EP4676440A2

EP4676440A2 - Oral composition based on active ingredients of natural origin and its use in therapy and in the treatment and prevention of oral mucosa disorders

Info

Publication number: EP4676440A2
Application number: EP24716461.9A
Authority: EP
Inventors: Miguel Angel Alonso Cohen; Sabrina CASELLA; Giulia BRAMATI; Bhavna KARNANI; Rossana SIDOTI; Marco Di Fulvio
Original assignee: Devintec Sagl
Current assignee: Devintec Sagl
Priority date: 2023-03-08
Filing date: 2024-03-07
Publication date: 2026-01-14
Also published as: WO2024184843A3; IT202300004341A1; WO2024184843A2

Abstract

The present invention relates to an oral composition based on active ingredients of natural origin and its use in therapy, particularly for the treatment and prevention of oral mucosa disorders, particularly but not limited to stomatitis, aphthae and oral ulcers.

Description

ORAL COMPOSITION BASED ON ACTIVE INGREDIENTS OF NATURAL ORIGIN AND ITS USE IN THERAPY AND IN THE TREATMENT AND PREVENTION OF ORAL MUCOSA DISORDERS

The present invention relates to a composition for oral use based on active ingredients of natural origin such as pea proteins and tamarind seed polysaccharides. In addition, the present invention also relates to a composition for oral use based on active ingredients of natural origin, such as pea proteins and tamarind seed polysaccharides, and to its use in therapy, particularly for use in a method for the treatment and/or prevention of oral mucosa disorders, particularly but not limited to stomatitis, aphthae and oral ulcers.

It is known that oral mucosa ulcers are quite common in the healthy population. Physiological symptoms, such as pain and burning sensation during daily activities such as drinking, eating or talking, are often accompanied by a worsening of the patient's quality of life. In some cases, occasional ulcers may become recurrent, known as aphthae or recurrent aphthous stomatitis. The prevalence of recurrent aphthous ulcer is higher in young adults, and the severity decreases with increasing age. The etiology of aphthous ulcer remains unclear. Possible causes include trauma to the oral mucosa, drug use, deficiency of vitamin B12, folic acid, iron, stress, hormonal changes, and metabolic diseases.

Topical agents such as local antibiotics, local antiseptics, topical NSAIDs, and topical corticosteroids are generally prescribed to relieve symptoms. Various approved drug formulations such as pills, mouthwashes, sprays, and pastes, such as vitamin B12, chlorhexidine mouthwash, steroid tablets, and local anesthetics are mainly suggested for the treatment of aphthous ulcers. These topical symptomatic treatments, such as corticosteroids, can reduce the severity of ulceration but are not always able to stop recurrences; moreover, longterm side effects are known. In clinical practice, dentists are often asked to recommend safe and rapid therapy to relieve the pain caused by mouth ulcers.

Patents EP3131568 B1 and EPO3348273 B1 describe a combination of xyloglucan extracted from tamarind and pea protein for the treatment of diarrhea and other intestinal infections and/or inflammation.

Patent application WO2017207223 A1 describes the use of protein-polysaccharide conjugates in the treatment of intestinal disorders.

Therefore, it is necessary to investigate alternative treatments to safely treat oral ulcers, including recurrent ones.

In recent years, naturally-derived compounds have attracted the interest of researchers as potential treatments for several diseases associated with the gastrointestinal tract, mainly because of their optimal safety profile.

In addition to this, in a social context of steadily increasing consumer interest in natural therapeutic solutions, there is a need for said therapeutic solutions to be as natural, preferably plant-based, as possible.

Therefore, it is a purpose of the present invention to provide a new composition of active ingredients of natural origin that can treat and prevent mouth disorders.

It is a further purpose of the invention to provide the use of the composition of the invention in therapy particularly, but not only, in the prevention and treatment of mouth disorders, such as stomatitis, mouth ulcers, and aphthae. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Results of the in vitro study: mucoadhesivity on reconstructed human oral epithelium.

Figure 2: Labeling index (LI) of BrdU-labeled cell; values are shown as mean ± SEM. *** P < 0.001.

Figure 3. MTT graph with percentage of viability; values shown are the mean±SEM.

Figure 4: Histological evaluation.

Figure 5, 5A and 5B: Effect of the composition of the invention on histopathological changes caused by phenol- induced oral ulcer and cytokine production in phenol-induced oral ulcer.

DESCRIPTION OF THE INVENTION

The Applicant, after a long and intensive research and development effort, has surprisingly found that an association or combination of specific active ingredients of natural origin, and particularly of plant origin, is particularly effective in the treatment and/or prevention of oral disorders, particularly disorders of the oral mucosa, preferably, but not limited to, stomatitis, aphthae, and ulcers.

Thus, according to one of its aspects, it is an object of the invention an oral topical composition comprising a mixture (I) that comprises or, alternatively, consists of:

(I) pea protein; and

(II) tamarind seed polysaccharide; possibly together with one or more conventional pharmaceutical or food grade vehicles and excipients.

Pea protein (PP) (or isolated pea protein) (I) occurs, for example, as a light cream-colored powder derived from Pisum sativum.

PPs are commercially available in powder form, generally light cream in color and with a bland odor and characteristic taste.

Preferably, PPs of the invention are high quality non-GMO.

PPs are known to be a good source of essential amino acids and fiber and are highly digestible. PPs are depot proteins composed mainly of albumins, legumins, and vicilins and are characterized by high solubility at alkaline pH, minimal solubility at the isoelectric point, and moderate solubility in acidic environments. In addition, globulins and vicilins impart gelling properties to PPs. Plant protein products are approved as food additives according to FDA (21CFR§170.3), and PPs received Generally Recognized Safe (GRAS) status in 2015 (GRN 608 Axiom Foods GRN 581 and GRN 788) after evaluation of absorption, distribution, excretion, bioavailability studies, toxicity, and mutagenicity.

Tamarind seed polysaccharide, also called "xyloglucan," is a hemicellulose that comes from the kernel, or endosperm, of the seeds of the tamarind tree (Tamarindus indica) and is produced by separating and refining the polysaccharide component.

Xyloglucan is commercially available as a white/whitish powder.

Because it is present in the primary cell walls of all vascular plants, it is naturally part of the human diet. Xyloglucan (also referred to here as "XG") is a nonionic, neutral, branched polysaccharide consisting of a cellulose-like backbone known to have high viscosity in aqueous solution, broad pH tolerance and high solubility in water. Its gel form can occur when xyloglucan is in aqueous phase under certain conditions. Branches of xylose and galactoxylose, together with secondary hydroxyl groups, give the polysaccharide a molecular structure similar to that of mucin, a glycoprotein that is a major component of the gastrointestinal mucosa, giving it mucoprotective and mucoadhesive properties. In addition, its high swelling capacity is critical for initiating bioadhesion activity. Xyloglucan is known to be noncytotoxic and not absorbed in the gastrointestinal tract. In addition, xyloglucan has also received Generally Recognized Safe (GRAS) status.

For example, tamarind seed extracts rich in xyloglucans are on the market from Indena SpA (Xilogel®) and DSP Gokyo Food & Chemical (Japan) (Glyloid®). For example, the molecular weight of xyloglucans contained in said products is preferably from 400,000 to 650,000 daltons.

The Applicant has surprisingly observed that compositions comprising pea protein and xyloglucan exert a significant beneficial effect on mucous membranes, particularly the oral mucosa.

In fact, as extensively demonstrated in the experimental section below, a representative composition of the invention has been shown to be very useful and effective in the treatment of stomatitis, aphthae, and mouth ulcers.

In addition to this, the composition of the invention has been shown to be safe and nontoxic in cytotoxicity, sensitization and irritation tests, and it has a remarkable ability to adhere to the mucous membranes of the mouth, as detailed in the experimental section below.

The composition of the invention thus represents an effective and natural therapeutic answer to oral cavity disorders, providing immediate pain relief through the formation of a protective barrier that also prevents the adhesion and proliferation of pathogens.

The composition of the invention is also highly effective for the treatment of oral disorders such as stomatitis, aphthae and ulcers, due to its properties of promoting mucosa integrity and enabling the restoration of physiological functions of epithelial cells, while protecting them from further external insults and thereby attenuating the inflammatory response associated with mouth injuries.

Thus, the composition of the invention exerts rapid action for the healing of stomatitis, aphthae and oral ulcers and oral mucosa disorders in general.

The subject to be treated with the composition of the invention is preferably a mammal, more preferably a human being.

The composition of the invention is non-toxic and can also be administered to children, pregnant women, the elderly and frail individuals.

As mentioned, the composition of the invention is for oral topical use.

The composition of the invention is therefore formulated for oral topical use, such as in the form of a paste, ointment, gel or other suitable preparation, preferably in the form of a gel suitable for application to the oral mucosa.

Preferably, the oral composition of the invention comprises at least one vehicle and/or excipient of conventional pharmaceutical or food grade. The types of excipients and vehicles of pharmaceutical or food grade used for preparing the composition of the invention, the content ratios of additives to active ingredients, and the methods for preparing the composition may be appropriately chosen by the expert in the field. Organic or inorganic substances, or solid or liquid substances may be used as excipients and vehicles, as long as they are of pharmaceutical or food grade.

Examples of excipients used in the preparation of gel form compositions include, for example, one or more excipients and vehicles selected from gums, e.g., xanthan gum; oils, e.g., castor oil; polymers, e.g., polyvinyl pyrrolidone; chelating agents, e.g., EDTA salts; organic acids, e.g., lactic acid; antiseptic agents, e.g., benzalkonium chloride; and the like.

The composition may also contain sweetening agents, preservatives agents, and flavoring agents.

Sweeteners can be one or more naturally occurring sugars, optionally reduced, such as sucrose, dextrose, xylitol, mannitol, or sorbitol, or a synthetic product such as sodium saccharin, aspartame, acesulfame k, or sucralose.

Flavoring agents are pharmaceutically acceptable flavors and tastes of synthetic oils or natural oils, the latter extracted from plants, flowers, fruits, and combinations thereof, such as cinnamon, mint, anise, and citrus leaves, bitter almonds, citrus fruits, especially orange and/or lemon oils, linden, vanilla, chocolate, and grapefruit. Flavorings of chocolate, vanilla or eucalyptus and fruit essences, especially apple, pear, peach, strawberry, apricot, orange, lemon and grape, can also be used advantageously.

The composition of the invention may also comprise other active components that are useful for use according to the invention.

The composition of the invention can be packaged in dosage units or multi-dose packages, according to conventional methods well known to the expert in the field.

Preferably, the composition of the invention is in the form of a gel to be applied to the injured parts of the oral mucosa, such as by means of a dropper.

Preferably, the composition of the invention comprises an amount by weight from 0.1% to 5% of pea protein, preferably an amount by weight from 0.5% to 3% , more preferably from 0.8% to 2%, even more preferably from 1.0% to 1.1%, advantageously about 1.05%; and an amount by weight from 0.01% to 3% of xyloglucan, preferably an amount by weight from 0.1% to 1%, more preferably from 0.2% to 0.8%, even more preferably from 0.3% to 0.5%, advantageously about 0.45%; preferably together with at least one conventional vehicle and/or excipient, said percentages being expressed by weight relative to the total weight of the composition.

For its use, it is possible to apply to apply a few drops of the composition of the invention, preferably in gel form, once or more times a day. Preferably, it is best to refrain from eating or drinking for a few minutes, such as from 5 to 20 minutes, preferably from 10 to 15 minutes, after application to allow the composition to form the protective barrier.

As mentioned, the composition of the invention is nontoxic, and treatment can therefore be continued for as long as necessary, generally a few days, e.g., from 2 to 10 days, more generally from 3 to 5 days, until symptoms disappear. The treatment can be repeated in case of recurrent oral disorders.

According to another of its aspects, it is an object of the invention an oral composition according to the invention for use in therapy.

According to another of its aspects, it is an object of the invention an oral composition according to the invention for use in a method for the prevention and/or treatment of stomatitis, aphthae and oral ulcers, and oral mucosa disorders in general.

According to another of its aspects, it is an object of the invention a method for the prevention and treatment of stomatitis, aphthae and oral ulcers and oral mucosa disorders in general, which comprises administering, to a subject in need, an effective dose of the composition according to the invention by topical oral application on the injured parts.

Experimental Section

Example 1

Representative composition according to the invention

Table 1

In the following examples, the composition of Example 1, also referred to as "sample" or "MD," was tested.

Example 2

IN VITRO EVALUATION OF MUCOADHESIVITY ON RECONSTRUCTED HUMAN ORAL EPITHELIUM

The mucoadhesivity of the composition of the tested Example 1 (also referred to here as "sample") was evaluated by measuring its ability to inhibit lectin-glycoprotein binding on reconstructed human oral epithelium (HOE).

One set of tissues was untreated (NO, negative control), one set was treated with a substance with known mucoadhesive activity (PC, positive control), and one set was treated with the test sample as such.

The results showed that the mucoadhesivity of the sample is 116.8%, which means that the product has significant mucoadhesive properties. The purpose of the test is to evaluate whether the tested product has mucoadhesive properties in a reconstructed human oral epithelium (HOE) model.

Bioadhesion can be defined as the condition in which two materials, at least one of which is biological in nature, are held together for long periods of time by interfacial forces. When one of the two adhesive surfaces is mucus or a mucous membrane, the phenomenon is called mucoadhesion.

Mucous membranes are the moist surfaces lining the walls of body cavities such as the gastrointestinal tract, vagina and urinary tract, lung, eye, etc. They are generally hydrophilic in that they contain high percentages of water (about 95%). The epithelial layer of the mucosa contains specific structures (specialized cells or glands depending on the type of epithelium) that secrete mucus directly onto the epithelial surfaces. Glycoproteins are the most important component of mucus, to which they impart the characteristic gel-like properties of cohesion and adhesion. The mucoadhesivity of a product intended for topical application to mucous membranes promotes a prolongation of contact time with the site of application itself.

The mucoadhesivity of a product intended for mucosa treatment can be determined by evaluating the percentage of inhibition of lectin-glycoprotein binding. For this purpose, biotinylated lectin (concanavalin-A), a protein found in some leguminaceous plants (Canavalia ensiformis) that has a high affinity for glucoside and mannoside residues in membrane glycoproteins, is used. The binding sites are then detected by the use of streptavidin peroxidase, which, due to its high affinity for biotin, forms the protein-glucosolectin-biotin-streptavidin-peroxidase complex. The complex is quantified due to the presence of peroxidase by the ortho-phenylenediamine oxidation reaction in the following reaction:

The intensity of the yellow-orange coloration of the solution (measured by a spectrophotometer at 450 nm) is proportional to the amount of glycoprotein-lectin bonds and thus to the amount of available sites (glycoproteins) for mucoadhesion. The decrease in absorbance value compared to the untreated control is found to be proportional to the ability of the test substance to "mucoadhere." Tissues

HOE tissue is an epithelium obtained from cells derived from oral mucosa squamous cells carcinoma cultured on an inert polycarbonate filter at the liquid/air interface in a specific medium. This model forms an epithelial tissue without stratum corneum, histologically similar to the mucosa of the oral cavity.

Experimental protocol

Tissues were allowed to equilibrate at room temperature for about 15 min and then transferred in growth medium and incubated (37°C, 5% CO2) overnight.

Treatment

After overnight incubation, the tissues were treated for 30 min with 70 pl of the tested sample or 70 pl of the controls.

Each experiment was conducted in triplicate.

Determination of mucoadhesivity

At the end of the treatment period, the tissues were treated successively with: 1. Concanavalin A

2. Strepatavidin peroxidase

3. o-phenylenediamine + H2O2

Between one administration and the next, the tissues were repeatedly washed with DPBS.

At the end of the test, the optical density (OD) reading was performed at 450 nm.

The mucoadhesivity of the sample is determined by measuring its ability to inhibit the binding between concanavalin A and glycosidic residues of cell membranes, so it is inversely proportional to the optical density measured at the end of the reaction and is calculated by the following formula: Mucoadhesivity (% of NC) = [(1 - OD sample ODNC) ■ 100] + 100 (Figure 1)

The absorbance (OD) measured at 450 nm is inversely proportional to the mucoadhesivity of the tested product. Values are expressed as mean ± standard deviation (SD). Statistical processing of the data was performed by Student's t test. Values of p<0.05 are considered significant. The percentages shown in the table were calculated from the OD values at 450 nm using the formula above and considering the OD of untreated tissues as 100% (NC, negative control); TS = tissues treated with the tested sample; PC = positive control (tissues treated with mucoadhesive-acting substance). * p<0.05 vs NC; ** p<0.01 vs NC.

The results obtained from the in vitro test show that the composition of Example 1 for oral use for the treatment of stomatitis (aphthae) has a MUCOADHESIVE effect in reconstructed human oral epithelium.

Example 3

SENSITIZATION ASSESSMENT

A biological evaluation was performed on the composition of Example 1 (referred to herein as "MD") aimed at identifying potential skin sensitization effects using the following tests:

Local lymph node assay (LLNA): BrdU-ELISA and histology according to ISO10993-10:2021 .

The local lymph node assay (LLNA) is a mouse model developed to assess the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and, in comparison, offers important animal welfare advantages. The test is based on the measurement of induced events during the induction phase of skin sensitization, particularly lymphocyte proliferation in draining lymph nodes, which is a hallmark of the skin sensitization response. The LLNA has been successfully validated and incorporated worldwide into regulatory guidelines. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization tests that not only provides reliable hazard identification information but also the data needed for effective risk assessment and management.

The basic principle of the Local Lymph Node Assay (LLNA) in mice is that sensitizers induce a primary proliferation of lymphocytes in the auricular lymph nodes draining the site of chemical application. This proliferation is proportional to the dose applied and provides a measure of sensitization.

CONSERVATION

The study program and all raw data are archived at the Experiment Center for ten years after the final report is issued.

At the end of the study, the residual sample was not preserved because it no longer had the characteristics of the initial sample, was no longer sterile, and its packaging was no longer sealed.

At the end of the conservation period, the Sponsor may request an extension of the conservation of all or part of the substances for a further period, or their return. In this case, an appropriate agreement will be drawn up. PROCEDURES

All procedures used during this study are recorded in the assay center's certified quality system.

The standards for performing the study were: -ISO 10993-1 :2010-ISO 10993-2:2006 ISO 10993-10:2021-180 10993-12:2021.

This study was conducted on behalf of the sponsor on the test described below and was performed at the University of Messina sperimentation facility at F. Stagno D'Alcontresn.31 Street, Messina.

TEST

"Local Lymph Node Assay" 07/09/202215/09/2022E.

Assay

Dosing details: MD was applied intradermally to the outer surface of the ear at a dose of 200mg/ml as suggested by 18010993-12:2021

-Vehicle used: saline solution

-Storage conditions of the formulation: room temperature

Species Characterization

Mice Strain: CD1 No.: 5 Sex: Male Weight: 25-30 g at the beginning of the test.

Supplier: Charles River-via Indipendenza 11-23885, Calco (Lecco), Italy

Animal experiments comply with Italian regulations on the protection of animals used for experimental purposes (650/2017-PR published August 21, 2017) and other scientific purposes (DM 116192) and with European regulations (OJEC L 358/1 of 18/12/1986).

JUSTIFICATION FOR THE ASSAY CHOICE

Rodent models provide a convenient means to study the attack and effusion of pathogens and simulate all the events observed in natural infections.

Caging

Each mouse was caged in stainless steel cages measuring 30x18x15 h cm equipped with an automatic washing cycle. The housing rooms were illuminated 12 hours a day. Room temperature and humidity were regulated by an air-conditioning unit and were constantly monitored. Records of housing conditions are kept in the records of Animal Facilities.

Cleaning and disinfection

The cages and animal rooms were cleaned periodically.

Food

The animals were fed a standard complete pelleted diet provided by the licensed breeder. Water

Tap water filtered from the local network was provided ad libitum.

Animal identification

A numbered tag placed across the edge of the right ear identified the animals selected for the study.

Quarantine

Before being used in this study, the animals were kept in a quarantine area for one week. At the end of the quarantine week, the animals were carefully examined to assess their suitability for the study.

Selection of animals

The animals used for this study were randomly selected from suitable animals available at that time. EXPERIMENTAL DESIGN

Mice, divided into groups (test sample and controls), were injected intradermally on the outer surface of the ear with the test samples and controls (positive and negative) once a day for three consecutive days (25pl for each ear). Twenty hours after the last treatment, the mice were treated intraperitoneally with a 10 mg/ml BrdU solution. Twenty-four hours after injection, the mice were sacrificed and the auricular lymph nodes were harvested. Throughout the in vivo study, animals were monitored for signs of systemic toxicity (described in Table 2). Mice in each group were weighed before the first application of the test and controls, and also on day 6, before being scarified. The animals were monitored daily for signs of local (irritation and/or necrosis at the application site) and systemic toxicity. A substance defined as a sensitizer in the literature, Eugenol (Sigma), was used as a positive control. The solution was prepared on the day of the test. As a negative control, a negative control was established consisting only of the solvent (vehicle) used for extraction: saline solution.

Clinical observations indicative of systemic toxicity

Table 2

Lymph node processing

The animals were sedated and their body weights were recorded. Draining (auricular) lymph nodes were excised, mechanically disaggregated and resuspended in sterile saline containing 0.9% NaCI. BrdU incorporation into cells was measured using the ELISA kit (Cell proliferation ELISA, BrdU (colorimetric) version 13.0 cat no 11647229001) and recorded as the mean of BLI (BrdU labeling index)/n mice treated with the sample, positive control and negative control. Each extract was loaded and processed in triplicate by ELISA KIT. A stimulation index (SI), relative to the vehicle, was obtained for each sample.

OBSERVATIONS

Elisa Kit Results

The data are depicted in Figure 2.

The labeling index (LI) of the BrdU-labeled cell in the lymph node was calculated by the number of stained spots BrdU-positive cells per 100 cells counted in the cortex, paracortex and marrow. In each lymph node, we counted cells at each standardized site in three sites of cortex, paracortex and marrow without prior knowledge of treatment. The LI of BrdU-labeled cells in the epidermis was calculated by the number of BrdU-positive cells stained per 100 epidermal cells counted from the epidermis for each section (Table 3), according to ISO 10993- 10.

Sensitization index classification system for MD.

Table 3

The stimulation index (SI) was calculated by the fold increase in the total number of BrdU-positive LNCs in a lymph node compared with that in the respective vehicle control groups (Table 4).

Stimulation index

Table 4

A substance was considered positive (irritant) if the SI value was greater than or equal to 3.

INTERPRETATION OF RESULTS

MD administered intradermally was considered NOT SENSITIZING.

CONCLUSION Based on the results, interpreted according to the procedures, the test item 0.2g/ml, according to ISO 10993- 12:2021, should be considered NOT SENSITIZING.

Example 4

EVALUATION OF CYTOTOXICITY BY DIRECT CONTACT

MTT tetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction test

A subconfluent L-929 cell culture was used for the direct contact-MTT cytotoxicity test, and a quantitative evaluation was performed. The amount of formazan (presumably directly proportional to the number of viable cells) is measured by recording changes in absorbance with a plate-reading spectrophotometer. Viable cells with active metabolism convert MTT to a purple formazan product with an absorbance maximum near 570 nm. When cells die, they lose the ability to convert MTT to formazan; therefore, the color formation serves as a useful and convenient marker of viable cells only.

After 24 hours of incubation at 37°C and in a 5% CO2 atmosphere, MTT solution was added and the cells were incubated for another hour. The cells were lysed with 100 pl of DMSO to allow solubilization of the dark blue crystals. The optical density (OD) of each well was measured with a microplate spectrophotometer.

Based on the results, interpreted according to ISO 10993-5:2009 and USP 87, the finished product MD should be considered NON-TOXIC.

The study program and all data are archived in the Analytical Institute's archive for one year after the issuance of the final report. At the end of the conservation period, the sponsor may request the extension of the conservation of all or part of the substances for an additional period or their return. In this case, an appropriate agreement will be made.

PROCEDURES

All procedures used during this study were recorded in the certified quality system of the test center. The following ISOs were complied with: -ISO 10993-5:2009 -ISO 10993-12:2021

This study was conducted on behalf of the sponsor on the test item described below and performed at the University of Messina Assay Center, via F. Stagno D'Alcontresn.31, Messina, Italy.

TEST METHOD

Dosage details:

-Dosing levels

- The tested composition is referred to here as "MD"

The product was tested at a dose of 0.2g as suggested by ISO 10993-12:2021 .

-Vehicles used: complete medium

-Storage conditions of the formulation: room temperature

Characterization

Murine fibroblasts L-929 (NOTO clone 929 [L cell, L929, L strain derivative] (ATCC® CCL1 ^TM) JUSTIFICATION FOR THE ASSAY CHOICE

For this assay, murine fibroblasts L-929 were used as reported in ISO 10993-5:2009. Identification and maintenance of test strains. Cultures of a cell line were kept frozen at -80°C in the corresponding culture medium containing DMSO 5% as cryoprotectant. Before use, DMSO was removed using complete culture medium and centrifuged. Using the complete culture medium corresponding to the cell line, sufficient numbers of cells were prepared to complete the assay, as described in Experimental Design.

EXPERIMENTAL DESIGN

The experimental design included a 96-well plate containing a monolayer of subconfluent cells (about 88% confluence) divided into the following groups:

Control group (n=9)

150 pl of complete medium

Samples

150 pl of test items in complete DMEM (D, n=9)

Reference control (n=9) inducing mortality

150 pl of complete medium containing SDS 1%

TREATMENT

Cells (4 x 10⁴/well) were plated on 96-well microtiter plates in a final volume of 150 pl. After 4 h of adhesion at 37 °C and 5% CO2, MD treatment was performed (final volume=150 pl). After twenty-four hours (24h), 200 pl of MTT (0.2 mg/ml) was added to each well and the cells were incubated for another 1 h. Next, the cells were lysed with 100 pl of DMSO to allow solubilization of the dark blue crystals. The optical density (CD) of each well was measured with a microplate spectrophotometer (Titertek Multiskan FC) equipped with a 570 nm filter. L929 viability in response to treatment was calculated as % viable cells = (CD treated/OD control) x 100.

OBSERVATIONS

Quantitative evaluation

OD value

Table 5 INTERPRETATION OF RESULTS

Viable cells with active metabolism convert MTT to a purple formazan product with a maximum absorbance near 570 nm. When cells die, they lose the ability to convert MTT to formazan; therefore, the color formation serves as a useful and convenient indicator of viable cells only. Cell viability (%) was expressed as a percentage compared with untreated control cells. The lower the Viab. % value, the greater the cytotoxic potential of the test item is. If the viability is reduced to < 70% of the blank, it has cytotoxic potential. The 50 % extract of the test sample should have a viability at least equal to or greater than that of the 100 % extract otherwise the test should be repeated. RESULTS

Acceptability criteria were met in accordance with the interpretation of results referred to ISO 10993-5. MD (0.2 g/ml) showed a viability of 85% (Figure 3).

CONCLUSIONS

Based on the results, interpreted according to ISO10993-5:2009 and 10993-12:2021, we can conclude that MD at the concentration of 0.2 g/ml was considered NON-CYTOTOXIC, maintaining the viability above 70%.

Example 5

ASSESSMENT OF IRRITATION

A biological evaluation was carried out on a sample of the composition of Example 1 to identify potential irritant effects using the following tests: -irritation of the oral mucosa according to ISO 10993-23:2021 (Special irritation test Annex D (D.3)).

Based on the results, interpreted according to ISO 10993-23:2021 (Special Irritation Test Annex D (D.3)), the sample should be considered non-irritating.

PROCEDURES

This study was conducted at the University of Messina Assay Center at 31 F. Stagno D'Alcontres Street, Messina, Italy.

TEST METHOD

Dosing details: the sample was applied topically on the cheek pocket of each mouse at a dose of 200mg/site, as suggested by ISO 10993-12:2021.

Vehicles used: saline solution

Formulation storage conditions: room temperature.

Species Characterization: Mice Strain:CD1 No.: 6

Sex: male Weight: 25-30 g at the beginning of the test.

Supplier: Charles River-via Indipendenza 11-23885, Calco (Lecco), Italy

Animal experiments comply with Italian regulations on the protection of animals used for experimental purposes (549/2018-PR published July 16, 2018) and other scientific purposes (DM 116192) and with European regulations (OJEC L 358/1 of 18/12/1986). JUSTIFICATION FOR THE ASSAY CHOICE

Rat models provide a convenient means to study pathogen attachment and effusion and simulate all the events observed in natural infections (2).

Caging

Each mouse was caged in stainless steel cages measuring 30x18x15 h cm equipped with an automatic washing cycle. The temperature and humidity of the room were regulated by an air conditioning unit and were monitored continuously. Records of housing conditions are kept in archives.

Cleaning and disinfection

The cages and animal rooms were cleaned periodically.

Food

The animals were fed a standard complete pelleted diet provided by the licensed breeder.

Water

Filtered tap water from the local network was provided ad libitum.

Animal identification

Quarantine

Before being used in this study, the animals were kept in quarantine for one week. At the end of the quarantine week, the animals were carefully examined to assess their suitability for the study.

Selection of animals

The animals used for this study were randomly selected from those suitable and available at that time.

Application

The test sample was administered on the cheek of 6 mice every hour for 4 hours.

One group was treated at cheek pocket with the test item (0.2 ml/site). The other group was treated with saline solution (vehicle), used as a control.

Histologic evaluation

RESULTS

Macroscopic assessment

After 24 hours from exposure, no erythema was present in all treated animals (NG=0).

Histologic evaluation

Histologic examination of the cheek pocket revealed mild characteristic pathologic changes, such as minimal leukocyte infiltration, after treatment with the test item. The histological score was made by an independent observer (Figure 4). The irritation index obtained was 0.94 (0=none).

Conclusions

Based on the results, interpreted according to ISO 10993-12:2021, the sample is to be considered NONIRRITATING to oral mucosal tissue.

Example 6 EVALUATION OF THE EFFECTS ON PHENOL-INDUCED ORAL ULCER.

In the present study, the composition of example 1 is referred to as MD

JUSTIFICATION FOR THE ASSAY CHOICE

The 90% phenol solution causes oral ulcers in rats, representing a convenient means to study the beneficial properties of the test item on oral ulcer.

METHODS

Wistar rats (180-220 g), purchased from Harlan (Milan, Italy), were used for the experiment.

Experimental group

Animal experiments comply with Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 11619) and other scientific purposes (DM 116192) and with EU regulations (OJEC L 358/1, 18.12.1986).

Cleaning and disinfection

The cages and animal room were cleaned periodically.

Food

Water

Tap water filtered from the local network was provided ad libitum.

Animal identification

Quarantine

Before being used in this study, the animals were kept in a quarantine area for one week. During this period, they were observed daily. At the end of the quarantine week, the animals were carefully examined to assess their suitability for the study.

EXPERIMENTAL DESIGN: phenol-induced oral ulcer.

All procedures used in this study were recorded in the assay center's certified quality system.

Assignment of animals to groups or randomization

Male Wistar rats (weight, 180-220 g) (Harlan, Milan, Italy) were housed in a controlled environment (22 ± 2 °C, 55 ± 15% relative humidity, 12-h light/dark cycle). After acclimatization for one week, the rats were fed a standard diet and water. The animal experiments complied with Italian legislation on the protection of animals used for experimental and other scientific purposes (DM 116) experimental and other scientific purposes (DM 116192) and with EU regulations (OJEC L 358/1, 18.12.1986).

The animals used for this study were randomly selected from suitable animals available at that time.

PHASE I: INDUCTION

Phenol-induced oral ulcer model

To induce oral ulcer, a small cotton ball was placed at one end of a glass tube with a diameter of 3 mm and then having the cotton ball immersed in a 900 g/l solution of pieno, burning the rats for 60 s on the left cheek. After 24 h, all rats had 3-mm-diameter ulcers on the oral mucosa. The oral ulcer tissues (left cheek pockets) of each group was processed for morphological, histological and biochemical analysis.

Wistar rats were randomly divided into six groups:

Group 1 (Sham + veh): the tampon was immersed in saline and placed for 60 s in the left cheek of the rat (n=12);

Group 2 (MD + veh): rats received MD topically on the oral mucosa (n=12);

Group 3 (phenol + veh): the tampon was dipped in phenol and placed for 60 s in the left cheek of the rat (n=12);

Group 4 (phenol + MD, 2 days): MD applied to oral mucosa once daily for two days starting from day 0 of oral ulcer induction (n=12).

Group 5 (Phenol + DM, 4 days): MD was applied to the oral mucosa once daily for four days starting from day 0 of oral ulcer induction (n=12).

Group 6 (Phenol + MD, 5 days): MD was applied to the oral mucosa once daily for five days starting from day 0 of oral ulcer induction (n=12).

Route of administration

Topical application on oral mucosa, once daily for 2, 4 and 5 days.

Quality control

All procedures followed ISO standards.

PHASE II: Sacrifice

Animals were sacrificed 2, 4 and 5 days after oral ulcer induction.

Phase III: RESULTS.

9.5.1 Effect of MD on the macroscopic analysis of the cheek pocket

The effects of MD on morphology, edema and hyperemia in the oral ulcer model were evaluated macroscopically, based on the maximum diameter of ulcer tissue observed (<1 mm, judgment of healing, >1 mm, judgment of nonhealing), edema and degree of hyperemia around the ulcer, according to Miao M et al., 2019 (1): Grade I, hyperemia diameter <1 mm; grade II, hyperemia diameter 1-2 mm; grade III, hyperemia diameter 2-3 mm; grade IV, hyperemia diameter >3 mm.

After oral ulcer induction following phenol administration, the ulcer mucosa was pitted and generally round, characterized by high hyperemia and edema (group Phenol + veh) compared with the control groups (Sham + veh and MD + veh). Interestingly, application of MD for 4 and 5 days significantly reduced the degree of hyperemia and edema (groups Phenol + MD, 4 days and Phenol + MD, 5 days) compared with the damaged group (group Phenol + veh). In contrast, MD applied topically on oral mucosa for only 2 days (groups Phenol + MD, 2 days) was unable to counteract edema formation and reduce mucosal degradation caused by phenol.

Table 6.

Effect of MD on macroscopic cheek pocket analysis. No edema and hyperemia were found in the cheek pocket of control rats (Sham+ veh and MD + veh). A greater degree of hyperemia and edema formation was found in the control pocket of phenol-damaged rats (phenol + veh) and in rats treated with MD for 2 days (phenol + MD, 2 days) than in the control groups. MD treatment administered for 4 and 5 days (Phenol + MD, 4 days and Phenol + MD, 5 days) significantly reduced the severity of macroscopic changes caused by phenol. *** p < 0.001 vs Sham + Veh; ### p < 0.001 and ## p < 0.01 vs phenol + veh.

Effect of MD treatment on histopathological changes caused by phenol-induced oral ulcer.

Cheeks were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. To detect changes in tissue architecture, the sections were stained with hematoxylin-eosin (H&E). All stained sections were observed under an inverted microscope with two CCD cameras (magnification, x2.5 and x20; Nikon, Tokyo, Japan). Inflammatory cell infiltration, vasodilatation, presence of hemorrhagic areas, edema, ulcerations, and abscesses were assessed in single-blind and classified, as previously described by Medeiros et al, 2011 (2). Scoring was as follows: score 1, normal epithelium and connective tissue without vasodilatation, absence of cellular infiltration or discrete cellular infiltration and absence of hemorrhagic areas, ulcerations or abscesses; score 2, discrete vasodilatation or areas of re-epithelialization, discrete inflammatory infiltration with mononuclear predominance and absence of hemorrhagic areas, ulcerations or abscesses; score 3, moderate vasodilatation, areas of hydropic epithelial degeneration, inflammatory infiltration with mononuclear prevalence hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema and possible ulceration and absence of hemorrhagic areas, edema and possible ulceration and absence of abscesses; score 4, severe vasodilatation and inflammatory infiltration with neutrophil prevalence.

As shown in Figure 5, 5A and 5B, the oral mucosa epithelium was intact and loose connective tissue in the control groups was restored (groups Sham + veh and MD +veh), while in the injured group (group Phenol + veh), the oral mucosa and squamous epithelium were found to be covered by necrotic tissue and infiltrated by a large number of inflammatory cells. Compared with the Phenol group (group Phenol + veh), the degree of injury was significantly reduced and positive staining of inflammatory cells decreased in animals treated with MD for 4 and 5 days (Phenol + MD, 4 days and Phenol + MD, 5 days). Interestingly, treatment with MD for 5 days also completely restored the squamous epithelium and the mucosa was closer to loose connective tissue. In contrast, treatment with MD for only 2 days (groups Phenol + MD, 2 days) did not restore the oral ulcer damage. (Figure 5, 5A and 5B).

No histological changes were found in the oral mucosa tissue collected from the control groups (groups Sham + veh and MD + veh). The oral mucosa was found to be destroyed and infiltrated by neutrophils in phenol -treated rats (group phenol + veh). MD treatment at 4 and 5 days restored histological changes and reduced neutrophil infiltration (Phenol + MD, 4 days and Phenol + veh, 5 days), whereas MD treatment for 2 days showed no improvement in counteracting phenol-induced damage (Phenol + veh, MD 2 days). *** p < 0.001 vs Sham + veh; ### p < 0.001 and ## p < 0.01 vs Phenol +veh; ND not detectable.

Effects of MD treatment on cytokine production in phenol-induced oral ulcer

Cytokine investigations reflect aphthae healing and local inflammatory cell infiltration in the oral ulcer. Specifically, the process observed in oral mucositis is likely initiated by antigenic stimulation of mucosal keratinocytes, causing secretion of T-cell activating cytokines and tumor necrosis factor-alpha (TNF-o), which causes inflammation by endothelial cell adhesion and neutrophil chemotaxis. TNF-o induces inflammation by stimulating the release of interleukins, such as interleukin-2 (IL-2), a glycoprotein that plays a critical role in regulating both cells and neutrophils. Binding of IL-2 to the IL-2 receptor on T lymphocytes induces cell proliferation and increased lymphokine secretion. Therefore, ELISA kits for TNF-o and IL-2 were performed in oral mucosa tissue. TNF-o and IL-2 levels were found to be in damaged group (group Phenol + veh) compared with controls (groups Sham + veh and MD + veh) (Figure 5A and 5B, respectively), confirming the role of TNF-o and IL_2 as important mediators in oral mucosa pathogenesis. Interestingly, treatment with MD at 4 and 5 days significantly reduced both TNF-o and IL-2 expression (groups Phenol + MD, 4 days and Phenol + MD, 5 days) (Figures 5A and 5B, respectively), suggesting a protective effect to counteract the inflammatory component related to aphthae. In contrast, MD administered for only 2 days (Phenol + MD, 2 days) was unable to reduce the levels of pro- inflammatory cytokines and TNF-o (Figures 5A and 5B, respectively).

TNF-o levels were significantly increased in phenol-damaged rats compared with control (A). These increased cytokine levels were significantly reduced by MD treatment for 4 and 5 days (A), in contrast to MD treatment for 2 days (A). IL-2 levels were significantly increased in phenol-injured rats, compared with control (B). Treatment with MD for 4 and 5 days (B), in contrast to treatment with MD for 2 days (B) significantly protected rats from injury. *** p < 0.001 vs. Sham + veh; ## p< 0.01 vs. Phenol + veh. *** p < 0.001 vs. Sham + veh; ## p < 0.01 vs. Phenol + veh.

CONCLUSIONS

The present study demonstrates the ability of MD to treat oral ulcer by reducing the degree of hyperemia and edema in the cheek tissue, significantly decreasing the levels of inflammatory cytokines edema in the cheek tissue, significantly decreasing the levels of inflammatory cytokines when administered for 4 and 5 days. This study highlights the role of MD in counteracting phenol-induced oral ulcer.

Claims

1 . An oral topical composition comprising a mixture (I) comprising or, alternatively, consisting of:

(i) pea protein; and

(ii) polysaccharides from tamarind seeds.

2. The composition according to claim 1, wherein said composition further comprises one or more excipients and/or vehicles of pharmaceutical or food grade.

3. The composition according to claim 1 or 2, wherein said composition comprises an amount by weight from 0.1% to 5% of pea protein, preferably from 0.5% to 3%, more preferably from 0.8% to 2%, even more preferably from 1.0% to 1.1%; and an amount by weight from 0.01% to 3% of xyloglucan, preferably from 0.1% to 1%, more preferably from 0.2% to 0.8%, even more preferably from 0.3% to 0.5%; said percentages being expressed by weight with respect to the total weight of the composition.

4. The composition according to claim 3, wherein said composition comprises about 1.05% by weight of pea protein and about 0.45% by weight of xyloglucan.

5. The composition according to any one of claims 1 to 4, wherein said composition is in the form of a paste, ointment or gel.

6. The composition according to claim 5, wherein said composition is in the form of a gel.

7. The composition according to any one of claims 1 to 6, wherein said excipients and/or vehicles of pharmaceutical or food grade include one or more of the following: gums, oils, polymers, chelating agents, organic acids, antiseptic agents, sweetening agents, preserving agents, and flavoring agents.

8. The composition according to claim 7, wherein said composition comprises one or more of the following substances: xanthan gum, castor oil, polyvinyl pyrrolidone, EDTA salts, lactic acid and/or benzalkonium chloride.

9. The composition according to any one of claims 1 to 8, for its use in therapy.

10. The composition for use according to claim 9, wherein said composition is for use in a method for the prevention and treatment of oral mucosa disorders, stomatitis, aphthae and oral ulcers.