Folate preparations

Abstract

The present invention relates to folate formulations and their use as topical agents for the treatment of epithelial tissue irritation and skin disorders.

Description

[Technical Field]

[0001] The present invention provides Folate formulations and in the topical treatment of inflammations and diseases, especially of the skin Folate It concerns the use of the preparation. [Background technology]

[0002] folic acid (folic acid) is a widely occurring growth factor with vitamin characteristics. Type folic acid (reduced folic acid) folate is needed for cells to produce their genetic material, DNA, and therefore for cells to divide properly. As a result, rapidly dividing cells and tissues, such as skin cells and intestinal cells, require folate. (folic acid) is directly affected by the state of

[0003] Folate naturally exists in the form of reduced folates, which contain mono- or polyglutamate groups. Rera Therefore, folate has vitamin properties. synthesis ) occurs only in microorganisms and plants. Folic acid itself is biologically inactive, and dihydrofolic acid Reductase By 7 , 8-dihydrofolic acid and then 5 , 6 , 7 , It must be enzymatically reduced to 8-tetrahydrofolate (THF), which is the biologically active form of folic acid. status THF acts as a carrier of the C1 unit, and the transfer is carried out by 5-methyl-tetrahydrofolate and 5 , 10-methylene-tetrahydrofolic acid, 5-formyl-tetrahydrofolic acid, 5-formimino-tetrahydrofolic acid, 10-formyl-tetrahydrofolic acid and 5 , 10- MethenilThe C1 unit is required for the synthesis of, for example, purine nucleotides and deoxythymidine-5'-monophosphate.

[0004] Many causes in folic acid salt Deficiency of but Cause There is a possibility that For example, folic acid, which is seen during pregnancy salt Increased demand for teeth , resulting in folic acid Salt leading to a state of deficiency There is a possibility In addition, dietary folate intake in celiac disease salt Impaired absorption of or , dihydrofolate in cancer treatment Reductase It is used as a competitive inhibitor of Rume Thotrexate or , intake of antimetabolites such as aminopterin Ya, a Alcohol abuse By , folic acid Salt Deficiency but Cause There is a possibility that Also, one or more folic acids salt Genetic dysfunction in metabolic enzymes By folic acid salt level but Leading to below standard There is a possibility that In addition, inflammatory symptoms This decrease in folate levels can be attributed to: Current folic acid Salt The required amount increase, and 、 Folic acid, which is necessary for the repair process of the tissue affected by the inflammation salt malabsorption of 、 Due to either of these Possible In the skin, folic acid salt A deficiency of vitiligo can lead to a condition called seborrheic eczema and is associated with vitiligo (loss of skin pigment). can be .

[0005] For example, just as division occurs in the metabolism of amino acids and nucleic acids, folic acid saltThe consequences of a deficiency in folate status are profound, since folate plays a role in metabolism. The latter is directly involved in the process of cell division. In rapidly dividing tissues such as bone marrow, this can lead to megaloblastic anemia and thrombocytopenia. Summary of the Invention [Problem to be solved by the invention]

[0006] Reduces homocysteine ​​levels (known as a risk factor for cardiovascular disease) Folate Plastic S Clinical trials have shown that 400 μg of folate is needed to achieve a positive effect, i.e., a reduction in blood homosteine ​​levels. typical It needs to be taken daily. [Means for solving the problem]

[0007] folic acid salt is found mainly in leafy vegetables, grains and liver, but is sensitive to heat and light, so it is quite difficult to get an adequate supply. It is difficult Therefore, the dietary intake of folic acid in healthy individuals salt The supply of necessarily It does not guarantee adequate supply. This is for individual people folic acid Salt reduction Condition attitude Other routes Folate intake via ,for example 、 skin via the leaves acid Salt intake or , skin self With your body of Folate The uptake is still not fully understood.

[0008] Therefore, folic acid Salt organization to Good to intake will be Like, folate New ways to use Folate preparations for , the current There is an ongoing need.

[0009] In a first aspect of the present invention, Inflammation ofand skin of Novel uses of folate as an active agent for the prevention and / or treatment of disease are provided. can .

[0010] Another object of the present invention is to provide a folate formulation in which the folate is readily absorbed by tissues.

[0011] This object is achieved by the use method according to claim 1 and by the formulation according to claim 6. Preferred embodiments of the invention are according to the dependent claims.

[0012] According to the present invention, a formulation containing at least one folate is administered to epithelial tissue. Inflammation of and epithelial tissue of Diseases, preferably skin Inflammation of and skin of This formulation is used for the treatment of diseases. The dosage is topical. Preferably, it is used on the skin. Inflammation of or skin of The disease is an inflammatory skin disease. The inflammation can be acute or chronic, the latter referring to a long-term inflammatory condition. one An example is psoriasis. Furthermore, treatment of chronic wounds can also be achieved by topical treatment with folate preparations, e.g., for 8 to 12 weeks. Such a period A wound that does not heal within the expected time frame is called a chronic wound. and Chronic wounds are thought to be caused by the one Or it appears to remain in multiple phases. of of disease one An example is dry eye syndrome (DES), This is also It is also known as keratoconjunctivitis sicca (KCS).

[0013] The folate preparations of the present invention contain a physiologically effective amount of folate. Na At least one Folate Contains this Folateis selected from the group consisting of arginine, choline, acetylcholine, 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine, and dimethylaminoethanol. The formulation further contains a conventional compound to form a support matrix.

[0014] this Folate The cation of 、 It may also be selected from the group consisting of calcium, magnesium, sodium, and zinc.

[0015] These cations are Arginine, choline, acetylcholine, 1 , Selected from the group consisting of 1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and dimethylaminoethanol The thing is preferable.

[0016] In a further embodiment, this Folate few Na The at least one folate is selected from the group consisting of the following folate compounds: 5-formyl-(6RS)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-methyl-(6RS)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, (6S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5-methyl-10-formyl-(6S)-tetrahydrofolic acid, and 5,10-diformyl-(6S)-tetrahydrofolic acid.

[0017] The formulation, in another embodiment, further comprises a pharmaceutically active compound. this The compounds include arginine, arginine esters, choline, acetylcholine, dimethylaminoethanol, vitamin B complex, and 、 It is preferably selected from the group consisting of vitamin D, especially vitamin D3.

[0018] In yet another embodiment, the formulation further comprises at least one compound selected from the group consisting of sodium gluconate, potassium gluconate, disodium glycerophosphate, and dipotassium glycerophosphate.

[0019] The folate preparation according to the present invention contains 0.01 to 2.5% by weight of the folate preparation. of Concentration of folic acid salt The compound is contained in a multiphase mixture having a non-aqueous phase or an aqueous phase.

[0020] Furthermore, the present folate formulation has a higher concentration relative to the total weight of the formulation. That is, High concentrations of folic acid, up to 5% or even 10% by weight salt It may contain compounds such as folic acid salt The compound is dissolved in an aqueous or non-aqueous solvent. can be solved The latter is e.g. 、 Glycerin. Folic acid salt The minimum amount of compound is 0.001% by weight of the total weight of the folate formulation.

[0021] In another embodiment, the formulation has thoroughly mixed phases: Phase A contains oil; Phase B contains glycerin and an emulsifier; and Phase C contains a physiologically effective amount of Folate and optionally a small amount selected from the group consisting of sodium gluconate, potassium gluconate, disodium glycerophosphate and dipotassium glycerophosphate. Na and at least one compound, such as, for example, tris(hydroxymethyl)-aminomethane. (TRIS) and optionally further compounds such as a pharmacologically acceptable buffer, e.g., and a pharmacologically acceptable antioxidant, e.g., glutathione. Optional Phase D contains one or more matting agents.

[0022] The folate preparation according to the present invention contains, in phase C, calcium, magnesium, sodium, zinc, arginine, choline, acetylcholine, 1-1-dimethylbiguanidine, phenylethylbiguanidine, and, The leaves Acid A small amount selected from the group consisting of dimethylaminoethanol salts Na At least one Folate The polar solvent preferably contains 0.1 to 1000 mg of the compound per 1 ml of the polar solvent. 、 Water, methanol, ethanol, n-propanol, isopropanol, glycerin, and dimethyl sulfoxide. Mixtures of these polar solvents can also be used. Arginine, choline, acetylcholine, 1,1-dimethylbiguanidine, phenylethylbiguanidine, and glucosamine 、 and 、 The leaves Acid The dimethylaminoethanol salt is preferred.

[0023] In another embodiment, the C phase is 1 mol Folate When phase C contains disodium glycerophosphate or dipotassium glycerophosphate, the concentration is preferably 0.4 to 5 molar.

[0024] The formulation according to the invention preferably contains a fat consisting of medium chain triglycerides in phase A. The fatty acids such as medium chain triglycerides are C6 to C8 12 The most preferred oil is caprylic / capric triglyceride. Optionally, it may also contain a dicarboxylic acid alcohol. The dicarboxylic acid chain length is C2 to C6. 10 and the alcohol is selected from the group consisting of methyl, ethyl, isopropyl, propyl, butyl, and pentyl alcohol.

[0025] The emulsifier used in phase B of the present formulation preferably has an HLB value of 5 or greater. Equilibrium HLB is a measure of the degree of hydrophilicity or lipophilicity and is determined by calculating the values ​​of various parts of the molecule as described by Griffin in 1954. The Griffin method for nonionic surfactants as described in 1954 is calculated as follows: HLB = 20 x M h / M, where: M h is the molecular weight of the hydrophilic portion of the compound, and M is the total molecular weight of the compound. An HLB value of 0 corresponds to a completely lipophilic / hydrophobic molecule, and an HLB value of 20 corresponds to a completely hydrophilic / lipophobic molecule. Emulsifiers with HLB values ​​in the range of 8 to 16 are suitable for stabilizing oil-in-water (o / w) emulsions.

[0026] Preferred emulsifiers are those with a fatty acid chain length of C 14 ~C 20 The sucrose ester should have an HLB value of 5 or greater. The most preferred is emulator sucrose stearate.

[0027] In another embodiment, the formulation according to the present invention is Inflammation of and skin of Folate preparations are used to treat inflammatory skin diseases, especially psoriasis. can be .

[0028] Additionally, the folate formulations are also used to treat wounds, especially chronic wounds. can be A wound is a type of injury that occurs relatively quickly, such as a tear, cut, or puncture (open wound) in the skin. and , where: Blunt injury teeth, This is the type of injury that causes a bruise (closed wound). this is, In pathology, it refers to a sharp injury that breaks the dermis of the skin.

[0029] In another aspect, Folate Sodium and potassium salts of gluconic acid and glycerophosphate may be used as penetration enhancers to increase penetration of the compound into the skin. [Brief explanation of the drawings]

[0030] For a better understanding of the present invention, the following examples are described in connection with the accompanying drawings, in which: [Figure 1]FIG. 1 shows the determination of effective doses of test articles or formulations folate cream and L-FTHF. [Figure 2] FIG. 2 shows the quantification of the effective doses of the test substances L-MTHF dicholine and L-FTHF diarginine. [Figure 3] FIG. 3 shows the growth of keratinocytes in media with various calcium concentrations. [Figure 4] FIG. 4 shows the results of the skin scraping assay. [Figure 5] FIG. 5 shows the results of the skin scraping assay. [Figure 5A] FIG. 5A shows the results of the skin scraping assay. [Figure 5B] FIG. 5B shows the results of the skin scraping assay. [Figure 5C] FIG. 5C shows the results of the skin scraping assay. [Figure 6] FIG. 6 shows the results of cell viability after exposure to folic acid. [Figure 7] FIG. 7 shows the results of cell viability after exposure to folic acid. [Figure 8A] FIG. 8A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 8B] FIG. 8B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 9] FIG. 9 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 10A] FIG. 10A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 10B] FIG. 10B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 11] FIG. 11 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 12A] FIG. 12A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 12B] FIG. 12B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 13] FIG. 13 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 14] FIG. 14 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 15A] FIG. 15A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 15B] FIG. 15B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 16] FIG. 16 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 17] FIG. 17 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 18A] FIG. 18A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 18B] FIG. 18B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 19] FIG. 19 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 20A]FIG. 20A shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in a skin model at various time points. [Figure 20B] FIG. 20B shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 21] FIG. 21 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 22] FIG. 22 shows the results regarding the bioavailability of folic acid, methylfolate, and formylfolate in the skin model at various time points. [Figure 23] FIG. 23 shows the results for the permeation of folinate in human skin. [Figure 24] FIG. 24 shows the course of treatment with folate cream in a patient with a skin disorder. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0031] An exemplary cream containing levofolinate calcium salt contains the following compounds: Cream formulation 1 [Table 1] Cream formulation 2 [Table 2] [Example]

[0032] Skin scraping evaluation analysis To assess the effect of various folate preparations on wound healing, an in vitro skin scratch assay was used, which involves inflicting a limited wound on the occlusive cell layer and subsequent regrowth of cells into the occlusive layer in the presence of the test substance compared to an appropriate control, i.e., untreated cells.

[0033] The experimental procedure for the scratch assay was as follows. Human primary skin keratinocytes (product code C-12005, Promocell, Heidelberg, Germany) were used. First, a dose of the test substance was administered that did not have a negative effect on the cells, such as a decrease in cell viability. Next, three different doses were used for the scratch assay. Furthermore, the cells were cultured in a growth-restricting medium to demonstrate the growth-promoting effect of the test substance. For the medium used (product code C-20011, Promocell, Heidelberg, Germany), growth restriction could be achieved by omitting or diluting epidermal growth factor (EGF) or a complex mixture of undefined factors derived from blood BPE. Growth-restricting medium was usually achieved by omitting EGF. For the actual scraping assay, keratinocytes were cultured in appropriate cell culture plates (6-well plates, product number 657160, Greiner BioOne, Frickenhausen, Germany) marked with position indicators. The cells were allowed to grow into a closed monolayer. The cells were then scratched along the position indicators with the tip of a pipette, creating a cell-free area (scratch, wound). The cell monolayer was washed to remove partially detached cells and then covered with medium containing the appropriate dose of the test substance. Cell regrowth into the cell-free area was observed over time at 0, 6, and 24 hours and documented photographically. Analysis was performed by displaying and calculating the cell-free area (software Image J) and comparing the calculated cell-free areas at different time points. This allowed conclusions regarding growth rates compared to the untreated control (cell culture medium without any test substance). Each dose of test substance was replicated three times, and the entire study was performed twice.

[0034] Test substance: [Table 3]

[0035] Instructions for lysing samples

[0036] The assay solutions were prepared as follows:

[0037] L-FTHF calcium (levoleucovorin calcium, levofolinate calcium, 5-formyl-(6S)-tetrahydrofolic acid calcium salt) (2.5%): Mix 125 mg of L-FTHF calcium with 125 mg of sodium gluconate and dissolve in 4.25 g of double-distilled water.

[0038] L-MTHF (2.5%): 125 mg of L-MTHF dicholine salt (5-methyl-(6S)-tetrahydrofolic acid dicholine salt) was added and dissolved in 4.875 g of double distilled water.

[0039] L-FTHF (2.5%): Add 125 mg of L-FTHF diarginine salt (5-formyl-(6S)-tetrahydrofolic acid di-L-arginine salt) to a 4.875 ml solution. g Dissolve in double distilled water.

[0040] Folate cream (0.25% calcium levofolinate): this The folate cream was used as delivered by the manufacturer and stored in a refrigerator.

[0041] Preliminary experiments have quantified effective doses. The cultures for the skin scraping assay are monolayer tissues that are significantly more sensitive than the organotypic skin models used for bioavailability, and therefore cannot be treated with undiluted solutions or formulations.

[0042] For this test, keratinocytes were seeded onto 96-well plates and incubated with five dilutions of the solution or cream for 24 hours. Subsequently, the vital dye was added, and finally, the color change caused by the conversion of the vital dye in the living cells was quantified photometrically.

[0043] Figures 1 and 2 show the determination of the effective dose of the test substance. Cytotoxicity test after viable cell conversion of the vital dye MTT, performed with keratinocytes in monolayer culture. Concentration data are based on undiluted solution or cream (=100%). Treatment time was 24 hours. Untreated cultures were used as negative control. SDS was used as positive control ("dead control"). N=6 + SEM

[0044] According to the results of dosage determination, this Folate cream is effective from a concentration of 0.00022% (viability = 70% of control). L-FTHF calcium is effective from a concentration of 0.13%. L-MTHF dicholine is effective from a concentration of 0.14% and L-FTHF diarginine is effective from a concentration of 0.12%. IC70 values ​​were calculated by linear regression (Hill slope, medical statistical analysis software GraphPad Prism 5.04). The term IC70 refers to the concentration at which the viability of treated cell cultures is reduced to 70% of that of untreated controls. IC70 is often cited as the boundary between toxicity and non-toxicity.

[0045] In preparation for the scratch assay, it was also examined whether the growth rate of keratinocytes was affected. A medium such as the serum-free medium used would allow each cell to proliferate at its highest possible rate, and no further acceleration of proliferation would be possible. However, it was found that the medium used did not lead to the maximum proliferation rate of the cells (see Figure 3). Therefore, further experiments were carried out using standard, unmodified medium.

[0046] For this study, keratinocytes were diluted and seeded into 96-well plates. After 24 hours, the standard medium was replaced with medium containing various calcium concentrations. After 24 hours, the number of viable cells was quantified by conversion of each of the vital dyes (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

[0047] Figure 3 shows a keratinocyte proliferation experiment. A change in the calcium content of the medium resulted in a partial increase in proliferation. N=6 + SEM

[0048] This indicated that an increased proliferation rate was possible compared to cells cultured in standard medium. Therefore, standard medium was chosen as the basis for supplementation in the scratch assay because depletion of the medium is not necessary to demonstrate growth promotion. Standard medium was also used as a control. As an additional control, bovine serum albumin (FBS) was added to the standard medium because its inherent growth factors are expected to promote keratinocyte proliferation and provide another measure.

[0049] Figures 4 and 5 show the cell-free area of ​​the induced scratch wound after 6 and 24 hours. Measurements are expressed as a percentage of the initial area for each treatment group. The line indicates the control (=standard medium) level. The smaller the bar, the faster the scratch, i.e., the cell-free area, was regenerated with cells. For each treatment group, three concentrations were tested in three separate cultures (N=3) + SEM.

[0050] After 6 hours, no significant increase has yet been achieved and the differences between treatment groups are minimal. this The folic acid solution was found to promote growth better than standard media, as was the FBS (fetal bovine serum) control. this Folate cream dramatically inhibited growth at the two highest concentrations, even resulting in the death of the culture at the highest concentration (no visible bar). This was unexpected, as only slight toxicity was observed after preliminary testing of the highest concentration. This is likely a methodological issue, as the stress caused by the injury appears to increase the sensitivity of the cultures. this Folate solutions also stimulated proliferation. Strikingly, L-FTHF diarginine (green) stimulated a dose-dependent reversal of repair. However, this difference was not significant, so this result may have occurred by chance.

[0051] Overall, the differences in lesion growth among the treatment groups were not significant, likely due to the relatively large variations among the three treatment groups.

[0052] Figures 5A, 5B, and 5C show photographs taken with 0.1% L-MTHF at 0, 6, and 24 hours, respectively. The drawn lines indicate the open wound surface defined for the analysis program. After 6 hours, no surface has grown too much, and after 24 hours, it becomes difficult to visualize the acellular area. [Example]

[0053] bioavailability To test the physiological availability in skin-like tissues, a reconstructed skin model (epiCS, product number CS-1001, CellSystems GmbH, Troisdorf) made from primary human keratinocytes was used. This model exhibits a barrier very similar to that of human skin. This model is regularly used as a test model for determining the corrosive and irritant properties of cosmetic ingredients. As the skin model is intended for consumer use, it is usually treated with the final product. However, to avoid overdosing, the tolerability of the skin model is assessed before treatment with the test product.

[0054] Skin models were cultured on a porous membrane as described above. Growing cells separate the apical compartment (containing the skin model) from the basal compartment (containing the nutrient medium). Mass transport from the apical to the basal compartment is thus possible almost exclusively through the cells. The only alternative is paracellular diffusion, which, due to the nature of tight junctions, is only possible for a few molecules and only to a very limited extent. To determine the physiological availability of the products, the barrier was tested for leakiness by resistance measurement before use. Models with electrical resistance between the apical and basal compartments below the average were not used in the experiments. Next, appropriate skin models were topically treated with the test products. After 4, 8, and 8 hours, medium samples were taken from the basal part of the model to determine the presence and concentration of folate. Each test product and control was tested at each of three replicate concentrations. The study was repeated until results from two separate runs were obtained. Quantification of folate content in the medium samples began with analysis of the 24-hour values. Since these already contained folate, further time points were subsequently examined to allow imaging of uptake dynamics, if necessary.

[0055] The culture medium samples were analyzed by a JC / MS method (High Performance Liquid Chromatography / Mass Spectrometry) established for commercial service providers. The sensitivity of this method is in the range of 1 nmol / L. Before starting the study, a feasibility study or pilot test was performed to be able to assess the validity of the method. The sensitivity of the method proved to be sufficient for the analysis of bioavailability.

[0056] In a preliminary experiment, solubilized test substances similar to those described in Example 2 were topically applied to each of three skin models. Only 100 μL of water was applied to each of the three models. The area of ​​the skin models was 0.6 cm2 each. 2After 24 hours of incubation, a sample of the medium was removed from the model and frozen for preliminary analysis. The model was then washed and incubated with a vital dye (resazurin) to quantify the number of viable cells in the model. This was done to determine whether the treatment had a negative effect on the cells. Of course, this could also impair the barrier function of the skin model.

[0057] Figures 6 and 7 show the relative vitality of skin model cells after topical treatment with 100 μL of test substance or water for 24 hours. Fluorescence values ​​of the vital dye (resazurin) were normalized to the control (=100%). Results for each run were N=3 + SEM.

[0058] The viability of the skin model is this It was found that the treatment during the run had no adverse effects. Treatment with the three test substances even appeared to slightly increase viability compared to the control. In the second run, a slight decrease in viability, 86% of the control, was observed following treatment with folate cream. However, this small decrease was not considered toxic. Thus, the viability of the skin model was not significantly affected by the treatment. Therefore, it should be assumed that the barrier function of the skin model was not impaired. Therefore, the model could be treated with the undiluted solution or cream for 24 hours. Compared to the irritation test with TG439 at 0ECD, in which the model was treated with the test substance for only 15 minutes, a 24-hour treatment is much more challenging. As mentioned above, the tested solutions and creams can be assumed to have excellent compatibility with human skin.

[0059] Preliminary analysis of some test samples indicated that very strong folate signals could be measured, potentially causing problems with the isolation column during analysis; therefore, dilution of the test samples was necessary prior to analysis. Additionally, the topically applied volumes were reduced to 50 μL each. Furthermore, in this study, the lower compartment was filled with 1 mL of Hank's balanced salt solution (without folic acid) rather than with medium (containing folic acid). Following the full 24-hour incubation with the test substances, the skin models were again incubated with resazurin, allowing quantification of potentially reduced model vitality. Two independent pathways were achieved in this study.

[0060] After incubation with L-MHTHF dicholine, methylfolate was measured in the medium. salt Peaks were outside the calibration line (Figures 9, 11, 13, 16, and 21). This was also true for formylfolate concentrations after 24 hours (Figures 14, 17, and 22). Although these peaks were interpolated, their absolute values ​​are considered uncertain. However, it is certain that they are very high.

[0061] For some samples measured in the first analytical run, formyl folate content was not considered (4-hour and 24-hour first round), and therefore these samples have no formyl folate content data or only one-pass data.

[0062] Figures 8A, 8B and 9 this Folic acid or methylfolate in compartments under the skin model during orchid salt The concentration of 100 μL of the solution was analyzed. N=3 + SEM.

[0063] The treated skin models showed little difference in terms of folate release. In contrast, methylfolate SaltSignificant differences in concentrations were measured between models: significantly higher concentrations were measured in models supplemented with L-MTHF dicholine than in models supplemented with L-FTHF or folate cream. this Folic acid concentration and Maximum Measured methylfolate Salt The difference between the concentrations is particularly large (Figure 9).

[0064] Figures 10A, 10B and 11 show the concentration of folic acid or methylfolate in the compartments under the skin model in the second round. salt The concentration of each compound is shown. In each case, 100 μl of solution was analyzed. N=3+SEM.

[0065] After 4 hours, both runs show very similar folate concentrations in the compartments (Figures 8A, 8B, and 10). The medium contained multiple folate models that were fed prior to treatment with the test substance. , more folate is provided topically The values ​​obtained here are assumed to always be derived in part from residual medium that is gradually released from the cells. Folate concentrations between the models are relatively similar after 4 hours, indicating that supplementation Is bright The differences are clearly independent. The small differences shown here (Figures 8, 8B and 10A, 10B) are not significant (analysis of variance via GraphPad Prism 5.04). In contrast, after 4 hours of treatment, the values ​​of methylfolate in the buffer are very different between the models (Figures 9 and 11). Based on this result, the different bioavailability of the folate formulations can be read off. The folate cream is 10 times less concentrated than the liquid product, but the methylfolate concentration in the dermal compartment is only 6-7 times lower.

[0066] Figures 12A, 12B, 13 and 14 show the concentration of folate, methylfolate, and methylfolate in the skin compartment 8 hours after the first run. salt , formyl folate salt Concentration measurements are shown. 100 μL of solution was analyzed in each case. N=3+SEM.

[0067] Figures 15A, 15B, 16 and 17 show the concentration of folic acid, methylfolic acid and methylfolic acid in the skin model 8 hours after the second run. salt , formyl folate salt Concentration measurements are shown. 100 μL of solution was analyzed in each case. N=3+SEM.

[0068] After 8 hours, the folate concentration in the skin model compartments barely exceeded the value after 4 hours (Figs. 12, 15 compared to Figs. 8A, 8B, and 10). In contrast, methylfolate salt The values ​​show at least a partial clear increase (Figs. 13 and 14). L-MTHF dicholine induces the highest concentration of formylfolate in the receptor compartment (basal). salt Concentrations were measured after supplementation with folic acid cream, L-FTHF calcium, and L-FTHF diarginine (Figures 14 and 17), and after supplementation with L-MTHF dicholine (Figures 13 and 17). salt The concentration is equivalent to that of formyl folate. salt Formyl folate was not found in the post-L-MTHF samples. salt Methylfolate salt They cannot be formed solely from or through energy-consuming metabolic pathways.

[0069] Figures 18A, 18B and 19 show the concentration of folic acid, methylfolate, and methylfolate in the compartments under the skin model 24 hours after the first round. salt Concentration measurements are shown. 100 μL of solution was analyzed in each case. N=3+SEM.

[0070] Figures 20A, 20B, 21 and 22 show the concentration of folic acid, methylfolic acid and methylfolic acid in the skin model compartments 24 hours after the second round. salt , formyl folate salt Concentration measurements are shown. 100 μL of solution was analyzed in each case. N=3+SEM.

[0071] After 24 hours (Figures 18A, 18B, 20A, and 20B), folic acid (folic acid) The concentrations of folic acid (partially) and methylfolate were both higher after 4 and 8 hours. (folic acid)The concentration is twice that of other skin models, which are similar in order. However, folic acid (folic acid) The concentrations are negligible compared to the measured methylfolate and formylfolate concentrations. The difference in methylfolate levels is quite high (Figures 19 and 21). The peak from the L-MTHF-treated sample was significantly higher than the highest standard. Also, the methylfolate concentration was relatively high between models treated with L-FTHF calcium. The solution concentration between models treated with L-FTHF diarginine was approximately half this, with the folate cream producing the lowest (uncontrollable) value of 108 nmol / L. The concentration was again significantly higher after 4 hours (approximately 3 nmol / L for folate cream) (Figures 19 and 21). Because formylfolate cannot be produced from methylfolate or through an energy-consuming metabolic pathway, formylfolate is not detectable in the samples after L-MTHF dicholine (Figure 22). On the other hand, it is easily measured in all other samples (with the exception of the control).

[0072] At the beginning of the bioavailability study, 50 μl of solution (25 mg / ml or 60 mmol / l) and 50 μl of cream (2.5 mg / ml or 6 mmol / l) were applied. In the lower compartment, 1 mL of HBSS (Hank's Balanced Salt Solution) was initially filled in each case. The results shown in the figures above were determined from 100 μL aliquots of this buffer.

[0073] The apical compartment was supplemented with 3 μmol of folate (solution) or 0.3 μmol of folate (in 50 μL).

[0074] In the basal compartment, folate was supplemented with L-FTHF calcium up to approximately 200 nmol / L after 24 hours. After 24 hours of supplementation, folate cream was measured at 29 nmol / L.

[0075] Methylfolate was measured at a maximum of 120 μmol / L (24-hour L-MTHF). After 24 hours of supplementation, 47 nmol / L of methylfolate cream was quantified.

[0076] Formylfolate was measured up to 150 μmol / L (24-hour L-FTHF calcium). At this time, 30.21 μmol / L of folic acid cream was measured.

[0077] In these skin scratch assays, supplementation with various folate preparations significantly accelerated the repair of artificial wounds after 24 hours. The studies were performed on monolayers of primary epithelial cells compared to untreated controls.

[0078] The physiological availability of methylfolate or formylfolate from the formulations could be demonstrated after supplementation of a reconstructed skin model. Topically administered folate derivatives are transported through the tissues of the model and partially metabolized by the cells. Part of topically administered formylfolate is metabolized to methylfolate, while the other part is transported through the tissues. Conversely, topically administered methyl formate is transported as such through the tissues of the skin model and is not metabolized to formylfolate. The intake effect was described as transport, but for formylfolate, it was two-fold superior from the folate cream than from a pure folate solution (L-FTHF calcium). Folic acid was also quantified in the basal compartment of the skin model. However, the measured concentrations were much lower than those of methylfolate and formylfolate. Thus, the measured folic acid 、 It probably originates from the medium used for the growth of the skin model and is likely to be released again by the cells during the course of the experiment. [Example]

[0079] Further experiments examined whether calcium levoleucovorin could penetrate the skin. Penetration depth was quantified by Raman spectroscopy. The measurement device was an inverted Raman spectrometer 3510 equipped with a 60x oil immersion lens and the evaluation software SkinTools from RiverD Rotterdam. The measurement method was 10 lateral images per application, 5 seconds of measurement time per measurement point, seven 2 μm steps, four 4 μm steps, a spectral range of 400–1800 cm-1, and a laser excitation wavelength of 785 nm. Signals were generated using the software SkinTools, which contains spectra (frequency ranges) specific to various skin types and devices. Users can add their own spectra to the library. Evaluation was performed via the CLP method (least squares fit). Signals were output as fit coefficients normalized to the fit coefficients of keratin.

[0080] To quantify the results, they must be calibrated with a standard column (see Tab 1 and Tab 2). First, the sensitivity of bovine serum albumin (BSA) is determined: the mass concentration of BSA in g / ml is plotted against the fit coefficient of BSA. A linear regression with a fixed zero gives the slope S. For this active substance, the molar concentration in mol / ml is plotted against the drug coefficient multiplied by the BSA fit coefficient. This gives the slope SA of the active ingredient. With respect to the slope, the so-called drug quantification factor can be multiplied (SA / S=C). Multiplying by the factor C, the fit coefficient of the active substance becomes the quantification value in mmol / g of keratin.

[0081] A standard example for determining the sensitivity of an analytical system to keratin is as follows:

[0082] Tab 1 [Table 4]

[0083] The standard sequence for determining the quantification factor of calcium levoleucovorin is as follows:

[0084] Tab 2 [Table 5]

[0085] Drug or placebo (sodium gluconate) application to the subject's skin was performed as follows: 20 μL was pipetted onto an allergy patch and applied to the subject's forearm for 30, 60, 120, and 240 minutes. At the end of the application period, the allergy patch was removed. Before determining the Raman profile, the treated skin area was rubbed with a dry paper towel.

[0086] The measured fit coefficients fi and quantification factors Ci are listed in the following table. [Table 6]

[0087] The slopes S and SA of BSA (bovine serum albumin) and the quantification coefficients Ci of the active substance are as follows: [Table 7]

[0088] Figure 23 shows calcium levoleucovorin profiles for application times of 30 to 240 minutes. There is a trend for calcium levoleucovorin concentrations to increase in the upper 5 μm and deeper layers of the skin. However, the standard deviations of the individual profiles, not shown, are large.

[0089] The table below shows the measured amount of active substance that penetrated the skin per area. The profile was evaluated from the skin surface to a depth of 30 μm. There is a trend towards an increase in calcium levoleucovorin concentration over time. Compared to the applied amount, the percentage of active ingredient in the skin is 10% after 240 hours of application. [Table 8] [Example]

[0090] The folate-containing cream described in Example 1 was used to treat five patients with different skin problems, namely neurodermatitis and psoriasis. Over the course of 21 days, symptoms significantly decreased. One patient had bald folliculitis and had been receiving conventional treatment for several years. Treatment with the folate cream for three weeks resulted in almost complete regression of symptoms, leading to skin healing, as shown in Figure 24. There was no recurrence for up to nine months, and the skin maintained a normal appearance.

[0091] A child suffering from neurodermatitis was treated with the folate preparation of the present invention for three weeks. During this treatment, the symptoms subsided and the skin completely healed. There was no recurrence after treatment was stopped.

Claims

1. 1. A formulation containing at least one folate for use in treating tissue inflammation and inflammatory skin diseases, wherein the formulation is a topical agent, the anion of the at least one folate being selected from the group consisting of 5-formyl-(6S)-tetrahydrofolic acid and 5-methyl-(6S)-tetrahydrofolic acid, and the cation of the folate being selected from the group consisting of arginine, choline, and calcium.

2. 2. The formulation according to claim 1, wherein the formulation contains at least one folate salt for use in the treatment of skin inflammation and skin disorders, characterized in that the skin disorder is psoriasis.

3. 2. The formulation according to claim 1, comprising at least one folate salt for use in the treatment of skin inflammation and skin disorders, characterized in that the skin disorders are dermatitis or neurodermatitis.

4. 2. The formulation according to claim 1, comprising at least one folate salt for use in the treatment of skin inflammation and skin disorders, characterized in that the skin disorder is a chronic wound.

5. 5. A formulation according to any one of claims 1 to 4, comprising at least one folate compound for use in the treatment of skin inflammation and skin disorders, characterized in that the formulation contains the folate compound in a concentration of 0.01 to 2.5 weight percent of the total weight of the formulation in a microemulsion, oil-in-water emulsion, water-in-oil emulsion, nanoparticle formulation, gel formulation, and / or spray formulation.

6. 6. A formulation according to any one of claims 1 to 5, comprising at least one folate salt for use in the treatment of skin inflammation and skin disorders, the formulation having a thoroughly mixed phase consisting of phase A, phase B, phase C and optionally phase D, The phase A contains oil, The layer B contains glycerol and an emulsifier, the C phase contains a physiologically effective amount of at least one folate; the optional D phase contains a matting agent; The preparation, characterized in that:

7. 7. The formulation of claim 6, wherein the formulation contains at least one folate for use in the treatment of skin inflammation and skin diseases, wherein the C phase of the formulation contains a physiologically effective amount of at least one folate, and the cation of the folate is selected from the group consisting of arginine and choline.

8. 8. A formulation according to any one of claims 1 to 7, comprising at least one folate salt for use in the treatment of skin inflammation and skin disorders, characterized in that the formulation also contains at least one additional compound selected from the group consisting of arginine, arginine esters, choline, acetylcholine, glucosamine, dimethylaminoethanol, B vitamins, and vitamin D.

9. 8. A formulation according to claim 6 or 7, containing at least one folate for use in the treatment of skin inflammation and skin diseases, characterized in that phase C contains 0.1 to 1000 mg of the folate per ml of polar solvent.

10. 10. A formulation according to any one of claims 1 to 9, containing at least one folate for use in the treatment of skin inflammation and skin disorders, characterized in that the formulation contains at least one compound selected from the group consisting of sodium gluconate, potassium gluconate, disodium glycerophosphate, and dipotassium glycerophosphate.

11. 7. A formulation according to claim 6, containing at least one folate, for use in the treatment of skin inflammation and skin disorders, wherein the oil of phase A is a medium chain triglyceride whose fatty acid is C ６ ~C １２ and optionally a dicarboxylic acid alcohol, wherein the dicarboxylic acid is C ２ ~C １０ wherein the alcohol is selected from the group consisting of methyl alcohol, ethyl alcohol, isopropyl alcohol, propyl alcohol, butyl alcohol, and pentyl alcohol.

12. 12. The formulation according to claim 11, comprising at least one folate for use in the treatment of skin inflammation and skin disorders, characterized in that the oil is caprylic triglyceride or capric triglyceride.

13. 8. A formulation according to claim 6 or 7, containing at least one folate, for use in treating skin inflammations and skin disorders, characterized in that the emulsifier of phase B has an HBL value of 5 or more.

14. 8. A formulation according to claim 6 or 7, containing at least one folate, for use in the treatment of skin inflammation and skin disorders, wherein the emulsifier is C １４ ~C ２０ and preferably sucrose stearate, and all of the sucrose fatty acid esters have an HBL value of 5 or greater.

15. 15. A formulation according to any one of claims 1 to 14, comprising at least one folate salt selected from the group consisting of 5-formyl-(6S)-tetrahydrofolic acid calcium salt, 5-methyl-(6S)-tetrahydrofolic acid dicholine salt and 5-formyl-(6S)-tetrahydrofolic acid di-L-arginine salt, for use in the treatment of skin inflammation and skin disorders.

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