WO2013171697A1

WO2013171697A1 - Oral composition comprising a low- availability active ingredient, for use as a medicament or a dietary/supplement/nutraceutical

Info

Publication number: WO2013171697A1
Application number: PCT/IB2013/053981
Authority: WO
Inventors: Andrea Fratter
Original assignee: Labomar S.R.L.
Priority date: 2012-05-16
Filing date: 2013-05-15
Publication date: 2013-11-21
Also published as: EP2849731B1; ITMI20120847A1; EP2849731A1; EP2849732A1; WO2013171270A1; ES2621235T3

Abstract

Oral compositions, having a low-bioavailability active ingredient, comprising a) the above-mentioned low-bioavailability active ingredient, b) a chitosan salt salified with N-acetylcysteine (NAC), and c) polysorbate, for use as a medicament or a dietary supplement/nutraceutical.

Description

"ORAL COMPOSITION COMPRISING A LOW-AVAILABILITY ACTIVE INGREDIENT, FOR USE AS A MEDICAMENT OR A DIETARY SUPPLEMENT/NUTRACEUTIC AL . "

FIELD OF THE INVENTION

The present invention relates to an oral composition, for use as medicament, a dietary supplement/nutraceutical , comprising a low-availability active ingredient.

STATE OF THE ART

Many substances, which are used in the pharmaceutical, nutraceutical formulations, and in the nutritional supplements show a reduced or poor bioavailability due to the failed enteric absorption and the metabolization or chemical denaturation that may occur in the gastro-enteric lumen, such as in liver cytochromes. Due to these physiological phenomena, the nutritional supplements and the pharmaceutical and nutraceutical formulations are often completely inefficient or markedly less efficient than what expected from in vitro data. Examples of such substances with a low bioavailability are generally vitamins, polyphenols, steroidal terpenes, non-steroidal terpenes, fatty acids, and coenzymes.

The absorption of hydrophilic substances through the intestinal mucous membrane is strongly limited by biophysical and biochemical factors that result in a reduced bioavailability.

Substances having a weak acid character, a lipophilic behavior and with a low molecular weight (<1000 Daltons) are absorbed even at the level of the gastric mucous membrane (acetylsalicylic acid) through active trans-cellular transport or aided diffusion mechanisms. Vice versa, those substances having a weak base character are ionized in the stomach, where the pH is of about 1.5, therefore they are not able to be absorbed through the gastric mucous membrane. Such weakly alkaline substances arrive to the ileum and the duodenum, where they are absorbed by virtue of a wider contact surface with the intestinal mucous membrane, and to the higher permeability of the cell membranes. The substances having a marked lipophilic character, such as liposoluble vitamins (A, D, AND, K, F), are assimilated only after being emulsified by bile salts, promoting the formation of micelles that facilitate the contact between the lipophilic substances and the surface of the intestinal villi. At the gastro-intestinal mucous membrane level, the main mechanisms that slow or compromise the trans-wall penetration of substances taken orally are: the thickness of the mucous secretions that may entrap the active ingredient and make it not available to the penetration;

the metabolization due to the CYP3A enzyme (present in the enterocyte of the villi);

- the extrusion by P-glycoprotein (present in the enterocyte of the villi);

the peptide nature of the substances (gastric acid-proteolytic hydrolysis); the reduced trans- wall flow by enteric tight junctions.

In fact, the presence of the anatomic barrier represented by the Tight Junctions, protein structures in the form of plaques, that keep the contact among epithelial cells, thus promoting an anatomic continuity of the epithelium and a reduced trans-wall flow of the exogenous substances therethrough, is extremely important to understand the mechanisms of enteric absorption for the active ingredients.

A possibility for the administration to a subject of substances that are poorly absorbed at the intestinal level is to formulate them through nano-emulsions (e.g., patent application IT2008VE00055) or via a trans-lingual absorption (e.g., patent application WO201 1161501).

However, for some substances and some therapeutic indications, it would be preferred to obtain an enteric release and absorption.

The substances of particular interest are curcumin, a polyphenol extracted from the of Curcuma longa root, and berberin, an isoquinoline alkaloid extracted from Berberis, which have shown interesting and well-documented anti-inflammatory and immune-modulant properties in the treatment of autoimmune inflammatory-related diseases, such as arthritis and colites.

Hornbach et al. disclose enteric absorption studies, for a possible use of an active ingredient such as tobramycin sulfate, i.e., an active ingredient in salt, therefore water-soluble, form, in oral formulations. On the other hand these studies, carried out on rat intestinal mucosae, provide for thiolated chitosan as an agent capable of enhancing mucosal adhesion. This thiolated chitosan is a compound obtained by formation of a covalent amide bond between the amine groups of the chitosan and the N-acetylcysteine carboxyl. According to what has been set forth in this article, also the presence of a further sulphurized derivative, i.e., glutathione, seems to be indispensable to obtain substantial results as regards the enteric mucous adhesion. The object of the present invention is, in particular, to provide formulations capable of promoting the inlet into the circulation of low-bioavailability active ingredients. Further object of the present invention is to provide formulations that:

I) are capable of releasing active ingredients with low or no bioavailability into the intestinal mucous membrane, such as in particular, curcumin and berberin, by modulating the main biophysical and biochemical mechanisms that degrade said substances or that oppose their crossing of the intestinal epithelium;

or

II) are capable of increasing the oral and lingual mucous permeability, to promote the inlet into the circulation of low-bioavailability active ingredients.

SUMMARY OF THE INVENTION

The Applicant has surprisingly found oral compositions, comprising

a) a low-bioavailability active ingredient,

b) a chitosan salt salified with N-acetylcysteine (NAC), and

c) a polysorbate.

The Applicant has surprisingly found that these compositions, by virtue of the concomitant presence of the components (b) and (c), are capable of promoting the inlet into the circulation of low-bioavailability active ingredients.

The Applicant has surprisingly found that, by virtue of the presence of the components (b) and (c), reduce the intestinal mechanisms opposing the absorption, such as cytochromes, PgP, and mucus viscosity, improving the inputting into the circulation of the active substances.

Therefore, preferred embodiments of the compositions that are the object of the invention are, in particular, compositions with oral mucosal release containing the above-mentioned components (a)-(c).

Further preferred embodiments are those having an intestinal mucosal release, comprising in particular a core (A) containing the above-mentioned components (a)- (c) on which a gastro resistant coating (B) is applied.

The oral compositions that are the object of the present invention may be employed in the preparation of medicaments, of dietary supplements/or nutraceutical formulations.

DETAILED DESCRIPTION OF THE INVENTION.

For the objects of the present invention, by the term "oral mucous membranes" are meant both the mucous membrane of the oral cavity and the sub-lingual mucous membrane.

Within the scope of the present invention, by "low-availability active ingredient" is meant an active ingredient with a bioavailability less than 30%, preferably less than 20%, still more preferably less than 10% or, according to a particularly preferred embodiment, less than 5% to the total weight of the active ingredient administered. The pharmaceutical active ingredients that are fundamental components of medicaments such as, for example anti-retroviral drugs, oligopeptide-based drugs, such as, for example: glutathione, hormonal or hormone-like substances, antiinflammatory analgesic drugs of the NSAID type, such as Ibuprofen, Diclofenac, Aceclofenac, Ketoprofen, etc.), drugs for pain therapy, drugs or active ingredients capable of opposing the toxic/inflammatory effects caused by anti-cancer drugs; finally, active ingredients for nutraceutical formulation/dietary supplements, such as, for example: Melatonin, Polydatin, Co-enzyme Q10, polyphenols from a number plant sources (polyphenols from green tea, Vitis vinifera, etc.), Trans-Resveratrol, Resveratrol, Polydatin, Co-enzyme Q10, Isoflavones such as, in particular, soja Isoflavones, Liposoluble vitamins A, D, for example, Vitamin D3, E, F, K such as, for example, Vitamin K2, Curcumin, Glutathione, alpha- and beta-keto-boswellic acids, amino acids such as L-Tryptophan, 5-Hydroxytryptophan, L-Glycine, Inositol, belong to this group.

For the objects of the present invention, by the term "supplements" are meant specific products intended to promote the intake of determined active ingredients in those cases in which the body needs them, or in the presence of an incorrect diet. For the objects of the present invention, by the term "medicament" is meant a formulation containing a pharmaceutical active ingredient for treatment and/or prevention of a disease in mammals, and still more preferably in humans.

For the objects of the present invention, by the term "nutraceuticals" are meant those formulations having proven beneficial characteristics as regards the health status of the subject, and containing active ingredients generally contained at lower concentrations in foods.

It has been surprisingly found that the formulations containing the components a)-c) according to the invention allow the release of the active ingredient and the absorption by the mucous membranes, in particular by the oral cavity, or by the intestinal mucous membranes of active ingredients that are normally poorly bioavailable.

Without wishing to be bound by theory, it is believed that the advantages of the composition of the present invention are at least partially related to the presence of a chitosan salt, which per se exerts a modulation action of the tight junctions of the mucosae epithelium, in particular, the oral and intestinal mucous membranes, with a substance, N-acetyl cysteine (NAC), having a mucolytic action.

The chitosan is a polysaccharide with a molecular weight exceeding 5000 D (Daltons, or molecular mass units, formed by repeating units of N-acetylglucosamine and D-glucosamine), For the objects of the present invention, chitosan preferably has a molecular weight exceeding 100,000 D and a de-acetylation degree above 90%, and can form macro-colloidal aqueous gels after acidification. Such gelification occurs following a protonation of the free amine groups of N-glucosamine, followed by the formation of an ammonium polycation; this process of polysalification and poly-cationization determines the chitosan hydrosolubility and its relative gelling ability.

NAC, having an acid behaviour, protonates the free amine group of the chitosan, thus forming the salt, in the absence of covalent bonds. The ionized form of chitosan exerts an interaction with the tight junctions that is much greater than that exerted by the non-ionic form.

The advantage in using this salt is precisely making the chitosan cationized, hence water-soluble and capable of acting as a mucolytic on the oral secretions, with a higher efficiency than conjugates (with covalent bonds between NAC and chitosan) of the prior art, such as, for example the one described in the above-mentioned article by Hornbach et al., set forth above.

In the chitosan salt with N-glucosamine in the composition according to the present invention, the weight ratio between Chitosan and NAC can range from 1 :1 to 3: 1, in particular from 2 to 1.

The polysorbate in the composition according to the present invention has an emulsifying action. The presence of an emulsifying agent allows promoting the penetration into the circulation of substances taken by mucosal absorption. Polysorbates, which are the polyoxyethylenated derivatives of sorbitano esters with fatty acids, are surfactants widely used in the formulation of oil-water emulsions, foaming products, and topic forms, since they act as absorption enhancers, by performing a micellization action of the water-insoluble or poorly water-soluble active ingredients, and acting as "cell surfactants", in practice altering the membrane permeability, thus contributing to make the active ingredient more freely available for absorption by the mucous membrane of the oral cavity.

The polysorbate contemplated in the composition that is the object of the present invention can be one of the commercially available polysorbates, such as polysorbate 20, 40, 60, 65, 80, and mixtures thereof.

Preferably, in the composition according to the invention, the polysorbate is polysorbate 80.

The oral mucous membrane release compositions are preferably in the form of adsorbed powders capable of giving rise to translucent aqueous dispersions for addition of water, and in the form of tablets obtained by compression of said adsorbed powders.

By the term adsorbed powders are meant microgranules in which the active ingredient and polysorbate are adsorbed in the chitosan salt and NAC.

These adsorbed powders are preferably administered in the form of an aqueous dispersion at the time of use by addition of water.

The above-mentioned aqueous dispersions must be held within the mouth for some minutes, then they can be swallowed, so as to promote the absorption of the active ingredients at the level of the oral and sub-lingual mucous membrane.

The thus-obtained aqueous dispersions are micellar dispersions that are stable and homogeneous when taken.

These micellar dispersions may also contain high-bioavailability active ingredients dissolved in the aqueous phase, such as water-soluble vitamins, enzymatic co-factors, mineral salts, and water-soluble amino acids. Only the low-bioavailability active ingredients are absorbed by oral mucosae, while the liquid phase containing substances with a normal intestinal absorption, such as water-soluble vitamins, magnesium, zinc, and potassium salts, or other substances with a proven intestinal bioavailability, can then be swallowed.

The oral mucosal compositions of the present invention can optionally contain further additives and/or excipients, such as, for example, polyols, preferably: sorbitol, mannitol, inositol, solvents/diluents such as caprylic/capric acid triglycerides, fillers such as starch and maltodextrins, glidants such as precipitated amorphous silica, flavoring substances and sweeteners, in particular rebaudiosid from Stevia rebaudiana.

This type of excipients has as its main purpose to help the technological manufacturing process, to make the formulation palatable, and to further reduce the possible drying or dehydrating action for the oral cavity of the oral mucosal compositions according to the present invention. The compositions in the form of tablets can optionally also contain agents promoting the disintegration thereof, such as, for example, cellulose derivatives, such as, for example, sodium carboxy methylcellulose, modified starches, polyvinyl pyrrolidone derivatives, for example, cross-linked polyvinyl pyrrolidone.

The oral mucosal release compositions according to the present invention are in particular prepared by a process comprising the following steps:

i) preparing a dispersion of the low-bioavailability active ingredient in polysorbate, optionally mixed with a caprylic/capric triglyceride, said active ingredient and said polysorbate being present in a concentration ranging between 1 and 30% by weight/total weight of the final preparation;

ii) adsorbing such liquid dispersion in a mixture of Chitosan and N-acetylcysteine in such amounts as to confer to the aqueous solution obtained by extemporaneous dispersion of the final powder a pH ranging between 3.5 and 4.5 and in which N- acetyl cysteine and Chitosan are in a concentration ranging between 0.01 and 3% by weight/total weight of the powder to obtain the formulation in the form of adsorbed powder;

iii) combining optional fillers, such as polyols, sugars, or other excipients known for this purpose in addition to sweeteners, flavoring agents, glidants, to obtain the formulation in the form of an adsorbed powder;

iv) compressing the adsorbed powder coming from the steps (ii) or (iii) to obtain the oral composition with oral mucosal release in the form of tablet.

v) optionally, dispersing the adsorbed powder thus obtained in the previous steps in water to obtain the formulation in the form of a translucent aqueous dispersion;

In particular, as detailed before, the oral compositions with release into the intestinal mucous membrane comprise:

A) a core comprising

a) a low-bioavailability active ingredient, b) a polysorbate,

c) and a chitosan salt acidified with N-acetylcysteine;

B) an enteric coating of said core.

Preferably, the core A) of the composition according to the invention further comprises Citrus paradisi extract titrated at at least 40% by weight/total weight of the extracted of Citrus x paradisi in bioflavonoids, and specifically titrated in Naringin and Naringenin, and a Piper nigrum extract titrated at 95% by weight/total weight of the Piper nigrum extract in piperine.

These extracts, in the form of a phytocomplex of grapefruit and black pepper, respectively, modulate the activity of various enzymes responsible for the bioconversion of xenobiotics, and in particular of the hepatic and intestinal cytochromes of the P450 and CYP3 A type.

In fact, it is known that some substances are capable of interacting with the P-gp pump, located in the enterocyte outer membrane by inhibiting it. Such effect results in an increased transit in the entero-portal circulation, and reduced metabolization by the hepatic and intestinal cytochromes, being an indication of a higher efficiency of the active ingredients taken orally. Such action mechanism is exerted in particular by some flavonoids that are contained in the Citrus genus.

These flavonoid complexes are also capable of inhibiting the enterocyte CYP-3A, and in some cases also the hepatic CYP-3A, thus reducing the pre-systemic bioconversion.

It has been found that, also by this mechanism, with the compositions according to the present invention, it is possible to induce an increase in the plasmatic concentrations of substances, that would be otherwise massively bioconverted already before the inlet into the circulation.

By a gastroresistant coating is meant a layer that is capable of protecting the composition against the action of gastric juices in the stomach, and of releasing the active ingredient in the intestine. Preferably, this coating is in the form of a film or of a rigid gastroresistant capsule. In the intestinal mucosal release formulations, it is crucial that the active substances and the substances intended to act as enhancers for the enteric absorption, are released only at the enteric level, but not at the gastric level. The early release of the chitosan into the gastric environment, in fact, would swell the polymer due to the strongly acid environment endowed with the presence of hydrochloric acid, and it would induce the formation of a gel, from which the active ingredients and the other substances could be released into the stomach to be subsequently decomposed.

The composition that is the object of the invention for the intestinal mucosal release is prepared by a process comprising the following steps:

i. preparing an aqueous solution or dispersion comprising at least one polysorbate in a concentration between 1 and 30% by weight/total weight of the solution, and a chitosan salt salified with N-acetylcysteine in such amounts as to confer to the aqueous solution containing 1% w/w Chitosan a pH ranging between 3.5 and 4.0, in which N-acetyl cysteine and chitosan are in a concentration between 0.01 and 3% by weight/total weight of the solution;

ii. wet granulating a composition comprising at least the low-bioavailability active ingredient with the aqueous solution or dispersion obtained in the step i.;

iii. drying up to a constant weight and sieving the granulate obtained in step ii.;

iv. optionally, compressing the granulate obtained in step iii. in the case that the intestinal mucosal release composition is in the form of a tablet;

v. applying the enteric coating of the core a).

When the coating B) is composed of a gastroresistant film step v. encompasses that the core coming from step iii. or iv. undergoes a filming process, for example, in a coating pan, with the dispersion of a substance capable of forming a film resistant to gastric juices in order to obtain a gastric protection. Preferably, the coating B) comprises or is composed of insoluble shellac (Shellac™), polyacrylates with an acid pH, or cellulose acetophthalate.

When the coating is composed of a gastroresistant capsule, the above process contemplates for the application of the gastroresistant capsule on the dried granulate coming from the step (iii).

The intestinal mucosal release compositions, in particular coated with a gastroresistant film, can in turn be in the form of tablets, of a gastroresitant granulate. The latter can be, in its turn, coated with a gastroresistant coating, preferably when the compositions that is the object of the invention are in the form of tablets, which are composed of filmed granules, preferably comprise a layer C) external to the coating B) comprising at least one excipient suitable for the compression. Preferably, said excipient of this external layer is selected from starch, microcrystalline cellulose, magnesium stearate, calcium phosphate, talc, silica, and mixtures thereof. In the composition according to the present invention, the core A), the coating B), and the optional outer layer C) can comprise further excipients and/or active ingredients in addition to those described above.

Non-limiting examples of further excipients are: calcium phosphate bibasic anhydrous, colloidal silica, amorphous silica, sorbitol, starch.

Examples of the preparation of the oral compositions that is the object of the present invention, and moreover the in vitro tests demonstrating the efficiency of the mucosal release compositions according to the present invention are set forth by way of illustrative, non-limiting purposes.

EXAMPLE 1 Oral mucosal release formulations

Some examples of oral mucosal compositions prepared according to the following steps are set forth herein below:

i) preparing a dispersion of the poorly bioavailable active ingredient in polysorbate 80 optionally mixed with a caprylic/capric triglyceride both in a concentration ranging between 1 and 30% by weight/total weight of the final preparation

ii) adsorbing such liquid dispersion in a mixture of Chitosan and N-acetylcysteine in such amounts as to confer a pH ranging between 3.5 and 4.5 to the aqueous solution obtained by extemporaneous redispersion of the final powder , and in which N-acetyl cysteine and Chitosan are in a concentration ranging between 0.01 and 3% by weight/total weight of the powder;

iii) adding fillers, such as polyols, sugars, or other excipients known for this purpose, in addition to sweeteners, flavoring agents, glidants;

iv) dispersing the adsorbed powder thus obtained in the previous steps in water to obtain a translucent aqueous dispersion.

Formulation 1

Melatonin 0.005 g

Polysorbate 80 0.050 g

Caprylic/capric triglyceride 0.050 g

Chitosan (100000 D, DG 95%) 0.050 g

N-Acetylcysteine 0.025 g

tribasic anhydrous Magnesium citrate 2.586 g

tribasic Zinc citrate 0.036 g Amorphous silica 0.075 g

Formulation 2

Melatonin 0.005 g

Polysorbate 80 0.050 g

Caprylic/capric triglyceride 0.050 g

Chitosan (100000 D, DG 95%) 0.050 g

N- Acetylcysteine 0.025 g

Anhydrous.tribasic Magnesium citrate 2.586 g

Zinc citrate 0.036 g

Amorphous silica 0.075 g

Fructose 0.541 g

Sorbitol 0.388 g

Rebaudiosid 98% 0.010 g

Orange flavor 0.125 g

EXAMPLE 2 - Intestinal mucosal release formulations

2.1 -Preparation of the granulate containing the active ingredient

Active ingredient:

Curcuma Longa (Dry extract, 95% Curcumin) 200 mg per tablet

2.2 Step 1

Core of the tablet to be granulated:

Preparing a mixture of dry powders, containing:

Curcuma Longa (D.E. 95% Curcumin) 100 g Chitosan HD 100 Mesh 75.0 g

N-AcetylCysteine 25.0 g

Grapefruit Seeds D.E. (50% BIOFLAVONOIDS) 25.0 g (optional)

Black pepper fruit D.E. (95% Piperine) 2.50 g(optional)

White maize starch 75.0 g

Tot. 302.5 g

2.3 Step 2

Granulating solution obtained as follows: Introducing 120 mL deionized water into a 250 mL flask by reverse osmosis and dispersing 2 grams Chitosan (MW>5000 D, DD>90%) under stirring on a magnetic plate; adding 1.0 g NAC so as to obtain a pH ranging between 3.5 and 4.0 as measured with a pHmeter, at the same time verifying the solubilization of the chitosan and the concomitant gelification. Adding to the thus-obtained solution, 29.0 grams POLYSORBATE 80 up to complete dispersion, thereby obtaining a straw yellow translucent system. Next, diluting up to a 200 mL volume with deionized water (reverse osmosis).

2.4 Step 3

Granulation

Placing the obtained mixture of powders (302.5 g) of step 1 in a plastic beaker and dispersing therein, under constant mechanical stirring the above granulating solution and obtained in Step 2 in successive volumes, while verifying the progressive start of the granulation process (total granulating solution added: 140 grams); once the granulated core is obtained, drying the same in a ventilated oven at 50° C until reaching a constant weight (anhydrification). Sieving the granulate. Finally, a powder weighing 414.5 g is obtained.

2.5 Step 4

Obtainment of the tablets

The granulate thus obtained (248.7 g) is then added with Microcrystalline cellulose (12.3 g), Dibasic Anhydrous Calcium Phosphate (Fujicalin) (60. Og), microcrystalline cellulose (Ceolus KG802) (30.0 g), Magnesium stearate (5.4 g), and Talc (3,6 g). The mixture of powders thus obtained is mixed in a special laboratory V mixer for 2 minutes.

The powder thus obtained in an amount of 359.3 g is tabletted, and the obtained tablets are then coated with a 25% shellac solution (ShellaC™) in a coating pan for a total of 0.06623 g, until obtaining 300 tablets for a final net weight of 360.0 g (300 tablets).

EXAMPLE 3 IN VITRO ABSORPTION OF THE GRANULATE OBTAINED AS DESCRIBED IN THE EXAMPLE 2 (steps 2.1-2.4)

The intestinal absorption is a complex process, which can be divided into a so-called transcellular and paracellular passive transport, and an active transport or transport with facilitated diffusion, which is mediated by specific receptors. The process can be affected by the intestinal metabolism, the action of outflow transporters, as well as by some characteristics of the chemical compound that is to be absorbed, among which the molecular weight, the size and configuration of the molecule, ionization degree, lipophilicity, solubility, and dissolution in the gastric content.

In order to predict the intestinal absorption and the transport through the mucous membrane, an in vitro study model was proposed, using the cell monolayer CaCo-2 and whose protocols are being validated as an alternative to trials on animals at ECVAM (Le Ferrec E. et al., 2001, Ecvam workshop 46).

The good in vivo/in vitro correlation for the passive transport of drugs characterizes the importance and diffusion of the CaCo-2 model when investigating the intestinal absorption.

The CaCo-2 line is widely used as an in vitro intestinal epithelium model for barrier toxicity studies, and for the qualitative screening of intestinal absorption, moreover by the pharmaceutical industry during the research and development steps for the identification and the optimization of lead compounds (Garate M.A. et al., 2000). Cells are cultured on specific porous supports housed in cell colture wells, which allow to have two well-separated compartments: an apical compartment, corresponding to the intestinal lumen in vivo, and a basolateral compartment, corresponding to the compartment of the body blood and lymphatic circulation .

The differentiation occurs within 21 days: differentiated CaCo-2 cells form at this time a continuous epithelium with functional tight junctions and microvilli.

The absorption test on the CaCo-2 model was applied for assessing the intestinal passive transit of water-dispersed Curcuma granulate (batch PGE23) at preset doses based on a preliminary cytotoxicity test.

2. EXPERIMENTAL DESIGN

Before carrying out the permeability assay for the test substance, the cytotoxicity was assessed on the CaCo-2 monolayer by a MTT test (in accordance with the internal procedures, 2 tests carried out with a 2hrs and lhr treatment, respectively) at different concentrations in triplicate to find the non-cytotoxic dose:

1) FIRST TEST : 0.25% / 0.5% / 1% > 2hrs treatment

2) SECOND TEST : 0.025% / 0.05% / 0.1% / 0.25% >lhr treatment The concentration selected for the intestinal absorption of curcumin is 0.25% curcuma granulate containing 0.052% curcumin (1.404 mM) with lhr treatment. The CURCUMA GRANULATE, i.e., the granulate obtained at the end of step 2-4 (step 3) described in the example 2, was tested while being dissolved in the 1% HBSS-MES buffer for the intestinal transit (buffer at a pH of 6.5 used in the apical part).

The transit through a monolayer of CaCo-2 cells of the test substance at a non- cytotoxic concentration was monitored at preset time points (0' - 15'- 30' and 60') - the liquid in the basolateral compartment was collected and the substance of interest (curcumin) was quantified by HPLC.

The following parameters were assessed to verify the monolayer integrity and to measure the changes in permeability occurring after the intestinal transit:

- barrier integrity test, by measuring the trans-epithelial electrical resistance (TEER); paracellular transport with toxicity assessment of the test substance on the integrity of the barrier function by using a marker of the paracellular transport

(Lucifer yellow) using the highest non-cytotoxic concentration in accordance with the MTT test results.

3. MATERIALS

3.1. ASSAY SYSTEM

3.1.1 CACO-2 CELL LINE

This line is composed of cells derived from intestinal cells (human colorectal adenocarcinoma) (ATCC).

CaCo-2 cells grow in a monolayer in DMEM medium containing 4.5 g/L glucose with the addition of 10% FBS, 1% non-essential amino acids, 4mM glutamine, 10 Mm Hepes, and antibiotics (Pen/Strep).

Cells reach confluence within about 6 days, and reach a steady state within 10 days. Differentiation is complete in about 21 days. Differentiated cells have high levels of alkaline phosphatase, isomaltase, and aminopeptidase.

The structural and functional differentiation of microvilli is associated to the monolayer polarization after confluence. Polarity is also apparent from the presence of tight junctions that are formed during differentiation.

The monolayer integrity is confirmed by the low permeability of the monolayer after the confluence. Polarity is also apparent from the presence of tight junctions that are formed during differentiation. The monolayer integrity is confirmed by the low permeability of the monolayer to mannitol or lucifer yellow (Le Ferrec E et al. 2001;

Hidalgo et. al. 1989)

3.2. 1 Equipment

Analytical balance XS 204(Mettler -Toledo)

Microplate autoreader M200-Infinite (Tecan)

Incubator C0₂ HERA CELL (HEREUS)

Microscope DM-THE (Leica)

Burker chamber (Marienfeld)

3.2.2.Reagents

DMEM 12-614F LONZA

GLUT AMINE BE 17-605 E LONZA

PEN/STREP DE 17-602E LONZA

NEAA SOLUTION 100X BE 13-114E LONZA

FBS DE14-701F LONZA

HBSS BE10-527F LONZA

HEPES 1M 17-737F LONZA

MES M2933 SIGMA

LUCIFER YELLOW L0144 SIGMA

HBSS - 1% MES APICAL SOLUTION (Prepared in laboratory and used within a week)

HBSS - 1% HEPES BASOLATERAL SOLUTION (Prepared in laboratory and used within a week)

3.1.2 CULTURE AND USE CONDITIONS OF THE ASSAY SYSTEM

Cells (150,000/insert, counted by a Burker chamber) are plated in inserts with a PET filter (Millipore, 10mm diameter with 0.4 mm pores) for 12-well plates, and are incubated at 37° C with 5% C02 and grown for 21 days. On top of the insert, 0.5mL medium is added, and 1.5 mL medium is added to the plate.

The medium is changed every second day. From day 21, the morphological analysis shows a complete differentiation of CaCo-2 cells.

4. METHODS

4.1 MTT assay: cytotoxicity of the test substances on a Caco-2 line

4.1.1 Principle of the method The potential cytotoxic effect of the substances is assessed on proliferating Caco-2 cells by a MTT assay.

The MTT test is considered as an international standard for quantifying cytotoxicity (ISO 10993-5). This method allows quantifying a cytotoxic effect induced by the product through the measurement of the cell viability. Cytotoxicity is quantified by measuring the decrease in cell viability with respect to an untreated negative control. It is based on the metabolization reaction occurring between the tetrazolium salt (MTT) and mitochondrial enzymes (succinate dehydrogenase) following the contact with the product for the various treatment times: only a viable cell can convert the tetrazolium salt into the insoluble derivative (Formazan), which reaction is characterized by the formation of a violet color at the insert base. The extraction of the violet colored derivative is carried out in isopropanol, and the quantification is carried out spectrophotometrically at 570 nm.

4.1.2 Procedure

The following concentrations were assessed:

3) 0.25%/0.5%/l% (FIRST MTT TEST): 2hrs treatment

4) 0.025%/0.05%/0.1%/0.25% (SECOND MTT TEST): lhr treatment

CaCo-2 cells were plated on 96- well plates (in triplicate) at a cell density of 120,000 cells per well (cell count by a Burker chamber).

After lhr or 2hrs incubation with the substance to be tested, the contents in formazan per well were assessed spectrophotometrically at a wavelength (λ) of 570

nm.

After subtracting the blank, the % viability is calculated with respect to the untreated control according to the formula:

% VIABILITY = (TREATED O.D./ UNTREATED O.D. CONTROL)* 100

4.2 Trans-epithelial electrical resistance (TEER) assay: assessment of the barrier function integrity

4.2.1 Principle of the method

Trans-epithelial electrical resistance (TEER) is a direct measurement of the skin barrier functionality: it reflects the total tissue resistance due to both its thickness and structure. It measures the barrier integrity at the level of the tight junctions.

The value of the monolayer trans-epithelial electrical resistance (TEER), consisting of differentiated CaCo-2 cells, represents an indication of the integrity level of the same monolayer, therefore of the formation of the tight junctions that are present between the enterocytes. The measurement of the trans-epithelial resistance value (expressed in Q*cm ) is carried out by using a volt-ohm-meter (ERS Millicell) (0- 2000Ω range).

The TEER values of the differentiated monolayer range as a function of the cell line, and they are acceptable from 150-200 Ω*αη .

4.2.2 Procedure

On a differentiated monolayer of CaCo-2 cells, after 21 days culture, TEER is assessed by carrying out a measurement of the insert with a special electrode, by positioning the shortest portion of the electrode within the insert in a 0.5 mL medium. The long portion of the electrode is immersed in 1.5 mL medium in the well containing the insert.

4.3 Lucifer yellow assay: assessment of the integrity of the cell junctions in the presence of the substance to be assessed

4.3.1 Principle of the method

Lucifer yellow is a fluorescent marker impermeable to the cell membrane. It is used to investigate the permeability of a substance at the paracellular level.

When junctions are integral, Lucifer yellow has a very low permeability.

4.3.2 Procedure

The transport experiments were carried out on a monolayer of CaCo-2 cells by applying, at the level of the apical compartment of the insert, 100 μη οΙ/L Lucifer yellow (LY) together with the substance to be tested (the highest non-cytotoxic at the MTT test) dissolved in HBSS-1% MES buffer (0.5 mL). At the basolateral level, 1,5 ml HBSS-1% Hepes buffer were added.

LY transport is assessed as the transit from the apical compartment to the basolateral one after a preset incubation period of lhr at 37° C.

Reading is carried out with a spectrofluorimeter (TECAN) with 428 nm excitation and 535nm emission. The measurement of fluorescence (RFU) is carried out at the apical and basolateral level, and the flow rate and permeability of the marker are calculated according to the following formulae:

LY FLOW = (RFU BL/RFU AP) x 100

Apparent permeability (PAPP, cm/sec)=

(BL Concentration / AP Concentration) x (BL Volume/Area x time) In order to get the LY concentration in the basolateral compartment, a standard curve is used, which is obtained starting from a number of LY concentrations in a 96-wells plate (ΙΟΟμΙ in triplicate):

LY: 0 - 6-12μΜ (low concentrations of standard)

LY: 0 - 6- 12-25 - 1 ΟΟμΜ (high concentrations of standard)

In an integral monolayer, the LY flow is less than 10% and the apparent permeability coefficient Papp is less than 2.3* 10-6 cm/sec (internal controls).

4.4 INTESTINAL TRANSIT ASSAY

4.4.1 Principle of the method

CaCo-2 cells grown as a monolayer on an artificial permeable support reproduce the intestinal epithelial layer (monolayer of cells that are polarized and coupled by tight junctions). The flow through the monolayer allows determining a substance permeability.

4.4.2 Procedure

After 21 days growth on an insert, differentiated cells exhibit an apical side, corresponding to the intestinal lumen in vivo, and a basolateral side, corresponding to the compartment of blood and lymphatic circulation in the body.

The culture medium was drawn from all the cell monolayers before carrying out the permeability experiments. All the cell monolayers were washed once with HBSS solution (Hank's Balanced Salt Solution) and let to equilibrate at 37° C for 20 min.

The test substance was prepared extemporaneously in HBSS IX containing MES 1%

(pH 6.5 in order to reflect the low duodenal acidity) at a 0.25% concentration. The solution test was added on the monolayer at the donor side (0.5 mL in triplicate), composed of the apical region. In parallel, the same amount of substance was fractionated, and it represents the sample at the time tempo 0 minutes APICAL (0'

AP).

At the receiver side of the monolayer, 1.2 mL HBSS IX -Hepes 1% (pH7.4) was added. The plates treated in this manner were positioned in an incubator at 37° C for the preset incubation period (15-30-60 minutes).

At each point of the preset kinetics, samples were collected from the receiving side of the monolayers (0.6 mL). The studies were carried out for a period of time of 60 min. Samples (defined as basolateral- BL) were taken at 15, 30, 60 minutes . The volumes of the basolateral compartment were kept constant by restoring HBSS IX after each sampling. At the end of the treatments, the amount of substance that was left at the apical level (T= 60' AP) was recovered, and the cells were solubilized in 0.5 mL bidistilled water, resuspended, and stored for analysis at 4° C and the definition of the Mass balance.

Analysis for curcumin amount was carried out by the HPLC-UV technique.

Apparent permeability coefficients Papp (cm/s) were determined by the following formula:

dQ

Papp

dt x A x Co

- dQ/dt was the slope of the substance permeability profile as a function time (seconds) through the monolayer of CaCo-2 cells (mass transferred per time unit) - A was the insert surface area (1 cm²)

- Co was the initial test concentration of the donor.

With the results obtained from the HPLC analysis, the mass balance calculation is carried out as follows:

% TOTAL MASS BALANCE=(C 60' AP *0.5ml* 100)/(Cd *0.5ml + C 60'BL + C pellet*0.5ml)

- C60' AP substance final concentration found in the apical compartment after 60 minutes;

- C60' BL substance concentration found in the basolateral compartment after 60 minutes;

- Cd initial (donor) concentration applied at the apical level, taking into account the dilution factor in the considered time lapse.

4.4.3 Calculation method

By using the mathematical formulae described by Arturrson and set forth in an Excel sheet, the ratios between the concentrations of substances in the basolateral (receiver) and apical (donor) compartments at each sampling time were calculated.

The values of "cumulative fraction transported drug" (cm) were calculated starting from concentration data at the basolateral level, as found experimentally (Tavelin S.; Le Ferrec E. 2001). The destination of the substance quantified in each compartment is set forth in the following table:

TABLE 1

The cell homogenate and the basolateral compartment contained the curcumin fraction absorbed by passive transport.

5. Statistical analysis

No statistical analysis was performed, except for the standard deviation calculation.

6. RESULTS

6.1 ASSESSMENT OF CYTOTOXICITY OF CURCUMIN GRANULATE AT 3 CONCENTRATIONS - MTT TEST

TABLE 2

The results of the MTT test after a 2-hour incubation with the composition of the core of the invention (obtained as described by carrying out all the process steps 2-1- 2.4, referred to also as CURCUMA GRANULATE ) are set forth in the table above: 0.25%/0.5%/l%

Cytotoxicity of CURCUMA GRANULATE was tested at three different concentrations for a 2hr treatment; in all the three concentrations, the product was cytotoxic, without showing a dose-dependent effect.

Since the intestinal transit also provides for use of the lhr treatment time, it was decided to treat the cells only during lhr with 4 lower concentrations than the above- mentioned CURCUMA GRANULATE. In this case also, borderline viability values were found, but higher than the lhr time and in any case above 50%. The results are set forth in the following table 3 TABLE 3

Based on these data, the highest non-cytotoxic concentration used to assess granulate intestinal transit is 0.25%.

The theoretical curcumin concentration within the granulate tested at 0.25% corresponds to 0.052% =1.404mM.

5.2 PARACELLULAR TRANSIT: ASSESSMENT OF TOXICITY OF THE TIGHT JUNCTIONS BY TEER AND LY

All the inserts prepared for the study showed initial TEER values of about 200 Q.*cm (as set forth in the Table 4 below); therefore, they are valid to assess toxicity of 0.25% CURCUMIN GRANULATE by LY.

The inserts were treated during lhr with CURCUMA GRANULATE/LY; the CN insert (untreated negative control) and the blank insert were treated only with LY.

The results are set forth in the following table 4.

Table 4

No difference in the TEER values was found after a curcuma granulate treatment compared to CN cells in HBSS-1% MES.

The results confirm a neutral action at the level of the structure of the tight junctions. In Table 5, the values of the paracellular Lucifer Yellow flow are set forth, as measured after lhr treatment with 0.25% CURCUMA GRANULATE.

Table 5 CN (negative CURCUMA ACCEPTABILITY control) GRANULATE VALUES

LY flow (%) 1.246±0.021 1.148±0.016 <10%

Papp 1.129* 10^"6 8.040* 10^"6 <2.3* 10^"6 The curcuma granulate at the tested concentration was confirmed as non-toxic for the junctions (Lucifer Yellow flow < 10% and Papp flow <2.3* 10-6 cm/sec ) (Tab. 5). 6.3 INTESTINAL TRANSIT

6.3.1 PERMEABILITY TEST FOR CURCUMIN ASSAYS

The results of the curcumin assay in HPLC-UV are summarized in table 4 set forth below.

Literature data showed that curcumin has a low bioavailability, due to the poor water solubility and a high metabolism (Wahlang B. et al.).

In order to assess linearity and stability of curcumin, the 95% curcuma rhizome was used and tetrahydrofuran (THF) had to be used as a solvent in a 1 :3 ratio with the solvent (1% citric acid), due to the very low solubility of the curcuma, as set forth in the table 6 herein below.

The analytic method used for the standard was precise and accurate for the concentrations used (LOQ 0.05mg/L, LOD 0.017 mg/L), but it did not prove sufficiently accurate to detect curcumin in the only presence of the buffers necessary for the absorption study, this behaviour partially limiting the curcumin solubility. The analytic results detected, in the solution of curcuma granulate 0.25% used for the study, an amount of curcumin that is less than the theoretical amount present in the sample used as set forth in the following table, where a loss of about 23 % in titration is shown.

This loss was to be attributed to the incomplete solubility of curcuma granulate in the buffers required for the study: in fact, the solution was very opaque and tended to precipitate.

Table 6 CURCUMA Theoretical amount Dosed amount of

DRY EXTRACT

of curcumin in the curcumin with a at 95%

curcumin solution 0.25% HPLC-UV

technique in the 0.25% solution

Concentrations 21.93g/100g 0.052%=1.404mM 0.04%=1.080mM The percent concentrations of CURCUMIN dosed in the different compartments analyzed in triplicate (3 inserts) after a treatment with CURCUMA GRANULATE during lh are set forth in the following table 4:

TABLE 7- CURCUMIN

It shall be apparent that the dosage of curcumin in the analyzed biological samples (apical= applied and residual solution after 60 min, basolateral at the preset times, and homogenate) was still and significantly affected by the low solubility of curcumin.

In fact, the % OF recovered values of curcumin in the granulate (from 52% -77%) show that a significant % curcumin was not quantified in the determined experimental conditions, thus not allowing a correct calculation of the final mass balance. However, the analysis of the distribution in the different compartments, while being underestimated due to the low solubility (example: in the insert 1, only 0.01% in the cell homogenate, and 0.01% at the apical level at the end of the incubation period was quantified, applying 0.04% substance as the initial amount), shows at 60 minutes a curcumin distribution that is equivalent to that between the apical compartment and that of the cell homogenate, this fact confirming the internalization thereof at the level of the intestinal epithelium.

On the other hand, no transit of curcumin was shown in the basolateral compartment. 7. CONCLUSION

The study of the intestinal transit in vitro on CaCo-2 cells of CURCUMIN GRANULATE tested at a 0.25% concentration defined based on the preliminary cytotoxicity values was carried out.

Both the data on paracellular transport with Lucifer yellow and the TEER measurement showed that curcumin granulate does not induce toxicity at the level of the intercellular j unctions .

In this study, the mass balance (Mass balance, MB), i.e., the percentage of curcumin recovered in the different compartments at the end of the intestinal transit could not be calculated due to the incomplete solubility of the curcuma granulate in the buffers used for the study.

A loss of 23% in titration in the applied solution was shown, with respect to the theoretical estimate; consequently, such loss can be also applied to the analysis of the samples generated in the study.

Then, the sum of the amount of recovered compound in the two compartments, the apical and the basolateral ones, was calculated, together with the fraction associated to the cell component (homogenate) with respect to the initial really applied amount (51.875% and 76.875% in 1 and 2 inserts, respectively).

In the present study, curcumin, a lipophilic molecule with a tendency to the intracellular accumulation thereof, showed a low permeability with a Papp (A-B) of: 8.040* 10^"7

These data are confirmed in literature (Wahlang B 2011), in fact, no curcumin transit was noted in the basolateral compartment.

However, the analysis of the distribution in the different compartments, although it is underestimated due to the low solubility of the curcumin in the buffers, shows already at 60 minutes a distribution of the curcumin equivalent between the apical compartment and the cell homogenate, this fact confirming the internalization thereof at the level of the intestinal epithelium.

Since the curcumin absorption, which forms an opaque solution containing particulate in water, is controlled by two steps: disintegration/dissolution and absorption through the mucosa, it is possible to hypothesize a quicker internalization of the granulated form if compared to other administration forms.

For curcumin, a lipophilic substance, the factor limiting the internalization thereof is, in fact, the solubility thereof in the aqueous solutions: the molecule, when rendered available, may internalize and build up more quickly at the level of the cell monolayer.

To support this behaviour, in literature it was shown , by using lysed cells, that 1 1.78% curcumin was metabolized during transport and accumulated in the cells (by kinetic studies on curcumin absorption and release). The accumulated amount was 20%. This datum can be compared with the accumulation results observed in the cell homogenate: 25% (on 2 inserts) and even 50% (on 1 insert).

10. BIBLIOGRAPHY

- Hidalgo et al. Characterization of the human colon carcinoma cell line (CaCo-2) as a model for intestinal epithelial permeability. Gastroenterology 96, 736-749 (1989). - Artursson P and Borchardt RT. Intestinal drug adsorption and metabolism in cell cultures: Caco-2 and beyond. Pharma. Res. 14, 1655-1658 (1997).

- Tavelin S. Application of epithelial cell culture in studies of drug transport. Methods in Molecular Biology vol. 188 Epithelial cell culture protocols.

- Le Ferrec E. In vitro models of the intestinal barrier. ECVAM workshop 46. ATLA 29, 649-668, 2001

- Wahlang B, Pawar YB, Bansal AK. Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model. Eur J Pharm Biopharm. 2011 Feb;77(2):275-82.

Claims

1. An oral compositions, comprising

a) a low-bioavailability active ingredient,

b) a chitosan salt salified with N-acetylcysteine (NAC), and

c) polysorbate.

2. The oral compositions according to claim 1 , wherein, in the chitosan salt with NAC, the weight ratio between Chitosan and NAC ranges from 1 : 1 to 3 : 1.

3. The oral compositions according to claim 2, wherein said weight ratio is 2 to 1.

4. The oral compositions according to any of the claims 1-3, wherein the polysorbate is selected from polysorbate 20, 40, 60, 65, 80, and relative mixtures.

5. The oral compositions according to any of the claims 1-5, wherein the polysorbate is polysorbate 80.

6. The compositions according to any of the claims 1-5, having an oral mucosal release.

7. The compositions according to claim 6, in the form of adsorbed powders.

8. The compositions according to claim 7, in the form of orodispersible tablets, obtained by compression of said adsorbed powders.

9. The compositions according to claim 8, comprising disintegrating agents selected from cellulose derivatives, modified starches, and polyvinyl pyrrolidone derivatives.

10. The compositions according to claim 7, in the form of translucent aqueous dispersions, obtained by adding an aqueous phase to said adsorbed powders.

11. The compositions according to claim 10, wherein said aqueous dispersions contain, dissolved in the aqueous phase, high-bioavailability active ingredients.

12. The compositions according to any of the claims 6-1 1, comprising at least one of the additives and/or excipients, selected from polyols, caprylic/capric acid triglycerides, starch and maltodextrins, precipitated amorphous silica, flavoring substances and sweeteners.

13. The compositions according to claim 12, wherein said flavoring agent/sweetener is rebaudiosid from Stevia rebaudiana.

14. A preparation process of the compositions according to any of the claims 6-13, comprising the following steps:

i) preparing an aqueous dispersion of the low-bioavailability active ingredient in polysorbate, optionally mixed with a caprylic/capric triglyceride, both in a concentration ranging between 1 and 30% by weight/total weight of the final preparation,

ii) adsorbing the liquid dispersion of the previous step i) in a Chitosan and N- acetylcysteine mixture in such amounts as to confer to the aqueous solution, obtained by extemporaneous redispersion of the final powder, a pH ranging between 3.5 and 4.5, and wherein N-acetyl cysteine and Chitosan are in a concentration ranging between 0.01 and 3% by weight/total weight of the adsorbed powder;

iii) optionally, adding at least one additive and/or excipient to the mixture of step ii); iv) optionally, compressing the mixture coming from step ii) or iii) to obtain the oral mucosal release composition in the form of a tablet.

v) optionally, dispersing the thus-obtained adsorbed powder in the previous steps in water to obtain the mucosal release composition in the form of a translucent aqueous dispersion.

15. The oral compositions according to any of the claims 1-5, in the form of an intestinal mucosal release composition, comprising:

A) a core, comprising

a) a low-bioavailability active ingredient,

b) a polysorbate,

c) and a chitosan salt acidified with N-acetylcysteine;

B) a gastroresistant coating of said core.

16. The oral compositions according to claim 15, wherein the core A) of the composition according to the invention further comprises a Citrus paradisi extract titrated at at least 40% by weight/total weight of the Citrus x paradisi extract in bioflavonoids, and specifically titrated in Naringin and Naringenin, and a Piper nigrum extract, titrated at 95% by weight/total weight of the Piper nigrum extract in piperine.

17. The oral composition according to any of the claims 15 or 16, wherein the coating (B) comprises a rigid gastroresistant capsule.

18. A process for preparing the intestinal mucosal release oral compositions according to any of the claims 15-17, comprising the following steps:

i. preparing a solution or aqueous dispersion comprising at least one polysorbate in a concentration between 1 and 30% by weight/total weight of the solution, and a chitosan salt salified with N-acetylcysteine in such amounts as to confer to the aqueous solution containing 1% w/w Chitosan a pH ranging between 3.5 and 4.0, wherein N-acetyl cysteine and chitosan are in a concentration between 0.01 and 3% by weight/total weight of the solution;

ii. wet granulating a composition comprising at least the low-bioavailability active ingredient with the solution or aqueous dispersion obtained in step L;

iv. applying the gastroresistant coating of the core A).

19. The oral compositions according to any of the claims 1-13 and 15-17, for use as a medicament.

20. The oral compositions according to claim 19, wherein the active ingredient is selected in the group consisting of: anti-retroviral drugs, oligopeptide-based drugs, analgesic and/or anti-inflammatory drugs, drugs for pain therapy, drugs or active ingredients capable of opposing the toxic/inflammatory effects caused by anti-cancer drugs.

21. Use of the oral compositions according to any of the claims 1-13 and 15-17, as a dietary supplement and/or a nutraceutical composition.

22. The use according to claim 21, wherein the active ingredient is selected from: Melatonin, Resveratrol, Polydatin, Co-enzyme Q10, Liposoluble vitamins (A,D,E,F,K) Tryptophan, 5-hydroxytryptophan, Isoflavones, Berberin, and curcumin.