Cetylated fatty acids, system for the preparation thereof and use thereof

Abstract

The present invention relates to a process for preparing a mixture of cetylated fatty acids and a system for carrying out said process. Furthermore, the present invention relates to a composition comprising, or alternatively, consisting of said mixture of cetylated fatty acids. Finally, the present invention relates to said composition for use in the treatment and/or prevention of: (i) rheumatoid arthritis of inflammatory and non-inflammatory origin, in particular osteoarthritis; (ii) other inflammatory joint conditions; (iii) psoriasis, lupus, periodontal diseases or cardiovascular or heart diseases; (iv) all post-traumatic osteoarticular pathologies including sports injuries; (v) all degenerative joint pathologies (arthrosis, gonarthrosis, coxarthrosis, etc.), and (vi) inflammatory-traumatic tendon and muscular conditions. Furthermore, it is envisaged that the composition of the present invention be used in the treatment and/or prevention of the above-mentioned pathologies and disorders (i)-(vi) in association with a rehabilitative therapy. The composition comprising said mixture is formulated in a pharmaceutical form for oral use (novel food, supplement or medical device), i.e. in the form of a pill, pastille, capsule, tablet, granules, dispersible powder, syrup, solution or sprayable solution; for topical use, i.e. in the form of a cream, unguent, ointment, gel or spray to be used as such for application on the skin, or else for transdermal use in the form of a patch.

Claims

1. A composition comprising a final refined mixture (MF) comprising a mixture of cetyl myristate and cetyl oleate, a vegetable oil and zinc metal, wherein the final refined mixture (MF) and the vegetable oil are in a ratio by weight 3:1, and, optionally, pharmaceutical or food grade additives and excipients.

2. The composition according to claim 1, wherein the final refined mixture (MF) is obtainable according to a process comprising the steps of: placing in contact, in a container (3) of a reactor (2), a mixture of myristic acid and oleic acid, with a cetyl alcohol and a zinc metal catalyst, in the absence of a solvent, so as to yield a reaction mixture (15); heating said reaction mixture (15) to a reaction temperature comprised from 150° C. to 200° C. and a reaction pressure of about 1 atmosphere, so as to give rise to an esterification reaction with the initial formation of esters of cetylated fatty acids and esterification water; allowing said reaction mixture (15) to react for a reaction time comprised from 1 hour to 8 hours until completion of said esterification reaction so as to obtain the complete formation of a mixture of cetylated fatty acids (MI) and the complete removal of said esterification water, the latter being achieved by introducing a flow of inert gas into the container (3) of said reactor (2) for the whole reaction time; subjecting the mixture of cetylated fatty acids (MI) to a refinement treatment, which comprises diatomaceous earth filtration in a filter press (23), so as to yield a filtered mixture Mf in which the zinc metal catalyst present therein is removed or greatly reduced in amount; treating the filtered mixture Mf in a reactor (27), at a temperature comprised from 150° C. to 200° C. and a pressure comprised from 5 mbar to 15 mbar in the presence of water vapour for a period of time comprised from 1 hour to 5 hours, so as to yield the final refined mixture (MF) based on cetylated fatty acids.

3. The composition according to claim 2, wherein the metal catalyst is zinc metal powder.

4. The composition according to claim 1, wherein the composition is formulated in a pharmaceutical form for topical use.

5. The composition according to claim 4, wherein said composition is in the form of a cream, unguent, ointment, gel or spray.

6. The composition according to claim 4, wherein said composition formulated for topical use is to be used as such for application on the skin or for transdermal use in the form of a patch.

7. The composition according to claim 1, wherein the composition is formulated in a pharmaceutical form for oral use.

8. The composition according to claim 7, wherein said composition is in the form of a pill, pastille, capsule, tablet, granules, dispersible powder, syrup, solution, sprayable solution.

Description

EXAMPLE 1

(1) Myristic acid (molecular weight 228), 50.0 g (0.219 moles). Cetyl alcohol (molecular weight 242), 53.0 g (0.219 moles). Catalyst: Zinc (Zn) metal powder, 0.1% (0.1 g). Temperature 180° C. At the end of the reaction the sample was filtered.

(2) TABLE-US-00001 TABLE 1 Residual pressure, Reaction time, h % acid % alcohol % wax mbar 1 16.2 16.3 67.4 600 2 9.8 9.6 80.5 500 3 5.9 6.2 87.9 300 5 2.3 2.0 95.7 200 7 1.7 1.5 96.9 5

EXAMPLE 2

(3) Myristic acid (molecular weight 228), 65.0 g (0.285 moles). Oleic acid 80% (molecular weight 274), 35.0 g (0.128 moles) [acidity 204.7 NS 200.5 mg KOH/g]. Total moles of acid: 0.413. Cetyl alcohol (molecular weight 242), 100.0 g (0.413 moles). Catalyst: Zn powder, 0.1% (0.2 g). Temperature 180° C. At the end of the reaction the sample was filtered. No occlusion of the condenser occurred in the initial phases of the reaction.

(4) TABLE-US-00002 TABLE 2 Residual pressure, Reaction time, h % acid % alcohol % wax mbar 1 17.9 18.3 67.9 600 2 10.4 10.8 78.7 500 3 6.6 7.2 86.2 300 5 4.2 4.9 90.9 200 7 2.4 2.5 95.1 5

EXAMPLE 3

(5) Cetyl alcohol (molecular weight 242), 100.0 g (0.413 moles). Oleic acid 80% (molecular weight 274), 112.0 g (0.409 moles). Catalyst: Zn powder, 0.1% (0.2 g). Temperature 180° C. At the end of the reaction the sample was filtered. No occlusion of the condenser occurred in the initial phases of the reaction.

(6) TABLE-US-00003 TABLE 3 Residual pressure, Reaction time, h % acid % alcohol % wax mbar 1 19.1 19.3 61.6 600 2 10.6 12.0 77.4 500 3 6.7 8.2 85.1 300 5 3.5 5.3 91.2 200 7 1.4 3.1 95.5 5

EXAMPLE 4

(7) Myristic acid (molecular weight 228), 160.0 g (0.701 moles). Oleic acid 80% (molecular weight 274), 88.0 g (0.321 moles) [acidity 204.7 NS 200.5 mg KOH/g]. Total moles of acid: 1.022. Cetyl alcohol (molecular weight 242), 250.0 g (1.033 moles). Catalyst: Zn powder, 0.1% (0.2 g). Temperature 180° C. Light flow of nitrogen in the reactor. At the end of the reaction the sample was filtered. No occlusion of the condenser occurred in the initial phases of the reaction.

(8) TABLE-US-00004 TABLE 4 Residual pressure, Reaction time, h % acid % alcohol % wax mbar 1 15.6 12.6 71.8 600 2 10.0 6.7 83.3 500 3 6.8 3.7 89.5 300 5 4.7 1.6 93.6 200 7 3.5 0.8 95.7 5

(9) Determination of the Melting Point of the Compositions Obtained According to NGD C27-1976

(10) Given the very characteristics of the fatty substances, the melting point is well defined by the measurement of the slip and clear points. These temperatures correspond to those at which the fraction of a substance in contact with the walls of the capillary tube begins to melt (slip point) and then slip and run inside the capillary tube itself (clear point). In order to measure them, the substance in question is placed in a special U-shaped tube of well-established dimensions (for thermal aspiration of the melted sample at a temperature of about 10° C. above the melting point) and allowed to solidify for a time of at least 16 hours and then the water bath it is immersed in is heated very slowly. A mixture consisting of cetyl myristate (75% by weight) from Example 1 and refined olive oil (25% by weight) has: a slip point of 44.9° C. and a clear point of 47.1° C. A mixture consisting of cetyl myristate and cetyl oleate (75% by weight) from Example 2 and refined olive oil (25% by weight) has a slip point of 44.4° C. and a clear point of 45.1° C. A mixture consisting of cetyl myristate and cetyl oleate (75% by weight) from Example 4 and refined olive oil (25% by weight) has a slip point of 44.2° C. and a clear point of 45.2° C.

(11) Experimental Design

(12) In vitro study of effectiveness—in vitro assessment of the anti-inflammatory activity of a sample of a mixture of cetylated fatty acids obtained with method I of the present invention on a cell culture. The aim of the present study was to assess, in an in vitro system, the capacity of said sample to modulate the inflammatory mechanisms induced in cultures of human synovial cells (fibroblast-like synoviocytes) (ATCC-HTB-93). The study of the anti-inflammatory activity was conducted via an assay, using the ELISA method, of several markers of inflammation, specifically, three pro-inflammatory cytokines: TNFalpha, IL1alpha and IL6.

(13) Preparation of the Samples and Method of Exposure

(14) Before being tested for effectiveness, the sample was heated to 50° C. in a temperature-controlled bath kept under stirring to obtain a homogeneous solution. Then the sample was emulsified with corn oil (37° C.) and a culture medium was added to it as follows: 0.1 g emulsified with 100 μl of corn oil, brought to a volume of 1 ml with culture medium (37° C.). Then successive dilutions were made in the culture medium. The sample was subjected to a preliminary cytotoxicity test for the purpose of selecting the most suitable concentrations for the final test. To this end, concentrations ranging from 10.00% to 0.08% were tested (1:2 serial dilutions). Based on an assessment of the test results, 1.00% samples were selected for carrying out the study of anti-inflammatory activity. For the purpose of carrying out the test, the cultures of human synovial cells (fibroblast-like synoviocytes) (ATCC-HTB-93) were treated for 24 hours with LPS (lipopolysaccharide from Escherichia coli, 1 μg/ml), a known irritant agent of a bacterial nature, to induce acute inflammatory stress and simultaneously treated with the samples to be tested at the concentration of 1% selected on the basis of the preliminary cytotoxicity test. At the end of monitored experimental period, the levels of the cytokines of interest were measured in the culture media via an ELISA assay. The results were compared with negative control cultures (untreated, CTR−) and positive control cultures (treated only with LPS, CTR+). Summarising, the experimental protocol provided for an assay of three pro-inflammatory markers (TNFalpha, IL1alpha and IL6) in: untreated cell cultures (negative control, CTR−); cell cultures in which an event of acute inflammation was experimentally induced (positive control, CTR+); cell cultures in which an event of acute inflammation was experimentally induced and which were simultaneously treated with the test samples at 1.00%.

(15) Assay of the Inflammation Markers (TNFalpha, IL1alpha and IL6)

(16) The culture media of the controls and of the cells treated with the test samples were used to assay the pro-inflammatory cytokines TNFalpha, IL1alpha and IL6 using the ELISA method. For this purpose, use was made of commercially available kits which exploit the competitive binding of an antigen (in this case the cytokine of interest) with its primary antibody. The immune complex (antigen-antibody) is in turn recognised by a secondary antibody conjugated to a peroxidase. The addition of the peroxidase substrate produces a colorimetric reaction with an intensity proportional to the quantity of immune complexes present and thus to the quantity of bound cytokines. The quantitative determination relies on a calibration curve constructed with known standard cytokine concentrations on an increasing scale.

(17) Results and Graphs

(18) The tables that follow show the results obtained in the present study. The results are reported as the quantity of cytokines released in the culture media during the experimental period (mean value±SD) and as a mean % variation compared to the controls.

(19) Anti-Inflammatory Activity—Assay of TNFalpha

(20) TABLE-US-00005 TABLE 5 Assay of the TNFalpha in the cell cultures CTR−, CTR+ and treated with a sample of a mixture of cetylated fatty acids obtained with the method I (sample R8P). The results are expressed as mean content ± SD (expressed in ng/l) and as mean % variation compared to the controls. Table 5. TNFalpha % Variation % Variation ng/l vs CTR− vs CTR+ CTR− 145.7 ± 6.4 — CTR+  185.7 ± 12.3 +27.5% — R8P 1.00% 156.7 ± 6.4 +7.5% −15.6%

(21) Anti-Inflammatory Activity—Assay of IL1alpha

(22) TABLE-US-00006 TABLE 6 Assay of the IL1alpha in the cell cultures CTR−, CTR+ and treated with the sample R8P. The results are expressed as mean content ± SD (expressed in ng/l) and as mean % variation compared to the controls. Table 6. IL1alpha % Variation % Variation ng/l vs CTR− vs CTR+ CTR− 115.6 ± 8.6 — CTR+ 144.1 ± 5.9 +24.7% — R8P 1.00% 110.1 ± 8.3 −4.7% −23.6%

(23) Anti-Inflammatory Activity—Assay of IL6

(24) TABLE-US-00007 TABLE 7 Assay of the IL6 in the cell cultures CTR−, CTR+ and treated with the sample R8P. The results are expressed as mean content ± SD (expressed in ng/l) and as mean % variation compared to the controls. Table 7. IL6 % Variation % Variation ng/l vs CTR− vs CTR+ CTR− 89.5 ± 9.1 — CTR+ 105.6 ± 5.9  +18.1% — R8P 1.00% 73.7 ± 4.5 −17.6% −30.2%

(25) Embodiments of the present invention are indicated below with FRn:

(26) FR1. A process for preparing a mixture of cetylated fatty acids (MI) comprising the steps of: placing in contact, in a container (3) of a reactor (2), at least one fatty acid selected from the group comprising or, alternatively, consisting of myristic acid, oleic acid or mixtures thereof, with a cetyl alcohol and a metal catalyst, in the absence of a solvent, so as to yield a reaction mixture (15); heating said reaction mixture (15) to a reaction temperature comprised from 150° C. to 200° C. and a reaction pressure of about 1 atmosphere, so as to give rise to an esterification reaction with the initial formation of esters of cetylated fatty acids and esterification water; allowing said reaction mixture (15) to react for a reaction time comprised from 1 hour to 8 hours until completion of said esterification reaction so as to obtain the complete formation of a mixture of cetylated fatty acids (Ml) and the complete removal of said esterification water, the latter being achieved by introducing a flow of inert gas into the container (3) of said reactor (2) for the whole reaction time.

(27) FR2. The process according to FR1, wherein said complete removal of esterification water is achieved by maintaining the reaction pressure constant at about 1 atm and introducing said flow of inert gas via a blowing means (7), into the portion of volume above the reaction mixture (15), thus allowing the esterification water to be drawn out of the container (3).

(28) FR3. The process according to FR2, wherein the esterification water drawn out of the container (3) during the esterification reaction at a constant reaction pressure is condensed in a horizontal condenser (11) and collected in a container (13); preferably, said condenser (11) is maintained at a temperature comprised from 10° C. to 40° C. and is connected to said container (3), in the upper portion (3a) thereof, via the conduit (12).

(29) FR4. The process according to FR1, wherein said complete removal of esterification water is achieved by using a vacuum program that applies a reduction in the reaction pressure in a non-linear manner and introducing said flow of inert gas, via the blowing means (7), into the reaction mixture (15), thus allowing the esterification water to be drawn out of the container (3).

(30) FR5. The process according to FR4, wherein the vacuum program preferably applies a reduction in the reaction pressure to 600 mbar after the first hour of reaction in a non-linear manner, preferably arriving at 5 mbar after a reaction time of seven hours.

(31) FR6. The process according to FR5, wherein the esterification water, drawn out of the container (3) during the esterification reaction with the vacuum program, is condensed in a horizontal condenser (11) and collected in a container (13) after having passed through a vertical condenser (16).

(32) FR7. The process according to FR6, wherein said condenser (11) is maintained at a temperature preferably comprised from 10° C. to 40° C. and is connected to said container (3) via the vertical condenser (16), which is maintained at a temperature preferably comprised from 70° C. to 90° C.

(33) FR8. The process according to any one of embodiments FR1-7, wherein said mixture of cetylated fatty acids (Ml) is subjected to a subsequent refinement treatment, which comprises diatomaceous earth filtration in a filter press (23), so as to yield a filtered mixture Mf in which the metal catalyst present therein is removed or greatly reduced in amount.

(34) FR9. The process according to FR8, wherein the filtered mixture Mf is treated in a reactor (27), at a temperature comprised from 150° C. to 200° C. and a pressure comprised from 5 mbar to 15 mbar in the presence of water vapour for a period of time comprised from 1 hour to 5 hours, so as to yield a final refined mixture (MF) based on cetylated fatty acids.

(35) FR10. A composition comprising a final refined mixture (MF) based on cetylated fatty acids obtained according to FR9, and a vegetable oil in a 3:1 ratio by weight; said composition being for use in the treatment and prevention of (i) rheumatoid arthritis of inflammatory and non-inflammatory origin, in particular osteoarthritis; (ii) other inflammatory joint conditions; (iii) psoriasis, lupus, periodontal diseases or cardiovascular or heart diseases; (iv) all post-traumatic osteoarticular pathologies including sports injuries; (v) all degenerative joint pathologies (arthrosis, gonarthrosis, coxarthrosis, etc.) and (vi) inflammatory-traumatic tendon and muscular conditions.