DE10043466A1 - Non-irritating topical preparation for treating or preventing skin disorders, e.g. atopic dermatitis, psoriasis or aging symptoms, containing manganese salt to stimulate urea synthesis by keratinocytes - Google Patents

Abstract

A preparation (A) for topical administration in the therapy and prophylaxis of pathological skin abnormalities contains effective amounts of a manganese compound (I), together with conventional carriers and auxiliaries.

Description

This invention relates to manganese compounds Tending pharmaceutical preparations for topical Application for therapy and prophylaxis sick severe changes to the skin, especially the Neurodermatitis (atopic dermatitis) and others Possible uses of manganese Links.

Urea (carbamide, urea) is used in the topical Therapy especially with the aim of hydration of the stratum corneum and serves as most important natural moisturizer. Beyond that but also the keratolytic, keratoplastic, proteo- and mucolytic, antimicrobial, antipruriginous and antiproliferative properties  used therapeutically by urea. In a Variety of pharmaceutical preparations is urea used as a penetration-promoting additive. Urea involves very complex interactions different structures of each epidermal Layers and influences the physicochemical Properties of the top layers of the epidermis in the In terms of buffer capacity and Acid mantle.

In humans, urea is the most important end product of protein metabolism and is especially renal eliminated. In the urea cycle of hepatocytes L-arginine (2-amino-5-guanidovaleric acid) by the L-isomer-specific enzyme arginase (A1, type I) to L- Ornithine and urea hydrolytically split.

For the human epidermis, urea is essential for the hydration of the stratum corneum is important. So becomes the water binding capacity of the horny layer crucially caused by urea. With regard results on the barrier function of the epidermis hence a central role for urea. Which Meaning of urea with regard to Barrier function of the epidermis in this context becomes clear when you look pathological Considered skin conditions where an epidermal Urea deficiency is detectable. This through Dryness, dandruff, lichenification and rhagades characterized subsequent symptoms as they for example in atopic dermatitis, in the Psoriasis or aging skin can be observed are an expression of a profound functional and epidermis differentiation disorder.

Urea is neosynthesized intraepidermally by keratinocytes, which express the L-isomer-specific enzyme arginase (A2, type II) cytoplasmic. The hydrolase is highly specific and has a high activity. It is largely present as a desmoenzyme and is therefore bound to high molecular weight insoluble carriers. The epidermal (extrahepatic) arginase represents by far the largest share in the distribution in the human body. It differs from the hepatic isoform in that it has an almost neutral pI value and a somewhat lower molecular weight (38,900 kDa). The tetrameric keratinocytic arginase is a metalloenzyme and binds as a cofactor Mn ²⁺ ions, which stabilize the molecule and regulate the activity. Crystallographic studies have shown that the manganese center in the arginase molecule consists of two coordinated Mn ²⁺ ions (MnA and MnB). These are linked together by water molecules and two aspartate residues. The absence of the metal ions causes the enzyme to dissociate into four inactive monomers. The addition of Mn ²⁺ ions leads to an increase in the enzymatic activity. The pH optimum is in the presence of Mn ²⁺ ions at pH 10. From pH 6 the tetramer dissociates reversibly via dimers to inactive monomers.

L-arginine is preferably carried through the Y + system carrier-mediated to intracellularly. The Y ⁺ system is a Na-independent, soluble, cationic amino acid transporter that transports L-arginine as well as L-lysine and L-ornithine. Expression of the transporter protein is regulated by the concentration of L-arginine, L-lysine and L-ornithine. In addition, the activity of the Y ⁺ system is increased by increased glucose, insulin or PGI2 concentrations. In addition, unspecific amino acid transport systems such as the A system and ASC system, which require a Na ⁺ gradient on the plasma membrane and the L system, which is independent of Na ⁺ , are known, but are probably only insignificant amounts of L-arginine can transport.

It is known that disorders or pathological Skin changes and aging skin are common with Disorders of the skin's barrier function go hand in hand. This manifests itself in the fact that the skin is excessive Water loses, unprotected harmful influences exposed and often with eczema responding. A common chronic skin disease, among the approximately 2-5% of the population is atopic dermatitis (atopic dermatitis, atopic eczema). An eczema skin essentially has the following symptoms: insufficient skin oiling, decreased water and Skin urea content, decreased skin through bleeding, decreased defense against infection of the Skin, excessive or weak sweating, increased Sensitivity to non-specific mechanical and chemical stimuli such as dust, soap, clothing and the like own sweat, weather dependency. The skin continues to be inflamed and occurs excruciating itching.

For the treatment or therapy of atopic eczema different methods are used. This includes the use of corticoids, antihistamines, the external use (topical application) of urea, Omega fatty acids, phototherapy and gamma Linolenic acid (e.g. contained in evening primrose seed oil, Black cumin oil, currant oil).  

The substances mentioned above have different ones Disadvantages. Cortisone therapy is for suitable for acute therapy, long-term use is, however, with side effects, for example one Thinning of the skin (atropy), connected. The stake of urea for the treatment of dry skin or Neurodermatitis leads to an improvement in The skin's water-binding capacity, however, has Urea often an irritant effect on the skin, especially on damaged skin. Antihistamines are not for topical application suitable because they have little effect wrinkles. Antihistamines are therefore commonly called Tablets, drops or syrup administered. On Another disadvantage of antihistamines is that the effects of taking it over a longer period of time samness decreases significantly. The effect of Evening primrose oil is controversial. There are studies no positive compared to placebos Effect could be demonstrated.

The use of manganese chloride and / or others manganese-containing compounds as an active ingredient for the topical application for the treatment of Skin diseases or for cosmetic use not described in the prior art.

Manganese has so far only been used as an active ingredient in medicine in the following form:

In addition to atopic eczema (neurodermatitis, atopic Dermatitis) is also common with many other skin types diseases, for example in general with chronic inflammatory skin disorders, included the psoriasis vulgaris, in primary or secondary Fibrosis and sclerosis of the skin, in wound healing, for blood circulation disorders in the skin as well as in cosmetics dehydration (dry and aging skin) or the impaired barrier function of the Skin a problem. Also in these areas  Urea, due to its ability to Water binding capacity of the skin or stratum corneums and thus the barrier function of the Used to improve skin, but how already mentioned, skin irritation as the most common Side effect occurs.

The object of the present invention is To provide preparations for topical application len, effective for therapy and prophylaxis pathological changes in the skin, namely the one above mentioned chronic inflammatory skin diseases, including atopic dermatitis and Psoriasis vulgaris, primary or secondary dry skin conditions, used in wound healing can be without the skin essentially too irritate.

The task is performed by a generic Preparation with the features of claim 1 solved. The sub-claims 2 to 8 show advantageous further training. The usage manganese-containing connections to therapeutic, prophylactic and / or cosmetic purposes described in claims 9 to 20.

According to the invention, the object is achieved by preparations for topical application on a manganese-containing compound in pharmaceutically active Concentration based, solved. In addition, in the preparations further excipients and auxiliaries contain.

Preferred are manganese-containing compounds manganese-containing salts used, in an even more preferred Manganese chloride is used.  

The concentration of the manganese-containing compounds in the preparation is preferably between 0.01 and 20 mass%. 0.1 to 10% by mass are preferred and most preferably 0.5 to 5% by mass.

As carriers and auxiliaries in the preparation are included in this invention for example to understand: diluents, Fillers, salts, buffers, stabilizers, etc. lubilizers, fragrances and other common Ingredients. The selection of these fabrics and possibly further carriers and auxiliary substances are known to the person skilled in the art common. Common galenical preparations can be found, for example, in the DAB or DAC.

The following vehicle systems can be used: gel, Lotio, cream, fat cream, ointment, paste, tincture, Solution, liposomal preparation, microemulsion, Nanoparticles.

According to the invention, z. B. may be included:

• - preservatives (e.g. ethanol, Citric acid, ammonia solution)
• - penetration enhancer (e.g. glycerin, Urea, DMSO)
• - fragrances (e.g. musk, rose oil, others Vegetable oils)

The preparation is preferably further Active ingredients added. Among the other preferred Active ingredients for the invention pharmaceutical preparation added and with the Active substances according to the invention can be used together are, for example, corticoids and theirs  Derivatives.

The preparations according to the invention for topical ap either hydrophilic, have amphiphilic or lipophilic properties.

Find preferred uses according to the invention manganese-containing compounds as active ingredients for Therapy and prophylaxis of chronic inflammatory skin diseases such as neurodermatitis (atopi dermatitis), psoriasis vulgaris, exogenous Eczema, such as allergic contact eczema or toxic irritative contact eczema and dysregulative microbial eczema. The aftercare of this chronic inflammatory diseases advantageous according to the invention.

Preferred uses according to the invention include also the therapy and prophylaxis of primary or secondary skin conditions caused by dryness (Xerosis), for example ichthyoses, keratoses and / or aging skin.

The use in the is also preferred Wound healing, e.g. B. the treatment of thermal wounds and scars, or use in leg ulcers.

In the cosmetic application, the manganese-containing compounds may also be suitable sam to increase the water retention capacity of the Skin, to improve the skin's barrier function and to improve the elastomechanical Properties of the skin to be used without that Essentially irritate skin.

The invention will now be illustrated by the following examples explained, but not the scope of the invention should limit.  

The experiments described below show that with topical application manganese chloride and / or other manganese-containing compounds on the skin well tolerated by healthy patients is found and that manganese chloride and / or other manganese compounds an increase hydration of the stratum corneum.

Furthermore, the experiments show that in the treatment of neurodermatitis patients a good effectiveness is achieved without in the case of urea occurring side effects.

example 1 Use of a manganese chloride cream for treatment neurodermatitis

• a) A manganese chloride cream was prepared which contained 5% by weight of manganese chloride, 5% by weight of glycerol monostearate, 40% by weight of wool wax alcohol ointment and 45% by weight of purified water.
  To test skin tolerance, the cream was applied to the skin of a healthy person. No skin irritation was observed. In the case of a person affected by neurodermatitis, a marked improvement was observed after a few days after using the cream and the person was symptom-free after the application. No skin irritation was observed.
• b) A 24-year-old patient suffers from itching and recurrent erythematosus  Skin changes on the flexor sides of the arms and in the area of the face. Because of the clinical Image, the distribution of skin symptoms and phenotypic stigma was assessed by a specialist the diagnosis for skin and venereal diseases "atopic dermatitis" posed. For monotherapy was a 5% cream containing manganese chloride like used in a) twice daily on the affected skin areas was applied. After 7 Extensive healing was observed for days become.

Example 2 Effect of manganese chloride on the barrier function of the skin

The following in vitro Study carried out:

1. Material and methods Detection and distribution of arginase in the epidermis

It is known that keratinocytes are the extrahepatic Arginase (A2, Type II) included. through immunohistology and Western blot analysis became the qualitative evidence on healthy skin and the intraepidermal layer-typical distribution examined semi-quantitative.

A Immunohistochemical detection

The immunohistochemical detection was more healthy human skin. The paraffin-fixed  Samples were made with Rothistol (Roth) and one descending alcohol series dewaxed and with distilled water and Tris buffer rehydrated. The treatment with Pronase was then carried out (Dako) and inoculation with polyclonal IgG Rabbit Anti-human arginase antibodies (Biogenesis, Poole, UK) in a dilution of 1: 1000. The coloring was carried out with corresponding secondary antibodies the LSAB method (Dako).

B Detection using the Western blot technique

The cells were first disrupted by 10 min Cooking at 95 ° C and a 5 min ultrasonic bath. 10 µl samples were mixed with 10 µl SDS buffer (with mercaptoethanol) mixed and in the gel pockets of the 12% polyacrylamide gel applied. The electrophoretic separation was at a voltage from 60-70 V. After transferring the gel into whatman paper was soaked in a buffer Current disturbance of 70 mA blotted over approx. 60 min. To The nitrocellulose membrane was blocked Incubation with the primary antibody (polyclonal IgG Rabbit anti-human arginase antibodies, 1: 10000; Biogenesis, Poole, UK) and after washing with the Secondary antibodies (monoclonal mouse anti-IgG Rabbit Antibody, 1: 5000; Dako). By Exposure of an x-ray film and a digital one The image analysis was carried out.

Cultivation of keratinocytes

Two keratinocytic cell culture systems were used for the cytological examinations. The immortal HaCaT cells (human adult low calcium high temperature keratinocytes, 23rd-35th passage), spontaneously transformed in the phenotype, served as the standard. In comparison, native keratinocyte cultures (2nd passage) isolated from human foreskins were used. The cultivation was carried out at 37 ° C, 10% CO ₂ and 90% humidity. We used arginine-free DMEM (Dulbecco's modified Eagle's medium) with an antibiotic cocktail (10,000 U / ml penicillin G, 10 mg / ml streptomycin, 25 µg / ml amphotericin B) as the medium. 10% inactivated fetal calf serum (FKS) was used as an additive up to and including the third day of culture. From the 4th day of culture, serum-free medium was used. The culture vessels used were 96-well microtitre plates (Greiner) with a cell seeding of 5 × 10 ³ cells / 200 μl medium / well, 24-well culture plates (Greiner) with a cell seeding of 25 × 10 ³ cells / ml medium / well or culture dishes (Greiner) with a Diameter of 50 mm with a cell seeding of 6 × 10 ⁴ cells / ml medium. The respective experiments were started on the 5th day of the culture.

Determination of the urea content using urease method

The determination of urea was carried out with a commercial test system (Biochemica: Boehringer, Mannheim) according to the principle of the Kjeldahl digestion. In the presence of the enzyme urease, urea becomes ammonia and carbon dioxide

split.

Ammonia sets in the presence of glutamate Dehydrogenase (GIDH) and reduced nicotinamide adenine dinucleotide (NADH) 2-oxoglutarate to L-glutamate around, consuming NADH.  

The NADH- Concentration is the ammonia concentration or half urea concentration proportional. The NADH concentration is due to its absorption Determine as a measured variable at 340 nm.

Evidence of urea stability

To demonstrate the stability of the urea in the Culture medium became a after 1, 24 and 48 hours Defined concentration using the urease method (340 nm).

Influence of manganese chloride on the Proliferation behavior of keratinocytes

Radioactively labeled ³ H-thymidine was used to investigate keratinocytic proliferation. The rate of incorporation of ³ H-thymidine in the DNA was defined as a measure of the proliferation. The influence of manganese chloride was determined at concentrations of 0.01; 0.05; 0.1; 1.0; 10.0; 50.0 and 100.0 µmol / l measured after 24 and 48 hours.

Cytotoxicity of manganese chloride

To objectify the cytotoxic effects of Manganese chloride was the number of vital cells using Thyrode-Eosin solution in the Fuchs-Rosenthal- Chamber at different concentrations Manganese chloride (0.01, 0.05, 0.1, 1.0, 10.0, 50.0 and 100.0 µmol / l) after 24 hours.  

Effect of manganese chloride on urea synthesis of keratinocytes

The urease method was used on the 5th day of culture Manganese chloride (10.0; 30.0; 50.0 µmol / l + 25 mmol / l L- Arginine) in FCS-free medium in different Concentrations for 24 or 48 hours Cell cultures added.

statistics

For biometric analysis of the different Average value groups from the normally distributed Populations with the same variance, the one-way Analysis of variance (one-way ANOVA) performed. About that In addition, the differences between the individual Groups with the assessment of linear contrasts Scheffé (or with pairwise comparisons with Bonferroni correction). To the distribution-independent comparison of the groups was made the Kruskal-Wallis test (H test) is used. Generally p <0.05 was defined.

Results Detection of arginase in the epidermis

The intracytoplasmic enzyme in the keratinocytes of the epidermis can be well represented in the immunohistological picture. It is striking that a concentration gradient in the direction of differentiation can be demonstrated. The concentration of the protein in the stratum basale is therefore significantly higher than in the stratum spinosum. No protein is detectable in the stratum corneum ( FIG. 1).

In the digital image analysis of FIG. 1 ( FIG. 2), the red intensity of the individual layers, which serves as a measure of the arginase concentration, also serves as a clear gradient in the concentration from the basement membrane zone in the direction of the vital epidermal portions. Western blot analysis can also be used to detect arginase in both HaCaT and native keratinocytes. The strong bands indicate a clear expression of the protein in the epidermis ( Fig. 3).

The influence of manganese chloride on the Proliferation behavior of keratinocytes

Overall, it was found that manganese chloride only has a proliferation-inhibiting effect in little relevant high concentrations (<10 μmol / l) ( FIG. 4).

Cytotoxicity of manganese chloride

The number of vital cells after incubation with Manganese chloride over 24 hours to one Concentration of 100 µmol / l shows none Changes. There is no evidence for Cytotoxicity.

Effect of manganese chloride on urea synthesis of keratinocytes

It turns out that with the addition of 50 μmol / l man chloride the extracellular urea concentration can already be increased at 25 mmol / l L-arginine ( FIGS. 5 + 6).

In summary it can be said that the Results an increase in keratinocytic Evidence of urea synthesis by adding manganese chloride. This is done by activating the urea-forming Arginase enzyme causes.

Manganese-containing preparations of this invention  can therefore be used wherever a Improved skin barrier function desired is. Examples of the application are u. a. the Be act drier or chronic due to moisture or sun exposure to stressed skin, the care therapy for inflammatory skin cranes kungen, a cosmetic treatment for "revitalization aging of the skin, to improve the rain generation and to stimulate cutaneous metabolism operations.

Claims (19)

1. Preparation for topical application for the Therapy and prophylaxis of pathological skin changes stanchions containing a manganese-containing compound in pharmaceutically effective amounts in addition to übli Chen carriers and auxiliary substances. 2. Preparation according to claim 1, characterized in that manganese salts are included. 3. Preparation according to claim 2, characterized in that manganese chloride ent hold is. 4. Preparation according to at least one of the previous existing claims, characterized in that 0.01-20% by weight the manganese-containing compound are included. 5. Preparation according to at least one of the previous existing claims, characterized in that 0.1-10 wt .-% of manganese-containing compound are included. 6. Preparation according to at least one of the previous existing claims, characterized in that 0.5-5 wt .-% of manganese-containing compound are included. 7. Preparation according to at least one of the previous existing claims,  characterized in that other active ingredients are included in the preparation. 8. Preparation according to at least one of the previous claims that thinners are used as auxiliary substances agents, fillers, salts, buffers, stabilizers sensors, solubilizers are used. 9. Use of compounds containing manganese for topical application for therapy and / or Prophylaxis of pathological changes in the skin. 10. Use of manganese-containing compounds topical application for therapy and / or Prophylaxis of inflammatory skin diseases th. 11. Use of manganese-containing compounds for topical application for therapy and / or Prophylaxis of neurodermatitis (atopic dermatitis tis). 12. Use of compounds containing manganese for topical application for therapy and / or Prophylaxis of psoriasis (psoriasis vulga ris). 13. Use of compounds containing manganese for topical application for therapy and / or Prevention of primary or secondary fibrosis and Sclerosis of the skin. 14. Use of compounds containing manganese for topical application for the therapy of Wun and scars, especially from burns  of the skin. 15. Use of manganese-containing compounds for topical application for the therapy of geal terter Haut and / or the prophylaxis of Hautal esterification processes. 16. Use of manganese-containing compounds for cosmetic application. 17. Use of compounds containing manganese for Increase the skin's water binding capacity. 18. Use of manganese-containing compounds for Improvement of the skin's barrier function. 19. Use of compounds containing manganese for Improvement of the elastomechanical property skin.