DESCRIPTION of the industrial invention having the title:

“Composition for use in the prevention and treatment of diseases of the gastrointestinal system and of the related symptoms”

DESCRIPTION

The present invention relates to a composition comprising hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica and chondroitin sulfate useful in the prevention and/or in the treatment of diseases of the gastrointestinal system.

Furthermore, forming an object of the present invention is a composition comprising an association or a combination or a mixture comprising or, alternatively, consisting of: (a) hyaluronic acid or a salt thereof, (b) an extract of Opuntia ficus-indica, and (c) chondroitin sulfate.

Furthermore, forming an object of the present invention is said composition for use in a method for the preventive or curative treatment of diseases of the gastrointestinal system, or of the symptoms related to said diseases, in a mammal.

The association or combination or mixture is particularly effective due to the synergistic action of the components thereof.

BACKGROUND OF THE INVENTION

STATE OF THE ART

There are many diseases of the gastrointestinal system and they differ depending on the anatomical site of interest: mouth, oesophagus, stomach, small intestine, large intestine, rectum, anus.

Diseases of the oesophageal and gastric tract among which one of the most common is gastroesophageal reflux are of particular interest for the following invention.

Gastroesophageal reflux is used to indicate “the involuntary and unintentional movement of part of the gastric contents into the oesophagus, without the involvement of the gastric and abdominal muscles". The oesophagus is a 25-30 cm long canal that connects the mouth with the stomach. Along the course thereof there can be observed two sphincter structures: the first between the hypopharynx and the cervical tract of the oesophagus (Upper Esophageal Sphincter, UES), the second Lower Esophageal Sphincter (LES), at the level of the esophagus-gastric junction. The latter is a high-pressure region which represents the main anti-reflux structure, thanks to the location thereof between the intrathoracic negative pressure region and the intraabdominal positive pressure region. Therefore, under normal conditions, an increase in abdominal pressure will affect the LES hindering the ingested material from returning to the oesophagus. Under physiological conditions the LES is closed and it is released for approximately 3-10 seconds after deglutition.

Besides the LES, other anatomical structures that contribute to the maintenance of the anti-reflux barrier are:

• His angle, an acute angle formed between the oesophagus and the gastric fundus;

• the phrenoesophageal ligament;

• the diaphragmatic collar, consisting of bundles of the diaphragm which, by being arranged in a scarf-like manner around the oesophagus, constrict the lumen thereof during the inspiratory phase.

The pathogenesis of the gastroesophageal reflux disease (GERD), includes numerous factors, such as for example:

1 . insufficient anti-reflux barrier of the lower esophageal sphincter, which has the purpose of mechanically preventing gastric juices from flowing back into the oesophagus.

2. delay in gastric emptying, for anatomical abnormalities or functional alterations: (I) anatomical abnormalities: pyloric stenosis (this is the terminal region of the stomach, which regulates the movement of gastric contents into the duodenum); (II) functional alterations: motor alterations of the fundus (region responsible for emptying liquids).

3. insufficient oesophageal clearing mechanism, which has the purpose of minimising contact between the oesophageal mucosa and gastric juices by acting both through oesophageal peristalsis and the neutralisation of acidic residues thanks to saliva.

4. gastric hyperacidity.

5. aggressiveness of the gastric contents that flow back into the oesophagus, through the action of hydrochloric acid.

6. duodenogastric reflux with the movement of pancreatic and biliary secretions into the stomach, which could cause more severe lesions in the event of gastroesophageal reflux.

Other predisposing factors include smoking, poor eating habits (abundant meals, high-fat foods, caffeine), drugs, pregnancy and obesity, which can exacerbate GERD. Hiatus hernia (extension of a portion of the stomach into the thorax through an opening in the diaphragm called the oesophageal hiatus) also frequently accompanies GERD and it can contribute to the prolonged exposure to gastroduodenal contents. In general, the walls of the oesophageal hiatus adhere closely to the oesophagus, but it can happen that the anchorage structures of the lower portion of the oesophagus lose tone, facilitating the ascent of a small part of the stomach into the thorax. In any event, whatever the cause may be, the frequent and repeated contact of regurgitated gastric material with the oesophageal mucosa exerts a harmful action on the latter, which is all the more serious the longer the contact time and the lower the pH of the reflux. Over time, the persistent phlogistic action affecting the oesophageal mucosa leads to an inflammatory reaction that can evolve into ulcerations, stenosis and so-called columnar metaplasia (or Barrett's epithelium, the single most important risk factor for the development of oesophageal adenocarcinoma). Symptoms considered to be typical are retrosternal pyrosis (defined by the patient as a burning sensation that starts at the stomach or the lower portion of the thorax and rises toward the neck) and regurgitation (perception of acidic and bitter tasting liquid inside the oral cavity), symptoms whose specificity for GERD is equal to 89% and 95%, respectively. Frequent albeit less specific symptoms are odynophagia, dysphagia, belching, epigastric pain, bloating and digestive difficulties. Some of these symptoms characterise the diagnosis of functional dyspepsia and it is known that between 10% and 17% of patients requiring medical intervention for dyspepsia have GERD.

GERD is one of the most common pathological conditions observed by gastroenterologists.

A study on the prevalence of the disease showed that GERD has a prevalence of 10-20% in western countries compared to only 5% observed in Asia; in particular, more cases were observed in North America, then in Northern Europe and in Southern Europe.

Scientific studies show that the symptoms of the disease have a strong impact on the quality of life given that persistent reflux symptoms are associated with reduced physical and mental well-being even during treatment with proton pump inhibitors (by way of example).

Given that it is a chronic disease, conventional treatment is almost always long-term and it consists, depending on severity, changes in lifestyle (eliminating chocolate, caffeine, alcohol, cigarette smoking, losing weight, etc.), in pharmacological treatments, surgical therapy.

Classes of drugs commonly used in GERD include: antacid drugs, histamine H2 receptor antagonists and proton pump inhibitors (PPIs), prokinetic agents.

Antacids are over-the-counter drugs that provide rapid relief of the symptoms of the disease but they are not capable of inducing a curative effect in erosive oesophagitis. These drugs contain carbonates or bicarbonates which reduce stomach acidity by reacting with hydrochloric acid to releasing carbon dioxide. H2-antagonist drugs such as ranitidine, famotidine, cimetidine, provide temporary relief of symptoms although with a slower onset time with respect to antacids. Prolonged use is not recommended given that patients may develop tolerance within 1-2 weeks, and the effect of these drugs is not of the curative type in any case.

PPI drugs (pantoprazole, lansoprazole, omeprazole, etc.) are the standard treatment in gastroesophageal reflux diseases. As a matter fact, the number of prescriptions for these drugs has doubled over the last 10 years. Often these prescriptions are associated with those of antiinflammatory drugs of a steroidal or non-steroidal nature. The mechanism of action of PPIs includes blocking the proton pump at the level of the stomach parietal cells: this hydrogen-potassium ATPase pump causes the release of hydrochloric acid into the stomach lumen. Compared to H2-antagonist drugs, these drugs have a faster action and, above all, they exert a curative effect on oesophagus lesions. The side effects most commonly observed in treatment with PPIs are nausea, diarrhoea, headache, insomnia and anaphylactic reactions.

Prokinetic agents, such as cisapride or metoclopramide, they activate serotonin or dopamine receptors capable of increasing oesophageal or gastric peristalsis. These drugs have a slow onset of action, a short duration, and they do not have a curative effect on the disease. Furthermore, they have various side effects such as tremors, dyskinesia, fatigue and increased cardiac adverse events, therefore the use thereof in the treatment of GERD is quite limited.

Besides the classic pharmacological remedies, alginates are also used for the symptomatic treatment of GERD. Alginates, such as sodium alginate, are natural polysaccharides which, in contact with the gastric environment, precipitate forming low density gel within a few minutes. The variation in pH triggered by bicarbonates and carbonates, also almost always present in the commercially available formulations, release carbon dioxide that is trapped inside the alginate gel, causing it to float on the gastric contents. Alginate gel forms in the portion of the stomach close to the gastroesophageal junction, right where the acid pocket develops. This allows to block and significantly reduce the backflow of acid from the stomach to the oesophageal canal.

WO 2017/055909 A1 discloses a combination of hyaluronic acid, chondroitin sulfate combined with at least one antacid agent for the prevention or the treatment of gastric hyperacidity and gastroesophageal reflux, and of the related diseases. EP 2 208 500 A1 discloses compositions based on polysaccharides from Opuntia Ficus Indica in fixed or therapeutic combination with other active ingredients and/or with other substances useful for the prevention or the treatment of gastroesophageal reflux disease, gastric oesophagitis and related diseases.

EP 3811 960 A1 relates to medicaments for the treatment of lesions and wounds of the skin and of the mucous membranes.

An object of the present invention to provide a composition alternative to those known in the known state of the art, useful in the treatment for the treatment of gastrointestinal diseases and, in particular, of gastric and oesophageal diseases such as gastroesophageal reflux disease both in humans and in animals.

SUMMARY OF THE INVENTION

The present invention is based on the research and identification of a novel combination of active ingredients which exert a synergistic and enhanced action of the various components of the combination subject of the invention.

The present invention relates to compositions comprising or consisting of a mixture of hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica, and chondroitin sulfate.

Furthermore, forming an object of the present invention is a composition comprising an association or a combination or a mixture comprising or, alternatively, consisting of: (a) hyaluronic acid or a salt thereof, (b) an extract of Opuntia ficus-indica, and (c) chondroitin sulfate, having the characteristics of the attached claims.

Furthermore, forming an object of the present invention is said composition for use in a method for the preventive or curative treatment of diseases of the gastrointestinal system, or of the symptoms related to said diseases, in a mammal, having the characteristics of the attached claims.

Furthermore, the present invention relates to such compositions for use in the treatment of gastrointestinal diseases, and in particular gastric and oesophageal diseases such as gastroesophageal reflux disease (GERD), laryngopharyngeal reflux (LPR), gastric ulcers, gastritis, dyspepsia, pyrosis, hyperacidity, oesophagitis, reflux oesophagitis, hiatus hernia both in humans and in animals.

The present invention provides - in a single composition - the following advantages: - hyaluronic acid or a salt thereof is an essential component of the extracellular matrix of fundamental importance in repair processes due to the ability thereof to facilitate cell turnover and repair damage to the mucous membranes induced by gastric diseases.

- thanks to the polysaccharide content, the extract of Opuntia ficus-indica is capable of adhering to the mucous membranes with a moisturising and soothing effect; the presence of enzymes stimulates the physiological digestive process, and the flavonoids present guarantee anti-inflammatory efficacy.

- chondroitin sulfate inhibits pepsin-induced damage at the level of the gastroduodenal mucosa protecting it by interacting with its amino group thereof.

The synergistic action of the composition subject of the present invention also determines an antioxidant activity and it increases mucoadhesion to the gastrointestinal walls in involved.

Other advantages and characteristics of the present invention will be apparent from the following detailed description.

GLOSSARY

The terms used in the present description are as generally understood by the man skilled in the art, unless otherwise indicated. In the context of the present description, the expression "extract” is used to indicate any product related to a herb or herbal matter including all products derived from mechanical treatments (pulverisation, crushing, mixing and/or other methods) and/or from extraction treatments (solvent extraction, distillation, and/or other specific methods) conducted on a herb or herbal matter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses a composition comprising - as main active ingredients - hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica and chondroitin sulfate.

HYALURONIC ACID

Hyaluronic acid is an important component of the synovial fluid and of the extracellular matrix. Chemically, it is a natural polymer consisting of alternating residues of glucuronic acid and n-acetyl-d- glucosamine and it belongs to the class of substances known as glycosaminoglycans (GAGs). In this class of compounds it represents the structurally simplest compound, the only one not covalently bound to a protein core and not sulfonated. Fibroblasts are the main cells that release hyaluronic acid in the extracellular matrix.

It takes part in important physiological processes: the repair and regeneration of wounds, morphogenesis and the structural organisation of the matrix. The biological role of hyaluronic acid is closely related to the hydrophilic and hydrodynamic properties thereof, which allow it to retain water and therefore play an important structural role in cells.

The molar mass may reach up to 10 ⁷ MDa and, thanks to the viscoelastic and rheological properties thereof, it is an interesting component for use in various medical applications. High molecular weight or low molecular weight hyaluronic acid may be used. The expression "high molecular weight” hyaluronic acid is used to indicate a hyaluronic acid having a molecular weight comprised from 1.0 million Daltons to about 4.0 MDa. For example, high molecular weight hyaluronic acid may have a molecular weight of about 2.0 MDa. In another example, hyaluronic acid may have a molecular weight of about 2.8 MDa. The expression "low molecular weight” hyaluronic acid is used to indicate a hyaluronic acid having a molecular weight lower than about 1.0 MDa. Low molecular weight hyaluronic acid may have a molecular weight comprised from about 200,000 Da (0.2 MDa) to less than about 1.0 MDa, for example from about 300,000 Da (0.3 MDa) to about 750,000 Da (0.75 MDa).

The topical application of hyaluronic acid allows, for example, to treat the aphthae of the oral mucosa with a rapid remission of symptoms thanks to the recognised anti-inflammatory properties thereof.

Through intra-articular injection route it is used in patients with knee osteoarthritis, through the ocular route it is instead used in ophthalmic preparations, especially in eye drops for dry eye syndrome due to the lubricating action thereof.

With regard to the present invention, hyaluronic acid is particularly interesting for the moisturising and soothing protective action thereof on the mucous membrane of the gastro-oesophageal tract. Furthermore, the local anti-inflammatory action thereof allows rapid relief of symptoms related to the burning sensation caused by the contact of hydrochloric acid with tissues. The ability of the compound to act on prostaglandins, metalloproteinases and other inflammatory mediators is known in literature. Hyaluronic acid and the salts thereof represent valid bioactive principles for the preparation of pharmaceutical, nutraceutical, dermocosmetic products or medical devices for the treatment of gastroesophageal reflux thanks to the moisturising, protective and regenerating action thereof on the tissues of the gastrointestinal mucosa.

OPUNTIA FICUS-INDICA

Opuntia ficus-indica is a tropical and subtropical plant belonging to the family of Cactaceae, to which more than 1500 species of cactus belong. Among these, the Indian fig is certainly the most known. Opuntia ficus-indica is a plant that can grow in arid and semi-arid climates, and it is more common, from a geographical point of view, in countries such as Mexico, Latin America, South Africa and the countries of the Mediterranean region, particularly in Sicily. The plant can reach 3 - 5 m high, with thick trunks ending in succulent and oblong stems called cladodes. After 1 or 2 years of life, cladodes produce flowers and fruits that have very different colours, ranging from pale green to deep red. The Indian fig fruit has very high nutritional value, given that it is rich in water and nutrients, and it is an important source of livelihood for populations in arid areas where it grows. The infusions, juice or oil of Indian fig seeds are widely used for nutritional, health and cosmetic purposes. It is also known and used as a medicinal remedy for various pathological conditions: in the sub-Saharan region, flowers and fruits are believed to have anti-ulcerative and anti-diarrhoeal properties; flowers are often used as a remedy for haemorrhoids through the oral route, and lymph of the cladode is used in the treatment of whooping cough.

Various scientific reports have investigated the chemical composition of the Indian fig. From the chemical analyses carried out it was observed that Opuntia has a very high content of polyphenols and flavonoids which confer them antioxidant and anti-inflammatory properties. In particular, the cactus flowers seem to be the main source of polyphenols and flavonoids and they therefore seem promising in the prevention of inflammatory diseases, cardiovascular and neurodegenerative diseases. Furthermore, the analyses showed that each part of the plant shows a specific compositional profile and stands out for the presence of a given nutrient. By way of example, flowers of Opuntia ficus-indica are particularly rich in gallic acid, kaempferol and quercetin; cladode contains nicotiflorin which is a potent neuroprotective agent and which has proven effective in reducing degeneration of the ganglia following damage induced by hypoxia, oxidative stress or glutamate.

Furthermore, more than any other part, the cladode is rich in polyunsaturated fatty acids. 90% of the total fatty acids contained in the extract is palmitic acid, oleic acid, linoleic acid and linolenic acid. Furthermore, the Indian fig is rich in minerals, especially potassium and zinc, and vitamins: the skin is rich in vitamin E and o-tocopherol. Recent studies have shown the high enzyme content present in the fruit of Opuntia ficus-indica,' in particular, the presence of proteolytic enzymes is of considerable interest given that they facilitate digestion and reduce the gastric emptying time. Additionally, it has been demonstrated that the presence of mucilage (mainly consisting of arabinogalactan and galacturonic acid) in Opuntia ficus-indica guarantees the fruit a gastro-protection action thanks to the formation of a layer which covers and protects the gastric mucosa. Mucilage of the aforementioned fruit are highly viscous given that the negative charges determine a strong molecular repulsion, and this results in an expansion of these molecules. It is believed that the change in shape of the molecules is actually responsible for the protection of the gastric mucosa. The extract of Opuntia ficus-indica is interesting from the nutritional and health point of view due to the very particular micronutrient content thereof. Among the substances present in the plant, flavonoids seem to be the most interesting.

In the context of the present invention, the extracts of Indian fig are interesting given that they exert a protective action at the gastroesophageal level and they can therefore be useful in the event of hyperacidity and reflux thanks to the protective action thereof and the ability thereof to improve and increase the mucosa defence factors.

Acknowledged scientific evidence shows that flavonoids stimulate the production of prostaglandins in gastric mucosal cells and, in this sense, they exert an anti-ulcerative action given that prostaglandins promote the secretion of bicarbonate and protective mucus, thus combating acid hypersecretion. Besides the aforementioned action, the anti-inflammatory and antioxidant action of flavonoids which - in the context of the present composition - reduces the inflammatory condition to which the subjects with gastric hyperacidity are continuously exposed, has been widely emphasised.

The antioxidant effect and the anti-ulcerative action of the juice of Opuntia ficus-indica was evaluated in a preclinical study. An anti-radical activity assay was carried out by mixing a standard solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with different concentrations of juice, in order to evaluate the antioxidant effect. The antioxidant action is measured by measuring the absorbance of the various samples to be tested with respect to the standard DPPH solution at 517 nm. The results obtained showed that the Indian fig juice resulted in a 50% decrease of the absorbance measured resulting in a marked antioxidant effect.

Anti-ulcerative activity was evaluated through an in vivo study in rats. The animals selected for the test were fed on a standard diet and divided into three groups before proceeding to the treatments: control group 1, group 2 in which mice were treated for 9 days with 3 ml of Indian fig juice, and group 3 in which mice were treated for 9 days with 100 mg/kg sucralfate. At the end of the treatment, all mice were treated with an ulcerative agent. After 60 minutes, the animals were sacrificed and the stomach of each was taken and opened along the greater curvature in order to carry out the necessary analysis. Macroscopic and microscopic analyses were carried out on the collected tissues.

Macroscopic observations have shown that treatment with the ulcerative agent causes the formation of disseminated ulcerative lesions, hyperaemia and thickening thereof. Pre-treatment with Indian fig juice has shown a protective effect on the mucosa with an epithelium very close to the physiological condition, significantly reduced areas of hyperaemia and low presence of lesions. The data were confirmed by microscopic observations: in the treated group the tissue shows areas of necrosis alternating with areas with hyperproduction of mucus as a compensatory response to the damage caused. In tissues taken from pre-treated mice, the epithelium shows the same characteristics as normal tissue.

The effect of a formulation containing an extract of Opuntia ficus-indica in symptoms of gastroesophageal reflux was tested in a clinical study. 118 subjects aged between 36 and 64 years were recruited for this study. Based on the pre-established selection criteria, healthy subjects but with reflux episodes disseminated for at least 2 or 6 times a week were selected. The study was conducted in double-blind and randomised with control. The recruited subjects were divided into two groups: placebo group (59) and study group (59) treated for 8 weeks with 6 g/day of the formulation of Mucosave (extract of Indian fig and olea europae). At the end of the treatment, the formulation containing an Indian fig extract is well tolerated and effective in controlling the symptoms associated with GERD. Furthermore, heartburn and acid regurgitation are reduced (resulting in a decrease in the abnormal acid exposure of the oesophagus).

CHONDROITIN SULFATE

Chondroitin sulfate is a glycosaminoglycan consisting of two monosaccharides: D-glucuronic acid and N-acetyl-D-galactosamine.

It is a component of proteoglycans that represent the second largest non-aqueous component of articular cartilage.

Like all glycosaminoglycans, chondroitin sulfate has a high density of negative charges that attract a cloud of cations, such as Na ⁺ , which are osmotically active, causing the inflow of large amounts of water into the cartilage extracellular matrix. The result is a tendency to swell which makes the matrix capable of withstanding the compression force.

Specifically due to this effect, chondroitin sulfate is used in the treatment of osteoarthritis, and studies show the effectiveness thereof in reducing pain and normalising the functions of the musculoskeletal system.

Numerous studies in literature show that besides stimulating the activity of Toll-like receptors 4 (TLR4 receptors) which inhibit the production of pro-inflammatory cytokines and inhibit the activation of nuclear transcription factor Nf Kb, chondroitin sulfate also carries out an anti-inflammatory action by inhibiting, in vitro, the synthesis of pro-inflammatory mediators such as cyclooxygenase 2, nitric oxide synthase and prostaglandins E2.

Further in vivo studies have confirmed the anti-inflammatory efficacy of chondroitin sulfate: reduction of levels of interleukin 6, a pro-inflammatory cytokine, and of TNF alpha. Chondroitin sulfate also increases the synthesis of proteoglycans and collagen, reducing the gene expression of metalloproteinases, enzymes responsible for the degradation of extracellular matrix proteins including collagen, elastin, the protein part of proteoglycans.

Studies also attest to the anti-apoptotic action of chondroitin, inhibiting the activation of Caspase 3 and Caspase 7.

Lastly, chondroitin sulfate also inhibits bone resorption. As a matter fact, studies in literature show that chondroitin sulfate significantly increases expression of osteoprotegerin and reduces that of RANKL (Receptor Activator of NF-KB Ligand).

More recent studies instead show that the supplementation of chondroitin sulfate, combined with hyaluronic acid, is effective in the symptomatic treatment of laryngopharyngeal reflux, gastroesophageal reflux, and non-erosive reflux disease.

The mechanism of action thereof in relation to such diseases is probably linked to the ability thereof to inhibit pepsin-induced damage at the level of the gastroduodenal mucosa protecting it by interacting with the amino group thereof.

The compositions subject of the present invention consists of a combination or mixture of hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica and chondroitin sulfate or a salt thereof for the prevention and/or treatment of diseases of the gastrointestinal system, and in particular for gastro- oesophageal diseases both in humans and in animals.

In particular, such diseases are gastroesophageal reflux disease (GERD), laryngopharyngeal reflux (LPR), gastric ulcers, gastritis, dyspepsia, pyrosis, hyperacidity, oesophagitis, reflux oesophagitis, hiatus hernia.

Forming an object of the present invention is a composition comprising an association or a combination or a mixture comprising or, alternatively, consisting of:

(a) hyaluronic acid or a salt thereof,

(b) an extract of Opuntia ficus-indica,

(c) chondroitin sulfate and, optionally food or pharmaceutical grade additives or excipients.

Said (a) hyaluronic acid or a salt thereof has an average molecular weight comprised from 1.0 MDa to 4.0 MDa, preferably comprised from 1.8 MDa to 3.2 MDa, more preferably comprised from 2.0 MDa to 2.8 MDa, or comprised from 0.1 MDa to 1.5 MDa, preferably comprised from 0.2 MDa to 1.0 MDa, more preferably comprised from 0.3 MDa to 0.75 MDa.

In a preferred embodiment, said (a) is preferably sodium hyaluronate.

In a preferred embodiment, said (b) extract of Opuntia ficus-indica, combined with said (a) and said (c) in said composition, is preferably an extract of fruits and/or cladodes of Opuntia ficus-indica, preferably of Opuntia ficus-indica fruits.

In a preferred embodiment, hyaluronic acid or a salt thereof is present in an amount comprised from 0.1 mg to 5000 mg, preferably in an amount comprised from 1 mg to 3500 mg, more preferably in an amount comprised from 5 mg to 2000 mg, even more preferably comprised from 6 mg to 100 mg, per dosage unit; the extract of Opuntia ficus-indica is present in an amount comprised from 0.1 mg to 5000 mg, preferably in an amount comprised from 1 mg to 3500 mg, more preferably in an amount comprised from 5 mg to 2000 mg, even more preferably in an amount comprised from 20 mg to 200 mg, per dosage unit; chondroitin sulfate is present in an amount comprised from 0.1 mg to 7000 mg, preferably in an amount comprised from 1 mg to 5000 mg, more preferably in an amount comprised from 5 mg to 3000 mg, even more preferably in an amount comprised from 20 mg to 200 mg, per dosage unit.

The dosage form may be a pharmaceutical composition or a complementary feed or a medical device or a cosmetic or a dietary supplement or a food for special medical purposes including the aforementioned active ingredients mixed to each other. In other words, said composition is preferably a medical device composition, a pharmaceutical composition, or a food for special medical purposes.

The preferred route of administration is oral or topical.

In other words, said composition is preferably for oral or topical use, more preferably in form of tablet, paste or gel, capsule, solution, suspension, powder, or granules.

The pharmaceutical forms that can be used according to the present invention are preferably tablets, divisible tablets, pastes, capsules, solutions, suspensions, powders, or granules.

In this description, the term "divisible tablet” is used to indicate a tablet having pre-cuts which allow to divide it into half tablets or quarters of tablets. Preferably:

(A) said composition is in form of tablet or divisible tablet and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 10 mg to 70 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 40 mg to 80 mg;

(c) the chondroitin sulfate in an amount comprised from 40 mg to 120 mg; or

(B) said composition is in form of paste or gel and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 6 mg to 20 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 60 mg to 120 mg;

(c) the chondroitin sulfate in an amount comprised from 30 mg to 80 mg; or

(C) said composition is in form of solution and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 6 mg to 20 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 80 mg to 130 mg;

(c) the chondroitin sulfate in an amount comprised from 50 mg to 90 mg;

Furthermore, forming an object of the present invention is said composition for use in a method for the preventive or curative treatment of diseases of the gastrointestinal system, or of the symptoms related to said diseases, in a mammal, wherein said mammal is preferably a human being or an animal.

Said diseases associated with the gastrointestinal system are preferably selected from the group comprising or, alternatively, consisting of: gastroesophageal reflux disease (GERD), laryngopharyngeal reflux (LPR), gastric ulcers, gastritis, dyspepsia, pyrosis, hyperacidity, oesophagitis, reflux oesophagitis, hiatus hernia.

The following examples are given purely by way of illustration and they do not limit the scope of protection of the invention as defined in the attached claims. Changes or variations to the embodiments exemplified herein, obvious to the person skilled in the art, are encompassed by the attached claims. Suitable excipients may be selected from those normally known in the state of the art and include, but are not limited to: diluents (for example dibasic calcium phosphate, lactose, microcrystalline cellulose and cellulose derivatives), thickeners (for example gums, hydroxypropyl methylcellulose and other cellulose derivatives), sweeteners (for example sorbitols, mannitol and other polyols, acesulfame K, aspartame, cyclamates, saccharin, sucralose), lubricants (for example magnesium stearate, stearic acid, waxes), dispersants, surfactants (for example sodium lauryl sulfate and polysorbates), flavour enhancement agents, adsorbents (for example silica gel, talc, starch, bentonite, kaolin), glidants and antiadherents (for example talc, colloidal silica, corn starch, silicon dioxide), colorants (for example iron oxides), opacifiers (for example titanium oxide), antioxidants, binders (for example gums, starch, gelatin, cellulose derivatives, sucrose, sodium alginate), disaggregating agents (starch, microcrystalline cellulose, alginic acid, crospovidone), plasticisers (for example ethyl cellulose and other cellulose derivatives, acrylates and methacrylates, glycerol and sorbitol), preservatives (for example parabens, sulfur dioxide), viscosifiers, emulsifiers, humectants, wetting agents, chelating agents and mixtures thereof.

Example 1 : tablets

Example 2: paste

Example 3: divisible tablets Example 4: tablets

Example 5: solution for oral use

EXPERIMENTAL PART

Various experimental models can be used to test the effectiveness of the combination of functional substances and to evaluate the synergistic action thereof.

The action of the individual components and of the combination subject of the present invention is evaluated by means of in vitro and/or in vivo tests.

The synergistic activity of the components can be evaluated in vitro by means of mucoadhesion tests conducted on cells (e.g. on epithelial cells of the buccal or gastric mucosa) or through other validated methods such as for example inclined plane with mucin.

The antioxidant activity of the composition comprising the three active substances of interest can be determined for example by following a radical scavenging evaluation method in which DPPH (2,2- diphenyl-1 -picrylhydrazyl) is used as reagent.

Ascorbic acid is prepared as standard sample. Different concentrations of the test formulation under examination to which a solution of DPPH in ethanol was added were tested and the mixture was incubated at room temperature away from light. The activity for scavenging the test substances under examination is determined by measuring absorbance at a wavelength of 517 nm. The percentage of scavenger activity is calculated using the following formula: Radical scavenging activity (%) = [1 -(A _sam pie / Abiank)] x 100. wherein' A _sam pie is the absorbance of the sample, andbiank is the absorbance of the blank.

Other tests for evaluating antioxidant activity known to the person skilled in the art may be used.

In vitro tests on cell cultures are useful to evaluate the protective effect of the composition subject of the present invention. For example, human gastric epithelial cells GES-1, cultured in DMEM growth medium completed and incubated at 37°C and in humid atmosphere at 5% of CO ₂ . Following a 24- hour treatment with different concentrations of the substances to be evaluated, an MTT test is carried out to evaluate the cell viability and the cytotoxicity of the active substances of interest. After a short incubation period with the reagent, the absorbance is measured at 570 nm.

In order to evaluate the antioxidant effect of the aforementioned composition, levels of intracellular ROS are evaluated by means of an assay with DCFH-DA (2,7-dichlorofluorescein diacetate); the differently treated cells are washed and incubated with DCFH-DA and the fluorescence intensity is measured.

The total antioxidant capacity (T-AOC) is evaluated by means of the ABTS method using an appropriate kit and measuring the optical density of the supernatant at 420 nm. Cells treated with the substances under examination at different concentrations are sonicated in order to analyse the levels of SOD, GSH-Px, MDA and LDH. The supernatant is collected and used to determine the levels of SOD, GSH-Px, MDA and LDH with appropriate kits.

An in vitro inflammation model can be used to evaluate the anti-inflammatory action of the composition subject of the present invention. For example, RAW264.7 murine macrophages stimulated with bacterial lipopolysaccharides (LPS) are used. The cells are cultured in DMEM growth medium complete and incubated at 37°C and in humid atmosphere at 5% of CO2. After 1 hour from treatment with different concentrations of the composition subject of the present invention, LPSs are added for a total time of 24 hours. At the end of this time, cell viability and cytotoxicity of the active substances of interest are evaluated through an MTT assay. Furthermore, the NO content in the growth medium is determined using the Griess reagent, and the NO concentration is evaluated by measuring absorbance at 540 nm and the calibration curve was obtained by diluting with NaNO2. Each treatment was carried out in triplicate. The cells were treated with different concentrations of the composition subject of the present invention for 1 hour to determine the anti-inflammatory effect, then LPSs were added and the cells were incubated again for 1 hour and for 18 hours. At the end of the treatment, the cells were harvested and protein extraction was carried out. The samples thus obtained were analysed by western blotting so as to determine the levels of inflammatory markers such as INOS, COX-2, TNF-o, IL-1p, p-NF-kB p65, p- IkBa.

An in vitro test for the inhibition of the H+/K+ ATPase enzyme, that is the gastric proton pump, may be carried out in order to verify the antiacid activity of the composition subject of the present invention comprising hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica and chondroitin sulfate. For example, in order to obtain the enzyme samples to be tested, gastric parietal cells were obtained from the stomach of the sheep (or from another source), they were homogenised and centrifuged for 10 minutes. The supernatant (enzyme extract) was used to determine the inhibitory activity of the substances under examination. The reaction mixture containing the enzyme extract and the composition subject of the present invention containing the three active substances of interest was preincubated for 60 minutes at 37°C. The reaction was induced by adding ATP (substrate), MgCl2 and KCI. After 30 minutes of incubation at 37°C, the reaction was stopped by adding a mixture containing 4.5% of ammonium molybdate and 60% of perchloric acid, it was subsequently centrifuged for 10 minutes and the inorganic phosphate produced by the reaction was spectrophotometrically measured at a length of 660 nm following the Fiske-Subbarow method. mQ water, ammonium molybdate and ANSA was added to the supernatant and the mixture was left for 10 minutes at room temperature. The absorbance of the released inorganic phosphate was measured at 660 nm. The enzymatic activity was calculated as micromoles of Pi released per hour at the various doses of the mixture of the two extracts (0-100 pg). The percent enzyme inhibition was calculated using the following formula:

Percent inhibition = [Activity(control) - Activity(test)/Activity(control)] x 100.

The effectiveness of the composition of the present invention may also be evaluated by means of an in vivo test on experimental animals in compliance with the directives of the European Union and the Ministry of Health and approved by an Ethics Committee. The tests are carried out on Sprague-Dawley mice pre-treated with the substances under examination. After pre-treatment, a high dose of an ulcerative agent was administered and after a variable incubation period, from 1 hour to 5 hours, the animals are sacrificed and the stomach of each is removed and opened along the greater curvature. The number and size of the ulceration areas (expressed in mm ² ) are evaluated on the gastric mucosa. The evaluation of the ulceration areas is carried out as described above. In order to evaluate gastric secretions, the volume of gastric juice is collected and used for measuring the pH, pepsin activity (by means of an appropriate kit) and total acidity by means of titration with sodium hydroxide. The amount of mucus produced may be evaluated by scraping the mucosa and the collected mucus is weighed using a precision electronic scale. Ex-vivo tests are carried out on the collected tissues. Isolated tissue sections are homogenised and the obtained samples are used for quantifying the levels of MDA, SOD, CAT and GPx. MDA levels are measured by analysing the levels of lipid peroxidation. To this end, a reactivity test is carried out using TBA (thiobarbituric acid) and by measuring absorbance at 532 nm with a UV-visible spectrophotometer. SOD levels are measured by means of an epinephrine test and by evaluating absorbance at 480 nm at the end of the test. CAT activity is evaluated using an appropriate kit. Free iron levels were evaluated on the blood samples taken from the animals subjected to the assays at the time of sacrifice by means of ferrozine-based colorimetric measurement.

A variant of the aforementioned assay provides for the use of EDTA as a reaction standard. Plasma calcium levels are measured with the colorimetric method using the reaction with cresolphthalein and measuring the absorbance of the complex formed at 570 nm. Furthermore, the anti-inflammatory action is evaluated on the plasma samples by determining the levels of TNF-o, IL-1 p, gastrin, NO and PGE2.

A further in vivo test on animals, useful to evaluate the gastro-protecting activity of the composition subject of the present invention, provides for the following protocol. The selected mice are starved for 18 hours prior to the operation to induce an oesophagitis condition. Subsequently, 2 hours prior to the operation, the mice were treated with the substances under examination. The mice were anesthetised and a 2 cm incision was carried out in the central part of the abdomen so as to expose the stomach, then the pylorus was bound to induce reflux, while keeping the vagus nerve intact. After 5 hours of operation, all mice were sacrificed. The oesophagus was immediately removed, washed with saline solution and photographed to evaluate mucosal lesions. The anti-inflammatory activity of the substances was evaluated by determining, through western blotting, the levels of inflammatory markers such as COX-2, IL-1p, TNFo and of IkBo and Nf-kB. Furthermore, in order to determine the histamine content, blood was collected from the supraorbital plexus through the microcapillary technique and the plasma was separated. The plasma was treated with perchloric acid and centrifuged for 30 minutes at 4°C. The supernatant was used to evaluate histamine content through HPLC, which was expressed in lU/mg of protein.

Furthermore, the effect of the composition subject of the present invention on the gastric emptying time is evaluated for example following the model described by Smits and Lefebvre (1996). Male mice were starved for 18 hours and they were subsequently fed 20 minutes prior to the start of the experiment so as to ensure that the food content in the stomach was as similar as possible. The composition subject of the present invention comprising the individual substances or the mixture thereof was administered orally; after 30 minutes, a marker (suspension containing 50 mg of phenol red in 100 ml of 1.5% carboxymethyl cellulose) was administered to the animals to evaluate the gastric emptying rate. After 20 minutes, the animals were sacrificed and the stomach was removed. The stomach was subsequently placed in tubes containing a saline solution, after 20 seconds under stirring, 1 M NaOH was added in each tube so as to obtain the maximum colorimetric intensity. At this point spectrophotometric analysis was carried out at the wavelength of 560 nm. The gastric emptying percentage was calculated using the following formula:

Gastric emptying (%) = [1 -(amount of phenol red present in the stomach after 20 min) I (amount of phenol red present in the stomach at time 0)] x 100.

Evaluation of gastric emptying may be carried out by means of spectroscopic magnetic resonance imaging (MRS) or other suitable method. This is a non-invasive technique which allows to carry out more than one experiment on the same animal. Mice should have a weight comprised between 275 and 315 g.

For the study, mice will be given a standard diet with the addition of an indigestible marker (titanium dioxide) and the treatment assigned for that group under study. Prior to the MRS analysis, the mice will be starved for 14 hours without food or water and 1 g of the diet assigned for each group will be given subsequently. The rats will be scanned every 25 minutes/150 minutes.

The evaluation of reflux and of the gastric secretion may be carried out after pylori binding, followed by the evaluation of the parameters in question. Before the start of the experiment, the animals will be starved for 24 hours; they will be allowed free access to water.

The animals will be anesthetised and subjected to a pylori binding intervention. In order to promote gastric reflux, a 1 cm-thick longitudinal myotonia will be carried out at the level of the gastroesophageal junction. The treatments will be administered immediately after the pylori binding intervention. The mice will be starved for 24 hours after intervention and subsequently sacrificed in a CO2 saturated atmosphere; removal of the oesophagus will follow.

The oesophageal mucosa will be separated from the muscle layer; the evaluation of the presence of possible lesions will follow. The area (mm ² ) of the oesophageal mucosa with lesions will be evaluated through microscopic analysis. After 24 hours from the pylori binding intervention, the animals will be sacrificed; the stomach content will be subsequently collected to evaluate the pH content and the total amount of gastric juice, and gastrectomy, to evaluate the presence of lesions at the level of the gastric mucosa. For the study, gastric juice will be added with 2 ml of distilled water and centrifuged at 5000 rpm for 15 minutes at 4°C. After centrifugation the supernatant will be used to evaluate the volume (mL/mouse), pH and acidity (mEq/L). The total acidity will be evaluated by means of titration with 0.01 N NaOH at pH = 7 (phenolphthalein will be used as an indicator). Total acidity will be expressed as mequiv. [H ⁺ ]/ml/4 h. The stomach will be opened by means of a cut on the greater curvature and placed on a polystyrene support to evaluate gastric lesions. The total area of gastric lesions will be measured, and the degree of severity of gastric lesions will be expressed by means of a gastric lesion index (mm ² ).

The proton pump inhibitory activity of the individual active ingredients and the combination thereof in the composition subject of the present invention can be evaluated using HGT-1 gastric parietal cells which, stimulated by histamine, release H ⁺ (histamine activates the proton pump).

After treatment with the samples under examination, as an indicator of proton secretion intracellular pH is measured by means of the fluorescent dye sensitive to pH 1.5 carboxy-seminaphto-rhodafluor acetoxymethylester (SNARF-1-AM).

Besides the aforementioned models, provided solely by way of non-limiting examples, modifications thereof and all the other in vitro or in vivo tests known to the person skilled in the art may be used in order to demonstrate the synergistic activity of the combination subject of the present invention.

Preferred embodiments E(n) of the present invention are illustrated below:

E1. A composition comprising a combination of hyaluronic acid or a salt thereof, an extract of Opuntia ficus-indica and chondroitin sulfate.

E2. Composition according to the preceding embodiment, wherein hyaluronic acid or a salt thereof is present in an amount comprised between 0.1 mg and 5000 mg more preferably in an amount comprised between 1 mg and 3500 mg even more preferably in an amount comprised between 5 mg and 2000 mg per dosage unit.

E3. Composition according to any one of the preceding embodiments, wherein the extract of Opuntia ficus-indica is present in an amount comprised between 0.1 mg and 5000 mg more preferably in an amount comprised between 1 mg and 3500 mg even more preferably in an amount comprised between 5 mg and 2000 mg per dosage unit. E4. Composition according to any one of the preceding embodiments wherein chondroitin sulfate is present in an amount comprised between 0.1 mg and 7000 mg more preferably in an amount comprised between 1 mg and 5000 mg even more preferably in an amount comprised between 5 mg and 3000 mg per dosage unit.

E5. Composition according to any one of the preceding embodiments, wherein the composition is a complementary feed, a medical device, a cosmetic, a dietary supplement, a nutraceutical, dietetic and nutritional composition, a foodstuff, a beverage, a nutraceutical product, a medicament, a medicated foodstuff, a pharmaceutical composition, a food for special medical purposes.

E6. Composition according to any one of the preceding embodiments effective in the prevention and/or in the treatment of diseases of the gastrointestinal system and of the related symptoms both in humans and in animals.

E7. Composition according to E1-E6, wherein the diseases associated with the gastrointestinal system are: gastroesophageal reflux disease (GERD), laryngopharyngeal reflux (LPR), gastric ulcers, gastritis, dyspepsia, pyrosis, hyperacidity, oesophagitis, reflux oesophagitis, hiatus hernia.

Further preferred embodiments F(n) of the present invention are illustrated below:

F1 . A composition comprising an association or a combination or a mixture comprising or, alternatively, consisting of:

(a) hyaluronic acid or a salt thereof,

(b) an extract of Opuntia ficus-indica, and

(c) chondroitin sulfate and, optionally food or pharmaceutical grade additives or excipients.

F2. The composition according to F1, wherein said (a) hyaluronic acid or a salt thereof has an average molecular weight comprised from 1.0 MDa to 4.0 MDa, preferably comprised from 1.8 MDa to 3.2 MDa, more preferably comprised from 2.0 MDa to 2.8 MDa, or comprised from 0.1 MDa to 1.5 MDa, preferably comprised from 0.2 MDa to 1.0 MDa, more preferably comprised from 0.3 MDa to 0.75 MDa; preferably wherein said (a) is sodium hyaluronate. F3. The composition according to F1 or F2, wherein said (b) an extract of Opuntia ficus-indica is an extract of fruits and/or of cladodes of Opuntia ficus-indica, preferably of fruits.

F4. The composition according to any one of F1-F3, wherein said composition comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 0.1 mg to 5000 mg, preferably comprised from 1 mg to 3500 mg, more preferably comprised from 5 mg to 2000 mg, even more preferably comprised from 6 mg to 100 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 0.1 mg to 5000 mg, preferably comprised from 1 mg to 3500 mg, more preferably in an amount comprised from 5 mg to 2000 mg, even more preferably in an amount comprised from 20 mg to 200 mg;

(c) the chondroitin sulfate in an amount comprised from 0.1 mg to 7000 mg, preferably comprised from 1 mg to 5000 mg, even preferably in an amount comprised from 5 mg to 3000 mg, even more preferably in an amount comprised from 20 mg to 200 mg.

F5. The composition according to any one of F1-F4, wherein said composition is for oral or topical use.

F6. The composition according to any one of F1-F5, wherein said composition is in form of tablet, paste or gel, capsule, solution, suspension, powder, or granules.

F7. The composition according to F6, wherein:

(A) said composition is in form of tablet or divisible tablet and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 10 mg to 70 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 40 mg to 80 mg;

(c) the chondroitin sulfate in an amount comprised from 40 mg to 120 mg; or

(B) said composition is in form of paste or gel and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 6 mg to 20 mg;

(b) the extract of Opuntia ficus-indica in an amount comprised from 60 mg to 120 mg;

(c) the chondroitin sulfate in an amount comprised from 30 mg to 80 mg; or

(C) said composition is in form of solution and it comprises, per dosage unit:

(a) the hyaluronic acid or a salt thereof in an amount comprised from 6 mg to 20 mg; (b) the extract of Opuntia ficus-indica in an amount comprised from 80 mg to 130 mg;

(c) the chondroitin sulfate in an amount comprised from 50 mg to 90 mg;

F8. The composition according to any one of F1-F7, wherein said composition is a medical device composition, a pharmaceutical composition, or a food for special medical purposes.

F9. The composition according to any one of F1-F8, wherein said composition is for use in a method for the preventive or curative treatment of diseases of the gastrointestinal system, or of the symptoms related to said diseases, in a mammal, wherein said mammal is preferably a human being or an animal.

F10. The composition according to F9, wherein said diseases associated with the gastrointestinal system are selected from the group comprising or, alternatively, consisting of: gastroesophageal reflux disease (GERD), laryngopharyngeal reflux (LPR), gastric ulcers, gastritis, dyspepsia, pyrosis, hyperacidity, oesophagitis, reflux oesophagitis, hiatus hernia.