FR2774292A1 - Pharmaceutical, cosmetic and food compositions for stimulating synthesis of pro-opiomelanocortin and its derivatives e.g. beta-endorphin, useful for treating e.g. inflammatory disorders - Google Patents

Abstract

Pharmaceutical, cosmetic and food compositions for stimulating or enhancing the synthesis of pro-opiomelanocortin (POMC) and its derivatives, e.g. beta -endorphin, by mammalian epithelial cells contain diatoms as the active ingredients and an inert vehicle or excipient.

Description

PHARMACEUTICAL, COSMETIC AND / OR FOOD COMPOSITIONS,
BASED ON DIATOMAS
The present invention relates to the field of pharmacy, cosmetics and / or food.

More specifically, it relates to pharmaceutical, cosmetic and / or food compositions based on diatoms.

Diatoms are one of the important classes of marine phytoplankton, they are small unicellular plants, usually 2-400 μm in length, with the exception of some forms up to 4 mm long. Diatoms are characterized by the presence of a siliceous protective capsule, the frustule, consisting of two parts nested one inside the other. Diatoms are usually divided into two orders, Centrales (Centric Diatoms) and Pennales (Pinnate Diatoms). They live in abundant quantities in the oceans and in large concentrations in the saline lakes found in the United States of America, Australia and especially in the Mediterranean area. They can be grown quite easily in test tubes, in tanks or in plastic tanks or any other suitable container.

Diatoms are susceptible to vegetative growth and sexual reproduction. Depending on the environment, they are able to produce messenger substances intended to attract or inform a congener.

According to the present invention, it has surprisingly been found that diatoms enable or significantly stimulate the synthesis of pro-opiomelanocortin (POMC) and its derivatives by mammalian cells and, more particularly, by epithelial cells.

Pro-opiomelanocortin (POMC) is a prohormone containing 265 amino acids; its synthesis has been demonstrated initially in the pituitary gland. The POMC, once synthesized, is then cleaved by specific convertases into a series of different hormones: ss-lipoprotein (ss-
LPH) containing 91 amino acids, B-endorphin containing 31 amino acids, corticotrophin (ACTH) containing 39 amino acids, met-enkephalin-5, melanocyte stimulating hormone (MSH):
MSH alpha, contained entirely in the structure of ACTH, MSH beta, contained in the structure of ss-LPH, MSH gamma, contained in a fragment of the first half of the
POMC.

Thus, diatoms allow not only the activation of the synthesis of POMC but also that of its derivatives such as ss-endorphin, MSH etc ...

Endorphins are also produced by other prohormones than the POMC. There are three main types of endorphin precursors: - pro-opiomelanocortin (POMC) o ss-endorphin and met-enkephalin, - pro-enkephalin <met-enkephalin and leu-enkephalin, - pro-dynorphin o dynorphine and leu-enkephalin.

Until recently, it was recognized that neurohormones (endorphins, enkephalins) were synthesized in the neuroendocrine organs such as the pituitary gland, the adrenal medulla, or in certain neuronal groups of the central nervous system. Specifically, it was known that ss-endorphin synthesis occurred in the pituitary gland and that of met-enkephalin and leuenkephalin in chromaffin cells of the adrenal medulla. It is now established that these neurohormones are also synthesized in the skin.

The skin is an organ continually exposed to aggression. During a break-in or a modification of the properties of the cutaneous barrier, warning signals (nervous system) are activated. The coordination of these two systems is facilitated by the neuroendocrine system. This narrow intricacy that exists between the nervous, immune and neuroendocrine systems is particularly demonstrative in the skin.

The cutaneous tissue is richly innervated. The different sensitivities (touch, epicritic, cold, heat ...) are provided by specialized nerve endings.

On the ways of the pain are installed inhibitory ways. Thus, at the level of the spinal cord, neurotransmission can be inhibited by endorphins contained and released by specialized neurons from the descending pathways of the spinal cord. It is now known that in the skin itself the transmission of the nerve impulses of the nociceptor may be inhibited by endorphins that may come from the bloodstream, brought by the inflammatory infiltrate, or synthesized in situ in the skin.

It has recently been shown that POMC and its derivatives are expressed and produced by keratinocytes. Thus, Schauer et al (1994), Chakraborty et al (1996), Slominski et al (1995),
Wintzen et al (1996) demonstrated the presence of MSH, ACTH and ss-endorphin in the supernatant of normal human keratinocytes or epidermal cancerous lineages. Among the other cells present in the skin, melanocytes also have the ability to synthesize POMC derivatives (Chakraborty et al., 1996, Slominski et al, 1995).

Pro-opiomelanocortin (POMC) and, consequently, derived hormones, are involved in many immune and inflammatory skin processes and are considered as messengers linking the nervous system to the immune system.

Thus, ss-endorphin and o-MSH play an important role in the control of inflammation and the immune system: monocyte migration, macrophage activation, production of cytokines and proinflammatory mediators.

The ss-endorphin modulates immunoglobulin synthesis, lymphocyte proliferation and natural tiller cell activity. The importance of endorphins is also verified at the dermatological level. Thus, serum and cutaneous serum endorphin levels are increased in psoriasis, which explains the alterations of the erythematous neurogenic response and abnormalities of sensory stimulus transmission. The ss-endorphin produced by the inflammatory cells present in the psoriasis plaques have an anti-nociceptive effect which explains the rarity of the pruritus in this affection.

Thus, the compositions according to the invention based on diatoms are intended in particular to treat pathologies in which inflammatory phenomena are associated. By way of example, mention may be made of: solar erythema, urticaria, psoriasis, insect bites, atrophic polychondritis, pruritus, articular involvement of chronic enteropatia, venous insufficiency, certain types of allopecia, etc.

During stress, POMC derivatives are released into the bloodstream.

Endorphins, by their analgesic effect, intervene in the control of the pain.

However, endorphins, also called pleasure hormones, are also involved in the field of pleasure. Thus, certain cutaneous stimulations induce the secretion of endorphins: the massage and the well being it provides, is accompanied by an increase in the rate of endorphins in the blood. Moreover, after irradiation induced by UV in humans or after intense exercise, the level of serum B-endorphin is stimulated.

Endorphins still control hair growth. Indeed, at the anagen phase of hair growth is associated an increase in the production of endorphins.

The POMC derivatives, including ss-endorphin, MSH, ACTH also have lipolytic activity.

Thus, the importance of endorphins can be measured both at the cosmetic level (pleasure and well-being, hair growth, lipolytic activity, etc.) and at the therapeutic level.

The present invention relates to pharmaceutical, cosmetic and / or food compositions intended in particular to stimulate or to enhance the synthesis of the proopiomelanocortin and its derivatives such as 3-endorphin, by the mammalian epithelial cells, characterized in that they contain, as active ingredient, diatoms, in combination or in admixture with any non-toxic excipient or inert carrier suitable for the intended route of administration.

By epithelial cells is more particularly meant keratinocytes, Malpighi cells, etc.

Diatoms used in the present invention include diatoms of the order of the
Centrals like Cyclotelles.

According to the present invention, the diatoms are in the form of ground material, extract, suspension or solution.

The diatoms, and more particularly the central diatoms such as cyclotella, are particularly present in the sub-Saharan regions of the Mediterranean region where we find lakes filled with very salty water which, at certain times of the year, do not are a crust of salts.

The diatoms are isolated by filtration of the water containing said diatoms or by sieving the salt containing said diatoms.

The diatoms thus obtained are then optionally subjected to grinding, and then the diatomaceous mash or the diatoms is extracted with one or more exhaustions using an organic solvent selected from the group consisting of lower alkanols ( ethanol ...), aliphatic ketones (acetone ...), alkanes (pentane, hexane, heptane ...), halogenated solvents (chloroform ...), aromatic solvents, esters (ethyl acetate ...) and / or the ethers.

According to the present invention, the organic extraction solvent will preferably be ethanol or acetone. An ethanolic or acetonic extract of diatoms is thus obtained.

Diatomaceous earthenware or diatoms may also be extracted with one or more water exhausts. If the solvent is water, a suspension of
Diatoms, which once heated then decanted or filtered, is in solution form.

The active ingredient content of the compositions according to the invention is quite variable according to the intended uses. Thus, the compositions in which the diatom content ranges from 0.001% to 10% by weight relative to the total weight of the composition, are in accordance with the invention.

When a diatom solids extract obtained by evaporation of the solvent is used, the concentration in the compositions according to the invention ranges from 0.01 μg / ml of culture medium to 10 μg / ml of culture medium.

In the more particular case of compositions in the form of creams, the content of active ingredient ranges from 0.1 mg / kg of composition to 10 g / kg of composition.

In the case of compositions intended for the oral route, the content of active principle ranges from 0.01 mg / kg of body weight to 200 mg / kg of body weight.

The suspension, the diatom solution or extract may, depending on the need, be concentrated, dried and optionally diluted in a solvent suitable for the intended use vegetable oil, mineral oil, sodium esters and the like. fatty acids and glycerol, organic liquid, wax or compound or fusible mixture.

The excipient or the inert carrier of the compositions according to the invention is one of those suitable for administration by the digestive, parenteral or topical route. By way of example, mention may be made of manufacturing aids (binders, fluidizers, etc.), flavoring agents, colorants, stabilizers, etc.

The compositions according to the invention are for example presented in the form of tablets, capsules, capsules, pills, solutes or injectable suspensions, patches, creams, gels or ointment.

The present invention also relates to the use of diatoms for the production of pharmaceutical, cosmetic and / or food compositions intended in particular for stimulating or enhancing the synthesis of pro-opiomelanocortin and its derivatives such as B-endorphin by epithelial cells. mammals.

The following examples illustrate the invention without limiting it. These examples are described taking into account the appended figures.

In these examples, the tests were carried out with an ethanolic extract of diatoms
Cyclotella. This extract will be called Cyclotella extract and represented by the initials EC.

EXAMPLE I
Cytocompatibility study of Cyclotella extract (EC)
It is shown, initially, that the Cyclotella extract (EC) is cytocompatible, that is to say that it affects, at the concentrations used, neither the proliferation nor the mitochondrial activity of the cells.

Cytocompatibility tests are essential to continue studies on an active ingredient. In addition to the knowledge of the cytocompatibility of the doses used, which is necessary for all products intended for a cosmetic skin application, these tests define the optimal concentrations during the evaluations of activity and avoid the introduction of ambiguous activity results.

The cytocompatibility of Cyclotella extract is evaluated by two biological tests performed on fibroblasts and keratinocytes: Mitochondrial activity and proliferation of cells.

1. TESTS OF MITOCHONDRIAL ACTIVITY
The mitochondrial activity is evaluated by the XTT test. The XTT test is a colorimetric test based on the reduction of a soluble tetrazolium salt XTT (XTT = 2,3-bis [2-Methoxy-4-nitro-5-sulfophenyl] -2H-tetrazolium-5 carboxyanilide) by NADPH reductase mitochondria of living cells in a water-soluble orange yellow formazan salt which solubilizes in the supernatant of the culture medium.

This colored solution is assayed spectrophotometrically using a microplate reader at 450 nm. Optical density, measured by the XTT assay, is directly correlated with mitochondrial activity or cytocompatibility. Good mitochondrial activity indicates good cytocompatibility. The test is performed with a constant number of cells; for this, the cells are incubated with the cyclotella extract at confluence to inhibit any proliferation.

A) Fibroblast test
Fibroblasts are cells that are easily cultured, these cells are the most abundant cell population of human skin.

The fibroblasts come from human skin taken from a 7 year old child. 3.105 fibroblasts are incubated for 24 hours in 200 μl of DMEM (Dubelcco Minimum Essential Medium) culture medium. The hyperconfluence cells, so that they do not divide, are in the 5th passage treated with various concentrations of ethanolic extract of
Cyclotella.

The results are given in Table 1 below.

Table 1: Mitochondrial activity (in OD) of fibroblasts cultured with concentrations of ethanol extracts of Cyclotella (EC1 ranging from 0.068 to 1.366 KLg / ml

<tb><SEP> EC: <SEP> Average <SEP> Variation <SEP> type
<tb><SEP> into <SEP> llg <SEP> / <SEP> ml <SEP> (DO)
<tb><SEP> Control <SEP> (1) <SEP> 1,027- <SEP>
<tb><SEP> Control <SEP> (1) <SEP> 1,027 <SEP> 1,147 <SEP><SEP> 1,145 <SEP> 1,090 <SEP> 1,207 <SEP> 1,123 <SEQ> 0,068
<tb> EC <SEP>: <SEP> 0.068 <SEP> (2) <SEP> 1.376 <SEP> 1.382 <SEP> 1.269 <SEP> 1.305 <SEP> 1.457 <SEP> 1.358 <SEP> 0.073
<tb> EC <SEP>: <SEP> 0.136 <SEP> (3) <SEP> 1.562 <SEP> 1.552 <SEP> 1.561 <SEP> 1.457 <SEP> 1.446 <SEP> 1.516 <SEP> 0.059
<tb> EC <SEP> 0.273 <SEP> (4) <SEP> 1.345 <SEP> 1.645 <SEP> 1.599 <SEP> 1.568 <SEP> 1.565 <SEP> 1.544 <SEP> 0.116
<tb> EC <SEP>: <SEP> 0.683 <SEP> (5) <SEP> 1.570 <SEP> 1.502 <SEP> 1.698 <SEP> 1.565 <SEP> 1.225 <SEP> 1.512 <SEP> 0.176
<tb> EC: <SEP> 1.366 <SEP> (6) <SEP> 1.363 <SEP> 1.514 <SEP> 1.464 <SEP> 1.443 <SEP> 1.609 <SEP> 1.467 <SEP> 0.112
<Tb>
Cyclotella extract does not alter the metabolism of fibroblasts: the differences are not statistically significant compared to the control. Cyclotella extract is cytocompatible with fibroblasts.

B) Test on keratinocytes
Keratinocytes are the most abundant cell population in the epidermis: it is the first cells that will be in contact with the outside world.

The keratinocytes come from human skin taken from a child aged 7 years. 2.105 keratinocytes are incubated for 24 hours in 200 μl of DMEM culture medium. The cells in hyperconfluence are at the 5th passage and are treated with various concentrations of ethanolic extract of Cyclotella.

The results are given in Table 2 below
Table 2: Mitochondrial activity (in OD) of keratinocytes cultured with concentrations of ethanolic extract of Cyclotella (EC) ranging from 0.068 to 1.366 g / ml

<tb><SEP> EC: <SEP> Average <SEP> Variation <SEP> type
<tb><SEP> into <SEP> llg <SEP> / <SEP> ml <SEP> (DO)
<tb><SEP> Control <SEP> (1) <SEP> 0.525 <SEP> 0.572 <SEP> 0.600 <SEP> 0.620 <SEP> 0.574 <SEP> 0.578 <SEW> 0.036
<tb> EC <SEP>: <SEP> 0.068 <SEP> (2) <SEP> 0.608 <SEP> 0.662 <SEP> 0.641 <SEP> 0.582 <SEP> 0.610 <SEP> 0.621 <SEP> 0.031
<tb> EC <SEP>: <SEP> 0.136 <SEP> (3) <SEP> 0.693 <SEP> 0.730 <SEP> 0.700 <SEP> 0.742 <SEP> 0.658 <SEP> 0.685 <SEP> 0.061
<tb> EC <SEP>: <SEP> 0.273 <SEP> (4) <SEP> 0.674 <SEP> 0.697 <SEP> 0.579 <SEP> 0.647 <SEP> 0.674 <SEP> 0.654 <SEP> 0.046
<tb> EC <SEP>: <SEP> 0.683 <SEP> (5) <SEP> 0.730 <SEP> 0 <SEP><SEP> 0.700 <SEP> 0.742 <SEP> 0.658 <SEP> 0.610 <SEP> 0.688 <SEP> 0.054
<tb> EC: 1.366 <SEP> (6) <SEP> 0.567 <SEP> 0.676 <SEP> 0.645 <SEP> 0.649 <SEP> 0.598 <SEP> 0.627 <SEP> 0.044
<Tb>
Cyclotella extract does not modify the keratinocyte metabolism: the values are not statistically significant compared to the control. Cyclotella extract is cytocompatible with keratinocytes.

2. PROLIFERATION TESTS
The proliferation of cells is one of the most important reflections of cellular behavior.

A pharmacological substance intended for this type of application must not cause excessive proliferation or significant reduction in cell division.

A constant number of cells are incubated in their respective medium in the presence of increasing concentrations of ethanolic extract of Cyclotella. An enumeration of the cells is carried out after 24 hours.

As previously, the concentrations of ethanolic extract of Cyclotella vary from 0.068 to 1.366 ssg / ml.

The effect of Cyclotella (EC) extracts on the proliferation of fibroblasts and keratinocytes is shown in Table 3 below.

<tb><SEP> E. <SEP> C: <SEP> | <SEP> Fibroblasts <SEP> (F) <SEP> Keratinocytes <SEP> (K)
<tb><SEP> in <SEP> g / ml <SEP>
<tb> 0 <SEP> = <SEP> control <SEP> (1) <SEP> 1,000 <SEP> 1,000
<tb><SEP> 0.068 <SEP> (2) <SEP> 1,120 <SEP> 1,098
<tb><SEP> 0.136 <SEP> (3) <SEP> 1.090 <SEP> 1.107
<tb><SEP> 0.273 <SEP> (4) <SEP> 1.075 <SEP> 1.114
<tb><SEP> 0.683 <SEP> (5) <SEP> 1.127 <SEP> 1.095
<tb><SEP> 1,366 <SEP> (6) <SEP> 1,114 <SEP> 1,112
<Tb>
This test was performed three times. There is no statistically significant enumeration between the different concentrations and with respect to the control. The Cyclotella extract does not modify the proliferation of fibroblasts and keratinocytes.

EXAMPLE II
Cyclotella Extract (EC) Activity on the Synthesis of a 15-Endorphin by Epithelial Cells
The epithelial cells in question here are human keratinocytes.

The test whose results are summarized in Table 4 below, is an immunofluorescence evaluation test, obtained with an anti-ss-endorphin antibody.

The fluorescence intensity reflects the rate of ss-endorphin synthesis by the keratinocytes, the higher the fluorescence intensity, the higher the rate of ss-endorphin synthesis.
The values given are arbitrary values corresponding to the fluorescence intensity.

<Tb>

<SEP> Keratinocytes <SEP> (Kc) <SEP> Kc <SEP> + <SEP> IL-1 <SEP> Kc <SEP> + <SEP> EC
<tb><SEP> (witness) <SEP>(<SEP> witness <SEP> enabled <SEP>) <SEP>
<tb> Synthesis <SEP> of <SEP> (3- <SEP> 0.120 <SEP> 1,000 <SEP> 1,650
<tb><SEP> endorphin
<Tb>
Keratinocytes in the presence of interleukin-1 (IL-1) synthesize about 10 times more ss-endorphin than the same resting keratinocytes.

The keratinocytes treated with the Cyclotella extract synthesized 16 times more p-endorphin than the same resting keratinocytes.

Thus, the synthesis of the pro-opiomelanocortin prohormone (POMC), and therefore the derived B-endorphin hormone, is strongly stimulated by the epithelial cells in the presence of Cyclotella extract.

Claims (10)

 Inert carrier or vehicle, non-toxic, suitable for the intended route of administration.  contain as an active ingredient diatoms, in association or in combination with any  ss-endorphin by mammalian epithelial cells, characterized in that they  stimulate or enhance the synthesis of pro-opiomelanocortin and its derivatives such as  1. Pharmaceutical, cosmetic and / or food compositions intended in particular to 2. Compositions according to claim 1, wherein the epithelial cells are the  keratinocytes. Compositions according to claim 1 or claim 2, wherein the diatoms  are of the order of Centrals like Cyclotelles. Compositions according to any one of claims 1 to 3, wherein the diatoms  are in the form of mash, extract, suspension or solution. Compositions according to claim 4, wherein the diatom extract is obtained by  extracting the crushed material of diatoms or diatoms by one or more burns using  an organic solvent selected from the group consisting of lower alkanols, ketones  aliphatics, alkanes, halogenated solvents, aromatic solvents, esters and / or  ethers. Compositions according to claim 5, wherein the organic solvent is ethanol or  acetone. Compositions according to claim 4, wherein the diatomaceous solution is obtained  by extraction of the crushed material of diatoms or diatoms by one or more  using water, then heating, and decantation or filtration of the suspension thus obtained. Compositions according to any one of claims 1 to 7, wherein the content of  Diatoms ranges from 0.001% to 10% by weight relative to the total weight of the  composition. Compositions according to any one of claims 1 to 8, wherein the excipient or  the inert vehicle is one of those suitable for digestive administration,  parenteral or topical. 10.Using diatoms for the production of pharmaceutical compositions  cosmetics and / or foodstuffs intended in particular to stimulate or amplify the synthesis of  pro-opiomelanocortin and its derivatives such as ss-endorphin by cells  epithelial mammals.