JPWO2020002714A5 - - Google Patents

Description

The present invention relates to folate salt preparations and their use in the topical treatment of inflammations and diseases, especially skin inflammations and diseases.

Folic acid is a ubiquitous growth factor with vitamin characteristics. Reduced folic acid is necessary for cells to divide properly, as they are necessary for the production of the genetic material, DNA. As a result, rapidly dividing cells and tissues, such as skin cells and intestinal cells, are directly affected by folic acid status.

Folate occurs naturally in the form of reduced folate that retains mono- or polyglutamate groups. Human metabolism cannot produce these folate compounds. Folate thus has vitamin properties. De-novo synthesis of folate compounds occurs only in microorganisms and plants. Folic acid itself is biologically inactive and is enzymatically reduced to 7,8 - dihydrofolate and further to 5,6,7,8-tetrahydrofolate (THF) by dihydrofolate reductase . There is a need. Tetrahydrofolic acid (THF) is the biologically active form of folic acid. THF acts as a carrier for the C1 unit and the transfer is 5 _- methyl-tetrahydrofolic acid, 5 , 10-methylene-tetrahydrofolic acid, 5-formyl-tetrahydrofolic acid, 5-formimino-tetrahydrofolic acid, 10-formyl-folic acid, respectively. Accomplished by tetrahydrofolic acid and 5,10- methenyl -tetrahydrofolic acid. The C1 unit is required, for example, for the synthesis of purine nucleotides and deoxythymidine _- 5'-monophosphate.

Many causes can cause folate deficiency. For example, an increased requirement for folate , such as that seen during pregnancy, can result in folate deficiency. In addition, folate malabsorption from food due to celiac disease , intake of antimetabolites such as methotrexate and aminopterin, which are used as antagonistic inhibitors of dihydrofolate reductase in cancer therapy, and alcohol abuse , Folate deficiency can be caused . Also, genetic malfunctions in one or more folate -metabolizing enzymes can lead to subnormal folate levels. In addition, inflammatory conditions can lower folate levels. This reduction in folate levels may be due to current increased folate requirements and/ or impaired absorption of folate required for repair processes in the affected tissue. There is In the skin, folate deficiency leads to a condition called seborrheic eczema and can be associated with vitiligo (loss of skin pigment).

Since folate plays a role in metabolism, for example, splitting occurs in the metabolism of amino acids and nucleic acids, the consequences of deficient folate status are numerous. The latter are directly involved in the process of cell division. In rapidly dividing tissues such as bone marrow, it can lead to megaloblastic anemia and thrombocytopenia.

Clinical trials have shown a positive effect of folate in reducing homocysteine levels (a known risk factor for cardiovascular disease). In order to achieve a positive effect, ie a reduction in homosteine levels in the blood, 400 μg of folate typically needs to be taken daily.

Folate is found mainly in leafy greens, cereals, and liver, but is sensitive to heat and light, making it rather difficult to supply adequately. Therefore, the dietary supply of folate to healthy individuals does not necessarily guarantee an adequate supply. Instead, it appears to be the main cause of folate -depleted conditions in individuals . Uptake of folate via other routes, such as folate uptake via the skin and folate uptake in the skin itself, is not yet well defined.

Therefore, there is an ongoing need for new uses of folate and formulations of folate that result in better tissue uptake of folate.

In a first aspect of the present invention there is provided a novel use of folate as an active agent for the prevention and/or treatment of skin inflammations and skin disorders.

Another object of the present invention is to provide a folate formulation in which folate is readily absorbed into tissues.

Said object is achieved by a use according to claim 1 and a formulation according to claim 6. Preferred embodiments of the invention are subject to the dependent claims.

According to the invention, at least one folate-containing formulation is used for the treatment of epithelial tissue inflammations and epithelial tissue diseases, preferably skin inflammations and skin diseases. The usage of this formulation is for topical use. Preferably, the skin inflammation or skin disease is an inflammatory skin disease. Inflammation may be acute or chronic. The latter applies to long-term inflammatory conditions. One example of a chronic inflammatory disease is psoriasis. Furthermore, treatment of chronic wounds can also be achieved by topical treatment with folate preparations. Wounds that do not heal within an expected period of time, such as 8 to 12 weeks, are considered chronic wounds. Chronic wounds appear to remain in one or more phases of wound healing. An example of a disease of the epithelium of the eye is dry eye syndrome (DES) , also known as keratoconjunctivitis sicca (KCS).

The folate formulation according to the invention contains a physiologically effective amount of at least one folate salt . The cation of this folate salt is selected from the group consisting of arginine, choline, acetylcholine , 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and dimethylaminoethanol. The formulation further contains conventional compounds to form a support matrix.

The cation of the folate salt may also be selected from the group consisting of calcium, magnesium, sodium, and zinc.

These cations are preferably selected from the group consisting of arginine, choline, acetylcholine , 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and dimethylaminoethanol.

In further embodiments, at least one folate of the folate salt is selected from the group consisting of 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.

The formulation, in another embodiment, further contains a pharmaceutically active compound. This compound is preferably selected from the group consisting of arginine, arginine esters, choline, acetylcholine, dimethylaminoethanol, vitamin B complex and vitamin D, especially vitamin D3.

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.

The folate formulation according to the invention contains folate compounds in a concentration of 0.01 to 2.5% by weight relative to the total weight of the folate formulation in a multiphasic mixture with a non-aqueous phase or an aqueous phase.

Furthermore, the main folate formulation may contain higher concentrations of the folate compound, ie up to 5% or even 10% by weight relative to the total weight of the formulation. Folate compounds can be dissolved in aqueous or non-aqueous solvents. The latter is, for example , glycerine. The minimum amount of folate compound is 0.001% by weight of the total weight of the folate formulation.

In another embodiment, the formulation has completely mixed phases. Phase A contains oil. Phase B contains glycerin and an emulsifier. Phase C comprises a physiologically effective amount of folate salt and optionally at least one compound selected from the group consisting of sodium gluconate, potassium gluconate, disodium glycerophosphate and dipotassium glycerophosphate. containing an aqueous folate composition having In addition, a pharmacologically acceptable buffer, such as tris(hydroxymethyl)-aminomethane (TRIS), and a further pharmacologically acceptable antioxidant, such as glutathione. A compound may optionally be included. Optional Phase D contains one or more matting agents.

The folate preparation according to the present invention comprises, in phase C, the group consisting of calcium, magnesium, sodium, zinc, arginine, choline, acetylcholine, 1-1-dimethylbiguanidine, phenylethylbiguanidine, dimethylaminoethanol salts of said folates. 0.1 to 1000 mg per ml of polar solvent of at least one folate salt selected from Polar solvents are, for example , water, methanol, ethanol, n-propanol, isopropanol, glycerin, dimethylsulfoxide. Mixtures of these polar solvents can also be used. Arginine, choline, acetylcholine , 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and the dimethylaminoethanol salts of said folates are preferred.

In another embodiment, Phase C contains 0.8 to 10 moles of sodium or potassium gluconate to 1 mole of folate salt . When phase C contains disodium glycerophosphate or dipotassium glycerophosphate , it is preferably 0.4 to 5 molar.

The formulations according to the invention preferably contain in phase A a fat consisting of medium chain triglycerols. Fatty acids such as medium chain triglycerols have chain lengths in the range of _C6 to _C12 . Most preferred are caprylic/capric triglycerol oils. Furthermore, a dicarboxylic acid alcohol may optionally be included. The chain length of this dicarboxylic acid is in the range of _C2 to _C10 and the alcohol is selected from the group consisting of methyl, ethyl, isopropyl, propyl, butyl, pentyl alcohol.

The emulsifier used in phase B of the formulation preferably has an HLB value of 5 or more. The hydrophilic-lipophilic balance of a surfactant is a measure of the degree of hydrophilicity or lipophilicity, and is quantified by calculating values for various parts of the molecule, as described by Griffin in 1954. be done. 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 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.

Preferred emulsifiers are sucrose esters with fatty acid chain lengths in the range of _C14 to _C20 . The sucrose ester should have an HLB value of 5 or greater. Most preferred is emulator sucrose stearate.

In another embodiment, the formulations according to the invention are used for the treatment of skin inflammations and skin disorders. This use is preferably topical. Folate preparations can be used to treat inflammatory skin diseases, especially psoriasis.

Furthermore, the folate preparation can be used to treat wounds, especially chronic wounds. A wound is a type of injury that develops relatively quickly, such as a tear, cut, or puncture of the skin (open wound), where blunt injury is similar to causing a bruise (closed wound). type of injury. In pathology, it refers to a sharp injury that breaks the dermis of the skin.

In another aspect, sodium and potassium salts of gluconic acid and glycerophosphate can be used as permeation enhancers to increase the penetration of folate salts into the skin.

For a further understanding of the invention, the following examples are described in connection with the accompanying drawings.
FIG. 1 shows quantification of effective doses of test article or formulation folate cream and L-FTHF. FIG. 2 shows quantification of effective doses of the test substances L-MTHF dicholine and L-FTHF diarginine. FIG. 3 shows keratinocyte growth in media with varying calcium concentrations. FIG. 4 shows the results of the scratch evaluation analysis. FIG. 5 shows the results of the scratch evaluation analysis. FIG. 5A shows the results of the scratch evaluation analysis. FIG. 5B shows the results of the scratch evaluation analysis. FIG. 5C shows the results of the scratch evaluation analysis. FIG. 6 shows the results of cell viability after exposure to folic acid. FIG. 7 shows cell viability results after exposure to folic acid. FIG. 8A shows results for bioavailability of folic acid , methylfolate and formylfolate at various time points in a skin model. FIG. 8B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 9 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 10A shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 10B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 11 shows the results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 12A shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 12B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 13 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 14 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 15A shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 15B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 16 shows the results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 17 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 18A shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 18B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 19 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 20A shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. FIG. 20B shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 21 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 22 shows results for bioavailability of folic acid, methylfolate and formylfolate at various time points in a skin model. Figure 23 shows the results for folinate permeation in human skin. FIG. 24 shows the course of treatment with folate cream in a patient with skin disease.

クリーム製剤２

An exemplary cream containing levofolinate calcium salt contains the following compounds.
Cream formulation 1

Cream formulation 2

Scratch Evaluation Analysis An in vitro scratch evaluation analysis was used to evaluate the effects of various folate formulations on wound healing. This in vitro formula induces a limited injury to the closed cell layer and the subsequent regrowth of cells into the closed layer in the presence of the test substance, using an appropriate control standard, i.e. the test substance. compared to untreated (untreated) cells.

The experimental procedure for the scratch evaluation analysis was as follows. The cells used were human primary epidermal keratinocytes (product code C-12005, Promocell, Heidelberg, Germany). First, a dose of test substance that does not have a negative effect on the cells, such as a reduction in cell viability. Three different doses were then used in the scratch evaluation analysis. In addition, cells were cultured in growth-restricting medium to demonstrate the growth-promoting effects of test substances. For the medium used (product code C-20011, Promocell, Heidelberg, Germany), either by omission of epidermal growth factor (EGF) or a complex mixture of indeterminate factors derived from blood BPE, or by dilution: Growth restriction could be achieved. A growth restricting medium was usually achieved by omitting EGF. For the actual scratch evaluation assay, the keratinocytes were cultured in appropriate cell culture plates (6-well plates from Greiner BioOne, Frickenhausen, Germany, product no. 657160) with position indication. was done. Cells grew into closed monolayers. Cells were then scratched with the tip of the pipette along the position mark, thereby creating a cell-free area (scratch, wound). Cell monolayers were washed to remove partially detached cells and then overlaid with medium containing the appropriate dose of test substance. Cell regrowth into cell-free areas was observed over time after 0, 6 and 24 hours and documented by photographs. Analysis was done 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 on the growth rate compared to the untreated control group (cell culture medium without any test substance). Three replicates were performed for each dose of test article. The entire test was performed in duplicate.

Test substance:

Instructions for dissolving the sample

The assay solution was made as follows.

4. L-FTHF calcium (levoleucovorin calcium, levofolinate calcium, 5-formyl-(6S)-tetrahydrofolic acid calcium salt) (2.5%): 125 mg L-FTHF calcium and 125 mg sodium gluconate; Dissolve in 25 g double distilled water.

L-MTHF (2.5%): Add 125 mg of L-MTHF dicholine salt (5-methyl-(6S)-tetrahydrofolic acid dicholine salt) and dissolve in 4.875 g of double distilled water.

L-FTHF (2.5%): Add 125 mg of L-FTHF di-arginine salt (5-formyl-(6S)-tetrahydrofolic acid di-L-arginine salt) and dissolve in 4.875 g of double-distilled water.

Folate Cream (0.25% levofolinate calcium): This folate cream was used as delivered by the manufacturer and stored in the refrigerator.

Preliminary experiments determined the effective dose. Cultures for scratch assay assays are significantly more sensitive monolayer tissue than the organotypic skin models used for bioavailability and should not be treated with undiluted solutions or formulations. It is possible.

For this test, keratinocytes were seeded on 96-well plates and cultured for 24 hours with 5 dilutions or creams. Subsequently, a vital dye was added and finally, the color change caused by the conversion of the vital dye in living cells was quantified photometrically.

Figures 1 and 2 show the determination of the effective dose of the test article. Cytotoxicity study after live cell conversion of the vital dye MTT performed with keratinocytes in monolayer culture. Concentration data are based on undiluted solution or cream (=100%). Processing time is 24 hours. Untreated cultured tissue was used as a negative control. SDS was used as a positive control (“dead control”). N = 6 + S.E.M.

Dose determination results show that 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 dicolin is effective from a concentration of 0.14% and L-FTHF diarginine from a concentration of 0.12%. IC70 values were calculated by linear regression (Hill slope, medical statistical analysis software GraphPat Prism 5.04). The term IC70 denotes the concentration at which the viability of treated cell cultures is reduced by 70% of untreated controls. IC70 is often cited as the boundary between toxicity and non-toxicity.

It was also examined whether keratinocyte growth rate was affected in preparation for the scratch assay. The medium, such as the serum-free medium, used would give the highest possible growth of each cell, in which case no further acceleration of growth would be possible. However, it was found that the medium used did not lead to maximal growth rate of the cells (see Figure 3). Therefore, further experiments were performed using standard, unmodified medium.

For this study, keratinocytes were diluted and seeded into 96-well plates. After 24 hours, the standard medium was replaced with medium with 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).

FIG. 3 shows keratinocyte proliferation experiments. Changes in the calcium content of the medium partially resulted in faster growth. N = 6 + S.E.M.

This indicated that an increase in 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. This is because media depletion is not required to show growth enhancement. Standard medium is also used as a control. As an additional control, bovine serum albumin (FBS) was added to the standard medium as the growth factors it contains are expected to promote keratinocyte proliferation and provide another measure.

Figures 4 and 5 show the acellular area 6 and 24 hours after induced scratching. Measurements were expressed as a percentage of initiation area for each treatment group. The line indicates the level of the control (=standard medium). The smaller the rod, the faster the scratch, ie the acellular area, was regenerated with cells. For each treatment group, triplicate concentrations were tested in three separate cultures (N=3) + SEM.

After 6 hours, no significant increase had yet been achieved and the differences between the treatment groups were only minor. After 24 hours, this folic acid solution was found to be more growth-promoting than standard media, like the FBS (fetal bovine serum) control. This folate cream surprisingly inhibited growth at two high concentrations. The highest concentrations even killed the medium (bars not visible). This was unexpected as only minor toxicity was observed after preliminary testing with the highest concentration. This is probably a methodological issue, as the stress caused by injury appears to increase the susceptibility of the cultures. This folate solution likewise promotes growth. Strikingly, L-FTHF diarginine (green) promoted a dose-dependent reversal of repair. However, this result may have occurred by chance, as this difference was not significant.

Overall, differences in growth of the injured area between treatment groups are not significant. This difference was probably due to the relatively large variation among the three treatment groups.

Figures 5A, 5B and 5C show, from left to right, photographs taken for L-MTHF 0.1% after 0, 6 and 24 hours. The drawn line indicates the open wound surface defined for the analysis program. After 6 hours it becomes difficult to reveal cell-free areas after 24 hours without overgrowing on any side.

Bioavailability A reconstructed skin model made from primary human keratinocytes (epiCS, CellSystems GmbH, Troisdorf, product number CS-1001) was used to test physiological availability in tissues such as skin. was done. This model shows a barrier that closely resembles human skin. This model is regularly used as a test model for approval determination of corrosive and irritant effects of cosmetic raw materials. Since the skin model is intended for use by consumers, it is usually processed with the final product. Nevertheless, to avoid overdosing, skin models are evaluated for tolerability prior to treatment with test products.

Skin models were cultured on porous membranes as described above. Proliferating cells separate the apical compartment (with the skin model) from the basal compartment (with the nutrient medium). Mass transport from the apical compartment to the basal compartment is thus possible almost exclusively through the cell. The only alternative for this is paracellular diffusion, which is only possible to a very limited extent for a few molecules due to functional tight junctions. To determine the physiological availability of the product, the barrier was tested for leaks by resistance measurement prior to use. Models with below-average electrical resistance between the apical and basal portions described above are not used in the experiment. A suitable skin model is then topically treated with the test product. After 4, 8 and 8 hours, samples of medium were taken at the base of the model for the presence and concentration of folate. Each test product and control is tested at three replicate concentrations of each. The study was repeated until results from two separate runs were obtained. Quantitation of folate content in media samples began with analysis of 24 hour values. Since these already contained folate, additional time points were subsequently examined so that uptake dynamics could be imaged, if necessary.

Media samples were performed 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. Feasibility calculations or preliminary tests were performed to assess the validity of the method before the investigation began. The sensitivity of the method proved sufficient for bioavailability analysis.

In preliminary experiments, solubilized test articles similar to those described in Example 2 were topically applied to each of the three skin models. Only 100 μL of water was applied to the three models. The area of the skin models is 0.6 cm ² each. After 24 hours of culture, media samples were removed from the model and frozen for preliminary analysis. Models were then washed and incubated with a vital dye (resazurin) to quantify the number of viable cells in the model. This was done to determine if the treatment had a negative effect on the cells. Of course, this could also impair the barrier function of the skin model.

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

It was found that skin model viability was not adversely affected by this in-run treatment. Treatment with the three test articles even appears to increase viability slightly compared to the control. In the second run, a slight reduction in viability is observed, 86% of control following treatment with folate cream. However, this low degree of reduction is not considered toxic. Thus, skin model viability was not significantly affected by treatment. Therefore, it should be assumed that the barrier function of the skin model is not compromised. Therefore, models could be treated with the stock solution or cream for 24 hours. Treatment over 24 hours is much more challenging when compared to the 0ECD TG439 irritation test where the model is treated with the test article for only 15 minutes. As mentioned above, a very good affinity for human skin can be assumed for the tested solutions and creams.

Preliminary analysis of some test samples showed that very strong folate signals could be measured during the analysis, which might cause problems with the isolation column. Dilution required. In addition, the volume applied topically was reduced to 50 μL each. Furthermore, in this study, the lower compartment was filled with 1 mL of Hank's Balanced Salt Solution (without folate) rather than with medium (with folate). Following a full 24 hour incubation period with the test article, the skin model was reincubated with resazurin. so that we can quantify the model's presumably reduced vitality. Two independent pathways were achieved in this study.

After incubation with L-MHTHF dicholine , the methylfolate peak quantified in the medium was outside the calibration line (Figs. 9, 11, 13, 16 and 21). This also applies to formylfolate concentrations after 24 hours (Figures 14, 17 and 22). Although these peaks are interpolated, their absolute values are considered uncertain. However, it seems certain that they are very high values.

Formyl folate content was not considered for some of the samples measured in the first analytical run (4h and 24h first round). Therefore, these samples have no formyl-folate content data or only one-pass data.

Figures 8A, 8B and 9 show the quantification of folic acid or methylfolate concentrations in compartments under the skin model during this run. In FIG. 8B, the bars of FIG. 8A are displayed at a different scale. Either way 100 μL of solution was analyzed. N=3+SEM.

The skin models of the treatment groups differed little in terms of folate release. In contrast, large differences were measured between the models in the concentrations of methylfolate. Significantly higher concentrations were measured among models supplemented with L-MTHF dicolin than in models supplemented with L-FTHF or folate cream. The difference between this folic acid concentration and the maximum measured concentration of methylfolate is particularly large (FIG. 9).

Figures 10A, 10B and 11 show the quantification of folic acid or methylfolate concentrations in the compartments under the skin model in the second round. In each case 100 μl of solution were analyzed. N=3+SEM.

After 4 hours, both runs show very similar folate concentrations in the compartments (Figures 8A, 8B and 10). Since this medium contains multiple models of folate fed prior to treatment with the test article , and more folate is provided locally, the values obtained here are It is assumed that it always derives partly from the residual liquid of the medium that is gradually released. Folic acid concentrations between the models are relatively similar after 4 hours and are therefore clearly independent of supplementation. The small differences shown here (FIGS. 8, 8B and 10A, 10B) are not significant (ANOVA through GraphPad Prism 5.04). In contrast, after 4 hours of treatment, we find very different values of methylfolate in the buffer between the models (Figures 9 and 11). Based on this result, different physiological availability of folate preparations can be read. Folate cream is 10 times less concentrated than the liquid product, but only 6 to 7 times less methylfolate concentration in the skin compartment.

Figures 12A, 12B, 13 and 14 show measurements of folic acid , methylfolate, formylfolate concentrations in the skin compartment 8 hours after the first run. 100 μL of solution was analyzed in each case. N=3+SEM.

Figures 15A, 15B, 16 and 17 show measurements of folic acid , methylfolate, formylfolate concentrations in the skin model 8 hours after the second run. 100 μL of solution was analyzed in each case. N=3+SEM.

After 8 hours, the folic acid concentration in the skin model compartment is almost no higher than the value after 4 hours (Figures 12, 15 compared to Figures 8A, 8B and 10). In contrast, methylfolate values show at least a partial clear increase (Figures 13 and 14). L-MTHF dicolin causes the highest concentrations. Formylfolate concentrations in the receiving compartment (basal) after supplementation with folic acid cream, L-FTHF calcium and L-FTHF diaarginine (Figures 14 and 17) and L-MTHF dicolin (Figures 13 and 17)16). equivalent to the methylfolate concentration of No formylfolate can be found in post-L-MTHF samples. Formylfolate cannot be formed from methylfolate or solely through energy-consuming metabolic pathways.

Figures 18A, 18B and 19 show measurements of folic acid, methyl folate concentrations in the compartment under the skin model 24 hours after the first round. 100 μL of solution was analyzed in each case. N=3+SEM.

Figures 20A, 20B, 21 and 22 show measurements of folic acid , methylfolate, formylfolate concentrations in skin model compartments 24 hours after the second round. 100 μL of solution was analyzed in each case. N=3+SEM.

After 24 hours (FIGS. 18A, 18B, 20A and 20B), both folic acid (partial) and methylfolate concentrations are higher than after 4 and 8 hours. Folic acid concentrations among skin models treated with L-FTHF calcium are twice those among other skin models. It is similar in order. However, folic acid concentrations are negligible when compared to the measured methylfolate and formylfolate concentrations. Differences in methylfolate levels are very high (Figures 19 and 21). The peak from the sample under L-MTHF was significantly higher than the highest standard. Also, methylfolate concentrations are relatively high among models treated with L- FTHF calcium. Solution concentrations between models treated with L-FTHF dialginine are about half this, with folate cream producing the lowest (uncontrolled) value of 108 nmol/L. Concentrations are again significantly higher after 4 hours (approximately 3 nmol/L folate cream) (FIGS. 19 and 21). Since formylfolate cannot be made from methylfolate, nor can it be made only through an energy-consuming metabolic pathway, formylfolate is not detectable in samples after L-MTHF-dicoline (FIG. 22). On the other hand, it can be easily measured in all other samples (with the exception of controls).

At the beginning of the physiological availability 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. The lower compartments were each initially filled with 1 mL of HBSS (Hank's Balanced Salt Solution). The results shown in the figure above were quantified from a 100 μL aliquot of this buffer.

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

In the basal compartment, folate was supplemented with L-FTHF calcium up to about 200 nmol/L after 24 hours. Folate cream 29 nmol/L was quantified after 24 h supplementation.

Methylfolate was measured up to 120 μmol/L (24 hour L-MTHF). 47 nmol/L of folate cream was determined after 24 hours of supplementation.

Formyl folate was measured up to 150 μmol/L (24 hour L-FTHF calcium). At this point, 30.21 μmol/L of folic acid cream was measured.

In these scratch evaluation analyses, supplementation with various folate formulations significantly accelerated repair of artificial wounds after 24 hours. This study was performed on monolayered lawns of primitive epithelial cells in comparison with untreated controls.

The physiological availability of methylfolate or formylfolate from formulations could be demonstrated after supplementation in a reconstructed skin model. Topically provided folate derivatives are transported through the tissues of the model and partially metabolized by the cells. Some of the topically provided formylfolate is metabolized to methylfolate and the other is transported through tissues. Conversely, topically provided methyl formate is transported through the tissues of the skin model as such and is not metabolized to formylfolate. The uptake effect, described as transport, is 2 times better from folate cream than from pure folate solution (L-FTHF calcium) for formyl folate. Folic acid was also quantified in the basal compartment of the skin model. However, the concentrations measured were much lower than those for methylfolate and formylfolate. Thus, the measured folic acid probably originates from the medium used for the growth of the skin model and is released again by the cells during the course of the experiment.

Further experiments examined whether calcium levoleucovorin could permeate the skin. Penetration depth was quantified by Raman spectroscopy. The instrumentation was an Inverse Raman Spectrometer 3510 equipped with a 60x oil immersion lens and the evaluation software SkinTools from RiverD Rotterdam. The measurement method was 10 lateral views per application, measurement time of 5 seconds per measurement point, 7 steps of 2 μm, 4 steps of 4 μm, spectral range from 400 to 1800 cm −1 , laser excitation wavelength of 785 nm. Signals were generated with the software SkinTools with spectra (frequency ranges) specific to various skins and devices. Users can add their own spectra to the library. The evaluation is done via the CLP method (least squares method). The signal is output as fit coefficients normalized to that of keratin.

To quantify the results, they must be calibrated against standard columns (see Tab 1 and Tab 2). First, the bovine serum albumin (BSA) sensitivity is quantified and the mass concentration of BSA in g/ml is plotted to the BSA fit coefficient. 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 active ingredient slope SA. For the slope, the so-called drug quantification factor can be increased (SA/S=C). Multiplied by a factor C, the fit coefficient for the active substance is the quantitative value of keratin in mmol/g.

A standard example for measuring the sensitivity of an analytical system to keratin is as follows.

Tab 1

The standard sequence for determining the quantification factor for calcium levoleucovorin is:

Tab 2

Application of drug or placebo (sodium gluconate) to the skin of subjects 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 time the allergy patch was removed. Prior to determining the Raman profile, the treated skin area was rubbed with a dry paper towel.

The measured fit coefficients fi and quantification factors Ci are listed in the following table.

The slope S and slope SA for BSA (bovine serum albumin) and the quantification coefficients Ci for active substances are:

Figure 23 shows the profile of calcium levoleucovorin with application times of 30 to 240 minutes. Calcium levoleucovorin concentrations tend to increase in the upper 5 μm and deeper layers of the skin. However, the standard deviation of the individual profiles--not shown here--is large.

The table below shows the measurements of the amount of active that penetrated the skin per area. Profiles were evaluated from the skin surface to a depth of 30 μm. Calcium levoleucovorin concentration tends to increase over time. Compared to the amount applied, the percentage of active ingredient in the skin is 10% for 240 hours of application.

The folate-containing cream described in Example 1 was used to treat 5 patients each suffering from a different skin problem, namely neurodermatitis and psoriasis. Over the course of 21 days of treatment, the symptoms were greatly reduced. One patient had folliculitis bald and had received conventional treatment for several years. Treatment with folate cream for 3 weeks resulted in almost complete regression of symptoms, leading to skin healing, as shown in FIG. There was no recurrence up to 9 months and the skin remained normal in appearance.

A child suffering from neurodermatitis was treated with a folate preparation according to the invention for 3 weeks. During this treatment the symptoms decreased and the skin healed completely. There was no recurrence after stopping treatment.

Claims (20)

A formulation containing at least one folate and used for treating inflammation and disease of epithelial tissue, characterized in that said formulation is a topical agent. A formulation containing at least one folate for use in treating epithelial tissue inflammation and epithelial tissue disease according to claim 1, characterized in that said tissue is the skin. . A formulation containing at least one folate used for treating skin inflammations and skin diseases according to claim 1 or 2, characterized in that said skin diseases are inflammatory skin diseases. The formulation. 4. A formulation containing at least one folate for use in treating skin inflammations and skin disorders according to any one of claims 1 to 3, wherein said skin disorder is psoriasis. The formulation, characterized in that: 4. A formulation containing at least one folate used for treating skin inflammations and skin disorders according to any one of claims 1 to 3, wherein said skin disorder is dermatitis or neurodermatitis. 4. A formulation containing at least one folate used for treating skin inflammations and skin disorders according to any one of claims 1 to 3, wherein said skin disorders are chronic wounds. The formulation, characterized in that A folate preparation, said folate preparation comprising:
- a physiologically effective amount of at least one folate salt, wherein the cations of said folate salt are arginine, choline, acetylcholine, 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and the folate salt selected from the group consisting of and dimethylaminoethanol;
- a conventional composite for forming a supporting matrix;
The folate formulation comprising 8. The folate formulation of claim 7, wherein the folate formulation comprises
- a physiologically effective amount of at least one folate salt , wherein the anion of said folate salt is 5-formyl-(6S)-tetrahydrofolic acid, 5-formyl-(6RS)-tetrahydrofolic acid , 10-formyl-(6R)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5-methyl-(6RS)-tetrahydrofolic acid, (6S)-tetrahydrofolic acid, 5 , 10-methylene-(6R) -tetrahydrofolic acid , 5,10-methenyl-(6R)-tetrahydrofolic acid , 5,10-diformyl-(6S)-tetrahydrofolic acid, 5-methyl-10-formyl-(6S)-tetrahydrofolic acid said folate salt ;
- a conventional composite for forming a supporting matrix;
The folate formulation comprising 9. The folate formulation according to claim 7 or 8, wherein said folate formulation comprises folate in a microemulsion, oil-in-water emulsion, water-in-oil emulsion, nanoparticle formulation and/or spray formulation. The folate formulation, characterized in that it contains a folate compound concentration of 0.01 % to 2.5% by weight of the total weight of the formulation. 10. A folate formulation according to any one of claims 7-9, said formulation having a well-mixed phase of phase A, phase B, phase C and optionally phase D,
- The A phase contains oil,
- The B layer contains glycerol and an emulsifier,
- said phase C contains a physiologically effective amount of at least one folate;
- the optional D phase contains a matting agent ;
said folate preparation. 11. The folate formulation of claim 10, wherein said Phase C of said formulation comprises a physiologically effective amount of at least one folate salt , wherein the cations of said folate salt are arginine, The folate preparation, characterized in that it is selected from the group consisting of choline, acetylcholine, 1,1-dimethylbiguanidine, phenylethylbiguanidine, glucosamine and dimethylethanol. 12. A folate formulation according to claim 10 or 11, wherein said Phase C of said formulation comprises a physiologically effective amount of at least one folate salt , the anion of said folate salt being 5-formyl-(6S)-tetrahydrofolic acid, 5-formyl-(6RS)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5-methyl-(6RS) )-tetrahydrofolic acid, (6S)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid , 5,10-diformyl-(6S)-tetrahydrofolic acid , 5-methyl-10-formyl-(6S)- The folate preparation, characterized in that it is selected from the group consisting of tetrahydrofolic acid. 13. A folate formulation according to any one of claims 7-12, wherein said formulation consists of arginine, arginine esters, choline, acetylcholine, glucosamine, dimethylaminoethanol, B vitamins and vitamin D. Said folate preparation, characterized in that it contains at least one additional compound selected from the group. 14. The folate formulation according to any one of claims 7 to 13, wherein the phase C comprises 0.1 to 1000 mg of arginine, choline, acetylcholine , 1,1- per ml of polar solution. Said folate preparation, characterized in that it contains dimethylpiguanidine, phenylethylbiguanidine, glucosamine and dimethylaminoethanol, the calcium, magnesium, sodium or zinc salts of said folate. 15. A folate formulation according to any one of claims 7 to 14, wherein said formulation is selected from the group consisting of sodium gluconate, potassium gluconate , disodium glycerophosphate and dipotassium glycerophosphate. The folate preparation, characterized in that it contains at least one compound of 16. The folate formulation of any one of claims _7-15 , wherein the oil of phase A is a medium chain triglycerol , the fatty acids of which have chain lengths of _C6 to C12. optionally, a dicarboxylic alcohol , wherein the dicarboxylic acid has a chain length of _C2 to _C10 , wherein the alcohol is methyl alcohol , ethyl alcohol , isopropyl alcohol , propyl alcohol , butyl alcohol , pentyl alcohol . 17. The folate formulation according to claim 16, characterized in that said oil is triglycerol caprylate or triglycerol caprate. 18. The folate formulation according to any one of claims 7 to 17, characterized in that the emulsifier of phase B has an HBL value of 5 or greater. 19. The folate formulation according to any one of claims 7 to 18, wherein the emulsifier is a sucrose fatty acid ester with saturated fatty acids of chain length _in the range of C14 to _C20 , preferably The above folate formulation, characterized in that it is sucrose stearate and all sucrose fatty acid esters have an HBL value of 5 or more. When using the gluconate or glycerophosphate in the folate preparation according to any one of claims 7 to 19 for the treatment of skin inflammations and skin diseases, the gluconate The use of said gluconate or said glycerophosphate salt , wherein said acid salt or said glycerophosphate salt acts as a penetration enhancer of said folate salt into said skin.