EA045244B1 - METHOD FOR PREPARING A COMPOSITION CONTAINING CETYLATED FATTY ACIDS - Google Patents

Description

The invention relates to a composition comprising a mixture containing, alternatively, consisting of at least one cetylated fatty acid or a mixture of cetylated fatty acids and a reduced percentage by weight antioxidant (for example, LIPOCET™), and the said composition is suitable for use in methods treatment of arthritis and inflammatory joint and musculoskeletal pain. The invention further relates to a composition comprising a mixture containing or alternatively consisting of at least one cetylated fatty acid or a mixture of cetylated fatty acids and optionally an antioxidant, said composition being intended for use in methods of treatment for protecting the gastric mucosa and to regulate blood glucose levels. Finally, the invention relates to a method for producing said at least one cetylated fatty acid or a mixture of cetylated fatty acids and producing compositions comprising said cetylated fatty acids and said antioxidant.

Compositions containing mixtures of cetylated fatty acids and their use in the treatment of arthritis and joint inflammation are known in the art. For example, patent documents WO 2004/084829 A2 and WO 2017/029580 A1 describe compositions containing cetylated fatty acids and optionally mixtures of tocophenols, such as antioxidants, contained in weight percentages ranging from 1 to 5% relative to the total weight compositions.

The prior art document WO 2004/084829 A2 does not disclose a process for producing at least one cetylated fatty acid or a mixture of cetylated fatty acids.

On the other hand, the prior art document WO 2017/029580 A1 illustrates a method for producing a mixture of cetylated fatty acids and a system for carrying out said method. However, the method known from the prior art document WO 2017/029580 A1 does not include stage (II), stage (IV), stage (V), stage (VI), stage (VII) and stage (VIII), discussed in more detail below .

Prior art US 4,113,881 A discloses a method for alleviating and suppressing the symptoms of inflammatory rheumatoid arthritis in mammals using synthetically produced cetyl myristoleate, or more preferably, as illustrated in Example 1, obtained by extraction from macerated tissues of mice. Therefore, prior art document US 4,113,881 A does not show a process in which at least one fatty acid, cetyl alcohol and a metal catalyst are brought into contact to produce a reaction mixture.

The prior art document WO 2010/079514 A1 illustrates a process for the preparation of two isomers of cetyl myristoleate (hexadecyl-cis-9-tetradecenoate and hexadecyl-cis-10-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 WO 2010/079514 A1 includes, inter alia, a solvent cooling step at a temperature of -70°C to -78°C, an ozonolysis step and an enzymatic transesterification step. Thus, such a method does not occur in the absence of a solvent, it is not carried out through the use of heating steps, and it involves steps that are fundamentally different from the method proposed in this document.

Finally, NZ 332959 A illustrates a process for preparing a mixture of cetylated myristic acid and cetylated palmitic acid, comprising the step of reacting the 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 in as a solvent. NZ 332959 A explains that the complete absence of solvent results in the inability to produce a significant amount of ester and, therefore, the method contains an obvious obstacle to operation in the absence of solvent so as not to suppress the formation of the ester, unlike the method that is the subject of the present invention .

The technical problem considered and solved by the invention is to create compositions containing cetylated fatty acids, improved in relation to known compositions, which are effective in the treatment of arthritis and inflammatory diseases of the joints and musculoskeletal system, as well as in protecting the gastric mucosa and regulating glucose levels in blood, have no side effects, are well tolerated, stable over time with respect to the action of oxidizing agents and, in addition, are economically beneficial for production.

In order to overcome these technical problems, the invention proposes compositions (pharmaceutical compositions, medical device compositions, new food products (foods or foods for special medical purposes (FSMP) or medical foods), food additives or cosmetic compositions, briefly: compositions according to invention) containing at least one cetylated fatty acid or a mixture of cetylated fatty acids and optionally at least one antioxidant (eg LIPOCET™).

- 1 045244

The presence of an antioxidant makes the compositions according to the invention stable over time, given that the titer of the active ingredient (cetylated fatty acids) remains practically unchanged over time, protecting the cetylated fatty acids from the action of oxidizing agents. Therefore, the presence of an antioxidant makes the compositions according to the invention advantageous in terms of effectiveness, transportation, storage, as well as the time and mode of storage from the moment the package containing the composition according to the invention is first opened.

In addition, the presence of an antioxidant with a lower weight percentage relative to prior art cetylated fatty acid compositions makes the composition according to the invention economical to produce on a large scale (i.e. using less antioxidant) and well tolerated, e.g. also when administered children.

These compositions of the invention (eg, LIPOCET™) have the ability to effectively and rapidly treat symptoms or disorders of the articular and/or muscular type, such as the diseases, symptoms or disorders listed from (i) to (vi) below in this description.

In addition, said compositions according to the invention have the ability to (i.i) protect the gastric mucosa, (i.ii) treat diabetes and (i.iii) treat diseases and/or disorders other than diabetes resulting from or associated with high blood glucose levels. with this level.

Finally, the invention provides a first method for producing said at least one cetylated fatty acid or mixture of cetylated fatty acids and a second method for producing said compositions according to the invention comprising said at least one cetylated fatty acid or mixture of cetylated fatty acids (obtained using said first method) and an antioxidant (for example, LIPOCET™).

These methods are easy to use and cost-effective.

Moreover, said first method - by the combined application of an inert gas flow throughout the ester formation stage and a vacuum program in the reactor chamber only in the last part of the said ester formation stage - preferably allows for complete acid conversion and excellent ester production yields. and economical working conditions.

Finally, said first method provides the production of cetylated fatty acids which are substantially free of catalyst traces and are 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 following detailed description, are achieved by the compositions, mixtures and methods of this invention due to the technical features claimed in the accompanying claims.

Detailed Description of the Invention

As a result of intensive research activities, the applicant has developed innovative compositions, their use in both therapeutic and non-therapeutic treatments, as well as methods for their preparation, as described in detail herein.

In fig. 1, 2 and 3 show an installation for carrying out a first method for producing (a) at least one cetylated fatty acid or a mixture of cetylated fatty acids.

In fig. 4a-4b-4c show cell culture assays of IL6 for the subject compositions according to Experimental Part 3.

In fig. 5a-5b-5c show LTC4 cell culture assays for the subject compositions according to Experimental Part 3.

In fig. 6a-6b-6c show PGE2 assays in cell cultures for the subject compositions according to Experimental Part 3.

The subject of this invention is a composition (in short, a composition according to the invention) (for example, LIPOCET™), comprising (I) and optionally (II), in which:

(I) is a mixture (in short, a mixture according to the invention) containing or alternatively consisting of:

(a) at least one cetylated fatty acid (or mixture of cetylated fatty acids), wherein said fatty acid has a number of carbon atoms from C6 to C21, preferably from C8 to C18, more preferably from C14 to C18, and it may be saturated or unsaturated, and optionally (b) at least one antioxidant, having a wt.% content of from 0.001 to 0.5%, or from 0.001 to 0.4%, or from 0.001 to 0.3%, or from 0.001 to 0 .2%, or from 0.001 to 0.1% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%; and (II) is at least one food grade or pharmaceutical grade additive and/or excipient.

The composition according to the invention may be a pharmaceutical composition,

- 2 045244 composition for a medical device, a new food product (food or drink or food/special medical purpose (FSMP) or medical nutrition) (for example, LIPOCET™) or a dietary supplement or a composition of a new food or dietary supplement or cosmetic compositions.

In a preferred embodiment, the composition according to the invention comprises (i) a mixture containing or alternatively consisting of said (a) at least one cetylated fatty acid (or mixture of cetylated fatty acids), wherein the fatty acid is C6-C21, C8-C18 or C14-C18 and it is saturated or unsaturated, and the specified (b) at least one antioxidant with a content of 0.001-0.5% or from 0.001 to 0.4%, or from 0.001 to 0.3%, or from 0.001 up to 0.2%, or from 0.001 to 0.1%, or 0.005-0.1%, or 0.01-0.08% by weight relative to the total weight of the composition.

Said (a) at least one cetylated fatty acid (or mixture of cetylated fatty acids) is a fatty acid esterified with cetyl alcohol (1hexadecanol, CH ₃ (CH ₂ ) ₁₅ OH) or a mixture of fatty acids esterified with cetyl alcohol.

Preferably, the fatty acid(s) of the at least one cetylated fatty acid is selected from the group consisting of, or alternatively, 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 containing 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 containing or alternatively consisting of cetylated myristic acid, cetylated oleic acid, cetylated linoleic acid and cetylated palmitic acid.

According to an embodiment of the composition of this invention containing (a), (b) and (c)-LIPOCET™ (according to any of the described embodiments), the mixture of fatty acids contains or, alternatively, consists of:

(a1) myristic acid: 30 to 55%, preferably 35 to 50%, more preferably about 41%;

(a2) oleic acid: 35 to 60%, preferably 40 to 55%, more preferably about 46%;

(a3) linoleic acid: 3 to 12%, preferably 5 to 10%, more preferably about 8%;

(a4) palmitic acid: 1 to 8%, preferably 2 to 6%, more preferably about 3%;

(a5-a9) various fatty acids (non-myristoleic acid): 1 to 4%, preferably 1.5 to 3%, more preferably about 1.8%; wherein the % is % by weight relative to the total weight of fatty acids from (a1) to (a9).

In one embodiment, said (a) contains a mixture of cetylated myristic acid and cetylated oleic acid, provided that said (a) does not contain cetylated myristoleic acid, in particular when the composition according to the invention is intended for use in methods of treating arthritis and inflammatory joint and musculoskeletal conditions. -motor pain.

In another embodiment, said (a) contains a mixture of cetylated myristic acid and cetylated oleic acid, which may also contain cetylated myristoleic acid, in particular when the composition according to the invention is intended for use in methods of treatment for protecting the gastric mucosa and regulating blood glucose levels.

Myristic acid is tetradecanoic acid ( _CH3 ( _CH2 ) _12COOH ), a saturated fatty acid with 14 carbon atoms, present in milk derivatives (butter, cream and cheese), coconut oil, palm kernel oil and some spices (nutmeg ). The myristic acid used in the invention can, for example, be selected from acids with 99% CAS 544-63-8 (EINECS 208-875-2) having the composition, % (GLC): lauric acid C12:0 - lower or equals 1; myristic acid C14:0 - greater than or equal to 99%; palmitic acid C16:0 - less than or equal to 1.

Oleic acid is cis-9-octadecenoic acid (CH ₃ (CH ₂ ) ₇ CH (CH ₂ ) ₇ COOH), a monounsaturated carboxylic acid with 18 carbon atoms, and is the most abundant component of most vegetable oils.

The oleic acid used in the invention can, for example, be selected from those in which at least 78% is oleic acid CAS 112-80-1 (EINECS 204-007-1), with a percentage composition (%GLC), for example: [lauric acid + myristic acid] C12:0+C14:0 - less than or equal to 0.5; oleic acid C 18:1 - greater than or equal to 78%; linoleic acid C18:2 - lower or

- 3 045244 is equal to 15, and others C18:3 are lower than or equal to 1.

The cetyl alcohol (1-hexadecanol) used in the invention can, for example, be selected from those corresponding to CAS 36653-82-4 (EINECS 253-149-0).

Preferably, when said (a) contains or consists of a mixture of cetylated myristic acid and cetylated oleic acid, the molar ratio between cetylated myristic acid and cetylated oleic acid is from 4:1 to 1:1, preferably it is from 3:1 to 1, 5:1, more preferably it is (2.0+0.2):1.

In a preferred embodiment, the composition according to the invention contains a mixture (I) containing, or alternatively consisting of:

(a) a mixture of cetylated myristic acid and cetylated oleic acid, preferably in a molar ratio of 4:1-1:1 or 3:1-1.5:1 or preferably (2.0 + 0.2):1, and (b ) at least one antioxidant containing 0.001-0.5% by weight, or from 0.001 to 0.3%, or from 0.001 to 0.2%, or from 0.001 to 0.1%, or 0.005-0.1% , or preferably 0.01-0.08% relative to the total weight of the composition.

The antioxidant contained in the composition of the invention together with (a) and optionally with (c) may be any antioxidant considered by one skilled in the art to be suitable for use in pharmaceutical compositions, medical device compositions, novel food products ( foods), additives or cosmetic compositions for the purposes presented in this description.

Antioxidants are often added to foods that primarily contain fat to delay the development of rancidity, which begins in the presence of oxygen. Natural antioxidants include flavonoids, polyphenols, ascorbic acid (vitamin C) and tocopherols (vitamin E). Synthetic antioxidants include butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ethoxyquinoline.

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

Tert-butylhydroquinone (TBHQ or tertiary butylhydroquinone) is used in the food industry as a preservative for unsaturated vegetable oils and many edible animal fats. TBHQ is approved as a food additive in the European Union with an acceptable daily intake (ADI) of 0.7 mg/kg body weight and is identified by code E319.

Aperoxide®TLA (registered trademark) is the trade name of an antioxidant effective in combating the phenomenon of rancidity of fat, used for example in the cosmetic field. Aperoxide®TLA is a mixture containing tocopherol ((±)-a-tocopherol) 10-15%, lecithin > 50%, ascorbyl palmitate 8-10% (an ester formed by ascorbic acid and palmitic acid, creating a fat-soluble form of vitamin C) and citric acid < 1% (INCI name: lecithin, tocopherol ((±)-a-tocopherol), ascorbyl palmitate, citric acid; CAS number: 8002-43-5, 10191-41-0, 137-66-6, 77 -92-9).

In a preferred embodiment, the composition according to the invention comprises a mixture (I) containing or alternatively consisting of said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids), wherein the fatty acid is C6-C21, C8-C18 or C14-C18 and is saturated or unsaturated, and (b) tert-butylhydroquinone (TBHQ), containing 0.001 to 0.5%, or 0.001 to 0.3%, or 0.001 to 0.2%, or 0.001 to 0.1%, or 0.005-0.1%, or 0.01-0.08% by weight relative to the total weight of the composition.

In a more preferred embodiment, the composition according to the invention comprises a mixture (I) containing, or alternatively consisting of:

(a) a mixture of cetylated myristic acid and cetylated oleic acid with a molar ratio of 3:1-1.5:1, preferably (2.0 + 0.2):1, and (b) tert-butylhydroquinone (TBHQ) containing weight 0.01% -0.08% relative to the total weight of the composition.

In another embodiment, in addition to (a) and optionally (b), mixture (I) according to the invention additionally contains (c) vegetable oil.

Preferably, said (c) vegetable oil is selected from the group consisting of, or alternatively consisting of, olive oil, sunflower oil, high oleic acid corn oil or mixtures thereof, any high oleic acid vegetable oil and mixtures thereof. Predominantly (c) is olive oil. Content by weight

- 4 045244 oleic acid and/or oleins in said oil is from 55 to 95%, preferably from 60 to

90%, even more preferably 65 to 85%, for example 70 to 80%.

In a preferred embodiment, mixture (I) according to the invention contains, or alternatively consists of, said (a) at least one cetylated fatty acid (or mixture of cetylated fatty acids), preferably a mixture of cetylated myristic acid and cetylated oleic acid, in a percentage by weight of 50 to 90% based on the total weight of the composition, preferably 60 to 85%, more preferably 70 to 80%;

of said (b) at least one antioxidant, preferably tert-butylhydroquinone (TBHQ), at a weight percentage of from 0.001 to 0.5% or from 0.001 to 0.3% or from 0.001 to 0.2% or from 0.001 up to 0.1% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%; and said (c) vegetable oil, preferably olive oil, in a weight percentage of from 9.5 to 50% relative to the total weight of the composition, preferably from 14.5 to 40%, more preferably from 19.5 to 30%.

In a preferred embodiment, mixture (I) according to the invention contains, or alternatively consists of:

(a) a mixture of cetylated myristic acid and cetylated oleic acid with a molar ratio of 4:1-1:1 or 3:1-1.5:1, preferably (2.0±0.2):1; preferably at a weight percentage of from 60 to 85% relative to the total weight of the composition, preferably from 70 to 80%;

(b) tert-butylhydroquinone (TBHQ) at a weight percentage of from 0.005 to 0.1% relative to the total weight of the composition, preferably from 0.01 to 0.08%; and (c) olive oil at a weight percentage of from 14.5 to 40% relative to the total weight of the composition, preferably from 19.5 to 30%.

In a more preferred embodiment, mixture (I) according to the invention contains, or alternatively consists of:

(a) a mixture of cetylated myristic acid and cetylated oleic acid with a molar ratio of (2.0 ± 0.2): 1 and a mass percentage of 70 to 80%;

(b) tert-butylhydroquinone (TBHQ) at a weight percentage of 0.01 to 0.08% based on the total weight of the composition; and (c) olive oil at a weight percentage of 19.5 to 30% based on the total weight compositions.

The subject matter of this invention is the compositions of the invention as defined herein (e.g., LIPOCET™), comprising: (i) a mixture of the invention containing or consisting of (a) and (b) and optionally (c) (as defined above ), and optionally (II) at least one food grade or pharmaceutical grade additive and/or excipient, said compositions being intended for use as a drug when administered, in a therapeutically effective amount, to a subject in need thereof.

The subject matter of this invention is compositions according to the invention (for example, LIPOCET™) containing (a) and (b) and optionally (c) (according to all embodiments specified in this description) for use in prophylactic and/or therapeutic and/or symptomatic treatment of diseases, symptoms or disorders, such as (i) rheumatoid arthritis of inflammatory and non-inflammatory origin, in particular osteoarthritis; (ii) inflammatory joint conditions other than rheumatoid arthritis; (iii) psoriasis, lupus, periodontal disease, or cardiovascular or heart disease; (iv) post-traumatic musculoskeletal and musculoskeletal disorders, including sports-related injuries such as pubalgia or sports pubalgia (pubalgia refers to chronic pain in the abdomen and groin); (v) degenerative joint diseases, preferably osteoarthritis, gonarthrosis, coxarthrosis and/or (vi) inflammatory/traumatic conditions associated with tendon and muscle, when administered to a subject in need thereof.

Preferably, the compositions of the invention for use in the method of treating diseases or disorders (i) to (vi) as above comprise (I) a mixture containing, or alternatively consisting of:

(a) mixtures of cetylated myristic acid and cetylated oleic acid with a molar ratio of 3:1-1.5:1, preferably (2.0±0.2):1, and (b) tert-butylhydroquinone (TBHQ) containing weight 0.01-0.08% relative to the total weight of the composition.

Preferably, the compositions of the invention for use in the method of treating diseases or disorders (i) to (vi) as defined above comprise (I) a mixture containing, or alternatively consisting of:

(a) mixtures of cetylated myristic acid and cetylated oleic acid with molar

- 5 045244 ratio (2.0±0.2): 1 and at a mass percentage from 70 to 80%;

(b) tert-butylhydroquinone (TBHQ) at a weight percentage of 0.01 to 0.08% based on the total weight of the composition; and (c) olive oil at a weight percentage of 19.5 to 30% based on the total weight compositions.

Said compositions of the invention for use in a method of treating diseases, symptoms or disorders listed in (i) to (vi) as defined above are administered to said subject preferably through the topical or transdermal route; preferably via local route. Preferably, the cetylated fatty acids (a1) to (a7) contained in mixture (a) have a molecular weight of 420 to 510 Da. This molecular weight range allows mixture(s) and thus the composition of the invention to effectively cross the cutaneous barrier (skin), facilitating its absorption through the local route.

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

The subject of this invention are compositions according to the invention (for example, LIPOCET™) containing (a) and (b) and optionally (c) (according to all embodiments given in this description) for use in a method of prophylactic and/or therapeutic and/ or symptomatic treatment of (i.i) symptoms and/or disorders due to or associated with damage to the gastric mucosa (treatment to protect the gastric mucosa), (i.ii) diabetes and disorders or symptoms arising from or related to diabetes, (i .iii) diseases and/or disorders other than diabetes arising from or associated with high blood glucose levels when administered to a subject in need.

Said (i.i) symptoms or disorders caused by or associated with damage to the gastric mucosa (protection of the gastric mucosa) are selected from the group including or alternatively consisting of: gastric ulcers, gastroesophageal reflux (GERD), heartburn.

The predominantly effective and long-lasting protection of the gastric mucosa exhibited by the use of the composition according to the invention in rats was observed in vivo in rats. The gastric mucosa is known to have a barrier effect and consists of a mucosal layer of about 0.2 mm that covers the gastric mucosa membrane. The purpose of this barrier is to protect the gastric epithelium from the damaging effects of HCl produced by the gastric parietal cells. It occurs due to the production of droplets of high molecular weight (hence, very viscous) mucoprotein by the cells of the surface of the stomach and due to the production of bicarbonate.

Thus, the subject of the invention is a composition (see Table 2 or samples 2-6) for use in a method of (prophylactic and/or therapeutic) therapy for a disorder or symptom caused by or associated with damage to the gastric mucosa, selected from the group consisting of or , alternatively, consisting of: stomach ulcers, gastroesophageal reflux (GERD), heartburn.

Said (i.iii) diseases and/or disorders other than diabetes, caused by or associated with high blood glucose levels, are selected from the group including or alternatively consisting of: hyperglycemia, chronic liver disease, obesity.

Advantageously, it has been found that by administering in vivo to rats a composition according to the invention, for example the composition of sample 4, at high doses, for example at doses above 1600 mg/kg, the blood glucose level of the exposed rats is statistically significantly reduced.

Thus, the subject of this invention is a composition (see Table 2 or Samples 2-6) for use in a method of treating diabetes or a disease and/or disorder other than diabetes caused by or associated with high blood glucose levels, e.g. cases of hyperglycemia, chronic liver disease and obesity.

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

Preferably, the compositions according to the invention for use in the method of treating diseases or disorders (i.i) to (i.iii) as indicated above contain (I) a mixture containing or, alternatively, consisting of:

(a) a mixture of cetylated myristic acid and cetylated oleic acid, preferably in a molar ratio of 4:1-1:1 or 3:1-1.5:1 or preferably (2.0±0.2):1, and (b ) tert-butylhydroquinone (TBHQ) with a content of 0.01%-0.08% by weight relative to the total weight of the composition.

The compositions of the invention may be formulated in a liquid form such as a solution, biphasic liquid system or emulsion, suspension, syrup, spray, ointment, oil or drink or alternatively in a semi-solid form such as a gel, soft gel, cream, foam, or alternatively in solid form such as powder, spray powder (spray-dried), granules, microgranules, flakes, aggregates, cheek soluble sticks, tablets, effervescent tablets, capsules, suppositories, bars or foods and equivalent forms known to one skilled in the art.

For example, the compositions of the invention may be formulated for oral administration in capsule form comprising: (a) a mixture of cetylated fatty acids of the invention, (b) at least one % antioxidant of the invention and excipients such as, for example, gelatin , glycerin and/or preservatives (eg LIPOCET™). In one embodiment, the composition is a 300 mg bovine gelatin capsule composition.

Advantageously, when the composition according to the invention is intended for oral administration, it is formulated in solid or liquid form, more preferably in the form of capsules or oil or powder or spray powder (spray drying) or spray liquid or beverage or food product.

Advantageously, when the composition according to the invention is intended for oral administration, it is formulated in solid or liquid form, more preferably in the form of an oil or powder or spray powder (spray drying) or spray liquid.

Advantageously, when the composition according to the invention is intended for topical use, it is formulated in a semi-solid form, more preferably in the form of a cream or gel.

Advantageously, when the composition according to the invention is intended for transdermal use, it is formulated in a form suitable for application by patch.

The subject matter of this invention is methods for treating the diseases, symptoms or disorders listed in (i) to (vi) above, preferably by topical or transdermal administration or, alternatively, for treating the diseases, symptoms or disorders listed in (i.i) - ( above). i.iii), preferably by oral administration, to a subject requiring an effective amount of one of the compositions according to the invention containing (a), or (a) and (b), or (a) and (b) and (c), described in this description.

The appropriate composition of the composition according to the invention depends, for example, on the condition to be treated/prevented, on the severity and course of the condition, on whether the composition is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the composition and discretion attending physician.

When the composition of the invention is administered by topical application, the amount of the composition administered is from about 1 to 15 mg/kg of the subject's body weight per day, preferably 3-10 mg/kg, more preferably 5-8 mg/kg. In addition, the composition according to the invention, for example a cream or gel, is applied by topical application to an area ranging from 1 to 25 cm in diameter on the treated area, depending on the anatomical area in question (for example, fingers: 3 cm, back: 20 cm).

When the composition of the invention is administered orally, the amount of the composition administered is from about 100 to 5000 mg/kg of the subject's body weight per day, preferably from 200 to 4500 mg/kg, more preferably from 300 to 4000 mg/kg.

The composition of the invention is suitably administered to the subject all at once or in multiple treatments.

The composition may be administered as a single treatment or in combination with other compositions or treatments (i.e., as a coadjuvant) useful for the prophylactic and/or therapeutic and/or symptomatic treatment of the diseases, symptoms and/or disorders listed in (i) - (vi) and/or listed in (i.i) - (i.iii) as described herein.

The subject of this invention is the non-therapeutic use of a composition according to the invention containing (a), or (a) and (b), or (a) and (b) and (c), for protecting the gastric mucosa, wherein said composition is preferably formulated to oral administration.

The composition according to the invention protects the gastric mucosa in such a way as to prevent its damage and thus the occurrence of disorders associated with the gastric region, such as, for example, gastric ulcers, gastroesophageal reflux (GERD), heartburn.

In the context of the invention, the expression subjects is used to refer to people or animals (eg, domestic animals such as dogs or cats or other mammals). Preferably, the compositions of the invention are for use in methods of treating humans.

The expression method of treatment in the context of the invention is used to denote an action involving the administration of a substance or mixture of substances or combinations thereof for the purpose of eliminating, reducing/reducing or preventing a pathology or disease and its symptoms or disorders.

The expression medical device in the context of the invention is used in accordance with the meaning established by Italian Legislative Decree No. 46 of 24 February 1997 (or according to the new Medical Devices Regulation (UE) 2017/745 (MDR)), that is, it indicates a substance or other product, used alone or in combination, intended by the manufacturer for use in humans for the diagnosis, prevention, control, therapy or mitigation of disease, and the product does not produce a primary effect in the human body or on the human body for which it is intended, either by pharmacological or immunological means or by a metabolic process, but its function can be coadjuvated by such means.

The compositions according to the invention optionally contain (II) at least one additive and/or excipient of food or pharmaceutical grade, such as a substance devoid of therapeutic activity suitable for pharmaceutical or food use, such as, for example, diluents, solvents (for example, water, glycerin, ethyl alcohol), solubilizers, thickeners, sweeteners, anti-caking agents, flavor enhancers, colorants, lubricants, surfactants, antimicrobials, antioxidants, preservatives, pH-stabilizing buffers, acidifiers and all excipients known to one skilled in the art.

In addition to (a), (b) and optionally (c), the compositions according to the invention may additionally contain other active ingredients, such as, for example, anti-inflammatory agents, probiotics, antacids, products for the treatment of joint and/or muscle disorders, vitamins groups B and E, mineral salts, painkillers, folic acid or folates, menthol, glucosamine, chondroitin, methylsulfonylmethane (MSM), essential oils.

Unless otherwise indicated, the indication that a composition contains one or more components or substances means that other components or substances may be present in addition to the one or those specifically indicated.

Unless otherwise indicated, the expression that the composition includes a component in an amount ranging from x to y is used to indicate that the specified component may be present in the composition in all amounts within the specified range, including, even if not stated, extreme values of the specified range.

The subject of this invention is a first method for producing said(a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids) as defined herein, comprising the steps (Fig. 1):

(I) contacting at least one fatty acid, cetyl alcohol and a metal catalyst in chamber 3 of reactor 2 in the absence of a solvent to obtain reaction mixture 15; followed by (II) saturating the container (3) with an inert gas, bringing the container 3 to a pressure P1 of approximately 1 atmosphere (0.1 MPa), feeding through said chamber 3 a flow of said inert gas, preferably a flow of from 5 to 0 .05 m ³ /h, more preferably 0.5 m ³ /h; followed by (III) first heating said reaction mixture 15 to a temperature T1 of 120 to 200°C, preferably 145 to 175°C, more preferably 160°C, at a pressure P1 of about 1 atm ( 0.1 MPa), and in the presence of a flow of inert gas to initiate the esterification reaction with the initial formation of the specified cetylated fatty acid or a mixture of cetylated fatty acids and esterification water; followed by (IV) maintaining said reaction mixture 15 under stirring at said temperature T1 and pressure P1 for a period of time ranging from 10 minutes to 2 hours, preferably from 30 minutes to 1.5 hours, more preferably for 1 hour; followed by (V) subjecting said reaction mixture 15 to a second heating until a temperature T2 of 201 to 260° C., preferably 210 to 240° C., more preferably 220° C. is reached, at a pressure P1 of about 1 atm ( 0.1 MPa), and in the presence of a flow of inert gas to continue the esterification reaction with the additional formation of the specified cetylated fatty acid or a mixture of cetylated fatty acids and esterification water; followed by (VI) maintaining said reaction mixture 15 with stirring at said temperature T2 and pressure P1 and in the presence of a flow of inert gas for a period of time ranging from 4 to 12 hours, preferably from 6 to 10 hours, more preferably for 8 h; preferably until the acidity of the reaction mixture 15, measured using the AOCS Official Method Cd 3d-63, is stable over time and ranges from 5 mg KOH/g to 8 mg KOH/g; followed by (VII) application in chamber 3 of a vacuum program which reduces the reaction pressure until a reduced pressure P2 of 100 to 10 mbar (10 to 1 kPa), preferably 80 to 30 mbar (8 to 3 kPa) is achieved , more preferably 50 mbar (5 kPa); followed by (VIII) maintaining said reaction mixture 15 with stirring at said reduced pressure P2 and in the presence of a flow of inert gas for a period of time ranging from 30 minutes to 4 hours, preferably from 1 to 2 hours, more preferably for 2 hours ; preferably until the acidity of the reaction mixture 15, measured using the AOCS Official Method Cd 3d-63, is between 4.5 mg KOH/g and 3.5 mg KOH/g, preferably 4 mg KOH/g,

- 8 045244 to achieve complete formation of said at least one unrefined cetylated fatty acid or a mixture of unrefined cetylated fatty acids (MI).

By the combined application of an inert gas flow during part or all of the ester formation stage and a vacuum program in the reactor chamber only in the last part of the said ester formation stage, this first method advantageously allows for the removal of esterification water and complete conversion of fatty acids to esters in excellent yield . This first method is easy to use and cost-effective, since there is no need to apply a vacuum program for the entire period of the esterification reaction.

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

In embodiment (FR2) of the said first method, said inert gas flow is supplied using a purge means 7 located in a portion of the volume of the chamber 3 located above the reaction mixture 15 (FIG. 1).

In embodiment (FR3) of the said first method, the esterification water removed from the chamber 3 during the esterification reaction by using the inert gas stream and the vacuum program described above is condensed in the horizontal condenser 11 and collected in a container 13 after flowing through the vertical condenser 16 (Fig. 2). and 3).

In embodiment (FR4) of the said first method, said horizontal capacitor 11 is maintained at a temperature ranging from 10 to 40° C., and it is connected to said container 3 by a vertical capacitor 16, which is maintained at a temperature ranging from 70 to 90° C. ( Fig. 3).

In embodiment (FR5) of said first method, in addition to steps (I) to (VIII), said first method further comprises the step (IX) of filtering said at least one unrefined cetylated fatty acid or a mixture of unrefined cetylated fatty acids (M1) obtained from step (VIII), using bleaching earth and/or filter earth in the filter press 23 (Fig. 3), to obtain at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf), in which the metal catalyst at substantially absent or present in an amount less than or equal to 2% by weight, preferably in an amount of from 0.01 to 1.5%, even more preferably in an amount of from 0.05 to 1%, for example 0.5% by weight relative 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 zinc metal, is preferably used in powder form, preferably in the form of powdered zinc metal. The amount of catalyst added is from 0.01 to 0.5% by weight relative to the total weight of the reaction reagents (i.e. fatty acids or a mixture of fatty acids + cetyl alcohol), preferably from 0.05 to 0.25% by weight, further more preferably 0.1% by weight.

In embodiment (FR6) of said first method, in addition to steps (I) to (IX), said first method further includes a step (X) (deodorization step) of treating said at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf ) in reactor 27 (FIG. 3) at a temperature T3 of 150 to 200°C, preferably 165 to 180°C, more preferably 180°C, and a reduced pressure P3 of 1 to 20 mbar (0 .1 to 2 kPa), preferably 3 to 5 mbar (0.3 to 0.5 kPa), in the presence of a stream of steam for a period of 1 to 5 hours, preferably 2 to 3 hours, for producing at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids (Mf).

The subject of this invention is a second method for preparing a composition according to the invention comprising said (a) at least one cetylated fatty acid (or a mixture of cetylated fatty acids) as defined herein, said second method comprising the step (XI) of mixing said at least at least one filtered cetylated fatty acid (Mf) obtained according to embodiment FR5 in combination with any of FR1-FR4 or, alternatively, said at least one refined cetylated fatty acid (Mf) obtained according to embodiment FR6 in combination with any of embodiments FR1 -FR5, with (b) at least one antioxidant (as defined in this invention), wherein said (b) at least one antioxidant is mixed in an amount by weight of from 0.001 to 0.5% or from 0.001

- 9 045244 up to 0.4%, or from 0.001 to 0.3%, or from 0.001 to 0.2%, or from 0.001 to 0.1%, relative to the total weight of the composition (composition according to the invention), preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%.

In a preferred embodiment, said second method of preparing a composition according to the invention comprises the step of mixing said at least one filtered cetylated fatty acid (Mf) or at least one refined cetylated fatty acid (MF) containing or alternatively consisting of a mixture of cetylated myristic acid and cetylated oleic acid, preferably at a molar ratio of 4:1-1:1 or 3:1-1.5:1 or (2.0±0.2):1 or about 1:1, and wherein (b) at least one antioxidant is tert-butylhydroquinone (TBHQ) with a wt.% content of from 0.001 to 0.5% or from 0.001 to 0.4% or from 0.001 to 0.3% or from 0.001 to 0.2 %, or from 0.001 to 0.1% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%.

In one 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, is mixed with said (c) before being introduced into the deodorization step (X). vegetable oil (as defined in this invention), preferably olive oil or corn oil.

In one embodiment, before deodorizing 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 step (XII) of mixing said (Mf) with said (c) vegetable oil, preferably with olive oil or corn oil, to form a mixture containing (Mf) and (c), which is then introduced into the deodorization step (X).

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

In principle, the method according to the invention can be summarized schematically with the following embodiments:

1) MI (stage I-VIII) -> Mf (stage IX) -> MF (stage X) + (b) (stage (XI)) + (c) (stage (XII)).

2) MI (stage I-VIII) -> Mf (stage IX) + (c) (stage XII) -> MF (stage X) + (b) (stage (XI)).

3) MI (stage I-VIII) -> Mf (stage IX) + (b) (stage XI) -> MF (stage X) + (c) (stage (XII)).

Said (a) at least one cetylated fatty acid (or mixture of cetylated fatty acids) is a fatty acid esterified with cetyl alcohol (1hexadecanol, CH ₃ (CH ₂ ) ₁₅ OH).

Preferably, the fatty acid of said (a) at least one cetylated fatty acid is selected from the group consisting of, 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 containing or alternatively consisting of cetylated myristic acid, cetylated oleic acid, cetylated linoleic acid and cetylated palmitic acid; even more preferably, said (a) contains, or alternatively consists of, a mixture of cetylated myristic acid and cetylated oleic acid.

According to one embodiment of the composition according to the invention, comprising (a), (b) (at least one antioxidant) and (c) (for example, LIPOCET™) (according to any of the described embodiments), the mixture of fatty acids contains or, alternatively, consists of :

(a1) myristic acid: 30 to 55%, preferably 35 to 50%, more preferably 40 to 45%; for example 41%, or 42%, or 43%, or 44%;

(a2) oleic acid: 35 to 60%, preferably 40 to 55%, more preferably 45 to 50%; for example, 46%, or 47%, or 48%, or 49%;

(a3) linoleic acid: 3 to 12%, preferably 5 to 10%, more preferably 6 to 9%; for example 7%, or 8%, or 8.5%;

- 10 045244 (a4) palmitic acid: from 1 to 8%, preferably from 2 to 6%, more preferably from 3 to 5%; for example 3.5%, or 4%, or 4.5%.

Additionally, said (a) + (b) + (c) may contain, together with one or more of (a1)(a4):

(a5) lauric acid from 0.1 to 0.3%, for example 0.2%;

(a6) palmitoleic acid from 0.3 to 0.5%; for example 0.4%;

(a7) stearic acid from 0.7 to 0.9%, for example 0.8%;

(a8) eicosanoic acid from 0.05 to 0.15%, for example 0.1%;

(a9) eicosenoic acid from 0.2 to 0.4%, for example 0.3%.

The total amount of said (a5) to (a9) is preferably 1 to 4%, preferably 1.5 to 3%; even more preferably 2 to 2.5%, for example 1.8% by weight.

Chromatographic separation of said mixture (a) + (b) + (c) results in a wax fraction (a1) - (a9) comprising (in wt.%) from 60 to 80%, preferably from 65 to 75%; with the remainder being the glyceride fraction. The chain length distribution in the specified wax fraction is as follows: 30 carbon atoms - from 60 to 70%; 32 carbon atoms - from 1 to 3%; and 34 carbon atoms - from 25 to 35%. In said wax fraction, the amount of myristic acid is from 60 to 70%, while the amount of oleic acid is from 25 to 35% by weight.

Embodiments of mixture (a)+(b)+(c) are shown below.

Sample 2: Composition according to the invention comprising a mixture of cetylated myristic acid and cetylated oleic acid at a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight relative to the total weight of the sample);

olive or corn oil (approximately 25% by weight relative to the total weight of the sample);

tert-butylhydroquinone (TBHQ) (0.02% by weight based on total sample weight) and optional excipients and/or additives.

Sample 3: Composition according to the invention, comprising a mixture consisting primarily of cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight relative to the total weight of the sample);

olive or corn oil (approximately 25% by weight relative to the total weight of the sample),

Aperoxide®TLA (0.05% by weight based on total sample weight) and optionally excipients and/or additives.

Sample 4: Composition containing a mixture consisting primarily of cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight based on the total weight of the sample), olive or corn oil (approximately 25% by weight based on the total sample weight), a mixture of tocopherols or tocopheryl acetate (0.001-0.3%), and optionally excipients and/or additives.

Sample 5: Composition according to the invention mixture: (a1)+(a2)+(a3) at a molar ratio of (a1):(a2):(a3)=4:4:l (approximately 80% by weight relative to the total weight sample);

olive or corn oil (approximately 20% by weight relative to the total weight of the sample);

a mixture of tocopheryl acetate and wheat germ oil (Tritucum vulgare) in a mass ratio of 1:25; and possibly excipients and/or additives.

Sample 6: Composition according to the invention mixture: (a1)+(a2)+(a3)+(a4) with molar ratio (a1):(a2):(a3):(a4)=4:4:1:0, 5 (approximately 85% by weight relative to the total weight of the sample);

olive or corn oil (approximately 15% by weight relative to the total weight of the sample);

a mixture of tocopheryl acetate and wheat germ oil (Tritucum vulgare) in a mass ratio of 1:25; and possibly excipients and/or additives.

Embodiments (An) in accordance with the first subject of the present invention are presented below:

Anl. A composition comprising (I) and optionally (II), in which:

(I) is a mixture comprising, or alternatively consisting of:

(a) at least one cetylated fatty acid, wherein said fatty acid is a saturated or unsaturated fatty acid having a carbon number of from C6 to C21, and (b) at least one antioxidant at a weight percentage of from 0.001 to 0, 5% relative to the total weight of the composition; and (II) represents at least one additive and/or excipient of a food or pharmaceutical

- 11 045244 tic variety.

An2. The composition according to An1, wherein said fatty acid(s) of at least one cetylated fatty acid is selected from the group consisting of, or alternatively consisting of:

myristic acid, oleic acid and mixtures thereof;

preferably (a) contains, or alternatively consists of, a mixture of cetylated myristic acid and cetylated oleic acid.

An3. Composition according to An1 or An2, wherein said (b) at least one antioxidant is selected from the group consisting of, or alternatively consisting of, tert-butylhydroquinone (TBHQ), a mixture containing tocopherol, lecithin, ascorbyl palmitate and citric acid (Aperoxide®TLA ), a mixture of tocopherols and natural rosemary extract; preferably (b) is tert-butyl hydroquinone (TBHQ).

An4. The composition according to any one of An1 to An3, wherein said (b) at least one antioxidant is present at a weight percentage of 0.005 to 0.1% relative to the total weight of the composition, preferably 0.01 to 0.08%.

An5. A composition according to any one of An1 to An4, wherein said (a) contains or alternatively consists of a mixture of cetylated myristic acid and cetylated oleic acid, wherein the molar ratio of said cetylated myristic acid to cetylated oleic acid is from 4:1 to 1: 1, preferably from 3:1 to 1.5:1, more preferably it is (2.0±0.2):1; and said (b) is tert-butylhydroquinone (TBHQ) and is present at a weight percentage of from 0.001 to 0.5% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0 .08%.

An6. A composition according to any one of An1-An5, where in addition to (a) and (b) said composition additionally contains (c) vegetable oil; preferably said vegetable oil is selected from the group consisting of, or alternatively consisting of, olive oil and high oleic acid sunflower oil.

An7. A composition according to any one of An1 to An6, wherein said composition contains said(a) at least one cetylated fatty acid, preferably a mixture of cetylated myristic acid and cetylated oleic acid at a weight percentage of from 50 to 90% relative to the total weight of the composition, preferably from 60 to 85%, more preferably from 70 to 80%;

said (b) antioxidant, preferably tert-butylhydroquinone (TBHQ), at a weight percentage of from 0.001 to 0.5% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0, 08%; and said (c) vegetable oil, preferably olive oil, in a weight percentage of 9.5 to 50% relative to the total weight of the composition, preferably 14.5 to 40%, more preferably 19.5 to 30%.

An8. A composition according to any one of An1 to An7, intended for use as a medicine.

An9. Composition for use according to An8, where said composition is intended for use in a method for the prophylactic and/or therapeutic and/or symptomatic treatment of (i) rheumatoid arthritis of inflammatory and non-inflammatory origin, in particular osteoarthritis; (ii) inflammatory joint conditions other than rheumatoid arthritis; iii) psoriasis, lupus, periodontal disease or cardiovascular or heart disease; iv) post-traumatic diseases of the musculoskeletal and musculoskeletal system, including sports-related injuries; (v) degenerative joint diseases, preferably arthrosis, gonarthrosis, coxarthrosis and/or (vi) inflammatory/traumatic conditions associated with tendons and muscles, by administering it to a subject in need.

An10. A composition for use according to An8 or An9, wherein said composition is prepared for topical or transdermal use; preferably for topical use.

Embodiments (Bn) in accordance with the second aspect of the present invention are presented below:

Bn1. Composition including:

(I) a mixture comprising, or alternatively consisting of (a) at least one cetylated fatty acid, and optionally (II) at least one pharmaceutical or food grade additive and/or excipient, wherein said composition is intended for use in a method of prophylactic and/or curative therapy (i.i) of a disorder or symptom caused by and/or associated with damage to the gastric mucosa, selected from the group consisting of or alternatively consisting of gastric ulcers, gastroesophageal reflux (GERD), heartburn;

(i.ii) diabetes mellitus;

- 12 045244 (i.iii) a disease and/or disorder other than diabetes, caused by or associated with high blood glucose levels, selected from the group consisting of, or alternatively consisting of:

hyperglycemia, chronic liver diseases, obesity;

by administering it to a subject in need.

Bn2. Composition for use according to Bn1, wherein said fatty acid(s) of at least one cetylated fatty acid is selected from the group containing or alternatively consisting of: lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid , linoleic acid, eicosanoic acid and mixtures thereof; preferably myristic acid, oleic acid or cetylated myristic acid and cetylated oleic acid.

Bn3. Composition for use according to Bn2, wherein in said mixture of cetylated myristic acid and cetylated oleic acid, the molar ratio of cetylated myristic acid to cetylated oleic acid is from 4:1 to 1:1, preferably from 3:1 to 1.5:1, more preferably it is (2.0 ± 0.2): 1.

Bn4. A composition for use according to any one of Bn1-Bn3, wherein said (I) mixture containing said (a) further contains (b) at least one antioxidant.

Bn5. A composition for use according to Bn4, wherein said (b) at least one antioxidant is selected from the group consisting of or alternatively consisting of tert-butylhydroquinone (TBHQ), a mixture containing tocopherol, lecithin, ascorbyl palmitate and citric acid (Aperoxide®TLA ), a mixture of tocopherols and natural rosemary extract; preferably (b) is tert-butyl hydroquinone (TBHQ).

Bn6. A composition for use according to Bn4 or Bn5, wherein said (b) at least one antioxidant is present at a weight percentage of from 0.001 to 0.5% relative to the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%.

Bn7. A composition for use according to any one of Bn1-Bn6, wherein said (I) mixture contains or alternatively consists of said mixture of cetylated myristic acid and cetylated oleic acid;

preferably wherein the molar ratio of cetylated myristic acid to cetylated oleic acid is from 4:1 to 1:1, preferably it is from 3:1 to 1.5:1, more preferably it is (2.0 ± 0.2): 1; and said (b) tert-butylhydroquinone (TBHQ);

preferably wherein (b) is present at a weight percentage of from 0.001 to 0.5% based on the total weight of the composition, preferably from 0.005 to 0.1%, more preferably from 0.01 to 0.08%.

Bn8. A composition according to any one of Bn1-Bn7, where in addition to (a) and (b) said composition additionally contains (c) vegetable oil; preferably, said vegetable oil is selected from the group consisting of, or alternatively, olive oil, high oleic sunflower oil, and mixtures thereof.

Bn9. A composition for use according to any one of Bn1 to Bn8, wherein said composition is prepared for oral administration.

Bn10. A composition for use according to any one of Bn1 to Bn9, wherein said composition is intended for use as a coadjuvant of one or more other compositions for use in prophylactic and/or therapeutic therapy (i.i), (i.ii) or (i.iii).

Embodiments (Cn) in accordance with the third aspect of the present invention are presented below:

Cn1. A method for producing (a) at least one cetylated fatty acid or a mixture of cetylated fatty acids, comprising the steps of:

(I) contacting at least one fatty acid, cetyl alcohol and a metal catalyst in the chamber (3) of the reactor (2) in the absence of a solvent to obtain a reaction mixture (15); followed by (II) saturating the container (3) using an inert gas, bringing the container (3) to a pressure P1 of approximately 1 atm (0.1 MPa) by introducing a stream of said inert gas through said chamber (3); followed by (III) first heating said reaction mixture (15) until a temperature T1 of 120 to 200° C. is reached, at a pressure P1 of approximately 1 atm (0.1 MPa) and in the presence of a flow of inert gas to initiating an esterification reaction to initially form said cetylated fatty acid or a mixture of cetylated fatty acids and esterified water; followed by (IV) maintaining said reaction mixture (15) under stirring at said temperature T1 and pressure P1 for a period of time ranging from 10 minutes to 2 hours; followed by (V) carrying out a second heating of said reaction mixture (15) until a temperature T2 of 201 to 260°C is reached, at a pressure P1 of approximately 1 atm (0.1 MPa), and in the presence

- 13 045244 flow of inert gas to continue the esterification reaction with the additional formation of the specified cetylated fatty acid or a mixture of cetylated fatty acids and esterification water; followed by (VI) maintaining said reaction mixture (15) under stirring at said temperature T2 and pressure P1 and in the presence of a flow of inert gas for a period of time ranging from 4 to 12 hours;

(VII) applying in the chamber (3) a vacuum program that reduces the reaction pressure until a reduced pressure P2 of 100 to 10 mbar (10 to 1 kPa) is achieved;

(VIII) maintaining said reaction mixture (15) with stirring at said reduced pressure P2 and in the presence of a flow of inert gas for a period of time ranging from 30 minutes to 4 hours to achieve complete formation of said at least one unrefined cetylated fatty acid, or mixtures of unrefined cetylated fatty acids (MI).

Cn2. Method according to Cn1, in which said step (VI) of maintaining said reaction mixture (15) with stirring at said temperature T2 and pressure P1 and in the presence of a flow of inert gas for a period of time ranging from 6 to 12 hours is carried out until the acidity value is reached reaction mixture (15), measured using AOCS Official Method Cd 3d-63, stable over time and ranging from 5 mg KOH/g to 8 mg KOH/g; and said step (VIII) of maintaining said reaction mixture (15) with stirring at said reduced pressure P2 and in the presence of a flow of inert gas for a period of time ranging from 30 minutes to 4 hours is carried out until the acidity value of the reaction mixture (15) is obtained, measured using the AOCS Official Method Cd 3d-63, ranging from 4.5 mg KOH/g to 3.5 mg KOH/g, to achieve complete formation of the at least one unrefined cetylated fatty acid or mixture of unrefined cetylated fatty acids ( MI).

Cn3. The method according to Cn1 or Cn2, wherein said at least one fatty acid is selected from the group consisting of, 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 myristic acid, oleic acid and a mixture of myristic acid and oleic acid.

Cn4. The method according to any of Cn1-Cn3, in which the specified flow of inert gas is applied using a purging means (7) located in a part of the volume of the chamber (3) located above the reaction mixture (15).

Cn5. Method according to any one of Cn1-Cn4, in which the esterification water withdrawn from the chamber (3) during the esterification reaction by applying an inert gas flow and a vacuum program is condensed in a horizontal condenser (11) and collected in a container (13) after flowing through a vertical capacitor (16).

Cn6. The method according to any one of Cn1-Cn5, further comprising step (IX) (filtration step) of filtering said at least one unrefined cetylated fatty acid (M1) obtained from step (VIII) on bleaching earth and/or filter earth in a filter press (23 ), to produce at least one filtered cetylated fatty acid (Mf), in which the metal catalyst is absent or present in an amount of less than 2% by weight relative to the weight of the filtered cetylated fatty acid or mixture of filtered cetylated fatty acids (Mf).

Cn7. The method according to any one of Cn1-Cn6, further comprising, after step (IX), step (X) (deodorization step) of treating said at least one filtered cetylated fatty acid (Mf) in a reactor (27) at a temperature T3 of 150 to 200° C., and a reduced pressure of 1 to 20 mbar (0.1 to 2 kPa), in the presence of a stream of steam for a period of 1 to 5 hours, to obtain refined cetylated fatty acid ( Mf).

Cn8. A method of producing a composition containing said (a) at least one cetylated fatty acid, comprising the step of mixing at least one filtered cetylated fatty acid (Mf) obtained according to any one of embodiments Cn1-Cn6, or, alternatively, said at least one a refined cetylated fatty acid (Mf) prepared according to any one of embodiments Cn1-Cn7, with at least one antioxidant, wherein said at least one antioxidant is mixed at a weight percentage of from 0.001 to 0.5% relative to the total weight of the composition, preferably 0.005 to 0.1%, more preferably 0.01 to 0.08%.

Cn9. A method for preparing a composition according to Cn8, wherein said at least one filtered cetylated fatty acid (Mf) or at least one refined cetylated fatty acid (Mf) contains or, alternatively, consists of a mixture of cetylated myristic acid and cetylated oleic acid acid, and wherein said at least one antioxidant is tert-butylhydroquinone (TBHQ).

Cn10. A method for producing a composition according to Cn8 or Cn9, in which, before step (X), said at least one filtered cetylated fatty acid (Mf) obtained from step (IX), preferably a mixture of filtered cetylated myristic acid and cetylated oleic acid, is subjected to step (XII ) mixing said at least one filtered cetylated fatty acid (Mf) with a vegetable oil, preferably olive oil, to form a mixture containing (Mf) and vegetable oil, which is then subjected to step (X).

Experimental part 1

In experimental part 1, the stability of the compositions according to the invention, including or not including an antioxidant, was analyzed. All compositions presented in table. 2 and samples 1, 2 and 3 were tested using the same method described below. In particular, the tests carried out on samples 2 and 3 (according to the invention) and on sample 1 (comparative sample) are presented by way of non-limiting examples. This is due to the fact that the stability results obtained with the compositions presented in table. 2 are mutually comparable with the results for samples 2 and 3 due to the presence of at least one antioxidant in the tested compositions.

Material

Initial condition: The mass percentage (%) for samples 1, 2 and 3 refers to the total mass of the sample.

Sample 1 (Comparative Composition): A composition comprising a mixture containing essentially cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight based on the total weight of the sample) , olive or corn oil (approximately 25% by weight relative to the total weight of the sample).

Sample 1 does not contain antioxidants.

Sample 2: composition according to the invention, comprising a mixture containing mainly cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight relative to the total weight of the sample);

olive or corn oil (approximately 25% by weight relative to the total weight of the sample); tert-butylhydroquinone (TBHQ) (0.02% by weight based on total sample weight) and optional excipients and/or additives.

Sample 3: composition according to the invention, comprising:

a mixture containing primarily cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight based on the total weight of the sample);

olive or corn oil (approximately 25% by weight relative to the total weight of the sample),

Aperoxide®TLA (0.05% by weight based on total sample weight) and excipients and/or additives.

Methodology

After a period of time T0, samples 1, 2 and 3 were kept at a controlled temperature of 18°C. Approximately every 40-45 days (ie, at times T1, T2, T3 and T4), a sample was taken from each sample and the peroxide value (PV) and Kreiss test were determined.

Peroxide numbers

The peroxide value (PV) was determined in a standard manner in accordance with the ISO 3960 method (fourth edition 2007-07-15, revised version 2009-05-15 prepared by Technical Committee ISO/TC 34/SC), as known to one skilled in the art.

This test is a classic analytical chemical method for determining the degree of rancidity of edible oil. The test quantifies the peroxide value (PV), which is an indicator of the amount of primary products of fatty acid autoxidation.

The method involves (briefly) the following: an oil sample is dissolved in isooctane and glacial acetic acid (glacial acetic acid: isoctane = 6:4 v/v) and potassium iodide is added. Iodine released from peroxides is determined by the iodometric method (visually) using a starch indicator and a standard solution of sodium thiosulfate (Na ₂ S ₂ O ₃ 0.01 N). The end point of the titration is determined by the iodometric method (visually).

PV is expressed in mEq of oxygen per kg of oil.

Given that determination of peroxide value (PV) is largely an empirical procedure, ISO3960:2007 sets sample weight at 5 g for PV greater than 1 and at 10

- 15045244 g for PV less than or equal to 1 and the applicability of this method is limited to animal and vegetable fats and oils with peroxide numbers from 0 to 30 mEq of active oxygen per kilogram.

Kreiss test

The Kreiss test is performed in a standard manner known to one skilled in the art.

The Kreiss test is a chemical test for qualitatively determining the degree of rancidity of edible oil. The test is used to identify secondary products of fatty acid autoxidation.

In fact, the organoleptic properties of rancid and rancid fats largely correlate with the presence of carbonyl products formed by the interaction between oxygen and unsaturated fatty acids.

Phloroglucinol (1,3,5-triphenol, or symmetric triphenol) is a chemical compound of the triphenol group, with the general formula C6H6O3, which crystallizes from its aqueous solutions in the form of a dihydrate (C ₆ H ₆ O ₃ -2H ₂ O).

The Kreiss test is based on a condensation reaction between carbonyl compounds (arising from rancidity of fat) with phloroglucinol in the presence of HCl, which produces a red compound. This is a qualitative method, and the result is considered positive when the color of the lower layer is more intense than the 0.0012% KMnO ₄ solution, and negative when the opposite occurs.

Kreiss test technique

1. 10 ml of oil and 10 ml of concentrated HCl are stirred for 30 s.

2. Add 10 ml of reagent: 0.1% solution of phloroglucinol in ethyl ether and stir for another 30 s.

After separating the layers, examine the color of the lower acid layer:

negative reaction ^ brownish or faded color; positive reaction ^ pink or red color (rancid oil).

results

As shown in table. 1, samples 2 and 3 (compositions according to the invention containing an antioxidant) have at time points T3 (that is, after about 150 days) and T4 (that is, after about 210 days) a peroxide value (PV) significantly lower than in sample 1 ( composition in the absence of an antioxidant).

In addition, samples 2 and 3 are negative on the Kreiss test at T3 and T4, while sample 1 is positive.

Table 1

THAT T1 in 30 days T2 after 60 days Technical specification in 60 days T4 after 60 days Sample 1 (in the absence of antioxidant) PV (mEq of active oxygen/kg not defined 6.51 6.92 24.38 27.47 Kreiss test not defined negative negative positive positive Sample 2 (with antioxidant) PV (mEq of active oxygen/kg not defined 5.62 5.4 6.55 4.39 Kreiss test not defined negative negative negative negative Sample 3 (with antioxidant) PV (mEq of active oxygen/kg not defined 4.77 6.47 8.64 5.34 Kreiss test not defined negative negative negative negative

Experimental part 2

In experimental part 2, an in vivo toxicity study was carried out on the compound according to the invention (sample 4).

In vivo study: 14-day oral toxicity study of the compound of the invention in Crl CD Sprague Dawley (SD) rats.

Compound tested:

Sample 4, a composition comprising a mixture containing primarily cetylated myristic acid and cetylated oleic acid, with a myristic acid:oleic acid molar ratio of about 2.1:1 (about 75% by weight based on the total weight of the sample), olive or corn oil (about 25% by weight relative to the total weight of the sample), at least one antioxidant at a weight percentage of from 0.001 to 0.3% (relative to the total weight of the sample), for example, a mixture of tocopherols or tocopheryl acetate.

Test system: 60 Sprague Dawley rats (30 males and 30 females) seven weeks old at the start of treatment.

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Group size: 5 male and 5 female rats per group.

Analysis levels: 0 - 1600 - 1900 - 2200 - 2600 - 4500 mg/kg body weight.

Administration volume: 10 ml/kg body weight.

Medicinal mixtures: prepared weekly and kept at 4°C.

Treatment plan: every day for 14 days in a row.

Treatment procedure: daily dose volume divided into two equal secondary fractions administered to each rat on an empty stomach at 60-minute intervals.

Observations: Mortality (daily); Clinical signs (daily); Body weight (twice a week); Food consumption (twice a week); Hematology (killing); Clinical chemistry (killing); Urinalysis (killing); Macropathology; Histology (if necessary).

Statistics: Data were analyzed using ANOVA, followed by Post hoc Dunnett's tests with JMP statistical detection software.

Results:

In the high-dose group, a statistically significant dose-dependent difference was recorded in relation to glucose levels, which was significantly lower (p<0.05) compared to the control group (inverse relationship between the administered dose of the compound according to the invention and the glucose level ). Unexpectedly, it was found that when rats were administered, for example, the composition of sample 4 in the highest doses, the level of glucose in the blood of the treated rats was statistically significantly reduced. Thanks to this result, the compositions according to the invention find actual application/use in the method of treating subjects suffering from diabetes or a non-diabetic disease and/or disorder caused by or associated with high blood glucose levels, as in the case of hyperglycemia, chronic liver diseases and obesity .

Complete hematology revealed no treatment-related differences between groups and no clinical signs during the 14-day treatment period.

There were no abnormalities reported in differential white blood cell counts or urinalysis between treatment groups.

Body weight gain and food intake were comparable in all groups, and no behavioral abnormalities were observed throughout the treatment period of the rats.

At the necropsy level, no abnormalities were recorded: all organs and tissues of various systems showed normal appearance, size, color and location.

Thus, Sample 4 (compound of the invention) was well tolerated at all doses tested, and blood glucose levels were statistically significantly lower in the high-dose group of rats (inverse dose-response relationship).

Surprisingly, effective and durable protection of the gastric mucosa was visually observed in the present in vivo study in rats. The gastric mucosa is known to have a barrier effect and consists of a layer of mucosa approximately 0.2 mm thick that covers the gastric mucosa membrane. The purpose of this barrier is to protect the gastric epithelium from the damaging effects of HCl produced by the parietal cells of the stomach. It is formed due to the production of high molecular weight (hence very viscous) droplets of mucoproteins by cells on the surface of the stomach and due to the production of bicarbonate. Therefore, the compositions according to the invention (see Table 2), for example samples 2-6, find actual application/use in a method of (prophylactic and/or curative) therapy for a disorder or symptom caused by or associated with damage to the gastric mucosa selected from the group including or alternatively consisting of gastric ulcers, gastroesophageal reflux disease (GERD), heartburn.

The in vivo toxicity study described above was repeated after 90 days. The results are comparable to those obtained after 14 days. In particular, at a dose of 4500 mg/kg body weight of the test rats (the maximum dose that did not produce toxic effects), it was unexpectedly observed that the test rats maintained and maintained good thyroid function without changes in diet and social behavior.

Experimental part 3

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

1. Research structure

The study described in Experimental Part 3 is designed to evaluate - in an in vitro system - the ability of the test products to modulate inflammatory mechanisms induced in human tenocyte cultures (ZEN BIO TEN-F, Lot # TENM012214F). The study of anti-inflammatory activity was carried out by analyzing - using enzyme-linked immunosorbent assay (ELISA) - some markers of inflammation, in particular pro-inflammatory cytokines IL6, prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), the latter products of cyclooxygenase and lipoxygenase - the main enzymes involved in inflammation cascade from arachidonic acid - activity, respectively.

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2. Products tested

Cn cream products (compositions according to the invention, according to table 2, C1-C6): concentration 0.050%, 0.025% and 0.010%.

Arnica 5.5%

Cream: concentration 0.070%, 0.035% and 0.014%.

Glucosamine 5.5%

Cream: concentration 0.070%, 0.035% and 0.014%.

All products tested are creams based on glycerin and/or glyceryl monostearate.

The products according to the invention (Cn cream products), tested in the specified in vitro study for assessing anti-inflammatory activity, have the following compositions presented in table. 2 (from C1 to C6). All products C1-C6 according to the invention gave similar experimental results. Therefore, only data for cream product C1 will be presented below, representing products C2-C6.

table 2

INGREDIENTS quantity, wt.% quantity,% wt. quantity,% wt. quantity,% wt. quantity,% wt. quantity,% wt. quantity,% wt. activity Range C1 C2 NW C4 C5 C6 Purified water 70-80 77.5 77.5 80 80 80 80 Solvent Mixture of cetylated fatty acids in vegetable oil 3-10 7.5 7.5 8 8 8 8 Lubricant Antioxidants: at least one selected from the group A-G 0.001- 0.03 A: Butylated hydroxytoluene - 0.005 - - - - - Antioxidant IN: Tocopheryl acetate - 0.008 0.01 - - - - Antioxidant C: Tert-butylhydroquinone (TBHQ) - - 0.05 0.05 - 0.05 - Antioxidant D: (Aperoxide®TLA) - - - - 0.05 - - Antioxidant E: Rosemary extract - - - - 0.01 Antioxidant F: Mixed tocopherols - - - - 0.01 0.05 Antioxidant G: Wheat germ oil (Triticum vulgare) - 0.025 0.01 - - - 0.05 Antioxidant Glycerin (Vegetable glycerin) 1-10 3 3 3 3 3 3 U humidifier Glyceryl monostearate 0.5-5 2.5 2.5 2.5 2.5 2.5 2.5 Emulsifier Butylene glycol 0.5-5 1.5 1.5 1.5 1.5 1.5 1.5 U humidifier Additives and/or excipients sufficient quantity up to (q.s.) 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 q.s. 100 -

(*) A mixture of cetylated fatty acids in vegetable oil (olive or corn or sunflower) contains, or alternatively consists of cetylated:

(a1) myristic acid: 30 to 55%, preferably 35 to 50%, more preferably about 41%;

(a2) oleic acid: 35 to 60%, preferably 40 to 55%, more preferably about 46%;

(a3) linoleic acid: 3 to 12%, preferably 5 to 10%, more preferably about 8%;

(a4) palmitic acid: 1 to 8%, preferably 2 to 6%, more preferably about 3%;

(a5-a9) various fatty acids (non-myristolic acid): 1 to 4%, preferably 1.5 to 3%, more preferably about 1.8%; where % is % by weight relative to the total weight of said mixture of cetylated fatty acids; where mass ratio (a):(c) = 4:1, 3:1, 2:1 or 1:1.

Before the samples were subjected to potency testing, they were prepared in a culture

- 18 045244 medium, starting from the following ratios: 0.05 g diluted in 1 ml of culture medium. Subsequent dilutions were carried out in culture medium.

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

3. Method

For this test, human tenocyte cultures (ZEN BIO TEN-F, Lot # TENM012214F) were treated for 48 h with interleukin-1 beta (IL-1e, 10 ng/ml - dose selected after range finding test), an agent involved in tendinopathic inflammatory conditions, and simultaneously with products tested at 3 concentrations selected from non-cytotoxic after a preliminary cytotoxicity test. At the end of the controlled experimental period, levels of inflammatory markers of interest in the culture medium were measured using ELISA. The results were compared with negative control cultures (untreated, CTR-) and positive control cultures (IL-1e-treated, CTR+).

Thus, the experimental protocol involved the analysis of three pro-inflammatory markers (IL6, LTC4 and PGE2) in untreated cell cultures (negative control, CTR-);

cell cultures in which an inflammatory event was experimentally induced (positive control, CTR+);

cell cultures in which an inflammatory event was experimentally induced and simultaneously treated with the test products.

4. Analysis of inflammatory markers (IL6, PGE2 and LTC4)

Culture media of control organs and cells treated with the tested products (point 2) were used to determine the inflammatory markers IL6, PGE2 and LTC4 using the ELISA method.

For this purpose, commercially available kits were used, in which competitive binding of an antigen (in this case, the cytokine of interest) is carried out with its primary antibody. The immune complex (antigen-antibody), in turn, is recognized by a secondary antibody conjugated to peroxidase. The addition of a peroxidase substrate causes a colorimetric reaction with an intensity proportional to the amount of immune complexes present and therefore to the amount of cytokine bound. Quantification is carried out using a calibration curve constructed with known and increasing concentrations of the standard cytokine.

5. Results

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

The results are presented as the amount of cytokine released in the culture medium during the experimental period (mean ± standard, incl.) and as the mean % deviation compared to control values.

IL6 assay results

Treatment of cell cultures with the test products (item 2) showed a decrease in the levels of IL6 released by cells after experimental induction of inflammation (Tables 3-5 and Figs. 4A, 4B and 4C). The tested samples more or less markedly modulate and inhibit the release of pro-inflammatory cytokine controlled during inflammation. All charts (FIGS. 4A, 4B and 4C) show a more or less clear dose-dependent trend in which the highest concentration tested has an assay result lower than IL6.

5.1. Analysis of IL6 in cell cultures CTR-, CTR+ and treated with the C1 cream product according to the invention (Table 2). The results are expressed as mean content ± standard, incl. (expressed in ng/l) and as the average % deviation compared to control values (Table a 3 and Fig. 4A).

Table 3

IL6 % deviation by % deviation by ng/l compared with CTR- compared with CTR+

CTR- 39.4 ±2.3 - CTR+ 53.8 ±3.2 +36.4% - CREAM Product C1 0.050% 38.0 ± 1.5 -3.5% -29.3% CREAM Product C1 0.025% 39.9 ±2.6 ±1.2% -25.8% CREAM Product C1 0.010% 41.3 ±3.0 ±4.9% -23.1%

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5.II. Analysis of IL6 in cell cultures of CTR-, CTR+ and Arnica cream 5.5%.

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 4 and Fig. 4B).

Table 4

IL6 % deviation by % deviation by ng/l compared with CTR- compared with CTR+

CTR- 39.4 ±2.3 - CTR+ 53.8 ±3.2 ±36.4% - ARNICA 5.5% CREAM 0.070% 49.9 ±2.1 ±26.7% -7.1% ARNICA 5.5% CREAM 0.035% 52.5 ± 1.6 +33.2% -2.4% ARNICA 5.5% CREAM 0.014% 52.9 ± 2.5 +34.4% -1.5%

5.III. Analysis of IL6 in cell cultures of CTR-, CTR+ and treated with Glucosamine 5.5% cream.

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 5 and Fig. 4C).

Table 5

IL6 ng/l % deviation compared to CTR- % deviation compared to CTR+ CTR- 39.4 + 2.3 - - CTR+ 53.8 + 3.2 +36.4% - GLUCOSAMINE 5.5% CREAM 0.070% 47.1 ± 1.1 + 19.7% -12.3% GLUCOSAMINE 5.5% CREAM 0.035% 48.7+1.0 +23.5% -9.5% GLUCOSAMINE 5.5% CREAM 0.014% 53.2+1.7 +35.0% -1.1%

LTC4 analysis results

Treatment of cell cultures with the test products (item 2) showed a decrease in the levels of LTC4 released by cells after experimental induction of inflammation (Tables 6-8 and Figures 5A, 5B and 5C). The test samples more or less significantly modulate and inhibit the release of a pro-inflammatory marker controlled during inflammation. All graphs (Figs. 5A, 5B and 5C) show a more or less obvious dose-dependent trend in which the highest concentration tested has an assay result lower than LTC4.

.IV. Analysis of LTC4 in cell cultures CTR-, CTR+ and treated with the C1 cream product according to the invention (Table 2).

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 6 and Fig. 5A).

.V. Analysis of LTC4 in CTR-, CTR+ and Arnica 5.5% cream-treated cell cultures.

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 7 and Fig. 5B).

Table 7

LTC4 nmol/l % deviation compared to CTR- % deviation compared to CTR+ CTR- 6.6 ±0.6 - - CTR+ 10.2 + 0.7 +55.7% - ARNICA 5.5% CREAM 0.070% 8.0 + 0.3 +22.3% -21.5% ARNICA 5.5% CREAM 0.035% 8.4 + 0.5 +28.9% -17.2% ARNICA 5.5% CREAM 0.014% 9.9+ 1.2 +51.4% -2.8%

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5.VI. Analysis of LTC4 in cell cultures of CTR-, CTR+ and treated with Glucosamine 5.5% cream.

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 8 and Fig. 5C).

Table 8

LTC4 nmol/l % deviation compared to CTR- % deviation compared to CTR± CTR- 6.6 ±0.6 - CTR+ 10.2 ±0.7 ±55.7% - GLUCOSAMINE 5.5% CREAM 0.070% 8.5 ± 1.0 ±30.0% -16.5% GLUCOSAMINE 5.5% CREAM 0.035% 9.3 ± 0.7 ±41.9% -8.9% GLUCOSAMINE 5.5% CREAM 0.014% 10.1 ±0.9 ±54.0% -1.1%

PGE2 analysis results

Treatment of cell cultures with the desired products (point 2) showed a decrease in the levels of PGE2 released by cells after experimental induction of inflammation (Tables 9-11 and Figs. 6A, 6B and 6C). The test samples more or less significantly modulate and inhibit the release of a pro-inflammatory marker controlled during inflammation. All charts (FIGS. 6A, 6B and 6C) show a more or less clear dose-dependent trend in which the highest concentration tested has an assay result lower than PGE2.

.VII. Analysis of PGE2 in cell cultures CTR-, CTR+ and treated with the C1 cream product according to the invention (Table 2).

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the average % deviation compared to control values (Table 9 and Fig. 6A).

Table 9

PGE2 % deviation by % deviation by ng/l compared with CTR- compared with CTR+

CTR- 109.8 ±4.6 - - CTR± 158.0 ±6.3 ±43.8% - CREAM Product C1 0.050% 134.0 ±5.2 ±22.0% -15.2% CREAM Product C1 0.025% 136.7 ±4.2 ±24.5% -13.4% CREAM Product C1 0.010% 153.6 ±9.2 ±39.9% -2.8%

.VIII. Analysis of PGE2 in cell cultures CTR-, CTR+ and treated with Arnica cream 5.5%.

Results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the average % deviation compared to control values (Table 10 and Fig. 6B).

Table 10

PGE2 % deviation by % deviation by ng/l compared with CTR- compared with CTR+

CTR- 109.8 ±4.6 - - CTR± 158.0 ±6.3 ±43.8% - ARNICA 5.5% CREAM 0.070% 131.7 ±6.2 ±19.9% -16.7% ARNICA 5.5% CREAM 0.035% 141.2 ±7.0 ±28.6% -10.6% ARNICA 5.5% CREAM 0.014% 155.7 ±9.7 ±41.7% -1.5%

5.IX. Analysis of PGE2 in cell cultures of CTR-, CTR+ and treated with Glucosamine 5.5% cream.

The results are expressed as mean content ± standard, incl. (expressed in ng/L) and as the mean % deviation compared to control values (Table 11 and Fig. 6C).

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Claims (12)

Table 11 PGE2 ng/l % deviation compared to CTR- % deviation compared to CTR± CTR- 109.8 ±4.6 - - CTR+ 158.0 ±6.3 ±43.8% - GLUCOSAMINE 5.5% CREAM 0.070% 137.3 ±6.6 ±25.0% -13.1% GLUCOSAMINE 5.5% CREAM 0.035% 149.1 ±3.9 ±35.8% -5.6% GLUCOSAMINE 5.5% CREAM 0.014% 154.0 ±5.4 ±40.2% -2.5% 6. Conclusions Considering the results obtained and reported in this report, and with reference to the experimental model used, all tested products modulated inflammatory mechanisms induced in cell culture, reducing the levels of controlled pro-inflammatory markers and, therefore, exhibiting anti-inflammatory activity. In particular, the C1 cream product, as well as the C2-C6 cream products of the invention, reduced the levels of IL-6, PGE2 and LTC4, thereby demonstrating the ability to modulate various enhancement mechanisms. CLAIM 1. A method for producing at least one unrefined cetylated fatty acid or a mixture of unrefined cetylated fatty acids, comprising the steps of: (I) contacting at least one fatty acid, cetyl alcohol and a metal catalyst in the chamber (3) of the reactor (2) in the absence of a solvent to obtain a reaction mixture (15), followed by (II) saturating the chamber (3) with using an inert gas, bringing the chamber (3) to a pressure P1 of approximately 0.1 MPa, introducing a stream of said inert gas through said chamber (3), followed by (III) first heating said reaction mixture (15) until the temperature is reached T1, ranging from 120 to 200°C, at a pressure P1 of approximately 0.1 MPa, and in the presence of a flow of inert gas to initiate the esterification reaction with the initial formation of the specified cetylated fatty acid or a mixture of cetylated fatty acids and esterification water, followed by (IV) maintaining said reaction mixture (15) under stirring at said temperature T1 and pressure P1 for a period of time ranging from 10 minutes to 2 hours, followed by (V) subjecting said reaction mixture (15) to a second heating until the temperature is reached T2, ranging from 201 to 260°C, at a pressure P1 of approximately 0.1 MPa, and in the presence of a flow of inert gas to continue the esterification reaction with the further formation of the specified cetylated fatty acid or a mixture of cetylated fatty acids and esterification water, followed by (VI) maintaining said reaction mixture (15) with stirring at said temperature T2 and pressure P1 and in the presence of a flow of inert gas for a period of time ranging from 4 to 12 hours; (VII) applying in the chamber (3) a vacuum program that reduces the reaction pressure until a reduced pressure P2 of 10 to 1 kPa is achieved; (VIII) maintaining said reaction mixture (15) under stirring at said reduced pressure P2 and in the presence of a flow of inert gas for a period of time ranging from 30 minutes to 4 hours to achieve complete formation of said at least one unrefined cetylated fatty acid or mixtures of unrefined cetylated fatty acids (Ml). 2. The method according to claim 1, wherein said step (VI) of maintaining said reaction mixture (15) with stirring at said temperature T2 and pressure P1 and in the presence of a flow of inert gas for a period of time ranging from 6 to 12 hours is carried out until the acidity of the reaction mixture (15), measured using the AOCS Official Method Cd 3d-63, is stable over time and ranges from 5 mg KOH/g to 8 mg KOH/g; and said step (VIII) of maintaining said reaction mixture (15) with stirring at said reduced pressure P2 and in the presence of a flow of inert gas for a period of time ranging from 30 minutes to 4 hours is carried out until the acidity value of the reaction mixture (15) is obtained, measured using the AOCS Official Method Cd 3d-63, ranging from 3.5 mg KOH/g to 4.5 mg KOH/g, to achieve complete formation of the specified at least one unrefined cetylated fatty acid or mixture of unrefined cetylated fatty acids (MI). 3. The method according to claim 1 or 2, wherein said at least one fatty acid is selected from the group consisting of lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, eicosanoic acid and their mixtures; preferably selected from myristic acid, oleic acid and a mixture of myristic acid and oleic acid. 4. The method according to any of the preceding paragraphs, in which the specified flow of inert gas is supplied using a purging means (7) located in a part of the volume of the chamber (3) located above the reaction mixture (15). 5. The method according to any of the preceding claims, in which the esterification water withdrawn from the chamber (3) during the esterification reaction by supplying an inert gas flow and a vacuum program is condensed in a horizontal condenser (11) and collected in a container (13) after flowing through vertical condenser (16). 6. The method according to any of the preceding claims, wherein said method further comprises the step (IX) of filtering said at least one unrefined cetylated fatty acid or mixture of unrefined cetylated fatty acids (MI) obtained from step (VIII) on bleaching earth and/ or filter earth in a filter press (23) to obtain at least one filtered cetylated fatty acid or mixture of filtered cetylated fatty acids (Mf), in which the metal catalyst used in step (I) is absent or present in an amount of less than 2% by weight relative to the weight of said filtered cetylated fatty acid or mixture of filtered cetylated fatty acids (Mf). 7. A method for producing at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids, where the method includes producing at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf) by the method according to claim 6 and step (X) (deodorization step) treating said at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf) in a reactor (27) at a temperature T3 of 150 to 200°C and a reduced pressure P3 of 0.1 up to 2 kPa, in the presence of a stream of steam for a period of time ranging from 1 to 5 hours, to obtain at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids (MF). 8. A method of producing a composition containing at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids, where the method includes obtaining at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf) by the method according to claim 6 and step mixing said at least one filtered cetylated fatty acid or a mixture of filtered cetylated fatty acids (Mf) with at least one antioxidant, wherein said at least one antioxidant is mixed in a weight percentage of from 0.001 to 0.5% relative to the total mass of the composition. 9. A method for producing a composition containing at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids, where the method includes producing at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids (MF) by the method according to claim 7 and step mixing said at least one refined cetylated fatty acid or a mixture of refined cetylated fatty acids (MF) with at least one antioxidant, wherein said at least one antioxidant is mixed in a weight percentage of from 0.001 to 0.5% relative to the total mass of the composition. 10. The method according to claim 8 or 9, wherein said at least one antioxidant is mixed in a weight percentage of 0.005 to 0.1%, preferably 0.01 to 0.08%, based on the total weight of the composition . 11. A method for producing a composition according to any one of claims 8 to 10, wherein said at least one filtered cetylated fatty acid (Mf) or at least one refined cetylated fatty acid (MF) contains, preferably consists of a mixture of cetylated myristic acid and cetylated oleic acid, and wherein said at least one antioxidant is tert-butylhydroquinone (TBHQ). 12. A method for producing a composition according to any one of claims 8 to 11, wherein before step (X) there is said at least one filtered cetylated fatty acid (Mf) obtained from step (IX), preferably a mixture of filtered cetylated myristic acid and cetylated oleic acid acid, is subjected to step (XII) of mixing said at least one filtered cetylated fatty acid (Mf) with vegetable oil, preferably olive oil -