Lipophilic active modulators in the form of dimethyl sulfone as transdermal penetration enhancers for active substances, compositions comprising the active modulators and methods for their preparation

Abstract

The object of this invention is a lipophilic activity modifier in the form of dimethyl sulfone (MSM) for use as a transdermal penetration enhancer for high molecular weight active substances with a molecular weight (Mw) > 500 Da. Another object of this invention is a cosmetic composition for transdermal application comprising an active substance and a transdermal penetration enhancer, characterized in that the active substance is encapsulated by the transdermal penetration enhancer, which is the lipophilic activity modifier according to claim 1, and the active substance is a high molecular weight active substance with a molecular weight (Mw) > 500 Da. Another object of the present invention is a method for preparing a composition according to the invention, characterized in that the method is carried out by thermal emulsification or cold emulsification, wherein thermal emulsification comprises the following steps: a) mixing dimethyl sulfone and a diluent in a reaction vessel; b) bringing the mixture to 70-85°C; c) stirring the mixture at 70-85°C; the start of homogenization depends on the stage of phase combination; d) cooling the mixture to 20-40°C, preferably 30°C; e) adding a high molecular weight active substance with Mw>500 Da and homogenizing the mixture, preferably for 1-3 min; while cold emulsification comprises the following steps: a) mixing dimethyl sulfone and a diluent in a reaction vessel and dissolving at a temperature maintained at 20-25°C; b) bringing the mixture to a temperature of 15-35°C, preferably ambient temperature or 25°C; c) stirring the mixture at a temperature of 15-80°C, preferably 25°C; the start of homogenization depends on the stage of phase combination; d) adding a high molecular weight active substance with Mw>500 Da and homogenizing the mixture, preferably for 1-3 min.

Description

[0001] The object of the present invention is a lipophilic activity modifier in the form of dimethyl sulfone (also known as methanesulfonylmethane, DMSO2, CAS No. 67-71-0) used as a transdermal permeation enhancer of an active substance, a composition comprising the activity modifier, and a method thereof for preparation thereof.

[0002] Cone snail venom consists of small peptides derived from the venom of the cone snail (Cone snail), which has evolved to capture prey and defend against predators. They represent a highly diverse class of bioactive molecules with varying structures and functions, exhibiting high selectivity for different ligands and voltage-gated ion channel subtypes. For example, venom from the cone snail *Cone shriveledus* (*Cone snail*)... Conus consors The μ-CnIIIC conospirin exhibits muscle relaxant activity by specifically blocking the Nav (Nav1.4) channel in skeletal muscle. This property can be used in cosmetics for daily anti-wrinkle treatments. Green BR, Bulaj G., Norton RS (2014). Structure and function of μ-conotoxins, peptide-based sodium channel blockers with anglesis activity. Future Medicinal Chemistry 6(15); 1677-1698. doi:10.4155 / FMC.14.107 Markgraf R., Leipold E., et al. (2012). Mechanism and molecular basis of the sodium channel subtype specificity of μ-conopeptide CnIIIC. British Journal of Pharmacology 167; 576-586. doi:10.1111 / j.1476- 5381.2012.02004.x ).

[0003] Cosmetics applied through the skin are a major category of anti-aging agents. Transdermal application of substances to produce systemic effects has many important advantages, including: avoiding the first-pass effect (hepatic metabolism); eliminating the potential breakdown of the therapeutic substance in the gastrointestinal tract; eliminating adverse effects of the substance on the gastrointestinal tract; eliminating interactions between the therapeutic substance and food and other orally administered medications; allowing therapeutic effects to be achieved after absorption of a lower dose; and allowing for a reduction in the frequency of administration of drugs with shorter biological half-lives (applicable to transdermal therapeutic systems), which is particularly important in the treatment of chronic diseases (applicable to transdermal therapeutic systems). Cal K, Stefanowska J. Methods to increase the permeation of therapeutic substances through the skin. Farm Pol, 2010, 66 (7): 514-520 ).

[0004] The epidermis, particularly the outer stratum corneum (SC), protects the skin from moisture loss and provides an airtight barrier for the penetration of other molecules, including active substances and irritants. It is known that the higher the number and molecular weight of polar groups in an active molecule, the greater the increase in hydrophilicity. This relationship can be approximated by the logP partition coefficient (or clogP, if this parameter has been theoretically determined). Active molecules with logP < 0 are considered to exhibit hydrophilicity, which hinders their solubility in nonpolar hydrophobic organic solvents. The epidermis exhibits hydrophobicity, which impedes the penetration of its hydrophilic active molecules.

[0005] It is estimated that only 1-2% of active substances applied to the skin can penetrate to the deeper layers, where they exhibit biological activity. This is due to the dense structure of the saturated fatty acid (SC), which resembles a brick wall with hydrophobic regions composed of lipids and hydrophilic regions composed of water and hydrated keratinocytes. To date, numerous methods and techniques have been developed to improve the permeability of active substances across the epidermis. These include specialized application systems (e.g., micropuncture), encapsulating active substances in lipophilic membranes (e.g., microvesicles), or using low-molecular-weight organic compounds, so-called transdermal permeability enhancers. The mechanism of action of transdermal permeability enhancers is not fully understood; some hypothesize the formation of specific channels that temporarily promote the passage of active substances through the SC. However, in the case of saturated fatty acid-based enhancers, it is hypothesized that they temporarily increase the permeability of SC lipids.

[0006] In commercially available cosmetic formulations, transdermal penetration enhancers are often oils, such as cholesterol succinate esters, or commercially available mixtures like Roelmi's Olifeel Skin or Innova's GPS-P and GPS-M. The principle behind these enhancers is to increase fluidity, i.e., the loosening of the skin layer. They bind to fatty residues on the epidermis, a passive rather than a biologically active action. Like dissolves like, so fat enhancers bind to fatty areas on the skin. This makes it more fluid and allows for greater passive diffusion. In contrast, these enhancers act only on fatty compounds. Other examples of transdermal penetration enhancers include, for example, alcohols (ethanol), glycols (propylene glycol), unsaturated fatty acids (oleic acid), terpenes (menthol), azones, sulfoxides (dimethyl sulfoxide), and surfactants (…). Cal K, Stefanowska J. Methods to increase the permeation of medicinal substances through the skin. Farm Pol, 2010, 66(7): 514-520 ).

[0007] The purpose of this invention is to provide a new and effective transdermal penetration enhancer for both water-soluble and fat-soluble active substances.

[0008] One element of the present invention is a lipophilic activity modifier in the form of dimethyl sulfone (MSM), used as a molecular weight (M... w Transdermal penetration enhancer of high molecular weight active substances with a concentration of >500 g / mol (Da, Daltons).

[0009] Advantageously, the active substance is selected from the group including peptides.

[0010] Advantageously, the active substance is a conotoxin having amino acid sequence No. 2 or No. 4.

[0011] Another element of the invention is a cosmetic composition for transdermal application, comprising an active substance and a transdermal penetration enhancer, characterized in that the active substance is encapsulated by the transdermal penetration enhancer, which is a lipophilic activity modifier according to the invention, and the active substance is M. wCompounds or mixtures of organic compounds with a value >500 Da.

[0012] Advantageously, the composition according to the invention comprises at least one cosmetically acceptable excipient, advantageously selected from the group consisting of emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

[0013] Advantageously, the compositions according to the invention are oil-in-water (o / w) or water-in-oil (w / o) emulsions.

[0014] Advantageously, the ratio of active substance to transdermal penetration enhancer is 1:1 to 1:50.

[0015] Advantageously, the composition contains at least one cosmetically acceptable excipient.

[0016] Advantageously, the active substance is cone snail toxin, and more particularly, µ-conotoxin.

[0017] Advantageously, µ-conotoxin is a conotoxin with the amino acid sequence Seq. no. 2.

[0018] Advantageously, µ-conotoxin is a conotoxin or its genetically modified form having an amino acid sequence No. 4.

[0019] Advantageously, the compositions according to the invention contain at least one additional active ingredient.

[0020] Another embodiment of the present invention is a method for preparing the composition according to the present invention, characterized in that the method is carried out by hot emulsification or cold emulsification, wherein hot emulsification includes the following steps: a) Mix dimethyl sulfone and diluent in a reaction vessel; b) Bring the mixture to 70-85°C; c) Stir the mixture at 70-85°C; the initiation of homogenization depends on the stage of phase combination; d) Cool the mixture to 20-40°C, preferably 30°C; e) Add a high molecular weight active substance with Mw>500Da and homogenize the mixture, preferably for 1-3 minutes; Cold emulsification involves the following steps: a) Mix dimethyl sulfone with the diluent in a reaction vessel and dissolve at 20-25°C. b) Bring the mixture to a temperature of 15-35°C, preferably to ambient temperature or 25°C; c) The mixture is mixed at a temperature of 15-80°C, preferably 25°C; the initiation of homogenization depends on the stage of phase combination.

[0021] d) Add a high molecular weight active substance with Mw>500Da and homogenize the mixture, preferably for 1-3 min.

[0022] Advantageously, in at least one step of the thermal emulsification step (ae), at least one additional active ingredient and / or at least one cosmetically acceptable excipient are added to the reaction vessel, said excipient being advantageously selected from the group consisting of: emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives or combinations thereof.

[0023] Advantageously, in at least one step of the cold emulsification step (ad), at least one additional active ingredient and / or at least one cosmetically acceptable excipient are added to the reaction vessel, said excipient being advantageously selected from the group consisting of: emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

[0024] This invention provides the following benefits: Lipophilic activity modifiers in the form of dimethyl sulfone (MSM, CAS No. 67-71-0) act as transdermal permeability enhancers, exhibiting dual hydrophilic or hydrophobic properties by generating unique biocompatible nanoshells stabilized by strong hydrogen and non-covalent bonds. This improves the permeability of high molecular weight active substances (Mw>500Da), particularly hydrophilic peptides, through the stratum corneum (SC). Compared to techniques typically used to encapsulate active substances in lipid membranes (microvesicles), the nanoshells are much smaller in size, depending on the mass and geometry of the active substance.

[0025] Dimethyl sulfone is attributed to its favorable physicochemical properties, such as low molecular weight (Mw=94.1 Da), high boiling point (T=238℃, does not evaporate from the skin surface), high water solubility (37g / 100g water), high biocompatibility (no known toxicity to organisms), and the ability to form strong and directional hydrogen interactions (through negatively polarized SO2 fragments) with cations, water molecules, and polar groups (-OH, -NH2, -P(=O)R) m. etc.) and to form non-covalent bonds with anions and lipophilic groups (through highly positively polarized methyl-CH3).

[0026] Due to their dual nature, dimethyl sulfone nanoshells containing active substances can be either hydrophilic (SO2 groups on the outside) or lipophilic (methyl groups on the outside). This allows for optimal and efficient transport of active substances through both the hydrophobic and hydrophilic regions of the SC. Furthermore, dimethyl sulfone molecules have a high affinity for water molecules, allowing some to bind to intercellular water, which further facilitates the transport of active substances through the SC.

[0027] After fulfilling its purpose, the dimethyl sulfone molecule is further absorbed into the body through the skin and safely excreted unchanged from the body. Furthermore, recent studies have shown that dimethyl sulfone exhibits antibacterial and anti-inflammatory properties on the skin. These characteristics, combined with current technologies for transdermal delivery systems of active ingredients (including drugs), make it highly desirable for therapeutic and (dermal) cosmetic applications.

[0028] The invention has been described in exemplary embodiments and accompanying drawings, wherein: Figure 1 The image shows the electrophoretic separation of *E. coli* S4B protein transformed with a plasmid containing the TRX+TIIIA gene on a 15% SDS-PAGE polyacrylamide gel. 1 is a Full-Range Rainbow Amersham standard: 225, 150, 102, 76, 52, 38, 31, 24, 17, 12 kDa; 2 is *E. coli* S4B lysate, *E. coli* S4B / pDM / TRX+TIIIA, with a mass of approximately 15 kDa. kDa, from a culture at 25°C; 3 is bacterial precipitate from a culture at 25°C after sonication; 4 is supernatant from a culture at 25°C after sonication; 5 is bacterial lysate of *E. coli* S4B / pDM / TRX+TIIIA from a culture at 24°C; 6 is bacterial precipitate from a culture at 24°C after sonication; 7 is supernatant from a culture at 24°C after sonication; 8 is bacterial lysate of *E. coli* S4B / pDM / TRX+TIIIA from a culture at 30°C; 9 is bacterial precipitate from a culture at 30°C after sonication; 10 is supernatant from a culture at 30°C after sonication. Figure 2The image shows the electrophoretic separation of *E. coli* S4B protein transformed with a plasmid containing the TRX+TIIIA gene on a 15% SDS-PAGE polyacrylamide gel. 1 is Amersham full-range rainbow molecular weight standards: 225, 150, 102, 76, 52, 38, 31, 24, 17, 12 kDa; 2 is *E. coli* S4B lysate, *E. coli* S4B / pDM / TRX+TIIIA, with a mass of approximately 15 kDa. kDa, from a culture at 37°C; 3 is bacterial precipitate from a culture at 37°C after sonication; 4 is supernatant from a culture at 37°C after sonication; 5 is lysate of *E. coli* S4B / pDM / TRX+TIIIA from a culture at 30°C; 6 is bacterial precipitate from a culture at 30°C after sonication; 7 is supernatant from a culture at 30°C after sonication; 8 is bacterial lysate of *E. coli* S4B / pDM / TRX+TIIIA from a culture at 18°C; 9 is bacterial precipitate from a culture at 18°C ​​after sonication; 10 is supernatant from a culture at 18°C ​​after sonication. Figure 3 The chromatogram of Escherichia coli transformed as in Example 1 is shown; Figure 4 The results of electrophoretic separation of E. coli proteins transformed with a plasmid containing the TRX+TIIIA gene in 15% SDS-PAGE after culturing at 30°C are shown. 1 is the E. coli S4B / pDM / TRX+TIIIA bacterial lysate with a mass of approximately 14.4 kDa; 2 is the supernatant after sonication; 3 is bacterial protein not captured on the Ni-NTA bed; 4 is the protein eluted with elution buffer; 5 is Amersham full-range rainbow molecular weight standards: 225, 150, 102, 76, 52, 38, 31, 24, 17, 12 kDa; and 6 is the sample 48 hours after dialysis (TRX::TIIIA fusion protein). Figure 5The image shows the electrophoretic separation of the fusion protein TRX::TIIIA after BrCN digestion on a 15% SDS-PAGE polyacrylamide gel. In the image, 1 represents undigested TRX+TIIA; 2 represents digestion with 0.1M HCl at +4°C, BrCN 100:1 for 24 hours; 3 represents digestion with 0.3M HCl at +4°C, BrCN 100:1 for 24 hours; 4 represents Amersham full-range rainbow molecular weight standards: 225, 150, 102, 76, 52, 38, 31, 24, 17, 12 kDa; 5 represents digestion with 0.1M HCl at +24°C, BrCN 100:1 for 3 hours; 6 represents digestion with 0.1M HCl at +24°C, BrCN 100:1 for 6 hours; 7 represents digestion with 0.1M HCl at +24°C, BrCN 100:1 for 24 hours; and 8 represents digestion with 0.3M HCl at +24°C. After digestion with 0.3M HCl at BrCN 100:1 for 3 hours, 9 indicates acid etching with 0.3M HCl at +24℃ for 6 hours at BrCN 100:1, and 10 indicates acid etching with 0.3M HCl at +24℃ for 24 hours at BrCN 100:1. Figure 6 The spectrum of synthetic cone snail toxin TIIIA without disulfide bonds is shown; Figure 7 The spectrum of the reaction mixture rich in synthetic fully reduced peptide (C) is shown, where peak A represents the signal of low-mass organic compound, peak B represents TIIIA with disulfide bonds, peak C represents synthetic doped TIIIA without disulfide bonds, and peak D represents the 12.85 kDa carrier protein and the 15.395 kDa undigested construct. Figure 8 The RP-HPLC chromatograms of the preparation stage are shown, where the signal at 19.1 min corresponds to TIIIA, the signal from 21 to 50 min corresponds to some digestion of the target and / or purification of the HIS tag, the peak between 50 and 56 min is the 12.8 kDa carrier protein, and 57–63 min is the undigested construct. Figure 9 Show Figure 10 A portion of the chromatogram, specifically referencing an amplified retention time of 17–22 min, where the signal from 18.12 to 20.00 min corresponds to 3 mg TIIIA; Figure 10The results of patch-clamp measurements of conotoxin activity in an oocyte model expressing the human sodium channel Nav 1.4 are shown for conotoxin TIIIA (toxin A), conotoxin SIIIA (toxin B), conotoxin TIIIAlaMut (toxin C), CnIIIC standard (toxin D), and conotoxin TIIIA (toxin E) extracted from the composition according to the invention. The left column shows the response of a single Nav 1.4 ion channel as an example of the observed inhibitory effect, while the right column shows the effect of the test substance on the conductivity curve (as a function of voltage and channel accessibility), where voltage and channel accessibility are two important parameters commonly used to assess Nav channel function. Figure 11 Concentration-response curves for TIIIA toxin, TIIIAlaMut toxin, and a standard (commercially available CnIIIC toxin) are shown; the effects were measured on human NaV1.4 ion channels, and the data were fitted to dose-response curves (Hill equation, assuming a single binding site) using Graphpad Prism 8 with fixed parameters: top = 0, bottom = 99, Hill N = -1. Error bars represent SEM.

[0029] This invention is presented by way of working examples, wherein all tests and experimental procedures described below were performed using commercially available test kits, reagents, and equipment, following the manufacturer's recommendations, unless otherwise expressly stated. All test parameters were measured using standard, well-known methods used in the art to which this invention pertains.

[0030] However, the embodiments described below are for illustrative purposes only and should not be construed as limiting the scope of patent protection.

[0031] Example 1 Design an E. coli expression system with pDM / TRX+TIIIA construct. To obtain recombinant soluble cone toxin TIIIA, a genetic construct encoding a TRX::TIIIA fusion protein was designed. This fusion protein contains the TIIIA cone toxin gene and the gene encoding the leader protein thioredoxin (TRX). The TRX protein provides reducing conditions to promote the correct folding of proteins with disulfide bonds and often results in the recombinant protein being expressed in a soluble form, which significantly shortens the purification process. In this study, the nucleotide sequence of the thioredoxin TRX gene was modified using a point mutation reaction, thereby removing the methionine (M) amino acid at position 37 and replacing it with lysine (K). This modification aimed to obtain the correct protein fragment after cyanogen bromide (BrCN) digestion, with the cleavage site being methionine. The nucleotide sequence of the TRX::TIIIA fusion gene (Sequence No. 1) was optimized for bacterial codon usage. Therefore, the constructed construct is controlled by a constitutive promoter (e.g., deoP1P2), which allows for the continuous synthesis of the recombinant protein in bacterial cells without the need for induced gene expression.

[0032] To enable protein purification on a NiNTA bed column, a sequence encoding six histidine residues (6His) and a short linker spanning the amino acid serine-glycine-serine (SGS) is added to the 5' end of the construct. The TRX::TIIIA fusion protein gene is inserted into an expression vector, which in this embodiment is a pDM expression vector digested with NdeI / XbaI restriction endonucleases. However, other vectors such as pDMR can also be used.

[0033] The nucleotide sequence of the cloned gene was confirmed, and an Escherichia coli S4B production strain (a derivative of any one of Escherichia coli strains DH10B, JM109, HB101, CSH50R, or DH10B) containing the pDM / TRX+TIIA construct (a single molecule of TIIIA cone snail toxin fused with thioredoxin) was constructed to produce a soluble peptide.

[0034] The procedure was similarly performed on the construct encoding the TRX::TIIIAlaMut fusion protein with nucleotide sequence Seq. no. 2.

[0035] Example 2 µ-conotoxin TIIIA was produced in Escherichia coli using an expression vector containing the construct according to Example 1. The method includes the following steps: Step (a) E. coli cells were transformed with an expression vector containing the construct of Example 1. In this example embodiment, a prokaryotic pDM / TRX+TIIA expression vector containing a construct encoding the TRX:TIIIA fusion protein with nucleotide sequence No. 1, encoded by electroporation, was introduced into an *E. coli* S4B expression strain, wherein the TRX:TIIIA fusion protein has amino acid sequence No. 2. Given this embodiment, the construct is controlled by the constitutive promoter deoP1P2, which allows for the continuous synthesis of the recombinant protein in bacterial cells without the need for induced gene expression.

[0036] Step (b) TRX::TIIIA fusion protein was expressed in transformed E. coli strains. To verify that the obtained protein was in a soluble form and to select the culture conditions that resulted in the highest possible expression of the gene encoding the recombinant fusion protein TRX::TIIIA, single colonies of material from the transformed *E. coli* strain were inoculated into liquid LB medium supplemented with tetracycline (100 µg / ml) and cultured at different temperatures of 18°C, 25°C, 30°C, and 37°C until an optical density of OD600≈1.0 was reached. The results are as follows: Figure 1-2 As shown.

[0037] Analysis showed that the recombinant protein was obtained in a soluble form, and the gene encoding the TRX::TIIIA fusion protein was expressed at its highest temperature of 30°C.

[0038] Subsequently, to optimize the expression of TIIIA cone snail toxin, laboratory-scale transformed *E. coli* strains were cultured in LB medium (10 g / L bacterial tryptone, 5 g / L yeast extract, 10 g / L NaCl) supplemented with the selection marker tetracycline (100 µg / ml) at 30°C and 150 rpm for 18 hours until an OD value of 3.2 to 3.5 was achieved. An appropriate volume of culture material (stock solution) stored at -70°C was used to inoculate the culture (500 µL stock solution per 500 ml LB medium). Stock solutions were prepared using a 1:1 ratio of bacterial culture (OD600 ≈ 0.6) and 20% glycerol. The bacterial material was stored in the submitter's strain library.

[0039] Step (c) Separation and purification TRX::TIIIA fusion protein Eighteen hours later, the culture prepared in step (c) and incubated at 30°C was centrifuged (8000 rpm). 1 L of the centrifuged biomass was resuspended in 50 ml of prepared rehydration buffer (50 mM TRIS HCl pH 7.8; 300 mM NaCl), sonicated for 1 hour and 15 min, and centrifuged twice at 12,000 rpm for 15 min each time. The supernatant was loaded onto a column pre-equilibrated with calibration buffer. The bed was then washed with wash buffer, and recombinant TRX::TIIIA protein was eluted with elution buffer. The flow rate for loading the sample was 1.0 ml / min, the column was washed at 1.5 ml / min, and elution was performed at 2.0 ml / min.

[0040] The following buffers are used for protein purification: ● Calibration buffer o 50mM phosphate buffer pH-7.8, 500mM NaCl, 10mM imidazole; ●Washing buffer o 50mM phosphate buffer pH-7.8, 500mM NaCl, 20mM imidazole; ● Elution buffer o 50mM phosphate buffer pH-7.8, 500mM NaCl, 150mM imidazole; Fractions were collected in 5 ml increments. Separation was performed using columns from the same company on a Bio-Rad Duo Flow System. The concentration of recombinant TRX::TIIIA protein was determined using the Bradford method: 10 µL sample, 990 µL 20 mM Tris-HCl buffer (pH 7.6), and 1 ml Bradford reagent. Absorbance was read at 595 nm on a spectrophotometer. Figure 3 The results of LPLC chromatography separation of 1 L of bacterial culture expressing TRX::TIIIA protein are shown on a Ni-NTASuperFlow affinity bed.

[0041] Step (d) The purified TRX::TIIIA fusion protein was treated with glutathione GSH / GSSG and then in buffer. Dialysis for disulfide bond folding Following chromatographic separation, approximately 30–35 ml of eluent with a protein concentration of approximately 1.0 mg / ml was obtained from 1 L of culture. After elution, up to 4 mM of reduced glutathione (GSH) and up to 1 mM of oxidized glutathione (GSSG) were added to the collected fraction to obtain proper disulfide bond assembly. The resulting sample was dialyzed in buffer (50 mM TRIS-HCl buffer, pH 7.8, 10% glycerol). Dialysis was performed at 4°C for 48 hours, with the buffer replaced after 24 hours.

[0042] The composition of the fractions obtained was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the recombinant TRX::TIIIA protein obtained from the prokaryotic system was analyzed by protein electrophoresis under SDS-PAGE denaturing conditions. The results are as follows: Figure 4 As shown.

[0043] Step (e) The TRX::TIIIA fusion protein containing complex disulfide bonds was digested with cyanide bromide. Cyanogen bromide (BrCN) digests proteins in an acidic environment. The digestion reaction is highly specific; BrCN reacts with the sulfur in the side chain of the amino acid methionine (Met), causing the peptide bond on the carboxyl group of methionine to break. Methionine is one of the rarest amino acids in proteins.

[0044] To digest the recombinant cone snail toxin from the leader protein thioredoxin, it was decided to perform digestion in an acidic medium with a 100:1 molar excess of BrCN (100 moles of BrCN per Met residue). The nucleotide sequence of the TRX:TIIIA fusion protein contains two methionine residues, followed by BrCN cleavage.

[0045] Calculate the amount of BrCN required for the reaction per methionine residue based on the molar mass of the fusion protein: Fusion protein: TRX::TIIIA Molar mass: 15403.67 g / mol BrCN excess relative to methionine residues: 100:1 For 1 mg of fusion protein, 1.375 mg of BrCN should be taken. This amount is contained in 2.60 μL of a solution of 5 M BrCN in CH3CN (d=1.093).

[0046] The 1.0 mg / ml TRX::TIIIA fusion protein, obtained after chromatographic separation and dialysis for 48 hours, was digested with BrCN containing different concentrations of HCl at +4°C and room temperature under light-protected stirring. Samples digested at room temperature were collected at 3, 6, and 24 hours to determine the most efficient digestion time.

[0047] The digested sample was transferred for LC-MS (liquid chromatography-mass spectrometry).

[0048] In a 15% SDS-PAGE polyacrylamide gel, the 1TRX::TIIIA fusion protein was separated by electrophoresis after BrCN digestion. Figure 5 As shown, in acidic medium with a BrCN molar excess of 100:1, the optimal digestion conditions for the TRX::TIIIA fusion protein are 0.1 M HCl, stirred at room temperature in the dark for 3 hours.

[0049] Step (f) Purification of digested TIIIA peptide In this example embodiment, the digested TIIIA peptide was purified by RP-HPLC. Following BrCN digestion, two main molecules were obtained from the TRX::TIIIA fusion protein with a molecular weight (MW) of 15395 Da: ● A TIIIA peptide with a molecular weight of 2426.836 Da; ● A TRX lead protein with a molecular weight of 12,855 Da.

[0050] The proportion of other non-specific products is variable.

[0051] To test the properties of a fully reduced peptide without disulfide bonds, a linear peptide (SEQ ID NO:5) identical in sequence to conotoxin TIIIA was synthesized. This linear peptide is more hydrophobic than peptides containing disulfide bonds and exhibits a later retention time compared to correctly folded peptides, such as... Figure 6-7 As shown.

[0052] for Figure 6-7 The quality control (QC) shown uses an analytical / semi-preparative HPLC kit (Waters).

[0053] Separation parameters: —C18 column, 250 x 10 mm, 100 Å, 5 µm —Buffer A - 0.1% TFA in water —Buffer B, 90% MeCN, 0.1% TFA, water —MeCN gradient 5-100% B, —Flow rate 2 ml / min, 5-100 buffer B, within 55 minutes.

[0054] For the purification process of TIIIA peptide, such as Figure 10-11 As shown, a preparative HPLC system (Knauer) was used.

[0055] Separation parameters: ●C18 column, 250 x 21.2 mm, 100 Å, 5 µm —Buffer A - 0.1% TFA in water —Buffer B, 90% MeCN, 0.1% TFA, water ●MeCN gradient 5-100% B, ● Flow rate 20 ml / min, buffer B 5-100, within 70 minutes.

[0056] One of the following two programs can be used for cleaning: Program I The BrCN digestion mixture was aliquoted into 25 ml aliquots and placed in 50 ml Erlenmeyer flasks. The aliquots were then frozen in liquid nitrogen and lyophilized at -80 °C and -3 mBar. The lyophilization time varied from 10 to 30 hours depending on the total volume of solvent to be removed. The dried residue after evaporation of the 25 ml reaction mixture was dissolved in 2 ml of 0.1% TFA and 5% MeCN and centrifuged at 15 kG for 20 min at room temperature. The sample was injected into HPLC, and semi-preparative separation was performed using a 2 ml loop, followed by preparative separation using a 5 ml loop. The sample was separated in a MeCN AB gradient from 5 to 100% in the presence of 0.1% TFA. The fraction containing TIIIA was collected, frozen in liquid nitrogen, and lyophilized. The dried sample was weighed.

[0057] Due to the large amount of material applied, separation on the preparation system resulted in the detection of more distinct signals. However, this did not affect the correct separation of TIIIA cone snail toxins.

[0058] Due to differences in column size and gradient length, undigested constructs and carrier proteins separate. Properly scaling up this method to larger scales (>100 mg) requires the introduction of additional preparation steps.

[0059] Program II Because the weight ratio of TIIIA peptide (2.4 kDa) to total TRX::TIIIA construct (15.3 kDa) and the second TRX product (12.8 kDa) obtained after BrCN digestion was unfavorable (1:6), and due to the limited volume of the HPLC column, it was advantageous to remove undigested protein and the second product before actual HPLC separation. A 10g column cartridge with a C18 bed, designed for FPLC chromatography, was used to bind the peptides and proteins present in the mixture after digestion. Salts and other small molecules were removed in the washing step with 2% MeCN and 0.1% TFA. The crude fraction containing TIIIA was eluted with 30% MeCN and 0.1% TFA, frozen and lyophilized in liquid nitrogen, and then subjected to the HPLC protocol for final purification. The C18 column was washed with 90% MeCN and 0.1% TFA, followed by washing with 80% MeOH and 0.1%. The presence of TIIIA peptide was checked in each fraction, and the purification procedure was repeated. Results are as follows. Figure 8-9 As shown.

[0060] The procedure was similarly performed on a vector containing the construct, which has nucleotide sequence No. 3 encoding the TRX::TIIIAlaMut fusion protein and amino acid sequence No. 4 encoding the fusion protein.

[0061] Example 3 Comparison of the effects of dimethyl sulfone (MSM) with known transdermal penetration enhancers The effectiveness of dimethyl sulfone (MSM) as a transdermal permeation enhancer for the µ-conotoxin (hereinafter referred to as the active molecule) according to Example 1 was investigated. The following transdermal permeation enhancers known in the prior art were selected for comparative analysis: ●Isosorbide dimethyl ether - Transdermal penetration enhancer A; ●Urea-transdermal penetration enhancer B; ●Salicylic acid - transdermal penetration enhancer C.

[0062] result: Isosorbide dimethyl ether - Transdermal Transmission Enhancer A Mechanism of action result Overview suggestion Urea-transdermal penetration enhancer B Mechanism of action: Urea acts as a promoter by forming strong hydrogen bonds. The NH2 group of urea forms hydrogen bonds only with -OH and >C=O groups from carboxylic acids or amides, for example. The formation of these bonds makes the entire urea molecule highly polar and non-lipophilic.

[0063] result Overview suggestion Salicylic acid - transition promoter C Mechanism of action: It works by exfoliating the skin (peeling increases skin permeability – exfoliation increases diffusion). This effect can only be achieved with large amounts of use, which can lead to over-exfoliation and irritation, making it unsuitable for our purposes.

[0064] result Overview suggestion The dimethyl sulfone (MSM) transdermal permeability enhancer according to the present invention Mechanism of action: MSM has two S=O groups that allow hydrogen bonding and two methyl groups (CH3-) that can form non-covalent bonds. This hydrogen bonding between the S=O groups and corresponding groups in the active molecule creates a highly polarized state on the outside, but forms a lipophilic region (i.e., the CH3 group), which facilitates the absorption of the encapsulated active molecule through the first layer of skin (epidermis). The degree of lipophilic and hydrophilic regions in the skin varies depending on the skin layer and location. The promoter can form a bond once via S=O (in this case, we have a lipophilic group on the outside), but when there is a hydrophilic region on the skin, it can also form a non-covalent bond via the CH3 group (in this case, we have a polar S=O group on the outside). Therefore, MSM acts as a lipophilic activity modifier depending on the different lipophilic / hydrophilic conditions in the skin layer it penetrates.

[0065] result Overview suggestion Example 4 In an embodiment of this invention, the composition according to the invention is a white, thick composition with a pH of 4.65±0.02 (measured at 25.0±0.2°C) and can be formulated into a cream.

[0066] The qualitative and quantitative composition of the composition is shown in Table 1 below.

[0067] Table 1. Examples of the qualitative and quantitative composition of the compositions according to the present invention, wherein the compositions according to the present invention are in the form of a cream containing the transdermal penetration enhancer according to the present invention.

[0068]

[0069] Example 5 In this embodiment, the composition according to the invention is in cream form, and the qualitative and quantitative composition of the composition is shown in Table 2 below.

[0070] Table 2 provides examples of the qualitative and quantitative composition of compositions according to the present invention, which are in cream form containing transdermal penetration enhancers according to the present invention.

[0071]

[0072] The compositions shown in Table 2 were prepared using the method according to the present invention, which includes the following steps: Add phase IA to the main mixer. Turn on the heat to 75-80°C and stir at -25 rpm. Homogenize for 1 min at 1800 rpm.

[0073] 2. Prepare phase IB in an auxiliary container. Mix thoroughly. Add to the main mixer. Mix at -25 rpm. Homogenize for 1 min at 1800 rpm.

[0074] 3. Weigh the components of Phase II into a mixing bowl. Heat to 75-80°C. Stir until homogeneous and dissolved.

[0075] 4. Once both phases reach a temperature of 75-80℃, add phase II to the main mixer. Mix at 35 rpm during the mixing process. Homogenize at 2500 rpm for 6 minutes. Stir at 20 rpm for approximately 15-20 minutes.

[0076] 6. Begin cooling to 40°C (2°C / min) while continuously stirring (40 rpm), and activate a 0.5 bar vacuum. 7. Prepare phases III and VIII in an auxiliary vessel. If there are problems dissolving phase III F, heat to 40-45°C.

[0077] 8. Turn on the 35℃ cooling.

[0078] 9. Add phase III at 35°C. Mix at 35 rpm for about 5 minutes.

[0079] 10. Add phase IV at 35°C. Stir at 35 rpm for about 5 minutes.

[0080] 11. Add phase V at 35°C. Mix at 35 rpm for about 5 minutes.

[0081] 12. Add phase VI at 35°C. Mix at 35 rpm for about 5 minutes.

[0082] 13. Add phase VII at 35°C. Stir at 35 rpm for about 5 minutes.

[0083] 14. Add phase VIII at 35°C. Stir at 35 rpm for about 5 minutes.

[0084] 15. Add phase IX at 35°C. Stir at 35 rpm for about 5 minutes.

[0085] Measure the pH value before adding phase X. Gradually add phase X components to adjust the pH value (4.3-5.7).

[0086] 17. Homogenize for 2 minutes at 2000 rpm, stir at 30 rpm for 10 minutes.

[0087] Example 6 The activity of recombinant μ-conotoxin against oocytes was determined in vitro using the patch-clamp method. In this embodiment, the activity of the μ-conotoxin with amino acid sequence No. 2 and sequence No. 4 according to the present invention relative to known μ-conotoxins SIIIA and CnIIIC was determined. Specifically, whether the novel recombinant conotoxins TIIIA and TIIIAlaMut possess sodium current blocking properties similar to those of the commercially available conotoxin CnIIIC was investigated. Table 3 shows a summary of the tested samples.

[0088] Table 3. Summary of samples tested for activity using molecular weight and effective concentration.

[0089]

[0090] The recombinant cone snail toxin was determined in Xenopus laevis using the patch-clamp method. Xenopus The activity of the Nav1.4 ion channel expressed on oocytes was studied. The Xenopus oocyte ion channel expression system is ideal for voltage-dependent ion channel electrophysiological properties due to the low background of endogenous channels and the large size of the oocyte. The patch-clamp method is a standard procedure in electrophysiology. The concept involves measurements on very small cell membrane patches (so-called patches). Electrodes are placed inside thin glass pipettes with a tip diameter of approximately 1 µm. The micropipette is filled with a solution similar in composition to the extracellular fluid, as the measurement is performed outside the cell. During the experiment, the tip of the micropipette is in direct contact with the membrane surface, forming a stable contact both mechanically and electrically. Under these conditions, the measuring electrodes record the current flowing through the membrane portion bound to the pipette tip. The number of ion channels present on this patch is small enough to distinguish the current flowing through individual channels.

[0091] method: The first step involves introducing mRNA encoding the Nav1.4 ion channel into oocytes via microinjection. Oocytes for microinjection are prepared as follows: 5-15 ml of oocytes are collected from Xenopus ovaries and divided into small clusters (approximately 10-20 oocytes per cluster). The oocytes are then dissociated using an enzymatic solution (at room temperature, 30-90 min) until some oocytes rupture. The cells are then washed at least 5 times with ND96 solution to remove residual enzymes. Healthy oocytes in stages V and VI (diameter > 0.8 mm) are selected under a microscope. Microinjection is performed on the day of collection. Approximately 100 oocytes are used for each test. The selected oocytes are then transferred to ND96 solution and injected with approximately 50 ml of cRNA solution (using an automated oocyte injector). The cultures are then incubated at 18°C ​​for 2-7 days, depending on the desired ion channel expression level. Oocytes thus prepared with the Nav1.4 ion channel expressed thereon are used to measure ion channel conductivity.

[0092] The response was measured at room temperature for 1–6 days post-cRNA injection and recorded at -70 mV using a software amplifier. Nav1.4 ion channels were activated using 1 μM of recombinant conotoxins SIIIA, TIIIA, TIIIAlaMut, and the synthetic standard CnIIIC, respectively. Rapid and reproducible solution exchange (<300 ms) was achieved using a 50 μL funnel-shaped oocyte chamber, combined with a rapidly flowing solution provided by a collector mounted directly above the oocytes. Agonist pulses were applied for 2 seconds at 4-min intervals. Voltage-current curves obtained on the same cells before and after application of the selected compounds to the extracellular environment were then compared.

[0093] result: Analysis results as follows Figure 10-11 As shown in Table 4.

[0094] Table 4. 50% inhibition (IC50) of sodium ion current generated in human NaV1.4 channels 50 ) and 99% inhibition (IC) 99 ) toxin concentration.

[0095]

[0096] The study showed that recombinant cone snail toxins TIIIA, SIIIA, and TIIIAlaMut at a concentration of 1 µM reduced the sodium current amplitude of voltage-gated sodium channels in oocytes. The results confirmed the activity of all recombinant cone snail toxins. Figure 10-11 For recombinant cone snail toxins TIIIA and TIIIAlaMut, the IC50 value, i.e., the concentration required to block 50% of Nav1.4 ion channels, is 1 µM. Figure 11 The results show that the ion channel blocking level of TIIIAlaMut cone snail toxin is approximately three times that of TIIIA cone snail toxin.

[0097] The activity of TIIIA cone snail venom extracted from the final product was also confirmed. The results confirmed that the activity level of TIIIA cone snail venom extracted from the final product was higher than that of pure TIIIA cone snail venom. This may be related to the fact that TFA acid was added during the extraction process, and TFA acid residues were present in the extract. According to literature data, cone snail venom exhibits higher activity in acidic environments. From the results obtained, it can be concluded that the final formulation of the cosmetic did not reduce the biological activity of TIIIA cone snail venom.

[0098] Example 7 Conotoxin encapsulated with MSM transdermal penetration enhancer was confirmed to be non-toxic.

[0099] Sequence List: Serial Number: 1 Sequence type: nucleotide sequence Length: 423 Note: The nucleotide sequence of the TRX+TIIIA construct according to this invention. sequence: ATGCATCACCATCATCACCATTCTGGTTCTTCTGACAAAATCATCCACCTGACCGACGACTCTTTCGACACCGACGTTCTGAAAGCTGACGGTGCTATCCTGGTTGACTTCTGGGCTGAATGGTGCGGTCCGTGCAAAAAGATCGCTCCGATCCTGGACGAAATCGCTGACGAATACCAGGGTAAACTGACCGTTGCTAAACTGAACATCG ACCAAACCCGGGTACCGCTCCGAAATACGGTATCCGTGGTATCCCGACCCTGCTGCTGTTCAAAAACGGTGAAGTTGCTGCAACCAAAGTTGGTGCACTGTCTAAAGGTCAGCTGAAAGAATTCCTGGACGCTAACCTGGCTATGCGTCATGGCTGCTGCAAAGGCCCGAAAGGCTGCAGCAGCCGTGAATGCCGTCCGCAGCATTGCTGC Serial Number (Sequ.): 2 Sequence type: amino acid sequence Length: 141 Note: The amino acid sequence of the recombinant protein encoded by the TRX+TIIIA construct according to this invention. sequence: MHHHHHHSGSSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKKIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLFKNGEVAATKVGALSKGQLKEFLDANLAMRHGCCKGPKGCSSRECRPQHCC Serial No.: 3 Sequence type: nucleotide sequence Length: 423 Note: Nucleotide sequence of the construct according to the present invention in the TRX+TIIIaMut variant. sequence: ATGCATCACCATCATCACCATTCTGGTTCTTCTGACAAAATCATCCACCTGACCGACGACTCTTTCGACACCGACGTTCTGAAAGCTGACGGTGCTATCCTGGTTGACTTCTGGGCTGAATGGTGCGGTCCGTGCAAAAAGATCGCTCCGATCCTGGACGAAATCGCTGACGAATACCAGGGTAAACTGACCGTTGCTAAACTGAACATCG ACCAAACCCGGGTACCGCTCCGAAATACGGTATCCGTGGTATCCCGACCCTGCTGCTGTTCAAAAACGGTGAAGTTGCTGCAACCAAAGTTGGTGCACTGTCTAAAGGTCAGCTGAAAGAATTCCTGGACGCTAACCTGGCTATGCGTCATGGCTGCTGCAAAGGCCCGAAAGGCTGCAGCAGCCGTGctTGCCGTCCGCAGCATTGCTGC Serial No.: 4 Sequence type: amino acid sequence Length: 141 Note: The amino acid sequence of the recombinant protein encoded by the construct according to the present invention in the TRX+TIIIaMut variant. sequence: MHHHHHHSGSSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKKIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLLFKNGEVAATKVGALSKGQLKEFLDANLAMRHGCCKGPKGCSSRACRPQHCC Sequence no. (Sequ. no.):5 Sequence type: amino acid sequence Length: 22 Description: A linear peptide with the same sequence as conotoxin TIIIA. Sequence: RHGCCKGPKGCSSRECRPQHCC. Claims (as amended under Article 19 of the Treaty) 1. A lipophilic activity modifier in the form of dimethyl sulfone (MSM), used as a transdermal transdermal enhancer for high molecular weight active substances including conopeptides with Mw > 2000 Da. 2. The activity regulator according to claim 1, wherein the active substance is a conotoxin having an amino acid sequence having sequence No. 2 or sequence No. 4. 3. A cosmetic composition for transdermal application, comprising an active substance and a transdermal penetration enhancer, characterized in that the active substance is encapsulated by the transdermal penetration enhancer, the transdermal penetration enhancer being a lipophilic activity modifier as defined in claim 1 or 2, and the active substance being a high molecular weight conopodide with a molecular weight greater than 2000 Da. 4. The composition according to claim 3, characterized in that it comprises at least one cosmetically acceptable excipient, said excipient being advantageously selected from the group consisting of: emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof. 5. The composition according to claim 3 or 4, characterized in that it is an oil-in-water (o / w) or water-in-oil (w / o) emulsion. 6. The composition according to any one of claims 3 to 5, characterized in that the ratio of the active substance to the transdermal penetration enhancer is 1:1 to 1:50. 7. The composition according to any one of claims 3 to 6, characterized in that it comprises at least one cosmetically acceptable excipient. 8. The composition according to any one of claims 3 to 7, characterized in that the active substance is conotoxin, preferably µ-conotoxin. 9. The composition according to claim 8, wherein the µ-conotoxin is a conotoxin having an amino acid sequence of Seq. no. 2. 10. The composition according to claim 8, wherein the µ-conotoxin is a conotoxin having an amino acid sequence having sequence No. 4 or a genetically modified form thereof. 11. The composition according to any one of claims 3 to 10, characterized in that it comprises at least one additional active ingredient. 12. A method for preparing a composition as defined in any one of claims 3 to 11, characterized in that the method is carried out by hot emulsification or cold emulsification, wherein the hot emulsification comprises the following steps: a) Mix dimethyl sulfone and diluent in a reaction vessel; b) Bring the mixture to 70-85°C; c) Stir the mixture at 70-85°C; the initiation of homogenization depends on the stage of phase combination; d) Cool the mixture to 20-40°C, preferably 30°C; e) Add high molecular weight active material with Mw > 500 Da and homogenize the mixture, preferably for 1-3 min; Cold emulsification involves the following steps: a) Mix dimethyl sulfone with a diluent in a reaction vessel and dissolve at 20-25°C; b) Bring the mixture to a temperature of 15-35°C, preferably to ambient temperature or 25°C; c) The mixture is mixed at a temperature of 15-80°C, preferably 25°C; the initiation of homogenization depends on the stage of phase combination; d) Add a high molecular weight active substance with Mw>500Da and homogenize the mixture, preferably for 1-3 min. 13. The method according to claim 12, characterized in that, in at least one of the thermal emulsification steps (ae), at least one additional active ingredient and / or at least one cosmetically acceptable excipient is added to the reaction vessel, said excipient being advantageously selected from the group consisting of emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof. 14. The method according to claim 12, characterized in that, in at least one of the cold emulsification steps (ad), at least one additional active ingredient and / or at least one cosmetically acceptable excipient is added to the reaction vessel, said excipient being advantageously selected from the group consisting of emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

Claims

1. A lipophilic activity modifier in the form of dimethyl sulfone (MSM), used as a transdermal transdermal accelerator for high molecular weight active substances with Mw > 500 Da.

2. The lipophilic activity modifier according to claim 1, characterized in that, The active substance is selected from the group including peptides.

3. The activity regulator according to claim 1 or 2, characterized in that, The active substance is a conospirin having an amino acid sequence of sequence No. 2 or sequence No.

4.

4. A cosmetic composition for transdermal application, comprising an active substance and a transdermal penetration enhancer, characterized in that, The active substance is encapsulated by a transdermal penetration enhancer, which is a lipophilic activity modifier as defined in claims 1-3, and the active substance is a high molecular weight active substance with Mw > 500 Da.

5. The composition according to claim 4, characterized in that, It contains at least one cosmetically acceptable excipient, which is advantageously selected from the group consisting of: emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

6. The composition according to claim 4 or 5, characterized in that, It is an oil-in-water (o / w) or water-in-oil (w / o) emulsion.

7. The composition according to any one of claims 4 to 6, characterized in that, The ratio of the active substance to the transdermal penetration enhancer is 1:1 to 1:50 (in 1 wag).

8. The composition according to any one of claims 4 to 7, characterized in that, It contains at least one cosmetically acceptable excipient.

9. The composition according to any one of claims 4 to 8, characterized in that, The active substance is conotoxin, preferably µ-conotoxin.

10. The composition according to claim 9, characterized in that, The µ-conotoxin is a conotoxin with the amino acid sequence Seq. no.

2.

11. The composition according to claim 9, characterized in that, The µ-conotoxin is a conotoxin or its genetically modified form having an amino acid sequence No.

4.

12. The composition according to any one of claims 4 to 11, characterized in that, It contains at least one additional active ingredient.

13. A method for preparing a composition as defined in any one of claims 4 to 12, characterized in that, The method is performed by hot emulsification or cold emulsification, wherein hot emulsification includes the following steps: a) Mix dimethyl sulfone and diluent in a reaction vessel; b) Bring the mixture to 70-85°C; c) Stir the mixture at 70-85°C; the initiation of homogenization depends on the stage of phase combination; d) Cool the mixture to 20-40°C, preferably 30°C; e) Add high molecular weight active material with Mw > 500 Da and homogenize the mixture, preferably for 1-3 min; Cold emulsification involves the following steps: a) Mix dimethyl sulfone with a diluent in a reaction vessel and dissolve at 20-25°C; b) Bring the mixture to a temperature of 15-35°C, preferably to ambient temperature or 25°C; c) The mixture is mixed at a temperature of 15-80°C, preferably 25°C; the initiation of homogenization depends on the stage of phase combination; d) Add a high molecular weight active substance with Mw>500Da and homogenize the mixture, preferably for 1-3 min.

14. The method according to claim 13, characterized in that, In at least one of the thermal emulsification steps (ae), at least one additional active ingredient and / or at least one cosmetically acceptable excipient are added to the reaction vessel, said excipient being advantageously selected from the group consisting of emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

15. The method according to claim 13, characterized in that, In at least one of the cold emulsification steps (ad), at least one additional active ingredient and / or at least one cosmetically acceptable excipient are added to the reaction vessel, said excipient being advantageously selected from the group consisting of emulsifiers, homogenizers, emollients, thickeners, distilled water, preservatives, or combinations thereof.

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