Note: Descriptions are shown in the official language in which they were submitted.
1
Vitamin D3 Compositions and Uses Thereof for the Treatment of Nasal Passages
Field of the Invention
A composition comprising vitamin D3 for the simultaneous washing and treatment
of the
nasal passages and paranasal sinuses of an individual and a method of
treatment thereof.
Background Art
Diseased sinuses that result from conditions such as Chronic Rhinosinusitis
(chronic
inflammation of the paranasal sinus mucosa), produce copious amounts of thick
tenacious mucus and may be associated with bacterial and/or fungal overgrowth.
The
use of nasal sprays or nasal drops to treat sinus disease has been shown to be
ineffective
[Mark Jorissen., (2004). Review article in Rhinology. "Post operative care
following
endoscopic sinus surgery"; Vol 42, pp 114-1201 and [Wormald PJ et al., (2004).
The
Laryngoscope. "A comparative study of three methods of nasal irrigation". Vol
114 pp
2224 -2227]. Thus, the current method of treatment for this condition is to
wash the sinus
cavities with large volumes of physiologic saline solution. This treatment is
designed to
thin the mucus and help the drainage of mucus through the sinus ostia
(drainage points).
However, physiologic saline solutions have a high ionic strength, and
therefore, are not
the optimal solutions with which to wash sinus cavities since it has been
suggested that
higher ionic strength solutions may have unwanted effects on the innate immune
system.
[Singh P K et al., (2000). American Journal of Physiol Lung Cell Molecular
Physiol
"Synergistic and additive killing by antimicrobial factors found in human
airway surface
liquid". Vol 279 L799 ¨L805)]. Innate immunity is the natural immune function
associated
with mucous membranes.
Nasal secretions contain innate immune defence proteins, including lysozyme,
lactoferrin,
human 8-defensin and secretory leukocyte protease inhibitor, which form an
important
component of innate immunity against inhaled antigens and micro-organisms.
Lysozyme
is the most abundant secreted innate immune defence protein from the paranasal
sinuses. Lysozyme is often characterised as
CA 2852591 2018-03-26
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 2 -
an antibacterial agent acting via its enzymatic muramidase activity but is
also a
cationic protein with its bactericidal action note solely dependent on its
enzymatic
activity. Interestingly, there does not appear to be a deficiency of lysozyme
protein in the sinus mucosa of chronic rhinosinusitis patients. In fact,
chronic
rhinosinusitis has been associated with an increase in the expression of
lysozyme
protein in the sinus mucosa.
Vitamin 03 is an active form of vitamin D and may stimulate the production of
natural innate immune components within nasal and sinus tissues. This vitamin
is
essentially a fat soluble vitamin and hence lipophyllic. Although vitamin D3
has
been used in the treatment of sinus disease, it is not useful in conditions
where it
is applied topically in the presence of thick mucus. That is, there is no
value
applying medication to the sinus cavities when they are full of thick mucus.
The
medication simply sits on the top of the mucus layer and exerts no effect on
the
tissues [Harvey R et al. (2009). Otolaryngology Head and Neck Surgery "Fluid
residuals and drug exposure in nasal irrigation". Vol 141 pp 757 -761].
Therefore, there exists a need to treat nasal and sinus mucosal tissue where
there is a thick deposit or build up of mucus.
Summary of the Invention
A method of washing the mucosal membrane and simultaneously delivering a
therapeutic amount of vitamin D3 to nasal and sinus tissue has been developed.
Chronic sinus disease is associated with disordered immune function of the
sinus
tissue. It has been shown in the published literature that vitamin 03 is
deficient in
some populations with this disease [Pinto J M et al., (2008) Journal of
Allergy and
Clinical Immunology. "Serum 25-hydroxyvitamin D levels are lower in urban
African Americans subjects with chronic rhinosinusitis." Vol 122 (2) pp 415-
417]. It
has also been demonstrated that local respiratory tissue enzyme (1 alpha
hydroxylase) is responsible for the activation of inactive vitamin D to its
active
form (vitamin 03). Failure of this to occur results in lowered local or innate
immune function. [Hansdottir S etal., (2008). Journal of Immunology.
Respiratory
Epithelial Cells convert inactive Vitamin 0 to its active form: Potential
effect on
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 3 -
host defence. Vol 181 pp 7090 -7099]. Hence both lowered serum vitamin D
levels and ineffective local 1 alpha hydroxylase activity may result in immune
disorders at the epithelial level. The placement of the active form of vitamin
D onto
local tissues may therefore be helpful in stimulating the appropriate immune
factors necessary for epithelial health. Preferably, the solution containing
the
active form of vitamin D is delivered using a positive pressure irrigation
device.
In one aspect of the present invention there is provided a method of treating
nasal
mucosal tissue and the mucosal tissue associated with the paranasal sinus
cavities comprising the step of topically administering a therapeutic amount
of
vitamin 03 in a large volume of delivery solution to the nasal tissue and
sinus
cavities.
Vitamin 03 has been associated with stimulating the innate immune system of
the
nasal and paranasal sinus mucosa. However to date, it has not been possible to
topically deliver a therapeutic amount of vitamin D3 to the nasal membranes
where there are thick deposits or a build up of mucus. Therefore, in a further
aspect of the present invention, there is provided a use of vitamin D3 in the
treatment of mucosal tissue of the nasal passage and paranasal sinus cavities,
wherein the vitamin D3 is topically delivered to the nasal passages and
paranasal
sinus cavities in a large volume of delivery solution using a positive
pressure
irrigation device.
In another aspect of the present invention, there is provided a composition
comprising a therapeutic amount of vitamin D3 in a low ionic strength delivery
solution.
The present invention provides for an individual to self administer a dose of
the
composition outside of a hospital or clinic environment. Therefore, in yet
another
aspect of the present invention there is provided a kit comprising: a
therapeutic
amount of vitamin 03 in a powdered form, powders to reconstitute to a low
ionic
strength delivery solution, a positive pressure irrigation device and
instructions for
their use. The kit may further comprise additional agents to aid in treatment
of
infection, inflammation or adhesion to the surface of the nasal passages.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 4 -
In another aspect, the present invention provides the use of a therapeutic
amount
of vitamin D3 in a low ionic strength solution in the preparation of a
medicament
for the treatment of diseased nasal and sinus tissue.
Brief Description of the Drawings
Figure 1 Example of a bottle for use in a positive pressure irrigation
device.
Figure 2 Example of a cap for attaching to the bottle of a positive
pressure irrigation device.
Figure 3 The SNOT 20 score system
Figure 4 Fungicidal activity of lysozyme is dependent on ionic strength
Figure 5 Commercial nasal irrigation solutions inhibit the
fungicidal
activity of lysozyme in vitro
Disclosure of the Invention
General
Those skilled in the art will appreciate that the invention described herein
is
susceptible to variations and modification other than those specifically
described.
It is to be understood that the invention includes all such variations and
modification. The invention also includes all of the steps, features,
compositions
and compounds referred to or indicated in the specification, individually or
collectively and any and all combinations or any two or more of the steps or
features.
The present invention is not to be limited in scope by the specific embodiment
or
examples described herein, which are intended for the purpose of
exemplification
only. Functionally equivalent products, compositions and methods are clearly
within the scope of the invention as describe herein.
5
Throughout the specification and claims, unless the context requires
otherwise, the word
"comprise" or variations such as "comprises" or "comprising", will be
understood to imply
the inclusion of a stated integer or group of integers but not the exclusion
of any other
integer or group of integers.
Other definitions for selected terms used herein may be found within the
detailed
description of the invention and apply throughout. Unless otherwise defined,
all other
scientific and technical terms used herein have the same meaning as commonly
understood to one of ordinary skill in the art to which the invention belongs.
The term "active agent" refers to a compound useful for effecting some
beneficial change
in the subject to which it is administered. For example, "active agents"
within the scope
of this definition include vitamin 03, steroids, mucoadhesives, antibiotics
and other
surface active agents.
The term "effective amount" or "therapeutic amount" as applied to "one or more
active
agents" refers to that amount which is sufficient to effect the desired change
in the subject.
It is within the knowledge and skill of a person skilled in the art to
determine the effective
amount of an active agent.
The term "treatment" as used herein covers any treatment of a disease in an
animal
(including a human), and includes: (i) preventing the disease from occurring;
(ii) inhibiting
the disease, i.e., arresting its development; (iii) relieving the disease,
i.e., causing
regression of the disease; or (iv) modifying normal biological activity.
The term 'disorders' as used herein covers any medical disorders such as but
not limited
to: Chronic Rhinosinusitis and other diseases which can lead to excessive
mucus build
up in the nasal passages and paranasal sinus cavities. Additional
CA 2852591 2018-03-26
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 6 -
diseases or disorders include non-allergic paranasal sinus disease, asthma
allergic rhinitis and Chronic Obstructive Pulmonary Disease. Mucus build up in
the nasal passages and paranasal sinus cavities may also result from surgery,
such as endoscopic sinus surgery.
Any solution that contains one or more salts has an ionic strength or electric
charge. The amount of a salt or different salts in solution can be defined by
its
concentration (mM or gU) or by its ionic strength (mM or mMol). For example, a
mM sodium phosphate buffer solution has an ionic strength of 21 mM. Normal
saline has a concentration of 9 g/L of sodium chloride and an ionic strength
of 154
10 mM. Commercially available irrigation solutions are often composed of a
number
of different salts, and have been estimated to have ionic strength ranging
from
130 mM to greater than 500 mM. An ionic strength between 0 to 35 mM is
considered to be ultra or very low ionic strength solution when used as a
delivery
solution.
The pharmaceutical preparation of the present invention may be administered to
any mammal. Preferably, the mammal is a human being.
Detailed Description of the Invention
The compositions and methods of the present invention are useful for the
treatment of the upper airways, including the nasal cavity and the paranasal
sinuses. Each side of the face has a set of nasal passages and include the
nostril, nasal cavity and paranasal sinuses. The paranasal sinuses are divided
into subgroups that are named according to the bones within which the sinus is
located. As such, the four subgroups are the maxillary sinuses, the frontal
sinuses, the ethmoid sinuses and the sphenoid sinuses. The paranasal sinuses
are joined to the nasal cavity by ostia (small orifices). The paranasal
sinuses,
nasal cavity and ostia often become blocked as a result of post-operative
surgery
complications, infection or disease.
Thus, the composition of the present invention is useful for the treatment of
nasal
and sinus problems and in particular problems which result from the blockage
of
the nasal and sinus passages as a result of tissue swelling and the build up
of
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 7 -
mucus. In order to ensure the nasal passages and sinuses of either or each
side
of the face are washed adequately, a sufficient volume of solution is
required. In
one embodiment, a volume of between about 40 ml to 150 ml is used to wash
each side of the face of the nasal and sinus passages of an individual. In a
preferred embodiment a volume of between 50 ml to 100 ml is used per side. In
a
highly preferred embodiment, a volume of between 70 ml to 90 ml is used to
wash
each side of the face of the nasal and sinus passages of an individual.
The composition of the present invention comprises vitamin D3 in a sufficient
amount to achieve a therapeutic effect at the mucus membranes of the nasal
passages and sinus cavities. Vitamin D3 has been shown to stimulate the innate
immune system associated with the mucous membranes in these areas. It is a fat
soluble vitamin and is therefore lipophyllic. Applicant
has found that the
lipophyillic nature of vitamin D3 allows binding of the vitamin D3 to mucus
and as
such may be more effective at acting on the mucus membranes so as to exert its
effect on the innate immune system within the upper respiratory tract.
The concentration of vitamin D3 in the composition is of a therapeutic amount
to
stimulate the innate immune system of the mucus membranes. In a preferred
embodiment, the amount of vitamin D3 in solution is between 0.05 p.g/m1 to 2.0
pg/ml. Preferably, the concentration of vitamin D3 in solution is between 0.10
pg/m1 to 0.15 pg/ml. In a highly
preferred embodiment, the vitamin D3
concentration is 0.125 p.g/ml.
To express the amount of vitamin D3 in another way, a preferred solution of
the
present invention is prepared by dissolving 1000 international units (i.u.) of
vitamin
D3 in 200 ml of fluid. The conversion of international units of Vitamin D3 to
metric
units is that 40 iu of vitamin D3 is equivalent to 1 pg. Therefore, in a
highly
preferred embodiment, the solution comprises 1000 i.u. (25 pg) of vitamin D3
in
200 ml of a fluid. Expressed as a percentage, this equates to 0.00000125%
vitamin D3 in solution.
Previous studies conducted on post operative patients have shown that no more
than 10% of a wash solution remains in the sinus cavity after washing
treatment.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 8 -
In most cases, only about 5-7% of the total wash volume remains in the nasal
passage. This amount of active vitamin D3 should be sufficient to activate
local
innate immunity and also increase serum levels of vitamin D3 as absorption
from
nasal tissues is excellent.
A solution with a low ionic strength is considered to be more beneficial for
the
immune function associated with mucous membranes. A low ionic strength
solution does not have the same adverse side effects on the innate immune
system of the mucous membranes as experienced when using solutions with a
high ionic strength. Low ionic strength solutions may assist innate immune
proteins, such as lysozyme, lactoferrin, human 6-defensin and secretory
leukocyte
protease inhibitor in maintaining their antibacterial and/or antifungal
activity in
diseased sinuses, such as chronic rhinosinusitis, hay-fever, common colds or
upper respiratory tract infections.
Furthermore, one advantage of the invention is the stimulation of the innate
immune system by vitamin D3, and as such, it is preferable that the function
of
vitamin D3 be enhanced and protected by the use of a delivery solution of a
low
ionic strength. Preferred ionic strengths of the delivery solution range from
20 to
30 mMol. In a highly preferred embodiment, the delivery solution is formulated
to
be of ultra low ionic strength of about 25.6 mMol.
The composition of the present invention may further comprise other active
agents. The addition of other agents is aimed at reducing inflammation in the
nasal or sinus passages or improving adhesion of the vitamin D3 to the
epithelium
in the nasal or sinus passages. Examples of active agents to reduce
inflammation
of the nasal passages include steroids and antibiotics.
Mucoadhesive agents for use in the compositions of the present invention
include
synthetic or natural polymers, which interact with the mucous membrane of the
nasal or sinus passages or the mucin molecules of mucus. Examples of
mucoadhesive agents include chitosan and hydroxyl propyl methyl cellulose.
Chitosan also has an antibacterial action and as such also aids to reduce
inflammation and bacterial overload in the nasal passages and paranasal
sinuses.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 9 -
A person skilled in the art would be aware of the addition of other known
agents
that aid in the adhesion of vitamin D3 to the nasal passages.
The composition of the present invention may be applied to the nasal or sinus
passages of either or both sides of the face. The large volume of delivery
solution
required to wash and treat the nasal passages and sinus cavities may be
administered by the use of a positive pressure irrigation device. Therefore, a
bottle has been designed to deliver the required amount of delivery solution
to the
nasal passages, and in particular to the paranasal sinuses. In addition, the
bottle
has been designed for ease of cleaning after use.
One problem with the bottles of the prior art is that the bottle becomes
infected
after initial use as there is often an overgrowth of pathogenic bacteria
associated
with the disease being treated. This occurs because the bottle cannot be
adequately cleaned and dried after use. The bottle of the present invention
has a
wide neck which permits the complete disassembly of the bottle components
which can be rinsed and thoroughly dried after use. This significantly
restricts the
overgrowth of pathogenic bacteria and the subsequent re-infestation of the
nasal
passages, including the paranasal sinuses.
Thus, in a highly preferred embodiment of the invention there is provided a
method of treating the nasal passages and paranasal sinuses by delivering
vitamin D3 in a low ionic strength solution using a large volume of solution
delivered using a positive pressure irrigation device.
The positive pressure device is operated by simply squeezing the bottle until
about 100mL of the delivery solution has been expelled via one nostril into
the
nasal and paranasal sinus cavities and exits from the opposite nostril. This
procedure is then repeated into the opposite nostril.
The methods and compositions of the present invention are capable of treating
tissue inflammation and a build up in mucus which results from acute or
chronic
disease processes or to clear mucus, dry blood, excised tissue and blood clots
resulting from nasal and sinus surgery conducted to facilitate aeration of the
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 1 0 -
paranasal sinuses in patients suffering from acute or chronic sinus disease
with or
without associated polyposis.
Exam pies
Example 1
The active form of vitamin D3 has been shown to be important in innate
immunity.
It has been shown that topically administering vitamin D3 to the treatment
site,
whilst simultaneously cleaning the nasal and sinus passages with the treatment
solution due to the positive pressure delivery system, also results in
improved
levels of defensins such as cathelicidin which form part of the innate defence
mechanism on mucous membranes.
Preparation of the vitamin D3 delivery solution
The concentration of vitamin D3 in the composition is of a therapeutic amount.
The amount of vitamin D3 in solution can be between 0.05 ig/m1 to 2.0 g/ml.
Preferably, the concentration of vitamin D3 in solution is between 0.10 pg/m1
to
0.15 g/ml. In this example, the vitamin D3 concentration is 0.125 g/ml.
Vitamin 03 was prepared by dissolving 1000 international units (i.u.) of
vitamin D3
in 200 ml of fluid. The conversion of international units of Vitamin D3 to
metric
units is that 40 lu of vitamin D3 is equivalent to 1 g. Therefore, the
solution
comprises 1000 i.u. (2514) of vitamin D3 in 200 ml of a fluid.
The vitamin D3 is dissolved in an ultra low ionic strength of about 25.6 mMol.
In
this example, the delivery solution is made up by reconstituting a sachet of
powders which contains sodium chloride, potassium chloride and Xylitol ¨ which
altogether have an ionic strength of 25.6 mMol when dissolved in 200mL of
water.
Treatment of a sub iect
The subject may be standing or seated during treatment. Preferably, the
subject
is located over a sink or bowl to ensure that the expelled delivery solution
is
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 1 1 -
contained after delivery to the nasal passages. The delivery solution is
applied to
the nostril by a positive pressure irrigation device which is operated by
simply
squeezing the bottle until about 100mL of the delivery solution has been
expelled
via one nostril into the nasal and paranasal sinus cavities and exits from the
opposite nostril. This procedure is then repeated into the opposite nostril.
To aid
in delivery of the solution to the nasal sinuses, the subject may place their
head
down, in a nose to the ground position for irrigation of the nasal and sinus
passages.
When used in children, a volume of about 50 ml per nostril should be
sufficient to
simultaneously cleanse and treat the mucous membranes. In adults, a volume of
between 70-90 ml is generally sufficient to cleanse and treat the mucous
membranes.
An example of the bottle for use in a positive pressure irrigation device is
shown in
Figure 1. The bottle is designed to ensure delivery of the solution to the
nasal and
sinus passages. The bottle of Figure 1 has a wide neck which permits the
complete disassembly of the bottle components which can be rinsed and
thoroughly dried after use. This significantly restricts the overgrowth of
pathogenic bacteria and the subsequent re-infestation of the nasal passages,
including the paranasal sinuses.
The bottle of the positive pressure irrigation device is fitted with a cap or
closure
that is suitable for the delivery of the solution to the nasal and sinus
passages via
the nostril. An example of such a cap is shown in Figure 2. The cap is
designed
to allow insertion into the nostril and to effect a seal against the nostril
so that the
irrigation fluid can effectively enter and progress through the nasal
passages.
The vitamin D3 preparation may be used on an ongoing basis, especially when
used to treat Chronic Rhinosinusitis which is a life long affliction. Thus the
treatment may be indicated once daily or twice daily at most. Alternatively,
the
use of the vitamin D3 preparation may be used as directed by a physician.
Results
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 12 -
The administration of vitamin D3 topically to the mucus of the nasal and sinus
passages in an ultra low ionic strength formulation whilst simultaneously
washing
the nasal and sinus passages with a large volume of delivery solution is
expected
to result in improved wound healing of the mucus membranes after surgery.
In addition, the vitamin D3 preparation is useful in the treatment of diseases
and
disorders such as Chronic Rhinosinusitis and other diseases which can lead to
excessive mucus build up and inflammation in the nasal passages and paranasal
sinus cavities. Examples of additional diseases or disorders include non-
allergic
paranasal sinus disease, asthma, allergic rhinitis and COPD (Chronic
Obstructive
Pulmonary Disease).
An improvement in the SNOT 20 scores in patients using the product is
anticipated by the use of the vitamin D3 composition. The SNOT 20 score is a
validated scoring system established to assess the severity of the condition
and
its impact on the patient's quality of life scores. Since the vitamin D3
composition
is designed to stimulate the innate immune system and to ensure the optimal
environment for the functioning of defensins patients would experience a
significant improvement in symptoms.
The SNOT 20 score system is represented in Figure 3.:
Example 2
The repeated use of saline irrigation or sprays may inhibit the antimicrobial
and/or
antifungal activity of important cationic innate defences, such as lysoyzme,
thereby facilitating microbial attachment to the sinonasal epithelium and
enabling
microbial colonisation. This study investigated whether the fungicidal
activity of
lysozyme is reduced in the presence of solutions with increasing ionic
strength,
and/or inhibited in the presence of commercial nasal irrigation solutions.
Method
A Colony Forming Unit (CFU) assay as described in Woods CM et al., "Human
lysozyme has fungicidal activity against nasal fungi". Am J Rhino! Allergy.
Jul-Aug
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 13 -
2011: 25 (4): 236-240, using Aspergillus fumigatus was utilised for this
study.
Aspergillus fumigatus is common fungi isolated from CRS patients.
Briefly, primary fungal cultures were prepared from freezer stocks and
subcultured
twice to ensure viability. Fungal conidia were collected from fresh, mature (7-
10
day old) cultures grown at 35 C on Sabouraud's dextrose (SAB) agar slopes
(Oxoid, Adelaide, Australia), by covering the fungal colonies with lml sterile
water
containing 0.25% Tween80 (Laboratory Supply) and agitating gently for a couple
of minutes before settling. The supernatant containing the conidia was removed
and diluted in SAB broth (Oxoid, Adelaide, Australia) to 106 conidia/ml.
Conidia
were allowed to germinate for 19 hours at 28 C (100% air, Heraeus Instruments,
model BB16). Germinated conidia were diluted 1000 fold with sodium phosphate
buffer (10mM, pH 7.4) and used in the assay.
Germinated fungal conidia were treated with recombinant human lysozyme
(Sigma Aldrich, code L1667; 5 M in a final volume of 6mL) and incubated at 35
C
(100% air, Heraeus Instruments, model BB16). The lysozyme was first dissolved
in freshly prepared 0.01% acetic acid to obtain a stock concentration of 100
M.
The negative control treatment (01.tM lysozyme) consisted of an equivalent
volume
of 0.01% acetic acid added to the germinated conidia. At 0 hour and 5 hours
2000 of solution was plated in triplicate onto Yeast Malt (YM) extract agar
plates
(Difco code#271210, prepared by Micromedia Laboratories, Melbourne,
Australia). After approximately 24 hour incubation at 35 C, the CFU were
enumerated visually.
1
= ¨ c,
The ionic strength / of a solution is calculated as 2
where Z is the
charge on the ion i present at a molar concentration c. Table 1 presents the
salt
concentrations and the calculated ionic strength of the sodium chloride
fortified
assay buffer, together with the ionic strength of commercial irrigation
solutions
(where formulation information was available) used in the following
experiments.
To determine the effect of ionic strength on the fungicidal activity of
lysozyme
against A. fumigatus the standard assay buffer was fortified with sodium
chloride
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 14 -
(0 ¨ 150mM, corresponding to ionic strength of 21 ¨ 171 mM, Table 1). The
assay
was replicated 5 times.
Buffer / commercial NaCI (mM) Ionic strength (mM)
irrigation solution
10mM Sodium 0 21
Phosphate Buffer 10 31
25 46
50 71
75 96
100 121
125 146
150 171
Normal saline 9g/L sodium chloride 154
0.9% NaCI
FLO Sinus Care sodium chloride, sodium 138
bicarbonate
potassium chloride,
glucose, calcium lactate
FESS Sinu-Cleanse 30mg/m1 NaCI > 500
Alkaline buffered
NielMed Sinus Rinse Isotonic sodium chloride > 154
Isotonic and sodium bicarbonate
NielMed Sinus Rinse Hypertonic sodium > 500
Hypertonic chloride and sodium
bicarbonate
Table 1
To determine the effect of nasal irrigation solutions on the fungicidal
activity of
lysozyme against A. fumigatus the standard assay buffer (sodium phosphate
buffer, 10mM, pH 7.4) was substituted. Commercially available nasal irrigation
solutions (preservative free) were prepared according to manufacturer
instructions
and used in the assay as described above.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 15 -
Nasal irrigation solutions tested were FESS Sinu-Cleanse (non-medicated
hypertonic saline solution; NaCI 30mg/m1 alkaline buffered; Care
Pharmaceuticals, Bondi Junction, NSW, Australia), FLO-Sinus Care (preservative
free and non-medicated; ENT Technologies, Malvern, VIC, Australia), NeilMed
Sinus Rinse (isotonic solution; NeilMed Pharmaceuticals Inc, Auburn, NSW,
Australia), NeilMed Sinus Rinse (hypertonic solution; NielMed Pharmaceutical),
and normal saline (0.9% sodium chloride for irrigation, isotonic, Baxter
Healthcare
Pty Ltd, Old Toongabbie, NSW, Australia). The assay was replicated 5 times for
each commercial nasal irrigation solution.
The fungicidal activity of lysozyme was calculated by the reduction of CFUs
using
the equation: [(Noh ¨ N5h)/Noh]x100, where N5h and Nob represent the number of
CFUs obtained after incubation in the presence of lysozyme after Oh(Noh) or
treatment for 5h (N5h). Therefore a fungicidal activity of 100% represents no
growth of fungi after treatment with lysozyme (i.e. all fungi are dead). A
fungicidal
activity of 0% represents growth equivalent to that observed at time 0 (i.e.
no
active growth/division of fungi). A negative fungicidal activity represents
active
growth of the fungi during the treatment period (i.e. fungi are not killed
during
treatment and continue to grow). For comparison, the same calculation was
applied to the control (acetic acid) treated fungi.
Statistical analyses utilised PASW Statistics 18 software (SPSS, Inc.). Non-
parametric statistics were utilised to determine statistical differences
between
responses of different assay buffer solutions (independent samples Kruskal-
Wallis
one-way ANOVA with multiple comparisons) or irrigation solutions compared to
the standard assay buffer (independent samples Kruskal-Wallis test).
Significance level was set at P<0.05.
Results
The fungicidal activity of 5 M lysozyme against A.fumigatus using the standard
assay buffer (ionic strength 21mM) was 94.7 2.1% (Figure 4). Increasing
ionic
strength was achieved by fortifying the standard sodium phosphate assay buffer
with NaCI. The fungicidal activity of lysozyme decreased with increasing ionic
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 16 -
strength, and was abolished at 46mM (P<0.05; Figure 4). Substitution of the
standard assay buffer with a commercial nasal irrigation solution (estimated
ionic
strength >120mM) abolished the fungicidal effects of lysozyme against A.
fumigatus, for each of the commercial nasal irrigation solutions tested
(P<0.05;
Figure 5).
Discussion
Patients with chronic rhinosinusitis experience repeated bacterial and/or
fungal
infections and often use commercial nasal irrigation solutions/sprays, mainly
for its
mechanical debridement effect. Nasal secretions contain a number of innate
cationic antimicrobial peptides, such as lysozyme. Lysozyme is known to have
bactericidal activity. More recently, lysozyme has also demonstrated
fungicidal
activity, with increased expression in the mucosa of chronic rhinosinusitis
patients.
The proposed mechanism of action of lysozyme's fungicidal activity involves
ionic
interactions with the fungal cell-wall, which may be inhibited by ionic
solutions
such as commercial sinus irrigation solutions/sprays. Using an in vitro assay
this
study determined that fungicidal activity of lysozyme is dependent on ionic
strength and commercial nasal irrigation solutions inhibit its fungicidal
capabilities.
The fluid secreted by sinonasal epithelium comprises a bilaminar mucous layer
with an outer viscous layer and a deeper aqueous periciliary layer (airway
surface
liquid). The airway surface liquid contains cationic antimicrobial proteins,
such as
lysozyme, lactoferrin, and defensins, which are important in eliminating
inhaled
microbes. Under normal conditions airway surface liquid has been found to be
hypotonic compared to plasma, with a pH of 5.5-6.5. In comparison, airway
surface liquid of patients with airway inflammation, infection or cystic
fibrosis is
isotonic, with a pH 7.2-8.3, suggesting changes in ion and water secretion
occur
during inflammatory processes. It may be possible that during the process of
inflammation and/or disease the salt concentration of airway surface liquid
increases, which has the potential to inhibit the functional activity of
cationic
antimicrobial peptides.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 17 -
Ionic strength is a measure of the concentration of all dissolved chemical
constituents and is important to consider because ions in solution have an
electrical charge that attract or repel against each other and are therefore
involved
in the ionic interactions between the cationic antimicrobial peptides and the
anionic fungal/microbial cell wall. This study demonstrates that the
fungicidal
activity of lysozyme is dependent on the ionic interactions with the fungi
cell wall
and ionic strength is a key factor. Using an in vitro assay we determined that
fungicidal activity was almost 100% in solutions with low ionic strength
(21mM),
but inhibited with increasing ionic strength (31mM), and abolished by an ionic
strength of 46mM. These ionic strengths are much lower than 'isotonic'
(usually
calculated at 9g/L NaCI) corresponding to ionic strength of 154mM.
Lysozyme's bactericidal activity is not solely dependent on its murimadase
activity
with non-enzymatic activity also being reported. It is likely that lysozyme's
fungicidal and bactericidal activity is reduced with inflammatory conditions
such as
chronic rhinosinusitis due to the presence of isotonic and hypertonic airway
surface liquid. We suggest that inflammatory processes such as upper
respiratory
tract infections, cystic fibrosis and chronic rhinosinusitis cause an
increased
tonicity of airway surface liquid which inhibits the antimicrobial activity of
these
peptides, leading to impaired innate immunity and ongoing bacterial/fungal
infection.
Use of commercial nasal irrigation solutions/sprays
Nasal and sinus irrigation solutions are recommended for the removal of
thickened nasal secretions in chronic rhinosinusitis, and often used to ease
sinus/nasal congestion associated with hayfever, common cold and upper
respiratory infections. There is evidence to suggest that aggressive douching
provides symptom relief through the physical action of removing thick
eosinophilic
mucin, crusts, and post-operative blood clots. However, these solutions are
isotonic or hypertonic, whereas normal airway surface liquid is reported to
have
much lower concentrations of ions.
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 18 -
This study found that commercially available sinus irrigation solutions
abolished
the fungicidal effects of lysozyme in vitro. Estimation of ionic strength of
these
solutions together with comparison of lysozyme's fungicidal activity versus
ionic
strength data suggests that the high ionic strength of these solutions
interferes
with the ionic interactions required to maintain fungicidal activity. It is
expected
that the ionic strength of these irrigation solutions would also inhibit the
antimicrobial activity of other innate cationic antimicrobial peptides present
in
sinonasal secretions. Furthermore, the action of sinus irrigation removes the
protective mucous layer exposing the epithelial cells to airborne pathogens.
It has been estimated that cationic antimicrobial peptides start to
reconstitute
themselves within 10-20 minutes after repeated lavages, but it may require 4-
24
hours before they re-achieve resting concentrations (normal pre-irrigation
airway
surface liquid levels), potentially leaving the epithelium with impaired
innate
immunity for a prolonged period. We hypothesise that repeated saline
irrigation
inhibits the antimicrobial activity of cationic antimicrobial peptides, thus
facilitating
microbial colonisation to the sinus epithelium, potentially contributing to
the on-
going inflammatory process in chronic rhinosinusitis patients. Lastly, it
should be
noted that the ionic environment affects bacterial susceptibility to cationic
antimicrobial peptides. The ionic composition of culture media can change
bacterial gene expression for a number of virulence genes (i.e. cell wall
thickness)
and bacterial susceptibility may be significantly enhanced in a mammalian
ionic
environment. From this, it can be postulated that bacteria may develop a
thicker
cell wall and be more resistant to antimicrobial peptides if grown in mucosa
constantly bathed in salt solutions that do not resemble normal airway surface
liquid.
In conclusion, the results presented here using an in vitro assay demonstrate
the
fungicidal activity of lysozyme is dependent on ionic strength and that
commercial
nasal irrigation solutions inhibit the fungicidal effects of lysozyme in
vitro. These
results have the potential to improve understanding of the pathophysiology of
chronic rhinosinusitis, and have implications for the routine use of nasal
irrigation
solutions/sprays in sinus disease. The ionic strength of these solutions
likely
interferes with the binding of lysozyme to fungi, thus inhibiting its
fungicidal
CA 02852591 2014-04-16
WO 2012/103575 PCT/AU2012/000074
- 1 9 -
capability. This could provide an environment that would facilitate microbial
attachment to the sinonasal epithelium and increases the likelihood of
microbial
colonisation, contributing to the on-going inflammatory process present in
chronic
rhinosinusitis.
