WO2012065651A1

WO2012065651A1 - A synergistic composition of plant extracts for treating skin diseases involving abnormal cell growth

Info

Publication number: WO2012065651A1
Application number: PCT/EP2010/067861
Authority: WO
Inventors: Ravi Shrivastava; Christiane Shrivastava
Original assignee: Vitrobio
Priority date: 2010-11-19
Filing date: 2010-11-19
Publication date: 2012-05-24
Also published as: EP2640409A1

Abstract

A composition of a non specific multiple cytokine and cell growth factor inhibitor suitable for use in treating skin diseases involving excessive skin cell growth containing preferably synergistic association of at least two tannins rich plant extracts such as those selected among the plant extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Vaccinium macrocarpon or Vaccinium myrtillus fruit, Quercus oak bark, Ginko biloba, Salix alba, Mimosa tenuiflora, Echinacea sp, Tenacetum parthenium, Camillia sinensis, or Sambucus nigra.

Description

A synergistic composition of plant extracts for treating skin diseases involving abnormal cell growth

The present invention concerns a synergistic composition of plant extracts, said association of plant extracts having synergistic inhibiting properties on skin cell growth stimulating proteins, particularly the cytokines. This synergistic composition can be used as a medicament or medical device for topical application to treat skin disorders involving abnormal skin cell growth such as Psoriasis, Eczema and atopic Dermatitis (PED) and other skin conditions involving disfunctioning of cell growth such as skin rashes, blisters of pemphigus and pemphigoid, dermatitis herpetiformis, erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, lichen planus, perioral dermatitis, pityriasis rosae, rosacea, and similar skin lesions, abbreviated as PED. The term topical includes all the external parts of the body such as the skin, mouth cavity, throat, upper respiratory tract, nasal cavity, vaginal mucosa, eye surface and the natural body openings. The application of this invention is therefore restricted to only those skin affections which are manifested on the body surface.

BACKGROUND ART

Skin and PED lesions: The skin is an organ composed of several layers of tissue and protects the body as an envelope. In humans, it is one of the most important organs of the body weighing about 5 kg and covering about 2m² body surface. Skin consists of three main parts: the outermost thinner layer called epidermis, the thickest middle part called dermis and the deeper, the hypodermis, which is traditionally not regarded as a layer of the skin. The skin is the fastest regenerating organ in the body; almost all the skin cells are replaced every 5-6 weeks. The basal layer of the epidermis multiplies continuously; the cells move outwards, die and shed as keratinocytes. A balance between the growing and the dying cells is necessary to maintain a normal skin physiology and morphology (McGrath, 2004).

Under certain pathological conditions such as in case of Psoriasis, Eczema, and different forms of Dermatitis (PED), an uncontrolled and excessive growth of the skin basal cell layer generates 2 to 3 times more cells compared to a normal skin cell regeneration leading to poor cell attachment and the appearance of scaly, dry, sloughing, and inflamed skin areas causing pain, itching, and aesthetic problems with or without secondary bacterial infection. Although excessive skin production is the main manifestation in all these diseases, according to the type or location of the lesion and the appearance, it may be presented as psoriasis, eczema or dermatitis. PED is very common skin diseases in human beings all over the world affecting between 1 and 3% of the world population (Griffiths, 2007).

The cause of PED type of skin lesions is not well understood but some immune- mediated disorders leading to excessive and uncontrolled growth in the dermal part of the skin is most accepted hypothesis. Although excessive skin production is the main manifestation in all these diseases, according to the type or location of the lesion and the appearance, it may be presented as psoriasis, eczema or dermatitis.

Psoriasis commonly causes red and scaly patches, called psoriatic plaques, which are inflammation areas due excessive skin production (Krueger, 2005). At these sites, skin rapidly accumulates and takes on a silvery-white appearance. Plaques frequently occur on the elbows and knees skin, but can affect any area including the scalp, hands palms, feet soles, and genitals.

In contrast to psoriasis, Eczema is often likely to be found on the flexor aspect of the joints. Eczema lesions are skin rashes characterized by one or more symptoms like redness, swelling, itching and dryness, sloughing, flaking, blistering, cracking, oozing, or bleeding.

Other skin lesions having red, flaky and very itchy skin is often grouped as dermatitis.

The skin folds are coarse thick and very dry. Lesions may often form reddish papules with vesicles formation which may generalize over the entire body surface.

The exact cause of these skin conditions leading to excessive cell growth is not yet known but suggested to be due to genetic factors, stress, immunological disorders, auto-immune diseases, allergic reactions, food allergens or even mental disturbances but the trigger causing excessive cell regeneration is not yet identified. Uncontrolled cell growth causes hyperplasia, hyperkeratosis and perakeratosis of the skin epidermis, which is further infected, leading to PED type of skin diseases.

The common feature in all the PED lesions is the abnormally excessive growth of skin dermal cells. Excessive skin cell growth in PED is always accompanied by an impressive high number of immuno-modulating cells such as the neutrophil granulocytes, macrophages, T cells, mast cells and dendritic cells and lymphocytes (Rachael, 2006). These cells are known to produce cytokines and Growth Factors (GFs) and this the reason why the concentration of many cytokines and GFs is extremely high on the surface of the PED lesions. Although the presence of these cells is essential for topical immunity, healing, immune-modulation and even cell growth; the reason for the presence of abnormally high number of these cells in the affected site is not defined. As these cells are involved in the production of cytokines and GFs, some of which trigger cell growth, we postulated that the abnormal and excessive cell growth in PED type of skin diseases is triggered due to excessive presence of these cell growth stimulating molecules. Therefore, neutralizing and rendering ineffective these molecules may constitute an excellent treatment for such diseases.

GFs are small proteins and their interaction with the skin dermal cells trigger cell division. Cytokines are peptides, proteins or glycoproteins with an average molecular weight of 8- 50 kDa and can be classified into three categories: interferons, interleukins and chemokines. Interleukins are cytokines grouped without biochemical or function relationship, but for convenience, classified according to the findings chronology. Today, there are 35 main cytokines called Interleukins (IL) which are further sub-classified. For example, there are already 11 identified members of the IL-1. Due to huge number of cytokines, due to their multiple functions, and due to their activity in extremely low concentrations, many cytokines are still not identified and many functions of the cytokines are still not known. Among the cell growth stimulating proteins, the role of certain GFs such EGF (epithelial cell GF), KGF (keratinocyte GF), PDGF (platelet derived GF), and FGF (fibroblast GF) and certain cytokines such as the Interferon γ, TNF (tumour necrosis factor) α/β, IL-2 (interleukin-2), IL-3, IL-5, IL-6, IL-8, IL-10, IL-12, IL-22, IL-23,TGF (tumour growth factor) α/β, Granulocyte Macrophage-colony Stimulating Factor (GM-CSF), SCF (Stem Cell Factor), and rhGM-CSF thrombopoietin are fairly well recognized by the scientific community (David Farni, 2009). An optimal level and a perfect balance between the activities of different types of cytokines ensure a healthy skin physiology. Therefore an ideal treatment to treat PED should normalize the concentration of topically available cytokines either by inhibiting the activity of cytokine producing cells or by reducing the amount of topically available cell growth stimulating cytokines and GFs in the lesion. Unfortunately, inhibiting the generation of cytokine producing cells requires oral administration of cytostatic drugs, which also influence other body functions and may have serious side effects. Another possibility consists of reducing the concentration of cytokines present topically on the surface of the PED lesions but this approach requires drugs, chemicals or monoclonal antibodies, capable to block all the cytokines at a time but the variability in the structure of these cytokines and the growth promoting factors makes this task nearly impossible.

This is the reason why all the currently available topical PED treatments are mostly symptomatic and have no effect on the cause of the origin of PED. Currently used drugs include corticosteroids as anti-inflammatory agents (Barnetson, 1992); Vitamin D (Calcipotrine) or Vitamin A (Tazarotene) to modulate keratinocyte growth; corticosteroids with vitamin D as antiinflammatory; salicylic acid preparations as analgesics; lactic acid preparations to normalize cell growth by peeling; urea preparations as humectants; glycolic acid to help faster detachment of old dead cells, and immuno-modulator creams to decrease the effect of topical allergens (tacrolimus, pimecrolimus, anthralin).

Oral and intravenous anti-inflammatory products such as acitretin (soriatane) which causes retinoid induced modulation of cutaneous inflammation or even cell growth blockers such as methotrexate as well as anti-fungal drugs to treat topical infection (cyclosporines, mycophenolate), are also employed to treat PED.

Injectable genetically engineered immuno-suppressants such as Alefacept (amevive), Adalimubab (humira), Infliximab (remicade), Etanercept (enbrel), and Ustekinumab; specific individual cytokine inhibitors such as TNF-a blockers which bind to TNF-a protein (Enbrel containing etanercept, Remicade containing infliximab, and Humira containing adalimubab); T-cell activators and T-cell movement blockers to prevent the activation of lymphocytes (raptiva, efalizumab); drugs that decrease number of T-cells (amevive, alefacept); drugs that interfere with chemical messengers of inflammation (ustekinumab) (Gandhi, 2010), UV light (Stern 1979), homeopathy, plant drugs, biotherapy, soaps, and thermal water (Halevy, 2001) also constitute treatments for PED but none of these treatments are directed to remove all the cytokines and the GFs which are involved in the development and maintenance of PED lesions.

Current research is focused to normalize body's immune system or the number of immuno-modulating cells in affected area or to block topical immune cell functions but these approaches are difficult as all the target cells are not known and cellular antagonists are not available. Such drugs may affect other cell functions and will have numerous side effects (Ma, 2010). Use of specific monoclonal antibodies to neutralize a specific cytokine is already practiced but there are many cytokines, antibodies against all the cytokines are not available, and such drugs have numerous side effects.

None of these treatments is considered effective and almost all the oral or systemic treatments have numerous side effects (David Farhi, 2009). Therefore, there is an urgent need to find a specific treatment, which can act on the cause(s) of PED without any toxic or side effects.

As there are many known and unknown cytokines and GFs, having an abnormally high concentration in the PED lesions acting in synergy to stimulate excessive and abnormal cell growth, the best approach consists of neutralizing all the cytokines and all the GFs so as to reduce their stimulating effect on the epidermal cell growth. The only common point in all these cell growth stimulating cytokines and GFs is that they are all proteins or peptides. Therefore, topical application of a non specific protein inhibitor was considered as one of the best approaches to treat PED. Surprisingly, the studies conducted in our laboratory showed that there is not only one but a group of specific cytokines involving IL-6, GM-CSF, SCF, IL-17A, IL-22, TNF-alpha, IL-1, IL-23, EGF and KGF, is essential to stimulate epidermal cell growth in reconstituted human epidermis model in vitro.

Therefore, reducing topical concentration of these cytokines was considered the best solution to treat skin diseases caused due to the excessive proliferation of localized cells.

As cytokines are proteins in nature, the aim of our research was to find some topical protein inhibitors so as to neutralize the cytokines and GFs.

The most common protein molecule binding substances are tannins. Tannins can be considered as any phenolic compound of sufficiently high molecular weight containing sufficient hydroxyls and other suitable groups (i.e. carboxyl) to form effectively strong bonds with most of the macromolecules and particularly with the protein molecules.

As most of the plants possess a large variety of tannins and the tannins have specific affinity for the proteins, topical application of any plant extract on an open skin lesion is therefore likely to induce some binding between the tannins and the free protein molecules or the cytokines present on the surface. Therefore most of the plants are likely to have some topical cytokine inhibiting properties but not enough to neutralize all the cytokines at a time and therefore not of much therapeutic use.

Due to these facts, the aim of our research was to first identify all the key cytokines and GFs involved in cell growth stimulation and then to find specific plant tannins capable to bind and to neutralize one or more cytokines or GFs so as to inactivate all the selected cell growth promoting GFs and cytokines present in PED type of lesions. As each plant contains hundreds of tannins and tannin are specific with respect to their binding affinity for proteins, we decided to test a variety of plant extracts, rich in tannins, to evaluate their anti-cytokine activity.

Topical application of a synergistic association of plant extracts containing specific tannins should neutralize cell growth stimulating cytokines and constitute an ideal treatment for the treatment of PED.

OBJECTIVES OF THE INVENTION Therefore, one of the objectives of the present invention was to verify whether the excessive amount of cytokines and GFs found in the PED type of lesions is effectively involved in PED and whether neutralizing these GFs constitute an ideal way to treat PED type of skin diseases,

Another objective of this invention was to suggest a method of treating topical skin affections due to abnormal growth of skin cells by neutralizing all the cytokines and the GFs involved in the development and progression of PED type of lesions.

Another objective of the invention is to provide a synergistic combination of plant extracts capable to bind all the cytokines and GFs present on the surface of the lesion so as to stop excessive and abnormal cell growth observed in many skin diseases.

Another objective of the invention is to provide a synergistic association of plant extracts rich in tannins, each capable to neutralize one or more cytokines or GFs involved in stimulating cell growth so as to stop the growth of skin epidermal cells.

Another objective of this invention is to provide a synergistic association of selected plant extracts capable to reduce or normalize the concentration of cell growth promoting cytokines and the GFs to treat skin diseases such as the Psoriasis, Eczema, and various forms of Dermatitis involving excessive cell growth such as dermatitis herpetiformis, skin rashes, blisters of pemphigus and pemphigoid, erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, lichen planus, perioral dermatitis, pityriasis rosae, rosacea, and similar skin lesions.

An another objective of this invention is to provide a specific association of plant extracts rich in specific protein inhibiting tannins capable to neutralize cytokines and GFs in skin lesions as a preventive or curative treatment for topical skin affections related to excessive cell growth and manifested as sloughing of the skin.

Another objective of this invention is to provide a topical cytokine and GF inhibiting synergistic association as a cosmetic or drug or a medical device which is safe, efficient and inexpensive for the treatment of excessive and abnormal cell growth related affections of the skin, oral mucosa, vaginal cavity and other natural openings of the body.

PHARMACOLOGY EXPERIMENTS

In vitro model of PED

The experiments were conducted on human reconstituted keratinocyte epidermis model as skin equivalents. In skin equivalents, epidermal keratinocytes are grown on a polycarbonate filter treated with type I collagen type of matrix, giving rise to almost normal skin tissue architecture with multilayered skin containing dermis and epidermis. Under controlled culture conditions, the number of cells and cell layers double every 5-6 days. Addition of any cell growth stimulating ingredient, accelerates cell growth which can be quantified.

Polycarbonate filter containing keratinocytes was placed in a serum-enriched culture medium (MCDB 153) in which different GFs or cytokines were added, either individually or in association, to study the effects on cell proliferation. The cell growth stimulating effect of a cytokine or cytokine can be quantified by staining the epidermal cell culture followed by histological examination to estimate the thickness and the number of cell layers in each epidermis. Increase in the thickness of the epidermis, in the number of cells and sloughing of cells indicate the cell growth stimulating properties of the test product.

Similarly, to study the cytokine or GF neutralizing properties of various purified tannins or plant extracts, the test product is added at different concentrations in the culture medium, either alone or in association, so as to evaluate effects on the cell growth compared to the corresponding cultures. Reduction in cytokine induced cell growth compared to corresponding controls in the presence of tannin or plant extract indicates binding with the cytokine (protein).

Selection of cytokine and GF inhibitors

The purified recombinant test cytokines were purchased from Peprotech France. On the basis of initial experiments, only those cytokines and GFs showing some epidermal cell growth stimulating properties were selected for further testing.

Commercially available recombinant cytokines and GFs such as EGF, KGF, PDGF, FGF, M-CSF, GM-CSF, SCF, Interferon γ, TNF α/β, IL-Ια/β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17D/F, IL-19, IL-22, IL-23, and rhGM- CSF thrombopoietin were selected after initial screening to evaluate cell growth promoting properties, either alone or in varios combinations with each other.

PLANT EXTRACTS & PREPARATION

222 dried plant extracts were initially tested for their cytotoxicity in vitro and for initial screening with respect to cytokine inhibition. On the basis of initial results, 110 plant extracts were used for complete anti-cytokine activity evaluation. Plant extracts can be prepared using any extraction method published in the literature or can be purchased for reliable suppliers supplying standardised tannin quantified plant extracts. For the experimental purpose, plant extracts were purchased from different suppliers in France (Greentech, Saint Beauzire; Pharmanager, Angers; Euromed, Gleize). The percentage of tannin in the dried extract varied between 15-34% depending upon the initial richness of the plant in tannins and the part of the plant used. These dried extracts were solubilised in culture medium (lOmg/ml) for all the experiments. Further dilutions were made in the culture medium or in excipients as per the requirements.

Due to cellular toxicity or problems of solubility, among 222 plants selected initially, only 110 were selected for initial screening and 32 were finally retained to test anti-cytokine activity either alone or in associations of 2 or more plants.

Different species of plant species in the same family were evaluated for their activities but only the results of the most active plant species are presented in the result section. For example, the plant extracts of Echinacea purpurea (E. purpurea), E. angustifolia, and E. pallida were evaluated for their anti-cytokine property but only the results of the most active species, E. purpurea are presented in the results. In total, 32 different plant extracts were retained for complete evaluation, the main being Sambucus nigra, Trigonella graecum, Vaccinium myrtilus fruit, Vaccinum macrocarpon fruit extract, Grape seed extract, E. purpurea (aerial parts), Mimosa tenuiflora (M. tenuiflora bark), Asculus hippocastanum (A. hippocastanum aerial parts), Salvia officinalis (S. officinalis, aerial flowered parts), Alchemilla vulgaris (A. vulgaris, aerial parts), Centella asiatica (C. asiatica, leaves), Camilla sinensis (Green tea, aerial parts), and Acacia catechu (A. catechu, bark gum), Vitis vinifera (leaves) , Prunella vulgaris (leaves), Tenacetum parthenium (aerial parts), Ribus nigrum, and Oak bark (Quercus alba, Quercus robur), Ginko biloba (leaves).

Initially, the maximum non cytotoxic concentrations (MNC) of each plant extract was determined by exposing cells to different concentrations of plant extracts. The concentrations used for further testing were 3%, 1%, 0,3%, 0,1%, 0,03% and 0,01% of the MNC for each test product.

Parameters studied

To identify the cell growth stimulating properties of different cytokines, a fixed non-cytotoxic concentration of each cytokine was added in the culture medium and effects on cell growth were compared with the corresponding control cell cultures (n = minimum 6 wells per test concentration). Those cytokines, which were found to stimulate cell growth, were associated at different combinations with each other employing the most active concentration to evaluate the effects of cytokine associations.

The results are presented as follows: Table 1 : Identification of individual cell growth stimulating cytokines.

Table 2: Identification of association of cytokines involved in stimulating cell growth.

Table 3 : Cytokine inhibitory effect of selected individual plant extracts and the association of plant extracts.

RESULTS

TABLE- 1 : C ELL GROWTH PROMOTING PROPERTIES OF INDIVIDUAL CYTOKINES ON HUMAN RECONSTITUTED EPIDERMIS COMPARED TO CONTROLS

These results prove that all cytokines and GFs found in the PED lesions do not participate in the cell growth but only some selected cytokines are involved in stimulating cell growth in vitro. T ey can be classified as: Highly active cytokines (>60% growth): EGF, TNF- a , GF- 7, GM-CSF, IL-1 a, IL-6, SCF; Moderately active cytokines (>40 & < 60%): FGF-21, IL- 17D, IL-22, IL-23. Less active cytokines ( < 40% and >9%): PDGF-AA, TGF- a, TNF-β, M- CSF, HB-EGF, IL-3, IL-10 and Inactive (<9%) TGF - β, IL - 1 β, IL - 2, IL - 4, IL-11 , IL- 12, IL-13, IL-15, IL-16, IL-17F, IL-19 and all others. TABLE-2: CELL GROWTH PROMOTING PROPERTIES OF ASSOCIATIONS OF THE MOST ACTIVE CYTOKINES ON HUMAN RECONSTITUTED EPIDERMIS COMPARED TO THE CORRESPONDING CONTROLS.

These results show that KGF is one of the important GFs to stimulate uncontrolled epidermal cell growth and interleukins, although implicated in the growth, requires the help of other cytokines to enhance cell growth in a synergistic fashion. The best association to stimulate proliferation of epidermal cells is EGF + TNFa + FGF-21 + KGF-7, + GM-CSF + IL-1 a + IL-6 + IL-17D + IL-22 + FGF-23 + SCF. This synergistic association of GFs/cytokines was used for further testing to evaluate the anti-cytokine effects of plant extracts. Note: the results of individual activity of cytokines vary to some extent in different experiments as the epidermal cultures used in each experiment may have different cell growth rate at the start of the experiment. Results are calculated compared to the controls in the same experiment (n=minimum 3 mean values). TABLE-3: CYTOKINE INHIBITORY EFFECTS OF PLANT EXTRACTS EITHER

ALONE OR IN ASSOCIATION

-IVE CONTROL EPIDERMS RECEICED NEITHER PLANT EXTRACTS NOR CYTOKINES,

+IVE CONTROL CULTURES RECEIVED ONLY THE ASSOCIATION OF CELL GROWTH STIMULATING CYTOKINES: EGF + TNFa + FGF-21 + KGF-7, + GM-CSF + IL-1 a + IL-6 + IL-17D + IL-22 + FGF-23 + SCF,

EXPERIMENTAL EPIDERMIS CULTURES RECEIVED CYTOKINE ASSOCIATION ALONG WITH A FIXED CONCENTRATION OF PLANT EXTRACTS (0.01% of each plant extract).

S. N° TEST MEAN % CHANGE % CHANGE

CYTOKINE AND GROWTH V/S -IVE V/S +

PLANT SCORE CONTROL CONTROL

EXTRACT

CODE

C - NEGATIVE 1.0 0.0 % OK

CONTROLS

c + POSITIVE 2.68 + 168.3% +168.3

CONTROLS

1 AC 2.03 + 103 % -38.8%

2 SO 2.05 + 105 % -38.7%

3 VV 1.61 + 61 % -63.76%

4 OB 2.38 + 138% -18.0

5 GB 2.06 + 106 % -37.01

6 V. SP 1.43 + 43 % -74.4

7 SA 1.57 + 57 % -66.13

8 MT 2.32 + 132 % -21.56

9 EP 2.35 + 135 % -19.79

10 TP 2.27 + 127 % -24.54

11 CS 1.77 + 77 % -54.25

12 SN 1.50 + 50% -70.29

ASSOCIATION

13 AC + VV 1.83 + 83 % -50.68

14 AC + MC 1.78 + 78 % -59.42

15 AC + EP 1.55 + 55 % -67.32

16 AC + CS 1.55 + 55 % -67.32

17 VV + GB 1.61 + 61 % -63.76

18 MC + EP 1.52 + 52 % -69.10

19 MC + CS 1.65 + 65 % -59.42

20 EP + CS 1.35 + 35 % -59.41

21 VV + V SP 1.18 + 18 % -89.30

22 VV + CS 1.25 + 25 % -85.15

23 VV + SN 1.30 + 30 % -82.17

24 VV + V SP + CS 1.14 + 14% -91.68

25 VV + MT + SN 1.38 + 38% -77.42

26 SN + V SP + CS 1.22 + 22% -86.93 1 = Acacia catechu; 2 = Salvia officinalis; 3 = Vitis vinifera; 4 = Oak Quercus robur bark; 5 = Ginko biloba; 6 = Vaccinuum sp; fruits; 7 = Salix alba; 8 = Mimosa tenuiflora; 9 = Echinacea purpurea; 10 = Tenacetum parthenium; 11 = Camillia sinensis; 12 =Sambucus nigra. Others = as given in the list of plants tested.

CONCLUSION

On the basis of these results it can be concluded that:

1. Cytokines stimulate epidermal cell growth,

2. A synergistic association of different cytokines is essential to enhance the skin cell growth above 100% which may lead to conditioned like psoriasis, eczema and dermatitis,

3. Certain plant extracts can reduce the cytokine induced cell growth,

4. The best plant extract associations are Vitis vinifera + Vaccinum sp. Fruit extracts or Vitis vinifera + Salix alba or Green tea or European Elder; and Sambucus nigra + Vaccinum sp fruit extract + Green tea leaf extract.

5. Topical application of these synergistic association of cytokine inhibiting plant extracts may help to reduce abnormally excessive cell growth responsible for skin diseases and can be used for topical treatment,

6. Although the exact mode of action of these plant extracts is not well understood, most of the active plant extracts were rich in different types of tannins and therefore specific tannin binding with cytokines is considered the most logical hypothesis involved in cytokine neutralization.

CLINICAL ASSESSMENT

To evaluate the clinical efficacy of the synergistic associations of the plant extracts, the most active formulation found in vitro experiments (Vaccinium macrocarpon + Vitis vinifera + Camellia sinensis), as given in the example 3, were prepared and filled in 50-ml plastic tubes for topical application on the PED type of skin lesions. The clinical trial was conducted at the Nexus clinical research centre in India on 60 patients each having visible lesions of psoriasis, eczema or dermatitis. The studies were conducted in accordance with the requirements of the current ICH guidelines on good clinical practice and declaration of Helsinki (Tokyo 2004).

The product was applied on 60 PED type of lesions in 52 patients (some patients had multiple lesions) for a period of 42-days. A similar preparation containing no plant extract was applied on other 30 lesions as placebo. According to the surface of the lesion, a thin layer of product was spread over the lesion, twice a day during the treatment period. Lesion healing was evaluated on a scale of 1 to 10 (1 = nearly healed and 10 = very severe lesions).

The results showed a mean reduction of 36% in the initial lesion score after 21 days of product application and 78% within 42 days of treatment compared to the corresponding controls. All other accompanying clinical signs were also reduced. No side effects were noted in any of the participants.

These results prove the topical application of cytokine and GF inhibitors is an excellent treatment for the skin diseases involving excessive epidermal cell growth. COMPOSITIONS

The compositions according to the invention must contain a synergistic association of at least 2 plant extracts having properties to inhibit specific cytokines involved in skin cell growth. The compositions and the medicament according to the invention can be applied topically, either as a gel, liquid, spray, and ointment, patch, as cotton gauze soaked with the composition according to the invention or as a power. The composition can be applied as a single or multiple applications per day, preferably 2-4 applications per day for a period of 30 to 90 days depending upon the size of the lesion and the presence of skin infection. The plant extracts ad subsequent compositions according to the invention can be prepared by mixing different ingredients employing any of the methods or procedures well known in the art.

The plant extracts can be prepared as liquid or dry plant extracts, either as a crude plant extract of the whole plant or of a specific part of the plant, or as a purified extract in the form of a liquid or power. Extracts can also be enriched in tannins using different methods well known in the art and can also be purified as a specific type of tannin. Although the experiments were conducted using the most active species of the plant, other species of the plant from the same family can also be used as the compositions of different species of plants in the same family is very identical.

The plant extracts can also be associated with a carrier system to improve the bioavailability or topical penetration as a powder or liquid. For example, the extract can be mixed with a carrier such as the glycerol, honey, clay, an ointment, water, alcohol, gel or a lotion. They can also be incorporated in a polymer film, in a hydro-gel, in a bandage like cotton bandage or in an antiseptic, anti-inflammatory, antibiotic or wound healing preparation, formulated according to usual methods.

The composition can be formulated as a drug as a medical device or as cosmetics for topical application and preferably as a medical device as the main active ingredients of the preparation act through their mechanical and physical properties on the free proteins (especially the cytokines and GFs) present on the surface of the injury without any pharmacological, biological or metabolic effects on the cells of the body.

The amount of product applied topically can be variable depending upon the surface of the lesion or the type of infection and the excipient used. For example a liquid preparation may be used in quantities varying between 0.1 to 50 ml per application and preferably between 2 to 5-ml per application. For example 3to 5 ml solution is essential to treat an eczema lesion of about 15 cm². To treat topical skin abnormalities, a liquid product can be spread directly over the lesion, 3-4 times a day at up to partial or complete recovery. A powdered preparation can be spread in quantities varying between 0.1 to 5g directly on the lesion based on the surface to be treated. For example, to treat a surface area of 5 cm2, about 0.5g powdered preparation can be spread over the lesion. The following examples illustrate the invention, but they should not be considered to limit the scope of the invention in any way. EXAMPLES

Examples of compositions according to the invention: In the absence of evidence to the contrary, the quantity of compounds comprised in different compositions is expressed in g/lOOg (w/w). Qsp signifies quantity sufficient to produce the amount indicated.

Any plant extract, rich in tannins, can be selected, particularly among the plant extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Quercus robur, Ginko biloba, Vaccinuum, Salix alba, Mimosa tenuiflora, Echinacea purpurea, Tenacetum parthenium, Camillia sinensis, and Sambucus nigra. Liquid or dried extracts or purified tannins cab be used.

Example 1: for the treatment of dermatitis

Liquid extract of Vitis vinifera seed (25.0% tannins): 5.4g

Liquid extract of Myrtille commune (12.0 % tannins): 2.6g

Water: qsp 100 g

(Apply a thin layer over the lesion, 3-4 times a day up to complete healing). Example 2: for the treatment of Psoriasis

Liquid extract of Vaccinium myrtillus (6.8% tannins): 3.5g

Liquid extract of Ginko biloba leaf (8.5% tannins): 1.5g

Glycerol qsp lOOg (Apply a few drops over the lesion, 3-4 times a day up to complete healing).

Example 3: for the treatment Eczema

Dry extract of Green tea leaves: 0.48g

Dry extract of Vitis viriifera seeds: 0.36g

Dry extract of Vaccinium macrocarpon fruits: 0,12g

Honey as thickening agent: 19,0g

Glycerol as buffer: QSP lOOg

(Apply a thin layer over the lesion, 3-4 times a day up to complete healing).

Example 4: for the treatment Eczema

Purified dried tannins from Camellia sinensis leaves: 0.3g

Purified dried tannins from Sambucus nigra fruit: 0.2g

Purified dried tannins from Vaccinium macrocarpon fruits: 0,12g

Honey as thickening agent: 19,0g

Glycerol as buffer: QSP lOOg

(Apply a thin layer over the lesion, 3-4 times a day up to complete healing).

Example 5: For the treatment of Eczema

Dry extract of Vitis vinifera seeds: l,5g

Dry extract of Simbuscus nigra fruits: 0,5 g

QSP ointment base: lOOg

(Apply a small amount of ointment, about lg per 5cm2 surface area, twice a day for 60 days).

Example 6: for the treatment of Psoriasis or Eczema

12% liquid hydroglycerinated extract of Vaccinium macrocarpon: 5.0g

12% liquid hydroglycerinated plant extract of Vaccinium myrtillus fruits: 3.0g

Glycerol as excepient qsp lOOg

Wg of this viscous liquid is sprinkled over 10cm2 cotton gauze, introduced into a air tight aluminium sachet, and used as a dressing over the lesion, one dressing per day up to complete healing). Example 7: Powder for the treatment of dermatitis

Dry powder of Vitis vinifera seeds: lO.Og

Dried powder of C. sinensis: 8.0g

Oak bark extract: 2.0g

Talc powder qsp lOOg

(Sprinkle about lg of powder over 10cm2 surface area, once a day up to complete healing).

Example 8: Hydrogel film for the treatment of atopic dermatitis

Dried fruit skin extract of Vitis vinifera: 0.74g

Dried leaf extract of C. sinensis 0.66g

Dried fruit extract of Simbuscus nigra: 0.12g

Xanthan gum: 0.4g

Water QSP lOOg

The gel (lOg) is applied on a semi-permeable protective film (5cm²), some water is evaporated by drying the film at 37°c for 48 hours and the part of the film containing the product is applied on the skin lesion, once every 3 days, up to complete healing).

The problem of many skin diseases involving excessive dermal cell growth such as the Psoriasis, Eczema and various types of Dermatitis (PED) is very common throughout the world. PED lesions appear on the skin surface causing skin desquamation, pain, itching, skin infection and aesthetic problem. The common feature in all such skin diseases is an excessive and uncontrolled growth of skin dermal cells leading to skin cracks, sloughing of skin cells, and subsequent infection. Until now the causes of PED were considered to be genetic, immunological, or psychological but exact reason for the development of PED was not yet understood.

We observed that certain types of immune-modulating cells are found abundantly at the site of PED infection without any apparent role. Some of these cells secrete specific protein molecules, the cell growth stimulating cytokines and growth factors, which causes uncontrolled cell growth leading to PED. Therefore blocking the activities of specific cytokines and cell growth factors involved in PED can be considered as the best solution to treat PED. We identified the cytokines and cell growth factors involved in excessive skin cell growth and observed that certain specific plant tannins bind specifically to these cell growth stimulating proteins. Due to multiplicity of these proteins, a synergistic association of different plant tannins is essential to neutralize all the cytokines and growth factors involved in PED.

This invention suggests the use of a specific synergistic association of plant extracts rich in tannins for the topical treatment of PED without any side effects.

This invention covers any composition of a non specific multiple cytokine and cell growth factor inhibitor suitable for use in treating skin diseases involving excessive skin cell growth.

This invention also covers such compositions of a non specific, multiple cytokine and growth factor inhibitor, containing synergistic association of at least two tannins suitable for use in treating skin diseases involving excessive skin cell growth by topically application.

This invention also cover such compositions for topical application for the treatment of skin affections caused due to excessive uncontrolled skin cell growth containing a synergistic association of at least two plant extracts among the extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Vaccinium macrocarpon or Vaccinium myrtillus fruit, Quercus oak bark, Ginko biloba, Salix alba, Mimosa tenuiflora, Echinacea sp, Tenacetum parthenium, Camillia sinensis, or Sambucus nigra.

This invention also covers such compositions for topical application for the treatment of skin diseases involving excessive cell growth such as the psoriasis, eczema, atopic dermatitis, skin rashes, blisters of pemphigus and pemphigoid, dermatitis herpetiformis, erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, lichen planus, perioral dermatitis, pityriasis rosae, rosacea, and similar skin lesions.

This invention also covers such compositions as a pharmaceutical, cosmetic or medical device for topical application on skin or mucus surfaces.

This invention also covers a method of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and cell growth factor inhibitor.

This invention covers such methods of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and growth factor inhibitor, containing synergistic association of at least two tannins.

This invention covers such methods of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and cell growth factor inhibitor containing synergistic association of at least two tannins rich plant extracts selected among the plant extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Vaccinium macrocarpon or Vaccinium myrtiUus fruit, Quercus oak bark, Ginko biloba, Salix alba, Mimosa tenuiflora, Echinacea sp, Tenacetum parthenium, Camillia sinensis, or Sambucus nigra.

Claims

1. A composition of a non specific multiple cytokine and cell growth factor inhibitor suitable for use in treating skin diseases involving excessive skin cell growth.

2. A composition according to claim 1 of a non specific, multiple cytokine and growth factor inhibitor, containing synergistic association of at least two tannins suitable for use in treating skin diseases involving excessive skin cell growth by topically application,

3. A composition according to claim 2 for topical application for the treatment of skin affections caused due to excessive uncontrolled skin cell growth containing a synergistic association of at least two plant extracts among the extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Vaccinium macrocarpon or Vaccinium myrtillus fruit, Quercus oak bark, Ginko biloba, Salix alba, Mimosa tenuiflora, Echinacea sp, Tenacetum parthenium, Camillia sinensis, or Sambucus nigra.

4. A composition for topical application according to the claim 3 for the treatment of skin diseases involving excessive cell growth such as the psoriasis, eczema, atopic dermatitis, skin rashes, blisters of pemphigus and pemphigoid, dermatitis herpetiformis, erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, lichen planus, perioral dermatitis, pityriasis rosae, rosacea, and similar skin lesions.

5. A composition according to anyone of the claims 1 - 4 as a pharmaceutical, cosmetic or medical device for topical application on skin or mucus surfaces.

6. A method of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and cell growth factor inhibitor.

7. A method according to claim 6 of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and growth factor inhibitor, containing synergistic association of at least two tannins. A method according to claim 7 of treating skin diseases involving excessive skin cell growth by topically application of a non specific, multiple cytokine and cell growth factor inhibitor containing synergistic association of at least two tannins rich plant extracts selected among the plant extracts of Acacia catechu, Salvia officinalis, Vitis vinifera, Vaccinium macrocarpon or Vaccinium myrtillus fruit, Quercus oak bark, Ginko biloba, Salix alba, Mimosa tenuiflora, Echinacea sp, Tenacetum parthenium, Camillia sinensis, or Sambucus nigra.