CA2852591C - Vitamin d3 compositions and uses thereof for the treatment of nasal passages - Google Patents

Abstract

A method of treating mucosal tissue of nasal passages and paranasal sinus cavities of an individual comprising the step of topically administering a therapeutic amount of vitamin D3 in a large volume of ultra low ionic strength delivery solution.

Description

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

- 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

- 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.

- 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

- 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

- 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.

- 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.

- 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 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 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 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 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.

= ¨ 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 (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 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.

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 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.

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.

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-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 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.

Claims (8)

The Claims Defining the Invention are as Follows:

1. Use of a therapeutic amount of vitamin D3 in a volume of delivery solution of 40 ml to 150 ml, wherein the delivery solution has an ionic strength of 0 to 35 mM, in the topical treatment of mucosal tissue of nasal passages and paranasal sinus cavities of an individual in need thereof.

2. Use of vitamin D3 and a positive pressure irrigation device in the treatment of mucosa] tissue of nasal passages and paranasal sinus cavities of an individual, wherein the vitamin D3 is for topical delivery to the nasal passages and paranasal sinus cavities in a volume of delivery solution of 40 ml to 150 ml, and wherein the delivery solution has an ionic strength of 0 to 35 mM.

3. The use according to claim 1 wherein the therapeutic amount of vitamin D3 is for delivery using a positive pressure irrigation device.

4. The use according to any one of claims 1 to 3 wherein the ionic strength of the delivery solution is between 20-35 m M.

5. A composition for intranasal administration comprising a therapeutic amount of vitamin D3 for the treatment of mucosal tissue of nasal passages and paranasal sinus cavities in a volume of delivery solution of 40 ml to 150 ml, and wherein the delivery solution has an ionic strength of 0 to 35 mM.

6. The composition according to claim 5 wherein the therapeutic amount of vitamin D3 is between 1 µg/ml to 2 µg/ml.

7. A kit for use in the treatment of mucosal tissue of nasal passages and paranasal sinus cavities comprising: a therapeutic amount of vitamin D3 in a powdered form, powders to reconstitute to a delivery solution having an ionic strength of 0 to 35 mM, wherein the delivery solution has a volume of 40 ml to 150 ml, a positive pressure irrigation device and instructions for their use.

8. Use of a therapeutic amount of vitamin D3 in a volume of delivery solution of 40 ml to 150 ml, wherein the delivery solution has an ionic strength of 0 to 35 mM, in the preparation of a medicament for the treatment of diseased nasal and sinus tissue.