EP0999831A1

EP0999831A1 - Use of petroselinum acid for treating inflammations

Info

Publication number: EP0999831A1
Application number: EP98935087A
Authority: EP
Inventors: Armand Malnoe; Markus Baur; Laurent Fay
Original assignee: Galderma SA
Current assignee: Galderma SA
Priority date: 1997-07-05
Filing date: 1998-07-02
Publication date: 2000-05-17
Also published as: JP2000511942A; AU8445998A; WO1999002149A1; CA2294817A1; BR9810121A; EP0888773A1

Abstract

The invention concerns the use of native or esterified petroselinum acid for preparing a composition for treating or preventing inflammations and/or modulating the metabolism of lipids in human superficial tissues.

Description

USE OF PETROSELINIC ACID FOR THE TREATMENT OF INFLAMMATION

The invention relates to a new use of a food, cosmetic and / or pharmaceutical composition containing petroselinic acid.

State of the art

Most cosmetic and / or dermatological compositions contain, at a more or less high concentration, a fatty phase generally consisting of at least one oil, a fatty material and / or a wax, for example in the form of emulsions of the water type. -in-oil or oil-in-water, oily gels, and oily solutions. Oils generally constitute a significant proportion of this fatty phase and can be of very varied origins. They may especially be vegetable, animal, mineral oils or even synthetic oils. Among these oils, vegetable oils constitute a class of choice because the current trend is more oriented towards the use of products of natural origin. These vegetable oils generally consist of triglycerides of fatty acids.

Vegetable essential oils are clearly distinguished from vegetable oils. Indeed, essential oils are odoriferous products obtained from a vegetable material, generally by steam entrainment. An essential oil extracted from an ombélliffère cannot therefore contain a vegetable oil, and in particular cannot contain petroselinic acid (Afifi et al., Vet. Med. J., Giza, 42 (3), 85-92, 1994; patent RU2027440 by A. Leontev et al .; patent CS24821 by O. Krejsa et al.).

In addition, the duration and extent of inflammation can be modulated by controlling the metabolism of leukotriene B4. For this, one can either control its degradation, or either interfere with the leukotriene B4 receptors.

To date, the actual activity and metabolism of petroselinic acid in human surface tissues is unknown. For example in keratinocytes, its action on the proliferation of peroxisomes involved in the degradation of fatty acids by β-oxidation, including leukotriene B4, is not not known. Similarly in keratinocytes, the action of petroselinic acid on leukotriene B4 receptors is not really known.

EP709084 (L'Oreal) describes that a cosmetic or dermatological composition containing a vegetable oil rich in petroselinic acid is particularly indicated for moisturizing dry skin. This hydrating effect has nothing to do with an anti-inflammatory effect, or even a modulator of lipid metabolism.

EPI 16439 (Suntory) discloses that certain fatty acids, including petroselinic acid, have a strong activity of inhibiting 5α-reductase from the bacterial flora of the scalp, and can therefore be used in tonic products for the hair treatment. The action of these fatty acids is therefore limited to the bacterial flora of the scalp. It is thus never suggested that petroselinic acid can act directly on the surface tissues of humans by preventing inflammation, or even by modulating the lipid metabolism of these tissues (thanks to the activation of catabolism not β- oxidation). As proof, EPI 16439 recommends using oleic acid for this purpose, while the latter is inactive against inflammation (see Example 1 below).

US4097604 (Oxford Hill) indicates that a salt of different fatty acids, in particular petroselinic acid, is active against pathogens of the oral cavity, thus making it possible to reduce the incidence of these bacteria on the appearance of periodontoses. The action of these fatty acids is therefore limited to the oral flora. It is thus never suggested that petroselinic acid can act directly on the surface tissues of man by preventing inflammation, or even by modulating the lipid metabolism of these tissues. For proof, US4097604 recommends to use for this purpose preferably oleic acid, while this fatty acid is inactive against inflammations (see Example 1 below).

EP355842 (Sansho Seiyaku) describes a care cream intended to prevent pigmentation caused by an overproduction of melanin, said cream possibly containing petroselinic acid. This effect on pigmentation, however, has nothing to do with an anti-inflammatory and / or modulating effect on lipid metabolism. FR2631235 (Sandoz) describes compositions suitable for dermal application in the treatment of an autoimmune skin disease or for promoting hair growth. These compositions contain as active principle of cyclosporine, and as vehicle of the active principle, a fatty acid including petroselinic acid.

Furthermore, Yagaloff et al. show that various fatty acids, including petroselinic acid and oleic acid, are antagonists of the leukotriene B4 receptor only in human neutrophil cells (Fatty acids, 52, 293-297, 1995). Yagaloff et al. not having worked on skin cells, it is doubtful to extrapolate these results to other cell types. As proof, by showing that oleic acid is also an antagonist of the leukotriene B4 receptor in neutrophil cells, Yagaloff et al. deduce that oleic acid could control the duration of an inflammatory response. However, oleic acid is now known to be completely inactive against inflammation in human keratinocytes (see Example 1 below).

Keller et al. (Proc. Natl. Acad. Sci. USA, 90, 2160-2164, 1993) explain that certain fatty acids, including oleic and petroselinic acids, have an activating effect on PPARα from Xenopus laevis in transfected human cancer cells. Keller et al. thus hypothesize that the activation of PPARα in the liver would regulate the β-oxidation of fatty acids. However, there is never any mention of the possibility of modulating an inflammatory response.

Devchand et al. show that the activation of PPARα in transfected liver cells makes it possible to stimulate the β-oxidation of fatty acids, and in particular the degradation of leukotriene B4 (Devchand et al., Nature, 384, 39-43, 1996). However, there is never any mention of petroselinic acid.

In the articles by Devchand et al. and Keller et al, no hypothesis has been put forward on the metabolism of petroselinic acid in superficial tissues, in particular in keratinocytes. It would however be doubtful to associate these two articles, and to project results on other cell types. As proof, oleic acid is now known to be completely inactive against inflammations in human keratinocytes (see example 1 below). after). In addition, it has still not been described that human surface tissues, and in particular keratinocytes, actually have receptors inducing the proliferation of peroxisomes (the PPARα system).

The prior art is silent about the activity and metabolism of petroselinic acid in human surface tissues, especially on the skin or keratinocytes. The present invention thus relates to the treatment of human surface tissues with petroselinic acid in order to reduce inflammatory responses, and / or even to modulate the metabolism of lipids.

Summary of the invention

The present invention thus relates to the use of petroselinic acid, native or esterified, for the preparation of a composition intended to be able to treat or prevent inflammation and / or modulate the metabolism of lipids in the surface tissues of the man.

Detailed description of the invention

The surface tissues of man can be the cells constituting the skin, the scalp, the eye, or the oral, buccal, nasal or vaginal mucous membranes, for example.

By “modulation of lipid metabolism” is meant more particularly the catabolism of lipid mediators linked to inflammation, differentiation, proliferation and / or the barrier function of surface tissues.

Likewise, inflammation of the surface tissues must be understood as the physiological phenomenon involving the production of pro-inflammatory cytokines, such as TNFα, by the cells of the surface tissues, for example keratinocytes and epithelial corneal cells, and the cells of the immune system contained in these tissues (lymphocytes, Langerhans cells, ect ...). Inflammation can result from infection, allergy, wound, autoimmune disease, and exposure to radiation and / or irritants and / or sensitizers, for example example. Petroselinic acid can thus be used in particular for the treatment or prophylaxis of diseases of the skin or scalp, in particular against inflammations linked to psoriasis, erythema (sunburn), eczema, seborrheic dermatitis , Valopecia areata, yeast, acne or other dermatoses, for example. Applications can also be extended to inflammations of the eye (inflammation of the cornea) and mucous membranes, in particular the oral, nasal, oral and vaginal mucosa.

The composition can be applied directly to surface tissue so that petroselinic acid can diffuse through and be assimilated therein. This composition can also be injected under the surface tissues, for example by means of a subcutaneous injection. In both cases, the application of petroselinic acid is considered to be topical, that is, applied directly to, or under, the surface tissue.

This composition can also be administered orally so that petroselinic acid can act on the mucous membranes (the oral, esophageal, stomach and intestinal mucosa), and / or it can pass into the blood circulation and be supplied directly to cells in the skin, eye, or mucous membranes, for example.

This composition can also be applied in the nasal passages, by means of a diffuser, a gel and / or a physiological liquid conventionally intended for the washing of the nasal passages.

Petroselinic acid, also called δ-6-cis-octadecenoic acid (18: ln-12), can be used in its native state, for example. However, petroselinic acid esters are also potentially activators of peroxisomal β- oxidation. Indeed, other fatty acids activators of peroxisomal β-oxidation, such as palmitic acid or oleic acid, in the native or esterified state, are recognized to be activators of peroxisomal β-oxidation (Schmidt and ai, Lipids, 3_1_, 1 115-1124, 1996). Petroselinic acid can thus be esterified with glycerol (mono-, di- or tri-acyl), an alcohol such as methyl and ethyl alcohols, a sugar, a tocopherol, a tocotrienol, a sterol or even a fatty alcohol, example. The composition can thus comprise an oil from an umbelliferous plant rich in petroselinic acid. The term "oil rich in petroselinic acid" means an oil containing at least 40% of petroselinic acid.

Umbelliferae are plants whose flowers are arranged in umbels. The species preferably used in the invention are coriander, chervil, carrot, celery, cumin and dill. The umbelliferous oil used according to the invention is extracted from the seed of this umbelliferous plant, for example by grinding or pressing, then refining. Umbelliferous oil has a petroselinic acid content which varies depending on the umbelliferous seed from which it is extracted. For the same umbelliferous plant, the petroselinic acid content also varies according to the country of origin of the umbelliferous plant and according to the extraction which can be more or less complete. Thus, coriander seed oil contains about 75 to 90% petroselinic acid, cumin seed oil contains about 55 to 65% o, carrot seed oil about 55 to 65% , parsley seed oil about 40 to 50%, fennel seed oil about 55 to 65%), chervil seed oil about 50 to 70%, seed oil celery about 45 to 60%, dill seed oil about 75 to 85%, these percentages being given relative to the total weight of the composition.

The oil can be used in the composition according to the invention at a concentration ranging from 0.01 to 50% by weight relative to the total weight of the composition, for example, and preferably from 0.2 to 20%.

It should be noted that, according to the galenical forms normally used for a topical or oral application, that is to say according to the food, cosmetic and / or pharmaceutical form, the quantity of petroselinic acid sufficient and necessary to observe an anti-inflammatory effect or Modulator of lipid metabolism can vary considerably. To this end, the invention also relates to the use of petroselinic acid in an amount sufficient for the treatment or prevention of inflammation and / or for the modulation of lipid metabolism in surface tissues.

In the field of human cosmetics, the subject of the invention is also the use of petroselinic acid, in particular contained in an umbelliferous oil, to prepare a dosage form normally used for topical application or for oral hygiene, and especially in the form of solutions oily, alcoholic or hydroalcoholic, gels, water-in-oil or oil-in-water emulsions, having the appearance of a cream or gel, optionally capable of foaming, in the form of an aerosol, or also in the form of vesicular dispersions containing ionic and / or nonionic lipids. These dosage forms are, in any case, prepared according to the usual methods of the cosmetic fields considered. This composition can in particular constitute a composition for cleaning, protecting, treating or caring for the scalp, for the face, for the neck, for the hands or for the body (for example in the form of day creams, night, make-up removing creams, sun creams or oils, oils for the body or the face, cleansing milks, cleansing milks, body milks), a makeup composition (for example foundation), an artificial tanning composition, a composition for the bath, a shampoo composition (treatment of the scalp), or a composition for oral hygiene (mouthwash, toothpaste, putty), for example.

In the field of human pharmacy, in particular dermatology, otolaryngology, ophthalmology. of gastroenterology and gynecology, this composition can be a capsule, a capsule, an emulsion, an ointment, an infectious composition under the skin, an ointment, a syrup, a diffuser, an eye drop, a shampoo or a bath mouth, containing petroselinic acid, in particular contained in an umbelliferous oil, intended for the treatment or prophylaxis of the skin, the eye or the mucous membranes, for example. The various possible dosage forms are, in any case, prepared according to the usual methods of the pharmaceutical fields considered.

Finally, in the field of human food, this composition can be any ingestible composition, liquid or solid, containing petroselinic acid, in particular contained in an umbelliferous oil. This composition can be a sauce such as a salad dressing, a table oil, a mayonnaise, an ice cream, a pastry composition, a filling or spread, for example.

The present invention is described in more detail by the examples presented below. It goes without saying, however, that these examples are given by way of illustration of the subject of the invention of which they do not in any way constitute a limitation. The percentages are given by weight unless otherwise indicated. EXAMPLE 1 Activation of the Keratinocytes

1) Anti-inflammatory effect: keratinocytes naturally express proteins and enzymes involved in inflammatory responses after treatment with pro-inflammatory agents, such as phorbol esters. Among the proteins and enzymes thus produced, one can thus count superoxide dismutase (SOD), and type I collagenase and tumor necrosis factor alpha (TNFα), for example.

In the tests which follow, primary or immortalized human keratinocytes in culture are subjected in vitro to pro-inflammatory treatment with 12-O-tetradecanoylphorbol acetate (TPA). The immortalized keratinocyte lines DK2, DK7, FK2 described in PCT / EP96 / 05812 (Nestlé Products Company) were used for this purpose. The FK2 and DK2 lines were also deposited, on October 5, 1995, under the Budapest Treaty, at the DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-38124 Braunschweig, Germany) and were seen assign the deposit numbers DSM ACC2240 and DSM ACC2238 respectively. The immortalized HaCaT keratinocyte line described by Boukamp et al was also used (J. Cell.

Biol, 106, 761-771, 1988).

A Petri dish (6 cm 0) containing 4 ml of NR-2 medium (Biofluids), described in PCT / EP96 / 05812, is inoculated with the above-mentioned primary or immortalized lines. Arrived at confluence, the medium is replaced by fresh NR-2 medium rich in calcium (CaCl2, 1.5 mM). Incubate for 2 days, and replace the medium again with fresh medium, and incubate again for 2 days. The cells are then fed for 2 days with NR-2 medium rich in calcium (CaCl2, 1.5 mM) containing 1 mg / ml of BSA and a given fatty acid (oleic or petroselinic acid) or an equivalent quantity of the solvent used for fatty acids (ethanol).

During the last 24 hours, 10 ng / ml of TPA or an equivalent amount of ethanol are added to the medium. The supernatants are then collected, and the TNFα content thus produced is measured by ELISA. Specific antibodies against

TNFα are commercially available (BioSource Inter., US, catalog number

KHC0012 and KHC3013). The results, part of which is presented in Table 1 below, show that the incubation of human keratinocytes with petroselinic acid leads to an inhibition of the production of TNFα by the cells subjected to an irritant agent. This inhibition is specific since oleic acid (control) has no inhibition effect. In addition, this inhibition is dependent on the dose applied in petroselinic acid.

Table 1 Inflammations of DK2 keratinocytes

(The control without TPA: 0.7 pg of TNFα / ml)

2) Catabolism of petroselinic acid: the fatty acid profile of the primary or immortalized keratinocytes is determined.

For this, the cells are incubated for 2 times 2 days (renewal of the medium after the first 2 days) in NR-2 medium rich in calcium (CaCl 2, 1.5 mM) containing 1 mg / ml of BSA and a fatty acid given (lOOμM of oleic acid 18: ln-9; lOOμM of petroselinic 18: 1 n-12) or an equivalent amount of 0 solvent used for fatty acids (ethanol: control).

The cells are then washed once in HBSS buffer (Hanks Balanced Sait Solution, Biofluids No. 325) containing 0.1% BSA, twice in HBSS buffer. The cells are then detached in 1 ml HBSS, centrifuged, and the cells D extracted in a mixture of hexane and isopropanol 2/1 (v / v) containing 0.01% of 2,6-di- tert-butyl-p-cresol. The phospholipids are then separated by thin layer chromatography (stlica gel 60) with a mixture of chloroform and acetone 96/4 (v / v) as eluent. The phospholipid fatty acids are then esterified by heating in a solution containing 10% BF3-methanol. The methyl esters are then separated, quantified by gas-liquid chromatography with detection by an ionized flame. Esters are identified by comparison with appropriate controls.

The identification of fatty acids is also confirmed by gas chromatography coupled to mass spectrometry, after derivatization with 4,4-dimethyl-oxazoline, as described by Fay et ai, J. Chromât., 541, 89-98, 1991 .

The results, some of which are presented in Tables 2 and 3 below, show that petroselinic acid is incorporated into the phospholipid fraction of the cells. It is also catabolized by β-oxidation (probably by peroxisomes) due to the presence in large quantity of delta-4-cis-hexadecenoic acid (16: ln-12). This metabolite and its counterpart, 16: ln-9 derived from β-oxidation of ac. oleic, are not detected when cells are incubated in the presence of oleic acid.

Table 2

* may contain traces of 16: 1 n- 10 ** may contain traces of 18: ln-9 nd: not detectable

Table 3

Example 2 Activation of colon cells

Human colon epithelial cells naturally express proteins and enzymes involved in inflammatory responses after treatment with pro-inflammatory agents, such as phorbol esters. Among the proteins and enzymes thus produced, one can thus count superoxide dismutase (SOD) and tumor necrosis factor alpha (TNFα), for example.

In the following tests, epithelial cells of the human colon in culture, primary and immortalized, are subjected in-vitro to an inflammatory treatment with TPA. The immortalized line is that described in EP96201064.1 (Nestlé Products Company) which was deposited, under the Budapest Treaty, with the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lb, D-38124 Braunschweig, Germany, on 16 April 1996, where it received deposit number DSM ACC2258. As described in Example 1, the effect of petroselinic acid on the production of TNFα by the cells of the colon is determined when these are subjected to an irritant treatment. Culture medium B50 (Biofluids), described in EP96201064.1, is used for this purpose. The results of the tests are comparable to those presented in Example 1.

Example 3 Activation of corneal cells

Epithelial cells of the human cornea naturally express proteins and enzymes involved in inflammatory responses after treatment with pro-inflammatory agents, such as phorbol esters. Among the markers of an inflammatory reaction, one can thus count collagenase I (easily detectable marker: Bazan et ai, Proc. Natl. Acad. Sci. USA, 90, 8678-8682, 1993), c-fos (see Bazan et ai), the arachidonic intermediaries 12-HETE and 12-HETrE (Conners et ai, Inves. Ophth. & Visu. Sci., 36, 828-840,

1995) and a new profile in cytokines and growth factors, for example.

In the following tests, epithelial cells of the human cornea in culture, primary or immortalized, are subjected in-vitro to an inflammatory treatment with TPA. The immortalized line is that described in

EP96203707.3 (Nestlé Products Company) which has been filed under the treaty of

Budapest, from the National Collection of Cultures of Microorganisms,

Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, October 22, 1996, where it received the deposit number CNCM 1-1777. The culture medium used is KGM-1 medium (= KBM medium from Clonetics, US, further comprising 0.15 mM CaCh, 30 μg / ml of bovine pituitary gland extract, 0.5 μg / ml of hydrocortisone , 5 μg / ml insulin, 10 μg / ml transferin, 10 ng / l murine epidermal growth factor (EGF), 10,000 U / ml penicillin and 10,000 U / ml streptomycin).

As described in Example 1, the effect of petroselinic acid on the production of collagenase I by the cells of the cornea is determined, when these are subjected to an irritant treatment. For this, the expression of collagenase can be detected by PCR, by Westem-Blot or by Elisa. To detect collagenase I by PCR, the RNA is extracted from the sensitized cells, and the presence of collagenase I mRNA is detected by PCR with a primer derived from the DNA sequence of collagenase I (Genebank: n ° X05231 ).

To detect collagenase I by Western-Blot, the sensitized culture medium is recovered, it is concentrated with an Amicon30® filter, the proteins are separated by electrophoresis on an SDS-Page polyacrylamide gel, they are transferred by Western-blot to a filter, the filter is subjected to a first anti-collagenase I antibody (Cortex Biochem, US, n ° 60338P), to a second antibody coupled to a peroxidase (Pierce, US, n ° 31400), then to the peroxidase substrate .

To detect collagenase I by Elisa, simply use the Amersham hMMP-1 kit (catalog, rpn 2610), following the supplier's recommendations.

The results of the tests are comparable to those presented in Example 1.

Example 4 Body milk (oil-in-water emulsion)

Oily phase:

Glyceryl stearate / PEG-100 stearate (Arlacel 165 sold by the company ICI) (emulsifier) 1%

Polysorbate 60 (emulsifier) 0.8%

2% hydrogenated polyisobutene

Stearic acid 1%

Coriander seed oil 8%

Aqueous phase :

Glycerin 3%

Carbomer (carbopol 941 sold by Goodrich) (gelling agent) 0.3% Triethanolamine (neutralizing agent) 0.3%

Preservative 0.3% Water qs 100% The emulsion is prepared by incorporating the oily phase into the aqueous phase with stirring. A body milk is obtained which provides good protection of the skin against inflammation.

EXAMPLE 5 Care Fluid (Oil-in-Water Emulsion)

Oily phase:

Methylglucose sesquistearate (emulsifier) 2% Cyclomethicone 13%

2% corandan granies oil

0.2% fragrance

PEG 20 methylglucose sesquistearate (emulsifier) 2%

Aqueous phase: Xanthan gums (gelling agent) 0.2% Polysacrylamide / isoparaffin C13-C14 / leureth-7 (Sepigel 305 sold by the company Seppic) (gelling agent) 0.8%

Preservative 0.3% Water qs 100%

The emulsion is prepared as in Example 4. A white fluid is obtained, which provides good protection to the skin against inflammation.

EXAMPLE 6 Care Cream (Water-in-Oil Emulsion)

Oily phase:

Polyglyceryl-4 Isostearate / Cetyl dimethicone copolyol / Hexyl laurate (Abil WE 09 sold by the company Goldschmidt) (emulsifier) 4%

Isohexadecane 5%

Coriander seed oil 10%

Cyclomethicone 3.5%

N-octanoyl-5-salicylic acid 1% Perfume 0.15 Aqueous phase:

Glycerin 10%

0.5% cellulose gum

Magnesium sulfate 0.65% Preservative 0.3%

Water qs 100% To prepare the emulsion, the constituents of phase A are heated to 80 ° C. until the various constituents are completely dissolved, then cooled to 65 ° C. Phase B is heated to 65 ° C and poured into phase A with stirring, then the mixture is cooled. A smooth, nourishing white cream is obtained, which provides good protection of the skin against inflammation.

Claims

claims

1. Use of petroselinic acid, native or esterified, for the preparation of a composition intended to be able to treat or prevent inflammation and / or modulate the metabolism of lipids in the surface tissues of man.

2. Use according to claim 1, characterized in that the composition makes it possible to treat or prevent inflammation and / or modulate the metabolism of lipids in surface tissues, via the activation of β-oxidation.

3. Use according to claim 1, characterized in that the composition is intended to modulate the cellular processes involving the cytokines.

4. Use according to claim 1, characterized in that the superficial tissues are the cells of a mammal constituting the skin, the scalp, the eye, or the oral, nasal, buccal or vaginal mucous membranes.

5. Use according to claim 1, characterized in that the composition is intended for topical or oral application.

6. Use according to claim 1, characterized in that the composition comprises an oil from an umbelliferous plant which is rich in petroselinic acid.

7. Use according to claim 5, characterized in that the oil comprises at least 40% of petroselinic acid.

8. Use according to claim 5, characterized in that the amount of oil ranges from 0.01 to 50% by weight relative to the total weight of the composition.

9. Use according to any one of claims 1 to 8, characterized in that petroselinic acid is present in an amount sufficient for the treatment or prevention of inflammation and / or for the modulation of lipid metabolism in surface tissues. lo

10. Use according to any one of claims 1 to 8, characterized in that the composition is a cosmetic, pharmaceutical and / or food composition.