FIELD OF THE INVENTION

The present invention relates to improved nasal spray formulation of beclomethasone dipropionate comprising; a film forming polymer, an antibacterial agent; a viscosity increasing agent having antibacterial properties with an advantageous effect on the nasal mucosa in case of allergic rhinitis and provided in the form of metered-dose nasal spray formulation.

TECHNICAL BACKGROUND

Nasal sprays known in the art where the examples of EP 0 216 917 B 1 refers to a therapeutic preparation for nasal administration that contains a thickening agent as methylcellulose. U.S. 5,843,881 A relates to a spray composition that contains alcohol, a polymer and an alcohol- masking perfume additive. The compositions are applied to the skin, the hair or the mucosa.

Both mucosal and glandular epithelial secrete a large array of molecules that are known to kill or neutralize microorganisms. The risk of colonization or infection by microorganisms might leads to chronic airway inflammation. Epithelial host defense molecules include small peptides that lyse bacteria. Lysozyme targets glycosidic bonds in the peptidoglycan cell wall of bacteria, typically leading to an enzymatic lysis, and is highly effective against many common upper airway gram-positive bacteria, such as streptococci.

Allergic rhinitis, nonallergic rhinitis, and chronic rhinosinusitis are described as inflammatory upper airway diseases with high prevalence and variations in their characteristics. The diseases are affected by each other, by lower airway diseases, by the composition of inhaled air, infections, the sensitization profile, and many genetic and lifestylerelated factors. Allergic rhinitis is an IgE-mediated symptomatic inflammation with a prevalence of 10-20 % in worldwide population. In the route of nasal passages, as a common rhinitides, allergic rhinitis (hay fever) causes to the nasal mucosa to swelling and lead to palpitations and respiratory disorders while changing its pH range to more alkaline environment, mostly greater than pH value of 6.5.

Regarding with the change in pH range in the case of allergic rhinitis, the effect of pH on the thermal stability of lysozyme enzyme became important. It is known from the literature that the maximum thermal stability of the Lysozym enzyme at pH 5.0 is due to minimum aggregation, and when the pH is further increased from 5.0 to 7.0, increase in aggregation decreases the thermal stability at pH 6.0 and 7.0 which leads to decrease in thermal stability of the enzyme causes inactivation of the ability to hydrolyzes glycosidic bonds between the monosaccharide units of bacterial glycoprotein cell wall, represented in Figure 1.

Figure 1. Lysozyme breaks some of the bonds between the peptidoglycans in the cell wall of a bacterium.

Lysozyme is an antibacterial enzyme found in body secretions, including saliva, mucus, tears, and human milk where acts as a part of body defence system against invading microbes. Lysozyme levels were decreased significantly in patients with perennial allergic rhinitis with recurrent sinusitis and reduced lysozyme concentrations can be critical factor which predispose the airways of patients with perennial allergic rhinitis.

Nasal mucosa has been known to carry amoeba and consequently could represent an important natural reservoir for Bacterial strains and act as a Trojan horse allowing bacteria to access the respiratory tract. Bacteria appears to result from various efflux pumps that efficiently remove antibiotics/or corticosteroid molecules from the cell, decreased contact of the drug molecule with the bacterial cell surface due to Bacterial ability to form biofilms, and changes in the cell epithelium to reduce the permeability of the membrane to the drug molecules like antibiotics and corticosteroid molecules.

Beclomethasone dipropionate is an anti-inflammatory corticosteroid having the chemical name 9-chloro-11b,17,21-trihydroxy-l6b-methylpregna-1,4-diene-3,2 0-doine17,21- dipropionate, beclomethasone dipropionate indicated in the relief of the symptoms of seasonal or perennial allergic and nonallergic (vasomotor) rhinitis having structure already defined in Patent No 3,312,590 which to use of anti-inflammatory compounds in topical preparations for the local treatment of inflammations.

Beclomethasone dipropionate is the active component of present dosage form, Nasal spray, in the invention having an anti-inflammatory steroid indication. The compositions of the prior art needs improvements in terms of antibacterial activity during allergic rhinitis between where give sufficient prevention of drug molecules from said bacterial biofilms leading to decrease the penetration of drug molecules in the application area.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a metered-dose nasal spray formulation comprising beclomethasone dipropionate, wherein the formulation is in the following antibacterial film forming agents content;

a) potassium sorbate in range of 0.1 - 10 mg per ml,

b) glycerol phosphate disodium salt hydrate in range of 0.05 - 5 mg per ml, c) hydroxyethylcellulose in the range of 0.01 - 1 mg per ml,

characterized by comprising micronized beclomethasone dipropionate (d90≤5μ) in the range of 0.05 to 0.1 % as weight per volume basis.

According to one embodiment, the composition comprises from 0.01 to 1 mg, preferably from 0.05 to 0.5 mg, more preferably from 0.1 to 0.3 mg of film forming polymer per 1 ml of nasal film composition. According to one embodiment, the film forming polymer is selected from the group of polymers which are versatile, organosoluble, and thermoplastic polymers, more preferably polymers derived from cellulose, ethylcellulose base done repeating anhydroglucose units.

According to one embodiment, the composition comprises from 0.05 to 5 mg, preferably from 0.1 to 3 mg, more preferably from 0.5 to 2 mg of viscosity increasing agent per 1 ml of nasal film composition.

According to one embodiment, the viscosity increasing agent is selected from polyol salts having cabability to adjust film forming efficiency with varying of its concentration in the prefered composition, preferably Glycerophosphate salts, more preferably Glycerol phosphate disodium salt hydrate.

According to one embodiment, the composition comprises from 0,1 to 10 mg, preferably from 0,2 to 5 mg, more preferably from 1 to 3 mg of antibacterial agent per 1 ml of nasal film composition.

According to one embodiment, the antibacterial agent is selected from the group of Alkali metal salts of sorbic acid considering of considered harmless to the motility of ciliated cellsin mucosa, more preferably potassium salt of sorbic acid, as Potassium sorbate.

According to one embodiment, the composition is a nasal spray, having capability of film forming characteristics.

The composition of the invention is notably for the improvement of antibacterial capabilities and pathologies of the upper airways.

The invention also provides a dispenser, preferably equipped with a spray pump, containing the nasal film composition of the invention.

The invention also relates to the potassium sorbate as an antibacterial agent, owing to its antibacterial characteristics in nasal film structure be able to prevent possible bacterial growth in alkali pH values in the case of allergic rhinitis during patient use. The compositions of the invention therefore, provides harmless, imperceptible, homogenous film on the nasal mucosa which protects it from possible bacterial growth between daily application of the doses.

An object of the present invention is to provide simulating the behaviour of Lysozyme enzyme which loses its activity in the symptoms of rhinitis due to the change of pH from acidic to alkali values, greater than pH 6.5. The invention also allows for improved anti-bacterial efficiency of the actuated product onto the mucosal surface without any damage to mucosal epitelhium. A dose would typically be one actuation into each nostril, and generally not more than 4 doses totally per day. The typical content of a dose is about 100μL. Table 1 represents the concentration of components in order to obtain Nasal Film composition,

Table 1. Stable Nasal Film Composition comprising Beclomethasone dipropionate

Results

The invention further provides advantages as will be apparent from the description.

The film-forming activity of the present invention provides sufficient time to drug molecules to penetrate into the nasal mucosal epithelium surface with forming uniform and elastic polymer film layer including antibacterial preservative, as Potassium sorbate, which prevents possible bacterial growth in alkaline pH values during allergic rhinitis with showing good stability in cell tolerance test performed with Calu-3 monolayers.

Antimicrobial Effectiveness Test (AET) were performed for nasal film suspension as represented in Figure 2, which justifies the requirements of antimicrobial effectiveness of the final Nasal film product conposition according to current European Pharmacopea monograph.

Figure 2. Antimicrobial Effectiveness Test (AET) for Nasal Film Suspension Composition The study focused on the Microbicidal effect of nasal film on indigenous microbes regarding with the intrinsic antimicrobial properties of nasal film against indigenous microbes with following method,

Method

Four isolates were used: Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus brasiliensis. Method were tested in order to evaluate the effectiveness of antimicrobial Nasal Film at 48 hours (test performed over 48 hours due to slow growth of Aspergillus brasiliensis and Candida albicans strains) following inoculation, bacteria were collected from film surfaces and enumerated. LoglO reductions were calculated by subtracting time point log 10 count on control and test surfaces from the initial inoculum. Figure 3 represents the antimicrobial effectiveness results for Nasal Film Surface.

Figure 3. The Antimicrobial Effectiveness Results for Nasal Film Surface, over 48 hours

Although initially all samples contained nearly 1x10 ⁶ CFU/mL, For Nasal Film samples, the CFU value gradually decreased during incubation and nearly diminished of Staphylococcus aureus and Pseudomonas aeruginosa at 24 hours, and at 48 hours for Candida albicans and Aspergillus brasiliensis.

Regarding with the determination of film forming behaviour of the Nasal Film composition, said film composition defined in the invention, Oscillatory tests were performed yielded results of higher storage modulus G' obtained rather than loss modulus G” which defines that formed Nasal Film showed chracteristics of viscoelastic solid in Figure 4.

Figure 4. Oscillatory Test Results for having ethylcellulose amounts of 0.05 mg/ml; 0.1 mg/ml and 0.2 mg/ml for Samples, Blue, Red and Blue, respectively The present study also investigate the local tolerance and the in-vitro nasal permeation of Beclomethasone dipropionate in nasal spray, nasal film formulations. For this purpose monolayers of the human bronchial cell line Calu-3 were used. Calu-3 cells represent a suitable model to study respiratory drug delivery in vitro in order to quantify the drug’s absorption the cumulative transport from the apical to the basolateral side of the cell monolayers. TEER (Trans epithelial electrical resistance) values for Calu-3 monolayers were determined. TEER values were constantly higher than 300 W-cm ² over the whole period of the transport studies. The adhesion during the 30 minute incubation phase was low but after 270 minutes no active ingredient be detected in the donor. Test formulations, nasal film, had suitable results in tolerance test with good stability of the active ingredient and yielded similar absorbtion behaviour with spray formulations.

The permeation experiments were performed with HBSS + 1 % MeOH + 0.25 % Tween 80 as acceptor medium with n=6 for each tested API.

The test compounds were diluted with transport medium (HBSS, pH 7.4 to appropriate yield concentrations) (see Table 2). The concentration of Beclomethasone transport solution was adjusted to the concentration of Beclomethasone dipropionate in the transport solution for test items A to C to ensure that the same theoretical concentration API was applied on the cell mon-olayers (see Table ).

Table 2. Preparation of the stock solutions of Beclomethasone dipropionate (pure API) for the permeation experiment

For all APIs a suspension was obtained which were mixed well and applied as suspension to the cells. In the following table the theoretical concentrations of the suspensions are given together with the measured concentrations of the supernatant of each transport solution. Therefore the suspension was diluted with ACN/H2075:25 (v/v; %). The concentrations were recalculated us-ing the dilution factor.Test item B and C refers Nasal Spray and Nasal Film compositions, respectively.

Table 3. Summary of in vitro permeability testing Beclomethasone dipropionate across Calu- 3 cell mono-layers.

After 30 minutes preincubation the amount API in the donor decreased tremendously from ca. 700 μg·mL ^-1 to 100 μg·mL ^-1 for Beclomethasone dipropionate in apical to basolateral transport direction and to ca. 50 μg·mL ^-1 for the basolateral to apical transport direction. This decrease may be caused by the adhesion of the lipophilic substance to plastic material of the Transwell system and possibly by an uptake of API in the cells. This adhesion process occurs normally within 30 minutes. During the further incubation of the monolayers with the transport solutions no further decrease of the donor concentration occurs. Indicating the adhesion resp., uptake was complete. Furthermore, it is obvious that no Beclomethasone has been transported to the acceptor chamber of the system. From the Rinoclenil solution the adhesion during the 30 minutes of pre-incubation was much lower. Nevertheless, no transport of Beclomethasone di-propionate could be detected. The experiments for Test item B and C had to be repeated with higher dilutions because the excipients interfered with the analytical method. The following table summarizes the repetition of the permeation experiments with the new dilu-tions (dil 1+29) of test item B and C resulting to final concentration of Beclomethasone dipropi-onate of nominal 33 μg·mL ^-1 .

Table 4. Summary of in vitro permeability testing Beclomethasone dipropionate across Calu- 3 cell mono-layers.

Figure 5. TEER values [%] of negative control HBSS, positive control HBSS + 0.1 % SDS and HBSS pH 7.4 + 1 % MeOH with different percentage shares of Tween 80, 0 min (after addition of the test substance), and after 60, 120 and 240 min Depicted are the mean values from three replicates and the standard deviation.

Figure 6. TEER values [%] of negative control HBSS, positive control HBSS + 0.1 % SDS, Rinoclenil in three concentrations and Test Item B and Test Item C with 500 μg mL-1, 0 min (after addition of the test substance), and after 60, 120 and 240 min. Depicted are the mean values from three replicates and the standard deviation.