A pharmaceutical composition with mucoadhesive properties and its use

Abstract

The object of the invention is a pharmaceutical composition with mucoadhesive properties, comprising an amphoteric absorption promoter, vegetable oil, preservatives, humectant and water, and at least one natural gum selected from a group comprising gellan gum, tragacanth gum, xanthan gum and a combination thereof, characterized in that the natural gum content is comprised in the range of 2 to 8% w / w and the ratio of the amphoteric absorption promoter: vegetable oil: preservatives: humectant expressed in % w / w is comprised in the ranges of 0.3 to 0.7: 1.7 to 2.3: 0.1 to 0.3: 3 to 7; wherein the water content constitutes a supplement to 100% w / w. The object of the invention further comprises a composition according to the invention for use in the alleviation and treatment of oral diseases.

Description

[0001] A pharmaceutical composition with mucoadhesive properties and its use.

[0002] The object of the present invention is a pharmaceutical composition with mucoadhesive properties and its use in the alleviation of the course and treatment of diseases of soft oral tissues.

[0003] Dental diseases, which include diseases of teeth, gums and oral mucosa, are a common social problem faced by both adults and children. The oral tissues, in particular the mucosa and gums, are delicate and vulnerable to mechanical trauma. In addition, constant contact of the oral cavity with microorganisms from the external environment increases the likelihood of damage and inflammation.

[0004] A common disease affecting patients of all ages is oromucosal infection. Depending on the cause, lesions can appear in a variety of forms: from redness with swelling to extensive erosions and ulcers sometimes covering almost the entire surface of the mucosa. The ailments occur as a result of systemic diseases (inter alia diabetes), immunodeficiency conditions (e.g., in oncology patients), long-term pharmacotherapy (e.g., with glycocorticosteroids) or injuries (e.g., during orthodontic treatment, after surgery). As a result of damage, the mucosa loses its protective barrier function, which promotes the development of viral, bacterial or fungal infections.

[0005] Oral candidiasis, is a fungal infection caused by yeasts (Candida sp.), which easily and quickly develop on the surface of the mucosa or in spaces between a dental plate and the mucosa, is a burdensome and recurrent condition. Children and persons with immunodeficiency are particularly susceptible to the development of the disease. Treatment of candidiasis usually begins with topical preparations containing antifungal agent. The lack of improvement or infection reoccurrence are the basis for the implementation of systemic treatment. Clotrimazole (CLO) belongs to a group of imidazole derivatives with antifungal activity. It is the most commonly used active substance in the treatment of candidiasis of the skin and oral and vaginal mucosa (Neil J. Khatter; Moien AB Khan. Clotrimazole. StatPearls 2022). Products with antifungal substances are available on the Polish pharmaceutical market in the form of creams, pastes, gels, rinsing liquids and unpleasantsuspensions with a bitter flavour for swabbing yeast lesions. In many cases, these preparations are intended to be applied to the skin, which means that they have a low capability to adhere to the membrane or are deprived of this capability at all. As a result, they flow off too quickly from the mucosal surface not being capable to keep the drug at the application site, which negatively affects therapeutic effectiveness and further reduces the comfort of use of the drug by the patient. On the European pharmaceutical market there are only preparations with CLO intended for skin and vaginal application. However, CLO in the form of lozengea (Clotrimazole troches, 10 mg, https: / / www.drugs.com / pro / clotrimazole-lozenge.html) is available on the world market.

[0006] A particular problem is posed by pre-cancerous conditions whose aetiology is mostly unknown. One of the most common chronic mucocutaneous disorders is lichen planus. It is estimated that the disease affects about 2-3% of the population, and the lack of a tangible cause of the disease makes the treatment only symptomatic, and therefore insufficiently effective. The lesions are accompanied by chronic pain and burning negatively affecting one’s everyday life . Available treatments, i.e., surgical removal of the lesion or topical and systemic corticosteroid therapy, lead only to temporary improvement. Shortly after surgical removal of the lesion or discontinuation of pharmacological treatment, relapses of the disease are observed. A long-term corticosteroid therapy also entails the risk of many adverse effects, including from the gastrointestinal tract or cardiovascular system.

[0007] An alternative to the above-mentioned methods is photodynamic therapy (PDT), which consists in the selective destruction of diseased tissues with a photosensitizing substance and radiation at appropriate wavelength. The only available photosensitizers are delta- aminolevulinic acid (ALA) in the form of hydrochloride and its methyl ester. Both substances are pro-drugs with cytotoxic and immunomodulatory effects, which are converted in the body to an appropriate sensitizer, protoporphyrin IX. The advantage of ALA is that after topical administration it accumulates in the mucosa and not in the submucosal or muscular membrane, which in turn increases the chances of selective destruction of cells without affecting tissue support structures.

[0008] In recent years, attempts have been made to use PDT in the treatment of precancerous lesions of the oral cavity. For example, in studies conducted at the MUB Department of Periodontal and Oral Mucosa Diseases, a significant remission of clinical symptoms of lichen planus was observed with a low risk of relapse even 12 months following the end of treatment (Sulewska M. et al., 2017). Despite the benefits of the use of a photosensitizing substance, limitations related to the form of the drug itself (a lipophilic base for ALA was used) were observed in the studies, i.e. difficulty in applying the drug and problems with maintaining it on the surface of the mucosa.

[0009] Therefore, it should be emphasized that there are no preparations with photosensitizing substances intended to be applied to mucosal tissue, hence in practice it becomes necessary to use products with photosensitizing substances intended to be applied to the skin (Ameluz (Biofronterra) and Metvix (PhotoCure) preparations not available in Poland). This problem is mainly due to the lack of suitable carriers exhibiting good mucoadhesive properties.

[0010] In the prior art, there are solutions related to the carriers themselves as well as their combinations with the active substance.

[0011] In recent years, mucoadhesive preparations without active substances intended for the care of the oromucosal tissue, i.e. MuGard liquid (https: / / www.rxlist.com / mugard-drug.htm) and Mucosit gel with plant extracts (https: / / www.herbapol.poznan.pl / pl / produkty / mucosit), have been put on the pharmaceutical market. The preparations comprise carbomer - a synthetic polymer of acrylic acid with a proven capability to interact with the mucosa in vivo, responsible for protecting and supporting the regeneration of the mucosa. Carbomer is available in a variety of types showing different variable physicochemical characteristics. Due to its sensitivity to pH changes and high likelihood of adverse interactions with active substances, it is difficult to obtain a stable preparation with the desired biopharmaceutical and quality characteristics.

[0012] In addition, WO 2020 / 248232 discloses a composition for oral applications comprising hyaluronic acid and a combination of polymers, among which natural gums, including tragacanth and xanthan gum, are also listed, with xanthan gum indicated as preferred. Such a composition, in particular, provides a rinse-free formulation having good consistency, which may be helpful for improved treatment efficacy of oromucosal tissue.

[0013] Prezotti et al., 2020 discloses mucoadhesive films with potential use as buccal drug delivery system comprising a combination of gellan gum and pectin with the addition of glycerol as a plasticizer. The films disclosed in the publication have a thickness comprised in the range of 18-30 μm, while being transparent, flexible and homogeneous. Further, a high content of gellan gum has been shown to improve the mechanical resistance and mucoadhesive properties of the films. In addition, the drug release rate, here being curcumin, which exhibits inter alia anti-inflammatory properties, was also tested.

[0014] Potas et al., 2020 discloses polyelectrolyte complexes of tragacanth gum and chitosan and their potential use in hydrogels additionally enriched with xanthan gum intended for use in oral mucosal preparations. The hydrogel preparations also contained an appropriate amount of propylene glycol, water and acetic acid, the presence of which resulted from the method of preparation of individual polymer components. These preparations were not tested for drug release but only for mucoadhesion and swelling. Despite a large variety of polymers used in the carriers, there are still no such combinations that would show good mucoadhesion, biocompatibility, and at the same time would be compatible with active substances intended for the treatment of oromucosal diseases and thus would allow to obtain preparations ensuring appropriate release of drugs and showing appropriate adhesion to the mucosa, which in turn would translate into the effectiveness of the preparations in the alleviation of the course and treatment of oromucosal diseases, respectively.

[0015] The object of the invention was to develop a pharmaceutical composition with good mucoadhesion, high biocompatibility and compatibility with pharmaceutically active substances and the one that, when combined with the active substance, would not lose its beneficial properties and would support the effect of the active substance. The object of the invention was also to develop compositions that would be suitable for use in the alleviation and treatment of oral diseases.

[0016] The object of the invention is a pharmaceutical composition with mucoadhesive properties comprising an amphoteric absorption promoter, vegetable oil, preservatives, humectant and water, and at least one natural gum selected from the group comprising gellan gum, tragacanth, xanthan gum and a combination thereof, characterized in that the natural gum content is comprised in the range of 2 to 8 % w / w and the ratio of the amphoteric absorption promoter: vegetable oil: preservatives: humectant expressed in % w / w is comprised in the range of 0.3 to 0.7: 1.7 to 2.3: 0.1 to 0.3: 3 to 7; wherein the water content constitutes a supplement to 100% w / w.

[0017] Preferably, the amphoteric absorption promoter is selected from a group comprising betaine and its derivatives, gelatin and lecithin.

[0018] More preferably, the amphoteric absorption promoter is lecithin.

[0019] Preferably, the vegetable oil is selected from the group comprising castor, peanut, cotton, safflower, linseed, almond, canola, sesame, soybean, sunflower, evening primrose, wheat germ, borage, olive oil and combinations thereof.

[0020] More preferably, the vegetable oil is castor oil.

[0021] Preferably, the preservatives are selected from a group comprising disodium edetate, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, benzalkonium chloride, and a combination thereof.

[0022] More preferably, the preservatives are a combination of disodium edetate and sodium benzoate in a 1 : 1 ratio. Preferably, the humectant is selected from the group comprising propylene glycol, glycerol, polyethylene glycol, and a combination thereof.

[0023] More preferably, the humectant is propylene glycol.

[0024] Preferably, gellan gum is a high acyl form.

[0025] Preferably, the viscosity of tragacanth measured for a 1% solution is 150 to 250 cPas at a temperature of 25°C ± 1°C.

[0026] Preferably, the viscosity of xanthan gum measured for a 1% solution is 1450 to 1550 cPas at a temperature of 25°C ± 1°C.

[0027] Preferably, the natural gum is tragacanth in an amount comprised in the range of 3 to 7% w / w. Preferably, the natural gum is a combination of tragacanth with xanthan gum in amounts expressed in % w / w comprised in the ranges of 3 to 7 % w / w for tragacanth and of 0.3 to 1.2 % w / w for xanthan gum, respectively.

[0028] Preferably, the natural gum is a combination of xanthan gum and gellan gum in amounts expressed in % w / w comprised in the ranges of 1.7 to 2.3 % w / w for xanthan gum and of 0.5 to 1 % w / w for gellan gum, respectively.

[0029] Preferably, the composition of the invention further comprises a pharmaceutically active substance for the treatment of oromucosal diseases selected from the group comprising antifungal substances and photosensitizing substances, characterized in that the active substance is present in an amount comprised in the range of 0.3 to 7% w / w.

[0030] More preferably, the antifungal substance is clotrimazole present in an amount of between 0.3 and 1.2% w / w.

[0031] More preferably, the photosensitizing substance is selected from a group comprising delta- aminolevulinic acid in the form of hydrochloride and its methyl ester in an amount comprised between 3 and 7% w / w, most preferably delta-aminolevulinic acid in the form of hydrochloride.

[0032] Preferably, the composition of the invention is in the form of gel, more preferably emulgel.

[0033] Preferably, the composition of the invention further comprises a liquid flavour selected from a group comprising cherry and mint flavours in an amount of 0.04% w / w.

[0034] The object of the invention further comprises a composition of the invention for use in the alleviation and treatment of oromucosal diseases.

[0035] Preferably, the treatment of oral diseases is photodynamic therapy.

[0036] More preferably, oromucosal diseases are selected from fungal diseases and precancerous conditions.

[0037] Even more preferably, a precancerous condition is lichen planus of the oral mucosa. Even more preferably, a fungal disease is oral candidiasis caused by Candida sp.

[0038] The compositions of the invention as well as their combination with an appropriate pharmaceutically active agent such as clotrimazole and delta-aminolevulinic acid are suitable for use in the alleviation and treatment of uncomplicated conditions of epithelial tissue, in particular the oral mucosa, as well as in supportive care after surgical, periodontal or implantology procedures, respectively. This is primarily due to the fact that, when in contact with the mucous membrane, the compositions and their combinations with active substances form a kind of protective layer evenly covering the application site, thus performing protective and coating functions. In addition, they exhibit a high capability to interact with the mucosa (mucoadhesive properties) enabling the drug to stay at the application site. In addition, the capability to improve the absorption of the drug and thus to increase its pharmacological efficacy was observed. The developed composition of the invention in combination with an antifungal substance such as CLO, owing to its capability to interact with the mucosa, allows for a longer contact time of the drug with the affected tissue, and additionally by partial dissolution of CLO particles within the composition, which in turn contributes to improved efficacy to treat oral candidiasis. In turn, the combination of the composition of the invention with ALA is a response to therapeutic needs of patients suffering from chronic precancerous conditions within the oral cavity, i.e. lichen planus, the standard treatment of which, i.e. local corticosteroid therapy, is not effective and the preparations used so far in therapy, if any, were intended for the treatment of other parts of the human body than the oral mucosa, which translated into their effectiveness, which in the case of the composition of the invention containing ALA was significantly better.

[0039] The object of the present invention is presented with the drawing, in which:

[0040] Figure 1 shows mucoadhesive properties expressed as work of mucoadhesion (Wad) of B1-B3 compositions of the invention, the compositions of the invention additionally containing delta-aminolevulinic acid in the form of hydrochloride (F1-F3 (5% ALA)) and the compositions of the invention additionally containing clotrimazole (C1-C3 (1% CLO)) compared to Anaftin preparation (mean± S.D.; n=4); * p< 0.05; ** p< 0.01

[0041] Figure 2 shows mechanical properties of: (a) hardness; (b) consistency; (c) cohesiveness of B1-B3 compositions of the invention, the compositions of the invention further comprising delta-aminolevulinic acid in the form of hydrochloride (F1-F3 (5% ALA)) and the compositions of the invention further comprising clotrimazole (C1-C3 (1% CLO)) compared to Anaftin dental preparation (mean± S.D.; n>4). Figure 3 illustrates the survival viability (expressed as % control) of the oromucosal tissue model after application of (a) the compositions of the invention (B1-B3) and (b) the compositions of the invention comprising 5% (w / w) ALA (F1-F3) (n=3, mean ± S.D.)

[0042] Figure 4 shows the concentration of interleukin Ibeta in the tissue medium after the application of the compositions (a) of the invention (B1-B3) and (b) the compositions of the invention comprising 5% (w / w) ALA (F1-F3) (n=3, mean ± S.D.)

[0043] Figure 5 shows a microscopic image of a section of an oromucosal tissue model (SkinEthic): (a) after 2 h and (b) after 5 h of incubation with the composition of the invention (B1-B3), compositions of the invention containing 5% (w / w) of delta-aminolevulinic acid in the form of hydrochloride (F1-F3) and NC control - water, and PS - sodium lauryl sulfate. Hematoxylin and eosin staining, x40 magnification.

[0044] Figure 6 shows the release profile of ALA (expressed as a percentage of the total drug dose in the Teflon extraction chamber) in the acceptor fluid from F1-F3 compositions compared to commercial Ameluz preparation (Referent) (mean ± SD; n = 3).

[0045] Figure 7 shows: (a) permeation of ALA into the acceptor fluid expressed in pg per unit area of the animal mucosal model by the composition of the invention used (B1-B3) compared to commercial Ameluz preparation (Referent); (b) oromucosal ALA retention determined after 3h of analysis relative to reference Ameluz preparation (n=5; mean ± S.D.)

[0046] In the course of studies on the development of compositions with mucoadhesive properties, various combinations of components belonging to a group of natural polymers were examined to obtain the best properties in the context of their use in the treatment and prevention of diseases of epithelial tissue, in particular of oral mucosa. The composition of the invention is in the form of emulgel with a consistency allowing easy and quick application to the tissue surface. Preservatives, stabilizing substances and absorption promoters present in the composition are excipients commonly used in preparations for topical administration.

[0047] The developed compositions of the invention are produced by a simple and repeatable technique with the use of a mechanical stirrer (or homogenizer). This method was tested under laboratory conditions using a mechanical stirrer (e.g. from Heidolph or Ika, or an unguator). The product with a weight not exceeding 200g was obtained at a time. To obtain a homogeneous composition of desired consistency, it is required to use an appropriate sequence in the process of the combining of ingredients. The compositions of the invention are based on natural macromolecular polymers, wherein the origin of raw materials is quite significant because, depending on the manufacturer, they are characterized by different properties, i.e. molecular weight, degree of purity, degree of acylation. Raw materials from CK Kelco (gellan gum in a high acyl (HA) form) and Sigma Aldrich (tragacanth with a viscosity of 1% solution being 200±15 cPas, xanthan gum with a viscosity of 1% solution being 1500±30 cPas) were used in the studies on prototypes. Viscosity testing was performed using a Viscotester 6 Plus ThermoHaake rotary viscometer (Thermo Scientific, Germany), equipped with a TL 7 rotor, at a temperature of 25°C ± 1°C. Measurements were made at a rotational speed of 30-60 / min. The delta-aminolevulinic acid used in the embodiments is in the form of hydrochloride. It is also acceptable to use delta- aminolevulinic acid methyl ester.

[0048] Example 1

[0049] Method for preparing the compositions of the present invention

[0050] On a laboratory scale, the compositions are made by homogenization using a mechanical stirrer (e.g. from Heidolph or Ika, or an unguator). To obtain a homogeneous composition of desired consistency, it is required to use an appropriate sequence in the process of the combining of ingredients.

[0051] In the first stage, a dispersion of tragacanth and xanthan gum (Bl composition) or only tragacanth (B2 composition) in water is prepared. It is recommended to gradually add polymers (in powder form) on the surface of the solvent at room temperature. In the case of Bl composition, both polymers can be simultaneously, in portions, dispersed in water. In separate vessels, an aqueous solution of preservatives (disodium edetate and sodium benzoate in a 1 : 1 ratio) and a solution of amphoteric absorption promoter, here lecithin in a humectant (here propylene glycol), are prepared. These solutions are successively added the polymer dispersion with continuous stirring. To the mixture thus prepared, vegetable oil, which in this case is castor oil, is added. Acceptable vegetable oils used in the pharmaceutical compositions are: castor, peanut, cotton, safflower, linseed, almond, canola, sesame, soybean, sunflower, evening primrose, wheat germ, borage, olive oils. At each stage, the time of gel carrier homogenization with the use of a compounding robot does not exceed 4-5 minutes (at a rotational speed of about 400-500 rpm). It should be added that the function of preservatives may be performed, for example, by disodium edetate, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, benzalkonium chloride, while the function of humectants may be performed by such substances as propylene glycol, glycerol, polyethylene glycol. In addition to lecithin, betaine and its derivatives and gelatin may be used as an amphoteric absorption promoter.

[0052] To obtain the B3 composition, it is necessary to preheat the solvent to 80°C to completely dissolve gellan gum. Xanthan gum is added only to a ready dispersion of gellan gum. In separate vessels, an aqueous solution of preservatives (disodium edetate and sodium benzoate in a 1 : 1 ratio) and a solution of lecithin in propylene glycol are prepared. A liquid flavour selected depending on the flavour of the medicinal substance, such as, for example, a cherry or mint flavour in an amount of 0.04% w / w, may be added to the mixture of lecithin in ethylene glycol. These solutions are successively added the polymer dispersion with continuous stirring. To the mixture thus prepared, vegetable oil, which in this case is castor oil, is added. At each stage, the time of gel carrier homogenization with the use of a compounding robot does not exceed 4-5 minutes (at a rotational speed of about 400-500 rpm).

[0053] The resulting composition is then combined with the active substance. The composition of the compositions of the invention is shown in Table 1.

[0054] Table 1: Composition of B1-B3 compositions of the invention

[0055] Example 2 Method for preparing the compositions of the invention further comprising an active substance in the form of delta-aminolevulinic acid and clotrimazole, respectively

[0056] To prepare the compositions of the invention further comprising an active substance which is water soluble (herein delta-aminolevulinic acid in the form of hydrochloride, ALA), the weighted active substance is ground with a humectant, (i.e. propylene glycol, a recommended ratio of ALA to propylene glycol is 3: 1, w / w), and then combined in portions with a suitable B1-B3 composition of the invention obtained according to the method described in Example 1. Homogenization is recommended, e.g. with the use of an unguator (time of 3-4 minutes, rotation speed of 400-500). The composition may further comprise a cherry flavour in an amount of 0.04% (w / w). The final ALA concentration in F1-F3 compositions is 5% (w / w) - Table 2.

[0057] Table 2: The composition of F1-F3 compositions of the invention comprising 5% (w / w) delta-aminolevulinic acid in the form of hydrochloride (ALA) and a suitable composition of the invention

[0058] When a pharmaceutical composition of the invention comprising an active substance taking the form of crystalline powder, sparingly soluble in water (here with clotrimazole, CLO), is prepared, the active substance is added to a solution of lecithin in propylene glycol (heating to 50°C is recommended). The resulting liquid suspension is homogenized with a gel base obtained according to the method described in Example 1, e.g., with the use of an unguator (4 minutes, rotation speed of 400-500). The composition may further comprise a mint flavour in an amount of 0.04% (w / w). The final CLO concentration in respective C1-C3 compositions of the invention is 0.5 and 1% (w / w), respectively - Table 3.

[0059] Table 3: The composition of C1-C3 compositions of the invention comprising 1% (w / w) clotrimazole (CLO). Example 3

[0060] Evaluation of B1-B3 compositions of the invention and respectively F1-F3 and C1-C3 compositions of the invention containing ALA and CLP, respectively, in terms of pharmaceutical quality and safety

[0061] The B1-B3 compositions of the invention with the composition specified in Table 1 were evaluated for pharmaceutical quality and safety of use in in vitro conditions using a 3D oral mucosa tissue model.

[0062] The testing of mucoadhesive properties was carried out using an TA.XT.Plus texture analyzer equipped with a 5 kg tensometric head and a upper G / Muc probe (Table 4). The composition in an amount of 1 ml was evenly applied with a syringe on the surface of the upper G / Muc probe, which was then mounted in the head arm. Freshly excised buccal mucosa from porcine cheek was attached to a thermostated heating plate with cyanoacrylic glue. Then, the upper G / Muc probe was lowered on the tissue surface at a speed of 0.5 mm / sec. The experimentally determined operating parameters of the texture analyzer were as follows: head contact force of 0.3 N, force holding time of 60 s, head lifting speed of 0.1 mm / s. The measurement of mucoadhesive properties was based on the assessment of the work necessary to interrupt the contact between the drug form and the biological material.

[0063] The compositions of the invention comprising ALA (F1-F3) and clotrimazole (C1-C3), respectively, were tested in an identical manner.

[0064] The tests carried out showed that all compositions of the invention in the form of emulgel are characterized by a high capability to adhere to the mucosa, which was confirmed ex vivo by a tensometric method using an animal model of buccal mucosa from porcine cheek compared to the control, i.e. the oromucosal preparation available on the market called Anaftin (Figure 1). The highest values of mucoadhesion work were found in the Bl composition, for which approx. 40% higher values of the mucoadhesion work parameter were reported compared to the Anaftin preparation and approx. 30% higher relative to the B2 and B3 compositions. The presence of the active substance did not significantly affect the mucoadhesive properties of the compositions containing active substances.

[0065] Table 4 M dh i i di i d i f h l b l l l i h li l

[0066] Example 4

[0067] Analysis of the mechanical properties of the compositions of the present invention and the compositions of the present invention comprising the active substances of the present invention.

[0068] The testing of the mechanical properties of B1-B3 compositions of the invention was carried out using a TA.XT.Plus texture analyzer equipped with an A / BE system for backwards extrusion with a diameter of 35 mm. The emulgel (30 g) was placed in the measuring vessel 2 h before the measurement, after which the system was lowered at a speed of 2 mm / s to a defined depth of 10 mm. In the analysis of the mechanical properties determining the application characteristics of the compositions of the invention, differences were shown between individual pharmaceutical compositions. The impact of the composition on the hardness (Figure 2a), consistency (Figure 2b) and cohesiveness of the composition (Figure 2c), i.e. the features affecting the ability to collect the composition from the package, spreading over the mucosal surface and its ability to adhere to tissue surface, was assessed.

[0069] The compositions of the invention were characterized by comparable hardness and cohesiveness values and a more compact consistency (almost three times higher values were observed) relative to the control, i.e. a reference oromucosal Anaftin product. A significant impact of the presence of the active substance, especially in the form suspended in the base (CLO), on the consistency and application properties of the compositions marked with C1 and C3 versus their corresponding Bl and B3 compositions (Figure 2), was observed.

[0070] The addition of ALA and CLO to the Bl composition significantly increased the values of analysed mechanical parameters (an almost 1.6-fold and 2.5-fold increase in the value of the hardness parameter for the F1 and C1 compositions, respectively, compared to the Bl composition, was reported), in contrast to the B3 composition, in which a decrease by approx. 30% in the hardness of the composition in the presence of ALA and CLO was observed, as well as a decrease by approx. 50% in the consistency parameter after dispersing ALA in this composition. In the case of the B2 composition, the introduction of CLO contributed to an almost twofold increase in the analysed consistency and cohesiveness parameters, while the presence of ALA resulted in a decrease in their value by approx. 20%.

[0071] Example 5

[0072] Assessment of irritation potential

[0073] The evaluation of the in vitro irritation potential of the compositions of the present invention and the compositions of the present invention containing, apart from the composition, an active substance, i.e. clotrimazole and delta-aminolevulinic acid, was carried out in accordance with the current legal standards for the evaluation of preparations for the skin and mucous membranes (OECD Guidelines for the Testing of Chemicals, In Vitro Skin and Mucosa Irritation: Reconstructed Human Epidermis Test Method, OECD / OCDE 439, 2015; EN ISO 10993-10:2013 Biological evaluation of medical devices - Part 10: Tests for irritation and skin sensitization (ISO 10993-10:2010)).

[0074] The compositions of the invention (B1-B3) and the compositions of the invention containing 5% (w / w) of ALA (F1-F3), a cytotoxic substance (classified in the ATC L01XD category: antineoplastic and immunomodulating drugs, cytostatics, other antineoplastic drugs according to the international anatomical therapeutic and chemical drug classification system) with a higher irritation potential compared to CLO (ATC D01A drugs for external use, imidazole and triazole derivatives) were selected for testing. A sufficient amount of the composition was applied to the surfaces of an oral epithelium model and incubated at 4 time points for 1, 2, 5 and 18 h to plot a response curve for the time of contact with the pharmaceutical composition and determine the limiting contact time at which cell viability is inhibited by 50% relative to cells in a control sample. The cytotoxic effect of the tested compositions on the viability of the tissue model was assessed with the MTT colorimetric test (testing of tetrazolium salt conversion by succinate dehydrogenase in metabolically active cells). In addition, the secretion of pro-inflammatory interleukins in the tissue medium was determined by using a BD FACSCanto II flow cytometer with FCAP Array v3 software (BD Bioscences Systems, USA). For this purpose, a commercial Human Inflammatory Cytokines Kit (BD Biosciences, San Jose, CA, USA) was used and the analysis was carried out in accordance with the protocol provided by the manufacturer and the procedure described in the literature [Czarnomysy R, Bielawska A., Bielawski K. Effect of 2nd and 3rd generation PAMAM dendrimers on proliferation, differentiation, and pro-inflammatory cytokines in human keratinocytes and fibroblasts. Int. J. Nanomedicine 2019, 14: 7123],

[0075] To assess the correlation between the results of in vitro tests and the in vivo effect, a prognostic model was used according to the European Centre for Validation of Alternative Methods (EVCAM), according to which the irritation potential of the preparation is predicted if the viability of the tissue model is below 50%, and the limiting concentration defined for II- 1 is ≥50 pg / ml.

[0076] The results of the tests of the viability of the reconstructed human oral epithelium upon incubation with the tested compositions without the active substance are presented in Figure 3(a), while after incubation with compositions containing 5% (w / w) ALA in Figure 3(b).

[0077] None of the compositions showed an irritative effect even after 18 h of incubation. The lowest influence on the viability of the tissue model cells was reported for the B3 composition of the invention and the composition additionally containing ALA (F3), although it should be emphasized that the differences as regards the effect of the B3 and F3 compositions were statistically insignificant (p>0.05). Importantly, the presence of ALA does not increase the irritation potential.

[0078] The secretion of interleukin Ibeta in the culture medium was measured cytometrically (Fig. 4).

[0079] The secretion of IL-1beta after incubation with compositions without the active substance (B1-B3) was low (below 5pg / ml) (Fig. 4a). The compositions of the invention containing ALA as an active substance (F1-F3) caused a moderate release of IL-1beta, although it should be emphasized that its level did not exceed 15 pg / ml and was significantly lower than that observed for the positive control, i.e. a surfactant with confirmed irritative effect (Figure 4b). Only in the case of the 5h time point, a higher concentration of IL-1beta in the medium was reported. According to the EU and GHS classification (R38 / Category 2 or no label), pharmaceutical compositions can therefore be considered non -irritative even after 18 h of incubation with a reconstructed human oral tissue model.

[0080] In addition, a histopathological analysis was performed based on the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), developed by associations of toxicologists and pathologists from Europe (ESTP), the United Kingdom (BSTP), Japan (JSTP), and the United States of America (STP). Ex vivo fragments of oral epithelium were fixed in formalin, followed by immersion in paraffin blocks. The epithelial tissue was assessed microscopically (an Axiolab 5 microscope equipped with an Axiocam camera and a ZEN 2.0 program (Zeiss) after staining of the biological material with the hematoxylline and eosin.

[0081] The study focused on the assessment of severity of pathological changes in cells in terms of keratinization (indicative of an irreversible degenerative process), atypia (indicative of early reversible degeneration of cells), the level of integrity of the connective tissue layer, apoptosis and necrosis. Pathophysiologically, the most serious changes are necrosis and apoptosis associated with cell death. Exemplary images are presented in Fig. 5.

[0082] In the case of compositions of the invention containing active substances, cytotoxic effects of moderate intensity were reported, with their occurrence over time being different. The most serious change found was atypia of moderate severity. Keratinization of epithelium and necrosis of insignificant severity, and the level of integrity of the connective tissue layer and apoptosis occurred to a minimum extent. The relatively most cytotoxic composition was shown to be F2. In the case of the F3 composition, however, a reduction in the severity of lesions over time was observed.

[0083] In the group of B1-B3 compositions of the invention, histopathological alterations did not occur (necrosis) or appeared in minimal extent (slight atypia and apoptosis, keratinization of slight severity). The Bl composition exhibited the smallest cytotoxic effects, while in the case of B2 and B3 compositions, an increase in cytotoxic changes was observed over the incubation time (time-dependent effect). It should be taken into account that lesions (especially as regards apoptosis) also occurred in the NC control group. This may result from the fact that the tissue which is in an ex vivo environment may react to environmental factors in a pathological way. In addition, the natural process in the case of the epithelium is the maturation and exfoliation of its cells, which under ex vivo conditions dominates over the processes of its regeneration. It is also worth noting that due to physiological conditions (saliva secretion, jaw movements), an average contact time of the preparation with the mucous membrane does not exceed 30-60 minutes. Therefore, the above results point to the safety of the compositions of the present invention when in contact with the oral mucosa.

[0084] Example 6

[0085] Dissolution studies and ALA permeation through excised buccal mucosa from porcine cheek for F1-F3 compositions of the invention

[0086] In addition, for the F1-F3 compositions of the invention (containing ALA), in vitro dissolution studies were performed in a Teflon extraction chamber using a pharmacopoeial paddle method and a test of ex vivo permeation through an buccal mucosa from porcine cheek using a flow- through apparatus equipped with thermostated diffusion chambers in accordance with current EMA In vitro skin permeation studies (IVPT).

[0087] In the dissolution test, a degassed simulated saliva fluid buffer, pH 6.8, in the amount of 100 ml was used as an acceptor medium. The experiments were carried out at a temperature of 37 °C ± 0.5°C. At specific time points, 2 ml of the acceptor fluid was collected, which, after filtering through cellulose syringe filters with a pore diameter of 0.45 μm, was diluted with an appropriate amount of PBS. ALA concentration was assayed by the HPLC method developed by Namjoshi et al. (2007). The loss of the medium was supplemented with a fresh acceptor fluid at a temperature of 37 °C.

[0088] During permeation testing, buccal mucosa from porcine cheek_obtained from animals weighing approximately 200-250 kg was used (Bost slaughterhouse, Turosn Koscielna, Poland). The biological material used is considered similar to human buccal mucosa in terms of its structure, lipid composition, as well as its ability to retain chemicals. The tissue, prepared immediately after the killing of the animal, was washed and frozen at -20 °C in physiological saline solution. Prior to the experiments, the material was thawed at room temperature, washed with a saline solution, and then the mucosa was separated from the muscle layer using surgical tools. The proposed ex vivo permeation test with the use of the buccal mucosa did not require an approval from a Local Ethics Committee. After placement in the diffusion chamber, the prepared mucosa was conditioned with an acceptor medium (PBS, pH 7.2) for 60 min (36±1 °C). The gel sample (corresponding to 5 mg ALA) was applied on the surface of the buccal mucosa. The concentration of the drug substance in the chamber (as recommended by the EMA In vitro skin permeation studies (IVPT)) did not limit the rate and did not affect the degree of mucosal permeation. The acceptor fluid washed the tissue from the side of the muscle layer. The permeation area was 0.81 cm2. The experiments were conducted at a temperature of 36± 1°C for 3h. At specific time points (i.e., 30, 60, 90, 120 and 180 minutes), the acceptor fluid was collected, and the loss of medium was supplemented with a fresh medium at 36 °C. Prior chromatographic analysis, the samples were filtered through cellulose syringe filters with a pore diameter of 0.22 μm. After 3h, the gel from the tissue surface was aspirated into a separate vessel and the tissue was washed with portions of SSF pH 6.8 (10 mL) to completely remove the drug substance from the surface of the biological material. The aspirate along with the washing fluid were shaken in a water bath (150 rpm, 30 °C). After filtration (0.45 μm nylon filters), the aspirate was diluted with PBS and quantitatively analysed by HPLC. At the end of the test, the tissue was cut into pieces, homogenized using the HPLC phase and incubated for 3h in a water bath (30°C, 150 rpm). After centrifugation (4000 rpm, 15 min) and filtering through nylon filters with a pore diameter of 0.45 μm, the amount of drug substance accumulating in the tissue was assayed by HPLC.

[0089] Some differences in the photosensitizing substance release profile were observed between the compositions of the invention (Figure 6). In general, the active substance was released most rapidly from Ameluz (77% of the initial dose of the drug was recorded in the acceptor fluid at 30 minutes of test). In the case of F1 and F3 compositions of the invention, approximately 80% of the dose of the drug was recorded at 90 minutes of the test. Already in the first 30 minutes of the test, the amount of ALA in the acceptor fluid (simulated saliva fluid, SSF, pH 6.8) exceeded 40% for all the tested formulations. The F2 composition was characterized by a prolonged release of the active substance.

[0090] Differences in the degree of drug permeation from the tested ALA compositions through the tissue model were also reported (Figure 7). The F1 and F3 compositions significantly improved the permeation of the active substance, especially in the second part of the test. The above observations correlate with results from dissolution studies (Figure 6). After 3h of the test, approx. 30% more active substance was reported in the acceptor fluid compared to the F2 composition. It should be emphasized that all compositions increased the permeation of ALA compared to the registered Ameluz preparation with ALA (Control), and the F1 and F3 compositions additionally increased the retention of the drug substance in the tissue (Figure 7).

[0091] The F1 composition, which helps achieve a rapid onset of drug action, while having a strong ability to enhance permeability to deeper epithelial layers, appears to comprise the most optimal carrier for a photosensitizer. Example 7

[0092] Tests of microbiological activity of C1-C3 compositions with clotrimazole according to the present invention.

[0093] The antifungal activity of C1-C3 compositions containing clotrimazole was assayed by the agar diffusion method according to the CLSI (Clinical and Laboratory Standards Institute) guidelines [Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, Approved Standard, CLSI document M-27-A3, 3rd ed.; Clinical and Laboratory Standards Institute (CLSI), Wayne, USA, 2008; Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast. Third Informational Supplement, CLSI document M27-S3; Clinical and Laboratory Standards Institute (CLSI), Wayne, USA, 2008.]. This method determines the susceptibility of the tested strain based on the size of the diameter of the growth inhibition zone around the well (disc) filled with the preparation. Standard strains of Candida albicans ATCC 10231, Candida parapsilopsis ATCC 22019 and Candida krusei ATCC 6528 were used for the test. The tested yeast strains were suspended in a sterile physiological saline solution (0.9% NaCl). Then they were seeded to plates with Sabourand medium to obtain a final density of 1.4 x 106cfu / ml (which corresponds to 0.5 on the McFarland standard scale). 100 mg of the tested C1-C3 compositions and controls (a clotrimazole cream preparation, Clotrimazolum GSK, 10 mg / g and a clotrimazole solution in dimethyl sulfoxide, 5 mg / g and 10 mg / g) were placed on the surface of the culture into hollow wells with a diameter of 8 mm under aseptic conditions. The plates were incubated at 37 °C ± 0.5 °C for 24h, following which the inhibition zone diameter reflecting inhibition of fungal colony growth around the wells was measured with an accuracy of up to 0.1 mm.

[0094] It has been shown that the C1-C3 compositions significantly inhibit the growth of all tested Candida sp. strains (p < 0.05) relative to the reference Clotrimazolum GSK cream preparation and show greater ability for emulgel to penetrate deep into the culture medium. There were slight differences in the size of growth inhibition zones between individual strains. Among the tested compositions, the C3 composition shows stronger activity against C. albicans and C. krusei, while the C2 composition inhibits the growth of C. parapsilopsis to the greatest extent. It should be emphasized that no significant differences in antifungal activity were observed between the compositions with clotrimazole at a concentration of 5 mg / g and 10 mg / g. The obtained results indicate that the antifungal effect of clotimazole may be related to the degree of its solubility in the composition. At the same time, it should be emphasized that the tested compositions with clotrimazole at a concentration of 5 mg / g are characterized by better in vitro activity against yeasts relative to the commercially available Clotrimazolum GSK cream preparation with clotrimazole at a concentration of 10 mg / g. The results are presented in Tables 5-7, respectively.

[0095] Table 5. Activities of C1-C3 compositions of the invention with clotrimazole and Clotrimzaolum GSK cream against the Candida albicans ATCC 10231 strain (mean ± S.D.; n

[0096] = 31

[0097] Composition Mean value of growth inhibition zone

[0098] Table 6. Activities of C1-C3 compositions of the invention with clotrimazole and Clotrimazolum GSK cream against the Candida parapsilopsis ATCC 6528 strain (mean ±

[0099] S D ; n = 3)

[0100] Mann-Whitney test; p < 0.05 relative to Clotrimazolum GSK cream

[0101] Table 7 Activities of C1 C3 compositions of the invention with clotrimazole and =

[0102] Example 8

[0103] Tests of the efficiency of Fl composition with delta-aminolevulinic acid of the present invention.

[0104] Based on the above results and sensory evaluation of a group of volunteers at the MUB Department of Periodontal and Mucosal Diseases, the F1 composition was selected for further studies on the volunteers.

[0105] 79 patients with histopathologically confirmed lesions of oral lichen planus (OLP) were enrolled in the study. The patients were randomized to two groups. In the first group (I), OLP foci were subjected to photodynamic therapy (5 sessions). In the second group (II), local pharmacotherapy was implemented, where a steroid was applied to the lesion twice a day for a period of 2 weeks. The patients from both groups were followed for a 6-month follow-up period.

[0106] The proprietary PDT protocol assumed the use of delta-aminolevulinic acid (ALA), which was applied to the saliva-deprived lesion and the surrounding mucosa (a layer of about 2mm) 40 minutes before irradiation with a FotoSan® 630 diode lamp delivering radiation at a wavelength of 630 nm from the end of the optical fiber. PDT sessions were repeated 5 times at weekly intervals.

[0107] Before the start of treatment and 1, 3 and 6 months after the implemented procedure (groups I and II), photographic documentation was prepared and the size of the lesions (in centimetres) was macroscopically assessed using a PCPUNC 15 periodontal probe (Hu-Friedy, IL, USA). The measurement concerned the length and width of the longest section connecting extreme points within a disease focus and a border of healthy mucosa.

[0108] 79 persons (14 men and 65 women) completed the 6-month follow-up period:

[0109] - group 1 - 46 persons (83 OLP foci)

[0110] - group 11 - 33 persons (50 OLP foci).

[0111] Of the 133 OLP lesions, 113 (84.96%) were located on the lining mucosa: 93 lesions on the cheeks, 18 lesions on the lateral surface of the tongue, and 1 lesion on the mucosa of the lower lip. Twenty-one foci occurred on the chewing mucosa: 17 foci on the gum, 3 foci on the palate, and 1 focus on the dorsal surface of the tongue.

[0112] 83 OLP foci were subjected to photodynamic therapy (group I): 70 foci located on the lining mucosa (55 foci on the cheeks, 15 foci on the lateral surface of the tongue), 13 foci on the chewing (keratinizing) mucosa (9 foci on the gums, 3 foci on the palate and 1 focus on the dorsal surface of the tongue).

[0113] An average size of OLP foci in group I prior to treatment was 2.29 cm2±1.65. An average size of lesions located on the non-keratinizing mucosa was larger (2.39 cm2±1.74) compared to lesions occurring on the chewing mucosa (1.74 cm2±0.84). Six months after PDT therapy, improvement was found in 72 sites, including 47 sites with complete resolution of lesions, which indicates that 86.75% of treated OLP foci responded (reacted) to therapy. An average reduction in the size of the lesions was 74.24%, with a reduction in an average size of the surface area to 0.59 cm2±1.06. After a 6-month follow-up period, a 79.08% reduction in the size of lesions located on the lining mucosa was reported (from 2.39 cm2±1.74 to 0.5 cm2±1.00, a reduction in an average size by 1.89 cm2). The OLP foci located on the chewing mucosa showed a reduction in their area by 0.62 cm2(from 1.72 cm2±0.84 to 1.12 cm2±1.26), with a 35.64% reduction in their size.

[0114] An average size of OLP foci in group II prior to treatment was 2.42 cm2±2.08. There were 42 lesions located on the lining mucosa (average size: 2.63 cm2±2.19), 8 lesions on the gum (average size: 2.02 cm2±1.32). After a six-month follow-up, improvement was found in 24 sites, including 12 sites with complete resolution of lesions. Of the 50 lesions subjected to topical steroid treatment, 26 sites (52%) showed no response to treatment, and in 2 of them an exacerbation and an increase in the expression of OLP lesions expressed by an increase in the size of the foci were observed. An average reduction of the size of the lesions was 36.37%. The reduction in the size of lesions located on the lining mucosa was 40.69% 5 (reduction in their surface area from 2.63 cm2±2.19 to 1.56 cm2±2.32). Lichen foci located on the chewing mucosa did not respond to treatment. There was an increase in their size by 11.36% (increase in their surface area by 0.15 cm2to 1.47 cm2± 1.47).

[0115] The obtained results indicate a higher efficacy of PDT therapy using the composition of the invention in the form of emulgel with ALA compared to topical corticosteroid therapy in the treatment of oral lichen planus.

[0116] In group I, a significantly higher reduction in the size of OLP foci was observed compared to group II (74.24% versus 36.37%) (p<0.001) (Table 8).

[0117] Table 8. Average size, standard deviation, and average reduction in OLP lesions in the study groups (group I and group II) prior to and following treatment.

[0118] References:

[0119] 1. Potas J. et al., Tragacanth gum / chitosan polyelectrolyte complexes-based hydrogels enriched with xanthan gum as promising materials for buccal application. Materials 2020

[0120] 2. Prezotti EG. Mucoadhesive fdms based on gellan gum / pectin blends as potential platform for buccal drug delivery. Pharm Dev Technol 2020

[0121] 3. Sulewska M. et al. A clinical evaluation of efficacy of photodynamic therapy in treatment of reticular oral lichen planus: a case series. Photodiagnosis Photodyn. Ther. 2019

[0122] 4. Khatter NJ, Khan MAB. Clotrimazole. [Updated 2022 Jul 11]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK560643 /

Claims

AMENDED CLAIMS received by the International Bureau on August 1 , 2024 (01.08.2024) Claims1. A pharmaceutical composition with mucoadhesive properties comprising an amphoteric absorption promoter, vegetable oil, preservatives, humectant and water, characterized in that comprise at least two natural gums selected from a group comprising gellan gum, tragacanth, xanthan gum and a combination thereof, wherein the content of natural gum is comprised in the range of 2 to 8 % w / w and the ratio of the amphoteric absorption promoter: vegetable oil: preservatives: humectant expressed in % w / w is comprised in the ranges of 0.3 to 0.7: 1.7 to 2.3: 0.1 to 0.3: 3 to 7; wherein the water content constitutes a supplement to 100% w / w.

2. The composition of claim 1, characterized in that the amphoteric absorption promoter is selected from a group comprising betaine and its derivatives, gelatin and lecithin.

3. The composition of claim 2, characterized in that the amphoteric absorption promoter is lecithin.

4. The composition of any one of claims 1 to 3, characterized in that the vegetable oil is selected from a group comprising castor, peanut, cotton, safflower, linseed, almond, canola, sesame, soybean, sunflower, evening primrose, wheat germ, borage, olive oils and a combination thereof.

5. The composition of claim 4, characterized in that the vegetable oil is castor oil.

6. The composition of any one of claims 1 to 5, characterized in that the preservatives are selected from a group comprising disodium edetate, sodium benzoate, benzoic acid, sorbic acid, potassium sorbate, benzalkonium chloride, and a combination thereof.

7. The composition of claim 6, characterized in that the preservatives are a combination of disodium edetate and sodium benzoate in a 1 : 1 ratio.

8. The composition of any one of claims 1 to 7, characterized in that the humectant is selected from a group comprising propylene glycol, glycerol, polyethylene glycol, and a combination thereof.

9. The composition of claim 8, characterized in that the humectant is propylene glycol.

10. The composition of any one of claims 1 to 9, characterized in that the gellan gum is in high acyl form.

11. The composition of any one of claims 1 to 10, characterized in that the viscosity of tragacanth measured for a 1% solution is 150 to 250 cPas at a temperature of 25°C ± 1°C.

12. The composition of any one of claims 1 to 11, characterized in that the viscosity of xanthan gum measured for a 1% solution is 1450 to 1550 cPas at a temperature of 25°C ± 1°C.

13. The composition of any one of claims 1 to 12, characterized in that the natural gum is a combination of tragacanth with xanthan gum in amounts expressed in % w / w comprised in the ranges of 3 to 7 % w / w for the tragacanth and of 0.3 to 1.2 % w / w for the xanthan gum, respectively.

14. The composition of any one of claims 1 to 12, characterized in that the natural gum is a combination of xanthan gum and gellan gum in amounts expressed in % w / w comprised in the ranges of 1.7 to 2.3 % w / w for the xanthan gum and of 0.5 to 1 % w / w for the gellan gum, respectively.

15. The composition of any one of claims 1 to 15, characterized in that it further comprises a pharmaceutically active substance for the treatment of oral diseases selected from a group comprising antifungal substances and photosensitizing substances, characterized in that the active substance is present in an amount comprised in the range of 0.3 to 7% w / w.

16. The composition of claim 16, characterized in that the antifungal substance is clotrimazole present in an amount of between 0.3 and 1.2% w / w.

17. The composition of claim 16, characterized in that the photosensitizing substance is selected from a group comprising delta-aminolevulinic acid in the form of hydrochloride and its methyl ester in an amount of between 3 and 7% w / w, preferably delta-aminolevulinic acid in the form of hydrochloride.

18. The composition of any one of claims 1 to 18, characterized in that it is in the form of gel, preferably emulgel.

19. The composition of any one of claims 1 to 19, characterized in that it further comprises a liquid flavour selected from a group comprising cherry and mint flavours in an amount of 0.04% w / w.

20. The composition of any one of claims 1 to 20, for use in the alleviation and treatment of oral diseases.

21. The composition of claim 21, characterized in that the treatment of oral diseases is photodynamic therapy.

22. The composition of claim 21 or 22, characterized in that the oral diseases are selected from fungal diseases and precancerous conditions.

23. The composition of claim 23, characterized in that the precancerous condition is oral lichen planus.

24. The composition of claim 23, characterized in that the fungal disease is oral candidiasis caused by Candida sp.

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