greater than a 90% (1
log) reduction in the number of one or more of microbes; [0029]
"active antimicrobial agent" means an antimicrobial agent that is
effective only or primarily during the active parts of a microbe's
lifecycle, e.g., cell division, and the activity of which involves
disruption of a cellular process; [0030] "biofilm" means a
community of microbes, particularly bacteria and fungi, attached to
a surface with the community members being contained in and/or
protected by a self-generated macromolecular matrix; [0031]
"entrenched biofilm" means a biofilm that has reached a steady
state mass after a growth period of two or more days; [0032]
"buffer" means a compound or mixture of compounds having an ability
to maintain the pH of a solution to which it is added within
relatively narrow limits; [0033] "buffer precursor" means a
compound that, when added to a mixture containing an acid or a
base, results in a buffer; [0034] "polyacid" means a compound
having at least two carboxyl groups and specifically includes
dicarboxylic acids, tricarboxylic acids, etc.; [0035] "solvate"
means the process of taking a solid material into solution in a
liquid; [0036] "sequestering agent" means a chemical that assists
in solvating a compound and in preventing the solvated form of that
compound from coming out of solution; [0037] "metal ion
sequestering agent" means a sequestering agent that works in
connection with one or more metal ions, particularly alkali and
alkaline earth metals; [0038] "chronic otitis media" means otitis
media with effusion or recurrent otitis media; [0039] "soil load"
means a solution of one or more organic and/or inorganic substances
added to the suspension of a test organism to simulate the presence
of body secretions, excretions, and the like; [0040] "inoculum"
means a solution containing bacteria, growth solution (e.g.,
tryptic soy broth) and protein soil load; and [0041] "substituted"
(in reference to a functional group) means containing a heteroatom
or functionality (e.g., hydrocarbyl group) that does not interfere
with the intended purpose of the group in question. [0042] "dwell
time" means the amount of time that an antimicrobial agent is
allowed to contact a bacterial biofilm; [0043] "biocompatible"
means presenting no significant, long-term deleterious effects on
or in a mammalian species; [0044] "ciliotoxic" means resulting in
significant cleavage or loss of function of cilia; and [0045]
"ciliacompatible" means not ciliotoxic.
[0046] Hereinthroughout, pH values of a liquid are those which can
be obtained from any of a variety of potentiometric techniques
employing a properly calibrated electrode, and effective solute
concentrations preferably are determined by latent heat of fusion
calculations from a properly calibrated DSC unit-produced scan
acquired over a temperature range that includes the melting
temperature of a given liquid composition.
[0047] Any numerical limitation used herein includes an appropriate
degree of uncertainty based on the number of significant places
used with that particular numerical limitation. For example, "up to
5.0" can be read as setting a lower absolute ceiling than "up to
5."
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is a perspective, schematic view of a disassembled
simplified multiwell array, of the type employed in a minimum
biofilm eradication concentration (MBEC) high throughput screening
assay.
[0049] FIG. 2 is a top view of an 8.times.12 multiwell array
employed in the testing of treating compositions of the present
invention.
[0050] FIG. 3 is a top view of a challenge plate matrix employed in
the testing of treating compositions of the present invention.
[0051] FIGS. 4 and 5 depict Air-Liquid Interface (ALI) model
ciliotoxicity testing results for a sinus treatment composition,
with active cilia area plotted against time in FIG. 4 and cilia
beat frequency plotted against time in FIG. 5.
[0052] FIG. 6 depicts averaged baseline hearing threshold data for
eight guinea pigs.
[0053] FIGS. 7 and 8 depict averaged threshold shifts in hearing
for eight guinea pigs, with FIG. 7 being based on data collected 7
days after liquid insertion and FIG. 8 being based on data
collected 28 days after liquid insertion.
DETAILED DESCRIPTION
[0054] Compositions such as those summarily described in the
preceding section can be used to break down, remove and/or disrupt
biofilms including, advantageously, bacterial biofilms located in
the middle or inner ear or the sinus cavities of an animal,
particularly a mammal. The compositions are biocompatible and safe
to use in and around the delicate tissues and structures of those
areas because they are free of constituent materials which might
harm such tissues or structures or unduly compromise long-term
hearing.
[0055] Embodiments of the composition have a sufficiently low
viscosity to enable delivery using techniques such as spray
application, lavage, misting, mopping, wicking and dripping. These
and other embodiments of the composition also can be easily removed
from the treatment site by subsequent flushing, rinsing, and
draining, although many such embodiments are sufficiently
biocompatible to allow for absorption.
[0056] While not wishing to be bound by theory, a metal ion
sequestering agent in the composition might complex or otherwise
bond with metal ions which crosslink, bridge or otherwise assist in
binding together polymer chains in the EPS/ECPS matrix of a
biofilm. Other components of the composition then might surround
the unbound polymer chains or fragments, breaking down the matrix,
solvating the uncrosslinked polymer chains or fragments, and
bringing them into solution or suspension so that they can be
flushed or otherwise removed from the treatment area using, for
example, additional amounts of the solvating system or a separate
rinsing agent.
[0057] The composition includes solvent and solute components.
[0058] The solvent component of the composition includes water and
at least one non-aqueous liquid.
[0059] Water has a high solute holding capability, good wetting
properties, excellent biocompatibility, environmental friendliness,
and low cost. Essentially any source of water can be used, although
those that are relatively free of bacteria without advance
treatment are preferred. The water need not be distilled,
deionized, or the like, although such treatments certainly are not
excluded, particularly where the water employed might include
undesirable solutes which might interfere with the intended purpose
of the composition. To enhance solubility of one or more of the
other components of the composition, the water can be heated.
[0060] The one or more non-aqueous liquids typically has/have a
.delta..sub.p value no higher than that of water, where
.delta..sub.p is the dipolar intermolecular force (polarity) Hansen
Solubility Parameter (HSP), a common method for predicting whether
one material will dissolve in another to form a solution; the HSP
values for most commonly used solvents are well documented.
[0061] Each component in a mixture or composition has three HSPs:
dispersion, dipole-dipole (polarity) interactions, and hydrogen
bonding. These parameters are generally treated as coordinates in
three dimensions, with HSP characterizations being visualized using
a spherical representation: the 3D coordinates are at the center of
the sphere with the radius of the sphere (R.sub.0 or "interaction
radius") indicating the maximum difference in affinity tolerable
for a "good" interaction with a solvent or solute. In other words,
acceptable solvents lie within the interaction radius, while
unacceptable ones lie outside it.
[0062] The distance between the HSPs of two materials in so-called
Hansen space (R.sub.a) can be calculated according to the following
formula:
(R.sub.a).sup.2=4(.delta..sub.d2-.delta..sub.d1).sup.2+(.delta..sub.p2-.-
delta..sub.p1).sup.2+(.delta..sub.h2-.delta..sub.h1).sup.2 (I)
where .delta..sub.d is the energy from dispersion forces between
the molecules, .delta..sub.p is the energy from dipole-dipole
intermolecular forces, and .delta..sub.h is the energy from
hydrogen bonds between molecules.
[0063] A simple composite affinity parameter, the Relative Energy
Difference (RED), represents the ratio of the calculated HSP
difference (R.sub.a) to the interaction radius (R.sub.0), i.e.,
RED=R.sub.a/R.sub.0. In situations where RED<1.0, the
solubilities of the molecules are sufficiently similar that one
will dissolve in the other. In situations where RED<1.0, the
solubilities of the molecules are not sufficiently similar for one
to dissolve the other. In situations where RED 1.0, partial
dissolution is possible.
[0064] The dipole-dipole interaction Hansen solubility parameter
for a particular solution or mixture of solvents can be calculated
according to the following formula:
.delta. p = n i = 1 .times. ( .delta. di .times. x di ) ( II )
##EQU00001##
where .delta..sub.di is the energy from dipolar intermolecular
force for solvent i, x.sub.di is the percentage of solvent i in the
solvent portion of the composition, and n is the total number of
solvent components.
[0065] Hereinthroughout, the .delta..sub.p value for a given
solvent or combination of solvents is determined at room
temperature (because solubility typically increases with increasing
temperature, meaning that the dissolution rate of the
macromolecular matrix and the bacterial cell wall proteins will
increase, the efficacy of the inventive composition is expected to
increase at higher temperatures) and pH values are those which can
be obtained from any of a variety of potentiometric techniques
employing a properly calibrated electrode.
[0066] More details about HSPs and related concepts can be found US
Pat. Publ. No. 2016/0073628.
[0067] The solvent component of the composition includes at least
one non-aqueous liquid, typically one with a .delta..sub.p value no
higher than that of water (.delta..sub.p.apprxeq.16.0 MPa.sup.1/2).
Exemplary composition intended for use in a sinus application can
employ 5-20% (w/v) dimethyl sulfoxide (DMSO) with an overall
.delta..sub.p.apprxeq.16.0 MPa.sup.1/2, while exemplary
compositions intended for use in an otic application can employ
5-15% (w/v) ethanol with an overall .delta..sub.p 15.4
MPa.sup.1/2.
[0068] In certain embodiments, preference can be given to those
organic compounds which are, or can be made to be, highly soluble
in water and to those which are ciliacompatible. Additionally,
preference can be given to any organic liquid that has been deemed
to be safe and "inactive" (by regulatory bodies) at intended usage
levels.
[0069] The composition includes at least 2% (w/v) of one or more
non-aqueous (organic) liquids, with the upper limit determined in
large part by the intended end use of the composition, i.e., the
upper limit for sinus applications probably is higher than that for
otic applications. In some embodiments, the lower limit can be 2.1,
2.2, 2.3, 2.4 or even 2.5%, with all of the foregoing being
presented in w/v format. Exemplary ranges include 2.25-15% (sinus)
or 2.0-10% (otic), with typical ranges being 2.5-13% (sinus) and
2.1-9% (otic) and preferred ranges being 2.5-12.5% (sinus) and
2.2-8% (otic), again with all of the forgoing being presented in
w/v format.
[0070] In addition to the solvent component, the composition also
includes as a primary component a solute component which can
contain as few as two sub-components: the dissociation product(s)
of at least one metal ion sequestering agent and at least one
effective enzyme. In embodiments not intended for otic
applications, anionic surfactant also can be included. The
dissociation product(s) of one or more salts also can be included
to increase effective solute concentration. Each of the foregoing
ingredients generally is considered to be biocompatible.
[0071] The metal ion sequestering agent can be an acid or base
capable of complexing or otherwise reacting with one or more metal
ions in the EPS/ECPS matrix of a biofilm. Metal ions of particular
interest, due to their likely involvement in the targeted biofilms,
include sodium, calcium and iron. The metal ion sequestering agent
desirably is water soluble, nontoxic and not prone to aggravate
long-term hearing loss.
[0072] Acids generally are preferred over bases, although either
type of sequestering agent can be used. Preference can be given to
those metal ion sequestering agents which are biocompatible.
Alternatively or additionally, preference can be given to those
metal ion sequestering agents which can act to chelate the metallic
cations ionic involved in crosslinking the macromolecular matrix of
a biofilm. The metal ion sequestering agent preferably is not
considered to be an oxidizer, particularly if it is an acid.
Additionally, strong preference can be given to those acids and
bases that have been deemed to be safe or "inactive" (by regulatory
bodies) at intended usage levels
[0073] Acidity is achieved by adding to water (or vice versa) one
or more acids, specifically strong (mineral) acids such as HCl,
H.sub.2SO.sub.4, H.sub.3PO.sub.4, HNO.sub.3, H.sub.3BO.sub.3, and
the like or, preferably, weak acids, particularly organic acids
and, preferably, organic polyacids. Examples of organic acids
include monoprotic acids such as formic acid, acetic acid and
substituted variants, propanoic acid and substituted variants
(e.g., lactic acid, pyruvic acid, and the like), any of a variety
of benzoic acids (e.g., mandelic acid, chloromandelic acid,
salicylic acid, and the like), glucuronic acid, and the like;
diprotic acids such as oxalic acid and substituted variants
(including oxamic acid), butanedioic acid and substituted variants
(e.g., malic acid, aspartic acid, tartaric acid, citramalic acid,
and the like), pentanedioic acid and substituted variants (e.g.,
glutamic acid, 2-ketoglutaric acid, and the like), hexanedioic acid
and substituted variants (e.g., mucic acid), butenedioic acid (both
cis and trans isomers), iminodiacetic acid, phthalic acid,
ketopimelic acid, and the like; triprotic acids such as citric
acid, 2-methylpropane-1,2,3-tricarboxylic acid,
benzenetricarboxylic acid, nitrilotriacetic acid, and the like;
tetraprotic acids such as prehnitic acid, pyromellitic acid, and
the like; and even higher degree acids (e.g., penta-, hexa-,
heptaprotic, etc.). Where a tri-, tetra-, or higher acid is used,
one or more of the carboxyl protons can be replaced by cationic
atoms or groups (e.g., alkali metal ions), which can be the same or
different. Preferred acids include mono-, di- or tri-protic citric
acid, acetic acid, octanoic acid and glutamic acid.
[0074] Basicity is achieved by adding to water (or vice versa) one
or more bases such as, but not limited to, alkali metal salts of
weak acids including acetates, fulmates, lactates, phosphates, and
glutamates; alkali metal nitrates; alkali metal hydroxides, in
particular NaOH and KOH; alkali earth metal hydroxides, in
particular Mg(OH).sub.2; alkali metal borates; NH3; and alkali
metal hypochlorites (e.g., NaClO) and bicarbonates (e.g.,
NaHCO.sub.3). Again, preference is given to those compounds which
are, or can be made to be, soluble in water and which are
biocompatible.
[0075] The concentration of metal ion sequestering agent added to
water, or vice versa, is relatively unimportant because of the
targeted effective solute concentration and hydronium ion
concentration, i.e., 6.ltoreq.pH.ltoreq.8. Thus, use of a strong
acid or base militates against addition of large amounts of that
acid/base. Additionally, the relatively moderate effective solute
concentration limits (discussed below) argue against significant
amounts of buffer precursor. Conversely, use of a very weak acid or
base permits addition of a much larger amount of the acid/base
and/or a much reduced amount of a buffer precursor.
[0076] Each of U.S. Pat. Nos. 8,940,792 and 9,314,017, as well as
U.S. Pat. Publ. Nos. 2010/0086576, 2014/0242188, and 2016/0073628,
suggests that a decrease or increase (depending on whether an acid
or base is used) in pH generally corresponds with enhanced
efficacy. However, because the present composition is desired to be
biocompatible and have minimal ciliotoxicity, the targeted pH range
is one log unit either side of neutral. Thus, a key
efficacy-enhancing variable from those prior teachings is not
available in compositions of the present invention. The present
composition has a targeted pH (t) which generally is on the order
of t=6.5.+-.v or t=7.5.+-.v where v represents 0.4, 0.3, 0.25, 0.2,
0.15, 0.1 or 0.05.
[0077] The amount(s) of acid(s) or base(s) necessary to reach a
given t value will, of course, depend on the strength of the
particular acid(s) or base(s) used. Because even small amounts of
those compounds considered to be weak acids or bases will adjust a
composition's pH, respectively, below or above the aforementioned t
values, the solute component almost always includes sufficient
amounts of a buffer precursor (discussed below) so as to provide a
composition having a desired t.
[0078] In addition to metal ion sequestering agent, each of the
documents set forth in the preceding paragraph requires
moderate-to-high levels of one or more surfactants. Because the
compositions of the present invention are intended to be used in
the presence of cilia and because many types of surfactant are
known to be ciliotoxic, inclusion of surfactants is
contraindicated. For embodiments of the composition intended for
otic applications, this means that separately added surfactants are
avoided altogether or strictly limited (e.g., less than 0.5, 0.41,
0.33, 0.25, 0.21, 0.17, 0.13, 0.09 or 0.05%, all w/v), while very
limited amounts of one or more anionic surfactants can be included
in embodiments intended for sinus applications. Again, this means
that an efficacy-enhancing option from the aforementioned prior
teachings is not available (or much less available) in compositions
of the present invention
[0079] The amount of anionic surfactant to be included in
embodiments intended for use in sinus-related applications
generally is less than 1.0%, commonly less than 0.75%, typically
less than 0.5%, preferably less than 0.4%, more preferably less
than 0.35%, and most preferably less than 0.3%, all presented in
w/v format. Where one or more anionic surfactant(s) is/are included
in a sinus-targeted composition, the total amount present generally
is from 0.02 to 0.67%, commonly from 0.03 to 0.55%, typically from
0.04 to 0.42%, preferably from 0.05 to 0.39%, even more preferably
from 0.06 to 0.36%, and still more preferably from 0.08 to 0.33%,
all again w/v.
[0080] Potentially useful anionic surfactants include, but are not
limited to, sodium chenodeoxycholate, N-lauroylsarcosine sodium
salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt,
sodium cholate hydrate, sodium deoxycholate, sodium dodecyl
sulfate, sodium glycodeoxycholate, sodium lauryl sulfate, and the
alkyl phosphates set forth in U.S. Pat. No. 6,610,314.
[0081] The enzyme sub-component of the solute component can be any
one or more which is/are capable of facilitating microbial cell
wall rupture by catalyzing and/or easing the breaking of chemical
bonds present in or between molecules in those cell walls. One
category believed to be particularly effective is glycosidase,
particularly the species lysozyme. Testing to date has shown that
lysozyme exhibits some efficacy at amounts as low as 80 ppm, i.e.,
0.08 g lysozyme per kg of composition. Commonly employed amounts of
enzyme(s) are at least 100 ppm, at least 125 ppm, and at least 150
ppm. Exemplary ranges include 85 to 500 ppm, 90 to 450 ppm, 95 to
400 ppm, 100 to 350 ppm, 105 to 300 ppm, 110 to 275 ppm, 115 to 250
ppm, 120 to 225 ppm, 125 to 200 ppm, 130 to 190 ppm, 135 to 185
ppm, and 140 to 180 ppm.
[0082] Composition efficacy generally increases with the presence
of at least moderate effective solute concentrations, which
generally increases in proportion with the amounts of solute
subcomponents employed. However, the amounts of two of those
subcomponents--metal ion sequestering agent and surfactant--are
severely limited.
[0083] To increase the effective solute concentration (tonicity) of
a composition, one or more types of other water soluble compounds
can be included in the solute component. Such compounds, upon
dissociation, increase the effective amount of solutes in the
composition without greatly impacting the molar concentration of
hydronium and hydroxyl ions.
[0084] Effective solute concentration of a composition can be
increased by adding large amounts of ionic compounds, particularly
electrolytes; see, e.g., U.S. Pat. No. 7,090,882. Essentially any
compound that at least partially dissociates in water and/or the
organic liquid(s) employed in the solvent component can be used to
achieve this effect, with exemplary compounds including, but not
being limited to, phosphates, acetates and any material deemed to
be an "inactive ingredient" in injections, gels, creams, lotions,
and/or ointments by governmental regulatory bodies.
[0085] A preferred method of increasing composition tonicity is
employing a buffer precursor as a subcomponent of the solute
component. For example, where the solute component includes one or
more acids, one or more salts of those or other acids can be
employed as solute subcomponent(s) which, in addition to increasing
tonicity of the composition, provides a pH buffer to it. Where x
moles of an acid are employed as a subcomponent of the solute
component, an excess (e.g., 2x-8x) of one or more salts of that
acid can be included as a separate subcomponent. (The same is true
for basic compositions, mutatis mutandis.) The identity of the
countercation of the acid salt (or counteranion of the base salt)
is not particularly important. Where the salt of a polyacid is used
as a buffer precursor, all or fewer than all of the carboxyl
hydrogen atoms can be replaced; for example, mono-, di- and
trisodium citrate all constitute potentially useful buffer
precursors but the latter provides a greater theoretical buffering
capacity than either of the other two. (Again, the same is true for
salts of a polybase, mutatis mutandis.)
[0086] Regardless of how achieved, the tonicity of the composition
is moderately high, with an effective solute concentration of from
100 to 300 mOsm/L being common and 200.+-.10 mOsm/L being typical.
Embodiments of the composition can exhibit minimum solute
concentrations of 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175 or 180 mOsm/L and maximum solute
concentrations of 275, 260, 250, 245, 240, 235, 230, 225, 220, 215,
210, 205, 200, 195, 190 or 185 mOsm/L. Ranges based on each of the
minimums and each of the maximums are envisioned, with some
exemplary options including, but not being limited to, 110 to 275,
125 to 250, 150 to 250, 160 to 240, 160 to 225, 175 to 250, 170 to
240, 170 to 230, 180 to 240, 180 to 235, and 180 to 220 mOsm/L.
[0087] Although unnecessary and typically contraindicated, any of a
variety of additives and adjuvants can be included in the solute
component to make a composition more amenable for use in a
particular end-use application with negatively affecting its
efficacy in a substantial manner. Examples include, but are not
limited to, fragrances, pigments, dyes, essential oils, foaming
agents, flavors, preservatives (e.g., antioxidants) and the
like.
[0088] The solute component thus provides to the composition a
near-neutral pH, typically from 6 to 8, and a moderate amount of
osmotically active solutes, often having an effective solute
concentration of no more than 250 mOsm/L and commonly no more than
200 mOsm/L. Some embodiments include no more than 0.5% (w/v) of one
or more anionic surfactants, while others include no added
surfactant(s). The composition also includes a relatively small
amount of one or more effective enzymes. The foregoing can be
tabulated as set forth below, with any value in a given row being
combinable with any value for each of the other subcomponent:
TABLE-US-00001 TABLE 1a otic compositions Solute subcomponents
metal ion sequestering 0.05-100, 0.1-50, 0.15-25, 0.2-10, 0.5-5
agent(s), g/L surfactant, g/L <2.5, <2, <1.5, <1,
<0.5, ~0 enzyme, ppm 60 to 400, 100 to 300, 125 to 250, 150 to
225 other, including salts 0.5-100, 0.6-75, 0.7-50, 0.8-30 1-25 of
sequestering agent(s),g/L Tonicity, mOsm/L 100-275, 125-250,
150-225, 175-220 C.sub.2-C.sub.4 alcohol, % (v/v) 1.8-10, 2.0-9,
2.1-8, 2.2-7, 2.3-6
TABLE-US-00002 TABLE 1b sinus compositions Solute subcomponents
metal ion sequestering agent(s), same as otic g/L surfactant, g/L
3-75, 6-70, 10-65, 12-60, 15-56 enzyme, ppm same as otic other,
including salts of 0.5-150, 1-125, 1.5-100, 2-75 3-50 sequestering
agent(s), g/L Tonicity, mOsm/L same as otic DMSO, % (v/v) 2.0-20,
2.2-17, 2.3-15, 2.4-13, 2.5-12.5
[0089] Various embodiments of the present invention have been
provided by way of example and not limitation. As evident from the
foregoing tables, general preferences regarding features, ranges,
numerical limitations and embodiments are to the extent feasible,
as long as not interfering or incompatible, envisioned as being
capable of being combined with other such generally preferred
features, ranges, numerical limitations and embodiments.
[0090] The composition can be prepared in a number of ways.
Description of an exemplary method follows.
[0091] Each of the solute subcomponents other than the enzyme(s)
can be added to sufficient water to constitute 60-90% of the
calculated desired volume. This solution can be stirred and/or
heated if desired. The desired amount of organic liquid(s) and
enzyme(s) then can be added. Once stirring, if used, is complete,
sufficient water is added so as to bring the composition to the
calculated tonicity and pH value. Advantageously, no special
conditions or containers are needed to store the composition for an
extended time, although refrigeration can be used if desired.
[0092] The composition conveniently can be provided as a solution,
although other forms might be desirable for certain end-use
applications. Accordingly, the composition can provided as a
soluble powder (for subsequent dilution, an option which can reduce
transportation costs), a slurry or emulsion, or a thicker form such
as a gel (including hydrogels, organogels and xero-gels) or paste
(i.e., a suspension in an organic base such as a fatty acid),
either of which might be particularly useful for providing
increased residence times. For the latter, the composition can
include additional ingredients such as a coalescent (e.g.,
polyvinylpyrrolidone). Salves or ointments, aerosols, foams, and
even suspensions also are possible.
[0093] An advantage of the composition described herein is an
ability to detach biofilms from the tissues to which they are
attached. Regardless of whether this occurs, the composition can
significantly reduce the number of viable bacteria remaining on or
around the affected tissue.
[0094] Even where use of a composition does not result in
detachment of a biofilm, embodiments of the composition can provide
large reductions in the number of bacteria, even with extremely
short residence times. For example, a 1, 2, 3 or 4 log (99.99%)
reduction in the number of bacteria in an entrenched biofilm with a
3, 4, 5, 7, 8, 9, or 10 minute residence time is possible.
[0095] The composition can act at least in part to interrupt or
break ionic crosslinks in the macromolecular matrix of a biofilm,
facilitating the passage of solutes and surfactant through the
matrix to bacteria entrained therein and/or protected thereby.
Disruption of the macromolecular matrix advantageously also can
result in detachment of the biofilm, alternatively or in addition
to treating bacteria entrained in that matrix.
[0096] The majority of the foregoing discussion has centered on
biofilms, particularly bacterial biofilms. This is unsurprising
given that a majority of sinus and otic issues have a bacterial
origin. However, the composition exhibits efficacy against
bacterial forms other than biofilms and against microbes other than
bacteria. Advantageously, the composition can kill and/or prevent
growth of microbes, regardless of their phase of life cycle.
[0097] With respect to bacteria, each of the lag phase (metabolic
protein production), log phase (reproduction) and stationary phase
(approximately equal amounts of dying, metabolizing and reproducing
bacteria) of a bacterium's lifecycle technically constitutes an
"active" phase. Regardless of whether an individual bacterium is
dormant, reproducing, or metabolizing, the composition can kill it
or prevent it from growing.
[0098] Bacteria that are part of biofilms often are dormant (not
metabolizing or reproducing), and this lack of cellular processes
(inactivity) often provides resistance to antibiotics, which
require active metabolism or reproduction for efficacy, and other
active antimicrobials.
[0099] Viruses technically are not living microbes because they
require a host for reproduction. Nevertheless, the composition is
capable of disrupting, penetrating and/or dissolving the protein
coating(s) on a virus. The ability to attack these protective
structures means that the composition exhibits efficacy against a
virus before it ever achieves the ability to reproduce after
cellular infection.
[0100] Ciliotoxicity generally increases with increasing surfactant
concentration, increasing tonicity, and/or departure of pH from
neutral. Given the foregoing description, the ordinarily skilled
artisan can provide a ciliacompatible composition that remains
effective against microbes in biofilm form.
[0101] The composition can be employed in a variety of ways.
[0102] For otic applications, it can be delivered to the targeted
areas of the ear during and/or after surgery. This might be as
simple as washing or rinsing the outer surface of a tympanic
membrane, for example, one on which a surgical procedure is to be
performed. (In such cases where the composition is not expected to
pass the tympanic membrane, a composition with more aggressive pH
and tonicity values can be employed.) For procedures involving
access of the middle/inner ear, the composition can be delivered
through a tympanostomy tube or via syringe inserted through a
perforation or incision in the tympanic membrane. In both cases, a
medical professional can continue to insert composition until
liquid backflow is observed. (A typical human middle ear holds 1 to
1.5 mL of liquid, by way of example.)
[0103] For sinus applications, the composition can be introduced to
the sinus cavity via a surgical technique such as trephination or
via a remote delivery mechanism such as, e.g., a Hydrodebrider.TM.
endoscopic sinus irrigation system (Medtronic; Minneapolis, Minn.)
or a Relieva Vortex.TM. sinus irrigation catheter (Acclarent, Inc.;
Irvine, Calif.). Regardless of delivery mechanism, a medical
profession can continue delivering composition to the targeted
cavity(ies) until returning effluent appears visually clear.
[0104] Regardless of where used, the composition can be permitted a
dwell of time of a few seconds up to several hours. The targeted
dwell time typically depends on the nature of the patient (e.g.,
ability to be sufficiently immobile to permit a long dwell time) as
well as the physiology of the area to be treated, e.g., whether
liquid introduced to that area naturally drains or pools.
[0105] As mentioned previously, flushing or rinsing of the treated
area typically is not necessary, although irrigation with a liquid
such as a normal saline solution certainly is possible.
[0106] The antimicrobial composition also or alternatively can be
used to provide sterility to pre- and post-surgical articles such
as sponges, topical wipes, bandages, pads, gauze, surgical packing,
and the like, particularly those intended or expected to contact
cilia-containing tissue.
[0107] Although sterilized, medical device implants such as
tympanostomy tubes can become colonized, prior to and during
implantation, with bacteria from the environment, from a healthcare
worker, or more commonly from bacteria present on the patient's own
skin. After insertion, these implants can become colonized from
systemic bacteria which make their way to the implant which
provides a surface for biofilm growth because the implant surface
is not protected by the host immune defenses. In addition,
currently employed sterilization techniques are not designed to
remove EPS/ECPS, the presence of which greatly facilitates
formation of a biofilm; therefore, even a sterilized device/article
that is properly implanted can have EPS/ECPS on its surface from
previous exposure.
[0108] If a biofilm forms on an implant, no currently available
treatment can eradicate it. Systemic antibiotics are ineffective
against such infections, certainly due to the inherent protection
by the EPS/ECPS but also perhaps due to limited blood supply at the
surface of the implanted article.
[0109] The aforedescribed antimicrobial compositions can be
effective topical treatments, applied to a to-be-implanted device
or article or can be used to wash the infected implant and
surrounding tissue to rid the body of a biofilm and/or
biofilm-forming materials such as EPS/ECPS.
[0110] The tympanic membrane where the implant is or was located
likewise can be treated with the previously described composition.
This can be done at the time of the original implantation (i.e.,
immediately following insertion of the article), and can be
followed with rinsing/irrigation, suctioning or both.
[0111] As has been mentioned several places above, deciliation is a
significant concern for any composition that is intended for use in
the sinus cavities and, particularly, the middle-inner ear. While
deciliation is preferably avoided altogether, no more than
.about.20%, preferably no more than .about.15%, and more preferably
no more than .about.10%, is acceptable for compositions intended
for use in sinus applications. For otic compositions, the
acceptable upper limit is that which results in measurable hearing
loss. Deciliation can be determined via scanning electron
microscopy, as described more fully below, and/or audiometric
testing.
[0112] The relevant portions of any specifically referenced patent
and/or published patent application are incorporated herein by
reference.
EXAMPLES
[0113] The relative efficacy of treating compositions was
determined using a MBEC high throughput screening assay, similar to
that used in the procedure described in ASTM E2799-12 (Standard
Test Method for Testing Disinfectant Efficacy against Pseudomonas
aeruginosa Biofilm Using the MBEC Assay). The assay employed a
multiwell assembly 10 of the type shown in FIG. 1, which includes
plate 12 having multiple wells 14 and lid 16 having multiple pegs
18. Each of plate 12 and lid 16 is made of a plastic such as
polystyrene or polycarbonate.
[0114] Bacteria is propagated in one or more of multiple wells 14
immediately after a plate 12 is removed from sterile packaging. In
the following examples, the plate included 96 wells in an array of
8 rows and 12 columns, as graphically represented in FIG. 2. Each
of 69 of the wells designated with an X received 150 .mu.L of the
10.sup.5 dilution inoculum. None of the wells in columns 9-11 were
used, while wells A12-C12 were reserved for use as sterility
controls. The five wells represented by D12-H12 served as bacterial
growth controls.
[0115] The bacteria used were S. aureus, ATCC 33592 (MRSA), and P.
aeruginosa, ATCC 15442. The surface of appropriate agar media were
inoculated with a recently grown stock culture of each bacteria. An
isolated colony was aseptically removed from the plate and
inoculated with 200 mL of soybean-casein digest medium; flasks were
incubated at 35.degree..+-.2.degree. C. and 150.+-.10 rpm (18-24
hours for staph, 16-18 hours for pseudomonas) with viable bacterial
densities being targeted at .gtoreq.10.sup.8 CFU/mL, checked by
serial dilution and plating. Into 100 mL portions of the growth
medium were pipetted 10 .mu.L aliquots from each incubation flask,
so as to adjust the bacterial densities to 10.sup.5 CFU/mL; these
were vortexed to achieve homogeneous distributions. Ten-fold serial
dilutions of the inoculums from the preceding sentence were
performed in triplicate, with 20 .mu.L aliquots of the serial
dilutions being spot plated on appropriate agar plates from
10.sup.0-10.sup.-7 before incubating the plates at
35.degree..+-.2.degree. C. (18-24 hours for staph, 16-18 hours for
pseudomonas).
[0116] Lid 16 was placed on plate 12, and assembly 10 was labeled
before being placed onto an orbital incubator/shaker set to
110.+-.10 rpm. The incubator/shaker was allowed to run at
35.degree..+-.2.degree. C. for the amount of time noted previously
as being appropriate for each bacterium type.
[0117] The bacterial growth pegs (D12-H12 in FIG. 2) were broken
off using flame sterilized pliers held flush against lid 14. Each
of those five pegs was placed into a separate sterile
microcentrifuge tube with 1.0 mL phosphate-buffered saline (PBS).
The microcentrifuge tubes then were placed on a stainless steel
tray floated in the center of a sonication device; the tray was
permitted to sonicate for 1800.+-.300 seconds. After sonication,
the solution in each microcentrifuge tube was serially diluted by
transferring 0.1 mL into a new sterile microcentrifuge tube
containing 0.9 mL PBS. The dilutions were spot plated onto agar
appropriate for the particular bacterium being tested.
[0118] One plate, designated the "Challenge Plate," is graphically
depicted in FIG. 3, where the symbols represent the following:
TABLE-US-00003 @ lysozyme-containing .star-solid. PBS composition
& 50:50 lysozyme-containing # lysozyme-free composition
composition/sterile neutralizer {circumflex over ( )} saline *
50:50 lysozyme-free composition/ = sterile neutralizer sterile
neutralizer .diamond-solid. sterile broth + 50:50 saline/sterile
neutralizer
[0119] Into well A12 of that plate was added 200 .mu.L sterile
broth, with that well serving as the device sterility control.
Next, 200 .mu.L sterile neutralizer was added to each well in
column 7 (neutralizer toxicity control) and well B12 (neutralizer
sterility control), while the wells in column 6 (neutralizer
effectiveness controls) received 100 .mu.L sterile neutralizer
followed by 100 .mu.L of a test composition: lysozyme-containing
composition in rows A-C, composition without lysozyme in rows D-F,
and normal saline in rows G-H. To each well in column 8 (untreated
control) and well C12 (PBS sterility control) was added 200 .mu.L
PBS. For columns 1-5, each well received 200 .mu.L of a test
composition: lysozyme-containing composition in rows A-C,
composition without lysozyme in rows D-F, and normal saline in rows
G-H.
[0120] The Challenge Plate was placed into a humidified incubator
set at 36.degree..+-.1.degree. C. for at least 60 minutes.
[0121] Also provided for each test was a Rinse Plate (200 .mu.L PBS
in each well), a Recovery Plate (200 .mu.L sterile neutralizer in
each well), and Quantification Plates 1-3 (100 .mu.L sterile
neutralizer in row A of each and 180 .mu.L PBS in rows B-H of
each).
[0122] The Challenge Plate was removed from the incubator. The
Growth Plate peg lid was rinsed for .about.10 seconds to remove any
planktonic microbes and transferred onto the Challenge Plate before
the combination was incubated at 36.degree..+-.1.degree. C. for the
appropriate time described above. Thereafter, the peg lid was
transferred to the Recovery Plate.
[0123] From the Challenge Plate was transferred 100 .mu.L of the
contents from each well of row A into the corresponding row A well
of the Quantification Plate 1, 100 .mu.L of the contents from each
well of row D into the corresponding row A well of the
Quantification Plate 2, and 100 .mu.L of the contents from each
well of row G into the corresponding row A well of the
Quantification Plate 3.
[0124] The Recovery Plate was placed in the stainless steel tray of
the sonicating device and permitted to sonicate for 1800.+-.300
seconds.
[0125] Dilution Plates 1-3 were prepared by adding 180 .mu.L PBS
into the wells of rows B-H of new 96-well multiwells. Following
sonication, 100 .mu.L of the contents from each well of row B of
the Recovery Plate were transferred into the corresponding well of
row A of Dilution Plate 1. Serial dilutions (10.degree. 40 were
achieved by transferring 20 .mu.L down each of the 8 rows, e.g., 20
.mu.L from cell A1 was put into B1, diluted and mixed, 20 .mu.L
from cell B1 was put into C1, diluted and mixed, etc. The contents
of each well of Dilution Plate 1 were mixed by pipetting up and
down, with the pipette tip being discarded after mixing each row.
The dilution series from the Dilution Plate 1 was spot plated on
appropriate agar for viable cell counts, described below.
[0126] Similarly, 100 .mu.L of the contents from each well of row E
of the Recovery Plate were transferred into row A of Dilution Plate
2, and 100 .mu.L of the contents from each well of row H of the
Recovery Plate were transferred into row A of Dilution Plate 3.
Dilution and spot plating for each were performed as with Dilution
Plate 1.
[0127] Each of Quantification Plates 1-3 were treated similarly to
Dilution Plates 1-3.
[0128] All agar plates were incubated at the appropriate
temperature and for the appropriate time in view of the type of
bacteria being tested.
[0129] Each plate was visually inspected, with any colony that was
visibly distinct from other colonies being counted as a colony
forming unit (CFU) and that number being used in the following
formula to determine relative efficacies of compositions at various
concentrations:
log.sub.10[10.sup.x(CFU/V.sub.pl)(V.sub.w/A.sub.pg)]
where V.sub.pl is the plated volume in .mu.L, V.sub.w is the volume
of a plate well (here, 200 .mu.L), A.sub.pg is the area of a lid
peg (here, 46.63 mm.sup.2), and x is an integer representing the
plate row, i.e., 1 for row A, 2 for row B, 3 for row C, etc. The
average of the values for each of columns 1-5 were determined, and
log reduction was determined by subtracting the average of treated
pegs from the average of untreated pegs.
[0130] As confirmations, pegs A12-C12 were checked to ensure that
they remained clear, pegs D12-H12 were checked to ensure the
presence of 10.sup.4-10.sup.6 CFU/mm.sup.2 of recovered microbe,
column 6 was checked to ensure neutralizer effectiveness, column 7
was checked to ensure neutralizer non-toxicity, and column 8 was
checked to ensure microbial growth.
Example 1: Otic Composition
[0131] Approximately 1 L of an exemplary solution for washing of
the middle ear was prepared from the following combination of
ingredients: [0132] 0.8 g/L anhydrous citric acid, [0133] 19.1 g/L
trisodium citrate dihydrate, [0134] 50 g/L ethanol, and [0135] 0.15
g/L lysozyme. The composition was calculated to have an effective
solute concentration of .about.200 mOsm/L, and its measured pH was
6.5.
[0136] When tested in a MBEC biofilm reactor, with a 5 minute
static application time, this composition was shown to result in a
0.8 log reduction in S. aureus and a 2.2 log reduction in P.
aeruginosa.
Example 2: Sinus Composition
[0137] Approximately 1 L of an exemplary solution for introduction
into the sinuses was prepared from the following combination of
ingredients: [0138] 0.75 g/L anhydrous citric acid, [0139] 14.25
g/L trisodium citrate dihydrate, [0140] 100 g/L DMSO, [0141] 0.15
g/L lysozyme, and [0142] 5 g/L sodium lauryl sulfate. The
composition was calculated to have an effective solute
concentration of .about.200 mOsm/L, and its measured pH was
7.0.
[0143] When tested in a MBEC biofilm reactor, with a 5 minute
static application time, this composition reduced S. aureus by 3
log.
Example 3: Ciliotoxicity Testing, Sinus--Human Sinonasal Epithelial
Cells
[0144] The ALI model, which uses epithelial tissue grown on
permeable filter supports submerged in culture medium, was used as
a screening test to determine whether the composition employed in
Example 2 exhibited significant ciliotoxicity.
[0145] Mucosal specimens acquired from residual clinical material
obtained during sinonasal surgery were transported to the
laboratory in saline placed on ice. ALI cultures were established
from human sinonasal epithelial cells enzymatically dissociated
human tissue using procedures described at, for example, M.
Ramanathan et al., "A comparison of experimental methods in
molecular chronic rhinosinusitis research." Am. J. Rhinol., vol.
21, pp. 373-77 (2007).
[0146] Cultures were grown to confluence in tissue culture flasks
(75 mL) with an appropriate proliferation medium before the
epithelium was allowed to differentiate five days later.
[0147] Mucosal samples prepared according to the procedure
described in M. B. Antunes et al., "Murine Nasal Septa for
Respiratory Epithelial Air-Liquid Interface Cultures,"
BioTechniques, no. 43, pp. 195-204 (2007) were placed in a glass
perfusion chamber.
[0148] Beating cilia on the edges of each sample were identified
using a Leica DMLFSA microscope set on an air table, using a water
immersion 63.times.objective and differential inter-ference
contrast optics (Leica Microsystems, Inc.; Bannockburn, Ill.). Once
beating cilia were observed, two seconds of video at a sampling
rate of 100 frames/second were captured with a high-speed
monochromatic digital video camera (Basler AG; Ahrensburg,
Germany), with the video images being routed into a PC workstation
for compression and storage. This process was repeated at 1-minute
intervals, with a 5-minute baseline beating rate being determined
before 20 .mu.L of the composition from Example 2 was pipetted onto
the apical surface of the mucosal sample and video imaging was
continued. Image files were analyzed with virtual instrumenta-tion
software customized to analyze ciliary beating. (For more
information on this type of video image analysis technique, see J.
H. Sisson et al., "All-digital image capture and whole-field
analysis of ciliary beat frequency," J. Microscopy, vol. 211, pp.
103-11 (2003).)
[0149] The targeted result was a finding that at least 50% of cilia
(as measured by active area) would continue beating through 30
minutes of exposure of a sample to the composition.
[0150] The actual results of the testing are shown in FIG. 4. The
median active area for the baseline period was .about.24.3%, while
the median active area as measured at 30-33 minutes was
.about.16.4%. Accordingly, the treating composition from Example 2
was deemed to have passed the ALI screening test.
[0151] The beat frequency of the cilia during exposure to the
treating composition from Example 2 also was measured, with those
results being shown in FIG. 5. The median cilia beat frequency for
the baseline testing was .about.4670 Hz, while the median for the
solution at 30-33 minutes of exposure was .about.3160 Hz. FIG. 5
also indicates that cilia beat frequency continues to increase
after exposure.
Example 4: Ciliotoxicity Testing, Sinus--Explant
[0152] The sinus treating composition from Example 2 also was
tested on three harvested mouse nasal septa. (Harvesting was
conducted as described in the M. B. Antunes et al. article
mentioned previously.)
[0153] Each harvested septal explant was held in sterile PBS before
being placed in a glass perfusion chamber held in place with a
nylon grid (1.5 mm), the outer frame of which snapped into the
inside of the perfusion chamber (Warner Instruments; Hamden,
Conn.). Each explant was tested at 27.5.degree. to 28.5.degree.
C.
[0154] A 3-minute baseline beating rate was determined before each
explant sample was given a 3-minute exposure to the sinus treating
composition from Example 2.
[0155] The video image analysis technique employed in Example 3
again was employed.
[0156] Tabulated below are the median cilia beat frequency for the
baseline period and 27-30 minutes after exposure.
TABLE-US-00004 TABLE 2 cilia beat frequency of mouse explants (Hz)
Baseline Post-exposure sample 1 4.8 8.0 sample 2 5.1 6.1 sample 3
6.6 8.9
[0157] The cilia beat frequency values after exposure are greater
than those before exposure, indicating no debilitating damage to
the cilia of the explant samples.
Example 5: Ciliotoxicity Testing, Sinus--In Vivo
[0158] The sinus treating composition from Example 2 also was
tested on ciliated mucosal surfaces in the sinuses of living
rabbits at the University of Sao Paolo.
[0159] Using the procedure described in E, Tamashiro et al., "In
vivo effects of citric acid/zwitterionic surfactant cleansing
solution on rabbit sinus mucosa," Am. J. of Rhinology &
Allergy, vol. 23, no. 6, pp. 597-601 (2009), indwelling catheters
were placed into the maxillary sinuses of female New Zealand white
rabbits, with 10 mL (at a rate of 0.33 mL/sec) of either 0.9%
normal saline or the treating composition from Example 2 being
instilled into the sinuses, followed by aspiration.
[0160] Test rabbits were anaesthetized and killed either one day or
seven days after irrigation. The 7-day timeframe permits evaluation
of possible long-term deciliation, while the 1-day timeframe guards
against the possibility of immediate deciliation followed by
regrowth in the intervening six days. (Sinus cilia often regrow
within 48 hours of being lost.)
[0161] Mucosa from both left and right maxillary sinuses were
harvested, with each mucosa sample being evaluated by scanning
electron microscopy (SEM) for morphological integrity of the
epithelium.
[0162] Analysis of the SEM images indicated the ciliotoxicity of
the treating composition from Example 2 equivalent to or even less
than that of normal saline, both after 1 day and after 7 days.
Example 6: Ciliotoxicity Testing, Middle Ear
[0163] Ototoxicity testing was conducted by the Department of
Otolaryngology in the University of Florida College of Medicine,
employing internally approved animal handling/testing procedures
which conform to the National Institutes of Health Guidelines for
the Care and Use of Laboratory Animals.
[0164] After an acclimation period of at least 5 days, each of
eight mature, albino male guinea pigs (Charles River; Wilmington,
Mass.) underwent a bilateral myringotomy, conducted with a blunt 27
gauge sterile needle. Sufficient liquid was injected so as to fill
the animals' middle ear spaces (.about.0.2 mL), with one ear
receiving a control 0.9% normal saline solution and the other
receiving the treating composition from Example 1. After injection,
the dorsal skull of each animal was tapped gently to ensure
thorough exposure of the entireties of the animal's middle and
inner ears to the liquids.
[0165] None of the test animals displayed evidence of
vestibulopathy during the study.
[0166] Cochlear action potential for each animal was measured
before solution injections, 7 days after injection, and 28 days
after injection, using electrocochleography as described by T. M.
Lo et al., "Hearing loss with stapedotomy and treated otitis
media," Otolayngol. Head Neck Surg., vol. 134(4), pp. 674-79 (April
2006). Electrocochleographic thresholds were measured for
calibrated tone bursts generated by an auditory electrophysiology
workstation using software from Tucker-Davis Technologies
(Gainesville, Fla.) and electrostatic speakers. Stimuli were
introduced through insert headphones placed in the animal's
external auditory canal. Tone bursts at frequencies of 4 kHz, 8
kHz, 16 kHz and 24 kHz were presented. Beginning at 100 dB,
auditory thresholds were evaluated by decreasing stimulus intensity
in 5 dB decrements until disappearance of the waveform. Waveforms
were averaged in response to 512 tone bursts at each tested
frequency/amplitude combination.
[0167] Hearing thresholds for each ear were compared using a
paired, two-tailed t-test (SAS Institute Inc.; Cary, N.C.).
Significance levels were determined at p<0.05.
[0168] A significant hearing loss for these experiments was
considered to be a 10 dB difference.
[0169] Averaged hearing thresholds for the test animals prior to
injection of any liquids can be seen in FIG. 6, while averaged
hearing threshold shifts for the test animals 7 days and 28 days
after liquid injection can be seen in, respectively, FIGS. 7 and 8.
(Bars represent standard error.)
[0170] In each case, hearing thresholds between treatments were not
different at all frequencies (p>0.05).
[0171] After final hearing assessments, each animal was euthanized
so that their cochlea could be examined by SEM so as to assess
outer hair cell (OHC) loss. When possible the cochlea were imaged
from all three turns, i.e., basal, middle and apical.
[0172] Thorough analysis of multiple images revealed no substantial
difference in the OHC loss of the middle ears receiving the
treating composition from Example 1 relative to the ears receiving
the control saline solution.
[0173] Based on both the comparative hearing data and the SEM
analysis, the report concluded that the treating composition
employed in Example 1 did not cause ototoxicity. The results were
deemed to be sufficiently definitive that a follow-on confirmative
study employing chinchillas was recommended to be foregone.
* * * * *