CN117205223A - Application of monogalactosyldiacylglycerogalactolipid in preparing drugs for treating psoriasis and pharmaceutical compositions thereof - Google Patents

Abstract

The application provides application of monogalactosyl diacylglycerol galactose in medicaments for treating and improving psoriasis, and a medicinal composition for treating and improving psoriasis. According to the application, the single galactosyl diacylglycerol galactolipid has a growth inhibition effect on human keratinocytes for the first time, animal experiment results show that compared with the back image of a positive control group, the administration group can obviously reduce erythema, infiltration and thickening, effectively inhibit proliferation of the keratinocytes, has a remarkable treatment effect on psoriasis according to the skin damage area and severity index (PASI) score and skin damage HE staining, and can effectively reduce the increase of MDSCs cell population, th1 cell population and Th17 cell population and the decrease of Tregs cell population, so that the MGDG external cream preparation provided by the application can effectively treat psoriasis.

Description

Application of monogalactosyldiacylglycerogalactolipid in the preparation of drugs for the treatment of psoriasis and pharmaceutical compositions thereof

Technical field

The present invention relates to the application of MGDG (Monogalactosyldiacylglycerol, monogalactosyldiacylglycerol galactolipid) in preparing drugs for treating psoriasis and its pharmaceutical composition, and belongs to the field of medicine.

Background technique

Psoriasis is a common chronic, relapsing, and inflammatory skin disease. Clinically, it mainly manifests as erythema and scale. Its histopathology is specific and manifests as excessive proliferation of epidermal keratinocytes, hyperkeratosis and parakeratosis. Acanthosis, sparse inflammatory cell infiltration around blood vessels in the superficial dermis, etc. The incidence rate of psoriasis in the natural population is 0.1% to 3%. Its pathogenesis is complex and it is easy to relapse. Most patients with psoriasis are mild to moderate, and topical drug treatment is the main treatment method and occupies an indispensable position.

80% of patients with psoriasis vulgaris have mild disease, so topical treatment is the preferred treatment option for psoriasis vulgaris. Currently commonly used topical drugs for psoriasis include glucocorticoids, retinoic acid, vitamin D derivatives, anthranol, tar preparations, and calcineurin inhibitors. The use of dithranol, tar preparations and glucocorticoids is limited due to serious side effects. The therapeutic effects of retinoic acid, calcineurin inhibitors and vitamin D derivatives are not very satisfactory. The recently launched calcipotriol betamethasone ointment, a compound preparation of glucocorticoids and vitamin D derivatives, is slightly more effective than calcipotriol, but its cost is high. The limited choice of topical drugs and the easy development of tolerance have always been the bottleneck of local treatment of psoriasis. Therefore, the development of new drugs for the treatment of psoriasis is of great significance.

MGDG (Monogalactosyldiacylglycerol, monogalactosyldiacylglycerogalactolipid) was originally isolated from plants more than 40 years ago and accounts for 80% of membrane lipids in green plant tissues. In contrast to the phospholipid-rich animal and yeast cell membranes, these lipids are the major components of the photosynthetic membranes of higher plants, algae, and bacteria. In animals, MGDG is present in low concentrations. The basic structure of MGDG is galactoseglycerol connecting two fatty acid chains. Research has found that MGDG extracted from plants has antibacterial, antiviral, antitumor and anti-inflammatory activities. (BRUNO A, ROSSI C, MARCOLONGO G, etal. Selective in vivo anti-inflammatory action of the galactolipidmonogalactosyldiacylglycerol[J]. Eur J Pharmacol, 2005, 524(1-3):159-168.)

The properties of MGDG in disease treatment have not yet been explored, so its application as an active ingredient in drugs has not been reported. It is necessary to further develop preparations of MGDG as an active ingredient to provide more options for topical drugs for the treatment of psoriasis. .

Contents of the invention

In view of the problems existing in the prior art, one of the purposes of the present invention is to provide the use of MGDG (Monogalactosyldiacylglycerol, monogalactosyldiacylglycerolgalactolipid) in the preparation of drugs for the treatment of psoriasis. The structural formula of the above-mentioned MGDG is shown in Formula I. :

Preferably, the above-mentioned psoriasis is psoriasis vulgaris.

Preferably, the above-mentioned drug is a drug that alleviates and improves psoriasis by inhibiting the growth of keratinocytes.

Preferably, the above-mentioned drugs are drugs that alleviate and improve psoriasis by inhibiting the increase of MDSCs, Th1, and Th17 and the decrease of Tregs.

Preferably, the drug is used to improve skin lesions, thereby alleviating and ameliorating psoriasis. More preferably, the drug can improve erythema, infiltration, and thickening of the skin.

The second object of the present invention is to provide a pharmaceutical composition, wherein MGDG is the active ingredient of the pharmaceutical composition.

Preferably, the above-mentioned medicine is an external medicine; a preferred embodiment is a cream for external use.

Preferably, the external medicine further includes an external base, and the external medicine is prepared after MGDG is mixed with the base.

More preferably, the base is selected from the group consisting of white petrolatum, stearyl alcohol, glyceryl monostearate, p-hydroxybenzoic acid, glycerin, sodium lauryl sulfate and water; wherein, white petrolatum, stearyl alcohol, monostearate Glyceryl esters and parahydroxybenzoic acid are the oil phase, and glycerin, sodium lauryl sulfate and water are the water phase.

More preferably, the mass percentage of the active ingredient MGDG in the external medicine is 0.2 to 98%, and the optimum is 0.2%.

the term

IMQ:Imiquimod, imiquimod

PASI: Psoriasis area and severity index, psoriasis area and severity index

MDSCs: Myeloid-derived suppressor cells, myeloid-derived suppressor cells

Tregs: Regulatory cells, regulatory T cells

Th1: Type 1helper T cells, type 1 helper T cells

Th17: Type 17helper T cells, type 17 helper T cells

Compared with the existing technology, the present invention has discovered for the first time that MGDG has a growth inhibitory effect on human keratinocytes. According to the results of animal experiments, compared with the pictures of the back of the administration group and the positive control group, erythema, infiltration, and thickening were all significant. Reduce and effectively inhibit the proliferation of keratinocytes. According to the Psoriasis Area and Severity Index (PASI) score and HE staining of skin lesions, it has a significant therapeutic effect on psoriasis. It can effectively reduce the MDSCs cell population and Th1 cells. The increase in the Th17 cell population and the decrease in the Tregs cell population indicate that the MGDG topical cream preparation provided by the present invention can effectively treat psoriasis. MGDG as a new topical drug provides a new direction for the treatment of psoriasis vulgaris.

Description of the drawings

Figure 1 is a photograph of the back of each group of mice in the external ointment administration group and the control group obtained in Example 1;

Figure 2 is a diagram of the PASI scores of the back skin lesions of mice in the IMQ control group and the external ointment administration group obtained in Example 1 from the first day to the seventh day;

Figure 3 is a statistical diagram of HE staining and epidermal thickness of the back skin lesions of mice in each group of mice in the external ointment administration group and the control group obtained in Example 1 on the seventh day;

Figure 4 is a diagram showing flow cytometric detection results of mouse back skin cells in each group of mice in the external ointment administration group and the control group obtained in Example 1;

Figure 5 is a comparison chart of experimental results of mouse therapeutic effects in the external cream administration group and the control group obtained in Examples 1-5;

Figure 6 is a photograph of the back of mice in the external cream administration group and control group obtained in Examples 1-5;

Figure 7 is a comparison chart of the growth inhibition experimental results of human keratinocytes in the Example.

Detailed ways

The present invention will be further described in detail and completely below with reference to the examples. The embodiments described below are illustrative and are only used to explain the present invention and are not to be construed as limitations of the present invention.

The experimental methods in the following examples, unless otherwise specified, usually follow conventional conditions such as those described in Pharmacology Experimental Manual, Third Edition, Science Press, 2002, in which imiquimod (IMQ) is modeled and The method of treating psoriasis is based on published literature ("Journal of the European Academy of Dermatology and Venereology" "Lysophosphatidylcholine facilitates the pathogenesis ofpsoriasis through activating keratinocytes and T cells differentiation viaglycolysis"), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be purchased through commercial channels.

The main materials involved in the embodiments of the present invention are as follows:

1. Experimental animals

Specific pathogen-free (SPF) BAL B/C male mice, 6-8 weeks old, weighing 20-23 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. and raised at the Experimental Animal Center of Central South University. This study has passed the experimental animal welfare ethics review of Central South University. During the experiment, the "3R" principle was strictly followed to minimize the pain and discomfort of the animals.

2. Experimental reagents

Example 1

1. Preparation of topical cream preparations

1. Use weighing paper to weigh 4 mg of MGDG and dissolve it in 20 μL of DMSO to prepare an MGDG solution for later use. Add 240mg white petroleum jelly, 160mg stearyl alcohol, 40mg glyceryl monostearate and 4mg p-hydroxybenzoic acid into a beaker, put it into a DF-101S collector-type constant temperature heating magnetic stirrer, heat and dissolve in a 70°C water bath, and stir for 10 Minutes, the oil phase of the cream is obtained; at the same time, add 140 mg glycerin, 20 mg sodium lauryl sulfate and 1.4 g water into another beaker and heat to dissolve in the same way, stir for 5 minutes, and obtain the water phase of the cream. Then slowly add the water phase to the oil phase and stir for 10 minutes. Finally, slowly add the MGDG solution and continue stirring for 3 minutes. Leave it at room temperature and quickly stir with a glass rod to a semi-solid state at room temperature to prepare the above-mentioned 0.2% MGDG cream preparation for treating psoriasis. , bottled for later use.

In Examples 2-10, creams were prepared using the proportions shown in Table 1. The preparation method is the same as that in Example 1 and will not be described again.

Example MGDG Example 2 0.1% Example 3 0.5% Example 4 2% Example 5 10%

Note: MGDG is percentage content.

Comparative example 1

The difference between this comparative example and Example 1 is that MGDG is not added as an active ingredient, and the dosage of cream excipients is the same as that of Example 1. Add white petroleum jelly, stearyl alcohol, glyceryl monostearate and p-hydroxybenzoic acid in proportion to the beaker, put it into the DF-101S collector-type constant temperature heating magnetic stirrer, heat and dissolve in a 70°C water bath, and stir for 10 minutes to obtain The oil phase of the cream; at the same time, add glycerin, sodium lauryl sulfate and water in proportion to another beaker and heat to dissolve in the same manner. Stir for 5 minutes to obtain the water phase of the cream. Then slowly add the water phase to the oil phase and stir for 10 minutes. Leave it at room temperature and quickly stir with a glass rod to a semi-solid state at room temperature to prepare a control cream preparation without MGDG.

Animal experiment

The following animal experiments evaluate the alleviating effects of different percentages of MGDG on imiquimod (IMQ)-induced psoriasis in mice.

1. Imiquimod (IMQ) modeling

The mouse model induced by imiquimod can well simulate psoriasis symptoms similar to humans in terms of skin thickening, abnormal keratinocyte-related proteins, inflammatory cell infiltration and related inflammatory cytokines. This model has become one of the most widely researched and applied psoriasis models due to its simplicity, ease of operation and stability.

Fifteen BALB/C mice with SPF level of 6-8 weeks were randomly divided into 3 groups, including 6 mice in the first group, 6 mice in the second group, and 6 mice in the third group. The shaved area on the back was 2cm × 2cm. Each group is processed as follows:

After each group was continuously treated for 7 days, the psoriasis-like symptoms of the mice were evaluated using the Psoriasis Area and Severity Index (PASI) score on the back skin lesions of the mice in each group. The mice were then weighed, photographed, and killed. The skin lesions were subjected to HE staining experiments to examine the thickness of the epidermis, i.e., the proliferation of keratinocytes, and cell flow cytometry to examine changes in the number of psoriasis-related immune cells. At the same time, the spleens of the mice were weighed and photographed.

Figure 1 shows the back photos of mice in each group on the 7th day. It can be seen that compared with the back skin lesions of the negative control group and the blank control group, there are obvious erythema, thickening, and scales, proving that the psoriasis after applying imiquimod The disease model was successfully induced. Compared with the negative control group, erythema, infiltration, and thickening in the administration group were significantly reduced, indicating that the 0.2% MGDG cream preparation can effectively treat psoriasis.

Figure 2 shows the PASI scores of the back skin lesions of mice in the IMQ control group and MGDG administration group from the first to the seventh day. Figure 2 shows that MGDG cream can effectively treat psoriasis.

Figure 3 is a statistical graph of HE staining and epidermal thickness of the back skin lesions of mice in each group on the seventh day. As shown in Figure 3, it can be seen that MGDG cream can effectively inhibit keratinocyte proliferation.

Figure 4 shows the results of flow cytometry of mouse back skin cells in each group. As shown in Figure 4, it can be seen that the number of MDSCs cell population, Th1 cell population, Th17 cell population and Tregs cell population in the IMQ control group were significantly increased compared with the drug administration group. The MGDG administration group could significantly inhibit the increase of MDSCs, Th1, Th17 and the decrease of Tregs induced by IMQ. Comparison of therapeutic effects of different dosages of creams

The same experimental method as above was used to compare the therapeutic effects of the MGDG creams of Examples 2 to 4. After continuous administration for 7 days, the 0.1% MGDG cream of Example 2 had no obvious therapeutic effect. There is no significant difference between the dosage of MGDG cream and Example 1. The specific experimental results are shown in Figures 5 and 6.

Growth inhibition assay of human keratinocytes

Extract primary keratinocytes from human foreskin tissue and culture them. When the cells are in good growth condition, 10,000 cells per well are seeded into a 96-well plate (keratinocytes are difficult and slow to adhere to). 100ul of cell suspension is added to each well. Each group is 6 duplicate wells, the same volume of PBS was added to the blank wells. Four time points were set to detect the cell proliferation rate: 0 hour, 24 hours, 48 hours, and 72 hours. After seeding the plate overnight, add 0.01uM, 0.1uM, 1uM, and 10uM MGDG solutions respectively (dissolve 1mgMGDG in 1.3mL DMSO to prepare a 1mM MGDG stock solution) until the cells adhere. The CCK8 method was used to measure the absorbance value of each well at a wavelength of 450 nm. The results are shown in Figure 7. Compared with the 0uM blank control, 1uM and 10uM MGDG had an inhibitory effect on keratinocyte growth.

The above experimental results show that only 0.2% MGDG cream can be used to treat psoriasis. As shown in the mouse animal experiment results, it can be seen that erythema, infiltration, and thickening are significantly reduced, and keratinocyte proliferation is inhibited. IMQ-induced increases in MDSCs, Th1, Th17, and decreases in Tregs, as well as PASI scores, confirm its effective therapeutic effect on psoriasis; in addition, in vitro experiments also confirmed that 1uM and 10uM MGDG can inhibit the growth of human keratinocytes; MGDG as a new Topical drugs provide new directions for the treatment of psoriasis vulgaris.

Finally, it is necessary to explain here that the above embodiments are only used to further explain the technical solution of the present invention in detail and cannot be understood as limiting the protection scope of the present invention. Any skilled person familiar with this profession will not deviate from the technical solutions of the present application. Within the scope of the solution, slight changes, modifications, substitutions, combinations, and simplifications made using the technical content disclosed above should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (10)

1. The use of monogalactosyldiacylglycerogalactolipid in preparing a drug for treating psoriasis, characterized in that the structural formula of the monogalactosyldiacylglycerogalactolipid is as shown in Formula I: 2. The use according to claim 1, characterized in that the psoriasis is psoriasis vulgaris. 3. The use according to claim 1, characterized in that the drug is a drug that alleviates and improves psoriasis by inhibiting the growth of keratinocytes. 4. The use according to claim 1, characterized in that the drug is a drug that alleviates and improves psoriasis by inhibiting the increase of MDSCs, Th1, and Th17 and the decrease of Tregs. 5. The use according to claim 1, characterized in that the drug is used to improve skin lesions and thereby alleviate and improve psoriasis. 6. Pharmaceutical composition, characterized in that the active ingredient of the drug is monogalactosyldiacylglycerogalactolipid, and the structural formula is as shown in formula I: 7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is an external drug. 8. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is a cream for external use. 9. The pharmaceutical composition according to claim 6, wherein the mass percentage of the active ingredient monogalactosyldiacylglycerogalactolipid in the external medicine is 0.2 to 98%. 10. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition for external use also includes an external base, and the external medicine is prepared by mixing monogalactosyldiacylglycerogalactolipid with the base. .