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1. (WO2019030629) A COMPOSITION FOR ORAL ADMINISTRATION IN THE THERAPEUTIC OR PREVENTIVE TREATMENT OF GASTROESOPHAGEAL REFLUX DISORDERS OR DISEASE
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"A COMPOSITION FOR ORAL ADMINISTRATION IN THE THERAPEUTIC OR PREVENTIVE TREATMENT OF GASTROESOPHAGEAL REFLUX DISORDERS OR DISEASE".

The present invention relates to a composition for use in a method for treating gastroesophageal reflux disorders or disease, preferably in a method for the symptomatic treatment of gastroesophageal reflux disorders or disease, wherein said composition is for oral use, in any form of administration pharmaceutically acceptable, and comprises a mixture comprising or, alternatively, consisting of an extract of Trigonella foenum graecum and a block copolymer of ethylene oxide and propylene oxide.

By gastroesophageal reflux is meant "the involuntary and unconscious passage of part of the gastric contents into the esophagus, without the joint participation of the gastric and abdominal muscles." The esophagus is a channel, 25-30 cm long, connecting the mouth with the stomach. Along its course are two sphincter structures: the first between the hypopharynx and the cervical tract of the esophagus (Upper Esophageal Sphincter, UES), the second at the level of the gastroesophageal junction (Lower Esophageal Sphincter, LES). The latter is a high pressure zone that represents the main anti-reflux structure, thanks to its location between the negative-pressure intrathoracic zone and the positive-pressure intra-abdominal zone. Therefore, under normal conditions, an increase in abdominal pressure has repercussions at the level of the LES, preventing the return of ingested material into the esophagus. Under physiological conditions the LES is closed and relaxes for a time of about 3-10 seconds following deglutition. Other anatomical structures, in addition to the LES, which contribute to maintaining the anti-reflux barrier are:

The angle of His, an acute angle formed between the esophagus and the gastric fundus;

The phrenoesophageal ligament;

The diaphragmatic collar, consisting of fasciae of the diaphragm, which wrap around the esophagus like a scarf and constrict the lumen during inspiration.

Numerous factors contribute to the pathogenesis of gastroesophageal reflux disease (GERD), including:

1. Insufficiency of the anti-reflux barrier of the lower esophageal sphincter, which has the purpose of mechanically preventing gastric juices from flowing up into the esophagus.

2. Delay in gastric emptying due to anatomical anomalies or functional alterations: (i) anatomical anomalies: pyloric stenosis (the terminal region of the stomach, which regulates the passage of the gastric content into the duodenum); (ii) functional alterations: motoric alteration of the fundus (the region responsible for draining fluids).

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

4. Gastric hyperacidity.

5. Aggressiveness of the gastric contents that flow back into the esophagus, due to the action of hydrochloric acid.

6. Duodenogastric reflux with the passage of pancreatic and biliary secretions into the stomach, which in the case of gastroesophageal reflux, can cause more severe lesions.

Other predisposing factors include smoking, improper dietary behaviour (abundant meals, high-fat foods, caffeine), drugs, pregnancy and obesity, which can exacerbate GERD. A hiatal hernia (extension of a portion of the stomach into the thorax through an opening in the diaphragm called the esophageal hiatus) also frequently accompanies GERD and can contribute to the prolonged exposure to gastroduodenal contents. In general, the walls of the esophageal hiatus adhere closely to the esophagus, but it can happen that the anchorage structures of the lower portion of the esophagus lose tone, favouring the ascent of a small part of the stomach into the thorax.

Whatever the cause may be, the frequent and repeated contact of regurgitated gastric materials with the esophageal 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 esophageal 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 esophageal 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).

The specificity of these symptoms for GERD is 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 pathological conditions most frequently encountered by gastroenterologists.

A study on the prevalence of the disease has shown that GERD has a prevalence of 10-20% in western countries, in contrast with just 5% in Asia. In particular, a larger number of cases have been recorded in

North America, followed by Northern Europe and Southern Europe.

Scientific studies have shown that the symptoms of the disease have a strong impact on quality of life, inasmuch as persistent symptoms of reflux, even during treatment with proton pump inhibitors, are associated with reduced physical and mental wellbeing.

Since the pathology is chronic, conventional treatment is almost always long term and consists, depending on the severity of the disease, in changing the subject's life style (elimination of chocolate, caffeine, alcohol, cigarettes, losing weight, etc.), pharmacological treatment and surgery.

The classes of drugs most commonly used to treat GERD include antacids, antagonists of the H2 histamine receptor and proton pump inhibitors (PPI), and prokinetic agents.

Antacids are over the counter drugs and offer rapid relief of the symptoms of the pathology, but have no curative effect in cases of erosive esophagitis. These drugs contain carbonates or bicarbonates which reduce the acidity in the stomach by reacting with the hydrochloric acid and releasing carbon dioxide, hb-antagonists like ranitidine, famotidine and cimetidine provide temporary relief of the symptoms, although they are slower acting than antacids. Long term use of these drugs is not recommended, since the subject may develop tolerance to them over 1-2 weeks, and in any case they have no curative effect. Proton pump inhibitors (pantoprazole, lansoprazole, omeprazole, etc.) are the standard drugs used in the treatment of gastroesophageal reflux pathologies. Indeed, the number of prescriptions for these drugs has doubled over the last 10 years. These drugs are often associated with prescriptions for steroidal and nonsteroidal anti-inflammatory drugs. The mechanism of action of the PPIs includes blocking of the proton pump in the parietal cells of the stomach; this hydrogen/potassium ATPase pump controls the release of hydrochloric acid into the lumen of the stomach. Compared with the H2 antagonists, these drugs are faster acting and, above all, exercise a curative action of the esophageal lesions. Like many of the drugs used to treat or reduce the symptoms of gastroesophageal reflux disorder (GERD), the PPIs also have side effects, especially when used in combination with steroidal drugs. The most commonly recorded side effects of treatment with PPIs are nausea, diarrhea, headache, insomnia and anaphylactic reactions. Prokinetic agents, such as cisapride and metoclopramide, activate the serotonin or dopamine receptors which increase esophageal or gastric peristalsis. These drugs have a slow onset of action, a short duration and are not curative of the pathology. They also have various side effects, including tremor, dyskinesia, fatigue and an increase in adverse cardiac events, so that they are not commonly used in treating GERD.

In addition to the classic pharmacological remedies, alginates are also used in the symptomatic treatment of GERD. Alginates, such as sodium alginate, are natural polysaccharides which precipitate in contact with the gastric environment, forming a low-density gel in just a few minutes. The reaction of carbonates and bicarbonates with the gastric hydrochloric acid releases carbon dioxide which is trapped in the alginate gel, forming a foam or gel (the "raft") which floats on top of the contents of the stomach. The alginate gel forms in the portion of stomach near the gastroesophageal junction, precisely where the acid pocket

develops. In this manner, the reflux of acid from stomach to the esophageal canal is blocked or greatly reduced. However, alginate-based drugs also fail to resolve the causes of gastroesophageal reflux.

Furthermore, patent WO2007/133082 A1 describes a composition comprising specific polysaccharides for use in the treatment of GERD. This document does not describe the presence in the composition of a block copolymer of ethylene oxide and propylene oxide (or poloxamer).

Patent PL218041 B1 describes a composition comprising poloxamer 407 and methylcellulose for use in the treatment of GERD.

Therefore, there is still a need for compositions or drugs for the treatment of gastroesophageal reflux that are well tolerated, effective and, if possible, free of side effects, especially in the case of individuals who have to take anti-reflux drugs or compositions for extended periods of time.

One object of the present invention is to provide a composition which overcomes the disadvantages listed above of the known compositions for use in a method for treating GERD.

As a solution to this requirement, the present invention provides a composition for use according to the appended claims.

The present invention relates to a composition (in short the composition of the invention) comprising or, alternatively, consisting of an effective amount of a mixture comprising at least or consisting of:

(a) an extract of Trigonella foenum graecum and

(b) a block copolymer of ethylene oxide and propylene oxide with the following structural formula:

HO-(CH2-CH2-0)x-(CH2-CH(CH3)-0)x-(CH2-CH2-0)x-H

wherein x and y are, independently of each other, a number comprised between 20 and 200.

In one embodiment, the composition according of the invention comprising an effective amount of a mixture consisting of:

(a) an extract of Trigonella foenum graecum and

(b) a block copolymer of ethylene oxide and propylene oxide with the following structural formula:

HO-(CH2-CH2-0)x-(CH2-CH(CH3)-0)x-(CH2-CH2-0)x-H

wherein x and y are, independently of each other, a number comprised between 20 and 200; with the condition that said composition and/or mixture does not comprise methylcellulose or derivatives thereof. In particular, the composition according to the present invention comprising an effective amount of a mixture consisting of:

(a) an extract of Trigonella foenum graecum and

(b) a poloxamer type block copolymer of ethylene oxide and propylene oxide, for instance poloxamer 407 and/or poloxamer 188, without the use of methylcellulose or derivatives thereof.

This invention further relates to a composition as defined above (the composition of the invention) for use in the therapeutic and/or preventive treatment of gastroesophageal reflux disorders or disease, and pathologies or disorders connected thereto, wherein said treatment comprises the oral administration of said composition to a subject.

Preferred embodiments of this invention will be evident from the detailed description given below and indicated in the appended claims.

Following extensive experimentation, the inventors have perfected a composition comprising an extract of Trigonella foenum graecum and a block copolymer (poloxamer) advantageous for use in the therapeutic or preventive treatment of gastroesophageal disorders and the pathologies and disorders associated with it. In particular, the use of the poloxamer increases the effectiveness of the composition without, however, increasing the viscosity of the gel to be administered to the patient. This is made possible by the temperature-dependent gelification of the poloxamer, which at room temperature has the form of a sol and creates low viscosity suspensions, but gelifies at body temperature, thus protecting the gastric and esophageal mucosa against the action of the acid. This makes it possible to maximise the compliance of the patient, by offering him/her a formulation which is not excessively viscous and thus easy to swallow. The Applicant has found that this advantageous effect is encountered only when the poloxamer is associated with the fibres of fenugreek, while associating the poloxamer with other fibres has not demonstrated any advantageous effect in relation to the viscosity of the resulting composition.

Furthermore, the Applicant has found that the use of methylcellulose and its derivatives in association with the poloxamer with the fenugreek fibres has no utility in relation to the aim of the present invention.

In the context of the present invention, by "method of treatment" of a pathology or disorder is meant a therapy intended to restore the conditions of health of a subject, maintain existing conditions or prevent the worsening of said conditions of health.

In the context of the present invention, by "prevention" of a pathology or disorder is meant a therapy intended to prevent the occurrence of such a pathology or disorder in a subject, also, but only, as a complication or effect of a pre-existing pathology or disorder.

If not otherwise indicated, in the context of the present invention the percentages and quantities of a component in a mixture refer to the weight of said component relative to the total weight of the mixture.

If not otherwise specified, in the context of the present invention, the ranges of numerical values of characteristics specified as "between X and Y" are inclusive of the limits of the range, i.e. X and Y, as well as all possible intermediate numerical values.

In the context of the present invention, by "composition/s" is meant a pharmaceutical composition, a composition for a food supplement, a composition for a food product or a composition for a medical device.

Fenugreek (Trigonella foenum-graecum) is a leguminous plant cultivated in India and North Africa. The plant belongs to the family of the Fabaceae and has various names depending on the language: Fenugrec (France), Methi (Hindi), Bockshornklee (Germany), Fieno greco (Italy), Alholva (Spain), etc.

The seeds of the plant are used worldwide as spices for condiments and in recipes for soups and pancakes, while the leaves are used as green leaf vegetables in food. The seeds have a bitter flavour and have been used for 2500 years for their medicinal properties, primarily as a remedy for anorexia and as a gastric stimulant.

Furthermore, the seeds are considered to have a carminative and antibacterial effect thanks to the components of which they are constituted. The principal component is fibres (50%), both insoluble (20%) and soluble (30%) in the form of galactomannan. The seeds also contain lipids (7.5%) in the form of triglycerides and phospholipids, steroidal saponins (4-8%) and alkaloids (1%) which contribute to their bitter flavour and stimulate the appetite. The remaining part of the seed is composed primarily of proteins (30%) and small amounts of vitamins and minerals.

The high concentration of fibre in fenugreek seeds make them one of the major natural sources of fibre; and, specifically, 30% of the fibres is soluble and able to form a gel like guar gum, oat bran and psyllium mucilage.

Another important characteristic of fenugreek fibres is their high stability, which gives them a long product shelf-life since they are not degraded by cooling or after cooking.

The most common indication for the use of fenugreek seeds is associated with the capacity of the fibres to increase intestinal regularity, reduce the risk of constipation without stimulating flatulence, and reduce the risk of diverticulitis and hemorrhoids.

Furthermore, the fibres are able to induce a sense of satiety and can contribute to weight loss without compromising an appropriate intake of protein.

Another property which has been documented in numerous studies is their capacity to reduce plasmatic glycemia and glycosuria. The literature includes numerous studies on the hypoglycemic action of fenugreek seeds in mice, rats and diabetic rats and dogs, as well as human subjects.

Numerous scientific studies have evaluated the beneficial effect of alimentary fibre in treating gastric acidity, and have found a correlation between improved symptoms of acidity and the intake of fibre.

For this reason, fenugreek seeds, and the fibre they contain, may be effective in treating gastric acidity and gastroesophageal reflux.

The composition according to the present invention comprises an extract of Trigonella foenum graecum, preferably a dry extract, and more preferably a dry extract of the seeds of Trigonella foenum graecum obtained by means of standard extraction methods known to the person skilled in the art, in particular water or hydroalcoholic extraction methods.

Preferably, in the composition of the present invention, the extract of Trigonella foenum graecum is present in an amount comprised between 10% and 95% by weight relative to the total weight of the composition, more preferably the extract of Trigonella foenum graecum is present in a concentration by weight comprised in a range of 15 to 90%, or 25% to 85%, or 30% to 80% relative to the total weight of the composition.

The Applicant has found that the action of fenugreek in vivo is further increased by its synergistic action with the poloxamer polymer. Said synergy does not benefit from the use of methylcellulose and derivatives thereof.

By "poloxamer" is meant a series of copolymers of ethylene oxide and propylene oxide (CAS number: 9003-11-6 or 106392-12-5). These are three block copolymers with the following structural formula:

HO-(CH2-CH2-0)x-(CH2-CH(CH3)-0)y-(CH2-CH2-0)x-H

All poloxamers have similar molecular structures, also indicated as PEG-PPG-PEG, with different molecular weights and compositions of the blocks of polyoxyethylene (x) and polyoxypropylene (y).

Of these polymers, the most commonly used are poloxamer 188 (in which x=80 and y=27), with a molecular weight ranging from 7680 to 9510 Da, and poloxamer 407 (approximately x= 101 , y=56), with a molecular weight ranging from 9840 to 14600 Da.

These polymers are widely used in pharmaceutical formulations as surfactants, emulsifiers, solubilizers, dispersing agents and enhancers of in vivo absorption (examples of tradenames: Pluronic®, Synperonic®, Kolliphor®).

Poloxamers are considered to be "functional excipients", since they are essential components and play an important role in such formulations.

One of the most interesting properties of poloxamers is their temperature-dependent sol-gel transition. It has been shown that the composition of the polymer influences the solubility and the sol-gel transition temperature in aqueous solutions: the solubility in water of the polyoxyethylene block is high at temperatures in the range 0-100°C, while the solubility of the polyoxypropylene block is reduced at temperatures above 15°C.

The gelification mechanism is based on the solubility of the polyoxypropylene block. Increasing the temperature above the micellisation temperature (which corresponds to a drastic reduction in the solubility of the polyoxypropylene block) leads to an aggregation of the central polyoxypropylene section of the polymer and the consequent formation of micelles, composed of a polyoxypropylene core and lateral polyoxyethylene chains. Further increasing the temperature leads to the gelification of the micelles. This derives from the stacking of the micellar structures.

Poloxamer 407 forms aqueous solutions with a sol-gel transition temperature at around 25°C, while solutions of poloxamer 188 have a sol-gel transition temperature above 40°C. Aqueous solutions of mixtures of the two polymers gelify at intermediate temperatures. For the purposes of this invention, the aqueous solution of a mixture of poloxamer 407 and poloxamer 188 with a ratio by weight of 2: 1 is the most advantageous, since it has a sol-gel transition temperature around 37°C, i.e. human body temperature. Using a mixture poloxamer 407: poloxamer 188 with a 2: 1 ratio will enable gelification in situ, providing highly effective reduction of gastroesophageal reflux without, however, increasing the viscosity of the pharmaceutical form, and thus maximising patient compliance.

The poloxamers have muco-adhesive properties which are very significant in technological terms for the creation of prolonged release pharmaceutical forms. The inventors have found that, due to its muco-adhesive properties, the mixture of one or more poloxamers adheres to the esophageal and gastric mucus membranes, preventing contact with the material deriving from gastroesophageal reflux and hence preventing damage to the mucus membranes themselves.

The combined action of the extract of Tngonella foenum graecum and the poloxamer, preferably a mixture of poloxamers, is particularly advantageous for the therapeutic or preventive treatment of the symptoms of GERD and its associated pathologies and disorders.

Preferably, in the composition of the present invention, the poloxamer is present in an amount comprised between 1% and 80% by weight relative to the total weight of the composition, more preferably the poloxamer is present in a concentration by weight of 1.5 to 70%, or 2.5% to 60%, or 5 to 50% relative to the total weight of the composition.

The present composition may be used for the treatment of gastric and esophageal pathologies, in particularly of gastroesophageal reflux disease. The effectiveness of the composition is derived from the following activity of its components:

The extract of a plant of the genus Tngonella (Tngonella foenum graecum), containing glucomannan in particular, gelifies in contact with the gastric fluids and forms a protective barrier which prevents the reflux of acid towards the esophagus.

The poloxamer has the capacity to bind with the mucus membranes of the esophagus and the stomach. In particular, the interaction of this polymer with the esophageal mucus membrane prevents contact with the reflux acid, thus protecting the mucus membrane against damage by the acid itself. In the present invention, the synergistic action occurs between the extract of a plant belonging to the genus Trigonella and at least one poloxamer, preferably poloxamer 188 or poloxamer 407 or a mixture thereof.

The synergy is enhanced when the extract of the plant of the genus Trigonella (Trigonella foenum graecum) is present to an amount comprised from 10 mg to 10000 mg and at least one poloxamer is present in an amount comprised from 10 mg to 10000 mg, preferably in a ratio of poloxamer 407 to poloxamer 188 respectively of 4:1 to 0.5:1 , preferably 2:1 to 1 : 1.

Preferably, in the composition according to the present invention, said extract of Trigonella foenum graecum (a) is in the form of a dry extract, preferably with a total fibre title of 50% to 95%, more preferably 60 to 90% or 75 to 85%, of the total weight of the extract.

Preferably, in the composition according to the present invention, the block copolymer (b) is a poloxamer. Preferably, in the composition according to the present invention, the block copolymer (b) is poloxamer 407, poloxamer 188, or a mixture thereof.

Preferably, in the composition according to the present invention, the block copolymer (b) is a mixture of poloxamer 407 and poloxamer 188, more preferably with a ratio by weight of 4: 1 to 0.5:1 , even more preferably with a ratio by weight of 2: 1 to 1 : 1.

The composition according to the present invention may be for use in human subjects or for veterinary use, for example, but without limitation, in pets like cats and dogs or in other mammals. Preferably, the composition according to the present invention is for use in human subjects.

In one embodiment, the composition is administered to the subject orally, for example in the form of tablets, pills, whether coated or not, capsules, solutions, suspensions, syrups, food containing the active ingredients or any other form known to a person skilled in the art.

It is understood that, in the method of treatment according to the invention, the administration of (a) and (b) may be simultaneous, for example in a single formulation, or in rapid succession, for instance by means of two or more formulations ingested by the subject in any order, in close succession (e.g. within 1 to 10 minutes) in two distinct compositions.

The composition or composition for use according to the present invention may comprise, in addition to (a) and (b), at least one inert ingredient, such as at least one excipient among those in common use and known to the person skilled in the art.

By "inert ingredient" is meant any substance, or combination of substances, auxiliary to the production of a pharmaceutical, food or nutraceutical form, to be found in the finished product and which is not the active ingredient, although it may modify its stability, release or other characteristics.

Non-limiting examples of such ingredients, as known to the person skilled in the art of pharmaceutical, nutraceutical or food formulations, are excipients such as diluents, absorbents, adsorbents, lubricants, glidants, colourants, surfactants, antioxidants, sweeteners, flavourings, binders, disintegrating agents, plasticisers, thickeners, emulsifiers, humectants, wetting agents, preservatives, chelating agents and the like.

In one embodiment, the composition or composition for use according to the present invention comprises, in addition to (a) and (b), at least an additional natural or synthetic active ingredient. Non limiting examples of said active ingredients are antacids, alginates and other muco-adhesive polymers, prokinetics and active ingredients capable of inhibiting the secretion of hydrochloric acid by the gastric parietal cells.

In the contest of the present invention, the term "medical device" is used in its meaning pursuant to Italian Legislative Decree 24 February 1997, n. 46, and Directive 93/42/EEC 14 June 1993, i.e. it indicates a substance or other product, used alone or in combination, intended by the manufacturer to be used in human subjects for the diagnosis, prevention, control, therapy or attenuation of a disease, said product not exercising the principle action, in or on the human body, for which it is intended, with pharmacological or immunological means nor via metabolic processes, but the function of which may be assisted by said means.

The composition (pharmaceutical composition, food supplement or composition for a medical device) of the present invention may be solid, liquid or semisolid, for example a suspension or gel, and may be in any form known to the person skilled in the art of food, pharmaceutical or nutraceutical compositions, by non-limiting example, in the form of capsules, tablets, at least partially hydrosoluble or orosoluble powders, granules, pellets, microparticles, optionally contained in a sachet or capsule or tablet (mini-tablet), liquid or semisolid preparation, gel, suspension, solution, two-phase liquid system and equivalent forms.

The present invention further provides a method of treatment, therapeutic or preventive, for gastroesophageal reflux disorders or disease and its associated pathologies and disorders in a subject in need thereof, wherein said method of treatment provides for the administration to said subject of an effective amount of the composition according of the invention in any of the embodiments described above, preferably said administration is oral and the composition of the present invention is in one of the above forms of administration.

The present invention relates also to the non-therapeutic use of the composition of the invention according to any of the embodiments described above for the non-therapeutic treatment of gastroesophageal reflux disorders and its associated disorders in a subject in need of said non-therapeutic treatment.

We give below some non-limiting examples of the present invention:

EXAMPLE 1


EXAMPLE 2

Active ingredient Daily dose

Trigonella foenum graecum, e.s. 400 mg

Poloxamer 407 100 mg

Poloxamer 188 50 mg

EXAMPLE 3


EXAMPLE 4


The following experimental part provides examples of practical embodiments of the invention, without limitation to its scope.

EXPERIMENTAL PART

Studies conducted on the composition according to the present invention for the treatment of gastroesophageal reflux.

An assessment was made, ex-vivo and in vitro, of the effect of a composition of the present invention and the individual constituents thereof (a) and (b) (in order to observe a synergistic effect among the individual components) (i) on gastric emptying, (ii) on reflux esophagitis, (iii) on gastric secretion (volume of gastric contents, pH and total acidity) and (iv) on gastric ulcer.

Materials and methods

Animals

Male mice of the ICR strain supplied by the company Charles River and weighing 20-25 g were used. The animals were housed in temperature-controlled rooms (temperature of 23 + 2°C, humidity 50 + 2%, 12-hour light-dark cycles). The mice had free access to water and food, which consisted of a standard diet supplied by the company Mucedola Mangimi (Settimo Milanese, Italy). All experiments were performed in observance of Legislative Decree no. 116 of 27 January 1992 and according to the guidelines of the Council of the European Union (86/609/EEC and 2010/63/EU).

Gastric emptying

Gastric emptying was evaluated with the method described by Smits and Lefebvre (Exp. Gerentol. 1996, 31 (5):589-96). For the purpose of determining the gastric emptying, the animals were orally administered a marker (0.2 ml/mouse of a suspension containing 50 mg of phenol red in 100 ml of 1.5% carboxymethylcellulose). After twenty minutes, the animals were sacrificed in a CO2 saturated atmosphere and the stomach was removed. The stomach was subsequently positioned inside a test tube containing 4 ml of saline solution; after 20 seconds of shaking, 2 ml of NaOH 1 M was added to each test tube in order to achieve the maximum colour intensity.

Spectrophotometric analysis (560 nm) was carried out on 1 ml of this solution. The percentage of gastric emptying was calculated according to the following formula: 100 X (1 - [amount of phenol red present in the stomach after 20 min) / (amount of phenol red present in the stomach at time 0]).

Reflux esophagitis and gastric secretion/ulcer

Reflux esophagitis and gastric secretion/ulcer were induced using the experimental model of pylorus ligation described by Shay and coll. (1945). The animals, fasted for 24 hours but with free access to water, were anaesthetised, the abdomen was opened and the pylorus was ligated. Four hours after the surgical procedure (time necessary to determine a submaximal lesion of the mucosa of the esophagus), the mice were sacrificed in a CO2 saturated atmosphere and the esophagus and stomach were removed for evaluation of: 1) the macroscopic esophageal and gastric damage, 2) the degree of esophageal and gastric inflammation (activity of myeloperoxidase) and 3) the characteristic parameters of gastric secretion (volume of the gastric contents, pH and total acidity).

1) Macroscopic esophageal and gastric damage

The esophagus (opened longitudinally) and stomach (opened along the greater curvature), were spread out on a polystyrene support and analysed with the aid of a microscope so as to identify lesions of the mucosa. The damage to the mucosa was determined using a scoring scale that takes into consideration the severity and extent of hyperaemia and haemorrhagic erosions.

2) Degree of inflammation of the esophagus and stomach

The degree of inflammation of the esophagus and stomach was assessed by determining the activity of myeloperoxidase (MPO). MPO is a protein that is found in the azurophilic granules of polymorphonuclear neutrophils and is used as a marker of leukocyte infiltration. In order to assess the activity of MPO, the tissues were subjected to three homogenisation cycles lasting 10 sec each, at maximum speed, in a suitable lysis buffer defined as MOPS (0.5% HTAB in MOPS 10 Mm), in a ratio of 50 mg of tissue/l mL of MOPS. The homogenates underwent centrifugation at 12,000 rpm for 20 min at 4°C. Subsequently, the supernatants were incubated with NaPP (sodium phosphate buffer pH 5.5) and TMB 16 mM (tetramethylbenzidine) and, after five minutes of incubation at room temperature, H2O2 diluted in NaPP was added. The reaction was blocked with cold acetic acid 2M and a reading of 1 ml of the reaction solution was taken on a spectrophotometer at a wavelength (X) of 650-655 nm. The values obtained were compared to a standard MPO curve and the results expressed as U/ml of MPO.

3) Evaluation of the characteristic parameters of the gastric secretion

The gastric contents were collected for the purpose of determining the pH value and total quantity of gastric juice (volume). Subsequently, 2 ml of distilled water was added to the gastric contents, which were centrifuged at 5000 rpm for 15 min for the determination of total acidity, carried out on the supernatant by titration up to pH=7 (using 2% phenolphthalein as an indicator) with NaOH 0.01 N. The total acidity was expressed as mequiv. [H+]/ml/4 h.

In vitro experimental part

The in vitro studies were run to demonstrate the advantageous effect of the combination of poloxamer and fibres of Trigonella foenum graecum (extract of Trigonella foenum graecum) in comparison with combinations of poloxamer and other fibres.

Preparation of the formulations

The combination of the poloxamers with the fibres was prepared using the cold method. Distilled water was added to a certain amount of poloxamer and vegetable fibre at a temperature of 4°C. The mixture was lightly stirred for approximately 18 hours to dissolve the polymers.

Measurement of the sol/gel transition temperature

A vial containing a magnetic stirring bar and the mixture of poloxamer and fibre was placed inside a thermostatically controlled bath. A thermistor or thermocouple temperature sensor was placed inside the vial. The vial was heated at a rate of 10C/min under continuous stirring at 30 rpm. The sol/gel transition temperature is that recorded by the temperature sensor when the rotation of the magnetic bar was stopped by the gelification of the mixture of poloxamer and fibre.

Preparation of the simulated gastric fluid

The simulated gastric fluid (SGF) was prepared to measure the muco-adhesion in accordance with the Italian Pharmacopoeia. 2.0 g of sodium chloride and 3.2 g of pepsin in powder form were dissolved in ultrapure water. To this was added 80 ml_ of HC1 1 M, and the solution brought to a final volume of 1000.0 ml with ultrapure water.

Rheological measurements

According to the prior art, the rheological analysis was conducted using a rotational rheometer equipped with a cone/plate combination as the measurement system. All samples were subjected to measurement of their viscosity at 25°C and 37°C using an increasing shear speed in the range 10-300 s 1.

A thermoregulation time of 900 s was employed.

All samples were subjected to measurement of their oscillation, which provides the application of a constant shear stress and to measure the viscoelastic response of the sample expressed by the storage modulus (GO) and loss modulus (GOO). Three independent variables were considered: temperature, time and frequency of the applied stress.

The oscillation measurements were conducted:

- at constant frequency (1 Hz) and at temperatures between 8°C and 40°C (heating rate: 1 °C / min) to assess the gelification temperature of the samples (temperature regulation time: 180 s);

- at constant temperature (37°C) and frequency (1 Hz) and increasing times, to determine the gelification time of the samples at body temperature (temperature regulation time: 0 s);

- at constant temperature (37°C) and increasing frequency between 0.5 Hz and 5 Hz, to assess the force of the gel (temperature regulation time: 900 s).

Muco-adhesion measurements

The muco-adhesive properties of the samples diluted with SGF (ratio by volume 1 : 4) were studied using the inclined plane test. The equipment consisted of an inclined plane (angle of inclination: 60 °, surface: 28 cm2) temperature controlled at 37°C, and electronic micro-scales connected to a PC. Pig gastric mucin was used as the biological substrate. The films of mucin were prepared directly on the plane: 2.5 ml of mucin dispersion at 8% p/p in a pH 4.5 acetate buffer was placed on the plane, which was held horizontal at 45°C for 45 minutes. The samples were placed on the plane previously coated with the biological substrate and held horizontal. The plane was then inclined and the amount of sample which fell onto the micro-scales recorded as a function of time.

Float time

The in vitro float time was determined by analysing the floating lag time and the variable duration for all compositions. The test was conducted by placing 5 mL of the raft systems (placed on the clock glass) in a 125 mL beaker containing 100 mL of HCI 0.1 N (pH 1.2) and the system was held at 37°C ± 0.5°C in a water bath. Their physical state was monitored for 20 hours. The following were measured: the time between the introduction of the dosage form (e.g. the composition according to the invention or its individual components) and its hydrostatic thrust on the HCI 0.1 N (FLt) and the time during which the dosage form remained floating (float time). Three repeat measurements were made for each composition.

Swelling index

The swelling index is the volume in mL occupied by 1 g of test material (e.g. the composition according to the present invention or its individual components) after swelling in an aqueous solution for 4 hours. The swelling index was compared using the method indicated in the European Pharmacopoeia 6.0.