WO1993009805A1 - Polar lipid composition of plant origin - Google Patents

Abstract

Polar lipid composition of plant origin for conveying an active agent and/or making it penetrate into a target cell, the composition being characterized in that it is comprised of an injectable, intra-articular, topical or ingestable aqueous emulsion of a polar lipid mixture rich in phospholipids, in glycolipids and in ceramides, having a composition which is substantially similar to that of the polar lipid constituents of lipid cytomembranes of cells and obtained from a plant compound such as cereal flour or an extract such as bran or lipids extracted from cereals by means of chlorinated solvants.

Description

 POLAR LIPID COMPOSITION OF PLANT ORIGIN

The present invention relates to a polar lipid composition of plant origin making it possible to transport an active agent and / or to make it penetrate into a target cell.

 For ecological reasons, we are currently trying to protect animals as much as possible and to avoid using them at laboratory level as much as possible: this current trend, encouraged by the various leagues for the protection of animals, means that the products of animal origin are more and more badly accepted.

 Independently of these philosophical concerns, the development of products from the animal kingdom could be hampered in the future: indeed, in recent years, we have seen the appearance in the animal kingdom, and in particular in ruminants, Prion-type viruses that attack the brain and nervous tissue and are responsible for extremely serious neurological damage.

However, it is not excluded that such viruses are likely to spread in humans; it is feared that their development could have dramatic consequences.

 Given this uncertainty, there is currently a prejudice against products from the animal kingdom.

 It should be noted in particular that certain products extracted from nervous tissues of bovine origin have already been banned by the Pharmacie Centrale des Hôpitaux de Paris.

 This new constraint is a source of difficulties linked to the fact that the products of animal origin are already in a partially or completely synthesized state, while the use of plant products always requires additional manipulations which can prove to be long and costly,

 Given this context, researchers working in fields such as dermatology or pharmacology are increasingly trying to use products from the plant kingdom or products of marine origin to replace animal products, despite increased difficulties thus encountered.

 This research has resulted, in accordance with document WO-A-92 00 182, with the development of a new process making it possible to obtain - from a plant compound such as cereal flour or an extract such as bran, or lipids extracted from cereals with chlorinated solvents - a lipid mixture rich in phospholipids, glycolipids and ceramides, and in particular, a basic lipid mixture having the following weight composition:

- Ceramides 90%

 - Lecithins 5%

- Galactolipids 5%. It has already been proposed to use this basic lipid mixture in fields such as cosmetology or dermatology.

 It has now been discovered, in accordance with the invention, that the basic lipid mixture of exclusively vegetable origin mentioned above can have numerous and important other applications, in particular in the field of pharmacology.

 Biologists have known for many years that all cells in the animal kingdom are surrounded by plasma membranes which constitute barriers with selective permeability; these cytomembranes, the texture of which is largely constant, are essentially constituted by a continuous double layer of lipid molecules arranged parallel to each other and united by their hydrophobic groups, in which various membrane proteins are embedded.

 Different constituents enter into the composition of these double lipid layers of the plasma membrane and, in particular, non-polar oils, or polar constituents of which there are three main classes:

 - phospholipids such as lecithin,

- glycolopids or galactolipids,

 - ceramides, sphingolipids and glycosphingolipids.

 Each type of cell has its own composition of polar lipid constituents, and specialists have been able to establish ranges of compositions specific to each type of cell.

Les chercheurs ont pu prouver que les constituants polaires des cytomembranes lipidiques ont un rôle déterminant sur la perméabilité cellulaire et donc influent sur les possibilités de pénétration des agents actifs à l'intérieur des cellules. The table below gives by way of example the approximate lipid compositions of different cell membranes.

Researchers have been able to prove that the polar constituents of lipid cytomembranes have a decisive role in cell permeability and therefore influence the possibilities of penetration of active agents inside cells.

 Now, in accordance with the invention, it has been realized that the above-mentioned basic lipid mixture has a composition which is largely identical to that of the polar lipid constituents used in the composition of the lipid double layers of the plasma membranes, and that, from from this basic mixture, it is possible to manufacture, by successive fractionations, a "second" lipid mixture whose composition is identical to that which is specific to a given type of cells.

 The idea underlying the invention is to use this identity to make, from the basic polar lipid mixture, a composition in which the lipid formula of the lipid cytomembranes is reconstituted to transport an active agent or facilitate its penetration into a target cell.

 To this end, the invention relates to a polar lipid composition of plant origin characterized in that it consists of an injectable, intra-articular, topical or ingestible aqueous emulsion of a polar lipid mixture rich in phospholipids, in glycolipids and ceramides, having a composition essentially identical to that of the polar lipid constituents of the lipid cytomembranes of cells and obtained from a plant compound such as cereal flour or an extract such as bran or lipids extracted from cereals with chlorinated solvents.

This composition, which can be pharmaceutical, cosmetic or even dietetic, can take on various forms without departing from the scope of the invention and, in certain cases, the composition may even constitute the active agent proper.

 For example, it is known that an excess of cholesterol can cause the deposition, on the internal wall of the arteries, of an atheroma plaque formed by a mixture of fat-laden cells and cholesterol crystals. The reaction of the wall opposite the atheroma plaque is variable: it begins with sclerosis (atherosclerosis) and is followed by calcifications (mediacalcosis). This can lead to thrombosis (blood clotting in the artery) and aneurysms (distention of the wall).

 However, we have already been able to demonstrate that the polar lipid mixtures in accordance with the invention and in particular the ceramides are emulsifiers for cholesterol: injection into the blood circuit of a patient whose arteries carry numerous plaques of 'atheroma of a composition according to the invention can facilitate the elimination of these deposits; this lipid mixture will indeed extract the cholesterol then facilitate its evacuation in the blood circuit.

 More precisely, to obtain the composition actually used in accordance with the invention, the concept of HLB or "Hyrophile-Lipophile Balance" is used which corresponds to a classification of emulsifiers according to their hydrophilicity or their lipophilicity. A low HLB (3 to 5) characterizes an emulsion with an oily continuous phase, a higher HLB (10 to 12) an emulsion with an aqueous continuous phase and beyond solubilization.

However, from this notion, many methods have been developed, both theoretical and experimental. to determine in each particular case the composition of a surfactant system giving the finest emulsion and therefore the optimum coupling of several surfactants.

 According to the invention, to determine in vitro the optimal composition for the emulsification of cholesterol, an experimental process was used based on research by measuring the conductivity of the amount of water causing the phase inversion of an emulsion .

 An HLB of 8 to 10 was thus obtained. According to a particularly advantageous variant of the invention, the emulsion has a composition essentially identical to that of the lipid cytomembranes of a target cell and contains an active agent coated in a sheath of the lipid mixture.

 Such a composition may, by way of example, contain a moisturizing agent associated, where appropriate, with additives such as vitamins

A or E and coated in a sheath having a composition identical to that of the lipid cytomembranes of the cells of the epidermis and in particular of the stratum corneum; the moisturizing agents present in these compositions can be liposoluble humectants or hygroscopic agents such as for example lanolin, polyunsaturated fatty acids in particular vitamin F, linoleic acid, gammalinolenic acid or eicosapentaenoic acid or alternatively water-soluble agents such as glycerin, mucopolysaccharides, allantoin derivatives, amino acids, urea, sodium, potassium.

In such compositions, which can be used for the fight against aging or the treatment of burns, the lipid mixture polar acts in fact on two levels: it improves the membrane permeability towards the active agent, but, at the same time contributes itself to the hydration of the cells; we have, in fact, recently been able to highlight the role in the hydration mechanisms of the skin of phospholipids and especially ceramides which are likely to contribute to better water retention, to restructure the epidermis and in particular the intercornéocytaire cement and to improve the resistance of the skin to external aggressions.

 The composition in accordance with the invention may also take the form of a medicinal composition intended for various therapeutic classes and the active agent of which may take various forms and in particular be chosen from the group formed by antibiotics, anti-inflammatories, cortidoids, antivirals, anticancer drugs and drugs for cardiovascular pathologies.

By way of example, one can more specifically cite the possibility of conveying the following active agents:

 Thanks to the choice of a polar lipid mixture whose composition corresponds to that of the lipid cytomembranes of the target cells which it is desired to treat, it is thus possible to inject the active agent directly on the desired site where it will "attack" selectively the target cells thus making it possible, for an improved effect and yield, to decrease its concentration and therefore to avoid side effects as much as possible.

 The above-mentioned examples are, of course, not limiting and one can envisage many other applications of the invention, consisting notably in bringing in an element such as sodium into a deficient cell.

 In all cases, the polar lipid sheath constitutes, thanks to its selective composition, a vector making it possible to improve the permeability of the target cell for the active product.

It is also possible, according to another characteristic of the invention, to incorporate into the composition, as active agent, not a compound strictly speaking "treating" but a toxic substance selective for a pathogenic agent, in particular a virus, a bacterium or a fungus.

 In this particular case, the polar lipid sheath acts as a "decoy" to trap the pathogen and facilitate its elimination.

 This aspect of the invention can be used for the fight against AIDS by coating AZT in a polar lipid sheath whose composition corresponds to that of the lipid cytomembranes of leukocytes: the AIDS virus can indeed recognize the lipids constituting the sheath which correspond to its normal path of penetration into the cells and thus come into contact with the AZT so as to promote its destruction.

 The polar lipid system of this patent can also be used as a vehicle for vaccine components necessary for strengthening humoral and cellular immunity.

 The characteristics of the polar lipid composition which is the subject of the invention will be detailed in the examples below:

EXAMPLE 1:

 A cosmetic composition was produced having the following weight formula:

Vitamin E acetate 0.5% Hydrogenated lecithin 0.5%

Ceramides 0.5%

Vitamin A 0.1%

(1,000,000 IU / g)

Preservatives + Water. This composition has proven to be very stable and has a particular feel that appeals to users.

 EXAMPLE 2:

 Report of a test carried out in the

Laboratories of the Faculty of Pharmacy of Chatenay Malabry (France) in order to verify the activity of a polar lipid mixture in accordance with the invention on protection by corticosteroids against a radical attack.

 The principle of this test consists in subjecting the red cells isolated from their plasma to an oxidative type aggression under controlled and standardized conditions so as to allow them to put all their enzymatic and molecular equipment into play to resist this aggression until modification of the cell membrane and bursting and lysis of the cell.

 More specifically, various preparations containing red cells were subjected to attack by organic free radicals produced by thermal decomposition of a particular nitrogen compound soluble in water: 2,2'-azo-bis-2 amidinopropane hydrochloride ( 100 mM), under an atmosphere of air at 37ºC. It was thus possible to produce a known and constant quantity of peroxide radicals.

 At regular intervals over time, a small volume of supernatant was taken from the preparation and its hemoglobin content was analyzed by spectrophotometry (λ = 540 nm or 405 nm).

 The resistance of the red blood cell population of each of the preparations tested was then expressed by the half-lysis time, that is to say the release time of 50% of the hemoglobin content.

To carry out this test, we chose isolated human red cells previously washed and resuspended in hematocrit (11%); these cells have, in fact, the advantage of having a relatively short half-life (around 110 days) and of having all the molecular and enzymatic equipment for anti-free radical protection, which allows them to be representative other cells in the body.

 To carry out this test, the following compositions according to the invention were previously prepared:

Composition A

 Vitamin E acetate 2%

1% hydrogenated lecithin

Ceramides 1%

Vitamin A acid 0.05%

Preservatives + Water.

Composition B

 Vitamin E acetate 2%

Hydrogenated lecithin 1% Ceramides 1%

Dexamethasone 0.02% Preservatives + Water.

 The following five test preparations were then made:

1: Red cells

 2: Red cells + Composition A (1/10)

3: Red cells + Composition B (1/10)

4: Red blood cells + Betneval (registered trademark)

 (1/10)

 5: Red cells + Effederm (registered trademark)

(1/10). Betneval and Effederm correspond to commercial drugs containing respectively dexamethasone and vitamin A acid at concentrations similar to those of compositions A and B in accordance with the invention.

 The table above gives the T50 half-lysis times expressed in minutes obtained for each of the preparations tested:

 These results show that the addition of the two commercial products only very weakly protects the life of red blood cells subjected to radical "stress", while the two compositions according to the invention which have been tested have made it possible to obtain much higher protection.

 EXAMPLE 3:

Report of a test carried out at the Connective Tissue Laboratory of the CNRS of Créteil (France) in order to verify the activity of a polar lipid mixture in accordance with the invention on the inhibition of a particular enzyme, the Human leukocyte elastase (ELH) which has the property of destroying elastin and therefore of causing cellular aging and also of altering the arterial walls. The activity of the mixture according to the invention was compared with that of oleic acid, reference substance.

The principle of this test consists in measuring the inhibition of the enzymatic activity by different preparations tested by following the kinetics at 410 nm of the degradation of a substrate S corresponding essentially to elastin le: Me-O-Suc-Ala ₂ -Pro- Val-pNa (N-Methoxysuccinyl-Ala-Ala-Pro-Val-p-Nitroanilide). In this test, we measure, over time, the evolution of the concentration of p-Nitroaniline which is released under the effect of the activity of the enzyme ELH on the substrate S. To perform these tests, we have used as T reaction buffers:

- either 0.1 M tris-HCl + 0.01% triton X-100 at pH 8,

 - or 0.1 M tris-HCL at pH 8.

In general, a better inhibition could be observed in tris-HCL, but a weaker enzymatic activity than in the buffer tris-HCL + 0.01% of triton X-100.

 In the context of this example:

 FIG. 1 represents the anti-elastatic activity of the reference oleic acid,

 FIG. 2 represents the anti-elastatic activity of preparation A,

 FIG. 3 represents the anti-elastatic activity of preparation B,

 FIG. 4 represents the anti-elastatic activity of preparation C,

- Figure 5 shows the anti-elastatic activity of preparation D.

 More precisely :

For each of the substances tested, the tanks 1 to 5 were prepared, the composition of which is listed in the table below:

Each of the tanks was then stirred for 30 min at 37 ° C so as to allow the enzyme time to act on its substrate.

 The p-nitro-aniline concentration was then read at 410 nm and the results obtained were plotted on curves 1 to 5 which correspond to the tanks of the same numbering. Curves numbered 1 correspond to the reference while curves 2 and 3 represent the kinetics in the absence of tested preparations and curves 4 and 5 in the presence of these preparations.

 On each of these curves, the time in minutes is plotted on the abscissa and, on the ordinate, the optical deviation DO read at 410 nm.

 The preparations tested were as follows:

- Preparation A: Ceramide 1% + hydrogenated lecithin

 1%

- Preparation B: Ceramide 1% + hydrogenated lecithin

 1% + Vitamin E acetate 2%

 - Preparation C: Ceramide 1% + hydrogenated lecithin

 1% + Vitamin E acetate 2% + Vitamin A acid 0.05%

- Preparation D: Ceramide 1% + hydrogenated lecithin

 1% + Vitamin E acetate 2% + Dexamethazone 0.02%.

For the reference oleic acid, we proceeded somewhat differently since we used seven measuring tanks from which we established, each time, the curves corresponding to the monitoring of the kinetics of the release of p-nitroaniline. Curves 1 to 3 correspond respectively, as for the preparations in accordance with the invention, to the reference curve and to the kinetics in the absence of oleic acid. Curves 4 and 5 correspond to an oleic acid concentration equal to 0.1 μg / ml while curves 6 and 7 correspond to an oleic acid concentration of 1 μg / ml.

 The curves corresponding to the reference oleic acid as well as to preparations A to D are attached in the appendix.

 On each of them, the percentage of inhibition of the anti-elastatic activity of each of the different preparations was measured after 10 min and the following results were found: Reference oleic acid at 0.1 μg / ml: 36%

 Reference oleic acid at 1 μg / ml: 84%

 Preparation A: 45%

 Preparation B: 24%

 Preparation C: 29%

Preparation D: 51%.

These results make it possible to clearly demonstrate the anti-elastatic activity of the compositions in accordance with the invention and, in particular, of the ceramides.

 This activity had so far never been demonstrated.

Claims

 R E V E N D I C A T I O N S  1) polar lipid composition of plant origin allowing to transport an active agent and / or to make it penetrate into a target cell, characterized in that it is constituted by an injectable, intra-articular, topical or ingestible aqueous emulsion of '' a polar lipid mixture rich in phospholipids, glycolipids and ceramides, having a composition essentially identical to that of the polar lipid constituents of the lipid cytomembranes of the cells and obtained from a plant compound such as cereal flour or an extract such as bran or lipids extracted from cereals by chlorinated solvents.  2º) Composition according to claim 1, characterized in that the lipid mixture constitutes the active agent.  3º) Composition according to claim 2, intended to facilitate the elimination of atheroma plaques, characterized in that it contains, as active agent, a lipid mixture of which the HLB is essentially between 8 and 10.  4 °) Composition according to claim 1, characterized in that the emulsion has a composition essentially identical to that of the lipid cytomembranes of a target cell and contains an active agent coated in a sheath of the lipid mixture.  5º) Composition according to claim 4, characterized in that the active agent is a moisturizing agent associated, where appropriate, with additives such as vitamins A or E. 6º) Drug composition according to claim 4, characterized in that the active agent is chosen from the group formed by antibiotics, anti-inflammatories, corticoids, antivirals, anticancer drugs and medicines for cardiovascular pathologies.  7º) Drug composition according to claim 4, characterized in that the active agent is a toxic substance selective for a pathogenic agent in particular a virus, a bacterium or a fungus.  8º) Drug composition according to claim 7, characterized in that the active agent is AZT.  9º) Composition according to any one of claims 1 to 7, more especially intended for the field of immuno allergology, characterized in that the allergen is conveyed by the glycolipid system to stimulate the immune defense.  10º) Composition according to any one of claims 1 to 9, characterized in that it constitutes a vector system of vaccine components to strengthen humoral and cellular immunity.