EP4110360A1

EP4110360A1 - Formulation based on natural ingredients for use in treating proctological disorders

Info

Publication number: EP4110360A1
Application number: EP21708576.0A
Authority: EP
Inventors: Marianna ALTIERI
Original assignee: Giuliani SpA
Current assignee: Giuliani SpA
Priority date: 2020-02-26
Filing date: 2021-02-22
Publication date: 2023-01-04
Also published as: WO2021170516A1; IT202000003934A1

Abstract

A composition for topical application, comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, honey, Centella asiatica and Hamamelis virginiana, for use in treating proctological disorders; a pharmaceutical formulation for topical application, comprising the above-mentioned composition and at least one pharmaceutically acceptable excipient, for use in treating proctological disorders is also described.

Description

Title: Formulation based on natural ingredients for use in treating proctological disorders

DESCRIPTION

Field of application

The present invention generally relates to the field of pharmaceutical industry. In particular, the invention relates to a natural base formulation for topical application for use in treating proctological disorders.

Prior art

It is known that proctology pertains the medical and surgical treatment of colorectal and anal disorders, including hemorrhoids, proctitis and anal fissures.

In particular, it is known that hemorrhoidal proctological disorders are disorders caused by a swelling and/or an inflammation of hemorrhoids, (i.e. cushions of vascular tissue and connective tissue, located in the anal canal) due to, for example, an abnormal dilation and distortion of vascular channels, vascular thromboses, degenerative processes of collagen fibers associated with supporting connective tissue alterations, loss of functionality of the anal subepithelial muscle.

Hemorrhoids may develop outside of or inside of the anal canal and, in the latter case, they may be classified according to appearance and prolapse level.

Typical symptoms of hemorrhoids comprise bleeding, itching and burning during defecation, constant and persistent pain in the most advanced stages.

It is also known that anal fissures are small cuts which typically form in the posterior margin of the anus, due to an excessive dilation of the same generally caused by the passage of hard bulky feces, as it often occurs during chronic constipation.

If fissures do not spontaneously heal, they may give rise to an ulcer, whose healing takes much longer and is more difficult.

One of the main causes that slow down the healing of the wound and lead to worsening of the fissure, there is a persistent and intense contraction of the involuntary sphincter, which hinders blood passage, causing small local ischemias with formation of ulcers. Since the anal area is rich in vessels and nerve endings, lesions in said area are so painful that they can impair normal daily life.

Fissure symptoms comprise: proctalgia during and after defecation, post defecation proctorrhagia, feeling of perianal weight, perianal itching and abrasions (which represent indicative symptoms of chronicization) .

It is also known that the rectal mucosa may be also subject to inflammations, known as proctitis, which may have several causes, including bacterial, viral, parasitic infections, traumata, drug use or as a consequence of medical treatments or of other pathologies. Clinical signs of proctitis are intense burning and pain during evacuation, lesion formation in the anal area, blood and pus discharge.

Currently, medical treatment for proctological disorders entails regularization of the intestinal tract, if it is not optimal, by an appropriate hydration, a fiber-rich diet (minimum 35 g fiber/day) and a good physical activity, as well as an appropriate local hygiene.

In particular, among the drugs used for the treatment of hemorrhoidal proctological disorders, the following are known: a) drugs by general route, including micronized diosmin-hesperidin and vitamin P, which have an anti-edema, protective action on capillaries, also reducing the perianal itching; and b) locally-acting drugs formulated in suppositories and ointments, typically based on heparin or substances with heparinoid action, cortisonoids and local anesthetics. Steroid drugs, administered chronically, may lead to hypotrophy of perianal skin and may promote the onset of or aggravate a perianal itching.

Some local anesthetics (especially cocaine derivatives and menthol) may have an allergic sensitization as side effect. Therefore, it is recommended to use them for short periods, especially in inflammatory or thrombotic manifestations. Said products further exert a short-lasting action and it is generally difficult to document their actual clinical efficacy; moreover, they may affect the symptoms (bleeding, burning, itching), but not the prolapse.

Various methods are also long been known for the outpatient treatment of hemorrhoids, including sclerosing therapy, infrared photocoagulation, elastic ligation, cryotherapy and selective, doppler-guided ligation of the vessels supplying the hemorrhoidal bulges.

It is also known that, depending on the hemorrhoids stage, their symptomatological impact and in particular the alteration of the life quality, on the patient’s conditions and age, a surgery may be necessary, which typically involves the exeresis of hemorrhoidal bundles or the correction of the prolapse.

As regards the therapy of anal fissures, primary treatments comprise, first of all, the optimization of the hygienic local conditions by using specific, non-irritating detergents, generally with a low pH, the daily practice of tepid hip baths (which contribute to reduce the tone of the sphincter and to improve lifestyle especially regarding physical activity). Other factors are also important, such as the introduction of fiber (including adding mucilage to the diet), the increase of liquids taken mainly with emollient tepid beverages (mallow, chamomile, berries tisane) or fruit juices- pear, kiwi, apricot - which facilitate intestinal regulation. As a support to topical therapy, the use of anal dilators which, used properly and for an appropriate time, could reduce the basal tone of the internal sphincter, is known; however, a proper use of dilators, up to several weeks, requires an expertness which the patient can hardly acquire, therefore the treatment is often not feasible.

The medical products most suitable for treating anal fissures involve formulations containing calcium antagonists or nitroglycerin, in the form of a cream, to be cyclically applied by topical route.

Among calcium antagonists, it is known the use of nifedipine, which however may cause various undesired side effects, including headache and flushing.

Topical nitrates (0.4% glyceryl trinitrate GNT) reduce the basal pressure of the internal sphincter, increase the local availability of nitric oxide and have a greater effect than the placebo (Cochrane review on 15 studies: 48.6% efficacy versus 37% of the placebo, p = 0.004), above all in the effective and rapid pain reduction, and should be used for about 6 weeks.

However, also these products have considerable side effects, including headache which may lead to treatment interruption in 25% of the cases.

From the state of the art regarding the pharmacological treatment of proctological disorders it is clear that there is a need for products assisting the symptomatological treatment of said disorders, which should be easy to handle and should also promote the restoration of a physiological, hydrated, and intact environment of the affected tissue.

In fact, most of the pharmacological products for topical use only contain active ingredients with specific anesthetic and/or analgesic action, without any ingredient which could act as a protectant of the area, reduce the rubbing due to feces passage or which indirectly acts also on the process of perianal skin and mucosa regeneration.

WO 2011/ 154418 describes a preparation for vaginal and rectal use, said preparation comprising micronized hyaluronic acid, with a particle size comprised between 50gm and 200gm and a molecular weight comprised between 1,000,000 Da and 1,800,000 Da. The preparation may also comprise a natural active substance selected from Aloe Vera, Centella Asiatica, Calendula, Tea tree essential oil and sweet almond oil.

“CELEVIS GEL”, a product produced and sold by Nathura S.p.a, is a formulation for topical application including, as active ingredients, hyaluronic acid, Centella asiatica extract, Hamamelis virginiana extract and bovine colostrum. Said formulation is particularly suitable for treating proctological disorders.

The technical problem underlying the present invention is to provide a formulation for treating proctological disorders, including hemorrhoids, proctitis and anal fissures, said formulation containing natural active ingredients, having an excellent tolerability, and being substantially free of the side effects of the prior art formulations, and having an improved action compared thereto.

Summary of the Invention

The present invention solves the above-mentioned technical problem by providing a composition for topical application, comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, honey, Centella asiatica and Hamamelis virginiana, for use in treating proctological disorders.

Preferably, the hyaluronic acid has a molecular weight comprised between 1300 kDa and 2200 kDa, conveniently comprised between 1500 kDa and 2000 kDa.

Preferably, the Hamamelis virginiana is in the form of a hydroglyceric extract containing 1-5% Hamamelis virginiana dry extract by weight of the total weight of the hydroglyceric extract.

Preferably, the Centella asiatica is in the form of a dry extract. Preferably, the honey is selected from the group comprising acacia honey, citrus honey, orange honey, wildflower honey, more preferably acacia honey.

The present invention also relates to a pharmaceutical formulation for topical application, comprising a composition for the use as defined above and at least one pharmaceutically acceptable excipient, for use in treating proctological disorders.

Preferably, the hyaluronic acid or a pharmaceutically acceptable salt thereof, more preferably sodium hyaluronate, is present in a quantity comprised between 0.1% and 2%, more preferably between 0.2% and 0.5%, by weight of the total weight of the formulation.

Preferably, the dry extract of Centella asiatica is present in a quantity comprised between 0.1% and 2%, more preferably between 0.2 % and 0.5%, by weight of the total weight of the formulation.

Preferably, the honey is present in a quantity comprised between 0.5% and 5%, more preferably between 1% and 3%, by weight of the total weight of the formulation.

Preferably, the Hamamelis virginiana hydroglyceric extract is present in a quantity comprised between 0.01% and 5%, conveniently between 0.5% and 3%, by weight of the total weight of the formulation.

Preferably, the at least one pharmaceutically acceptable excipient is selected from the group comprising sweet almond oil, glycerine, decyl oleate, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, capiylic glycol, hexane- 1,2-diol, cross-linked polyacrylic acid, ethylhexyl glycerol, sorbitan isostearate, polysorbate 60, lecithin, lactic acid, phytic acid, sodium hydroxide, tocopherol, ascorbyl palmitate, citric acid, trisodium disuccinate of ethylenediamine 37%.

Preferably, the at least one pharmaceutically acceptable excipient is present in a quantity comprised between 2% and 10%, more preferably between 5% and 8%, by weight of the total weight of the formulation.

Preferably, the above-mentioned pharmaceutical formulation is in the form of a gel or a O/W emulsion, more preferably a hydrophilic gel.

Advantageously, the composition of the present invention contains natural ingredients with protective, hydrating, lenitive and lubricating action, which make it particularly suitable, in conjunction with conventional pharmacological therapies, for treating the most frequent proctological disorders, such as for example hemorrhoids, proctitis and anal fissures, alleviating symptoms of burning, itching and rubbing- induced pain related to said disorders.

Advantageously, the present composition comprises high-molecular- weight hyaluronic acid (preferably 1500-2000 kDa), a highly hygroscopic molecule which promotes re-epithelialization and lubricates the anal epithelium. Moreover, thanks to its anti-inflammatory properties, hyaluronic acid also contributes to wound scarring, as well as to the reduction of pain and burning associated with anal disorders.

Advantageously, the Centella asiatica extract promotes the maintenance of the integrity of the venous wall and microcirculation in compensation conditions.

Said properties counteract phenomena of localized weakness of the venous wall and of organization of the mucosa in cushions of highly vascularized tissue, which are typical of hemorrhoidal proctological disorders (in fact, mechanical stresses by hardened fecal masses play certainly a role in stressing the hemorrhoidal vascular plexus).

Advantageously, the present composition also comprises Hamamelis virginiana, in particular in the form of a hydroglyceric extract, whose properties, within the scope of treating proctological disorders, comprise the impermeabilization action of the most superficial layers of the skin and the mucosae, promoting regeneration of the damaged tissues, as well as the vasoconstrictor action on the superficial vessels of the skin.

The Hamamelis virginiana extract has also a strong antioxidative action, protects cellular hyaluronic acid from depolymerization by hyaluronidases, and protects endothelial cells and hyaluronic acid from free radicals. Moreover, topical application of said extract allows to considerably reduce irritating and inflammatory processes of the skin.

The composition of the present invention advantageously comprises also acacia honey.

In fact, thanks to its hydrating properties, with high osmotic activity (due to a sugar content of 80%) and the ability to modulate the pH, honey promotes a rapid formation of a moist interface on the wound, protecting it from the external environment.

Moreover, honey advantageously has antibacterial, antifungal, antiviral and antioxidant properties, and it is also able to enhance production of nitric oxide, which promotes scarring and capillary circulation.

A further advantage of the present invention is that the composition disclosed herein has an excellent tolerability on the skin and does not elicit side effects, so it is possible to use it when required or for a prolonged time without adverse effects, till normalization of the symptoms associated with proctological disorders.

The composition according to the invention may be applied by topical route both externally, in the perianal and/or anal area, with a light massage directly in the affected area, or internally (rectum), by using a rectal applicator.

The present invention also relates to a formulation for topical application comprising the above-mentioned composition and at least one pharmaceutically acceptable excipient, for use in treating proctological disorders. Brief description of the figures

Figure 1A shows a bar graph concerning HaCaT-cell viability in the presence of Formulation A (1.5% honey and 0.2% hyaluronic acid) and of Formulation B (1.5% bovine colostrum and 0.2% hyaluronic acid), tested at concentrations of 10 mg/ml, 100 mg/ml and 1000 mg/ml, under basal conditions.

Figure IB shows a bar graph concerning HaCaT-cell viability in the presence of Formulation A (1.5% honey and 0.2% hyaluronic acid) and Formulation B (1.5% bovine colostrum and 0.2% hyaluronic acid), tested at concentrations of 10 mg/ml, 100 mg/ml and 1000 mg/ml, after treatment with 3mM methylprednisolone.

Figure 2 shows a bar graph concerning aquaporin 3 (AQP3) concentration in the homogenate of intestinal epithelium (colon) of the positive control sample (Ctr +, epithelium not treated with honey and subjected to dehydration), the negative control (Ctr-, epithelium not treated with honey and not subjected to dehydration) and the sample subjected to dehydration and treated with 1.5% acacia honey. Statistical analyses were performed according to t-test: variations relative to CTR- are considered significant for *p<0.05, variations relative to CTR+ are considered significant for °p<0.05.

Figure 3 shows a bar graph concerning cell viability of cells from the intestinal epithelium (colon) reconstructed in vitro in the presence of 1% SDS. The data obtained for the negative control (Ctr-, intestinal epithelium not exposed to SDS and not treated with honey), the positive control (Ctr+, intestinal epithelium exposed to SDS and not treated with honey) and the sample exposed to SDS and treated with 1.5% acacia honey, were statistically analyzed by t-test. Variations relative to CTR- are considered significant for *p<0.05, variations relative to CTR+ are considered significant for °p<0.05.

Detailed description The present invention relates to a composition for topical application which comprises, as active ingredients, hyaluronic acid, acacia honey, Hamamelis virginiana and Centella asiatica, for use in treating proctological disorders.

The expression “proctological disorders”, as used herein, means the set of diseases and disorders affecting the last segment of the digestive system, namely rectum and anus, and said expression comprises: hemorrhoids, anal fissures, anal and perianal fistulae, abscesses and proctitis.

This association of components allows to use the composition as a scarring, lubricating and anti-inflammatory agent, thus counteracting the main symptoms associated with said disorders, including itching, inflammation, burning and rubbing-induced pain, and promoting the restoration of a physiological, hydrated, elastic and intact environment.

The present composition is suitable for topical application since it may be formulated so as to allow its application on the skin, for example in the form of a gel or of a cream, thereby allowing local application in a simple and functional way.

The expression “topical application”, as used herein, refers to an administration of the present composition by direct contact with the action site to which it is intended, typically the skin and/or the mucosae, thereby performing a local action.

Moreover, the composition of the invention allows to obtain a combined and synergistic action of the ingredients constituting the same, performing, in particular, scarring function and immunomodulatory function, which are increased and effective even at low concentrations.

The composition of the invention comprises hyaluronic acid, or a pharmaceutically acceptable salt thereof, preferably having high molecular weight, i.e. having a molecular weight of at least 1300 kDa, preferably between 1500 kDa and 2000 kDa. Hyaluronic acid is able to bind different types of biological molecules, including water, lipid substances such as membrane phospholipids, positively charged substances (tertiary ammonium), proteins with which form proteoglycans are formed, as well as components of the extracellular matrix.

In particular, the high hygroscopicity of high-molecular-weight hyaluronic acid is essential for carrying out its action of tissue hydration and maintenance of the extracellular space, thereby facilitating the transport of nutrients and ions in solution.

On the skin, hyaluronic acid plays an important role as free-radical scavenger and as an antioxidant; in particular, high-molecular-weight hyaluronic acid advantageously forms a viscous layer around the cells in a dose-dependent manner, slowing down the movement of reactive oxygen species (ROS).

During scarring stages, hyaluronic acid is involved in various cellular responses activated by receptor binding: in the first inflammatory stage, it is accumulated in the damaged tissue, inducing the release of inflammatory cytokines thanks to the interaction with CD44, and improves the infiltration of cells which migrate to the damage site; during the granulation stage, the high hyaluronic acid levels promote cell proliferation and migration of, for example, keratinocytes, and angiogenesis.

Moreover, hyaluronic acid is able to attract fibroblasts to the wound site, facilitating the synthesis of new collagen, carrying out the so-called gap- filler function.

Hyaluronic acid is also directly involved in scar formation and in tissue remodeling itself; in particular, high-molecular-weight hyaluronic acid carries out a structural function.

Thanks to the above-mentioned properties, hyaluronic acid is particularly suitable for treating the symptoms related to proctological disorders.

In particular, said compound contributes to the wound scarring, the reduction of the pain associated with wounds with difficult healing, and the reduction of the itching or the burning associated with anal disorders. Another natural active ingredient contained in the present composition is Centella asiatica, used in the form of a dry extract, which contains different active substances, the most important among them being asiaticoside, madecassoside and centelloside, which belong to the chemical class of the triterpene compounds. According to the European Pharmacopeia, in order to have a therapeutic effect, the extract should contain at least 6% of total triterpene derivatives, expressed as asiaticoside; advantageously, the dry extract of Centella asiatica used in the composition according to the present invention is titrated to 10% triterpenes. The extract of Centella asiatica stimulates collagen production by human fibroblasts, improving the connective structure of the vessel wall. In fact, the triterpene fraction of Centella induces an increase of production of fibronectin, a structural protein important for supporting the wall of venous vessels, in human fibroblast cultures. A further effect of the Centella asiatica extract is the improvement of scarring processes, which is fundamental to alleviate the symptoms associated with anal fissures.

Asiaticoside promotes fibroblast proliferation and synthesis of new extracellular matrix, playing a role in cell-cycle progression, in extracellular-matrix synthesis and in cell proliferation, with increase of the proteins involved in the synthesis of new extracellular matrix in fibroblasts.

Moreover, asiaticoside has an anti-inflammatory property, probably due to the inhibition of nitric oxide (NO) synthesis, thereby facilitating scarring of ulcers, as well as an anti-itching and anti-inflammatory activity.

The present composition also comprises Hamamelis virginiana, in particular in the form of a hydroglyceric extract. The main chemical constituents of the dry extract of Hamamelis virginiana are tannins, gallotannins and hamamelis tannins (2% to 10%), and triterpenes, diterpenes and flavonoids (quercetin and kaempferol) are also present.

The properties are mainly astringent, but also flebotonic and analgesic, which makes said extract particularly suited for treating hemorrhoids.

The biological effects of tannins are connected to their ability to form complexes with macromolecules, in particular proteins, thanks to the affinity of tannins for proline amino acid residues.

When applied topically, tannins contained in the Hamamelis virginiana extract impermeabilize the most superficial layers of the skin and mucosae, thereby promoting regeneration of the damaged tissues, and have an important vasoconstrictor action on the superficial vessels of the skin.

The astringent and anti-inflammatory properties of the Hamamelis virginiana extract are due to the inhibition of the enzymes responsible for the degradation of connective tissue; in fact, vascular integrity is impaired by the increase of the activity of these enzymes. The anti inflammatory action, in particular, is due to proanthocyanidins, such as catechin and epicatechin. Moreover, the active compounds of said extract inhibit self- association of ascorbic acid, increase capillary resistance, reduce capillary permeability, increase venous-lymphatic tone and stabilize collagen.

The present composition also comprise honey, preferably acacia honey. This ingredient has a number of biological activities which are extremely useful for treating proctological disorders, including for example an important action of tissue hydration, as well as an action as a barrier for protecting a superficial wound (for example, fissures), as shown in detail in the Examples.

Moreover, honey is able to adjust pH, increase nitric oxide production, exert an antioxidant action, as well as counteract the establishment and diffusion of foreign microorganisms (for example, bacteria, fungi, molds and viruses), thereby promoting and accelerating the process of scarring.

The present invention also relates to a formulation for topical application, said formulation comprising the above-mentioned composition and one pharmaceutically acceptable excipient, for treating proctological disorders as defined above.

In an embodiment, the formulation of the invention comprises, in percentage by weight of the weight of the total composition, the following active ingredients:

Hyaluronic acid 0.1-2%

Centella asiatica dry extract 0. 1-2%

Hamamelis virginiana hydroglyceric extract 0.01-5%

Acacia honey 0.5-5%

In a further embodiment, the formulation of the invention comprises, in percentage by weight of the weight of the total composition, the following active ingredients:

Hyaluronic acid 0.2%

Centella asiatica dry extract 0.5%

Hamamelis virginiana hydroglyceric extract 2%

Acacia honey 1.5%

Unless specified otherwise, the percentages stated in the present invention should be understood as percentages by weight of the total weight of the formulation of the invention.

The pharmaceutically acceptable excipients which can be used in the formulation according to the present invention comprise: sweet almond oil (prunus dulcis), glycerine, decyl oleate, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, caprylic glycol, hexane- 1,2-diol, cross-linked polyacrylic acid, ethylhexyl glycerol, sorbitan isostearate, polysorbate 60, lecithin, lactic acid, phytic acid, sodium hydroxide, tocopherol, ascorbyl palmitate, citric acid, trisodium disuccinate of ethylenediamine 37%.

The above-mentioned excipients are present in the formulation in an overall amount comprised between about 2% and 10% w/w, preferably between 5% and 8% by weight of the formulation, the rest consisting of water.

Preferably, the present formulation for topical application is in the form of a gel or of an O/W emulsion, more preferably in the form of a hydrophilic gel, even more preferably having a pH between 7 and 8.

Advantageously, the formulation in the form of hydrophilic gel not only allows to have an easily applicable on the skin product, as it is rapidly absorbed, but also it allows an application that is not greasy, since it is a water-based formulation.

The present formulation may also be advantageously formulated as a cream for topical application.

Thanks to the presence of the above-mentioned natural active ingredients contained therein, the formulation of the invention is able to assist the treatment of the symptoms associated with proctological disorders, including hemorrhoids, proctitis and anal fissures, significantly reducing burning, dryness, rubbing-induced pain, improving the elasticity and tone of the tissues and promoting the restoration of a balanced and lubricated environment.

Besides exert the regenerative actions of the skin, the formulation of the invention also allows to exert an improved lenitive and anti-inflammatory action, associated with a very low cytotoxicity, thanks to the combination of the natural active ingredients, as described above in detail and as reported in the experimental section herein.

It should also be noted that, besides exert the regenerative actions of the skin, the formulation of the invention also allows to exert an improved lenitive and anti-inflammatory action, associated with a low cytotoxicity, thanks to the combination of its natural active ingredients, as described above.

Furthermore, the present composition is also convenient due to the ease of preparation.

In this regard, it constitutes a further aspect of the invention a process for the preparation of the above-mentioned formulation for topical use as described in detail above, said process comprising the preparation of an aqueous phase comprising hyaluronic acid, acacia honey and Centella asiatica extract, followed by subsequent addition of the Hamamelis virginiana hydroglyceric extract to said aqueous phase.

The invention will now be described with the following experimental part, intended to better understand the content, without however limiting the scope.

EXAMPLE 1

A detailed example of a formulation according to the present invention is provided below:

EXAMPLE 2 - Comparative test on the effect of human fibroblast migration ("scratch test”) between a composition according to the present invention and the product “CELEVIS GEL”. The test was performed in vitro, using a dermal human fibroblasts primary culture, cultured in DMEM medium (supplemented with 10% fetal bovine serum (FBS), 2mM glutamine, penicillin (100 IU/ml) and streptomycin (100 pg/ml)) in an incubator at 37°C and in 5% CO2 atmosphere. Materials and methods

The following samples were prepared:

LI: commercial product “CELEVIS GEL” containing, as active ingredients, hyaluronic acid (0.2%), colostrum (1.5%), Centella asiatica extract (0.5%), Hamamelis virginiana extract (2%). - L2: composition according to the invention, consisting of hyaluronic acid (0.2%), acacia honey (1.5%), witch hazel extract (2%), and Centella asiatica dry extract (0.5%); L3: composition consisting of the following excipients, as defined in Example 1: SEPINOV EMTIO (3 %), glycerine 99.8% PF (2%), SENSIVA (0.3%), SYMDIOL68 (1%), NATRLQUEST E30 (0.4%), lactic acid (0.01%), purified water (q.s. to 100).

L4: combination hyaluronic acid (0.2%) and acacia honey (1.5%) in aqueous solution of lactic acid (100 mM);

Control: growth medium DMEM as defined above.

The above percentages of the single ingredients are understood as meaning by weight of the total weight of the respective samples.

The above-mentioned thus composed samples are the starting samples from which the dilutions used for the present test were obtained.

In particular, to obtain said dilutions, 5 g of each starting sample were weighed, subsequently dissolved in 10 ml PBS at 4°C under stirring (in the L2 starting sample, a basic aqueous solution (10 mM NaOH) was also added to facilitate sample solubilization), thereby obtaining the corresponding stock solutions.

In particular, the thus-obtained solutions of LI, L2 and L4 contained a final concentration of 1 mg/ml hyaluronic acid.

Preparation of the incubation medium

For each solution of LI, L2, L3 and L4, 6 wells (of 2 ml volume each) were prepared to make two distinct series of experiments, which differ in the percentage of hyaluronic acid (HA) with which the culture medium is enriched.

In particular, in the cell culture plates to be treated with samples LI, L2, L4, two known volumes of the stock solution of each sample were separately added to the conventional culture medium, carrying out a 1: 100 dilution and a 1: 10 dilution of said solutions, thus obtaining the following two series of incubation medium: (a) First series: incubation medium at 10 pg/ml hyaluronic acid (corresponding to 5 mg/ml starting sample, and to 10 mg starting sample per well);

(b) Second series: incubation medium at 100 pg/ml hyaluronic acid (corresponding to 50 mg/ml starting sample and 100 mg starting sample per well).

In the cell culture plates to be treated with L3, two known volumes of the L3 stock solution were separately added to the conventional culture medium, carrying out a 1: 10 dilution and a 1: 100 dilution of said solution.

“Scratch test”

A scratch was realized with a sterile tip on the confluent fibroblast monolayer (time TO), after which the following were performed:

- a first experiment, wherein the cells in culture were treated with the first series of incubation media as defined above, for each sample respectively (LI, L2, L3, L4 and control);

- a second experiment, wherein the cells in culture were treated with the second series of incubation media as defined above, for each sample (LI, L2, L3, L4 and control);

After 24 hours (T24), the cells were photographed, calculating the percentage of cut scarring on the cell monolayer using the software Image J. The extent of the closing of the scratch of the cell monolayer is expressed as a percentage with respect to TO of the same culture.

Statistical analysis of the data was performed with the program GraphPad.

Results and comments

The results are shown in the following table:

Generally, in the presence of a scratch, the fibroblasts undergo cell migration until they are all confluent, to close the lesion formed on the cell layer, thereby simulating the process of epithelium scarring. The control sample was thus considered as an ideal reference parameter, because, consisting only of culture medium and 10% fetal serum, it stimulated cell growth (and thus promoted the scratch scarring), without ever causing inhibition of the cells.

From the results reported in the above-mentioned table, the following can be observed.

Sample L2 did not show any cytotoxic behavior in vitro at both tested dosages (10 mg/ well and 100 mg/ well) in the two series of experiments.

On the contrary, sample LI and sample L3 showed an inhibitory action on cell migration with the increase in dose; in particular, this behavior is more evident for sample LI, probably because of the hyper- stimulating action of colostrum, very rich in growth factors, which action becomes a cytotoxic-like effect on the cells. This result shows that the composition of the present invention, which contains the same active ingredients in the same quantities as sample L2, has a scarring effect considerably higher than that of the composition contained in the known commercial product “CELEVIS GEL”.

Moreover, it was observed that the effect of sample L4 was greater than that of sample LI, meaning that the combination of hyaluronic acid and honey is more stable and more effective than the combination of hyaluronic acid and colostrum contained in sample LI.

Finally, it was observed that the efficacy of the combination of hyaluronic acid and honey is enhanced in the presence of the Centella asiatica extract and the Hamamelis virginiana extract, as can be shown from the comparison of the results obtained from samples L2 and L4.

EXAMPLE 3 - Comparative tests between the combination of hyaluronic acid and honey, and the combination of hyaluronic acid and colostrum.

In the light of the above-mentioned better scarring effect of the combination of hyaluronic acid and honey compared to the combination of hyaluronic acid and colostrum, on which the commercial product “CELEVIS GEL” is based, the Applicant performed a series of comparative tests to establish whether the combination of hyaluronic acid and honey exhibited further better therapeutic properties compared to the above- mentioned known product.

The comparative tests performed were the following:

Test of viability of human keratinocytes, and Antioxidant-activity test.

For both tests, the following samples were assayed: a) Formula A containing honey (1.5%), hyaluronic acid (0.2%) b) Formula B containing colostrum (1.5%), hyaluronic acid (0.2%) Test of viability of human keratinocytes

The test was performed on an immortalized cell line of human keratinocytes (HaCaT), under two different conditions: a basal condition and a stressful condition, wherein the keratinocytes were exposed to 3mM methylprednisolone (MET), an antiproliferative agent.

Methylprednisolone is able to inhibit cell proliferation and can thus create a condition where cell turnover is impaired, mimicking a difficult- to-heal wound condition.

The test was performed at three different times (3 hours, 6 hours and 24 hours), using different concentrations of the studied samples in the range comprised between 0.1 mg/ml and 100 mg/ml.

Method

Cell Counting Kit-8 (Sigma Aldrich) was used for cell viability evaluation.

Briefly, cells were incubated in the presence of WST-8 (2 —(2 methoxy 4 nitrophenyl) 3 —(4 nitrophenyl) 5 —(2,4 disulfophenyl) 2H tetrazolium, monosodium salt), which was reduced by cellular dehydrogenase to the orange formazan product that is soluble in in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells.

Results

Results at 24 hours under basal condition (Figure 1A) show that Formulation A, at the concentration of 1000 mg/ml, significantly improves cell viability by about 50% compared to control (p<0.01 vs control), while Formulation B did not give any statistically significant results with respect to the control.

Results at 24 hours under stressful conditions (Figure IB) show that Formulation A is surprisingly effective in counteracting cell metabolic dysregulation, proving to be better than Formulation B at the concentrations of 10 mg/ml, 100 mg/ml and 1000 mg/ml.

The results therefore showed that Formulation A, based on honey and hyaluronic acid, such as the composition of the present invention, showed the ability to improve cell viability in a time period of 24 hours, and also showed to be able to counteract the anti-proliferative effects induced by methylprednisolone, demonstrating an immunomodulatory capacity even at low concentration better than that showed by Formulation B, based on colostrum and hyaluronic acid, such as the product “CELEVIS GEL”.

Antioxidant-activity test

The antioxidant-activity test using the DPPH radical is one of the fastest techniques for evaluating the antioxidant activity of a substance, and allows to measure the reducing activity of antioxidant molecules against DPPH, which is characterized by a purple red color and decolourises in the presence of an antioxidant agent.

Method

Formulation A and formulation B were tested in seven 1:2 serial dilutions in water, using ascorbic acid (gradual concentrations between 25 and 0.78 pg/ml), a known antioxidant agent, as a positive control.

All the samples were mixed with a DPPH (1, 1 -diphenyl-2 -picrylhydrazyl) solution (1: 19), transferred into cuvettes (optical path 1 cm) and incubated for 30 minutes at room temperature in the dark.

The inhibition of DPPH was calculated according to the following formula:

% inhibition = [(Abs_c-Abs_e)/Abs_c] x 100 wherein: - Abs_c is the absorbance of the positive control determined at 517 nm

- Abs_e is the absorbance of the tested samples determined at 517 nm Results

Both formulations A and B showed maximum antioxidant activity at the concentration of 10 mg/ ml, according to the values showed in the Table below.

Formulation A showed to have a higher antioxidant activity than Formulation B; this is probably due to the fact that the honey contained in Formulation A contains polyphenols and other secondary antioxidant metabolites.

The comparative tests illustrated in this Example show that the combination of honey and hyaluronic acid, on which the composition of the present invention is based, is more effective than the composition of colostrum and hyaluronic acid, on which the product “CELEVIS GEL” is based, in promoting cell viability over time, also more effectively counteracting the reduction of cell viability in keratinocytes treated with methylprednisolone .

Moreover, the combination of hyaluronic acid and honey showed better antioxidant properties than the combination of colostrum and hyaluronic acid.

In the light of this, the combination of hyaluronic acid and honey, on which the composition according to the present invention is based, proves to be highly effective in the treatment of difficult-to-heal wounds, where cell turnover and local immunity are impaired, which are typical conditions of proctological disorders.

EXAMPLE 4 - Hydrating properties of honey The study described below evaluated the ability of acacia honey to facilitate and preserve tissue hydration.

The acacia honey tested was purchased from Apicoltura Vangelisti s.r.l. (Miele Vangelisti).

The experimental model was an intestinal epithelium (colon) reconstructed in mtro, starting from human cells, which were experimentally subjected to a dehydration condition by exposing the cells to laminar air flow (under hood) for 6 hours.

The marker relating to the hydration state was the Aquaporin 3 protein (AQP3), a channel protein which facilitates the rapid flow of water molecules in tissues. Under the experimental conditions, the expression of this protein was induced by a stressful condition of dehydration to facilitate the flow of water between the intra- and extracellular compartments and counteract tissue hydration.

The biochemical tests related to tissue dehydration assessments were followed by histological analysis at the end of the treatment with honey .

The study (performed in triplicate) considered:

- tissues of negative control (CTR-, i.e. non-altered intestinal epithelium)

- tissues of positive control (CTR+, i.e. experimentally dehydrated intestinal epithelium) - treated tissues (i.e. intestinal epithelium contextually treated with 1.5% acacia honey and subjected to dehydration). The results obtained were subjected to statistical analysis according to t- test: variations relative to CTR- are considered significant for *p<0.05, variations relative to CTR+ are considered significant for °p<0.05.

The results, showed in Figure 2, relate to the concentration of Aquaporin 3 determined in the homogenate of the epithelium of the positive control, the negative control and the sample treated with acacia honey. Statistical analysis showed a significant increase in the expression of AQP3 compared to CTR- under all the experimental conditions, except in the cells previously treated with 1.5% acacia honey (t=3 hours).

This indicates that the treatment with acacia honey of the human intestinal epithelium (colon) reconstructed in vitro preserved the tissue from induced dehydration and the associated cell degeneration for 3 hours; moreover, said treatment limited the effects of the induced dehydration for 6 hours.

EXAMPLE 5 - Protective properties of honey

The study described below evaluated the “barrier effect” of the honey used in Example 4 by evaluating its ability to protect the intestinal epithelium reconstructed in tro from the action of an irritating agent, in particular a solution of 1% SDS (sodium dodecyl sulphate).

In fact, this solution is able to induce a significant damage to the tissue, inducing a high cell mortality.

The study (performed in triplicate) considered:

- tissue of negative control (CTR-, i.e. tissue not exposed to SDS)

- tissue of positive control (CTR+, i.e. tissue exposed to SDS)

- treated tissue (i.e. tissue treated with 1% SDS and previously treated with 1.5% acacia honey).

The obtained results were subjected to statistical analysis according to t- test: variations relative to CTR- are considered significant for *p<0.05, variations relative to CTR+ are considered significant for °p<0.05.

The results shown in Figure 3 showed a significant reduction in the vitality of the positive control, i.e. the tissue exposed to the irritating agent and not treated with acacia honey.

Instead, the tissue on which a homogeneous layer of honey was applied showed a better cell viability compared to the positive control, meaning that honey has an important tissue protection activity against irritating agents, highlighting a significant “barrier effect”.

EXAMPLE 6 - Method for producing the formulation according to the invention

In a turboemulsifier provided with a system of external and internal blades, 81 g purified water were heated to 75°C.

After activating the external blades at a speed of 20 rpm, the internal blades at a speed of 40 rpm, setting up a mixing speed at 1400 rpm, 2 g vegetable glycerin, 1.5 g acacia honey, 10 g Sepinov EMTIO and 0.2 g hyaluronic acid sodic salt were added.

Once homogeneity was reached, the thus-obtained aqueous phase was cooled to 25°C.

Separately, in another turboemulsifier, 8 g water were heated to 40°C.

After activating the external blades of the turboemulsifier at a speed of 20 rpm, the internal blades at a speed of 40 rpm, setting up a mixing speed at 1400 rpm, 0.5 g Centella asiatica dry extract were added. After 40 minutes mixing, once homogeneity was reached, the aqueous phase so obtained was cooled to 25°C.

The aqueous phase containing the Centella asiatica extract was added to the above-mentioned aqueous phase containing hyaluronic acid and acacia honey, mixing for 30 minutes till homogeneity. Subsequently, 1 g SYMDIOL, 0.3 g SENSIVA SC 50, 0.4 g NATRLQUEST E30 and 2 g Hamamelis virginiana hydroglyceric extract were added.

To control if the pH value of the formulation so obtained was comprised between 7 and 7.8, a sample was taken and its pH was measured using a pH meter. When the pH needed to be adjusted, a (80 %) lactic acid solution was added to the formulation till the desired pH value was obtained.

BIBLIOGRAPHY

Alternative medicine review volume 12, number 1, 2007.

Community herbal monograph in Hamamelis Virginiana L., Folium, 2008.

Douglas et al., 2001. Hemorrhoids and Varicose Veins: A Review of Treatment Options. Alternative medicine review, vol.6, number 2, 2001.

Garni, 2011. Hemorrhoids a common ailment among adults, causes and treatment: a review. International journal of pharmacy and pharmaceutical Sciences. Vol.3, Suppl.5.

F. Guarniero, G. Sansonetti et al., 1986. Giorn. It. Angiol- vol.VI n. 1/86.

Frenkel et al., The role of hyaluronan in wound healing. Int Wound J. 2012.

George et al., 2009. Antiallergic, antiprurito, and anti-inilammatory activities of Centella asiatica extracts. Afr. J. Trad. CAM 6 (4): 554-559.

H.J. Gohil, Pharmacological review on Centella asiatica: a potential herbal cure-all. Indian J pharm Sci. 72 (5): 546-556.

Jamil et al., 2007. Centella asiatica (linn.) Urban a review. Natural product radiance, vol. 6(2) pp. 158- 170.

Joksimovic et al., Efficacy and tolerability of hyaluronic acid, tea tree oil and methylsulfonyl-methane in a new gel medical device for treatment of haemorrhoids in a double-blind, placebo-controlled clinical trial. Updates Surg. 2012;64(3): 195-201.

Maharjan et al., High and Low Molecular Weight Hyaluronic Acid Differentially Regulate Human Fibrocyte Differentiation. October 2011 | Volume 6 | Issue 10.

Odukoya et al., 2009. Hemorrhoid Therapy with medicinal plants: astringency and inhibition of lipid peroxidation as key factors. International journal of biological chemistry.

Schouten et al. Ischaemic nature of anal fissure. Br J Surg 1996;83:63- 5 mentioned in the document “La patologia anorettale non neoplastica al primo livello di cura Parte 2: la ragade anale” Alberto Bozzani Area Gastroenterologica SIMG.

Stefanelli et al., Can we decrease the acute proctitis in prostate cancer patients using hyaluronic acid during radiation therapy: a prospective historically controlled clinical study. Eur Rev Med Pharmacol Sci. 2012 May; 16(5):639-45.

Vazquez JR et al., Outcomes of hyaluronan therapy in diabetic foot wounds. Diabetes Res ClinPract. 2003; 59(2): 123-7.

Waili et al., Honey for Wound Healing, Ulcers, and Burns; Data Supporting Its Use in Clinical Practice. Review The Scientific World JOURNAL (2011) 11, 766-787.

Weindl et al. Hyaluronic acid in the treatment and prevention of skin diseases: molecular biological, pharmaceutical and clinical aspect. Skin Pharmacol Physiol. 2004; 17:207-213.

WHO Monography: Herba Centellae. Wolff, M. Kieser, 2007. Hamamelis in children with skin disorder and skin injuries: results of an observational study. Eur J Pediatr 166:943- 948.

Claims

1. A composition for topical application, comprising hyaluronic acid or a pharmaceutically acceptable salt thereof, honey, Centella asiatica and Hamamelis virginiana, for use in treating proctological disorders.

2. The composition for the use according to claim 1, wherein said hyaluronic acid has a molecular weight comprised between 1300 kDa and 2200 kDa, preferably comprised between 1500 kDa and 2000 kDa.

3. The composition for the use according to claim 1 or 2, wherein said Hamamelis virginiana is in the form of a hydroglyceric extract containing 1-5% Hamamelis virginiana dry extract by weight of the total weight of said hydroglyceric extract.

4. The composition for the use according to any one of claims 1 to 3, wherein said Centella asiatica is in the form of a dry extract.

5. The composition for the use according to any one of claims 1 to 4, wherein said honey is selected from the group comprising acacia honey, citrus honey, orange honey, wildflower honey, preferably acacia honey.

6. A pharmaceutical formulation for topical application, comprising a composition for the use according to any one of claims 1 to 5, and at least one pharmaceutically acceptable excipient, for use in treating proctological disorders.

7. The pharmaceutical formulation for the use according to claim 6, wherein said hyaluronic acid or a pharmaceutically acceptable salt thereof is present in a quantity comprised between 0.1% and 2%, preferably between 0.2% and 0.5%, by weight of the total weight of said formulation.

8. The pharmaceutical formulation for the use according to claim 6 or 7, wherein said dry extract of Centella asiatica is present in a quantity comprised between 0.1% and 2%, preferably between 0.2% and 0.5%, by weight of the total weight of said formulation.

9. The pharmaceutical formulation for the use according to any one of claims 6-8, wherein said honey is present in a quantity comprised between 0.5% and 5%, preferably between 1% and 3%, by weight of the total weight of said formulation.

10. The pharmaceutical formulation for the use according to any one of claims 6-9, wherein said Hamamelis virginiana hydroglyceric extract is present in a quantity comprised between 0.01% and 5%, preferably between 0.5% and 3%, by weight of the total weight of said formulation.

11. The pharmaceutical formulation for the use according to any one of claims 6-10, wherein said at least one pharmaceutically acceptable excipient is selected from the group comprising sweet almond oil, glycerine, decyl oleate, hydroxyethyl acrylate/ sodium acryloyldimethyl taurate copolymer, caprylic glycol, hexane- 1,2-diol, cross-linked polyacrylic acid, ethylhexyl glycerol, sorbitan isostearate, polysorbate 60, lecithin, lactic acid, phytic acid, sodium hydroxide, tocopherol, ascorbyl palmitate, citric acid, trisodium disuccinate of ethylenediamine 37%.

12. The pharmaceutical formulation for the use according to claim 11, wherein said at least one pharmaceutically acceptable excipient is present in a quantity comprised between 2% and 10%, preferably between 5% and 8%, by weight of the total weight of said formulation.

13. The pharmaceutical formulation for the use according to any one of claims 6-12, characterized by being in the form of a gel or a O/W emulsion, preferably a hydrophilic gel.