WO2007105864A1

WO2007105864A1 - Pharmaceutical composition comprising silymarin for prevention and treatment of irritant contact dermatitis

Info

Publication number: WO2007105864A1
Application number: PCT/KR2007/000659
Authority: WO
Inventors: Hwan Mook Kim; Song-Kyu Park; Jong Soon Kang; Mi Hwa Han; Kiho Lee; Sang-Bae Han; Ki Hoon Lee; Chang Woo Lee
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2006-03-14
Filing date: 2007-02-07
Publication date: 2007-09-20
Also published as: KR20070093574A

Abstract

The present invention relates to a pharmacological composition containing silymarin for the prevention and cure of irritant contact dermatitis. More specifically, the inventive composition containing silymarin or its main ingredients, silybin, silydianin and silychristin suppress symptoms of irritant contact dermatitis induced from an application of 1-chloro-2,4-dinitrobenzene on mouse ears such as swelling and increases in the water contents of the ears, weight increases in the lymph nodes and upregulated expression of TNF-α The pharmacological compositions of this invention are thus useful in the prevention and cure of irritant contact dermatitis.

Description

PHARMACEUTICAL COMPOSITION COMPRISING

SILYMARIN FOR PREVENTION AND TREATMENT OF

IRRITANT CONTACT DERMATITIS

Technical Field

[1] The present invention relates to a pharmacological composition comprising silymarin for prophylactic and therapeutic treatments for contact dermatitis.

[2]

Background Art

[3] Contact dermatitis is an acute or chronic inflammatory disease caused by skin contact with irritant substances. Contact dermatitis causes most trouble in workplaces in which a high risk of field exposure to dermatitis-inducing substances is present. According to a recent report (Saary, J. et al., J. Am. Acad. Dermatol. 53(5):845~855, 2005), between 24 to 170 out of 100,000 workers develop contact dermatitis and 29% to 72% of them leave their jobs, although results may vary according to survey methods and standards and many factors including the work environment. Contact dermatitis is classified into irritant contact dermatitis and allergic contact dermatitis. It has been reported that up to 70-80 % of contact dermatitis occurring in the workplace is irritant contact dermatitis, whereas 20-25% is allergic (Nixon, R. et al., Aust. F am. Physician 34(5):327~333, 2005). The main cause of irritant contact dermatitis is a breach in the skin barrier from skin lesions caused by contact with irritants. Any person can develop dermatitis when exposed to irritants potent enough to induce skin lesions. Allergic contact dermatitis, however, is essentially an allergic response upon exposure, albeit in trace amounts, to allergy inducers, and only occurs in people sensitized by previous exposures to them.

[4]

[5] Tumor necrosis factor α (TNF-α is an inflammatory cytokine whose expression is rapidly upregulated when an inflammatory response is induced, although it is expressed under normal circumstances as well. Receptors for TNF-α are known to be expressed in virtually all cells of the human body and accordingly, most cells are affected by TNF-α. TNF-α has been reported to play an important role in various inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Furthermore, it is well established that TNF-α is also involved inflammatory skin diseases including allergic and irritant contact dermatitis and that suppressing TNF-α activity greatly helps in curing such diseases (LaDuca, J. R. et al., Dermatol. Clin. 19(4):617~635, [6]

[7] Silymarin is a flavolignan complex belonging to the flavonoid family. It is a mixture of three major components of silybin, silydianin and silycristin as well as various flavonoids such as dehydrosilybin. Silymarin is extracted from milk thistle ( Silybum marianurri), a plant that belongs to the family Compositae and native to southern Europe and north Africa, but has spread into the Americas and now grown as a garden plant or for medicinal purposes. The use of milk thistle as a herbal tea for promoting breast milk flow in nursing mothers has been well known since old times. Johannes Rademacher, a 19 century German physician and herbalist, added to such recognition of milk thistle as a medicine by demonstrating its efficacy against hep- atosplenic diseases such as jaundice. Even in this modern age, silymarin still finds use as a supplement in the therapy for liver diseases and there has been ongoing progress in the efforts to decipher its mechanism of action.

[8]

[9] Most prominently, silymarin has been found to stabilize the membranes of liver cells as to block the inflow of harmful substances and upregulate protein synthesis in the liver to promote liver regeneration. Recently, a study implicated an inhibition of the expression of inflammatory factors in the liver protection by silymarin wherein the inhibition is mediated through downregulation of Kupper cells, a macrophage found inside liver tissues (Dehmlow, C. et al., Hepatology 23(4):749~754, 1996). Silymarin was also reported to provide protection from inflammatory diseases such as sepses by suppressing the activity of macrophages and preventing inflammation (Kang, J. S. et al ., J. Pharmacol. Exp. Ther. 302:138-144, 2002). No studies have been reported, however, on the effects of silymarin against irritant contact dermatitis.

[10]

Disclosure of Invention Technical Problem

[11] The objective of the present invention is to provide a novel use of silymarin as a prophylactic and therapeutic agent. To achieve this object, the present invention provides a pharmaceutical composition for the treatment and prevention of irritant contact dermatitis that includes silymarin or its major ingredients, i.e., silybin, silydianin and silychristin.

[12] Another objective of the invention is to provide a cosmeceutical for treating and preventing the same disease containing said composition as the active ingredient.

[13] Still another objective of the invention is to provide novel uses of silymarin or its main ingredients of silybin, silydianin and silychristin in the pharmaceutical manufacture of prophylactic and therapeutic agents for irritant contact dermatitis. [14] It is yet another objective of the present invention to provide a treatment method for irritant contact dermatitis comprising a step for administering said composition in therapeutically effective amounts to patients of the same disease.

[15]

Technical Solution

[16] In the following, the present invention is described in detail.

[17]

[18] The present invention provides a pharmacological composition for treating and preventing irritant contact dermatitis, which contains silymarin or its main ingredients of silybin, silydianin and silychristin. The term "silymarin" as used in this invention includes commercially available compositions of the flavolignan complex as well as extracts obtained from the seeds and fruits of plants belonging to the family Compositae, including dandelion (Taraxacum platycarpurri) and milk thistle (Silybum marianurri).

[19]

[20] To demonstrate the efficacy of the silymarin composition of this invention, B ALB/c mice were either first given an application of the inventive composition and then after 30 minutes an application of l-chloro-2,4-dinitrochlorobenzene (DNCB)(subject group), or the DNCB alone (control group) in their ears to induce irritant contact dermatitis. When the mice's ears were checked for swelling, there was swelling in the mice of the control group; the subject group, however, exhibited a gradual reduction in the swollen width of their ears (See FlG. 1), demonstrating a dose-dependent suppression of irritant contact dermatitis by silymarin.

[21] The present inventors also looked into the weight of the lymph nodes of these

B ALB/c mice after giving identical treatments as above to them. When a local immune response is triggered, immune cells gather around lymph nodes near to the site of reaction, thereby increasing the weight of the lymph nodes. We chose auricular lymph nodes for examination since these nodes were closest to the mice's ears, the site of dermatitis induction. The results shown in FlG. 2 indicate a dose-dependent weight reduction of the lymph nodes by silymarin.

[22]

[23] The present inventors also examined whether this suppression of DNCB-induced dermatitis by silymarin requires pre-treatment with silymarin before DNCB. B ALB/c mice were treated with silymarin either 30 minutes before or 15 minutes after the DNCB application and examined for swelling in their ears. It turned out that post-insult treatment of silymarin is capable of suppressing swelling in the ears on a level similar to pre-insult treatments (FlG. 3). [25] Corresponding weight increases in the lymph nodes were examined for B ALB/c mice after similarly receiving silymarin treatments either 30 minutes before or 15 minutes after the DNCB application. Again, a reduction in DNCB -induced weight increase was observed for those lymph nodes that received silymarin after DNCB, and their reduction was on a level similar to those received treatments before DNCB (FlG. 4)

[26]

[27] To examine edemata arising from irritant contact dermatitis, the present inventors measured water content of the swollen ears treated with silymarin either 30 minutes before or 15 minutes after the application of dinitrochlorobenzene. The increase in the water contents of edemata from the applied DNCB was shown to be suppressible with silymarin regardless of the order of its application (FlG. 5). These results strongly demonstrate that silymarin can actively suppress symptoms of irritant contact dermatitis even after an exposure to irritants provided it is administered before the symptoms become too severe.

[28]

[29] In another aspect of the invention, silymarin characteristically keeps TNF- from being overproduced. The present inventors applied the inventive pharmacological composition to B ALB/c mice 30 minutes before the application of DNCB to trigger dermatitis, and measured the TNF-α levels of the mice. The measured TNF-α levels confirmed silymarin s capability to repress DNCB-induced expression of TNF-α(FlG. 6).

[30]

[31] The inventive pharmacological composition for the treatment and prevention of irritant contact dermatitis contains silymarin, or its major ingredients silybin, silydianin and silychristin in an amount of 0.02 to 90 weight parts per 100 weight parts of the composition.

[32]

[33] The pharmacological composition of this invention may additionally comprise one or more active ingredients that exhibit equivalent or similar function to silymarin or its three main ingredients.

[34]

[35] In addition, the pharmacological composition of this invention, which contains silymarin or its main ingredients of silybin, silydianin and silychristin, may additionally comprise excipients conventionally used in the field of the invention.

[36]

[37] The pharmacological composition containing silymarin, or its main ingredients, silybin, silydianin and silychristin, as described in this document may additionally comprise pharmaceutically allowable carriers, excipients and diluents that suit the purposes. The inventive composition may be formulated into any pharmaceutically allowable form via methods well known in the field of pharmaceutics: for example, oral preparations including liquids, powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols; external preparations such as ointments; suppositories and sterile injections.

[38]

[39] Examples of carriers, excipients and diluents for the inventive composition containing silymarin may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methyl hydroxybezoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[40]

[41] Preparations may be formulated using routine diluents and excipients such as fillers, binders, humectants, disintegrants, and surfactants.

[42]

[43] The appropriate dose for the inventive composition of silymarin or its main ingredients of silybin, silydianin can be determined by taking account of absorption and excretion rates for the active ingredients, patient s age, weight, gender, condition and severity of the symptoms. In general, an internal preparation containing 0.001-30% active ingredients can be applied to affected areas once or several times a day for adults. This dose can be adjusted according to sex, age and condition of the patient. Furthermore, the administration dose for silymarin is subject to variation according to the route of administration, severity of the condition, sex and age. Thus, under no circumstances should the dose described above be understood as limiting the scope of the present invention.

[44]

[45] In still another aspect of this invention, the present inventors provide cosme- ceuticals containing silymarin or its main ingredients, silybin, silydianin and silychristin as the active ingredients. The cosmeceutical of the present invention may additionally comprise one or more active ingredients equivalent or similar in activity.

[46]

[47] The inventive cosmeceutical containing silymarin or its main ingredients may be formulated into conventional emulsions and solubilized formulations. Examples for emulsions include moisturizers, nourishing creams, cosmetic essences and moisturizing essences. Examples for solubilized formulations include skin softeners. Furthermore, the silymarin-containing cosmeceutical of this invention may additionally comprise dermatologically-allowed vehicles and media to support formulation into topical or systemic cosmetic supplements routinely used in the cosmetics industry.

[48]

[49] Examples of suitable cosmetic formulations include solutions, gels, anhydrous solids or pastes, oil-in-water emulsions, suspensions, microemulsions, microcapsules, microgranules, ionic (liposomes) or non-ionic vesicular dispersions, creams, lotions, moisturizing lotions, powders, ointments, sprays and concealing sticks. In addition, foams and aerosols with compressed propellants are contemplated as possible formulations.

[50]

[51] In addition to silymarin or its main components, the cosmeceutical of the present invention may further comprise cosmetically or dermatologically-allowed conventional additives whose examples include: fats, organic solvents, solubilizer, thickeners, gellifiers, softeners, anti-oxidants, suspension agents, stabilizers, foaming agents, aromas, surfactants, water, ionic and non-ionic emulsifiers, extenders, metal ion se- questerers and chelators, preservatives, vitamins, screening agents, humectants, essential oils, dyes, pigments, hydrophilic or lipophilic activating agents and lipid vesicles. These additives may be introduced in amounts conventionally used in derma- tological applications.

[52]

[53] More specifically, the cosmeceutical of the present invention can be formulated into skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nourishing lotions, massaging creams, nourishing creams, hand creams, nourishing essences, packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, pressed powders, loose powders and eye shadows.

[54]

[55] In yet another aspect of this invention, the present inventors provide uses of silymarin or its main ingredients in the manufacture of therapeutic and prophylactic agents against irritant contact dermatitis.

[56]

[57] It is yet another objective of the present invention to provide a treatment method for irritant contact dermatitis comprising a step for administering said composition in therapeutically effective amounts to patients of the same disease.

[58] The pharmacological composition of this invention can be administered to patients of irritant contact dermatitis to cure or improve the condition of the disease. [59] The daily dose for the administration of the inventive composition is 1-500 mg, or preferably 10-300 mg for adults. The actual dose, however, would be dependent on multiple factors such as age and sex of the patient, the exact nature of the illness, and its severity. Also the administration route can be varied according the condition of the patient and the severity of the symptoms.

[60]

[61] Furthermore, in still another aspect of this invention, the present inventors provide.

Brief Description of the Drawings

[62] FIG. 1 shows the effect of silymarin pre-treatment on the swelling of mouse ears induced by dichloronitrobenzene (DNCB).

[63] FIG. 2 exhibits the effect of silymarin pre-treatment on the weight increase of mouse lymph nodes induced by DNCB.

[64] FIG. 3 contrasts the effects of silymarin treatments before and after the DNCB treatment on the swelling of mouse ears induced by the latter.

[65] FIG. 4 contrasts the effects of silymarin treatments before and after the DNCB treatment on the weight increase of mouse lymph nodes induced by the latter.

[66] FIG. 5 contrasts the effects of silymarin treatments before and after the DNCB treatment on the DNCB-induced increases in the water content of mouse ears.

[67] FIG. 6 exhibits the effect of silymarin pre-treatment on the DNCB-induced up- regulation of TNF-α expression in treated mice.

[68]

Best Mode for Carrying Out the Invention

[69] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

[70]

[71 ] <Example 1> The effect of silymarin on DNCB-induced irritant contact dermatitis

[72] [Subexample 1-1] Suppression of the swelling in mice ears induced by DNCB

[73] 5 week-old female B ALB/c mice purchased from Orient Bio (Korea) were acclimatized until the 6th week. Silymarin was purchased from Sigma (USA). The mouse model for irritant contact dermatitis was established by referring to the methods of Kuriyama (Kuriyama, K. et al., Inflamm. Res. 51(10):483~489, 2002) and Baolin (Baolin, L. et al., Planta Med. 71(5):424~428, 2005). Irritant contact dermatitis was induced in mice by applying 20 D of 1% dichloronitrobenzene (DNCB) in 4:1 mixture of acetone and olive oil (Sigma). A serial dilution of silymarin (0.05, 0.5, 5, 50 D per mouse ear) was applied 30 minutes before inducing dermatitis with DNCB. Swelling of ears was measured 4 hours after the DNCB induction. [74] The results, as shown in FlG. 1, demonstrate a dose-dependent suppression of

DNCB-induced dermatitis by silymarin treatments. [75] [Subexample 1-2] Suppression of the DNCB-induced weight increase in mouse lymph nodes [76] After carrying out steps as described in subexample 1-1, auricular lymph nodes were taken out of the affected mice and their weights were recorded. [77] Data shown in FlG. 2 demonstrate that DNCB-induced increases in the weight of lymph nodes are suppressed when mice are treated with silymarin. [78] [79] <Example 2> Comparing the effects of silymarin treatments before and after the DNCB treatment [80] The present inventors examined whether treating silymarin before DNCB was necessary for suppressing induced dermatitis or a silymarin treatment after the exposure to DNCB was sufficient for suppression.

[81] [Subexample 2-1] Suppression of ear swelling induced by DNCB

[82] The experiment was carried out using methods similar to subexample 1-1. 50 D of silymarin was treated to mice either 30 minutes before or 15 minutes after the DNCB treatment to monitor swelling in the ears induced by the latter. [83] As shown in FlG. 3, the present inventors were able to observe suppression of swelling in mouse ears even when silymarin was applied after DNCB just as we were able to observe in silymarin treatments in advance of DNCB. [84] [Subexample 2-2] Suppression of the weight increase in mouse lymph nodes induced by DNCB [85] The experiment was carried out using methods similar to subexample 2-1. Auricular lymph nodes were taken out of the affected mice and their weights were recorded. [86] As shown in FlG. 4, the present inventors were able to observe suppression of

DNCB-induced increase in the weights of mouse lymph nodes even when silymarin was treated after DNCB just as we were able to observe in silymarin treatments prior to DNCB. [87] [Subexample 2-3] Suppression of the DNCB-induced increase in the water contents of mouse ears [88] In order to examine edemata arising from irritant contact dermatitis, the present inventors measured the water contents of mouse ears. The water content measurements started with taking the weights of mouse ears and then allowing them to dry in a drying oven to completely remove water. The weight differences between wet and dry ears were then calculated to obtain the water content. [89] Induction of dermatitis with DNCB in mice was carried out similarly as described in subexample 2-1. The water content measurements were conducted as described above by removing ears from the affected mice. [90] As shown in FlG. 5, the present inventors were able to observe suppression of

DNCB-induced increase in the water contents of swollen mouse ears even when silymarin was treated after DNCB just as we were able to observe in silymarin treatments prior to DNCB. [91]

[92] <Example 3> Suppression of TNF-α expression by silymarin

[93] Tumor necrosis factor alpha (TNF-α is a well-known mediator of the inflammatory response. The present inventors therefore looked into the influence of silymarin on the

DNCB-induced expression of TNF-α. [94] The experiment was carried out similarly as in subexample 1-1 to remove the ears.

These ears were ground to obtain a protein extract. An enzyme-linked immunosorbent assay (ELISA) was carried out on the extract to measure the TNF-α contents. [95] From the data shown in FIG. 6, the present inventors concluded that silymarin treatments were able to suppress the DNCB-induced increases in the expression of

TNF-α. [96] We now provide below example formulations for the pharmacological compositions and cosmeceuticals of the present invention. [97]

[98] <Example formulation 1> Manufacturing an external preparation for the skin

[99] Unless otherwise indicated, all parts are by weight.

[ 100] 1. Formulation for a skin cream

[101] Cetostearyl alcohol 2.8 parts

[102] Beeswax 2.6 parts

[ 103] Stearic acid 1.4 parts

[104] W/O glyceryl monostearate 2 parts

[ 105] PEG- 100 stearate 1 parts

[106] Sorbitan sesquioleate 1.4 parts

[107] Jojoba oil 4 parts

[108] Squalane 3.8 parts

[ 109] Poly sorbate 60 1.1 parts

[110] Macadamia oil 2 parts

[111] Tocopherol acetate 0.2 parts

[112] Methyl polysiloxane 0.4 parts

[113] Ethyl paraben 0.1 parts

[114] Propyl paraben 0.1 parts

[115] Euxyl K-400 0.1 parts [116] 1,3-butylene glycol 7 parts

[117] Methyl paraben 0.05 parts

[118] Glycerine 6 parts

[119] d-panthenol 0.2 parts

[120] Silymarin 4.6 parts

[121] Triethanolamine 0.2 parts

[122] p-H O 46.05 parts

[123]

[124] 2. Formulation for a skin lotion

[125] Cetostearyl alcohol 1.6 parts

[126] Stearic acid 1.4 parts

[127] W/O glyceryl monostearate 1.8 parts

[128] PEG-100 stearate 2.6 parts

[129] Sorbitan sesquioleate 0.6 parts

[130] Squalane 4.8 parts

[131] Macadamia oil 2 parts

[132] Jojoba oil 2 parts

[133] Tocopherol acetate 0.4 parts

[134] Methyl polysiloxane 0.2 parts

[135] Ethyl paraben 0.1 parts

[136] Propyl paraben 0.1 parts

[137] 1,3-butylene glycol 4 parts

[138] Methyl paraben 0.1 parts

[139] Xanthan gum 0.1 parts

[140] Glycerine 4 parts

[141] d-panthenol 0.15 parts

[142] Allantoin 0.1 parts

[143] Silymarin 3.5 parts

[144] Carvone (2% aq. Sol) 4 parts

[145] Triethanolamine 0.15 parts

[146] Ethanol 3 parts

[147] P-H₂O 48.3 parts

[148]

Industrial Applicability

[149] The silymarin of the present invention or its main ingredients of silybin, silydianin and silychristin are active in suppressing symptoms arising from irritant contact dermatitis such as swelling of ears, weight increases in lymph nodes, increases in the water contents of ears and the upregulated expression of TNF-α The inventive pharmacological compositions containing silymarin or its main ingredients are thus useful for preventing and curing irritant contact dermatitis

Claims

[1] A pharmacological composition containing silymarin or its main ingredients, silybin, silydianin and silychristin for the prevention and therapy of irritant contact dermatitis.

[2] The pharmacological composition according to Claim 1, wherein the silymarin or its main ingredients of silybin, silydianin and silychristin is capable of suppressing the upregulated expression of tumor necrosis factor alpha (TNF-α).

[3] The pharmacological composition according to Claim 1, additionally comprising pharmaceutically allowed vehicles, excipients and/or diluents.

[4] A cosmeceutical for irritant contact dermatitis containing silymarin or its main ingredients of silybin, silydianin and silychristin.

[5] The cosmeceutical according to Claim 4, wherein the silymarin or its main ingredients of silybin, silydianin and silychristin is capable of suppressing the up- regulated expression of tumor necrosis factor alpha (TNF-α.

[6] The cosmeceutical according to Claim 5, wherein the formulation of said cosmeceutical is selected from the following list: skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nourishing lotions, massaging creams, nourishing creams, moisturizing creams, hand creams, cosmetic essences, nourishing essences, packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, pressed powders and loose powders.

[7] A use of silymarin or its main ingredients, silybin, silydianin and silychristin for the production of therapeutic and prophylactic agents against irritant contact dermatitis.

[8] A therapeutic method for irritant contact dermatitis comprising the step of administering the pharmacological composition of Claim 1 in therapeutically effective amounts to a patient suffering irritant contact dermatitis.