Patent Description:
Berberine chloride (or BERC), an isoquinoline alkaloid extracted from plants of the Berberis genus, is known to be as one of the most effective substances with a high nutraceutical potential. This substance has shown interesting and well-documented anti-inflammatory and hypoglycaemic properties in the treatment of diseases such as metabolic syndrome or in cardiovascular prophylaxis [<NUM>-<NUM>].

The association of BERC with substances capable of creating pharmacological synergies in terms of reducing cholesterol plasma fractions, in particular LDL, Apo B and fasting glycaemia, and promoting cardioprotection against the processes of ageing and oxidative stress on the cardiac muscle and vascular endothelium is particularly interesting.

For example, another mixture of natural substances from the tocopherol family and natural tocotrienols, extracted from various plants including the main one Elaeis Guineensis, has demonstrated interesting cholesterolaemic-lowering properties [<NUM>, <NUM>, <NUM>], trophic properties of the arterial endothelium [<NUM>] and above all a cardioprotective activity [<NUM>].

It is known that polysorbate <NUM> (PS <NUM>), an ester of polyoxyethylenated sorbitan with oleic acid, has shown to enhance the bioavailability of several pharmacologically active ingredients and nutraceutical substances through a mechanism of increasing diffusibility thereof through the mucopolysaccharide layer lining the enteric epithelium (UWP) and by inhibiting the P-gP (glycoprotein P) pump [<NUM>].

A pharmaceutical issue related to BERC and demonstrated in the studies on CACO-<NUM> is the high rate of expulsion from the enterocyte by the action of the P-gP pump [<NUM>,<NUM>] (<FIG>). Such phenomenon appears to be the main cause of the low oral bioavailability of BERC in humans [<NUM>].

In addition, derivatives of the vitamin E family also have bioavailability problems. In fact, they are lipophilic and natural substances in the form of oils that show limited enteric bioaccessibility, due to a wide individual variability in bile secretion and overall in biophysical processes preparatory to the emulsification of dietary fats in the first intestine. Such a phenomenon contributes to a bioavailability of these substances that is not always adequate and subject to wide inter-individual variation.

In view of these phenomena, especially as a function of those of biotransformation and lack of enterocytic internalisation, it is necessary to improve the bioavailability and bioaccessibility profile of these active substances.

In addition, it is desirable to find a composition or formulation or combination or mixture that solves the aforementioned technological-pharmaceutical issues, and that is also able to best express its pharmacological and therapeutic potential.

XP55607781 (URL: https//www. iganutraceuticals. com/product/eulipid) discloses Eulipid®, which is a pharmaceutical preparation, in soft-gel capsule form, used to restore normal cholesterol levels, comprising a combination of Berberis aristata extract with bioflavonoids, resveratrol, policosanols, alpha lipoic acid, coenzyme Q10 and vitamin E.

<CIT> discloses a solid oral nutraceutical composition for the treatment of metabolic syndrome, comprising Berberis aristata extract, Cynaria scolimus extract, a polyoxyethylenated sorbitan ester (polysorbate <NUM>) and a Piper nigrum extract. The D2 composition appears to be able to improve the bioavailability profile of active berberine chloride (or BERC). In addition, the D2 composition contains vitamin B1, but not vitamin E.

<CIT> relates to a composition for the control and reduction of blood cholesterol levels that may include berberine and/or an extract of the Berberis family together with hydroxytyrosol. The composition may also comprise the antioxidant vitamin E or alpha-tocopherol.

<CIT> discloses a formulation with detoxifying properties that also includes a powdered extract of Berberis aristata and Tween <NUM>, i.e. polysorbate <NUM>.

A first object of the present invention is a powdered mixture comprising:.

A second object of the invention is an oral formulation comprising the above powdered mixture in combination with suitable excipients and/or diluents.

Finally, a further object is a method of preparing the aforementioned powdered mixture, which comprises the following steps:.

The powder mixture according to the invention meets the need to simultaneously increase the bioavailability of two different active ingredients, namely the dry extract of Berberis Aristata, i.e. the berberine chloride comprised therein, and the vitamin E derivative.

This is made possible by the use of promoters of absorption that are able to generate an immediate fine micellar hydro-dispersion that preludes a functional contact with the enteric epithelium, preferably at the first intestinal tract level. Precisely, the absorption promoters, in association with each other, create the conditions for forming mixed micelles together with bile salts which promote the emulsification of the vitamin E derivative and, at the same time, improve the dispersion of BERC, preventing its intraluminal crystallisation thereof. This aids the action of bile salts, which are not always sufficient to promote adequate aqueous dispersion of the active substances and consequent effective contact with the absorbing epithelium; furthermore, the association of promoters of absorption interferes with the enterocytic P-gP pump, reducing its ability to expel the active substances towards the enteric lumen.

Thus, compared to the known art, in this scenario, not only is an increase in the bioavailability of the Berberis Aristata extract and the active berberine chloride promoted simultaneously, but also that of the other active ingredient, namely the vitamin E derivative, present in the formulation due to the association of the two enteric absorption promoters.

The Applicant believes that, thanks to this intuition, it is possible to maximise the efficacy of these two active substances of nutraceutical use, creating a useful and new mixture or association capable of intervening in the normalisation of lipidemic and glucometric parameters, the alteration of which may be a prelude to major dysmetabolic and cardiovascular illnesses such as the metabolic syndrome and the risk of heart attack and cerebral vascular events, especially in subjects with mild to moderate cardiovascular risk and in primary prevention. The same composition (or mixture) may also be used in formulations for use in the treatment of hepatic steatosis and for use in the prevention of its further progression to more severe forms, such as cirrhosis of the liver.

Further advantages will become apparent from the detailed description and the hereinafter examples.

Hereinafter, the Applicant describes the various objects of the invention in more detail.

Extracts are generally understood to be products of natural origin that are obtained from different types of plants and medicinal herbs. There are several parts of the plant that can be used to obtain these products: leaves, flowers, fruits (pulp and/or peel), seeds, roots, rhizomes, bark, etc. They may be preparations in liquid form (fluid extracts), solid form (dry extracts) or of intermediate consistency (soft extracts), obtained by suitable extraction processes; the latter ones include the use of appropriate solvents and the use of maceration or percolation, or other suitable processes.

A dry extract means a solid preparation obtained by means of an extraction process using solvents that extract the active ingredients contained in the extract itself, and a following step of evaporation of the solvent. Generally, the dry extract is characterised by a dry residue of not less than <NUM>% by mass and has a moisture content generally less than or equal to <NUM>% by mass.

The powdered mixture comprises the active ingredient dry extract of Berberis Aristata preferably in an amount comprised between <NUM>% and <NUM>% by weight, preferably between <NUM>% and <NUM>% by weight, even more preferably between <NUM>% and <NUM>% by weight of the total weight of the mixture (%w/w).

The powdered mixture comprises as an active ingredient the vitamin E derivative preferably in an amount comprised between <NUM>% and <NUM>% by weight, preferably between <NUM>% and <NUM>% by weight, preferably comprised between <NUM>% and <NUM>% by weight of the total weight of the mixture (%w/w).

Preferably, the vitamin E derivative is an oil comprising a mixture of natural tocopherols and tocotrienols with a content of said tocopherols and tocotrienols comprised between <NUM>% and <NUM>% by weight of the total weight of the derivative. Even more preferably, the vitamin E derivative is a mixture of α, β, γ isomers of tocotrienols and α isomers of tocopherols.

Preferably, the vitamin E derivative is in oil form. Preferably, the vitamin E derivative is a mixture of tocopherols or a mixture of tocopherols and tocotrienols.

The powdered mixture comprises as promoters of absorption: a polyoxyethylenated sorbitan, preferably polysorbate <NUM>, and a sucrose ester of fatty acids.

Absorption promoter (singular) means a substance that may be considered as an excipient because it has no therapeutic biological activity in itself, but that is capable, by one or more biological mechanisms, of promoting absorption, especially enteric absorption for the purposes of the present invention, of ingredients or active ingredients.

The class of substances used to inhibit the action of the P-gP protein is preferably the sorbitan polyoxyethylenate class, preferring in particular polysorbate <NUM>. The class of substances capable of promoting a ready and uniform emulsion of the vitamin E derivative is preferably that of polysorbates and sucrose esters with fatty acids.

The total content of the promoters of adsorption comprised in the powdered mixture of the invention is preferably comprised between <NUM>% and <NUM>% by weight, preferably between <NUM>% and <NUM>% by weight, even more preferably between <NUM>% and <NUM>% by weight of the total weight of the mixture (%w/w).

Preferably, polyoxyethylenated sorbitan is at least selected from: polysorbate <NUM>, polysorbate <NUM>, polysorbate <NUM> and polysorbate <NUM>. Preferably, polyoxyethylene sorbitan is polysorbate <NUM> (PS80) or polyoxyethylene sorbitan monooleate (from oleic acid). Preferably polyoxyethylenated sorbitan is E433 according to the European classification.

The amount of polyoxyethylenated sorbitan, preferably polysorbate <NUM>, comprised in the powdered mixture is preferably comprised between <NUM>% and <NUM>% by weight, more preferably comprised between <NUM>% and <NUM>% by weight.

Preferably, the sucrose ester with (of) fatty acids is selected from: palmitic ester, stearic ester or mixtures thereof. Preferably sucrose ester with fatty acids is E473 according to European regulations.

The amount of sucrose ester of fatty acids is preferably comprised between <NUM>% and <NUM>% by weight, more preferably comprised between <NUM>% and <NUM>% by weight.

For the purposes of the present invention, sugar (sugars, plural) means a polyol or a monosaccharide or a polysaccharide. Preferably, the sugar is selected from the group consisting of sorbitol, mannitol, xylitol, isomalt, erythritol, maltodextrins, fructose, and mixtures of the foregoing.

Said sugar comprised in the powdered mixture according to the invention is preferably in an amount comprised between <NUM>% and <NUM>% by weight, more preferably between <NUM>% and <NUM>% by weight, even more preferably between <NUM>% and <NUM>% by weight of the total weight of the mixture (%w/w).

For the purposes of the invention, the Applicant considers sugar to be essential in terms of pharmaceutical technology for obtaining the powdered mixture according to the invention.

The present invention is thus characterised by a powdered mixture based on plants of the Berberis genus, more specifically Berberis Aristata, and the vitamin E derivative, associated with the aforementioned absorption-promoting substances that are able to inhibit the P-glycoprotein located on the enterocyte membrane and promote the emulsification of the vitamin E derivative, preferably Tocopherols and/or Tocotrienols and/or mixtures thereof, into the aqueous secretions of the enteric lumen.

Method of preparing the powdered mixture comprising the following steps:.

Optionally, the last step of adding the sugar to the wet homogeneous paste e) of the method of preparing the powder mixture may be followed by other steps, such as the step of adding other excipients, such as amorphous silica, mixing and stirring to obtain an even, flowable powder; if necessary, the powder is sieved.

The second object of the invention is an oral formulation comprising the powder mixture as described above in combination with suitable excipients and/or diluents.

For merely descriptive and non-limiting purposes, excipients and/or diluents known to the state of the art and suitable for preparing formulations, preferably solid formulations (powders, tablets and/or capsules), are: bulking agents, gliding agents, lubricating agents, flow agents, disintegrating agents, flavouring agents, pH correcting agents, sweetening agents.

The oral formulation according to the invention may be prepared by following the method of preparing the powdered mixture and including further suitable excipients and/or diluents according to addition and mixing methods known to the person skilled in the art. Optionally, the last step of adding the sugar to the wet homogeneous paste e) of the method of preparing the powder mixture may be followed by other steps, such as the step of adding other excipients, such as amorphous silica, mixing and stirring to obtain an even, flowable powder; if necessary, the powder is sieved. At this point, any other excipients among those known to the person skilled in the art are added to improve the chemical-physical parameters.

Preferably the oral formulation may be in solid form, even more preferably in the form of a granulate, tablet, capsule (with animal or vegetable gelatin), or single-dose sachet/bag. The methods for obtaining oral solid formulations, e.g. tablets and capsules, are already known to the person skilled in the art based on the knowledge of general pharmaceutical technologies.

Preferably, the oral formulation may include the active ingredient dry extract of Berberis Aristata in a content varying between <NUM>% and <NUM>% by weight, more preferably between <NUM>% and <NUM>% by weight of the weight of the oral formulation (%w/w).

The oral formulation may comprise the vitamin E derivative in an amount varying between <NUM>% and <NUM>% by weight, more preferably between <NUM> and <NUM>% by weight of the weight of the oral formulation (%w/w).

Preferably, the oral formulation comprises promoters of absorption in an amount comprised between <NUM>% and <NUM>% by weight of the total weight of the oral formulation.

Preferably, the oral formulation may include polyoxyethylenated sorbitan in an amount comprised between <NUM>% and <NUM>% by weight, more preferably between <NUM>% and <NUM>% by weight of the total weight of the oral formulation (%w/w).

The oral formulation comprises sucrose ester of fatty acids preferably in an amount comprised between <NUM>% and <NUM>% by weight, more preferably between <NUM>% and <NUM>% by weight of the weight of the oral formulation (%w/w).

The oral formulation preferably comprises the sugar in amounts varying between <NUM> and <NUM>% by weight, more preferably between <NUM>% and <NUM>% by weight of the weight of the oral formulation (%w/w).

The oral formulation according to the invention comprising the powder mixture is preferably in the form of a dietary supplement, food supplement, food for special medical purposes, nutraceutical, medicinal products for human or animal use.

Dietary supplement means a product or formulation that may be used either as a sole nutritional source or as a supplement to the patient's diet, under the supervision of a physician. This is a food product for special medical purposes.

A food supplement means a formulation that falls under the definition underlying Directive <NUM>/<NUM>/EC as amended. In this regulation, food supplements are precisely defined as: "food products intended to supplement the common diet and constituting a concentrated source of nutrients, such as vitamins and minerals, or other substances having a nutritional or physiological effect, in particular, but not exclusively, amino acids, essential fatty acids, fibre and extracts of plant origin, both mono- and multi-compound, in pre-dosed forms".

Foods for Special Medical Purposes (or FSMPs) mean food products expressly prepared or formulated for managing the diet of patients with specific nutritional needs, including infants, to be used "under medical supervision". FSMPs are intended for the complete or partial feeding of patients who have a limited or impaired ability to take in, digest, absorb, metabolise or eliminate common foods or certain nutrients contained therein, or having specific nutritional needs determined by clinical conditions. FSMPs differ from other food products and food supplements themselves in a number of characteristics, such as: they require medical supervision; they may replace - totally or partially - the normal diet; they are intended for specific pathological conditions. With regard to the composition, FSMPs are regulated by Regulation <NUM>/<NUM>.

Nutraceutical means one or more components or active ingredients of food that have positive effects on health, prevention and treatment of diseases.

Medicinal product means a pre-packaged pharmaceutical form, industrially manufactured and authorised on the basis of a report containing the chemical, biological, pharmaceutical, pharmaco-toxicological and clinical experimental results relating to the drug, which is marketed under a special name (registered trademark).

Preferably, the oral formulation according to the invention is for use in the treatment and prevention of metabolic syndrome and related cardiovascular diseases and/or for use in the treatment of hepatic steatosis.

Metabolic syndrome means a pathological state characterised by: high blood pressure, impaired fasting glycemia or insulin resistance and dyslipidaemia, and a marked waist circumference (due to excess abdominal fat).

Relevant cardiovascular diseases mean diseases or altered cardiovascular conditions that may be caused by or result from a state of metabolic syndrome, such as, for example, increased cardiovascular risk, atherosclerosis, atheromas, arterial hypertension.

Hepatic steatosis (or fatty liver) means a pathological state characterised by a lipid content of more than <NUM>% of the total weight of the liver. It derives from an altered lipid metabolism. This situation may be caused by many different disorders and diseases. For instance, risk factors for hepatic steatosis are: diabetes, obesity (especially abdominal obesity -android or apple obesity-), an unbalanced diet, non-balanced and excessively rich in fats, anaemia, alcoholism. In addition, certain drugs, hormonal imbalances, nutritional imbalances (Kwashiorkior), carnitine deficiency, prolonged fasting and excessive exposure to toxic substances may favour the accumulation of triglycerides in the liver.

Even more preferably, the oral formulation is for use in the treatment and prevention of a disease condition selected from the group consisting of: metabolic syndrome, increased fasting glycaemia, glucose metabolism disorders, impaired glucose tolerance, hypercholesterolaemia, primary and secondary hypertriglyceridaemia, intolerance to statins and other cholesterolaemic drugs, increased cardiovascular risk, atherosclerosis, atheromas and mixtures of the foregoing.

In <FIG> and <FIG>, it is possible to appreciate the different dispersion behaviour in FaSSIF at <NUM> of the powder mixture according to the invention of Table <NUM> compared to a conventional preparation as shown in Table <NUM> per tablet with the only difference being that it does not contain the promoters of adsorption contained in the powdered composition according to the invention.

The method of preparing the powder mixture formulation of Table <NUM> of the invention is the same as that described for Example <NUM> below (see Examples section).

The formulation of Table <NUM> was used for the simulation study in FaSSIF (Fasted State Simulated Intestinal Fluid) (<FIG> and <FIG>), at a temperature of <NUM>. Specifically, for this study, the behaviour of the tablet powdered mixture according to the invention shown in Table <NUM> below was compared with that of a conventional preparation, the formulation of which is shown in Table <NUM>.

It should be noted that the aforesaid conventional preparation consists of the same active ingredients at the same concentrations as those of the composition of the invention but without the absorption promoters required in the powdered composition according to the present invention.

From <FIG> and <FIG>, it may be appreciated, on the one hand, the clear and complete hydro-dispersion with an even orange colour in the beaker on the left (BATCH N°SE0014), comprising the mixture according to the invention of Table <NUM>; on the other hand, the sedimentation of Berberine chloride and superficialisation preferably of the vitamin E derivative in the beaker on the right having the composition of Table <NUM> (BATCH N°SE0013), wherein neither the promoters of absorption nor sugar are present. Furthermore, considering the view from above, it may be seen in the beaker on the right without the excipients of the composition of the invention the presence of deep red oil droplets floating on the surface of a clear liquid (visible the anchor at the bottom), indicative of a lack of dispersion in the aqueous medium of the vitamin E derivative. In the beaker on the left, on the other hand, it may be seen the absence of such droplets, indicating that emulsification in the enteric aqueous medium and the disappearance of the clarity of the medium have taken place.

In this sense, the Applicant considers that the association of the active ingredients, Berberis Aristata extract and vitamin E derivative, in the presence of at least one polysorbate and at least one sucrose ester, ensures an excellent and ready emulsification of the two active ingredients in the FaSSIF and at the same time ensures a partial inhibition of the P-gP pump capable of maximising the bioavailability of BERC, preventing its enterocyte "pumping off" towards the lumen.

The Applicant provides the hereinafter examples for merely illustrative and non-limiting purposes.

Step A - mixing together Berberis Aristata extract with sucresters in plastic beakers until a homogeneous powder is obtained (sieving if necessary).

Step B - combining the mixture of vitamin E derivatives with polysorbate <NUM> and mixing until homogeneous and clear liquid step (deep red colour).

Proceeding to wet step A with step B until a wet powder (paste) is obtained. Combining step C with step A+B at room temperature (<NUM>-<NUM>) under manual or mechanical stirring until a wet, homogenous powder (granule) is obtained. Proceeding to sieve. Combining step A+B+C with step D (silica) and mixing until a fine, uniform, non-wetting powder is obtained.

Step B - combining the vitamin E derivative with polysorbate <NUM> and mixing until a homogeneous and clear liquid step (deep dark red colour).

Proceeding to wet step A with step B until a wet powder (paste) is obtained. Combining step C with step A+B at room temperature (approx. <NUM>-<NUM>) under manual or mechanical stirring until a wet, homogenous powder (granule) is obtained. Proceeding to sieve. Combining step A+B+C with step D (silica) and mixing until a fine, uniform, non-wetting powder is obtained.

Claim 1:
Powdered mixture comprising
- a dry extract of Berberis Aristata, with a berberine chloride content comprised between <NUM>% and <NUM>% by weight of the total weight of the extract (%w/w), and
- a vitamin E derivative selected from: tocotrienols, tocopherols and mixtures of the foregoing,
and as promoters of absorption:
- a polyoxyethylenated sorbitan, preferably polysorbate <NUM>, and
- a sucrose ester of fatty acids,
and one sugar.