Patent Description:
Compositions comprising mixtures of cetylated fatty acids and use thereof in the treatment of arthritis and joint inflammation are known in the prior art. For example, patent documents <CIT> and <CIT> disclose compositions comprising cetylated fatty acids and, optionally, mixtures of tocophenols such as antioxidants at a percentage by weight comprised from <NUM>% to <NUM>%, with respect to the total weights of the compositions.

Prior art document <CIT> does not disclose a method for preparing at least one cetylated fatty acid or a mixture of cetylated fatty acids.

On the other hand, prior document <CIT> illustrated a process for preparing a mixture of cetylated fatty acids, and a system for carrying out said process. However, the method known from prior document <CIT> does not provide for a step (II), a step (IV), a step (V), a step (VI), a step (VII) and a step (VIII) as discussed in greater detail below.

The prior art document <CIT> discloses a method for relieving and inhibiting the symptoms of inflammatory rheumatoid arthritis in mammals using synthetically obtained cetyl myristoleate, or more preferably - as illustrated in Example <NUM> - obtained by means of an extraction from macerated mice tissues. Therefore, the prior art document <CIT> does not illustrate a method in which at least one fatty acid, cetyl alcohol and a metal catalyst are placed at contact to obtain a reaction mixture.

The prior art document <CIT> illustrates a process for the preparation of two isomers of cetyl myristoleate (hexadecyl cis-<NUM>-tetradecenoate and hexadecyl cis-<NUM>-tetradecenoate), and the therapeutic use of the latter in the treatment of osteoarthritis and other inflammatory diseases. The process discussed in the prior art document <CIT> comprises - inter alia - a cooling step in the presence of a solvent at a temperature comprised from -<NUM> to -<NUM>, an ozonolysis step and an enzymatic transesterification step. Thus, such process does not take place in the absence of solvent, it is not carried out through the application of heating ramps, and entails steps that are basically different from the process proposed herein.

Lastly, document <CIT> illustrates a process for the preparation of a mixture of a cetylated myristic acid and a cetylated palmitic acid, comprising a step of reacting myristic acid and palmitic acid with cetyl alcohol at high temperatures in the presence of at least one acid catalyst and at least one aromatic hydrocarbon acting as solvent. Document <CIT> explains that a complete absence of solvent causes an impossibility to produce substantial amounts of ester, and thus contains an explicit disincentive to operate in the absence of solvent in order not to depress the formation of ester, contrary to the process subject of the present invention.

The technical problem addressed and solved by the present invention lies in providing compositions comprising improved cetylated fatty acids with respect to the known compositions, that are effective in the treatment of arthritis and inflammatory joint and musculoskeletal disorders, as well as in the protection of the gastric mucosa and in the regulation of blood glucose levels, free of adverse effects, well-tolerated, stable over time with respect to the action of the oxidising agents and, furthermore, economically advantageous to produce.

In order to overcome said technical problems, the present invention provides compositions (pharmaceutical compositions, medical device compositions, novel foods (food or food for special medical purpose (FSMP) or medical foods), dietary supplements or cosmetic compositions, in brief: compositions produced by the method of the invention) comprising at least one cetylated fatty acid or a mixture of cetylated fatty acids and, optionally, at least one antioxidant (for example, LIPOCET™).

The presence of the antioxidant makes the compositions stable over time, given that the titre of the active ingredient (cetylated fatty acids) remains almost unvaried over time, protecting the cetylated fatty acids from the action of the oxidising agents. Consequently, the presence of the antioxidant makes the compositions advantageous with respect to efficacy, transport, storage as wel as preservation time and mode from the moment of first opening of the package containing the composition.

Furthermore, the presence of the antioxidant at a smaller percentage by weight with respect to the compositions based on cetylated fatty acids of the prior art makes the composition economically advantageous to produce on a large scale (i.e. use of a lower amount of antioxidant) and highly tolerated, for example, also when administered to paediatric subjects.

Said compositions (for example, LIPOCET™) are capable of effectively and rapidly treating symptoms or disorders of the joint and/or muscular type such as the diseases, symptoms or disorders listed from (i) to (vi) hereinafter in the present description.

Furthermore, said compositions are capable (i. i) of protecting the gastric mucosa, (i. ii) of treating diabetes and (i. iii) of treating diseases and/or disorders other than diabetes deriving from or related with high blood glucose levels.

The present invention provides a first method for preparing said at least one cetylated fatty acid or mixture of cetylated fatty acids and a second method for preparing said compositions comprising said at least one cetylated fatty acid or mixture of cetylated fatty acids (produced by means of said first method) and the antioxidant (for example, LIPOCET™).

Said methods are easy to apply and economically advantageous.

Furthermore, said first method - through the combined application of an inert gas flow over the entire duration of the step for forming the esters and a vacuum program in the chamber of the reactor only in the last part of the step for forming said ester - advantageously allows a complete conversion of acids and an excellent yield of the production of esters under simple and economical operating conditions.

Lastly, said first method ensures the production of cetylated fatty acids which are basically devoid of traces of catalyst and, thus, safe for health and well-tolerated by the subjects to whom they are administered.

These and other objects which will be clear from the detailed description that follows, are achieved by the compositions, by the mixtures and by the methods of the present invention thanks to the technical characteristics claimed in the attached claims.

Following an intense research activity, the Applicant has developed innovative compositions, their uses in both therapeutic and non-therapeutic treatment methods and processes for their preparation, as reported in detail in the present description.

A composition is produced (for example, LIPOCET™) comprising (I) and, optionally, (II), wherein:.

The composition may be a pharmaceutical composition, a medical device composition, a novel food (food or beverage or food/beverage for special medical purpose (FSMP) or medical food) (for example, LIPOCET™) or a dietary supplement or a composition for a novel food or a dietary supplement or a cosmetic composition.

In a preferred embodiment, the composition comprises the (I) mixture comprising or alternatively, consisting of:.

Said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids) is a fatty acid esterified with cetyl alcohol (<NUM>-hexadecanol, CH<NUM>(CH<NUM>)<NUM>OH) or a mixture of fatty acids esterified with cetyl alcohol.

Preferably, the fatty acid of (a) at least one cetylated fatty acid is selected from among the group comprising or, alternatively, consisting of: lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, eicosanoic acid, and mixtures thereof; more preferably said (a) at least one cetylated fatty acid is a mixture of cetylated fatty acids comprising, or alternatively, consisting of a mixture of cetylated myristic acid and cetylated oleic acid; even more preferably said (a) at least one cetylated fatty acid is a mixture of cetylated fatty acids comprising or, alternatively, consisting of cetylated myristic acid, cetylated oleic acid, cetylated linoleic acid and cetylated palmitic acid.

According to an embodiment of the composition comprising (a), (b) and (c) - LIPOCET™ (according to any of the described embodiments), the fatty acid profile comprises or, alternatively, consists of:.

In an embodiment, said (a) comprises a mixture of cetylated myristic acid and cetylated oleic acid provided that said (a) does not comprise cetylated myristoleic acid, in particular, when the composition is for use in methods for treatment of arthritis and of inflammatory joint and musculoskeletal pain.

In a further embodiment, said (a) comprises a mixture of cetylated myristic acid and cetylated oleic acid which may also comprise cetylated myristoleic acid, in particular when the composition is for use in treatment methods for protecting the gastric mucosa and regulating blood glucose levels. Myristic acid is tetradecanoic acid (CH<NUM>(CH<NUM>)<NUM>COOH), a saturated fatty acid with <NUM> carbon atoms present in milk derivatives (butter, cream and cheese), coconut oil, palm seed oil and in some spices (nutmeg).

The myristic acid used in the present invention may be, for example, selected from among those at <NUM>% <NPL> (EINECS <NUM>-<NUM>-<NUM>) having a composition % (GLC): lauric acid C12:<NUM> lower than or equal to <NUM>; myristic acid C14:<NUM> higher than or equal to <NUM>%; palmitic acid C16:<NUM> lower than or equal to <NUM>.

Oleic acid is cis-<NUM>-octadecenoic acid (CH<NUM>(CH<NUM>)<NUM>CHCH(CH<NUM>)<NUM>COOH), a monounsaturated carboxylic acid with <NUM> carbon atoms and it is the most abundant constituent of most vegetable oils.

The oleic acid used in the present invention may be, for example, selected from among those having at least <NUM>% in oleic acid <NPL> (EINECS <NUM>-<NUM>-<NUM>) with a composition percentage (%; GLC) for example: [lauric acid + myristic acid] C12:<NUM>+C14:<NUM> lower than or equal to <NUM>; oleic acid C18:<NUM> higher than or equal to <NUM>%; linoleic acid C18:<NUM> lower than or equal to <NUM> and other C18:<NUM> lower than or equal to <NUM>.

The cetyl alcohol (<NUM>-hexadecanol) used in the present invention may be, for example, selected from among those having <NPL> (EINECS <NUM>-<NUM>-<NUM>).

Advantageously, when said (a) comprises or consists of a mixture of cetylated myristic acid and cetylated oleic acid, the mole ratio between the cetylated myristic acid and the cetylated olieic acid is comprised between <NUM>:<NUM> and <NUM>:<NUM>, preferably it is comprised between <NUM>:<NUM> and <NUM>:<NUM>, more preferably it is (<NUM>±<NUM>):<NUM>.

The antioxidant comprised in the composition together with (a) and, optionally, with (c) may be any antioxidant considered - by the man skilled in the art - suitable for use in pharmaceutical compositions, medical device compositions, novel foods (foods), supplements or cosmetic compositions for the purposes reported in the present description.

Antioxidants are often added to foods predominantly containing fats, so as to delay the development of the rancidity that starts in the presence of oxygen. Natural antioxidants include flavonoids, polyphenols, ascorbic acid (vitamin C) and tocopherols (vitamin E). Synthetic antioxidants include butylhydroxyanisole (BHA), butylhydroxytoluene (BHT) and ethoxyquinoline.

Preferably, said (b) at least one antioxidant is selected from among the group comprising or, alternatively, consisting of: tert-butyl-hydroquinone (TBHQ), Aperoxid® TLA (as defined in the present invention), a tocopherol (for example tocopheryl acetate) or a mixture of tocopherols, a natural rosemary extract, wheat germ oil (Triticum vulgare), butylated hydroxytoluene and mixtures thereof, wheat germ oil (Triticum vulgare); more preferably (b) it is tert-butyl-hydroquinone (TBHQ).

Tert-butyl-hydroquinone (TBHQ or tertiary butylhydroquinone) is used inthe food industry as a preservative for unsaturated vegetable oils and many edible animal fats. TBHQ is authorised as a food additive in the European Union with an acceptable daily intake (ADI) of <NUM>/kg of body weight and it is identified with the code E319.

Aperoxid® TLA (registered trademark) is the trade name of an antioxidant effective at combating the phenomenon of fat rancidity, used for example in the cosmetic field. Aperoxid® TLA is a mixture comprising tocopherol ((±)-α-tocopherol) <NUM> - <NUM> %, lecithin > <NUM> %, ascorbyl palmitate <NUM> - <NUM> % (an ester formed by ascorbic acid and palmitic acid creating a liposoluble form of vitamin C) and citric acid < <NUM> % (INCI NAME: lecithin, tocopherol ((±)-α-tocopherol), ascorbyl palmitate, citric acid; <NPL>, <NPL>, <NPL>, <NPL>).

In a preferred embodiment, the composition comprises the (I) mixture comprising, or alternatively, consisting of:.

In a more preferred embodiment, the composition comprises the (I) mixture comprising, or alternatively, consisting of:.

In a further embodiment, besides (a) and, optionally, (b), the (I) mixture further comprises (c) a vegetable oil.

Preferably said (c) vegetable oil is selected from among the group comprising or, alternatively, consisting of olive oil, sunflower oil, corn oil or mixtures thereof with high content of oleic acid, any vegetable oil with high content of oleic acid and mixtures thereof. Advantageously, (c) is olive oil. The content by weight of oleic acid and/or oleins in said oil is comprised from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%, even more preferably from <NUM>% to <NUM>%, for example from <NUM>% to <NUM>%.

In a preferred embodiment, the (I) mixture comprises or, alternatively, consists of
said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids), preferably a mixture of cetylated myristic acid and cetylated oleic acid, at a percentage by weight comprised from <NUM>% to <NUM>% with respect to the total weight of the composition, preferably from <NUM>% to <NUM>%, more preferably from <NUM>% to <NUM>%;.

In a preferred embodiment, the (I) mixture comprises, or alternatively, consists of.

In a more preferred embodiment, the (I) mixture comprises, or alternatively, consists of.

Forming an object of the present invention is a method to manufacture the compositions reported in the present description (for example, LIPOCET™) comprising: (I) the mixture of the invention, comprising or consisting of (a) and (b) and, optionally, (c) (as defined above), and, optionally, (II) at least one food or pharmaceutical grade additive and/or excipient, said compositions being for use as medicament when administered, at a therapeutically effective amount, to a subject in need.

Forming an object of the present invention is a method to manufacture compositions (for example, LIPOCET™) comprising (a) and (b) and, optionally, (c) (according to all embodiments described in the present description) for use in a preventive and/or curative and/or symptomatic treatment of diseases, symptoms or disorders such as (i) rheumatoid arthritis of inflammatory and non-inflammatory origin, in particular osteoarthritis; (ii) joint inflammatory conditions other than rheumatoid arthritis; (iii) psoriasis, lupus, periodontal diseases or cardiovascular or heart diseases; (iv) post-traumatic osteoarticular and musculoskeletal diseases including sports-related traumas, for example pubalgia or athletic pubalgia (pubalgia refers to a chronic pain of the abdomen and groin); (v) degenerative joint diseases, preferably osteoarthritis, gonarthrosis, coxarthrosis, and/or (vi) tendon and muscle-related inflammatory/traumatic conditions, when administered to a subject in need.

Preferably, the compositions for use in a method for treatment of diseases or disorders from (i) to (vi) as identified above comprise the (I) mixture comprising or, alternatively consisting of:.

Said compositions for use in a method for treatment of diseases, symptoms or disorders listed from (i) to (vi), as defined above, are administered to said subject preferably through the topical or transdermal route; more preferably through the topical route. Advantageously, the cetylated fatty acids from (a1) to (a7) comprised in the mixture (a) have a molecular weight comprised from <NUM> Da to <NUM> Da. This molecular weight range allows the mixture (a), and thus, the composition to effectively overcome the dermal barrier (skin) facilitating the absorption thereof through the topical route.

Alternatively, said compositions for use in a method for treatment of diseases, symptoms or disorders listed from (i) to (vi), as defined above, are administered to said subject preferably through the oral route.

Forming an object of the present invention is a method to manufacture the compositions (for example, LIPOCET™) comprising (a) and (b) and, optionally, (c) (according to all embodiments described in the present description) for use in a method for preventive and/or curative and/or symptomatic treatment of (i. i) symptoms and/or disorders deriving from or related with damage to the gastric mucosa (treatment for protecting the gastric mucosa), (i. ii) diabetes and disorders or symptoms deriving from or related with diabetes, (i. iii) diseases and/or disorders other than diabetes deriving from or related with high blood glucose levels, when administered to a subject in need.

i) symptoms or disorders deriving from or related with the damage of the gastric mucosa (protection of the gastric mucosa) are selected from among the group comprising or, alternatively, consisting of: gastric ulcers, gastroesophageal reflux (GERD), heartburn.

Advantageously, effective and lasting protection of the gastric mucosa exerted by the use of the composition of the present invention in rats was observed in vivo in rats. Gastric mucosa is known to exert a barrier effect and it consists of a mucosal layer of about <NUM> which covers the mucous membrane of the stomach. The purpose of this barrier is to protect the gastric epithelium from the damaging action of the HCl produced by the parietal cells of the stomach. It derives from the production - by the gastric surface cells - of high molecular weight (thus very viscous) mucoprotein droplets and from the production of bicarbonate.

The Compositions produced are (see Table <NUM> or Samples <NUM>-<NUM>) for use in a method for the (preventive and/or curative) treatment of a disorder or symptom deriving from or-related with the damage of the gastric mucosa selected from among the group comprising or, alternatively, consisting of: gastric ulcers, gastroesophageal reflux (GERD), heartburn.

iii) diseases and/or disorders other than diabetes deriving from or related with high blood glucose levels are selected from among the group comprising or, alternatively, consisting of: hyperglycaemia, chronic liver diseases, obesity.

Advantageously, it was found that through the in vivo administration to rats of a composition for example the composition of Sample <NUM>, at high doses, for example at doses higher than <NUM>,<NUM>/Kg, the glucose levels in the blood of the treated rats are statistically significantly reduced.

Therefore, forming an object of the present invention is a composition (see Table <NUM> or Samples <NUM>-<NUM>) for use in a method for treatment of diabetes or of a disease and/or disorder other than diabetes deriving from, or related with, high blood glucose levels, such as in the case of hyperglycaemia, chronic liver diseases and obesity.

Said compositions for use in a method for treatment of diseases, symptoms or disorders listed from (i. iii), as defined above, are administered to said subject preferably through the oral route.

Preferably, the compositions for use in a method for treatment of diseases or disorders from (i. iii) as identified above comprise the (I) mixture comprising or, alternatively, consisting of:.

The compositions may be formulated in a liquid form, such as solution, biphasic liquic system or emulsion, suspension, syrup, spray, ointment, oil or beverage or, alternatively, in a semisolid form, such as gel, soft-gel, cream, foam, or, alternatively, in a solid form, such as powder, spray powder (spray drying), granules, microgranules, flakes, aggregates, buccal soluble sticks, tablets, effervescent tablets, capsules, suppositories, bars or food and equivalent forms known to the man skilled in the art. For example, the compositions of the invention can be formulated for oral use as capsules comprising (a) a mixture of cetylated fatty acids according to the invention, (b) at least one antioxidant at the % according to the invention and excipients such as, for example, gelatine, glycerol and/or preservatives (for example, LIPOCET™). In an embodiment, the composition is in the form of <NUM> capsules of bovine gelatine.

Advantageously, when the composition is for oral use, it is formulated in solid or liquic form, more preferably in the form of capsules or oil or powder or spray powder (spray drying) or spray liquid or beverage or food.

Advantageously, when the composition is for oral use, it is formulated in solid or liquic form, more preferably in the form of oil or powder or spray powder (spray drying) or spray liquid. Advantageously, when the composition of the invention is for topical use, it is formulated in semisolid form, more preferably in the form of cream or gel.

Advantageously, when the composition is for transdermal use, it is formulated in form suitable to be applied by means of a patch.

The composition are used in treatment of diseases, symptoms or disorders listed above from (i) to (vi), preferably by means of administration through the topical or transdermal route, or, alternatively, for the treatment of diseases, symptoms or disorders listed above from (i. iii), preferably by means of oral administration, to a subject in need of an effective amount of one of the compositions comprising (a) or (a) and (b) or (a) and (b) and (c) described in the present description.

The appropriate assay of the composition will depend, for example, on the condition to be treated/prevented, on the severity and course of the condition, on the fact that the composition is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the composition and at the discretion of the treating physician.

When the composition is administered through the topical route, the amount of the administered composition is of about from <NUM>/kg to <NUM>/kg of body weight of said subject per day, preferably <NUM>-<NUM>/kg, more preferably <NUM>-<NUM>/kg. Furthermore, the composition of the invention, for example a cream or gel, is applied through the topical route on an area comprised in the range from <NUM> to <NUM> in diameter on the area to be treated, depending on the anatomical area in question (for example, fingers: <NUM>, back: <NUM>).

When the composition is administered through the oral route, the amount of the administered composition is of about from <NUM>/kg to <NUM>/kg of body weight of said subject per day, preferably from <NUM>/kg to <NUM>/kg, more preferably from <NUM> to <NUM>,<NUM>/kg.

The composition is suitably administered to the subject all at once or on severa treatments.

The composition may be administered as a single treatment or in combination with other compositions or therapies (i.e. as coadjuvant) useful in the preventive and/or curative and/or symptomatic treatment of the diseases, symptoms and/or disorders listed from (i) to (vi) and/or listed from (i. iii) as described in the present description.

The non-therapeutic use of the composition comprising (a) or (a) and (b) or (a) and (b) and (c) for the protection of the gastric mucosa, wherein said composition is preferably formulated for oral use is described.

The composition protects the gastric mucosa so as to prevent the damaging thereof and thus the onset of disorders related with the gastric region such as, for example, gastric ulcers, gastroesophageal reflux (GERD), heartburn.

In the context of the present invention, the expression "subjects" is used to indicate human subjects or animal subjects (e.g. pets, such as dogs or cats or other mammals). Preferably, the compositions are for use in treatment methods for human subjects.

The expression "treatment method" in the context of the present invention is used to indicate an action, comprising the administration of a substance, or mixture of substances or combination thereof, with the aim of eliminating, reducing/decreasing or preventing a pathology or disease and its symptoms or disorders.

The expression "medical device" in the context of the present invention is used according to the meaning laid down by the Italian Legislative Decree n° <NUM>, dated <NUM> February <NUM> (or according to the new Medical Devices Regulation (UE) <NUM>/<NUM> (MDR)), i.e. it indicates a substance or another product, used alone or in combination, designated by the manufacturer to be used in humans for the diagnosis, prevention, control, therapy or attenuation of a disease, the product not exercising the main action, in or on the human body, for which it is designated, neither using pharmacological or immunology means nor by means of a metabolic process but the function thereof can be coadjuvated by such means.

The compositions optionally comprise (II) at least one food or pharmaceutical grade additive and/or excipient, such as a substance devoid of therapeutic activity suitable for pharmaceutical or food use, such as, for example, diluents, solvents (e.g. water, glycerine, ethyl alcohol), solubilizers, thickeners, sweeteners, anti-caking agents, flavour enhancers, colourants, lubricants, surfactants, antimicrobials, antioxidants, preservatives, pH stabilizing buffers, acidifiers and all auxiliary substances known to the man skilled in the art.

Besides said (a), (b), and, optionally, (c), the compositions may further comprise other active components such as, for example, anti-inflammatories, probiotics, antacids, products for the treatment of joint and/or muscle-related disorders, vitamins of group B and E, mineral salts, pain killers, folic acid or folates, menthol, glucosamine, chondroitin, methylsulfonylmethane (MSM), essential oils. Unless specified otherwise, the indication that a composition "comprises" one or more components or substances means that other components or substances can be present besides the one, or the ones, indicated specifically.

Unless specified otherwise, the expression composition comprises a component at an amount "comprised in a range from x to y" is used to indicate that said component may be present in the composition at all the amounts present in said range, even though not specified, extremes of the range comprised.

Forming an object of the present invention is a first method for preparing said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids) as defined in the present description comprising the steps of (<FIG>):.

By means of the combined application of an inert gas flow for part or the entire duration of the step for forming the esters and a vacuum program in the reactor chamber only in the last part of the step for forming said ester, said first method advantageously allows the removal of the esterification water and the complete conversion of fatty acids into esters with excellent yield. Said first method is simple to apply and economically advantageous, since it is not necessary to apply a vacuum program for the entire duration of the esterification reaction.

In an embodiment (FR1) of said first method, said at least one fatty acid is selected from among the group comprising or, alternatively, consisting of lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, eicosanoic acid and mixtures thereof; preferably selected from among myristic acid, oleic acid and a mixture of myristic acid and oleic acid; more preferably a mixture of myristic acid and oleic acid, even more preferably a mixture of myristic acid and oleic acid provided that myristoleic acid is not comprised in said mixture.

In an embodiment (FR2) of said first method, said inert gas flow is applied using the blowing means <NUM> positioned in the volume portion of the chamber <NUM> overlying the reaction mixture <NUM> (<FIG>).

In an embodiment (FR3) of said first method, the esterification water, drawn out of the chamber <NUM> during the esterification reaction by applying the inert gas flow and the vacuum program described above, is condensed in a horizontal condenser <NUM> and collected in a container <NUM> after flowing through a vertical condenser <NUM> (<FIG> and <FIG>).

In an embodiment (FR4) of said first method, said horizontal condenser <NUM> is kept at a temperature comprised from <NUM> to <NUM> and it is connected to said container <NUM> by means of a vertical condenser <NUM> which is kept at a temperature comprised from <NUM> to <NUM> (<FIG>).

In an embodiment (FR5) of said first method, besides steps (I)-(VIII), said first method further comprises the step (IX) of filtering said at least one unrefined cetylated fatty acid or mixture of unrefined cetylated fatty acids (Ml) obtained from step (VIII) on bleaching earth and/or filtering earth in a filter press <NUM> (<FIG>), to obtain at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf) wherein the metal catalyst is substantially absent or present at an amount less than or equal to <NUM>% by weight, preferably at an amount comprised from <NUM>% to <NUM>%, even more preferably at an amount comprised from <NUM>% to <NUM>%, for example <NUM>% by weight, with respect to the weight of said filtered cetylated fatty acid or mixture of filtered cetylated fatty acids (Mf).

The metal catalyst (zero oxidation state metal), preferably metal zinc, is preferably used in powder form, preferably powdered metal zinc. The amount of catalyst added is comprised in the range from <NUM>% to <NUM>% by weight with respect to the total weight of the reaction reagents (i.e. fatty acid or mixture of fatty acids + cetyl alcohol), preferably from <NUM>% to <NUM>% by weight, even more preferably <NUM>% by weight.

In an embodiment (FR6) of said first method, besides steps (I)-(IX), said first method further comprises the step (X) (deodorisation step) of treating said at least one filtered cetylated fatty acid or mixture of filtered cetylated fatty acids (Mf) in a reactor <NUM> (<FIG>) at a temperature T3 comprised from <NUM> to <NUM>, preferably from <NUM> to <NUM>, more preferably at <NUM>, and a reduced pressure P3 comprised from <NUM> mbar to <NUM> mbar, preferably from <NUM> mbar to <NUM> mbar, in the presence of water vapour flow for a period of time comprised from <NUM> hour to <NUM> hours, preferably from <NUM> hours to <NUM> hours, to obtain at least one refined cetylated fatty acid or mixture of refined cetylated fatty acids (Mf).

Forming an object of the present invention is a second method for preparing a composition comprising said (a) at least one cetylated fatty acid (or mixture of cetylated fatty acids) as defined in the present description, wherein said second method comprises the step (XI) of mixing said at least one filtered cetylated fatty acid (Mf), obtainable according to the embodiment FR5 in combination with any one of FR1-FR4 or, alternatively, said at least one refined cetylated fatty acid (MF), obtainable according to the embodiment FR6 in combination with any one of embodiments FR1-FR5, with (b) at least one antioxidant (as defined in the present invention), wherein said (b) at least one antioxidant is mixed at a % by weight comprised from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% with respect to the total weight of the composition (composition of the invention), preferably from <NUM>% to <NUM>%, more preferably from <NUM>% to <NUM>%.

In a preferred embodiment, said second method for preparing a composition comprises the step of mixing said at least one filtered cetylated fatty acid (Mf) or at least one refined cetylated fatty acid (MF) comprising or, alternatively, consisting of a mixture of cetylated myristic acid and cetylated oleic acid, preferably at a mole ratio of <NUM>:<NUM>-<NUM>:<NUM> or <NUM>:<NUM>-<NUM>:<NUM> or (<NUM>±<NUM>):<NUM> or about <NUM>:<NUM>, and wherein said (b) at least one antioxidant is tert-butyl-hydroquinone (TBHQ) at a % by weight comprised from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% or from <NUM>% to <NUM>% with respect to the total weight of the composition, preferably from <NUM>% to <NUM>%, more preferably from <NUM>% to <NUM>%.

In an embodiment, said at least one filtered cetylated fatty acid (or mixture of cetylated fatty acids) (Mf), preferably a mixture of filtered cetylated myristic acid and cetylated oleic acid, before being subjected to the deodorisation step (X), is added with said (c) vegetable oil (as defined in the present invention), preferably olive oil or corn oil.

In an embodiment, before the deodorisation step (X), said at least one filtered cetylated fatty acid (Mf), preferably a mixture of filtered cetylated myristic acid and cetylated oleic acid, is subjected to the step (XII) of adding said (Mf) with said (c) a vegetable oil, preferably olive oil or corn oil, to form a mixture comprising (Mf) and (c) which is subsequently subjected to the deodorisation step (X).

In a preferred embodiment, deodorisation step (X) of the first method to obtain at least one refined cetylated fatty acid (MF), preferably a mixture of refined cetylated myristic acid and cetylated oleic acid, and the step (XI) of mixing said at least one refined cetylated fatty acid (MF) with at least one antioxidant, preferably TBHQ, of the second method and, optionally, the step (XII) of adding the at least one filtered cetylated fatty acid (Mf) with (c) the vegetable oil, are carried out in the same reactor.

Basically, the method of the present invention can be summarised schematically with the following embodiments:.

Said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids) is a fatty acid esterified with a cetyl alcohol (<NUM>-hexadecanol, CH3(CH2)15OH).

Preferably, the fatty acid of said (a) at least one cetylated fatty acid is selected from among the group comprising or, alternatively, consisting of: (a1) myristic acid, (a2) oleic acid, (a3) linoleic acid, (a4) palmitic acid, (a5) lauric acid, (a6) palmitoleic acid, (a7) stearic acid, (a8) eicosanoic acid, (a9) eicosenoic acid and mixtures thereof; more preferably said (a) at least one cetylated fatty acid is a mixture of cetylated fatty acids comprising, or alternatively, consisting of cetylated myristic acid, cetylated oleic acid, cetylated linoleic acid and cetylated palmitic acid; even more preferably said (a) comprises, or alternatively, consists of a mixture of a cetylated myristic acid and a cetylated oleic acid.

According to an embodiment of the composition comprising (a), (b) (at least one antioxidant) and (c) (for example, LIPOCET™) (according to any of the described embodiments), the fatty acid profile comprises or, alternatively, consists of:.

Furthermore, said (a)+(b)+(c) may comprise together with one or more said (a1) to (a4):.

The total amount of said from (a5) to (a9) is preferably comprised from <NUM>% to <NUM>%, preferably comprised from <NUM>% to <NUM>%; even more preferably from <NUM>% to <NUM>%, for example <NUM>% by weight.

The chromatographic separation of said mixture (a)+(b)+(c) leads to obtaining a wax fraction from (a1) to (a9) (at % by weight) comprised from <NUM>% to <NUM>%, preferably from <NUM>% to <NUM>%; the remaining part being the glyceride fraction. The chain length distribution in said wax fraction is of the type: <NUM> carbon atoms from <NUM>% to <NUM>%; <NUM> carbon atoms from <NUM>% to <NUM>%; and <NUM> carbon atoms from <NUM>% to <NUM>%. In said wax fraction, the amount of myristic acid is comprised from <NUM>% to <NUM>%, whereas that of oleic acid is comprised from <NUM>% to <NUM>% by weight.

Embodiments of the mixture (a)+(b)+(c) are reported hereinafter:.

The invention relates to methods of preparation. The examples referring to a composition "according to the invention" are for comparison purposes between compositions obtained by the method of the invention and prior art's compositions. EXPERIMENTAL PART <NUM>.

In experimental part <NUM> the stability of compositions of the invention comprising or not comprising an antioxidant was analysed. All the compositions reported in Table <NUM> and Samples <NUM>, <NUM> and <NUM> were tested using the same method described hereinafter. In particular, the tests conducted on Samples <NUM> and <NUM> (according to the invention) and on Sample <NUM> (comparative sample) are reported by way of non-limiting example. This is because the stability results obtained with the compositions reported in Table <NUM> are mutually comparable to those of Samples <NUM> and <NUM>, thanks to the presence of at least one antioxidant in the tested compositions.

Premise: the percentages (% by weight of Samples <NUM>, <NUM> and <NUM> refer to the total weight of the sample.

Sample <NUM>: (comparative composition) composition comprising:.

Sample <NUM> does not comprise antioxidants.

Sample <NUM>: composition according to the invention comprising:.

After a time T0, samples <NUM>, <NUM> and <NUM> were stored at a controlled temperature of <NUM>. Approximately every <NUM>-<NUM> days (i.e. at times T1, T2, T3 and T4), a sample was taken from each sample and the peroxide value (PV) and the Kreiss' test were determined.

The peroxide value (PV) was determined in a standard way according to the ISO3960 method (fourth edition <NUM>-<NUM>-<NUM>, corrected version <NUM>-<NUM>-<NUM>, prepared by the Technical Committee ISO/TC <NUM>/SC), as known to the man skilled in the art.

Said test is a classical analytical chemistry method for determining the degree of rancidity of a food oil. The test quantitatively determines the peroxide value (PV), which is an index of the amount of primary autoxidation products of fatty acids.

The method provides for (briefly): the oil sample is dissolved in isooctane and glacial acetic acid (glacial acetic acid: isooctane = <NUM>:<NUM> v/v) and potassium iodide is added. Iodine released from the peroxides is determined iodometrically (visually) with a starch indicator and a standard solution of sodium thiosulphate (Na<NUM>S<NUM>O<NUM> <NUM> N). The titration endpoint is determined iodometrically (visually)
The PV is expressed as mEq of oxygen per kg of oil.

Given that determination of the peroxide value (PV) is a highly empirical procedure, ISO3960:<NUM> set the mass of the sample at <NUM> for PV greater than <NUM> and at <NUM> for PV less than or equal to <NUM>, and limited the applicability of this method to animal and vegetable fats and oils with peroxide values from <NUM> mEq to <NUM> mEq of active oxygen per kilogram.

The Kreiss' test is performed in a standard manner, as known to the man skilled in the art.

The Kreiss' test is a chemical test for qualitatively determining the degree of rancidity of a food oil. The test is used to detect the secondary products of the autoxidation of fatty acids.

As a matter of fact the organoleptic properties of rancid and rancidifying fats are significantly correlated to the presence of carbonyl products formed by interaction between oxygen and unsaturated fatty acids.

Phloroglucinol (<NUM>,<NUM>,<NUM>-triphenol or symmetrical triphenol) is a chemical compound of the triphenol group, of the brute formula C6H6O3 which crystallises from the aqueous solutions thereof as dihydrate (C6H6O3·2H2O).

The Kreiss' test is based on the condensation reaction between carbonyl compounds (arising from fat rancidity) with phloroglucinol in the presence of HCl which gives rise to a red compound. This is a qualitative method and the result is considered positive when the colouration of the lower layer is more intense than a <NUM>% solution of KMnO<NUM> and negative when the opposite occurs.

After stratification, the colouration of the lower acid layer is examined:.

As reported in Table <NUM>, samples <NUM> and <NUM> (compositions according to the present invention comprising an antioxidant) show at times T3 (i.e. after about <NUM> days) and T4 (i.e. after about <NUM> days) a peroxide value (PV) significantly lower than in sample <NUM> (composition in the absence of antioxidant).

Furthermore, samples <NUM> and <NUM> are negative to Kreiss' test at times T3 and T4, while sample <NUM> is positive.

An in vivo toxicity study of a compound according to the invention (Sample <NUM>) was conducted in experimental part <NUM>.

In vivo study: <NUM>-day oral toxicity study, in Crl CD Sprague Dawley (SD) rats, of a compound according to the invention.

Thus, Sample <NUM> (compound according to the invention) is well tolerated at all the tested doses and the blood glucose levels are statistically significantly lower in the group of high-dose rats (inverse dose-effect relationship).

Surprisingly, an effective and durable gastric mucosal protection was visually observed through the present in vivo study in rats. Gastric mucosa is known to exert a barrier effect and it consists of a mucosal layer of about <NUM> which covers the mucous membrane of the stomach. The purpose of this barrier is to protect the gastric epithelium from the damaging action of the HCl produced by the parietal cells of the stomach. It derives from the production - by the gastric surface cells - of high molecular weight (thus very viscous) mucoprotein droplets and from the production of bicarbonate. Therefore, the compositions of the present invention (see Table <NUM>), for example Sample <NUM>-<NUM>, find valid application/use in a method for the (preventive and/or curative) treatment of a disorder or symptom deriving from or- related with the damage of the gastric mucosa selected from among the group comprising or, alternatively, consisting of: gastric ulcers, gastroesophageal reflux (GERD), heartburn.

The in vivo toxicity study reported above was repeated at <NUM> days. The results are comparable to those obtained at <NUM> days. In particular, at the dose of <NUM>/Kg of body weight of the tested rats (maximum dose which did not give toxicity effects) it is surprisingly observed that the tested rats maintained and preserved a good thyroid function without alteration of food and social behaviour.

In vitro efficacy study - in vitro evaluation of the anti-inflammatory activity of products according to the present invention and comparative products on cell culture.

The study described in experimental part <NUM> was intended to evaluate - in an in vitro system - the ability of the tested products to modulate inflammatory mechanisms induced in human tenocyte cultures (ZEN BIO TEN-F, Lot # TENM012214F). The study of anti-inflammatory activity was conducted through the assay - using the ELISA method - of some inflammation markers, in particular proinflammatory cytokines IL6, prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the latter products of cyclooxygenase and lipoxygenase - the main enzymes involved in the inflammatory cascade from arachidonic acid - activity respectively.

GLUCOSAMINE <NUM>% CREAM: concentration <NUM>%, <NUM>% and <NUM>%.

The tested products are all creams based on glycerine and/or glyceryl monostearate.

The products according to the invention (Cream Cn products) tested in said in vitro anti-inflammatory activity evaluation study have the following compositions reported in Table <NUM> (from C1 to C6). All the C1-C6 products according to the present invention gave similar experimental results. Thus, only the data of the Cream product C1, representing products C2-C6, will be reported hereinafter.

Before being subjected to the efficacy test, the samples were prepared in culture medium, starting from the following ratios: <NUM> diluted in <NUM> with culture medium. Subsequent dilutions in culture medium.

The products were subjected to preliminary cytotoxicity test aimed at selecting the most suitable concentrations for the final test. After evaluating the results of the cytotoxicity test, the above reported concentrations were selected for conducting the anti-inflammatory activity study.

For the test, human tenocyte cultures (ZEN BIO TEN-F, Lot # TENM012214F) were treated for <NUM> hours with interleukin-<NUM> beta (IL-1β, <NUM> ng/ml - dose selected following a range finding test), an agent involved in the tendinopathy inflammatory conditions, and simultaneously with the products tested at <NUM> concentrations, selected from among the non-cytotoxic ones following a preliminary cytotoxicity test. At the end of the monitored experimental period, the levels of the inflammatory markers of interest in the culture media were measured using ELISA. Results were compared with negative control cultures (untreated, CTR-) and positive control cultures (treated with IL-1β, CTR+).

In summary, the experimental protocol provided for the assay of three pro-inflammatory markers (IL6, LTC4 and PGE2) in:.

The culture media of the controls and of the cells treated with the products subjected to the test (paragraph <NUM>) were used for the determination of the inflammatory markers IL6, PGE2 and LTC4 using the ELISA method.

Commercially available kits - which exploit the competitive binding of an antigen (the cytokine of interest in this case) with the primary antibody thereof - were used for this purpose. The immune complex (antigen-antibody) is in turn recognised by a secondary antibody conjugated to a peroxidase. The addition of the peroxidase substrate produces a colorimetric reaction with intensity proportional to the amount of immune complexes present and thus to the amount of bound cytokine. Quantitative determination exploits a calibration curve constructed with known and increasing concentrations of standard cytokine.

The following tables and charts show the results obtained in the present study.

The results are reported as the amount of cytokine released in the culture medium during the experimental period (mean value ± std. ) and as a mean % variation as compared to the controls.

Treatment of cell cultures with the tested products (paragraph <NUM>. ) showed a reduction in levels of IL6 released by the cells following the experimental induction of inflammation (Tables <NUM>-<NUM> and <FIG> and <FIG>). The tested samples more or less markedly modulate and inhibit the release of the pro-inflammatory cytokine monitored during inflammation. All the charts (<FIG> and <FIG>) show a more or less evident dose-dependent trend, in which the highest concentration tested has the assay lower than IL6.

Assay of IL6 in the CTR-, CTR+ cell cultures and treated with the Cream Product C1 according to the present invention (Table <NUM>). The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of IL6 in the CTR-, CTR+ cell cultures and treated with ARNICA Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of IL6 in the CTR-, CTR+ cell cultures and treated with GLUCOSAMINE Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Treatment of cell cultures with the tested products (paragraph <NUM>. ) showed a reduction in levels of LTC4 released by the cells following the experimental induction of inflammation (Tables <NUM>-<NUM> and <FIG>, <FIG>). The tested samples more or less markedly modulate and inhibit the release of the pro- inflammatory marker monitored during inflammation. All the charts (<FIG>, <FIG>) show a more or less evident dose-dependent trend, in which the highest concentration tested has the assay lower than LTC4.

Assay of LTC4 in the CTR-, CTR+ cell cultures and treated with the Cream Product C1 according to the present invention (Table <NUM>). The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of LTC4 in the CTR-, CTR+ cell cultures and treated with ARNICA Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of LTC4 in the CTR-, CTR+ cell cultures and treated with GLUCOSAMINE Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Treatment of cell cultures with the tested products (paragraph <NUM>. ) showed a reduction in levels of PGE2 released by the cells following the experimental induction of inflammation (Tables <NUM>-<NUM> and <FIG> and <FIG>). The tested samples more or less markedly modulate and inhibit the release of the pro-inflammatory marker monitored during inflammation. All the charts (<FIG> and <FIG>) show a more or less evident dose-dependent trend, in which the highest concentration tested has the assay lower than PGE2.

Assay of PGE2 in the CTR-, CTR+ cell cultures and treated with the Cream Product C1 according to the present invention (Table <NUM>). The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of PGE2 in the CTR-, CTR+ cell cultures and treated with ARNICA Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Assay of PGE2 in the CTR-, CTR+ cell cultures and treated with GLUCOSAMINE Cream <NUM>%. The results are expressed as mean content ± std. (expressed in ng/l) and as mean % variation as compared to the controls (Table <NUM> and <FIG>).

Claim 1:
A method for preparing (a) at least one cetylated fatty acid or a mixture of cetylated fatty acids comprising the steps of:
(I) placing at contact at least one fatty acid, cetyl alcohol and a metal catalyst in a chamber (<NUM>) of a reactor (<NUM>) in absence of solvent, to obtain a reaction mixture (<NUM>); followed by
(II) saturating the container (<NUM>) using an inert gas bringing the container (<NUM>) to a pressure P1 of about <NUM> atmosphere, applying a flow of said inert gas through said chamber (<NUM>); followed by
(III) applying a first heating ramp to said reaction mixture (<NUM>) up to reaching a temperature T1 comprised from <NUM> to <NUM> at a pressure P1 of about <NUM> atmosphere and in the presence of the inert gas flow to initiate an esterification reaction with initial formation of said cetylated fatty acid or mixture of cetylated fatty acids and esterification water; followed by
(IV) keeping said reaction mixture (<NUM>) under stirring at said temperature T1 and pressure P1 for a period of time comprised from <NUM> minutes to <NUM> hours; followed by
(V) applying a second heating ramp to said reaction mixture (<NUM>) up to reaching a temperature T2 comprised from <NUM> to <NUM> at a pressure P1 of about <NUM> atmosphere and in the presence of the inert gas flow to continue the esterification reaction with further formation of said cetylated fatty acid or mixture of cetylated fatty acids and esterification water; followed by
(VI) keeping said reaction mixture (<NUM>) under stirring at said temperature T2 and pressure P1 and in the presence of the inert gas flow for a period of time comprised from <NUM> hours to <NUM> hours;
(VII) applying a vacuum program in the chamber (<NUM>) which reduces the reaction pressure up to reaching a reduced pressure P2 comprised from <NUM> mbar to <NUM> mbar;
(VIII) keeping said reaction mixture (<NUM>) under stirring at said reduced pressure P2 and in the presence of the inert gas flow for a period of time comprised from <NUM> minutes and <NUM> hours to obtain complete formation of said at least one unrefined cetylated fatty acid or mixture of unrefined cetylated fatty acids (MI).