CN113995742B - Whitening composition containing valproic acid and application thereof - Google Patents

Abstract

The invention belongs to the technical field of skin external medicines, and specifically relates to a whitening composition containing valproic acid and its use for skin whitening. The present invention provides a composition comprising valproic acid (VPA) or a pharmaceutically acceptable salt thereof, characterized in that the composition is used for skin whitening. The inventor unexpectedly discovered that valproic acid, a neurology drug, can inhibit the expression of tyrosinase and inhibit melanin production, and can be used for the treatment of pigmentation diseases or as a whitening active ingredient.

Description

Whitening compositions containing valproic acid and uses thereof

Technical field

The invention belongs to the technical field of external skin drugs, and specifically relates to a whitening composition containing valproic acid and its use.

Background technique

Due to the increasingly harsh natural environment, increased life and work pressure, and increasing age, people's skin often suffers from pigmentary diseases such as dark yellow, pigmentation, and spots. Improving skin color has gradually become an area of widespread concern, and products with whitening effects Products also receive much attention. The color of skin is determined by the content of melanin, carotenoids in the epidermis and oxyhemoglobin, hemoglobin, bilirubin and other pigments in the dermis, with melanin being the main influencing factor. Melanin is a high molecular polymer mainly synthesized and secreted by melanocytes (melanoncyte, MC). There is a specific organelle in melanocytes - melanosome, which contains melanin granules, and the synthesis, storage and transport of melanin granules are all carried out in melanosomes. Excessive deposition of melanin in the skin can cause dull skin color and even cause hyperpigmentation diseases. Therefore, the ability and efficiency of melanocytes to synthesize, transport and degrade melanin have a direct impact on skin color.

There are many clinical types of pigmentary dermatoses, and methods such as laser and drugs cannot satisfy the majority of beauty seekers. Tranexamic acid is a synthetic amino acid antifibrinolytic drug that can competitively inhibit the binding of lysine in fibrin to plasmin, thereby inhibiting the lysis of fibrin clots and producing hemostasis. Previous clinical studies It is mainly used for various types of bleeding caused by hyperfibrinolysis. It is FDA approved for the treatment of menorrhagia and the prevention of bleeding. Existing clinical studies have shown that oral and/or topical tranexamic acid has good efficacy in the treatment of chloasma with minimal adverse reactions. It can be treated alone or in combination with other drugs or methods. It is currently the drug of choice for the treatment of chloasma. However, the mechanism of action of tranexamic acid in treating melasma remains uncertain. Currently, there is still a certain recurrence rate for melasma treated with tranexamic acid. Moreover, because tranexamic acid is highly hydrophilic, it is difficult to pass through the barrier of the stratum corneum of the skin. It is necessary to encapsulate the drug through penetration-enhancing technology such as liposomes to promote transdermal permeability.

Therefore, there is an urgent need to develop skin whitening products, especially whitening products that can improve the effects or medication methods of pigment diseases such as dark yellow skin, pigmentation, and spots.

Contents of the invention

In order to improve the above technical problems, the present invention provides a composition containing valproic acid (VPA) or a pharmaceutically acceptable salt thereof, characterized in that the composition is used for skin whitening.

According to an embodiment of the present invention, the "whitening" means whitening the color of the skin, including but not limited to reducing the natural coloration, pigmentation, dark yellow, spots, etc. of the skin.

According to an embodiment in the context of the present invention, "natural coloration" means coloration induced by factors such as external conditions (such as natural light, eg irradiation of UVA and/or UVB) and/or due to chronic aging of the skin.

According to embodiments in the context of the present invention, "pharmaceutically acceptable salts" include, but are not limited to, valproate with alkali metal (eg, lithium, sodium, potassium) ions, alkaline earth metal (eg, magnesium or calcium) ions, aluminum ions , a salt formed from at least one ammonium ion (NH4 ⁺ ), or an addition salt selected from at least one selected from valproic acid and at least one organic base selected from the following: alkylamine, for example, ethanolamine, diethanolamine, triethylamine Ethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine, or an addition salt selected from valproic acid and at least one amino acid selected from the following : Glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine Acid, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine.

According to an embodiment of the present invention, the composition may be a whitening agent or an anti-pigmentation agent, for example, the composition may be a cosmetic composition or a pharmaceutical composition, such as a cosmetic preparation or a pharmaceutical preparation.

According to embodiments of the present invention, the composition may further comprise cosmetically acceptable excipients or pharmaceutically acceptable excipients.

According to an embodiment of the present invention, the composition is a cosmetic composition comprising cosmetically acceptable excipients.

According to an embodiment of the present invention, the composition is a pharmaceutical composition, which contains pharmaceutically acceptable excipients.

According to an embodiment of the present invention, the composition may be a topical or oral preparation, preferably a topical preparation, such as a preparation suitable for topical application, in particular to the skin.

According to an embodiment of the present invention, the term "cosmetically acceptable excipient" is intended to mean one that does not have an unpleasant odor or appearance and does not cause any unacceptable stinging to the user when applied topically to the skin or skin appendages, Tightening or redness medium, for example selected from common cosmetic additives including but not limited to: water, solvents, oils, waxes, pigments, fillers, surfactants, cosmetic or dermatological actives, UV sunscreens, polymers substances, gelling agents, preservatives and fragrances.

According to embodiments of the present invention, the "pharmaceutically acceptable excipients" may be selected from, for example, excipients. Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup and methylcellulose. The preparations may also contain: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and hydroxypropyl benzoate; sweetening and flavoring agents. The compositions of the present invention may be formulated to provide immediate, sustained or delayed release of the active ingredient following administration using methods known in the art.

According to embodiments of the present invention, the composition may have a form selected from the following depending on the mode or site of administration: tablets, pills, powders, lozenges, sachets, cachets, elixirs, mixes Suspensions, emulsions, solutions, syrups, aerosols (solid or dissolved in liquid solvents), pastes (such as ointments), soft gelatin capsules, hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

According to embodiments of the present invention, the composition may also contain or not contain other whitening active ingredients, for example, it may or may not contain additional other active ingredients with whitening effect.

As an example, the whitening active ingredient of the composition is only valproic acid and/or a pharmaceutically acceptable salt thereof.

According to an embodiment of the present invention, the composition comprises a whitening effective amount of valproic acid and/or a pharmaceutically acceptable salt thereof.

As an example, the composition may be a single-dose composition or a multi-dose composition, wherein in each dose of the composition, valproic acid and/or a pharmaceutically acceptable salt thereof is present in a whitening effective amount. For example, the content of valproic acid and/or a pharmaceutically acceptable salt thereof in each dose of the composition may be about 1 to 1000 mg, such as 5 to 800 mg, such as 100 to 500 mg. It should be understood that the actual amount of valproic acid and/or a pharmaceutically acceptable salt thereof administered can generally be determined according to specific circumstances, which may include the skin condition of the subject, the route of administration selected, and the actual amount of valproic acid administered. compounds, the age, weight and sensitivity of the individual, etc.

According to embodiments of the present invention, the compositions of the present invention may be sterilized by known sterilization techniques or by filterable sterilization or the like. For example, the composition may be packaged for use as is, or may be lyophilized and the lyophilized formulation mixed with a sterile aqueous vehicle prior to administration. The pH of the composition is generally between 3 and 11, more preferably between 5 and 9, and most preferably between 7 and 8. It is understood that the use of certain excipients, carriers or stabilizers may cause valproic acid to form salts.

The present invention also provides a preparation method of the composition, which includes mixing valproic acid or a pharmaceutically acceptable salt thereof with other components in the composition.

The present invention also provides the use of valproic acid, a pharmaceutically acceptable salt thereof or a composition thereof as described above, wherein the valproic acid or a pharmaceutically acceptable salt thereof is used for at least one selected from the following Usage: For whitening, for inhibiting the generation of unwanted cells, for inhibiting the expression of tyrosinase, for inhibiting the expression of tyrosinase-related protein-1 gene, for inhibiting tyrosinase-related protein -2 gene expression.

The present invention also provides the use of the above-described valproic acid, pharmaceutically acceptable salts thereof or compositions thereof for preparing cosmetics or medicines, wherein the cosmetics or medicines can be used for at least one purpose selected from the following : Used for whitening, used to inhibit the generation of unwanted cells, used to inhibit the expression of tyrosinase, used to inhibit the expression of tyrosinase-related protein-1 gene, used to inhibit tyrosinase-related protein- 2 gene expression.

The present invention also provides a method for whitening skin, comprising applying valproic acid, a pharmaceutically acceptable salt thereof or a composition thereof as described above to the skin.

The present invention also provides a method for improving skin color, comprising applying to the skin valproic acid, a pharmaceutically acceptable salt thereof, or a composition thereof as described above.

The present invention also provides a method for preventing and/or treating pigmented skin diseases, comprising administering valproic acid, a pharmaceutically acceptable salt thereof or a composition thereof as described above to a subject in need thereof, such as a patient, preferably a human .

According to the method of the present invention, the site of application is skin.

According to an embodiment of the present invention, the pigmented skin disease is selected from at least one of dark yellow skin, pigmentation, stains, etc.

The present invention also provides methods of inhibiting undesirable cells, comprising administering valproic acid, a pharmaceutically acceptable salt thereof, or a composition thereof as described above to a subject in need thereof.

According to an embodiment of the present invention, the undesirable cells are selected from cells causing skin darkening or pigmented skin diseases, such as melanocytes, for example selected from the group consisting of human melanoma cells (MNT1), human primary melanocytes Cells (HEMn) or human immortalized keratinocytes (HaCaT) and human melanoma cells (MNT1) co-cultured with KRT5 gene silenced by lentivirus.

Those skilled in the art will understand that the above-described methods and uses of the present invention may be for therapeutic or non-therapeutic purposes depending on the conditions of the subject.

beneficial effects

The inventor unexpectedly discovered that valproic acid, a neurology drug, can inhibit the expression of tyrosinase and inhibit melanin production. It can be used for the treatment of pigmentation diseases and can be used as a whitening active ingredient. Human melanoma cells (MNT1), human primary melanocytes (HEMn), HaCaT cells with lentiviral silenced KRT5 gene and human melanoma cells (MNT1) were co-cultured and treated with VPA, tyrosinase (TYR) The expression of KRT5 gene haploinsufficiency HaCaT cells (KRT5-1/0) edited by CRISPR/Cas9 was co-cultured with human primary melanocytes (HEMn), and the melanin in the valproic acid-treated group was significantly reduced. reduce. Valproic acid is a small molecule drug and is expected to be made into a drug for external use, which is safer for external use.

Description of the drawings

Figure 1 is a comparison chart of the immunoblotting experiment results of human melanoma cells (MNT1) in Example 1.

Figure 2 is a comparison chart of the immunoblotting experimental results of human primary melanocytes (HEMn) in Example 1.

Figure 3 is a comparative chart of immunoblot experiment results of co-culture of HaCaT cells with KRT5 gene silenced by lentivirus and human melanoma cells (MNT1) in Example 1.

Figure 4 is a comparison chart of laser confocal experiment results of HaCaT cells (KRT5-1/0) edited by CRISPR/Cas9 with haploinsufficiency of the KRT5 gene in Example 1 and co-cultured with human primary melanocytes (HEMn).

Detailed ways

The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following examples are only illustrative and explain the present invention and should not be construed as limiting the scope of the present invention. All technologies implemented based on the above contents of the present invention are covered by the scope of protection intended by the present invention.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

Example 1: Cell immunoblotting experiment

1. Experimental methods

(1) Cell culture and passage: MNT1 cells are grown in DMEM medium containing 20% fetal calf serum, placed in a 37°C, 5% carbon dioxide incubator, and passaged once every 2-3 days, with 1 passage each time 3 proportions were carried out; the MNT1 cells in the experimental group added VPA dissolved in DMSO to their culture medium to make the final concentration of VPA 0.1mM. The MNT1 cells in the control group added an equal volume of DMSO solvent to the culture medium of the experimental group. The control group was cultured according to the above culture conditions for 72 hours; human primary melanocytes (HEMn) were grown in MelM medium, placed in a 37°C, 5% carbon dioxide incubator, and passaged once every 2-3 days, with 1 passage each time. 2 proportions; the HEMn cells in the experimental group added VPA dissolved in DMSO to their culture medium to make the final concentration of VPA 0.1mM. The HEMn cells in the control group added an equal volume of DMSO solvent to the culture medium of the experimental group. The control group was cultured according to the above culture conditions for 72 h.

(2) Protein extraction from cells: Wash the cells three times with PBS, add an appropriate amount of RIPA lysis buffer, let stand on ice for 30 minutes, centrifuge at high speed for 30 minutes and then take the supernatant. After quantitating the extracted protein by BCA method, add SDS heats and deforms the protein and then packages it.

(3) Western blotting experiment: Install the precast gel together with the glass plate into the electrophoresis tank, add the electrophoresis buffer and start preparing to load the sample. Use a micro-injector to absorb the sample against the wall. Aspirate the sample without sucking in air bubbles. Insert the needle of the sample container into the sampling hole and slowly add the sample, then connect the electrophoresis tank to the electrophoresis instrument for electrophoresis. Cut the PVDF membrane according to the size of the glue and soak it with pure methanol for 1 minute to activate. Assemble the transfer sandwich: 2 layers of filter paper sponge sponge, 2 layers of filter paper sponge. After each layer is placed, remove the bubbles and place the transfer tank in an ice bath. , put the sandwich (black side with black side), add transfer solution, plug in the electrode, 300mA, 2h. After the transfer is completed, cut off the power, take out the membrane, block it with quick blocking solution for 5-10 minutes, cut the target band according to the position of the marker, add primary antibody of appropriate dilution and incubate, overnight at 4°C, and wash 3 times with TBST. , add horseradish peroxidase (HRP)-labeled secondary antibody at an appropriate dilution, incubate at room temperature with slow shaking for 1.5 hours, wash 3 times with TBST, dilute A and B luminescent solutions at a 1:1 ratio, mix and develop.

(4) CRISPR/Cas9-edited HaCaT cells with KRT5 gene haploinsufficiency and human primary melanocytes (HEMn) were inoculated into laser confocal culture dishes, added VPA and cultured for 72 hours, then discarded the medium, and added the control group, etc. volume of DMSO, wash three times with PBS, add 1 ml of 4% paraformaldehyde to each dish to fix the cells, let stand at room temperature for 30 minutes, then wash three times with PBS, permeate with 0.2% Triton X-100 for 10 minutes, and wash three times with PBS. The primary antibody was washed three times with PBS overnight at 4°C, then the secondary antibody was added and placed at 37°C for 2 hours in the dark. The cells were washed three times with PBS, stained with DAPI, washed three times with PBS, and observed under a laser confocal microscope.

2.Experimental results

The experimental results are shown in Figure 1-4, where:

Figure 1 shows that western blot experiments verified that after human melanoma cells (MNT1) were treated with VPA for 72 hours, TYR expression was reduced compared with the control group.

Figure 2 shows that Western blotting experiments verified that after 72 hours of VPA treatment in human primary melanocytes (HEMn), TYR expression was reduced compared with the control group.

Figure 3 shows that Western blotting experiments verified that HaCaT cells with lentivirus-silenced KRT5 gene were co-cultured with human melanoma cells (MNT1). Compared with the control group, melanogenesis-related proteins were up-regulated (MITF, TYR increased), while in the VPA-treated group Reversing this effect, MITF and TYR decreased.

Figure 4 shows that laser confocal experiments verify that CRISPR/Cas9 edits HaCaT cells (KRT5-1/0) with haploinsufficiency of KRT5 gene, co-cultured with human primary melanocytes (HEMn), KRT5-1/0 group and control Compared with the Negative group, the red fluorescence of Pmel17 was enhanced, indicating an increase in melanin, while the red fluorescence of Pmel17 in the VPA-treated group was weakened, indicating a decrease in melanin.

The embodiments of the present invention have been described above through exemplary embodiments. However, the present invention is not limited to the above-described embodiment. Any modifications, equivalent substitutions and improvements made by those skilled in the art within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

Claims (2)

1. The use of valproic acid for preparing pharmaceutical preparations, wherein the pharmaceutical preparation is used to inhibit melanogenesis of human melanoma cells MNT1 and human primary melanocytes HEMn; Wherein, the active ingredient of the pharmaceutical preparation is only valproic acid. 2. The use according to claim 1, wherein the pharmaceutical preparation further contains pharmaceutically acceptable excipients.